Botanical Research: Series Editors

Advances in
BOTANICAL RESEARCH
Series Editors
JEAN-CLAUDE KADER Laboratoire Physiologie Cellulaire
et Moléculaire des Plantes, CNRS,
Université de Paris, Paris, France
MICHEL DELSENY Laboratoire Génome et

Développement des Plantes,
CNRS IRD UP, Université de
Perpignan, Perpignan, France
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CONTRIBUTORS TO VOLUME 59
CLAUDE ALBAN Laboratoire de Physiologie Cellulaire Végétale, CNRS,

UMR5168; CEA, DSV, iRTSV; INRA, UMR1200; Université Joseph
Fourier, Grenoble, France
GILLES J. BASSET Center for Plant Science Innovation, University of
Nebraska-Lincoln, Lincoln, Nebraska, USA
DEAN DELLAPENNA Department of Biochemistry and Molecular
Biology, Michigan State University, East Lansing, Michigan, USA
ROLAND DOUCE Laboratoire de Physiologie Cellulaire et Végétale,
CNRS, UMR5168; CEA, iRTSV; INRA, UMR1200; Universite´
Joseph Fourier, F-38054 Grenoble, France
ANNE-LISE DUCLUZEAU Center for Plant Science Innovation,
University of Nebraska-Lincoln, Lincoln, Nebraska, USA
TERESA B. FITZPATRICK Department of Botany and Plant Biology,
Sciences III, University of Geneva, Geneva, Switzerland
FABIENNE FURT Center for Plant Science Innovation, University of
Nebraska-Lincoln, Lincoln, Nebraska, USA
LAURENT MÈNE-SAFFRANÉ Department of Biochemistry and Molec-
ular Biology, Michigan State University, East Lansing, Michigan, USA
FABRICE RÉBEILLÉ Laboratoire de Physiologie Cellulaire et Végétale,
CNRS, UMR5168; CEA, iRTSV; INRA, UMR1200; Universite´
STÉPHANE RAVANEL Laboratoire de Physiologie Cellulaire et Végé-
tale, CNRS, UMR5168; CEA, iRTSV; INRA, UMR1200; Universite´
NICHOLAS SMIRNOFF Biosciences, College of Life and Environmental
Sciences, University of Exeter, Exeter EX4 4QD, United Kingdom
CHLOË VAN OOSTENDE Center for Plant Science Innovation,
University of Nebraska-Lincoln, Lincoln, Nebraska, USA
JOSHUA R. WIDHALM Center for Plant Science Innovation, University
of Nebraska-Lincoln, Lincoln, Nebraska, USA
PREFACE
VITAMINS: A PLANT AFFAIR

All organisms need to synthesize, transform and interconvert a myriad of
molecules to enable them to grow and reproduce. All these reactions are
catalysed by enzymes (the living tools) which facilitate chemical modifica-
tions of substrates owing to their specific binding properties. In many cases,
suitable coenzymes (nicotinamide adenine dinucleotide [NADþ], nicotin-
amide adenine dinucleotide phosphate [NADPþ], flavin adenine dinucleotide
[FAD], flavin mononucleotide [FMN], pyridoxal 50 -phosphate, biotin, coen-
zyme A, etc.) may assist in biochemical transformations. Some of these
coenzymes may be more or less tightly bound to enzymes as part of prosthet-
ic groups (biotin, FMN, etc.). Coenzymes may also be loosely bound to
enzymes as detachable molecules. In that case, they are acting as substrates,
being often recycled through other set of reactions (NAD(P)þ, folates,
ascorbate, etc.).
Vitamin (a combination word from vita and amine) are by definition
dietary substances required for good health and normal development that
are only synthesized by microorganisms and plants. During the course of
animal evolution, the ability to biosynthesize these compounds has been lost
and, instead, elaborate uptake mechanisms have been developed. There are
13 recognized vitamins, involved in various catalytic functions. The largest
number of vitamins serve as precursors to coenzymes (vitamins B1 [thiamine],
B2 [riboflavin], B3 [niacin], B5 [pantothenic acid], B6 [pyridoxine], B9 [folic
acid]) or as coenzymes themselves (vitamins B8 [biotin], B12 [cobalamin], C
[ascorbic acid], K [phylloquinone, menaquinone]). Some of these vitamins,
especially the hydrophobic (vitamins A [retinol, pro-vitamin A carotenoids],
E [tocopherols, tocotrienols] and D [ergocalciferol, cholecalciferol]), cannot
be truly considered as coenzymes: vitamins A and D display hormonal effects
in the human body, and vitamin E has a protective role in membranes by
scavenging free radicals. Vitamins are involved in almost all important
cellular functions, displaying protective (antioxidant) functions or participat-
ing to numerous metabolisms, including the energetic metabolism (respira-
tion, photosynthesis) and the metabolisms of sugars, amino acids, fatty acids
and nucleic acids. The daily amount of vitamins required for a good health
depends on the considered vitamin and fluctuates widely, from a few micro-
grams (B12, D, K) to several milligrams (B3, B5, C). Vitamin deficiencies are
xii PREFACE
quite common in low-resource countries but also occur in developed

countries due to bad food habits. Well-known vitamin-related diseases in-
clude, among others, blindness (vitamin A), beriberi (vitamin B1), pellagra
(vitamin B3), anaemia (vitamins B6 and B9), scurvy (vitamin C), rickets
(vitamin D) or neural tube defects (vitamin B9). In addition, antioxidant
vitamins (such as A, C, E and B6) have protective roles as efficient quenchers
of reactive oxygen species.
Plants synthesize an impressive diverse array of natural products including
vitamins, and plants are considered as a major nutritional source for these
essential molecules. Plants are able to synthesize 12 out of the 13 vitamins.
Indeed, plants have no cobalamin-dependent proteins and use for methio-
nine synthesis an alternate catalytic mechanism that does not need vitamin
B12. Vitamin B12 is only synthesized in prokaryotes, and humans primarily
obtained it from animal food, thanks to the intestine flora of herbivores. Two
of the vitamins (vitamins A and D) have ‘hormonal’ functions in animals,
which functions do not exist in plants. Plants do not synthesize vitamin A,
but carotenoids. Some of these carotenoids are pro-vitamin A, which are
transformed in retinol once assimilated by animals. Vitamin D (D2 and D3) is
formed from the precursors ergosterol (mainly present in fungal cells) and
cholesterol (mainly present in mammalian cells) following sun exposure (UV
radiation). Although vitamins D2 and D3 can be found in low amounts in the
membranes of some Solanaceous plants, higher plants are not considered as a
source of vitamin D and plant food cannot compensate insufficient synthesis
in the human body. Thus, the plant kingdom is a recognized dietary source
for 11 out of the 13 vitamins.
As many vitamins are only required in trace quantities, their biosynthesis is
normally strictly controlled and the involved enzymes are generally produced
in very small amounts. This is why it has been extremely difficult to elucidate
their complete biosynthetic pathways, and it still remains the case that several
steps within the biosynthesis of vitamins are poorly understood (e.g. thiazole
ring scaffolding). However, the advent of modern recombinant DNA tech-
niques, coupled with the completion of many genome projects, made possible
to decipher pathways in plants, thus allowing now a more complete under-
standing of how these molecules are made. The general picture emerging
from these recent data indicates that the metabolic web represented by these
molecules is of a rare complexity. Indeed, not only may the synthesis of
vitamins require some 10 enzymatic steps but also several of these metabolic
routes are split between various compartments of the plant cell, adding a
further level of complexity when compared to prokaryotes. Since all cell
compartments need their vitamins, this situation implies transport and
trafficking of intermediates and end products of the pathways. Today,
there is no explanation for such compartmentalization.
The actual understanding of how these biosynthetic pathways operate can
be exploited for health and wealth creation. Vitamin synthesis is largely
PREFACE xiii
restricted to plants and microorganisms, a biochemical feature that can be

harnessed for the development of specific pesticides (bactericides, herbicides,
fungicides, etc.). Taking into account the health problems related to vitamin
deficiencies, together with an increase in the use of vitamin supplements for
human and animal nutrition, there is also a requirement, from a nutritional
and commercial standpoint, to enhance the production of many of these
vitamins. Overproduction of the vitamins can be achieved in a number of
ways, by removing transcriptional controls, overproduction of key enzymes
that represent bottlenecks in the pathways of biosynthesis, suppression of
metabolic feedbacks, limitation of the catabolism and increase of the storage.
It is clear that the ‘optimization’ of these systems requires a complete under-
standing of (i) their endogenous regulation and (ii) their integration within
the metabolism as a whole.
This book includes comprehensive and authoritative reviews from leading
experts on vitamins in plants, and we are thankful for their time and effort.
The aim of this book is to collect and interpret the rapid growing experimen-
tal information on vitamins in plants, especially in the challenging area of
their biosynthesis. We also hope that this book may be useful as a starting
point for those graduates and undergraduate students and researchers
wishing to pursue special studies in this field.
FABRICE RÉBEILLÉ AND ROLAND DOUCE

CONTENTS OF VOLUMES 35–58
Series Editor (Volumes 35–44)
J.A. CALLOW
School of Biosciences, University of Birmingham,
Birmingham, United Kingdom
Contents of Volume 35
Recent Advances in the Cell Biology of Chlorophyll Catabolism

H. THOMAS, H. OUGHAM and S. HORTENSTEINER
The Microspore: A Haploid Multipurpose Cell

A. TOURAEV, M. PFOSSER and E. HEBERLE-BORS
The Seed Oleosins: Structure Properties and Biological Role

J. NAPIER, F. BEAUDOIN, A. TATHAM and P. SHEWRY
Compartmentation of Proteins in the Protein Storage Vacuole:

A Compound Organelle in Plant Cells
L. JIANG and J. ROGERS
Intraspecific Variation in Seaweeds: The Application of New Tools

and Approaches
C. MAGGS and R. WATTIER
Glucosinolates and Their Degradation Products

R. F. MITHEN
xvi CONTENTS OF VOLUMES 35–58
PLANT VIRUS VECTOR INTERACTIONS

Edited by R. Plumb
Aphids: Non-Persistent Transmission

T. P. PIRONE and K. L. PERRY
Persistent Transmission of Luteoviruses by Aphids

B. REAVY and M. A. MAYO
Fungi
M. J. ADAMS
Whitefly Transmission of Plant Viruses

J. K. BROWN and H. CZOSNEK
Beetles
R. C. GERGERICH
Thrips as Vectors of Tospoviruses

D. E. ULLMAN, R. MEIDEROS, L. R. CAMPBELL,
A. E. WHITFIELD, J. L. SHERWOOD and T. L. GERMAN
Virus Transmission by Leafhoppers, Planthoppers and Treehoppers

(Auchenorrhyncha, Homoptera)
E. AMMAR and L. R. NAULT
Nematodes
S. A. MacFARLANE, R. NEILSON and D. J. F. BROWN
Other Vectors
R. T. PLUMB
CONTENTS OF VOLUMES 35–58 xvii
ANTHOCYANINS IN LEAVES
Edited by K. S. Gould and D. W. Lee
Anthocyanins in Leaves and Other Vegetative Organs: An Introduction

D. W. LEE and K. S. GOULD
Le Rouge et le Noir: Are Anthocyanins Plant Melanins?

G. S. TIMMINS, N. M. HOLBROOK and T. S. FEILD
Anthocyanins in Leaves: History, Phylogeny and Development

D. W. LEE
The Final Steps in Anthocyanin Formation: A Story of

Modification and Sequestration
C. S. WINEFIELD
Molecular Genetics and Control of Anthocyanin Expression

B. WINKEL-SHIRLEY
Differential Expression and Functional Significance of

Anthocyanins in Relation to Phasic Development in
Hedera helix L.
W. P. HACKETT
Do Anthocyanins Function as Osmoregulators in Leaf Tissues?

L. CHALKER-SCOTT
The Role of Anthocyanins for Photosynthesis of Alaskan Arctic

Evergreens During Snowmelt
S. F. OBERBAUER and G. STARR
Anthocyanins in Autumn Leaf Senescence

D. W. LEE
A Unified Explanation for Anthocyanins in Leaves?

K. S. GOULD, S. O. NEILL and T. C. VOGELMANN
xviii CONTENTS OF VOLUMES 35–58
An Epidemiological Framework for Disease Management

C. A. GILLIGAN
Golgi-independent Trafficking of Macromolecules to the Plant Vacuole

D. C. BASSHAM
Phosphoenolpyruvate Carboxykinase: Structure,

Function and Regulation
R. P. WALKER and Z.-H. CHEN
Developmental Genetics of the Angiosperm Leaf

C. A. KIDNER, M. C. P. TIMMERMANS, M. E. BYRNE
and R. A. MARTIENSSEN
A Model for the Evolution and Genesis of the Pseudotetraploid

Arabidopsis thaliana Genome
Y. HENRY, A. CHAMPION, I. GY, A. PICAUD,
A. LECHARNY and M. KREIS
Cumulative Subject Index Volumes 1–38
Starch Synthesis in Cereal Grains

K. TOMLINSON and K. DENYER
The Hyperaccumulation of Metals by Plants

M. R. MACNAIR
Plant Chromatin — Learning from Similarities and Differences

J. BRZESKI, J. DYCZKOWSKI, S. KACZANOWSKI,
P. ZIELENKIEWICZ and A. JERZMANOWSKI
CONTENTS OF VOLUMES 35–58 xix
The Interface Between the Cell Cycle and Programmed Cell Death in
Higher Plants: From Division unto Death
D. FRANCIS
The Importance of Extracellular Carbohydrate Production by Marine

Epipelic Diatoms
G. J. C. UNDERWOOD and D. M. PATERSON
Fungal Pathogens of Insects: Cuticle Degrading Enzymes and Toxins

A. K. CHARNLEY
Multiple Responses of Rhizobia to Flavonoids

During Legume Root Infection
JAMES E. COOPER
Investigating and Manipulating Lignin Biosynthesis

in the Postgenomic Era
CLAIRE HALPIN
Application of Thermal Imaging and Infrared Sensing in Plant

Physiology and Ecophysiology
HAMLYN G. JONES
Sequences and Phylogenies of Plant Pararetroviruses, Viruses, and

Transposable Elements
CELIA HANSEN and J. S. HESLOP-HARRISON
Role of Plasmodesmata Regulation in Plant Development

ARNAUD COMPLAINVILLE and MARTIN CRESPI
xx CONTENTS OF VOLUMES 35–58
Chemical Manipulation of Antioxidant Defences in Plants

ROBERT EDWARDS, MELISSA BRAZIER-HICKS,
DAVID P. DIXON and IAN CUMMINS
The Impact of Molecular Data in Fungal Systematics

P. D. BRIDGE, B. M. SPOONER and P. J. ROBERTS
Cytoskeletal Regulation of the Plane of Cell Division: An Essential

Component of Plant Development and Reproduction
HILARY J. ROGERS
Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration,

and Coordination with Reactions in the Cytosol
ALYSON K. TOBIN and CAROLINE G. BOWSHER
Defensive and Sensory Chemical Ecology of Brown Algae

CHARLES D. AMSLER and VICTORIA A. FAIRHEAD
Regulation of Carbon and Amino Acid Metabolism: Roles of Sucrose

Nonfermenting-1-Related Protein Kinase-1 and General Control
Nonderepressible-2-Related Protein Kinase
NIGEL G. HALFORD
Opportunities for the Control of Brassicaceous Weeds of Cropping

Systems Using Mycoherbicides
AARON MAXWELL and JOHN K. SCOTT
Stress Resistance and Disease Resistance in Seaweeds: The Role of

Reactive Oxygen Metabolism
MATTHEW J. DRING
Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus

ANNA AMTMANN, JOHN P. HAMMOND,
PATRICK ARMENGAUD and PHILIP J. WHITE
CONTENTS OF VOLUMES 35–58 xxi
Angiosperm Floral Evolution: Morphological

Developmental Framework
PETER K. ENDRESS
Recent Developments Regarding the Evolutionary

Origin of Flowers
MICHAEL W. FROHLICH
Duplication, Diversification, and Comparative Genetics of Angiosperm

MADS-Box Genes
VIVIAN F. IRISH
Beyond the ABC-Model: Regulation of Floral Homeotic Genes

LAURA M. ZAHN, BAOMIN FENG and HONG MA
Missing Links: DNA-Binding and Target Gene Specificity of Floral

Homeotic Proteins
RAINER MELZER, KERSTIN KAUFMANN
and GÜNTER THEIßEN
Genetics of Floral Development in Petunia

ANNEKE RIJPKEMA, TOM GERATS and
MICHIEL VANDENBUSSCHE
Flower Development: The Antirrhinum Perspective

BRENDAN DAVIES, MARIA CARTOLANO and
ZSUZSANNA SCHWARZ-SOMMER
Floral Developmental Genetics of Gerbera (Asteraceae)

TEEMU H. TEERI, MIKA KOTILAINEN, ANNE UIMARI,
SATU RUOKOLAINEN, YAN PENG NG, URSULA MALM,
EIJA PÖLLÄNEN, SUVI BROHOLM, ROOSA LAITINEN,
PAULA ELOMAA and VICTOR A. ALBERT
Gene Duplication and Floral Developmental Genetics of Basal Eudicots

ELENA M. KRAMER and ELIZABETH A. ZIMMER
xxii CONTENTS OF VOLUMES 35–58
Genetics of Grass Flower Development

CLINTON J. WHIPPLE and ROBERT J. SCHMIDT
Developmental Gene Evolution and the Origin of Grass

Inflorescence Diversity
SIMON T. MALCOMBER, JILL C. PRESTON, RENATA
REINHEIMER, JESSIE KOSSUTH and ELIZABETH A. KELLOGG
Expression of Floral Regulators in Basal Angiosperms and the Origin and

Evolution of ABC-Function
PAMELA S. SOLTIS, DOUGLAS E. SOLTIS, SANGTAE KIM,
ANDRE CHANDERBALI and MATYAS BUZGO
The Molecular Evolutionary Ecology of Plant Development: Flowering

Time in Arabidopsis thaliana
KATHLEEN ENGELMANN and MICHAEL PURUGGANAN
A Genomics Approach to the Study of Ancient Polyploidy and

Floral Developmental Genetics
JAMES H. LEEBENS-MACK, KERR WALL, JILL DUARTE,
ZHENGUI ZHENG, DAVID OPPENHEIMER and
CLAUDE DEPAMPHILIS
Series Editors (Volume 45– )
JEAN-CLAUDE KADER
Laboratoire Physiologie Cellulaire et Moléculaire des Plantes, CNRS,
Université de Paris, Paris, France
MICHEL DELSENY
Laboratoire Génome et Développement des Plantes,
CNRS IRD UP, Université de Perpignan,
Perpignan, France
RAPESEED BREEDING
History, Origin and Evolution

S. K. GUPTA and ADITYA PRATAP
CONTENTS OF VOLUMES 35–58 xxiii
Breeding Methods
B. RAI, S. K. GUPTA and ADITYA PRATAP
The Chronicles of Oil and Meal Quality Improvement in Oilseed Rape

ABHA AGNIHOTRI, DEEPAK PREM and KADAMBARI GUPTA
Development and Practical Use of DNA Markers

KATARZYNA MIKOLAJCZYK
Self-Incompatibility
RYO FUJIMOTO and TAKESHI NISHIO
Fingerprinting of Oilseed Rape Cultivars

VLADISLAV ČURN and JANA ŽALUDOVÁ
Haploid and Doubled Haploid Technology

L. XU, U. NAJEEB, G. X. TANG, H. H. GU, G. Q. ZHANG,
Y. HE and W. J. ZHOU
Breeding for Apetalous Rape: Inheritance and Yield Physiology

LIXI JIANG
Breeding Herbicide-Tolerant Oilseed Rape Cultivars

PETER B. E. MCVETTY and CARLA D. ZELMER
Breeding for Blackleg Resistance: The Biology and Epidemiology

W. G. DILANTHA FERNANDO, YU CHEN and
KAVEH GHANBARNIA
Development of Alloplasmic Rape

MICHAL STARZYCKI, ELIGIA STARZYCKI and JAN PSZCZOLA
Honeybees and Rapeseed: A Pollinator–Plant Interaction

D. P. ABROL
xxiv CONTENTS OF VOLUMES 35–58
Genetic Variation and Metabolism of Glucosinolates

NATALIA BELLOSTAS, ANNE DORTHE SØRENSEN,
JENS CHRISTIAN SØRENSEN and HILMER SØRENSEN
Mutagenesis: Generation and Evaluation of Induced Mutations

SANJAY J. JAMBHULKAR
Rapeseed Biotechnology
VINITHA CARDOZA and C. NEAL STEWART, JR.
Oilseed Rape: Co-existence and Gene Flow from Wild Species

RIKKE BAGGER JØRGENSEN
Evaluation, Maintenance, and Conservation of Germplasm

RANBIR SINGH and S. K. SHARMA
Oil Technology
BERTRAND MATTHÄUS
INCORPORATING ADVANCES IN PLANT PATHOLOGY
Nitric Oxide and Plant Growth Promoting Rhizobacteria: Common Features

Influencing Root Growth and Development
CELESTE MOLINA-FAVERO, CECILIA MÓNICA CREUS, MARÍA
LUCIANA LANTERI, NATALIA CORREA-ARAGUNDE, MARÍA
CRISTINA LOMBARDO, CARLOS ALBERTO BARASSI
and LORENZO LAMATTINA
How the Environment Regulates Root Architecture in Dicots

MARIANA JOVANOVIC, VALÉRIE LEFEBVRE, PHILIPPE
LAPORTE, SILVINA GONZALEZ-RIZZO, CHRISTINE
LELANDAIS-BRIÈRE, FLORIAN FRUGIER, CAROLINE
HARTMANN and MARTIN CRESPI
CONTENTS OF VOLUMES 35–58 xxv
Aquaporins in Plants: From Molecular Structure to Integrated Functions

OLIVIER POSTAIRE, LIONEL VERDOUCQ and
CHRISTOPHE MAUREL
Iron Dynamics in Plants

JEAN-FRANÇOIS BRIAT
Plants and Arbuscular Mycorrhizal Fungi: Cues and Communication in the

Early Steps of Symbiotic Interactions
VIVIENNE GIANINAZZI-PEARSON, NATHALIE
SÉJALON-DELMAS, ANDREA GENRE, SYLVAIN
JEANDROZ and PAOLA BONFANTE
Dynamic Defense of Marine Macroalgae Against Pathogens: From Early

Activated to Gene-Regulated Responses
AUDREY COSSE, CATHERINE LEBLANC and
PHILIPPE POTIN
INCORPORATING ADVANCES IN PLANT PATHOLOGY
The Plant Nucleolus

JULIO SÁEZ-VÁSQUEZ AND FRANCISCO JAVIER MEDINA
Expansins in Plant Development

DONGSU CHOI, JEONG HOE KIM AND YI LEE
Molecular Biology of Orchid Flowers: With Emphasis on Phalaenopsis

WEN-CHIEH TSAI, YU-YUN HSIAO, ZHAO-JUN PAN, CHIA-
CHI HSU, YA-PING YANG, WEN-HUEI CHEN AND
HONG-HWA CHEN
xxvi CONTENTS OF VOLUMES 35–58
Molecular Physiology of Development and Quality of Citrus

FRANCISCO R. TADEO, MANUEL CERCÓS, JOSÉ M.
COLMENERO-FLORES, DOMINGO J. IGLESIAS, MIGUEL A.
NARANJO, GABINO RÍOS, ESTHER CARRERA, OMAR
RUIZ-RIVERO, IGNACIO LLISO, RAPHAË L MORILLON,
PATRICK OLLITRAULT AND MANUEL TALON
Bamboo Taxonomy and Diversity in the Era of Molecular Markers

MALAY DAS, SAMIK BHATTACHARYA, PARAMJIT SINGH,
TARCISO S. FILGUEIRAS AND AMITA PAL
Molecular Mechanisms Underlying Vascular Development

JAE-HOON JUNG, SANG-GYU KIM, PIL JOON SEO
AND CHUNG-MO PARK
Clock Control Over Plant Gene Expression

ANTOINE BAUDRY AND STEVE KAY
Plant Lectins
ELS J. M. VAN DAMME, NAUSICAA LANNOO
AND WILLY J. PEUMANS
Late Embryogenesis Abundant Proteins

MING-DER SHIH, FOLKERT A. HOEKSTRA
AND YUE-IE C. HSING
Phototropism and Gravitropism in Plants

MARIA LIA MOLAS AND JOHN Z. KISS
CONTENTS OF VOLUMES 35–58 xxvii
Cold Signalling and Cold Acclimation in Plants

ERIC RUELLAND, MARIE-NOELLE VAULTIER,
ALAIN ZACHOWSKI AND VAUGHAN HURRY
Genome Evolution in Plant Pathogenic and Symbiotic Fungi

GABRIELA AGUILETA, MICHAEL E. HOOD,
GUISLAINE REFRÉGIER AND TATIANA GIRAUD
Aroma Volatiles: Biosynthesis and Mechanisms

of Modulation During Fruit Ripening
BRUNO G. DEFILIPPI, DANIEL MANRÍQUEZ,
KIETSUDA LUENGWILAI AND MAURICIO GONZÁLEZ-AGÜERO
Jatropha curcas: A Review

NICOLAS CARELS
You are What You Eat: Interactions Between Root Parasitic

Plants and Their Hosts
LOUIS J. IRVING AND DUNCAN D. CAMERON
Low Oxygen Signaling and Tolerance in Plants

FRANCESCO LICAUSI AND PIERDOMENICO PERATA
Roles of Circadian Clock and Histone Methylation in

the Control of Floral Repressors
RYM FEKIH, RIM NEFISSI, KANA MIYATA,
HIROSHI EZURA AND TSUYOSHI MIZOGUCHI
xxviii CONTENTS OF VOLUMES 35–58
PAMP-Triggered Basal Immunity in Plants

THORSTEN NÜRNBERGER AND BIRGIT KEMMERLING
Plant Pathogens as Suppressors of Host Defense

JEAN-PIERRE MÉTRAUX, ROBERT WILSON JACKSON,
ESTHER SCHNETTLER AND ROB W. GOLDBACH
From Nonhost Resistance to Lesion-Mimic Mutants:

Useful for Studies of Defense Signaling
ANDREA LENK AND HANS THORDAL-CHRISTENSEN
Action at a Distance: Long-Distance Signals in Induced Resistance

MARC J. CHAMPIGNY AND ROBIN K. CAMERON
Systemic Acquired Resistance

R. HAMMERSCHMIDT
Rhizobacteria-Induced Systemic Resistance

DAVID DE VLEESSCHAUWER AND MONICA HÖFTE
Plant Growth-Promoting Actions of Rhizobacteria

STIJN SPAEPEN, JOS VANDERLEYDEN AND YAACOV OKON
Interactions Between Nonpathogenic Fungi and Plants

M. I. TRILLAS AND G. SEGARRA
Priming of Induced Plant Defense Responses

UWE CONRATH
Transcriptional Regulation of Plant Defense Responses

MARCEL C. VAN VERK, CHRISTIANE GATZ
AND HUUB J. M. LINTHORST
CONTENTS OF VOLUMES 35–58 xxix
Unexpected Turns and Twists in Structure/Function of PR-Proteins

that Connect Energy Metabolism and Immunity
MEENA L. NARASIMHAN, RAY A. BRESSAN,
MATILDE PAINO D’URZO, MATTHEW A. JENKS
AND TESFAYE MENGISTE
Role of Iron in Plant–Microbe Interactions

P. LEMANCEAU, D. EXPERT, F. GAYMARD,
P. A. H. M. BAKKER AND J.-F. BRIAT
Adaptive Defense Responses to Pathogens and Insects

LINDA L. WALLING
Plant Volatiles in Defence

MERIJN R. KANT, PETRA M. BLEEKER, MICHIEL VAN WIJK,
ROBERT C. SCHUURINK AND MICHEL A. HARING
Ecological Consequences of Plant Defence Signalling

MARTIN HEIL AND DALE R. WALTERS
Oxidation of Proteins in Plants—Mechanisms and Consequences

LEE J. SWEETLOVE AND IAN M. MØLLER
Reactive Oxygen Species: Regulation of Plant Growth and Development

HYUN-SOON KIM, YOON-SIK KIM, KYU-WOONG HAHN,
HYOUK JOUNG AND JAE-HEUNG JEON
Ultraviolet-B Induced Changes in Gene Expression and Antioxidants in Plants

S. B. AGRAWAL, SURUCHI SINGH
AND MADHOOLIKA AGRAWAL
xxx CONTENTS OF VOLUMES 35–58
Roles of -Glutamyl Transpeptidase and -Glutamyl Cyclotransferase in

Glutathione and Glutathione-Conjugate Metabolism in Plants
NAOKO OHKAMA-OHTSU, KEIICHI FUKUYAMA
AND DAVID J. OLIVER
The Redox State, a Referee of the Legume–Rhizobia Symbiotic Game

DANIEL MARINO, CHIARA PUCCIARIELLO, ALAIN PUPPO
AND PIERRE FRENDO
Arabidopsis Histone Lysine Methyltransferases

FRÉDÉ RIC PONTVIANNE, TODD BLEVINS,
AND CRAIG S. PIKAARD
Advances in Coffea Genomics

ALEXANDRE DE KOCHKO, SÉLASTIQUE AKAFFOU, ALAN
ANDRADE, CLAUDINE CAMPA, DOMINIQUE CROUZILLAT,
ROMAIN GUYOT, PERLA HAMON, RAY MING,
LUKAS A. MUELLER, VALÉRIE PONCET,
CHRISTINE TRANCHANTDUBREUIL, AND SERGE HAMON
Regulatory Components of Shade Avoidance Syndrome

JAIME F. MARTÍNEZ-GARCÍA, ANAHIT GALSTYAN,
MERCÈSALLA-MARTRET, NICOLÁS CIFUENTES-ESQUIVEL,
MARC¸ AL GALLEMÍ, AND JORDI BOU-TORRENT
Responses of Halophytes to Environmental Stresses with Special

Emphasis to Salinity
KSOURI RIADH, MEGDICHE WIDED, KOYRO HANS-WERNER,
AND ABDELLY CHEDLY
Plant Nematode Interaction: A Sophisticated Dialogue

PIERRE ABAD AND VALERIE M. WILLIAMSON
CONTENTS OF VOLUMES 35–58 xxxi
Optimization of Nutrition in Soilless Systems: A Review

ELISA GORBE AND ÁNGELES CALATAYUD
Pollen Germination and Tube Growth

HUEI-JING WANG, JONG-CHIN HUANG,
AND GUANG-YUH JAUH
Molecular Mechanisms of Sex Determination in Monoecious

and Dioecious Plants
GEORGE CHUCK
The Evolution of Floral Symmetry

HÉLÈNE CITERNE, FLORIAN JABBOUR, SOPHIE NADOT,
AND CATHERINE DAMERVAL
Protein Turnover in Grass Leaves

LOUIS JOHN IRVING, YUJI SUZUKI, HIROYUKI ISHIDA,
AND AMANE MAKINO
Carpel Development
CRISTINA FERRÁNDIZ, CHLOÉ FOURQUIN,
NATHANAEL PRUNET, CHARLIE P. SCUTT, EVA SUNDBERG,
CHRISTOPHE TREHIN, AND AURÉLIE C. M.
VIALETTE-GUIRAUD
Root System Architecture

PAUL A. INGRAM AND JOCELYN E. MALAMY
xxxii CONTENTS OF VOLUMES 35–58
Functional Genomics of Cacao

FABIENNE MICHELI, MARK GUILTINAN, KARINA PERES
GRAMACHO, MIKE J. WILKINSON, ANTONIO VARGAS DE
OLIVEIRA FIGUEIRA, JÚLIO CÉZAR DE MATTOS CASCARDO,
SIELA MAXIMOVA, AND CLAIRE LANAUD
The Ecological Water-Use Strategies of Succulent Plants

R. MATTHEW OGBURN AND ERIKA J. EDWARDS
Nodule Physiology and Proteomics of Stressed Legumes

M. I. QURESHI, S. MUNEER, H. BASHIR, J. AHMAD,
AND M. IQBAL
Molecular Aspects of Fragrance and Aroma in Rice

APICHART VANAVICHIT AND TADACHI YOSHIHASHI
Miscanthus: A Promising Biomass Crop

EMILY A. HEATON, FRANK G. DOHLEMAN, A. FERNANDO
MIGUEZ, JOHN A. JUVIK, VERA LOZOVAYA, JACK WIDHOLM,
OLGA A. ZABOTINA, GREGORY F. MCISAAC, MARK B. DAVID,
THOMAS B. VOIGT, NICHOLAS N. BOERSMA,
AND STEPHEN P. LONG
Plant Adaptations to Salt and Water Stress: Differences and Commonalities

RANA MUNNS
Recent Advances in Understanding the Regulation of Whole-Plant Growth

Inhibition by Salinity, Drought and Colloid Stress
PETER M. NEUMANN
CONTENTS OF VOLUMES 35–58 xxxiii
Recent Advances in Photosynthesis Under Drought and Salinity

MARIA M. CHAVES, J. MIGUEL COSTA AND
NELSON J. MADEIRA SAIBO
Plants in Extreme Environments: Importance of Protective Compounds

in Stress Tolerance
LÁSZLÓ SZABADOS, HAJNALKA KOVÁCS, AVIAH ZILBERSTEIN
AND ALAIN BOUCHEREAU
Ion Transport in Halophytes

SERGEY SHABALA AND ALEX MACKAY
The Regulatory Networks of Plant Responses to Abscisic Acid

TAISHI UMEZAWA, TAKASHI HIRAYAMA, TAKASHI
KUROMORI AND KAZUO SHINOZAKI
Molecular Mechanisms of Abscisic Acid Action in Plants and Its Potential

Applications to Human Health
ARCHANA JOSHI-SAHA, CHRISTIANE VALON
AND JEFFREY LEUNG
Signalling Strategies During Drought and Salinity, Recent News

TIJEN DEMIRAL, ISMAIL TURKAN AND A. HEDIYE SEKMEN
An Overview of the Current Understanding of Desiccation Tolerance in the

Vegetative Tissues of Higher Plants
MONIQUE MORSE, MOHAMED S. RAFUDEEN AND
JILL M. FARRANT
Root Tropism: Its Mechanism and Possible Functions in Drought Avoidance

YUTAKA MIYAZAWA, TOMOKAZU YAMAZAKI, TEPPEI
MORIWAKI AND HIDEYUKI TAKAHASHI
xxxiv CONTENTS OF VOLUMES 35–58
Roles of Circadian Clock in Developmental Controls and Stress Responses in

Arabidopsis: Exploring a Link for Three Components of Clock Function in
Arabidopsis
RIM NEFISSI, YU NATSUI, KANA MIYATA, ABDELWAHED
GHORBEL AND TSUYOSHI MIZOGUCHI
Engineering Salinity and Water-Stress Tolerance in Crop Plants: Getting

Closer to the Field
ZVI PELEG, MARIS P. APSE AND EDUARDO BLUMWALD
Drought Stress: Molecular Genetics and Genomics Approaches

MELDA KANTAR, STUART J. LUCAS AND HIKMET BUDAK
Carotenoids
ABBY J. CUTTRISS, CHRISTOPHER I. CAZZONELLI,
ELEANORE T. WURTZEL AND BARRY J. POGSON
Vitamin B1 (Thiamine): A Cofactor for Enzymes Involved in the Main

Metabolic Pathways and an Environmental Stress Protectant
MARIA RAPALA-KOZIK
Biosynthesis of Vitamin B2 and Flavocoenzymes in Plants

MARKUS FISCHER AND ADELBERT BACHER
Biosynthesis of NAD and Its Manipulation in Plants

GRAHAM NOCTOR, JUTTA HAGER AND SHENGCHUN LI
Pantothenate Biosynthesis in Higher Plants

MICHAEL E. WEBB AND ALISON G. SMITH
Vitamin B6 in Plants: More Than Meets the Eye
TERESA B. FITZPATRICK1
Department of Botany and Plant Biology, Sciences III,

University of Geneva, Geneva, Switzerland
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
A. The Discovery of Vitamin B6............................................... 3
B. Currently Known Forms of Vitamin B6 and its Derivatives .......... 3
II. Biological Functions and Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
A. Vitamin B6 as an Enzyme Cofactor ....................................... 5
B. The Role of Vitamin B6 as an Antioxidant .............................. 11
C. The Importance of Vitamin B6 to Human Health ...................... 13
III. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
A. Intracellular and Within Plant Tissues .................................... 14
B. Examples of Food Content ................................................. 14
IV. Biosynthesis and Cellular Location of the Pathways . . . . . . . . . . . . . . . . . . . . . 15
A. De Novo Biosynthesis of Vitamin B6 ...................................... 15
B. Biosynthesis of Vitamin B6 Through Salvage Pathways ............... 20
C. Cellular Localization of the Pathways .................................... 21
V. Regulation, Turnover and Catabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
A. Regulation and Turnover of Vitamin B6 Biosynthesis ................. 22
B. Degradation of Vitamin B6 in Plants ..................................... 25
VI. Impact of the Vitamin on Plant Physiology and Development . . . . . . . . . . . 25
VII. Comparison with Other Autotrophic Non-Plant Organisms . . . . . . . . . . . . . 29
VIII. Engineering the Pathway for Nutritional Enhancement . . . . . . . . . . . . . . . . . . 30
IX. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
1
Corresponding author: E-mail: theresa.fitzpatrick@unige.ch
Advances in Botanical Research, Vol. 59 0065-2296/11 $35.00

Copyright 2011, Elsevier Ltd. All rights reserved. DOI: 10.1016/B978-0-12-385853-5.00006-4
2 T. B. FITZPATRICK
ABSTRACT
Vitamin B6 is derived primarily from plant sources and is an essential nutrient in the
human diet. While it is well established as a cofactor for numerous metabolic
enzymes, more recently, vitamin B6 has been implicated as a potent antioxidant.
The term vitamin B6 is generic for the compounds pyridoxine (PN), pyridoxal,
pyridoxamine and their phosphorylated derivatives (vitamers). The de novo biosyn-
thesis pathway of the vitamin in plants has recently been unravelled and involves only
two proteins, PDX1 and PDX2, that directly synthesize the cofactor vitamer, pyri-
doxal 50 -phosphate. There is also a salvage pathway that can interconvert the six
vitamers. The isolation of mutants in either the salvage or de novo biosynthesis
pathway has provided enormous insight into the role of this vital set of compounds
in metabolic, physiological and developmental processes in plants. Due to both its
cofactor and antioxidant role, the vitamin has been implicated in both abiotic and
biotic stress responses in plants. The dual role of the vitamin is beginning to provide
insight into the homeostatic maintenance of this set of compounds with exciting new
areas of research being uncovered. Here an impression of the vital roles this vitamin
plays as well as the general properties of the vitamin in plants is provided.
I. INTRODUCTION
Vitamin B6 is well established as one of nature’s most versatile cofactors

where it participates in a wide variety of biochemical reactions. The vitamin
was discovered almost a century ago and has been studied for decades.
However, it is only with the advent of the genomic era that its biosynthesis
could be unravelled. Indeed, despite plants being one of the most important
sources of this vitamin in the human diet, it is only recently that de novo
biosynthesis of vitamin B6 has been deciphered in plants. With this knowl-
edge, it became increasingly clear that the role of the vitamin goes beyond
that of cofactor. Not only is this vitamin a potent antioxidant but also recent
studies, incorporating forward and reverse genetics as well as physiological
and biochemical approaches, have been able to demonstrate the essential role
this compound plays in metabolic, physiological and developmental process-
es. Vitamin B6 is vitally important in human nutrition because humans
cannot synthesize it de novo. Thus, knowledge of the enzymes that participate
in the biosynthesis in plants could be used to enrich recombinant plant tissues
with vitamins for improved human nutrition and beneficial effects. In addi-
tion, because humans lack most enzymes required for vitamin B6 biosynthe-
sis, the inhibitors of those enzymes might be effective as herbicides, with
potentially little effect on human metabolism. Here, a comprehensive over-
view of the currently understood biological importance of vitamin B6 and its
mechanism as well as the distribution, biosynthesis pathways in addition to
homeostatic maintenance and regulation is provided. The focus is almost
VITAMIN B6 IN PLANTS: MORE THAN MEETS THE EYE 3
entirely on plants and the fundamental studies that have been performed in
recent years to uncover the roles of this important compound therein.
A. THE DISCOVERY OF VITAMIN B6
In the 1930s, after the discovery of thiamin, riboflavin, niacin and pantothe-
nate, the search began for what was thought to be a missing member of the
family of B vitamins. In particular, the search was linked to a cure for acrodynia
in young rats that presents as growth retardation and skin lesions (similar to
pellagra in humans). It was already known that adding niacin to the diet cured
human pellagra but this did not work on rats. Moreover, in 1934, Paul György,
a Hungarian born physician who had immigrated to the United States, noted
that a yeast concentrate missing vitamins B1 and B2 was unable to prevent and
cure acrodynia in rats either (György, 1934). Four years later, five separate
groups of researchers including György isolated a crystalline material from
yeast that could cure acrodynia (Lepkovsky, 1938). The compound was duly
named vitamin B6. Subsequently, the chemical structure was identified as
2-methyl-3-hydroxy-4,5-di-(hydroxymethyl)pyridine and its chemical synthesis
was established soon after (Harris and Folkers, 1939). Given its structural
homology to pyridine, the name pyridoxine (Fig. 1) for the free alcohol
was proposed and became generally accepted. Later on, two more related
compounds were identified, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethyl-
pyridine (pyridoxal, PL) and 2-methyl-3-hydroxy-4-amino-methyl-5-hydroxy-
methylpyridine (pyridoxamine, PM; Snell, 1944). Also in 1944, the first enzyme
dependent on vitamin B6 for activity was discovered, tyrosine decarboxylase
from Streptococcus faecalis (Gunsalus and Bellamy, 1944) and set a precedent
for dozens more that were to be found in subsequent years.
B. CURRENTLY KNOWN FORMS OF VITAMIN B6 AND ITS DERIVATIVES
The basic structure of vitamin B6 consists of a pyridine ring substituted with

a methyl, hydroxyl and hydroxymethyl group at C-2, C-3 and C-5, respec-
tively (Fig. 1). Depending on the substituent at C-4, the compound is char-
acterized as pyridoxal (PL, R¼¼CHO), pyridoxamine (PM, R¼¼CH2NH2)
or pyridoxine (PN, R¼¼CH2OH). Each of the latter compounds can also
exist as the corresponding monophosphate esters (linked to the hydroxy-
methyl side-chain at C-5) and are classified as PLP, PMP and PNP, respec-
tively (Fig. 1). In addition, the vitamin can occur in a glycosidic linkage with
one to several sugar molecules, predominantly glucose, which can be at C-50
of PN leading to pyridoxine 50 --D-glucoside (PN-50 --G) or its stereoisomer
PN-50 --D-G, or at C-40 giving PN-40 --G. The latter two occur in
4 T. B. FITZPATRICK
Biologically active forms of vitamin B6

CHO CH2NH2 CH2OH
HO HO HO
OH OH OH
N N N
Pyridoxal Pyridoxamine Pyridoxine
CHO CH2NH2 CH2OH

4¢
HO HO HO
4 5¢ OPO32- OPO32- OPO32-
3 5
2
1 6
N N N
2¢
Pyridoxal 5¢-phosphate Pyridoxamine 5¢-phosphate Pyridoxine 5¢-phosphate
Biologically inactive/antagonistic forms of vitamin B6

COOH CH2OCH3 CH2OH
CH2OH
HO HO HO OH
HO
OH OH OH OH
O
HO
N N N N
4-pyridoxic acid 4-deoxypyridoxine 4¢-O-methylpyridoxine Pyridoxine 5¢-b-D-glucoside

(Ginkgotoxin)
Fig. 1. The predominant forms of vitamin B6. Pyridoxal, pyridoxamine, pyridox-

ine and their phosphorylated analogues pyridoxal 50 -phosphate (cofactor form),
pyridoxamine 50 -phosphate and pyridoxine 50 -phosphate. 4-pyridoxic acid is a degra-
dation product of the free forms in bacteria, plants and animals. 4-deoxypyridoxine is
a potent antagonist of the cofactor form, whereas pyridoxine 50 --D-glucoside is an
example of glycosylated derivatives. The accepted numbering of the ring atoms is
shown for pyridoxal 50 -phosphate.
microorganisms, whereas PN-50 --G is predominantly found in plants and

can constitute 5–70% of the total vitamin B6 pool (Gregory and Ink, 1987).
As PN-50 --G is poorly metabolized by humans, due to the absence of an
enzyme able to hydrolyze the -glycosidic linkage, the bioavailability of
plant-derived vitamin B6 can thus be reduced substantially. It is thought
that the glycosylated forms maintain the intracellular pool by enhancing the
stability against heat, light and UV irradiation (Gregory, 1998; Nushimura
et al., 2008). It should be noted that a potent antagonist of active B6 vitamers
has been identified in animals, 4-deoxypyridoxine (4-dPN, Fig. 1). In vitro
studies have demonstrated that the phosphorylated form, 4-deoxypyridoxine
50 -phosphate, competes with PLP (the cofactor form, see below) for the
active site of enzymes dependent on vitamin B6, such as tyrosine decarbox-
ylase or aspartate transaminase (Scountzou et al., 1989). Indeed, another
vitamin B6 analog, 40 -O-methoxypyridoxine (also known as Ginkgotoxin;
Fig. 1) that is present in seeds and leaves of Ginkgo biloba has neurotoxic
properties as it can induce epileptic seizures (Kaye et al., 2002; Samuels et al.,
2008). This finding has been related to the reduced availability of PLP for
glutamate decarboxylase, which is involved in the formation of -aminobu-
tyric acid (GABA; Kästner et al., 2007).
II. BIOLOGICAL FUNCTIONS AND REQUIREMENTS
A. VITAMIN B6 AS AN ENZYME COFACTOR
Probably the most well-defined biological role of vitamin B6 to date is as a

vital cofactor for a large number of essential enzymes not only in plants but
also in all living organisms. Indeed, in its form as PLP (Fig. 1), the vitamin is
necessary for 148 biological reactions, representing ca. 4% of all enzymatic
activities that have been assigned so far. A recent review has annotated at
least 44 of these enzyme activities in plants based on the availability of the
Arabidopsis genome and the vitamin B6 database (http://bioinformatics.
unipr.it/cgi-bin/bioinformatics/B6db; Mooney and Hellmann, 2010). These
include many of the well-known amino acid transformations, for example,
glycine dehydrogenase and cysteine synthase, in addition to enzymes of
ethylene and auxin biosynthesis, for example, 1-aminocyclopropane-1-car-
boxylate (ACC) synthase and tryptophan synthase, respectively. Enzymes
dependent on vitamin B6 as a cofactor can be classified according to their
enzymatic activity or according to structural similarities as outlined below.
1. Activity-based classification of dependent enzymes

Enzymes dependent on vitamin B6 represent the largest and most diverse
group among cofactor-dependent catalysts. They occur within five of the six
general enzyme classes (Table I) as described by the Enzyme Commission
(EC) of the Nomenclature Committee of the International Union of Bio-
chemistry and Molecular Biology (NC-IUBMB; http://www.chem.qmul.ac.
uk/iubmb/enzyme/). Transaminases (EC 2.6.1) within the group of trans-
ferases are the largest group among them. While a few exceptions are related
to carbohydrate and lipid metabolism, the majority of enzymes dependent on
vitamin B6 as a cofactor act in the intra- and extracellular metabolism of
amino acids. The reactions catalyzed can be divided according to the position
at which the net reaction of the respective compound occurs. Reactions at the
-position include transamination, decarboxylation, racemization, elimina-
tion and replacement of an electrophilic group, while those at the - or -
position comprise elimination and replacement reactions (Drewke and
Leistner, 2001; Percudani and Peracchi, 2003).
6 T. B. FITZPATRICK
TABLE I
Activity-Based Classification of Vitamin B6-Dependent Enzymes (As Recommended by
the Enzyme Commission (EC))
Classification (EC) Example Total score

1. Oxidoreductases 1
1.4.4.2 Glycine dehydrogenase
2. Transferases 80
2.4.1.1 Phosphorylase
2.5.1.47 Cysteine synthase
2.6.1.27 Tryptophan transaminase
3. Hydrolases 2
3.5.99.7 1-Aminocyclopropane-1-carboxylate-deaminase
3.7.1.3 Kynureninase
4. Lyases 52
4.1.1.15 Glutamate decarboxylase
4.2.1.20 Tryptophan synthase
4.2.3.1 Threonine synthase
5. Isomerases 13
5.1.1.13 Aspartate racemase
5.4.3.8 Glutamate-1-semialdehyde 2,1-aminomutase
6. Ligases 0
The complete list can be found in Percudani and Peracchi (2003).
2. Structural classification of dependent enzymes

In addition to an activity-related taxonomy, an evolutionary-based classifi-
cation according to sequence and structural similarities is recently being
preferred for the characterization of vitamin B6-dependent enzymes
(Christen and Mehta, 2001; John, 1995). Sequence analyses of all enzymes
requiring vitamin B6 identified so far revealed that no overall sequence
identity exists among them: Size, oligomeric state, catalytically active form
and stability vary widely (Christen and Mehta, 2001; John, 1995; Kern et al.,
1999). However, unique regions exist which are involved in the binding of the
cofactor, in particular (Fig. 2). A conserved lysine residue in the enzyme’s
PLP-binding site (which forms an internal aldimine with PLP) and a glycine-
rich loop with the general motif GXGXXG (e.g. GNGLLGNG in glycogen
phosphorylase) have been identified (Marceau et al., 1990). Moreover, amino
acids functioning as hydrogen bond donors (e.g. Ser, Asp, Thr, Arg) interact
with the phosphate group of PLP. Further, an aspartic acid (or a tyrosine or
methionine residue) can frequently form a salt bridge to the N-1 of the
cofactor and is thought to maintain the protonation of the cofactor and
thus its functionality (Eliot and Kirsch, 2004; John, 1995).
Lys
R2
o HN
c
R1
Gly-rich loop
Internal aldimine N H
O
HO P
O O- Hydrogen bond with Ser
O- and/or Arg, Asn or Thr
H
8–9 residue N+ O
H CO R3
pocket
O O-
HN
Salt bridge with Asp R4
H
N or Tyr or Met
R6
Asp
CO
R5
Fig. 2. The conserved PLP-binding site present in enzymes dependent on vitamin

B6 as a cofactor. Despite a low level of overall sequence similarity, many amino acids
interacting with the coenzyme are conserved and arranged in a similar pattern. The
cofactor is presented in its imine form (black) bound to lysine. Residues indicated as
R1 to R6 illustrate neighbouring amino acids in the enzyme (shown in grey).
The structural classification is based on the relative orientation of the

above-mentioned motifs to each other as well as on the folding characteristics
of the protein and has led to the annotation of seven groups, Class I–VII
(Table II). A polyphyletic origin has been proposed that leads to the
well-characterized fold-types I–V, which includes a variety of the common
reaction types. Although the structures of several enzymes belonging to fold-
types VI and VII have been solved, they have not yet been conclusively
specified (Eliot and Kirsch, 2004). However, rather confusingly, it should
be noted that each fold-type includes a variety of reaction types, and yet a
single reaction type can cover more than one structural class. For example,
the bacterial alanine racemase belongs to fold-type III, whereas serine race-
mase and the fungal alanine racemase are classified in fold-types II and I,
respectively (Eliot and Kirsch, 2004).
3. The mechanism of vitamin B6 as a cofactor

Despite the fact that vitamin B6-dependent reactions are diverse, they share a
common principle in the first step of catalysis (Fig. 3A): PLP forms an
internal aldimine with the E-amino group of a conserved lysine residue in
TABLE II
Classification of Vitamin B6-Dependent Enzymes Into Fold-Types According to Sequence and Structural Similarities
Fold-type Structural characteristics Representatives

I N-term — Gly-rich domain — hydrophobic TA: Class I: aspartate aminotransferase (2.6.1.1)
-strand — Aspa — Lys — C-term Class II: glycine acetyltransferase (2.3.1.29)
Planar -sheets at N-term Class III: -alanine-pyruvate transaminase (2.6.1.18)
Class V: phosphoserine transaminase (2.6.1.52)
DC: prokaryotic ornithine decarboxylase (4.1.1.17)
Lyases: cystathionine -synthase (2.5.1.48)
II N-term — loop structure — Lys — Gly-rich Synthases: tryptophan synthase -subunit (4.2.1.20);
domain — Asp/Glu/Ser — C-term threonine synthase (4.2.3.1)
Planar -sheets at N-term Lyases: D-serine ammonia lyase (4.3.1.18)
Regulatory domain
III N-term — hydrophobic -strand — Lys — Gly-rich DC: eukaryotic ornithine decarboxylase (4.1.1.17)
domain — Insertionb — C-term Racemases: alanine racemase (5.1.1.1)
()8 barrel structure at N-term
IV N-term — Lys — Glu — Asn — C-term TA: Class IV: D-alanine transaminase (2.6.1.21)
V Lactate dehydrogenase fold at C-term GTA: glycogen phosphorylase (2.4.1.1); starch phosphorylase (2.4.1.1)
VI n.s. TA: succinyldiaminopimelate transaminase (2.6.1.17)
VII n.s. TA: valine-pyruvate transaminase (2.6.1.66)
‘—’ indicates conserved residues, strands, helices, barrels and/or loops in the protein structure. The EC-numbers are given in parentheses. Abbreviations:
C-term, C-terminus; DC, decarboxylases; GTA, glycosyltransferases; N-term, N-terminus, n.s., not specified; TA, transaminase.
a
Asp can be replaced by Ala, Ser, Tyr, Val, Ile or Met.
b
Insertion: domain boundary between the N- and C-terminus is often encoding the catalytic active site.
– –
OOC R –
OOC R –
OOC R
A
Deprotonation +HN +HN +HN
–
Protein
HO HO
OPO32– HO
Lys Protein H H+ OPO32– OPO32–
–
OOC R
+
H 2N
O
Lys
NH+ Amino acid +
HN
N+
H
N+ ·N·
substrate H H
HO HO Quinonoid
OPO32– HO
OPO32– OPO32–
N N N
H+ H+ Protein lysine H+ H – R H R H R
Pyridoxal 5¢-phosphate Internal aldimine External aldimine
CO2 +HN +HN +HN
–
HO HO HO
Decarboxylation OPO32– OPO32– OPO32–
N
H+ N+ ·N·
H H
Quinonoid
B
H HOOC R
HOOC R H
R H HOOC
Fig. 3. The mechanism of pyridoxal 50 -phosphate catalyzed reactions. (A) Pyridoxal 50 -phosphate is bound to a lysine residue of the
protein as an internal aldimine. The incoming substrate displaces the lysine residue to form an external aldimine with pyridoxal 50 -phosphate.
In the case of transformations at the -carbon of the substrate, the external aldimine can proceed via either the elimination of the -proton
(deprotonation, upper panel) or elimination of the -carboxy group (decarboxylation, lower panel) depending on which bond is perpendicu-
lar to the plane of the pyridine ring. Note the external aldimine shown is in the conformation for deprotonation. (B) The Dunathan
stereoelectric hypothesis. The particular reaction specificity of a pyridoxal 50 -phosphate-dependent enzyme can be explained by the relative
orientation of the substrate to the pyridine ring of the cofactor. The bond at C of the substrate that is to be cleaved is aligned with the
-orbitals of the cofactor exemplified for primary reaction types, that is, deprotonation and decarboxylation. R refers to the side-chain of the
substrate.
10 T. B. FITZPATRICK
the active site of the enzyme. In the presence of the substrate, the link to the
protein via the internal aldimine is displaced by the incoming substrate to
form an external aldimine occurring between the aldehyde group of PLP and
the amino group at C of the substrate. Although the bond between the
lysine residue and the cofactor is broken upon formation of the external
aldimine, the cofactor remains bound to the enzyme by various amino acid
residues throughout catalysis (Fig. 2). Once the external aldimine is formed,
the subsequent reaction specificity is a function of the properties of the
particular enzyme, that is, its tertiary structure coordinates the relative
orientation and properties of the binding folds for both PLP and the sub-
strate. The phenomenon has been exquisitely explained by Dunathan’s
hypothesis for diverse reactions that can occur at C of an amino acid
substrate (Dunathan, 1966) in which the pyridine ring of PLP acts as an
electron sink. The hypothesis proposes that the enzyme coordinates the
relative orientation of substrate and cofactor in such a way that the bond
at C of the substrate that is to be cleaved lies parallel to the -orbitals of the
pyridine ring, thus achieving maximum overlap. This leads to a lowering of
the transition state energy and to an increase in the reaction rate leading to
the release of Hþ (transamination and racemization reactions), CO2 (decar-
boxylases) or the R-group, respectively (Fig. 3B; Dunathan, 1966; Eliot and
Kirsch, 2004; Toney, 2005). The bond breakage results in the generation of
an -carbanion. The pyridine ring moiety of PLP serves primarily to reso-
nance-stabilize this anion by conjugation with the extended -system of the
cofactor defining the next stage of the mechanism, the quinonoid intermedi-
ate. Reprotonation at the -position followed by displacement of the
product and its release from PLP completes one round of the reaction.
Enzymes that catalyze PLP-dependent reactions at the - and -position
are found in the trans-sulphuration pathway (Fig. 4), which metabolically
links the sulphur-containing amino acids L-cysteine, L-homocysteine and
L-methionine (Brosnan and Brosnan, 2006). Although, these reactions can
occur, albeit very slowly, in the absence of an enzyme, only the enzyme can
provide the unique environment to coordinate substrate and reaction speci-
ficity (John, 1995). In contrast, glucan phosphorylases, that is, glycogen
phosphorylase and starch phosphorylase, depend on the phosphate group
at C-50 rather than on the aldehyde group at C-40 (Palm et al., 1990). These
enzymes are found in all organisms and tissues from bacteria, higher plants
and mammals where they play an essential role in carbohydrate metabolism.
So far, only a single enzyme has been described that apparently uses PMP
rather than PLP as a cofactor: CDP-6-deoxy-D-glycero-L-threo-4-hexulose-
dehydrase. This enzyme catalyzes an unprecedented one-electron redox reac-
tion in the course of forming 3,6-dideoxy sugars, for example, ascarylose,
COO–
+H
3N S
L-methionine
COO–
+ SH
H3 N
L-homocysteine
Pyruvate + NH3 Cystathionine

b-elimination
b-lyase
COO–
NH3+
+H
3N S
COO–
L-cystathionine
O-succinyl Cystathionine
g -replacement
L-homoserine g -synthase
COO–
SH
+H
3N
L-cysteine
Fig. 4. The trans-sulphuration pathway as an example of a PLP-dependent reac-

tion where catalysis occurs at the - or -position of the substrate.
paratose, abequose and tyvelose, which play an important role in bacterial

pathogenicity (Rubenstein and Strominger, 1974).
B. THE ROLE OF VITAMIN B6 AS AN ANTIOXIDANT
Recently, vitamin B6 has been postulated as a new candidate to efficiently

quench reactive oxygen species (ROS) comparable to ascorbic acid (vitamin
C) and -tocopherol (vitamin E). The link between vitamin B6 and ROS
was first established in the phytopathogenic fungus Cercospora nicotianae
where it was shown that mutant strains were sensitive to their own toxin
Cercosporin, a photosensitizer producing singlet oxygen in the light (Jenns
et al., 1995). Further studies revealed that the mutated genes were involved in
vitamin B6 biosynthesis (Ehrenshaft et al., 1999). There has been some
debate as to whether the antioxidant capacity is a direct or indirect effect,
that is, can the vitamin quench ROS in vivo (singlet oxygen, in particular) or
are the observations, a result of modulation of enzymes, involved in ROS

scavenging that are dependent on the vitamin as a cofactor. Arguments in
favour of vitamin B6 as a direct antioxidant include the fact that in vitro it is
rapidly degraded in the presence of singlet oxygen (Bilski et al., 2000; Ehrenshaft
et al., 1998, 1999; Osmani et al., 1999). Moreover, it has recently been shown
that singlet oxygen levels increase in a mutant (pdx1.3) that is impaired in de
novo biosynthesis of vitamin B6 and as a consequence the mutant is photosensi-
tive (Titiz et al., 2006). Indeed, combining the latter pdx1.3 mutation with a
photosensitive double mutant vte1 npq1 (mutations in tocopherol and caroten-
oid biosynthesis, respectively) augmented photosensitivity suggesting interplay
between vitamin B6, tocopherol and carotenoids in maintaining the antioxidant
pool in plastids (Havaux et al., 2009). Further, the maximum efficiency of
photosystem II (PSII) photochemistry (Fv/Fm) decreases concomitantly with a
reduction in the D1 protein (a marker for singlet oxygen stress) in pdx1.3
mutants (Titiz et al., 2006). In addition, exogenously applied vitamin B6 has
been shown to protect plant protoplasts from singlet oxygen-induced cell death
(Danon et al., 2005), and similar studies have been performed on yeast and
animal cell cultures (Chumnantana et al., 2005; Jain and Lim, 2001; Kannan
and Jain, 2004). Interestingly, a recent study has shown that pdx1.3 mutants
exposed to high light have lower levels of tocopherol as well as higher singlet
oxygen production and lipid peroxidation (Havaux et al., 2009). To date, the
role (if any) of vitamin B6-dependent enzymes in ROS scavenging has not been
explored. However, the studies mentioned above suggest that vitamin B6 does
indeed contribute directly to the antioxidant pool in the plastids. The question
now is whether this role is redundant with tocopherols and carotenoids or
complementary.
By definition, an antioxidant is a molecule with a low reduction poten-
tial that can donate either electrons or hydrogen atoms thereby prevent-
ing, in low concentrations (i.e. in the magnitude microgram per gram),
the oxidation of other molecules (Asensi-Fabado and Munné-Bosch,
2010). Theoretical studies suggest that singlet oxygen and, to some extent,
hydroxyl radicals can be attacked by vitamin B6, in particular, PN, PL
and to a lesser extent PLP, forcing the subtraction of a hydrogen atom
from either C-50 or C-40 (Matxain et al., 2006, 2007). This evidence would
additionally support the direct role of the vitamin in quenching ROS
particularly singlet oxygen. It has been noted that the Trolox equivalent
antioxidant capacity (TEAC) assay gives low values for vitamin B6. In
this assay, an antioxidant is added to a free radical-generating system,
and the inhibition of the free-radical action is measured and related to
that of the vitamin E analog, Trolox. Therefore, this is not surprising if
the predominant role of vitamin B6 is not in trapping free radicals but is
in quenching or scavenging singlet oxygen (a non-free radical form of

oxygen; Asensi-Fabado and Munné-Bosch, 2010).
C. THE IMPORTANCE OF VITAMIN B6 TO HUMAN HEALTH
It is because of its vital role in so many metabolic reactions that vitamin B6 is

regarded as being involved in more bodily functions than any other single
nutrient and is required for the maintenance of both physical and mental
health (Gengenbacher et al., 2006). According to the European Academy of
Nutritional Sciences (EANS), a daily intake of 2.0 mg of vitamin B6 is
sufficient for 97–98% of healthy individuals in each stage of life and in both
gender groups. As the value strongly depends on the amount of protein taken
up, a general model by Die Deutsche Gesellschaft für Ernährung (DGE)
predicts an uptake of 0.02 mg vitamin B6 per gram of ingested protein [http://
www.dge.de/pdf/ws/ReferenceValues.pdf]. Supplementation of vitamin B6 in
the human diet is rarely required but may be essential under various physio-
logical conditions ranging from weakness, convulsive seizures, appetite,
growth depression, anaemia and mental confusion to dermatitis caused by
a long-term deficiency. Hypovitaminosis is related to a low quality and
unbalanced diet. In particular, it can occur in alcoholics, elderly people,
epileptics, asthmatics and people dependent on medication against tubercu-
losis, Parkinson’s disease or cancer (Merrill and Henderson, 1987). Many
diseases like epilepsy, diabetes, autism (in combination with magnesium) and
Parkinson’s disease have been positively affected by pharmacological doses
of this vitamin. Symptoms of hypervitaminosis have been described following
long-term daily intake of 200 mg or more of vitamin B6 such as dermatitis,
numbness in the extremities and neurological disorders (Lieberman and
Brunig, 2003). A variety of biochemical consequences that can occur after
extensive intake of vitamin B6 have been described, such as a decline in
antibody circulation and in the biosynthesis of nucleic acids and proteins.
These effects are explainable by the central role of PLP predominantly in
amino acid metabolism (Linkswiler, 1967). Apart from this, elevated intra-
cellular levels of vitamin B6 in animal systems have a broad effect on systemic
homeostasis. The repression of glucocorticoid hormone receptors (Tully
et al., 1994) and the inhibition of various genes, either related or completely
unrelated to vitamin B6, for example, cytosolic aspartate aminotransferase,
tyrosine aminotransferase and serum albumin, have been described. In par-
ticular, the expression of proteins carrying a binding fold for phosphorylated
substrates or effectors, like both RNA and DNA polymerase or reverse
transcriptase, is inhibited by PLP (Oka, 2001).
III. DISTRIBUTION
A. INTRACELLULAR AND WITHIN PLANT TISSUES
The highest concentration of vitamin B6 in plant tissues is often in the range

of microgram per gram of fresh weight tissue. In particular, the vitamin is
found in leaves, flowers, fruits, roots, tubers and bulbs with a significantly
stronger accumulation in seeds compared to other organs. The subcellular
compartmentalization of vitamin B6 has not yet been elucidated. This is an
important point to be addressed not only at the general level of vitamin
distribution across membranes but indeed how the vitamers themselves are
distributed. As several metabolic enzymes are dependent on PLP as a cofac-
tor in the cytosol, mitochondria and chloroplast, this vitamer must be
available for integration into the corresponding enzymes to ensure catalytic
activity. It is known that de novo biosynthesis of vitamin B6 takes place in the
cytosol, which results in the formation of PLP. As a corollary from this, how
this vitamer is transported across the intracellular membranes is an impor-
tant point that has not been reported on. For example, it is not known if it is
first dephosphorylated and passed through the membranes by diffusion or if
a specific transport system is in operation. A number of vitamin B6 trans-
porters have been identified in other organisms, but no transporter has been
identified to date in planta (Rodionov et al., 2009; Stolz and Vielreicher,
2003). Enzymes of the salvage pathway are reported to be present in the
plastid (see below) suggesting that once a vitamer passes the plastid mem-
brane it can be converted to an appropriate biologically active form. Indeed,
recently, vitamin B6 has been identified in the chloroplasts of tobacco
(Havaux et al., 2009). However, a comprehensive analysis of the intracellular
distribution remains to be established. Moreover, whether the vitamin is
present in free form or a storage form or synthesized simultaneously with
the enzymes reliant on it is not known.
B. EXAMPLES OF FOOD CONTENT
Foods considered as excellent sources of vitamin B6 include meats, cereal

grains, vegetables and nuts. However, bioavailability is highly variable and
poorly documented. Generally, animal products contain more bioavailable
forms of vitamin B6 compared to plant food sources (Kabir et al., 1983a,b).
It has been recognized that vitamin B6 exists in protein and non-protein
bound forms. Especially in plants, PN can exist in quite significant amounts
as a -glucoside (Yasumoto et al., 1977). For example, more than 50% of the
total vitamin B6 content was observed to be in the PN-glucoside form in
TABLE III
Vitamin B6 Content of Various Plants of Nutritional Interest
Plant food Vitamin B6 (mg/100g)

Barley 0.33
Broccoli 0.20
Carrot 0.12
Cassava 0.09
Kale 0.35
Oat 0.14
Rice (unpolished) 0.50
Rice (polished) 0.11
Sesame seeds 0.79
Soybeans 0.42
Spinach 0.22
Tomato 0.33
Wheat bran 1.38
A comprehensive list of the content of various food sources can be
found at http://www.foodcomp.dk/v7/fcdb_details.asp?FoodId¼0674
and at USDA National Nutrient Database: http://www.ars.usda.gov/
ba/bhnrc/ndl.
broccoli, cauliflower, carrots and cooked soybeans (Kabir et al., 1983a,b).

Studies on the bioavailability of PN-glucoside have demonstrated that the
glucoside is poorly available to humans and animals as a source of
vitamin B6. The vitamin B6 content of commonly used plant foods is listed
in Table III.
IV. BIOSYNTHESIS AND CELLULAR LOCATION

OF THE PATHWAYS
A. DE NOVO BIOSYNTHESIS OF VITAMIN B6
The biosynthesis of vitamin B6 de novo was first explored in Escherichia coli

in the 1960s, as it was the model organism of the time and was elucidated
therein over the course of the following three decades. Notably, the pathway
involving seven enzymatic steps culminating in the production of PNP was
assumed to be ubiquitous amongst all organisms that can synthesize this
compound. The E. coli biosynthetic pathway has been extensively reviewed
recently in Fitzpatrick et al. (2007), and the reader is directed to it for
reference purposes. Despite the importance of the vitamin, its biosynthesis
had not undergone a thorough investigation or validation in any other
organism. It was only in 1999, through serendipitous but pioneering work
from Margaret Daub and colleagues, during a screen for proteins that
conferred singlet oxygen resistance to the phytopathogenic fungus C. nico-

tianae, that they discovered a gene they had previously named SOR1 (for
Singlet Oxygen Resistance 1) which was in fact involved in vitamin B6
biosynthesis (Ehrenshaft et al., 1998, 1999). An independent study by Ste-
phen Osmani and colleagues identified a homologous gene in Aspergillus
nidulans (Osmani et al., 1999). Due to the involvement of this gene in vitamin
B6 biosynthesis, it was renamed Pdx1. Subsequent studies identified an
associated gene that was named Pdx2 (Ehrenshaft and Daub, 2001). It then
became clear from the increasing amount of information available from
genomic studies that both these genes are widely distributed and are in fact
found in all archaea, fungi, plants and most bacteria (Ehrenshaft et al., 1999;
Mittenhuber, 2001). Moreover, neither Pdx1 nor Pdx2 showed homology
with any gene involved in the E. coli biosynthesis pathway. Indeed, a genomic
analysis revealed that the two key genes of the E. coli pathway, PdxA and
PdxJ, are present predominantly in only a small subset of the -division of
proteobacteria (Ehrenshaft et al., 1999; Mittenhuber, 2001; Osmani et al.,
1999). Thus it became accepted that the majority of organisms must have a
pathway of vitamin B6 biosynthesis completely different from the one estab-
lished for E. coli. Because of these observations, renewed interest in vitamin
B6 was initiated, and several studies appeared on defining the nature of this
‘alternative’ pathway. Many reports using genetic approaches confirmed the
presence and involvement of Pdx1 and Pdx2 in vitamin B6 metabolism in
various organisms including plants (Belitsky, 2004; Dong et al., 2004;
Ehrenshaft et al., 1999; Gengenbacher et al., 2006; Mittenhuber, 2001;
Osmani et al., 1999; Sakai et al., 2002; Tambasco-Studart et al., 2005).
Furthermore, the precise function of the proteins was resolved and recon-
stituted in vitro by two independent groups (Burns et al., 2005; Raschle et al.,
2005). It is now known that Pdx1 and Pdx2 function together as a glutamine
amidotransferase directly producing the cofactor PLP in the presence of
glutamine, ribose 5-phosphate and glyceraldehyde 3-phosphate (Tambasco-
Studart et al., 2005; Fig. 5). The pentose and triose phosphate sugar isomers,
ribulose 5-phosphate and dihydroxyacetone phosphate, respectively, can
also be used as substrates (Tambasco-Studart et al., 2005). In order to
distinguish the substrate specificity of the two routes, the E. coli-type path-
way is referred to as ‘deoxyxylulose-dependent’ and the alternative pathway
‘deoxyxylulose-independent’ (Tambasco-Studart et al., 2005). In the ‘deox-
yxylulose-independent’ pathway, which is the one present in plants, Pdx2 is a
glutaminase that hydrolyzes glutamine passing the ammonia released onto
Pdx1. The latter, however, performs the remarkable polymorphic transfor-
mation of ammonia as well as the pentose phosphate and triose phosphate
sugars into PLP. The mechanism behind this multi-step reaction is both
De novo pathway Salvage pathway
CH2NH2 CH2NH2
HO HO
OPO32− OH
R5P P-ase
O N PMP N PM
HO SOS4
OPO32−
PDX3 T-ase
HO OH
CHO CHO
HO HO
Gln
PDX2 PDX1 OPO32− OH
NH3
P-ase
Glu
N PLP N PL
SOS4
PDX3 PLR
OH
O OPO32− CH2OH CH2OH
HO HO
2−
G3P OPO3 OH
P-ase
N PNP N PN
SOS4
Fig. 5. Biosynthesis pathways of vitamin B6. Pyridoxal 50 -phosphate is produced

de novo from ribose 5-phosphate (R5P), glyceraldehyde 3-phosphate (G3P) and
glutamine (Gln) through the action of PDX1 and PDX2. Enzymes of the salvage
pathway can interconvert the vitameric forms, that is, PN (pyridoxine), PL (pyridox-
al), PM (pyridoxamine) or their phosphorylated derivatives (PNP, PLP, PMP, respec-
tively) as depicted. Only the PN/PM oxidase (PDX3) and PN/PL/PM kinase (SOS4)
have been identified in Arabidopsis. T-ase, P-ase and PLR refer to transaminase,
phosphatase and PL reductase, respectively. R5P and G3P refer to ribose 5-phosphate
and glyceraldehyde 3-phosphate, respectively.
intriguing and challenging to enzymologists and has been the subject of

fertile study for the past few years. The most up to date review, at the time
of writing, of the studies performed on this aspect can be found in Fitzpatrick
et al. (2010) and will therefore not be reiterated here.
All plants have at least one homolog of PDX1 and one homolog of PDX2.
For example, Nicotiana tabacum contains two homologs of PDX1 and a
single copy of PDX2 (Denslow et al., 2005), whereas in Arabidopsis, three
homologs of PDX1, that is, PDX1.1 (At2g38230), PDX1.2 (At3g16050),
PDX1.3 (At5g01410) and a single homolog of PDX2 (At5g60540) exist
(Tambasco-Studart et al., 2005). Currently, the most information available
on the properties of these genes is from those of Arabidopsis and will be the
focus of the discussion here. In accordance with Class I glutaminases, PDX2
contains the signature catalytic triad Cys, His and Glu residues, and activity
has been reconstituted in vitro in the presence of PDX1. The three mentioned
residues are essential for coordinating the mechanism of glutamine hydroly-
sis by PDX2 where the Cys residue functions as a nucleophile, the His as the
proton donor in the hydrolytic reaction and the Glu maintains the orienta-
tion and tautomeric state of the His residue (Zalkin and Smith, 1998). A key
point in the mechanism is the formation of an oxyanion hole that promotes
the stabilization of transient negative charges generated during glutamine
hydrolysis. The oxyanion hole is formed by two backbone amide groups, one
from the residue following the nucleophile and the second from an adjacent
-strand, referred to as the ‘oxyanion strand’ (Zalkin and Smith, 1998). The
structure of Pdx2 (an / triple layer sandwich, Fig. 6) from various bacterial
sources (Strohmeier et al., 2006; Zein et al., 2006) as well as that from
Plasmodium falciparum (Gengenbacher et al., 2006), in addition to site-
directed mutagenesis of these as well as the Arabidopsis enzyme
(Tambasco-Studart et al., 2007), has confirmed several aspects of the mecha-
nism. Notably, glutaminase activity is only observed in the presence of
PDX1, where interaction with the latter coordinates the reorientation of
the peptide necessary for formation of the oxyanion hole (Strohmeier et al.,
2006; Tambasco-Studart et al., 2007).
However, of the three PDX1 homologs in Arabidopsis, only PDX1.1 and
PDX1.3 display catalytic activity. PDX1.2, however, while expressed does
not catalyze the biosynthesis of PLP. Moreover, all the residues required for
PDX2
90 Å 40 Å
90 °C
PDX1
Fig. 6. The crystal structure of the Bacillus subtilis Pdx1/Pdx2 complex (PDB ID:
2NV2) is shown as viewed from the side (left), the top (centre) and a single protomer
of PDX1 (green) and PDX2 (grey) from Thermotoga maritima (PDB ID: 2ISS;
centre). The alpha helix at the N-terminus of PDX1, coordinating the interaction
between the two proteins, is shown in yellow. The stick representations (pink) are
bound ribose 5-phosphate and a phosphate ion (PDX1) and bound glutamine
(PDX2).
catalytic activity have been substituted in PDX1.2 (Tambasco-Studart et al.,

2007). It has been proposed that this homolog may have a regulatory func-
tion whereby it affects the activity of either of the catalytic homologs
(Tambasco-Studart et al., 2007). Indeed, reports utilizing yeast two-hybrid
analyses and overexpression fusion protein constructs have suggested that
PDX1.2 can interact with either PDX1.1 or PDX1.3 (Wagner et al., 2006) but
the biochemical and physiological meaning behind the interaction have not
been elucidated. The structure of PDX1 homologs from bacteria and yeast
have been resolved to date (Neuwirth et al., 2009; Strohmeier et al., 2006;
Zein et al., 2006; Zhu et al., 2005) but there is no report so far of a plant
homolog. In all cases, the architecture is that of a classic (/)8 barrel but is
unusual in that the bacterial enzyme assembles as a dodecamer with the
subunits forming a cylinder composed of two opposing rings of six (/)8
barrels with an internal cavity that is 40 Å in diameter (Fig. 6; Strohmeier
et al., 2006; Zein et al., 2006). Notably, the yeast enzyme, on the other hand,
is composed of just one hexameric ring of (/)8 barrels (Neuwirth et al.,
2009). Our own studies using gel-filtration and static light scattering show
that the Arabidopsis enzymes assemble as dodecamers similar to their bacte-
rial counterparts (Moccand, C, Kaufmann, M. & Fitzpatrick T.B.). The
structure of the entire de novo PLP biosynthesis machinery has also been
solved from bacterial sources. The architecture of the bacterial complex is
composed of the core of 12 PDX1 molecules arranged in two hexameric rings
to which 12 PDX2 subunits attach like the cogs of a cogwheel (Strohmeier
et al., 2006; Zein et al., 2006; Fig. 6). These studies in addition to several
structural-based biochemical analyses (Hanes et al., 2009; Raschle et al.,
2007, 2009) provided a wealth of information on the mechanism of vitamin
B6 biosynthesis (reviewed in Fitzpatrick et al., 2010). Moreover, there is
precedence that the mechanism is conserved in the plant enzymes
(Tambasco-Studart et al., 2007). The active sites of the glutaminase
(PDX2) and synthase (PDX1) domains are remote from each other, implying
the existence of an ammonia tunnel (Strohmeier et al., 2006). The interaction
between the two subunits is coordinated by a novel N-terminal -helix on the
synthase domain (Fig. 6, shown in yellow) and is in turn necessary for the
conformational change in PDX2 priming its activation (through correct
assembly of the oxyanion strand as mentioned above). However, it should
be noted that a comparison of the amino acid sequences of PDX1 from
Bacillus subtilis and Arabidopsis revealed that a methionine residue in the
B. subtilis enzyme (M13) is replaced by a leucine in the Arabidopsis enzymes.
The latter affords a much tighter coupling of glutaminase and synthase
reactions and thus a higher catalytic efficiency for the use of ammonia by
Arabidopsis PDX1 (Tambasco-Studart et al., 2007). Substrate utilization is
another feature in which the PLP synthase homologs differ from each other.
In Arabidopsis, dihydroxyacetone phosphate can only be efficiently used as a
substrate in the presence of PDX2 (Tambasco-Studart et al., 2007), which is
not the case for the bacterial enzyme (Raschle et al., 2005).
B. BIOSYNTHESIS OF VITAMIN B6 THROUGH SALVAGE PATHWAYS
As stated above, PN, PL, PM and their respective 50 -phosphoesters are all
included under the term ‘vitamin B6’. All these vitamers can coexist in any
one organism. Importantly, in addition to de novo biosynthesis of vitamin B6
from carbohydrate precursors, a second pathway is in existence that results
in the interconversion of the different vitamers such that a particular one is
made available when required (Fig. 5). The salvage pathway is active in all
organisms identified so far but here the pathway as it has been defined in
plants to date will be described. The first salvage pathway enzyme to be
identified in plants was SOS4 in Arabidopsis. The latter got its name from a
search for Salt Overly Sensitive mutants, one of which (sos4) rather surpris-
ingly showed high sequence homology to E. coli PL kinase (Shi et al., 2002;
Shi and Zhu, 2002). The functionality of SOS4 as a PL kinase was confirmed
by complementation studies in E. coli but its activity has not been reconsti-
tuted in vitro and it is not known if it additionally phosphorylates either of
the other vitamers (PN or PM). However, the ability to rescue the sos4
mutant with PN and not PL would corroborate exclusive phosphorylation
of the latter. Therefore, if SOS4 is exclusively phosphorylating PL, the fact
that the sos4 mutant can be rescued by PN supplementation implies that
there must be a PN kinase awaiting identification. The product of the latter,
PNP, can be converted to PLP by an oxidase (see below). However, no
specific phosphatase has been identified so far that carries out the dephos-
phorylation of PNP, PLP or PMP in order to restore the free forms but is
thought to be catalyzed by unspecific phosphatases (Mittenhuber, 2001).
The conversion of either PNP or PMP to PLP is catalyzed by a specific
oxidase called PDX3. The functionality of Arabidopsis PDX3 has been
confirmed in vitro and in vivo by complementation studies in yeast (Sang
et al., 2007). The enzyme is dependent on FMN as a cofactor and appears to
utilize either PNP or PMP as substrates but exhibits a higher specificity for
PNP. Molecular oxygen is used as an electron acceptor, and hydrogen
peroxide is released during catalysis. Rather unusually and apparently spe-
cific to the plant PDX3 homologs is that they carry a so-called ‘Yjef_N’
domain at the N-terminus (Sang et al., 2011). The Yjef_N domain shows
homology to human apolipoprotein A–I binding protein that is involved in
the regulation of vesicle fusion in the endosomal/lysosomal route as well as
showing homology to the human TGR-CL10C thyroidal receptor for

N-acetylglucosamine (Sang et al., 2007). While intriguing, the function of
the Yjef_N domain remains to be elucidated. All PDX3 homologs in over 30
organisms examined including red algae, land plants, charaphyte algae and
chlorophyte algae have both Yjef_N and PN oxidase domains. It has there-
fore been suggested that acquisition of the Yjef_N domain and its fusion with
the PN oxidase domain in plant PDX3s may have begun with the endosym-
biotic acquisition of the chloroplast. PLP can also be derived from PMP by
the activity of unspecific transaminases in a so-called shuttle mechanism of
coenzyme action. In addition, an apparently specific PL reductase (PLR) has
been identified in yeast that is dependent on NADPH and produces PN
(Nakano et al., 1999). While there are homologs of this protein in plants
(own observations), no report of PLR activity has appeared to date. The
significance of where all of these enzymes are localized should not be under-
estimated, as a high level of coordination between de novo and salvage
pathway biosynthesis can be expected. Thus, it remains to be determined if
more than one homolog of each of these genes exists and if a deferential
location can be confirmed.
C. CELLULAR LOCALIZATION OF THE PATHWAYS
At the subcellular level, several groups have investigated the localization of

the de novo biosynthesis pathway proteins. However, not all are in agree-
ment. In the first instance, the transient expression of all Arabidopsis PDX1
homologs and PDX2 fused to GFP displayed an exclusive localization to the
cytosol by confocal microscopy (Tambasco-Studart et al., 2005). However,
independent analysis of plants stably expressing PDX1.3 fused to GFP
suggested that, in addition to the cytosol localization, this protein likely
exists also in association with cellular membranes (plasma membrane,
nuclear envelope and chloroplast outer membranes; Chen and Xiong,
2005). A subsequent study by the same group concluded that PDX2 fused
to GFP is found in the cytosol in addition to being associated with the plasma
membrane (Chen and Xiong, 2009b). However, Denslow and co-workers
localized GFP-PDX2 to the periphery of epidermal cells and guard cells and
the nuclei of guard cells of transgenic plants (Denslow et al., 2007). There-
fore, while it is clear that the proteins of de novo biosynthesis are at least in
the cytosol, there is also evidence for membrane association. It should be
noted that there is, however, no evidence for translocation into an organelle
corroborated by the fact that the full-length proteins do not harbour a transit
peptide and are entirely soluble and functional as recombinant proteins in
activity assays (Tambasco-Studart et al., 2005, 2007). The physiological
relevance for instigating or establishing an association with organelle mem-

branes remains to be established.
The salvage pathway genes on the other hand have not yet undergone such
an extensive investigation at the subcellular level. The PDX3 gene encodes a
protein carrying a clear N-terminal extension characteristic of plastid target-
ing, and the localization has indeed recently been confirmed in Arabidopsis
protoplasts (Sang et al., 2011). However, a splice variant is predicted (http://
www.arabidopsis.org) which would lack the plastid transit peptide but has
not yet undergone investigation. There are also apparently two splice var-
iants of the SOS4 gene in Arabidopsis, both predicted to encode cytosolic
proteins (Shi et al., 2002). Moreover, both splice variants fully complemented
an E. coli PN/PM/PL kinase knockout mutant suggesting that they do not
contain a target peptide. Therefore, it appears that there is evidence that the
vitamers can be interconverted in the plastid at least. However, PLP is
needed as a cofactor not only in the chloroplast but also in the mitochondria,
which begs the question of how a particular vitamer is translocated across
intracellular membranes. While the non-phosphorylated vitamers may dif-
fuse across membranes at appropriate concentrations, this mechanism will
not support the movement of the polar phosphorylated vitamers. Moreover,
it is likely that the levels of vitamers are tightly controlled to prevent non-
specific inactivation of enzymes not dependent on the vitamin as a cofactor as
well as the ability of the aldehyde forms of the vitamin to fortuitously react
with free amines in the cell, potentially inactivating cellular components.
Therefore, transport systems for the vitamin can be anticipated but remain
to be defined. Although none have been described in plants so far, systems
that are apparently specific for the vitamin have been described in bacteria
and yeast (Rodionov et al., 2009; Stolz and Vielreicher, 2003).
V. REGULATION, TURNOVER AND CATABOLISM
A. REGULATION AND TURNOVER OF VITAMIN B6 BIOSYNTHESIS
Several studies have investigated the regulation of the genes involved in de

novo vitamin B6 biosynthesis at the transcriptional level in response to
various abiotic stresses. There is evidence that the transcription of both
PDX1 and PDX2 is enhanced in response to environmental stresses that
are accompanied by release of ROS such as high light, chilling and drought
in Arabidopsis (Denslow et al., 2005, 2007), wounding as well as the intracel-
lular concentration of hormones such as ethylene and gibberellins in bean
(Phaseolus vulgaris; Graham et al., 2004), in addition to ethylene and salicylic
acid in the rubber tree (Hevea brasiliensis; Sivasubramaniam et al., 1995).

Further, an in silico analysis of the three PDX1 genes from Arabidopsis
suggests differential regulation of the homologs. In particular, a Genevesti-
gator analysis of PDX1.2 indicated a regulation disparate from either
PDX1.1 or PDX1.3 (Titiz et al., 2006). Indeed, a recent investigation has
suggested that this homolog is up-regulated by ozone in contrast to the other
two paralogs (Denslow et al., 2007). Moreover, differential potential regu-
latory elements have been identified in the immediate upstream regions of
PDX1.1 and PDX1.3 in Arabidopsis, suggesting distinctions in their regula-
tion but have not been tested to date. While it has been established that
PDX1.3 is more requisite than PDX1.1 in Arabidopsis (Titiz et al., 2006)
based on a higher expression level of the former, the controlling elements
maintaining the different expression level of each paralog remains to be
established. Interestingly, a G-Box motif (CACGTG) has been identified
exclusively in the upstream region of PDX1.2 from Arabidopsis. This motif
is specific for G-box-binding transcription factors involved in the phosphor-
ylation-dependent regulation of light-responsive promoters and putatively in
seed maturation (Jakoby et al., 2002). However, PDX1.2 has not been shown
to respond strongly to light (Titiz et al., 2006). Somewhat surprisingly, there
is no available knockout of PDX1.2 from the vast collections available, thus
whether its functionality, once determined, is essential to Arabidopsis
remains to be elucidated.
However, all these studies have been conducted at the transcriptional level.
The corresponding modulation at the protein level and moreover any adjust-
ment in the vitamin B6 content has not been investigated thoroughly. Indeed,
in this context, there are several anomalies that need to be dissected to
correlate vitamin B6 content with the particular phenotype being observed.
For example, both sos4 and pdx1.3 are sensitive to high light stress and salt
stress, yet both show a remarkable resistance to drought (González et al.,
2007). Interestingly, SOS4 is predicted, based on publically available micro-
array data, to be down-regulated by salt, osmotic stress and drought. How-
ever, although both sos4 and pdx1.3 show reduced total vitamin B6 content,
the PLP level is increased strongly in sos4 (ninefold) while it would be
expected to be decreased in pdx1.3 (although the latter has not been directly
determined). Moreover, a single point mutation in pdx1.3 (rsr4-1) results in a
more severe phenotype than the complete knockout of the gene (Wagner
et al., 2006). Further, sos4, pdx3 and pdx1.3 are all sucrose sensitive pheno-
types while rsr4-1 is not. In all these mutants, there are variations in the levels
of the vitamers examined, but the complete profile including phosphorylated
vitamers has not been determined for all of them. It is expected that there will
be a strong interaction between the salvage and de novo pathways of vitamin
B6 biosynthesis with one perhaps compensating for a loss of the other in a

particular tissue, depending on the developmental stage and environmental
status. Therefore, it is imperative that all of the vitamer levels are determined
for each mutant so that hypotheses can be formulated and validated. Only
with such vitamer profiles can a direct correlation between phenotype and
vitamer content be drawn.
There is evidence for post-transcriptional modification and tight regula-
tion of the enzymes involved in de novo biosynthesis of vitamin B6. For
example, we have recently shown that the endogenous Arabidopsis PDX1.1
protein can be overexpressed either alone or in combination with its partner
protein PDX2 (Raschke et al., 2011). However, while substantial PDX1.3
overexpression can be achieved at the transcriptional level, no increase is
observed at the protein level suggesting tight post-transcriptional regulation
of this paralog. Indeed, a recent proteomic study has demonstrated that
AtPDX1.3 is an ubiquitination target (Manzano et al., 2008). The covalent
modification of a protein with ubiquitin is well documented as a powerful
system to regulate the stability and function of such proteins
(Mukhopadhyay and Riezman, 2007). Interestingly, AtPDX1.1 and
AtPDX1.3 are also predicted to differ in their half-life times, being 220 and
5–31 h, respectively (Meinnel et al., 2005).
In addition, it is expected that the pentose and triose phosphate pool in the
cell and the equilibrium between the metabolic pathways that feed and drain
them; Calvin cycle and pentose phosphate pathway, glycolysis in addition to
vitamin B6 biosynthesis will constitute a level of control for determining the
fluxes of precursors towards different branches. Indeed, the regulation can be
expected to be bidirectional because not only does the cell require the sugars
to make the vitamin de novo but also conversely several other vital pathways
in the cell are in turn dependent on the compound as a cofactor. Some key
examples include starch breakdown, where the initial step requires the activ-
ity of the PLP-dependent enzyme -glucan phosphorylase, which catalyzes
the liberation of glucose-1-phosphate from the non-reducing ends of
-1,4-linked glucan chains (Zeeman et al., 2004); phytohormone biosynthesis
as previously mentioned, which are key regulators of plant growth and
development, for example, in auxin biosynthesis, not only does tryptophan
synthase require PLP as a cofactor but also the recently identified amino-
transferase (TAA1) that catalyzes the formation of indole-3-pyruvic acid
from L-tryptophan and ethylene (Stepanova et al., 2008). The requirement
of PLP for ethylene biosynthesis and degradation has been mentioned above
where ACC synthase mediates the conversion of S-adenosylmethionine
to ACC, a final precursor to ethylene, and ACC deaminase degrades ACC
to 2-oxobutyrate and ammonia (McDonnell et al., 2009). PLP is also
required for glucosinolate biosynthesis. The latter are a group of compounds

that are involved in plant defence and impart the flavour in cruciferous
vegetables. PLP-dependent C-S-lyases catalyze the step from S-(alkylaceto-
hydroxy imoyl)-L-cysteines to the corresponding thiohydroximic acids which
are intermediates in the biosynthesis pathway (Mikkelsen et al., 2004).
B. DEGRADATION OF VITAMIN B6 IN PLANTS
To date, there has been no report on the mechanism of vitamin B6 degrada-

tion in plants. However, it has been investigated, albeit to a limited extent, in
bacteria and animals. According to the available literature, the oxidative
degradation of vitamin B6 predominantly occurs at the level of the free
forms, in particular, PL. Both PN and PM are converted to PL by oxidation
and transamination, respectively. The latter is converted to 4-pyridoxic acid
(4-PA; Fig. 1) in subsequent reactions, but in animals, 4-PA is not converted
further and is excreted in the urine (Burns and Conney, 1960; Stanulović
et al., 1976). In bacteria, for example, Pseudomonas MA-1 (ATCC 33286)
and Arthrobacter sp., 4-PA can either be first isomerised to 5-pyridoxic acid
(isopyridoxine) or directly degraded to ammonia, carbon dioxide and acetic
acid (Jong et al., 1986; Lee et al., 1986; Nelson and Snell, 1986). Thus, several
bacteria can use vitamin B6 as the sole source of carbon and nitrogen.
VI. IMPACT OF THE VITAMIN ON PLANT

PHYSIOLOGY AND DEVELOPMENT
With the elucidation of both de novo and salvage pathways of vitamin B6
biosynthesis, several studies have appeared documenting the effect of mutat-
ing any one of the genes involved. These mutations have provided a wealth of
information on the effect of vitamin B6 on plant physiology and develop-
ment. For example, a mutation in the genes encoding either PDX1.1 or
PDX1.3 results in impaired growth and development (Chen and Xiong,
2005; Tambasco-Studart et al., 2005; Titiz et al., 2006; Wagner et al., 2006).
However, the impairment is much more pronounced in pdx1.3 mutant plants
with severe retardation of root growth, in particular, when grown on sterile
medium in the absence of a source of the vitamin (Fig. 7). The roots of pdx1.3
have a stunted growth phenotype due to impairment in both cell division and
cell elongation (Chen and Xiong, 2005). This is similar to what happens with
the vtc1 mutant impaired in vitamin C biosynthesis (Conklin et al., 2000).
However, supplementation of pdx1.3 with vitamin C did not rescue the
phenotype (Chen and Xiong, 2005). Evidence has been provided that the
pdx1.3
pdx1.1
WT
Fig. 7. Growth of wild type (WT), pdx1.1 and pdx1.3, respectively, on sterile
medium in the absence of vitamin B6. The picture was captured 9-days after germi-
nation. A retardation of root growth, in particular, is clearly visible in the mutant
lines and is more pronounced in pdx1.3.
phenotype is more related to a defect in auxin biosynthesis (Chen and Xiong,

2009a,b) rather than a redox imbalance. This is plausible given that the
vitamin is required as a cofactor for auxin and ethylene biosynthesis, both
of which are required for root growth. It has been shown in Arabidopsis that
PDX1.3 is more requisite than PDX1.1, as mentioned above. Indeed,
PDX1.3 appears to be expressed at a higher level than PDX1.1, and mutation
of PDX1.3 results in a more severe depletion in vitamin B6 content (Titiz
et al., 2006). The latter point was corroborated by analyses of the single-copy
mutant lines pdx1.1/pdx1.1/PDX1.3/pdx1.3 and PDX1.1/pdx1.1/pdx1.3/
pdx1.3, which could be used to demonstrate a gene dosage effect where the
level of PDX1.3 could be correlated with the total vitamin B6 content (Titiz
et al., 2006). It is interesting to note that the phenotype of pdx1.3 is less
pronounced on soil leading to the suggestion that there is a source of the
vitamin in soil (Titiz et al., 2006). Mutating both PDX1.1 and PDX1.3
together or indeed the single PDX2 is lethal for Arabidopsis with develop-
ment being arrested at the globular stage of embryo growth, consistent with
the vital role the vitamin plays in the plant (Tambasco-Studart et al., 2005;
Titiz et al., 2006). In 2006, Hellmann and collaborators published data on the
identification of a novel mutant carrying a point mutation in the PDX1.3
gene that specifically causes an amino acid exchange from Gly to Ser (G54S;
Wagner et al., 2006). This mutant, named rsr4-1 (for reduced sugar
response), was originally identified in an ethyl-methanesulfonate-based
screen to generate mutants having a reduced activation of a sugar-responsive
patatin class I promoter (Wagner et al., 2006). rsr4-1 is characterized by
aberrant root and leaf growth, delayed flowering and a significantly lower PL
content as compared to wild-type plants, which is surprisingly even more
pronounced than for the pdx1.3 null mutant. The mutant can be rescued by
supplementation with vitamin B6, which restores normal development. The
rsr4-1 mutant is broadly affected in its metabolism, in that amino acids,
raffinose and shikimate contents as well as trichloroacetic acid cycle inter-
mediates are altered (Wagner et al., 2006). However, metabolomic data
demonstrated that amino acids such as Ile and Asp are increased, which is
contrary to the expectation that reduced vitamin B6 content would correlate
with a reduction in amino acid biosyntheses that involves PLP-dependent
enzymes.
The effects of mutation in either of the two genes that have been identified
as part of the salvage pathway (SOS4 and PDX3) have also been documen-
ted. The sos4 mutant has an abnormal root phenotype characterized by
growth rates slower than in wild-type plants and the absence of root hairs
in the maturation zone (Shi and Zhu, 2002; Shi et al., 2002). In addition, an
independent study has noted that sos4 plants are chlorotic, have a reduced
biomass and display early flowering (González et al., 2007). Interestingly,
sos4 plants have a dramatically increased level of PLP (ninefold), which has
been assigned as due to an increase in de novo biosynthesis of the vitamin.
Whether this physiological trait is indirectly related to the phenotype has not
been established. Two pdx3 mutants have been characterized which show
close to normal development in soil (González et al., 2007). However, it
should be noted that the latter are not null mutants so the effect of complete
knockout/knockdown of PDX3 remains to be verified.
Given the recent annotation of vitamin B6 as an antioxidant, it is of no
surprise that several studies have appeared associating the vitamin with
environmental stress. For example, mutants in Arabidopsis that are deficient
in vitamin B6 are susceptible to several forms of abiotic stress (high light, salt,
osmotic stress, oxidative stress, UV-B; Chen and Xiong, 2005; Denslow et al.,
2007; Titiz et al., 2006). PDX1 expression is enhanced by light (Titiz et al.,
2006), and pdx1.3 mutants are hypersensitive to high light and rose bengal (a
photosensitizer that can generate singlet oxygen upon illumination; Chen
and Xiong, 2005) but not superoxide or hydrogen peroxide (Havaux et al.,
2009). Further, as mentioned earlier, the maximum quantum efficiency of
PSII photochemistry (Fv/Fm) decreases concomitantly with a reduction in the
D1 protein in pdx1.3 mutants that are exposed to high light (Titiz et al.,
2006). Therefore, a fraction of the vitamin B6 pool would be expected to be

located close to PSII for efficient quenching of singlet oxygen at its site of
production and for prevention of D1 oxidation. Indeed, a recent study in
tobacco has reported that vitamin B6 (PN and PM, in particular) can be
found in the chloroplast (Havaux et al., 2009). However, only the depho-
sphorylated vitamers were examined, and due to the aqueous protocol used,
it cannot be excluded that there was transport of the vitamers between
organelles while the experiment was being conducted. Nevertheless, an Ara-
bidopsis triple mutant (vte1 npq1 pdx1.3) that is deficient in tocopherols,
zeaxanthin and vitamin B6 exhibited increased sensitivity to high light and
showed increased lipid peroxidation (Havaux et al., 2009). In the same study,
it was demonstrated that pdx1.3 mutants exposed to high light have lower
tocopherol levels as well as higher singlet oxygen production and lipid
peroxidation suggesting that more tocopherols are consumed in vitamin
B6-deficient plants to counteract the imbalanced antioxidant capacity. It
must be mentioned that it has also been shown that although pdx1.3 mutant
plants have reduced chlorophyll and carotenoid levels (15–20%), this did not
significantly affect photosynthetic performance (Havaux et al., 2009). Chlo-
rophyll loss is associated with an increase in the chlorophyll a/b ratio and a
selective decrease in the abundance of several PSII antenna proteins
(Lhcb1/2, Lhcb6). These changes depend on light intensity with high light
amplifying the difference between pdx1.3 and wild type. These phenomena
are only observed in young leaves and disappear in mature, well-developed
leaves. Like pdx1.3, pdx3 plants are also susceptible to high light intensities
(González et al., 2007) but unlike pdx1.3 have not undergone a thorough
investigation. It has also been shown that SOS4 (pyridoxal kinase) is required
for salt tolerance in Arabidopsis (Shi and Zhu, 2002; Shi et al., 2002). In
particular, the sos4 mutation leads to increased sensitivity towards the ions
Naþ, Kþ and Liþ and high sucrose levels. Addition of ACC, a precursor in
ethylene biosynthesis, and to a greater extent, the synthetic auxin 2,4-
dichlorophenoxy acetic acid was sufficient to restore normal root growth,
indicating that the sos4 mutation is upstream of ethylene and auxin biosyn-
thesis in the development of root hairs (Shi and Zhu, 2002).
It would therefore seem that PLP, as an important cofactor, controls
essential processes of plant growth and development as well as plant
responses to stress, while the free forms of the vitamin B6 group, which
show the strongest antioxidant properties (Bilski et al., 2000), are vital to
contribute to the antioxidant defence network in plants. It remains to be
deciphered how vitamin B6 contributes to the antioxidant pool in the chlo-
roplast, in particular, where carotenoids and tocopherols are playing a major
role and have been studied extensively.
VII. COMPARISON WITH OTHER AUTOTROPHIC

NON-PLANT ORGANISMS
In addition to plants, bacteria and fungi have the ability to biosynthesize

vitamin B6 de novo. Due to the several studies carried out by various groups
over the past decade, in particular, it is now clear that the majority of organ-
isms capable of synthesizing this vital nutrient de novo do so through the
so-called ‘DXP-independent’ pathway employing the PLP synthase complex
made up of PDX1 and PDX2. There is a substantial body of evidence to
support the fact that the biochemistry behind this pathway is highly similar
in all organisms that utilize this route and has been described here in detail for
plants. However, the notable exception to utilization of the latter pathway is
E. coli and several members of the -division of proteobacteria. Even though it
was tacitly assumed for decades that all organisms that can synthesize vitamin
B6 de novo employ the same route as E. coli. The E. coli-type pathway involves
two branches with seven enzymatic steps (Hill and Spenser, 1986; Hill et al.,
1996; Lam and Winkler, 1990, 1992; Zhao and Winkler, 1994, 1995, 1996;
Zhao et al., 1995). In one branch, the sequential action of the enzymes GapA,
PdxB and PdxF results in the conversion of erythrose 4-phosphate into
4-phosphohydroxy-L-threonine (Lam and Winkler, 1990; Yang et al., 1998).
The latter then undergoes oxidation and decarboxylation by PdxA to form
3-hydroxy-1-aminoacetone phosphate (Banks and Cane, 2004; Cane et al.,
1998). In the other branch, DXP is derived from glyceraldehyde 3-phosphate
and pyruvate by the action of DXP synthase (Cane et al., 1998; Sprenger et al.,
1997). The products of the two branches, that is, 3-hydroxy-1-aminoacetone
phosphate and DXP, are then condensed by PdxJ to form PNP (Cane et al.,
1999; Laber et al., 1999), which must undergo oxidation, catalyzed by PdxH,
to form the cofactor vitamer, PLP (Di Salvo et al., 1998). This latter pathway is
referred to as ‘DXP-dependent’ to distinguish it from the pathway in plants.
The fact that different organisms utilize different precursors is quite common
in de novo vitamin biosynthesis. This may be based on an evolutionary accom-
modation to their host cells as well as on the specificities of the particular
organism’s life cycle. It should be mentioned again in this context that on the
other hand all organisms have the ability to interconvert the different vitamer
forms through the salvage pathway as described above. For example, human
cells, while not autotrophic, can synthesize PLP upon acquiring vitamin B6
through the diet. The 50 -phosphorylated derivatives of PN, PL and PM are in
fact dietary sources of vitamin B6, but phosphatases in the human intestine
dephosphorylate these compounds before absorption (Said and Mohammed,
2006). Specific kinases are present to biosynthesize the phosphorylated vita-
mers within the cell.
VIII. ENGINEERING THE PATHWAY FOR

NUTRITIONAL ENHANCEMENT
In general, the vitamin content of plant-derived foods can be increased in

several ways: Either through optimization of growth conditions, breeding or
through the use of transgenic techniques. Increasing the level of vitamin B6 in
plants is not only important for biofortification but also because the vitamin
displays an important antioxidant function, which may improve redox bal-
ance and therefore benefit crop growth and resistance to both biotic and
abiotic stresses. However, it could be anticipated that plants having enhanced
levels of the vitamin could provide important insights into homeostatic main-
tenance of this vital metabolite. So far, attempts to increase the vitamin B6
content in plants have employed the transgenic approach. However, the first
few trials to generate such plants met with limited success. A first effort to
overproduce the vitamin was attempted in tobacco through heterologous
expression of the PDX genes from the phytopathogen C. nicotianae but
resulted in only a 1.2-fold increase in its level (Herrero and Daub, 2007). A
more substantial increase was impeded by a combination of acute down-
regulation of the endogenous genes and limited transgene expression. This
outcome provoked the conclusion that vitamin homeostasis is too tightly
regulated in plants to permit its enhancement. Another study employing a
seed-specific promoter to drive overexpression of the Arabidopsis PDX genes
achieved a twofold increase in the vitamin B6 content of seeds (Chen and
Xiong, 2009a). The most recent study has achieved vitamin B6 levels up to
sevenfold greater than those in wild type through overexpression of the
endogenous Arabidopsis PDX1.1 gene either alone or in combination with
its partner gene PDX2 (Raschke et al., 2011). In this study, the vitamin was
observed to accumulate in seeds. Interestingly, as a consequence of the
enhanced levels of vitamin B6, there was a considerable enlargement of all
aerial organs including the seeds (twofold). The enlargement was shown to be
due to an increase in cell size in organs examined, although development was
retarded compared to wild-type plants. Moreover, it could be shown that the
enhanced vitamin levels led to an increase in available nutrients, that is,
protein, lipid and carbohydrate (Raschke et al., 2011). Furthermore, these
plants were more resistant than wild type to oxidative stress. Therefore, these
vitamin B6-enhanced lines provide an important tool to explore the facets of
this vitamin in more detail. Importantly, the latter study provides an impetus
to apply the same strategy to a crop plant to increase the vitamin content not
only for biofortification but also because of the potential to withstand various
types of abiotic stress. There has been no report of increasing the vitamin B6
content of a crop plant to date but it will be interesting to see if the latest
approach can achieve just that in a plant used for food.
IX. CONCLUSIONS
Vitamin B6 is an important regulator of cellular metabolism in plants, as a

cofactor in several enzymatic reactions as well as being a potent antioxidant.
Studies on the distribution, occurrence and, in particular, trafficking of this
vitamin in plants will enhance our understanding of the contribution to each
of these roles in the future. Research has, and is sure in the future, to reveal
several crossing points among the enzymes and pathways that are involved in
both de novo as well as salvage pathway biosynthesis, which in turn will help
to manipulate a particular vitamer in foods for beneficial effects. However,
the complexity of trafficking of these vitamers between organelles remains to
be deciphered and makes this goal particularly challenging. Plastids appear
to be the organelle that has several methods for combating oxidative stress
and include the use of vitamin B6; given their importance in sustaining life
through oxygenic photosynthesis, such a protective arsenal is not surprising.
It should be kept in mind that vitamin B6 is also necessary as a cofactor in
several hormone biosynthesis pathways (auxin and ethylene) which will in
turn effect the phenotypes observed upon manipulation if these vitamins and
moreover reflect expected complex interactions between hormones and vita-
mins that have yet to be explored.
ACKNOWLEDGEMENTS
The generous support of the Swiss National Science Foundation (SNF) grant
PP00A_119186 to T. B. F. is gratefully acknowledged. I would also like to
extend gratitude to the insightful discussions and subject accounts of various
students that have passed through the laboratory, which have assisted in
formulating the ideas and concepts put forward in this chapter, namely Drs.
Thomas Raschle, Olca Titiz, Marina Tambasco-Studart and Maja Raschke.
Dr. Céline Roux and Svetlana Boycheva are acknowledged for their help
with Figs. 6 and 7, respectively, and Dr. Nicolas Szydlowski for critical
reading of the chapter.
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Biotin (Vitamin B8) Synthesis in Plants
CLAUDE ALBAN*,{,{,},1
*Laboratoire de Physiologie Cellulaire Végétale, CNRS,

UMR5168, Grenoble, France
{
CEA, DSV, iRTSV, Grenoble, France
{
INRA, UMR1200, Grenoble, France
}
Université Joseph Fourier, Grenoble, France
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
A. Significance ...................................................................... 41
B. Distribution and Nutritional Aspects ....................................... 41
C. Biotin-Containing Proteins ................................................... 43
II. The Biosynthetic Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
A. The Origin of Pimeloyl-CoA.................................................. 47
B. 7-Keto-8-Aminopelargonic Acid Synthase ................................. 48
C. 7,8-Diaminopelargonic Acid Synthase—Dethiobiotin Synthetase...... 49
D. Biotin Synthase ................................................................. 51
III. Protein Biotinylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
IV. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
ABSTRACT
Biotin, also known as vitamin H or B8, is an essential cofactor for CO2-manipulating
enzymes found in all three domains of life. The past few years have seen decisive
progress accomplishments on the elucidation of biotin metabolism in plants, at both
1
Corresponding author: E-mail: claude.alban@cea.fr

40 C. ALBAN
the molecular and cellular levels, and several unique features are emerging. Notice-
ably, biotin synthesis in plants is split between cytosol and mitochondria. Biotin-
utilizing enzymes are also quartered between different compartments of the plant cell.
Among these compartments, mitochondria play a central role. In this review, I will
summarize the most recent discoveries about the synthesis, manipulation and com-
partmentalization of biotin in plant cells. These advances open challenging prospects
for plant biotechnology purposes through a better understanding of regulation,
storage and utilization of the vitamin. Understanding how the biotin biosynthetic
pathway interacts with other metabolic pathways and the emerging involvement of
mitochondria in plant growth and development, through its intimate implication in
vitamins synthesis are also particularly challenging.
ABBREVIATIONS
50 -UTR 50 -untranslated region
AdoMet S-adenosyl-L-methionine
AdoMTOB 4-(methylthioadenosyl)-2-oxobutanoate
ADR adrenodoxin reductase
ADX1 adrenodoxin 1
DAPA 7,8-diaminopelargonic acid
DTB dethiobiotin
KAPA 7-keto-8-aminopelargonic acid
PLP pyridoxal 50 -phosphate
uORF upstream open reading frame
I. INTRODUCTION
Biotin, also known as vitamin H or B8, is a cofactor for some carboxylases,

decarboxylases and transcarboxylases dealing with crucial metabolic pro-
cesses such as fatty acid and carbohydrate metabolism (Alban et al., 2000;
Knowles, 1989). In mammals, biotin is also known to regulate gene expres-
sion through different ways including histone biotinylation (Beckett, 2007;
Zempleni, 2005). Despite its essential functions, de novo synthesis of this
vitamin is restricted to bacteria, a few fungi and plants. All animals including
humans cannot synthesize biotin as part of their normal metabolism and
therefore rely on the supply of biotin from exogenous sources. Biotin is a
fusion of an imidazolinone ring with a tetrahydrothiophene ring bearing a
valeric acid side chain (Fig. 1). There are three chiral carbon atoms in biotin,
leading to eight possible stereoisomers. However, only one is biologically
active; this isomer is denoted (þ) or (D)-biotin. The absolute stereochemistry
of D-biotin established by X-ray crystallography revealed that the
BIOTIN (VITAMIN B8) SYNTHESIS IN PLANTS 41
HN
NH
S Ureido ring
HOOC
H H Tetrahydrothiophene ring
Valeric acid
H
Fig. 1. The structure of biotin.
imidazolinone and the tetrahydrothiophene rings are fused in a cis configu-

ration, producing a bottle structure (Fig. 1).
A. SIGNIFICANCE
Biotin was discovered in the search for the nutritional factor that prevents
egg white injury in experimental animals, and the use of the biotin antagonist
in egg white, the biotin-binding protein avidin, was further useful in produc-
ing biotin deficiency in animal models (Kögel and Tönnis, 1936). The detri-
mental effect of feeding high doses of raw egg white most often involves
dermatologic lesions such as dermatitis or alopecia. This explains the name
of vitamin H (Haut, German word for skin) given to biotin at that time. In
addition to primary deficiencies of the vitamin, genetic disorders in biotin
metabolism have been identified. These are rare, affecting infants and chil-
dren, but usually having serious consequences (neurologic abnormalities
such as hypotonia, altered consciousness, seizures and ataxia, and skin
damages such as rash and alopecia) (Baumgartner and Suormala, 1999).
Congenital defects fall into two major categories. The first involves the
absence of a biotin apoenzyme. In the second, multiple carboxylases have
defective activities due to absence of biotinidase, the enzyme responsible for
biotin recycling, or altered holocarboxylase synthetase (HCS), the enzyme in
charge of biotin-dependent carboxylases activation by biotinylation (Fig. 2).
These last congenital disorders usually respond to high doses of biotin.
B. DISTRIBUTION AND NUTRITIONAL ASPECTS
Biotin exists under two forms in living cells, free or covalently bound to
proteins. In bacterial and animal cells, free biotin content is low or even
undetectable. In Escherichia coli, for example, free biotin never accumulates
above a nanomolar concentration range. In contrast, plant cells contain a
large pool of free biotin. In pea leaves, for instance, free biotin accumulates in
the cytosol of mesophyll cells to a concentration of about 11 M (Baldet
42 C. ALBAN
Dietary biotin
Protein-bound Free
150–300 mg/day
Biotinidase
O
Holocarboxylase
HN NH synthetase
COOH
S
Biotinidase Biotin
O
HN NH
O
Apocarboxylases
COOH
(PCC, MCC, PC, ACC)
N
S H
Biocytin NH2
Proteolytic HN NH
O
Degradation N
S H
Holocarboxylases
Congenital disorders
Proteins Lipids Carbohydrates
Aminoacid Fatty acid Gluconeogenesis
catabolism synthesis
Fig. 2. The biotin cycle in mammalian cells.
et al., 1993a). The pool of protein-bound biotin associated to biotin-depen-

dent carboxylases is mainly present within organelles (1.2 M within chloro-
plast stroma and 13 M within mitochondrial matrix). The free/bound-biotin
ratio in the whole cell is > 6 (Baldet et al., 1993a). In Arabidopsis cultured
cells, the free biotin pool is somewhat lower with a ratio free/bound of
around 1.5 (Claude Alban and Virginie Pautre, unpublished observation).
To date the precise fate of free biotin in plant cells is still poorly understood
but it could behave as a reserve pool for maintaining biotin-dependent
carboxylases activity and thus cell viability under stress conditions affecting
biotin synthesis or availability. This is well illustrated in the following exam-
ple. After 3 days of treatment of Arabidopsis cultured cells with sublethal
concentrations of acidomycine, an inhibitor of biotin synthesis, the pool of
free biotin was found to be drastically reduced while that of bound biotin and
the activity of biotin-dependent carboxylases were poorly affected. After
6 days of treatment, the pool of bound biotin and biotin enzyme activities
were, in turn, significantly reduced with as consequences a global alteration
of respiration, photosynthetic activity and cell division. These effects
were reversed by supplementation with free biotin (Claude Alban and
Virginie Pautre, unpublished data).
Because of the dual nature of biotin in living cells, dietary biotin is

present in two forms: free and protein bound (Fig. 2). The protein-bound
form, in which vitamin is covalently linked to polypeptides through a
specific lysine residue, is degraded by digestive proteases to the biotinyl-
lysine adduct biocytin. Then, biotinidase is thought to be responsible for
the cleavage of biocytin, liberating the free vitamin that can be absorbed by
the intestine and then transferred to the cytosolic space of other tissues
where it is directly assimilated (Hymes and Wolf, 1996). The existence of
biotinidase in the plant kingdom has not yet been reported. Since, in
general, foodstuffs of plant origin have a greater free biotin content
than foodstuffs of animal origin (with the exception of milk), plant biotin
is more rapidly absorbed and metabolized than animal sources of the
vitamin.
In humans, the daily requirement of biotin has been estimated between 150
and 300 g. Biotin is widely distributed in foods and feedstuffs, although the
absolute amount of biotin present in even the richest dietary sources are low.
Milk, liver, egg yolk and a few vegetables (nuts, fruits and unpolished rice)
are the most important natural sources for human nutrition (Table I).
The oilseed or alfalfa meals and dried yeasts are the most important natural
sources for the feeding of nonruminant animals. A second potential source of
biotin for higher organisms is the microbial synthesis by gut microflora. As a
consequence, simple deficiencies of biotin in animals or humans are extreme-
ly rare. Few cases of biotin deficiencies have been reported in humans. Most
of these involved nursing infants whose mothers’ milk contained inadequate
supplies of the vitamin, chronic ingestion of egg white, or patients receiving
incomplete parenteral nutrition (Combs, 1998a). It is also thought that
marginal biotin status plays a causative role in the aetiology of sudden infant
death syndrome.
C. BIOTIN-CONTAINING PROTEINS
Biotinylated proteins are not widespread in nature. For example, the only
biotin-dependent carboxylase in E. coli is acetyl-CoA carboxylase (EC
6.4.1.2), a multisubunit enzyme, in which one of the subunits is biotinylated
and corresponds to the biotin carboxyl carrier protein (BCCP). Other bacte-
ria contain one to no more than three biotinylated proteins (Fall, 1979).
Eukaryotic cells appear to contain a slightly greater number of biotinylated
proteins. For example, Saccharomyces cerevisiae contains four or five bioti-
nylated proteins depending on growth conditions (Lim et al., 1987), whereas
mammals (Jitrapakdee and Wallace, 2003) are reported to contain four
44 C. ALBAN
TABLE I
Biotin Contents of Food
Food Biotin (g/100 g)

Dairy products
Milk 2
Cheeses 3–5
Meats
Beef 3
Liver 52
Calf kidney 100
Cereals
Barley 14
Sorghum 29
Rice 25
Oilseed
Rapeseed 99
Soybean 27
Vegetables
Carrots 3
Cauliflower 17
Potatoes 0.1
Soybeans 60
Nuts
Peanuts 34
Walnuts 37
Others
Eggs 20
Brewers’ yeast 80
Alfalfa meal 54
Compilation of values by Combs (1998b) and Bonjour (1991).
biotinylated proteins. All these enzymes play crucial cellular housekeeping

functions. More specifically, acetyl-CoA carboxylase that catalyses the
ATP-dependent carboxylation of acetyl-CoA is recognized as the regulatory
enzyme of lipogenesis; methylcrotonoyl-CoA carboxylase (EC 6.4.1.4) cata-
lyses the conversion of methylcrotonoyl-CoA to methylglutaconyl-CoA, a
key reaction in the degradation pathway of leucine; propionyl-CoA carbox-
ylase (EC 6.4.1.3) is a key enzyme in the catabolic pathway of odd-numbered
fatty acids and the amino acids, Ile, Thr, Met and Val; and pyruvate carbox-
ylase (EC 6.4.1.1) has an anaplerotic role in the formation of oxaloacetate.
The common feature of these reactions is the transfer of a carboxyl group
from bicarbonate to an acceptor substrate, utilizing biotin as a carboxyl
carrier. The reactions catalysed by these enzymes take place in two steps (1)
and (2), resulting in the overall reaction (3):
HCO
3 þ Enzyme - Biotin þ ATP - Mg
! Enzyme - Biotin CO2 þ ADP - Mg þ Pi ð1Þ
Enzyme - Biotin CO

2 þ Acceptor
! Acceptor CO 2 þ Enzyme - Biotin ð2Þ
HCO
3 þ Acceptor þ ATP - Mg ! Acceptor CO2
ð3Þ
þ ADP - Mg þ Pi
The features that distinguish the reactions of each of these enzymes are the
acceptor substrates. The family of biotin enzymes also includes oxaloacetate,
methylmalonyl-CoA and glutaconyl-CoA decarboxylases that are involved
in sodium transport in anaerobic prokaryotes (Dimroth, 1985) as well as
transcarboxylase (EC 2.13.1) that participates in propionic acid fermentation
in Propionibacterium shermanii (Wood and Kumar, 1985). The latter two
classes of enzymes do not require ATP as a substrate. In all biotin enzymes
described to date, the biotin is covalently linked to the e-amino group of a
specific Lys residue located within a highly conserved (Ala/Val)-Met-Lys-
(Met/Leu) tetrapeptide motif.
Plant acetyl-CoA carboxylase has been documented since 1961 (Hatch and
Stumpf, 1961). Investigations of plant acetyl-CoA carboxylases increased in
the late 1980s because of its regulatory role in fatty acid biosynthesis and also
because this enzyme is the molecular target of powerful herbicides in use
since the early 1980s and effective against grasses (the Graminaceae) includ-
ing grass weeds (Harwood, 1988). Since then, other biotin-containing pro-
teins with variable structure and subcellular localizations have been
discovered in plants. These include two structurally distinct isoforms of
acetyl-CoA carboxylases in cytosol and plastids (Alban et al., 1994), a
geranyl-CoA carboxylase in plastids (Guan et al., 1999), a methylcroto-
noyl-CoA carboxylase in mitochondria (Alban et al., 1993) and a cytosolic
seed storage biotin-protein (SBP) with an atypical biotinylation motif (Duval
et al., 1994). Comprehensive information on the structure, regulation and
function of plant biotin-containing proteins are available on leading reviews
(Alban et al., 2000; Nikolau et al., 2003) and will not be detailed here. In this
chapter, I have attempted to summarize the recent advances about
biotin biosynthesis and protein biotinylation processes in higher plants and
their implications for industry, for example, the rational design of new
herbicides.
46 C. ALBAN
II. THE BIOSYNTHETIC PATHWAY
In all known microbes, biotin is synthesized from pimeloyl-CoA through

four enzymatic steps comprising 7-keto-8-aminopelargonic acid (KAPA)
synthase (EC 2.3.1.47), 7,8-diaminopelargonic acid (DAPA) synthase (EC
2.6.1.62), dethiobiotin synthetase (EC 6.3.3.3) and biotin synthase (EC
2.8.1.6) encoded by bioF, bioA, bioD and bioB genes, respectively (Fig. 3).
Enzymes encoded by these genes in E. coli, Bacillus sphaericus and more
recently in Bacillus subtilis or Mycobacterium tuberculosis have been totally
or partially characterized biochemically, and/or structurally, and their reac-
tion mechanisms elucidated (Dey et al., 2010; Schneider and Lindqvist, 2001;
Streit and Entcheva, 2003). In plants, the biosynthetic pathway appears to
follow the same pattern as identified for bacteria (Fig. 3). This was deduced
from measuring pools of the different intermediates of biotin biosynthesis,
employing lavender (Lavandula vera) cells cultures treated with radiolabelled
B. sphaericus A. thaliana
B. subtilis Gram + Plants
? ?
E. coli
bioI Gram –
Pimelic acid
Malonyl-CoA (ACP)
bioW bioC, FabH-G-Z-I-B, bioH

Pimeloyl-CoA (ACP)
L-ala bioF (BIO4), KAPA synthase
7-keto-8-aminopelargonic acid (KAPA)

bioA (BIO1), DAPA synthase
7,8-diaminopelargonic acid (DAPA)
bioD (BIO3), DTB synthetase
Dethiobiotin (DTB)
bioB (BIO2), biotin synthase
Biotin
Fig. 3. The biotin biosynthetic pathway in bacteria and plants. The bacterial gene
names are given in lower case letters; the respective plant homologs are shown in
bracketed uppercase letters.
precursors (Baldet et al., 1993b). An alternative approach used to study

biotin synthesis in plants has been the isolation and characterization of
auxotrophic mutants. The Arabidopsis bio1 mutant was the first plant auxo-
troph shown to result in embryo lethality (Schneider et al., 1989). Seeds
homozygous for the mutation failed to develop unless exogenous biotin,
DTB or DAPA was supplied to the plant (Shellhammer and Meinke,
1990). The E. coli bioA gene, which codes for DAPA synthase, could geneti-
cally complement the bio1 mutation, demonstrating that bio1/bio1 mutant
plants are defective in this enzyme (Patton et al., 1996). Later on, a second
biotin auxotroph mutant of Arabidopsis was identified, defective in the final
step of biotin synthesis, that is, the conversion of DTB to biotin (Patton et al.,
1998). Genetic and phenotypic characterization of this bio2 mutant also
showed embryo lethality as a consequence of the BIO2 gene knockout and
efficient phenotypic reversion on addition of exogenous biotin (Arnal et al.,
2006). Thus, biotin biosynthesis is an indispensable procedure for plant
growth and inhibition of the enzymes of the pathway is potentially an
attractive target for herbicide development (Alban et al., 2000). As a proof,
inhibition of KAPA synthase reaction by triphenyltin acetate (TPTA),
DAPA synthase reaction by KAPA analogs or biotin synthase reaction by
acidomycin is lethal for the plant (Baldet et al., 1993b; Hwang et al., 2010;
Nudelman et al., 2004). However, until the past decade, none of these
enzymes had been characterized.
A. THE ORIGIN OF PIMELOYL-COA
If the last four steps of biotin biosynthesis, from pimeloyl-CoA to biotin, are
common to most bacteria, fungi and plants, the origin of this precursor is
much less clear. Alternate pathways to pimeloyl-CoA seem to coexist in
nature (Fig. 3). The gram-positive bacteria such as B. sphaericus or B. subtilis
are capable of forming pimeloyl-CoA from pimelic acid with a single gene
encoding a pimeloyl-CoA synthetase (EC 6.2.1.14; bioW; for a review, see
Streit and Entcheva, 2003). Further, in Bacillus species, the bioI gene, which
appears to be restricted to these organisms, encodes a cytochrome P450
family member that makes the pimeloyl moiety by cleaving long-chain
acyl-ACPs precursors (Stok and De Voss, 2000). Gram-negative bacteria
like E. coli do not synthesize pimeloyl-CoA from pimelic acid. Genetic
analysis in E. coli identified two genes essential for pimeloyl-CoA synthesis,
bioC and bioH whose exact function remained unknown for more than
15 years (Ifuku et al., 1994). Recently, the group of Cronan demonstrated
that the pimeloyl moiety in E. coli is synthesized by a modified fatty acid
synthetic pathway in which !-carboxyl group of a malonyl-thioester is
48 C. ALBAN
methylated by BioC, which allows recognition of this atypical substrate by

the classical fatty acid synthetic enzymes. The malonyl-thioester methyl ester
enters fatty acid synthesis as the primer and undergoes two reiterations of the
fatty acid elongation cycle to give pimeloyl-ACP methyl ester, which is finally
hydrolysed to pimeloyl-ACP and methanol by BioH (Lin et al., 2010). This
work also suggests that pimeloyl-ACP rather that pimeloyl-CoA is the
physiological substrate for biotin synthesis. In eukaryotes, our knowledge
of the origin of the pimeloyl moiety is still fragmentary. Recently, the protein
encoded by the BIO1 gene in yeasts was found to have the function of a
pimeloyl-CoA synthetase (Hall and Dietrich, 2007). In plants, labelled pime-
lic acid has been efficiently incorporated into biotin using lavender cell
cultures, suggesting the existence of pimeloyl-CoA synthetase activity also
in plants (Baldet et al., 1993b). However, to date, no plant gene homologous
to gram-positive bacterial bioW or yeast BIO1 genes has been identified.
B. 7-KETO-8-AMINOPELARGONIC ACID SYNTHASE
KAPA synthase, the first committed enzyme in the pathway, catalyses the
decarboxylative condensation of pimeloyl-CoA and L-Alanine to produce
KAPA, CoASH and carbon dioxide:
Pimeloyl - CoA þ L - alanine ! KAPA þ CoA - SH þ CO2
The structure and reaction mechanism of KAPA synthase place it in the
subfamily of -oxoamine synthases, a small group of pyridoxal 50 -phosphate
(PLP)-dependent enzymes of the -family (Alexeev et al., 1998; Ploux and
Marquet, 1996; Webster et al., 2000). Searches of the Arabidopsis genome
database detected a single gene (here named AtBIOF or BIO4) encoding a
predicted protein with 27–32% identity to protein sequences of well-charac-
terized bacterial KAPA synthases (Pinon et al., 2005). Despite the relatively
low overall amino acid identity with its bacterial counterparts, the plant
protein was able to complement an E. coli bioF-mutant and to catalyse
KAPA synthase reaction when assayed using pimeloyl-CoA and L-Ala as
substrates. Biochemical, kinetic and spectroscopic studies of purified recom-
binant enzyme evidenced high substrate specificities and allowed determina-
tion of the reaction mechanism. Essential steps of this mechanism are
formation of an external aldimine between PLP cofactor and the substrate
L-Ala. Abstraction of the C2-H proton of the aldimine, possibly by Lys-319,
leads to a quinonoid intermediate, which then attacks the thioester carbonyl
of pimeloyl-CoA. Release of CoASH produces a -ketoacid aldimine, which
after decarboxylation is converted into the product (Pinon et al., 2005). More
importantly, the salient fact of this study concerned the surprising cellular
distribution of KAPA synthase as determined by two independent methods.

Both GFP-fusions targeting experiments and subcellular fractionation stud-
ies showed that this initial step in biotin synthesis in Arabidopsis takes place
in the cell cytosol, which contrasts with the mitochondrial location of the
remaining pathway (see below).
C. 7,8-DIAMINOPELARGONIC ACID SYNTHASE—DETHIOBIOTIN SYNTHETASE
The antepenultimate step in the biotin biosynthetic pathway, the conversion

of KAPA to DAPA, is catalysed by DAPA synthase, another PLP-depen-
dent enzyme. In most bacteria, the enzyme uses S-adenosyl-L-methionine
(AdoMet) as amino group donor, an unusual feature among aminotrans-
ferases (Breen et al., 2003; Izumi et al., 1975; Mann and Ploux, 2006):
KAPA þ AdoMet ! DAPA þ AdoMTOB
Interestingly, B. subtilis uses L-Lysine, another unusual amino donor for
the reaction (Van Arsdell et al., 2005). The chemical TPTA is a potent
inhibitor of KAPA synthase reaction in Arabidopsis with strong herbicidal
activity (Hwang et al., 2010). The supplement of biotin or biotin biosynthesis
intermediate such as DTB, DAPA, KAPA þ AdoMet, but not KAPA alone
rescued germination and plant growth inhibited by TPTA, suggesting that
AdoMet might be an essential amino group donor for the synthesis of DAPA
in plants, as well. DTB synthetase carboxylates DAPA to form the ureido
ring of DTB in an ATP-Mg-dependent penultimate step of the pathway
(Alexeev et al., 1995; Huang et al., 1995):
DAPA þ ATP þ CO2 ! DTB þ ADP þ Pi
In Arabidopsis, genetic studies revealed that DAPA synthase (BIO1; BioA
ortholog) and DTB synthetase (BIO3; BioD ortholog) are encoded in adja-
cent genes defining a single genetic locus and are expressed in both single and
chimeric BIO3–BIO1 transcripts, through alternative splicing events
(Muralla et al., 2008). One of the fused transcripts is monocistronic and
encodes a bifunctional protein capable of complementing the orthologous
auxotrophs of E. coli (bioD and bioA). The second one includes 10 more
nucleotides that introduce a premature stop codon. As a consequence, this
splice variant is bicistronic, with distinct but overlapping reading frames.
This bicistronic transcript is potentially capable of producing separate BIO3
and BIO1 proteins. The existence of a monocistronic BIO3–BIO1 transcript
is not a unique feature of Arabidopsis. Homology searches among eukaryotic
and prokaryotic genomes revealed the presence of a bifunctional BIO3–BIO1
homologue gene in others flowering plants, mosses, green and red algae and
50 C. ALBAN
in most ascomycete and basidiomycete fungi (Hall and Dietrich, 2007;

Magliano et al., 2010; Muralla et al., 2008; Fig. 4). These data suggest that
a fusion event between prokaryotic bioD and bioA ancestor genes occurred
Vitis vinifera (XM_002270515.1)

Arabidopsis thaliana (EU089963.1)
Brassica rapa subsp. pekinensis (AC189479.2)
Medicago truncatula (barrel medic) (AC174353.16)
Sorghum bicolor (sorghum)(XM_002468273.1)
Zea mays (BT065649.1)
Plants
Oryza sativa Japonica Group (NM_001067889.1)
Physcomitrella patens subsp. Patens (XM_001764409.1)
Ostreococcus lucimarinus CCE9901 (XM_00T422822.1)
Micromonas sp. RCC299 (XM_002503155.1)
Chlamydomonas reinhardtii (XM_001690622.1)
Cyanidioschyzon merolae strain 10D (CMG023C)
Aspergillus nidulans FGSC A4 (XM_659156.1)
Aspergillus oryzae RIB40 (XM_001816971.1)
Aspergillus flavus NRRL3357 (XM_002382995.1)
Aspergillus terreus NIH2624 (XM_001209788.1)
Bifunctional
Aspergillus niger CBS 513.88 (XM_001396701.1)
Aspergillus fumigatus Af293 (XM_74618.1) Fungi
Neosartorya fischeri NRRL 181 (XM_001257570.1)
Aspergillus clavatus NRRL 1 (XM_001270182.1)
Penicillium chrysogenum Wisconsin 54-1255 (XM_002563776.1)
Uncinocarpus reesii 1704 (XM_002541682.1)
Coccidioides immitis RS (XM_001247545.1)
Penicillium marneffei ATCC 18224 (XM_002143123.1)
Talaromyces stipitatus ATCC 10500 (XM_002479411.1)
Aiellomyces dermatitidis SLH14081 (XM_002627316.1)
Aiellomyces capsulatus NAm1 (XM_001538071.1)
Phaeosphaeria nodorum SN15 (XM_001805235.1)
Pyrenophora tritici-repentis Pt-1 C-BFP (XM_001930446.1)
Sclerotinia sclerotiorum 1980 UF-70 (XM_001590649.1)
Botryotinia fuckeliana B05.10 (XM_001558049.1)
Podospora anserina DSM 980 (XM_001903481.1)
Podospora anserina (CAP61291.1)
Gibberella zeae PH-1 (anamorph: Fusarium graminearum) (XM_389224.1)
Yarrowia lipolytica CLIB122 (XM_504233.2)
Malassezia globosa CBS 7966 (XM_001729066.1)
Ustilago maydis 521 (XM_753969.1)
Crytococcus neoformans var. neoformans JEC21 (XM_569073.1)
Laccaria bicolor S238N-H82 (XM_001880692.1)
Coprinopsis cinerea okayama7#130 (XM_001836166.1)
Schizosaccharomyces iaponicus yFS275 (XM_002171908.1)
Hydrogenobaculum sp. Y04AAS1 (ACG57182.1)
Hydrogenobaculum sp. Y04AAS1 (YP_002121160.1)
Aquifex aeolicus (O66557.1)
Hydrogenivirga sp. 128-5-R1-1 (EDP73255.1)
Sulfurihydrogenibium sp. YO3AOP1 (ACD66647.1)
Acidithiobacillus ferrooxidans ATCC 53993 (ACH83818.1)
Bacteria
Acidithiobacillus ferrooxidans ATCC 53993 (YP_002220025.1)
Methanocaldococcus jannaschii (Q58696)
Kurthia sp. 538-KA26 (BAB39453.1)
Staphylococcus carnosus subsp. carnosus TM300 (YP_002635311.1)
Bacillus subtilis (P53555.1)
Brevibacillus brevis NBRC 100599 (BAH46441.1)
Helicobacter pylori J99 (Q9ZKM5.1)
Helicobacter pylori (025627.1)
Helicobacter pylori B38 (YP_003057676.1)
Helicobacter acinonychis str. Sheeba (CAJ99442.1)
Herminiimonas arsenicoxydans (CAL60336.1)
Azoarcus sp. BH72 (YP_933392.1)
Lysinibacillus sphaericus (P22805.1)
Rhizobium leguminosarum bv. viciae 3841 (CAK12322.1)
Rhodopirellula baltica SH 1 (NP_865422.1)
Zymomonas mobilis subsp. mobilis ZM4 (AAV90542.1)
Mycobacterium tuberculosis (P0A4X6.1)
Mycobacterium bovis (P0A4X7.1)
Mycobacterium leprae (P45488.1)
Monofunctional
Corynebacterium glutamicum (P46395.2)
Thiomicrospira crunogena XCL-2 (ABB40873.1)
Haemophilus influenzae 86-028NP (AAX88383.1)
Haemophilus influenzae 86-028NP (YP_249043.1)
Haemophilus influenzae (P44426.1)
Neisseria meningitidis 8013 (CAX50480.1)
Neptuniibacter caesariensis (ZP_01167088.1)
Campylobacter hominis ATCC BAA-381 (ABS52358.1)
Lachancea thermotolerans (CAR23468.1)
Buchnera aphidicola (Baizongia pistaciae) (Q89AK4.1)
Buchnera aphidicola str . Bp (Baizongia pistaciae) (AAO26998.1)
Buchnera aphidicola (Schizaphis graminum) (Q8K9P0.1)
Buchnera aphidicola (Acyrthosiphon pisum) (P57379.1)
Pichia stipitis CBS 6054 (EAZ63280.2)
Debaryomyces hansenii (CAR66048.1)
Saccharomyces cerevisiae (P50277.1)
Zygosaccharomyces rouxii (CAR30704.1)
Hemiascomycetes
Kluyveromyces lactis (CAH01942.1)
Escherichia coli (P12995.2)
Escherichia vulneris (P53656.1)
uncultured bacterium pCosAS1 (AAG53588.1)
Salmonella thyphimurium (P12677.2)
uncultured bacterium pCosHE1 (AAG60563.1)
Erwinia pyrifoliae DSM 12163 (CAY74978.1)
Serratia marcescens (P36568.1)
Serratia odorifera 4Rx13 (ZP_06189072.1)
Providencia rustigianii DSM 4541 (EFB74154.1)
Providencia alcalifaciens DSM 30120 (ZP_03320306.1)
Providencia stuartii ATCC 25827 (EDU58416.1)
Proteus penneri ATCC 35198 (ZP_03806407.1)
Photorhabdus luminescens subsp. laumondii TTO1 (CAE13777.1)
Mesorhizobium loti MAFF303099 (BAB52209.1)
Xanthobacter autotrophicus Py2 (ABS68156.1)
Gemmatimonas aurantiaca T-27 (YP_002762353.1)
Prochlorococcus marinus MED4 (CAE19931.1)
Prochlorococcus marinus subsp. marinus str. CCMP1375 (AAQ00670.1)
Thermosynechococcus elongatus BP-1 DNA (BAC09487.1)
Flavobacterium johnsoniae UW101 (ABQ03844.1)
Capnocytophaga ochracea DSM 7271 (ACU93703.1)
Pedobacter heparinus DSM 2366 (ACU04770.1)
Chlamydophila pneumoniae LP CoLN (ACZ32943.1)
Fig. 4. Phylogenetic analysis of eukaryotic bifunctional DTB synthetase/DAPA

synthase and monofunctional DAPA synthase prokaryotic orthologs. Protein
sequences were first aligned using CLUSTALW. The phylogenetic tree was con-
structed using the protpars and neighbour modules from the PHYLIP package and
the BLOSUM 62 similarity matrix.
early in the evolution of modern-day eukaryotes. By contrast, in the large

majority of species in the Saccharomycotina and Schizosaccharomycetes
classes, the bioA and bioD gene orthologues (named BIO3 and BIO4 in
yeasts) are separate (Hall and Dietrich, 2007; Magliano et al., 2010). Hall
and Dietrich (2007) propose that much of the biotin pathway was lost in
Saccharomycotina ancestors of Saccharomyces and Candida and that the
bioA and bioD orthologs were reacquired through a separate, horizontal
gene transfer from an unidentified prokaryotic donor. Noticeably, although
numerous bacteria have neighbouring bioD and bioA genes in the same
orientation, a bacterial gene fusion event does not seem to have occurred.
The ability to produce a bicistronic transcript through differential splicing
appears to have been a more recent event because it is limited to selected
angiosperms (Muralla et al., 2008). Biotin biosynthesis in plants thus pro-
vides an intriguing example of a bifunctional locus that catalyses two sequen-
tial reactions in the same metabolic pathway. This complex locus exhibits
several unusual features that distinguish it from biotin operons in bacteria
and from other genes known to encode bifunctional enzymes in plants
(Muralla et al., 2008). Interestingly, the BIO3–BIO1 protein contains an
N-terminal sequence that is predicted to target the protein to mitochondria
by all the major prediction programs for intracellular localization of plant
proteins (Muralla et al., 2008). This localization is supported by experimental
proteomic data in the case of Chlamydomonas reinhardtii BIO3–BIO1 protein
(Chlamydomonas Mitochondrial Proteome; Atteia et al., 2009). It therefore
appears that in plants, both DAPA synthase and DTB synthetase activities
take place in mitochondria. Finally, the capacity of a single plant enzyme to
convert KAPA into DAPA and then DTB may have implications for ongo-
ing efforts to design herbicides that interfere with biotin production and with
biotechnological strategies to increase biotin levels in crop plants (for bio-
fortification or phytofarming aims; Muralla et al., 2008).
D. BIOTIN SYNTHASE
Biochemical and molecular characterization of the biotin biosynthetic path-

way in plants has dealt primarily with biotin synthase, the final enzyme of the
pathway, because this reaction is a rate-limiting step and also because its
mechanism still remains an enigma for chemists and biologists. Biotin
synthase is an AdoMet-dependent radical enzyme, undoubtedly the most
complex of the pathway generating biotin. Its activity aims to sulphur
insertion at the C6 and C9 position in DTB and the intimate chemistry of
the underlying reaction is not yet fully understood.
52 C. ALBAN
DTB þ AdoMet þ ‘‘S’’ ! Biotin þ 50 - deoxyadenosine þ L - methionine

Also, the role of specific proteins and cofactors, and the ultimate source of
sulphur are still matter of debate (for a review, see Jarrett, 2005a). In E. coli, the
conversion of dethiobiotin to biotin is catalysed by a complex involving at least
three proteins (including flavodoxin, flavodoxin reductase and MioC) in addi-
tion to biotin synthase which alone is not able to support this reaction (Sanyal
et al., 1996). Interestingly, plant biotin synthase (here named BIO2) seems to
accommodate these bacterial partners of the reaction, yielding a functional
biotin synthase complex. This capacity accounted for the cloning of the Arabi-
dopsis BIO2 gene by functional complementation of an E. coli bioB mutant
(Baldet and Ruffet, 1996). In addition, subcellular fractionation studies and
Western blot analyses using antibodies raised against the plant recombinant
enzyme clearly demonstrated its mitochondrial location (Baldet et al., 1997).
As its bacterial counterparts (Berkovitch et al., 2004), purified BIO2
protein is a homodimer that, in its active reconstituted form, coordinates a
[2Fe–2S]2þ and a [4Fe–4S]2þ cluster per monomer (Daouda Traoré, Antoine
Picciocchi and Claude Alban, unpublished data; Fig. 5). The purified enzyme
alone is not able to support biotin synthesis. Combination experiments using
purified BIO2 protein and extracts from pea leaf or potato tuber organelles
(plastids and mitochondria) showed that only mitochondrial fractions could
A B
0.45 BIO2 [2Fe–2S]/[4Fe–4S]

330
2Fe/2S
0.35
Absorbance
410
4Fe/4S
0.25
540
0.15
A 280 = 0.69
0.05
N (Pro44) 0
AdoMet 300 400 500 600 700
DTB Wavelength (nm)
Fig. 5. (A) Structure of Arabidopsis BIO2 monomer modelized by Swiss-model

program (www.expasy.ch) using E. coli biotin synthase structure as a matrix
(Berkovitch et al., 2004). AdoMet, S-adenosyl-L-methionine; DTB, dethiobiotin.
(B) UV–visible spectrum of purified recombinant BIO2 protein incubated with excess
iron and sulphide under strict anaerobiosis. The spectrum profile is consistent with
the presence on the enzyme of both a [2Fe2S] and a [4Fe4S] cluster (Daouda
Traoré, Antoine Picciocchi, Claude Alban, Lilian Jacquamet, unpublished data).
elicit biotin formation at catalytic rates in the plant reconstituted system, in

keeping with the specific location of BIO2 protein (Picciocchi et al., 2001).
A biochemical screening of potato mitochondrial matrix (fractionation of
proteins onto chromatographic columns and in vitro reconstitution experi-
ments of biotin synthase activity) together with a genomic-based search in
the Arabidopsis genome database resulted in the identification of an adreno-
doxin-like protein (ADX1), an adrenodoxin reductase (ADR) and a cysteine
desulphurase (NFS1) as essential components for the reaction (Picciocchi
et al., 2003). ADX1 and ADR form a physiological reduction system, fuelling
the reaction with electrons from NADPH. The role of this system is to
sustain the reductive cleavage of S-adenosylmethionine (AdoMet), an oblig-
atory cofactor of the reaction, through the [4Fe4S]2þ centre of BIO2. This
two-step reaction, which generates AdoMet radical intermediates, is involved
in the activation of the CH bonds of the DTB substrate where the sulphur
atom is to be introduced, with production of 9-mercaptodethiobiotin as an
intermediate (Baldet et al., 1993b; Taylor et al., 2008; Tse Sum Bui et al.,
2004). Thus, BIO2 is part of the group of ‘Adomet-radical’ enzymes to which
belongs a variety of enzymes using such radical intermediates, but for quite
different purposes (for a review, see Jarrett, 2005a). The in vitro stimulation
of biotin synthase activity by the NFS1 protein strongly supports the idea
that cysteine is the initial sulphur donor for biotin in plant mitochondria. The
desulphurase is thought to take part in the recycling of BIO2 activity by
providing a renewable source of sulphur for the reaction. The sulphur atom
may be attached to DTB via the [2Fe2S]2þ cluster of BIO2, suggesting that
BIO2 is actually a real catalyst (Picciocchi et al., 2001, 2003), in contrast to
what was originally suggested from in vitro studies with bacterial biotin
synthase (Choi-Rhee and Cronan, 2005a,b; Jarrett, 2005b). In vitro forma-
tion of biotin at catalytic rates by biotin synthase has been recently confirmed
(Farrar et al., 2010). This work demonstrates that low in vitro biotin synthase
activities usually reported in the literature are mainly due to strong synergic
inhibition by both 50 -deoxyadenosine, an end-product of biotin synthase
reaction, and S-adenosyl-L-homocysteine, a major contaminant commonly
present in commercial AdoMet preparations. Different mechanisms have
been hypothesized to explain sulphur insertion into DTB. One mechanism
proposes that sulphide from the [2Fe–2S]2þ cluster is attached in a stepwise
manner to the C9 and C6 positions of DTB, with concomitant reduction and
loss of the residual cluster (Ugulava et al., 2001). An alternate mechanism
suggests that reduction and loss of the cluster precedes catalysis, and that
sulphur insertion is from an enzyme-bound cysteine persulphide that is
formed either during cluster degradation (Jameson et al., 2004) or via the
action of a cysteine desulphurase (Ollagnier-de-Choudens et al., 2002).
54 C. ALBAN
In addition to their implication in biotin synthase reaction, mitochondrial

ADX1/ADR redox system and NFS1 protein could be also involved in the
synthesis of the lipoate cofactor which also occurs in plant mitochondria
(Douce et al., 2001), the lipoate synthase and biotin synthase reactions being
mechanistically related. Also, ADX1, ADR and NFS1 and the yeast homol-
ogous proteins (namely Yah1p, Arh1p and Nfs1p, respectively) have been
identified as key components of mitochondrial iron–sulphur cluster (ISC)
assembly machinery (Balk and Lobreaux, 2005; Lill and Muhlenhoff, 2005).
Consequently, these proteins could have a dual function, a specific function
in the biotin synthase and lipoate synthase reactions and a more general role
in biosynthesis of Fe–S clusters for other redox enzymes. Interestingly,
attempts to complement a bio2 mutant with a truncated version of BIO2
lacking the mitochondrial targeting sequence failed, even with provision
of the substrate DTB, suggesting that biochemical constraints, and the
apparent close connection with the mitochondrial FeS machinery,
may account for the reaction being retained within mitochondria (Arnal
et al., 2006).
III. PROTEIN BIOTINYLATION

As mentioned in Section I, biotin is a cofactor for some carboxylases dealing
with crucial metabolic processes such as fatty acid synthesis and carbohy-
drate metabolism. The biotinylation of these enzymes is a post-translational
modification allowing the transformation of inactive apo-proteins into their
active holo forms. Therefore, this original and very specific post-translational
modification can be considered as the ultimate step of the biotin biosynthetic
pathway, and as such, it was included in this review. The covalent attachment
of biotin is catalysed by biotin-protein ligase (BPL) also called HCS (EC
6.3.4.11-15). D-Biotin is attached to a specific Lys residue of newly synthe-
sized apo enzyme, via an amide linkage between the biotin carboxyl group
and a unique e-amino-group of Lys residue (Samols et al., 1988). It occurs in
two steps (4) and (5) as follows:
D - Biotin þ ATP ! D - biotinyl 50 -AMP þ PPi ð4Þ
D - Biotinyl 50 -AMP þ apocarboxylase ! holocarboxylase þ AMP ð5Þ

In plants, four different biotin-dependent carboxylase activities have
been identified, two acetyl-CoA carboxylase activities, one in cytosol
and one in plastids; one geranoyl-CoA carboxylase in plastids; and one
methylcrotonoyl-CoA carboxylase in mitochondria (Alban et al., 2000).

Moreover, sequencing of the Arabidopsis genome confirmed the occurrence
of two genes encoding two distinct isoforms of acetyl-CoA carboxylase and
one gene for methylcrotonoyl-CoA carboxylase (Nikolau et al., 2003). As a
result, plants offer a unique case of triple compartmentalization of biotin-
dependent carboxylases. HCS activity localization in plant cell parallels this
complexity. In pea leaf cells, HCS activity was mainly located in cytosol of
fractionated protoplasts, but a significant activity was also identified in both
highly purified chloroplasts and mitochondria (Tissot et al., 1997). In Arabi-
dopsis cultured cells, HCS activity was also essentially recovered in cytosol of
fractionated protoplasts and, to a lesser extent, in chloroplasts and mito-
chondria (Puyaubert et al., 2008). This suggests that carboxylases are bioti-
nylated in their compartment of residence. Two HCS genes have been
evidenced in Arabidopsis (Tissot et al., 1997). Firstly, HCS1 cDNA has
been isolated by functional complementation of an E. coli mutant (Tissot
et al., 1997). Subsequently, the systematic sequencing of Arabidopsis genome
enabled the identification of HCS1 gene. Moreover, it confirmed the exis-
tence of a second HCS gene (HCS2), localized in the pericentromeric region
of chromosome 1 (Arabidopsis Genome Initiative, 2000). HCS1 and HCS2
genes present very large similarities and probably result from the duplication
of a common ancestor gene (Denis et al., 2002). HCS1 presents a broad
specificity of substrates and is able to biotinylate efficiently in vitro all
recombinant biotin-dependent apo-carboxylases identified in Arabidopsis
and E. coli, as well as the seed-specific biotinyl protein, SBP, albeit to a
lower extent (Denis et al., 2002; Puyaubert et al., 2008; Tissot et al., 1996,
1998). Interestingly, HCS2 expression produces a highly diverse family of
alternatively spliced mRNAs (Denis et al., 2002). However, none of the
putative HCS2 proteins, produced by alternative splicing of HCS2, were
active in vitro when overproduced in E. coli, nor rescued an E. coli mutant
affected in protein biotinylation (Denis et al., 2002). Moreover, reverse
genetics studies evidenced that HCS1 gene is essential for plant viability,
whereas disruption of HCS2 gene in Arabidopsis does not lead to any
obvious phenotype when plants are grown under standard conditions.
These findings suggested that HCS1 is the only protein responsible for
HCS activity in Arabidopsis cells, including the cytosolic, mitochondrial
and plastidial compartments. A close scrutiny of HCS1 gene expression
and splicing enabled Puyaubert et al. (2008) to propose an original mecha-
nism to account for this multiplicity of localizations. Located in HCS1
mRNA 50 -untranslated region, an upstream open reading frame (uORF)
regulates the translation initiation of HCS1 and the subsequent targeting
of HCS1 protein. Moreover, an alternative splicing of HCS1 mRNA can
56 C. ALBAN
regulate the presence and absence of this uORF, thus controlling organelle
versus cytosolic localization of HCS1 gene product (Fig. 6). This provides a
possibility for fine molecular regulation and, beyond the specific issue of
HCS1 protein, unveils the general complexity of plant metabolism compart-
mentalization. The physiological role of HCS2 gene is much less clear. HCS2
gene does not seem to bear any fundamental function in carboxylases bioti-
nylation in plants. It has been proposed that HCS2 could be an inactive
pseudogene in Arabidopsis or may have a regulatory function as a non-
coding RNA (Puyaubert et al., 2008). Alternatively, HCS2 proteins might
be involved in histones biotinylation. Indeed, beside its classical role in
carboxylases biotinylation, evidence is emerging that HCS in mammalian
cells nuclei participates in the epigenetic control of chromatin structure and
gene expression, through biotinylation of histones (Narang et al., 2004;
Zempleni, 2005). However, these conclusions are matter of debate and
controversy, and it is not clear whether histones are truly biotinylated
in vivo or not. Indeed, to date, no direct evidence for the existence of natural
biotinylated histones, from mass spectroscopic analyses, for example, has
been provided. All available data rely on secondary detection systems such as
streptavidin–HRP, and/or on in vitro biotinylation assays using recombinant
mammalian HCS. A recent study has called into question the reliability of
streptavidin detection of biotin on histones. It concluded that binding
of streptavidin to histones occurs independently of the biotin-binding site
on streptavidin (Bailey et al., 2008). Also, Healy et al. (2009) critically
examined a number of methods used to detect biotin attachment on histones,
including [3H]-biotin uptake, Western blot analysis of histones and mass
spectrometry of affinity-purified histone fragments with the objective of
determining if the in vivo occurrence of histone biotinylation could be defini-
tively established. Their conclusion was that ‘biotin is not a natural histone
modification’. Our initial efforts to demonstrate in vivo plant histones bioti-
nylation have also not been successful (Claude Alban, unpublished data).
For example, treatment of Arabidopsis cultured cells with [3H]-biotin specifi-
cally labelled biotin-dependent carboxylases, but no [3H]-biotin incorpora-
tion by histones could be evidenced (Fig. 7A). On the other hand, plant
histones were poor substrates for in vitro biotinylation by Arabidopsis HCS,
compared to carboxylases. Further, since similar low levels of biotin incor-
poration into unrelated basic proteins (with pKa > 9, i.e. comparable to those
of histone proteins), such as lysosyme (Fig. 7B), RNAse A or cytochrome c,
were also measured, this suggested that in vitro biotinylation of histones by
plant HCS is also artefactual. Collectively, these data suggest that the well-
established regulatory impact of biotin on gene expression in eukaryotes
must be through alternate mechanisms. For example, in mammals an
Chromosome 2
Nucleus
Transcription and splicing
HCS1
ATG HCS1 gene
0 1 2 TGA
Transcription
1 2 3 4 5 6 7 8 9 10
AUG
0 1 2 HCS1 mRNAs 1 2
Splicing
HCS1.un HCS1.s
HCS1.un HCS1.s
Cytosol
Translation and targeting
HCS1.un HCS1.s
1 2
Translation 0 1 2
0 100% HCS1 proteins

Re-initiation HCS1 TP HCS1
HCS1 TP-HCS1
Targeting
HCS1 HCS1 HCS1
Cytosol Plastids Mitochondria
= UpstreamORF
HCS1.un = Unspliced HCS1 mRNA
HCS1.s = Spliced HCS1 mRNA
TP = Transit peptide
= Ribosome
Fig. 6. A model of uORF-mediated translational control and HCS1 compart-

mentalization in Arabidopsis cells. HCS1 gene is represented in chromosome 2 of
Arabidopsis thaliana. Following its transcription, alternative splicing produces two
mRNA variants HCS1.un (unspliced) and HCS1.s (spliced). After their export into
the cytosol, HCS1.un and HCS1.s are translated. HCS1.un produces a short protein
starting at AUG2, which by eluding the transit peptide, leads to a cytosolic localiza-
tion. HCS1.s produces a longer protein starting at AUG1 and dual-targeted into the
plastids and mitochondria. Boxes figure a schematic view of the molecular mechan-
isms controlling this sketch of events in the nucleus and the cytosol. When HCS1 50 -
UTR is unspliced, the persistence of the uORF (starting at AUG0) disengages the
ribosomes from the mRNA. They fail to reinitiate at the close AUG1: translation
starts from AUG2 and produces a cytosolic HCS1. When HCS1 50 -UTR is spliced
out, uORF inhibition on translation initiation at AUG1 is abolished. Translation
starts from AUG1 and produces a HCS1 protein headed by a transit peptide.
58 C. ALBAN
BC zyme
BC zyme
A B
Lys nes
Lys es
2
2
ton
CP
CP
to
o
o
H is
H is
eins
eins
prot
prot
s
s
one
one
ble
ble
Solu
Solu
Anti-biotin-HRP
Hist
Hist
M M
97
66
45
31
21 Ponceau stain
14
Coomassie blue 3H-phosphor
15 min 2h
Incubation time with HCS1
Fig. 7. Attempts in biotinylation of plant histones by Arabidopsis HCS1 (Claude

Alban, unpublished data). (A) [3H]-biotin uptake into Arabidopsis cultured cells.
Arabidopsis cells were treated for 4 days with 0.5 mM acidomycin, an inhibitor of
biotin synthesis, in order to deplete the endogenous pool of biotin, and then cultured
for 8 days in the presence of 0.1 M [3H]-biotin (25 Ci/mmol). Cells were then washed,
resuspended into fresh culture medium supplemented with acidomicine and finally
cultured for 10 more days. Total soluble proteins and histone proteins were extracted
and analysed by SDS-PAGE, electrotransfer onto PVDF membrane and exposition
of the membrane to a tritium-specific phosphor screen for 11 weeks. Detection of
radioactive bands was performed by scanning the screen on a phosphoimager. The
molecular weight of the observed bands matches that of carboxylases. No radioactive
bands for histones were detected. Molecular mass markers (M) values are given on the
left in kDa. (B) Substrate specificity of Arabidopsis HCS1. The enzyme was incubated
for 15–120 min in the presence of biotin and apo-BCCP2 (the biotinyl subunit of
Arabidopsis acetyl-CoA carboxylases in its apo-form), extracted Arabidopsis histones
or lysosyme as protein substrates. Biotinylated proteins were analysed by western
blotting using anti-biotin-HRP as a probe and chemifluorescence detection.
alternative route involving a cGMP-dependent signalling cascade has been

proposed. This mechanism requires HCS, guanylate cyclase and cGMP-
dependent protein kinase (Solorzano-Vargas et al., 2002).
IV. CONCLUDING REMARKS
The past few years have seen dramatic advances in our understanding of the
enzymes that manipulate biotin in plants, including the characterization
of biotin-containing carboxylases, biotin synthesizing enzymes and BPLs.
Most of these proteins have been purified and/or their genes cloned and
characterized. With these achievements, a better understanding of the regu-
lation and of the interconnection between these different pathways is now
possible. A new challenge will be also to discover other cell functions for
biotin, especially in regard to the identification of novel biotinylated proteins
differing from the well-characterized carboxylases.
As it was underlined in this review, enzymes involved in biotin metabolism
are scattered among cell compartments, with mitochondria playing a central
role (Fig. 8). Such complex situation involving several compartments of the
plant cell is also found in other plant vitamin pathways such as those of
folates, ascorbate, pantothenate, niacin or phylloquinones (Lunn, 2007;
Rébeillé et al., 2007). This highlights the complexity and the peculiarity of
plant metabolism. The complex compartmentalization of biotin, biotin-
mediated reactions and biotin synthesis in the plant cell implies an intracel-
lular trafficking of biotin and precursors (Fig. 8). Biotin synthesis requires at
Cytosol Mitochondrion
Pimeloyl-CoA KAPA KAPA

BIO4 BIO1
AdoMet
DAPA
Plastid BIO3
ADR Dethiobiotin
Biotinyl-protein ADX1
BIO2 AdoMet
NFS1
HCS1 Biotin
Biotin HCS1
Biotinyl-protein
Biotinyl-protein biotin
HCS1
HCS1 gene
Nucleus
Fig. 8. Compartmentation of biotin metabolism in the plant cell. Biotin synthe-

sizing enzymes are KAPA synthase (BIO4); bifunctional DAPA synthase (BIO1)/
dethiobiotin synthetase (BIO3) (BIO3–BIO1 protein); biotin synthase (BIO2) asso-
ciated with stimulatory proteins as redox partners ADR and ADX1, and cysteine
desulphurase NFS1. Protein biotinylation in both the organelles and the cytosol is
mediated by HCS1 protein variants originating from the same gene (HCS1)
60 C. ALBAN
least one mitochondrial transporter to permit the counter-exchange of

KAPA (synthesized in the cytosol) and biotin. Indeed, once synthesized,
biotin must be exported outside mitochondria to cytosol and to chloroplasts
for protein biotinylation reactions. In plants, only one biotin transporter has
been identified so far. Arabidopsis AtSuc5 is a plasma membrane sucrose/
biotin co-transporter, possibly involved in biotin uptake and allocation
between the various plant tissues (Ludwig et al., 2000). Intracellular traffick-
ing of biotin and intermediates is, however, largely unknown, and the
proteins responsible for these activities have never been isolated or identified
hitherto. Interestingly, searches of the Arabidopsis genome database detected
a gene (At2g01170) encoding a predicted protein with about 45% similarity
to the protein sequence of S. cerevisiae Bio5p (Claude Alban, unpublished
observation). Bio5p is a KAPA/DAPA translocator found in biotin auxo-
trophic yeast strains (Phalip et al., 1999). The expression of the plant protein
into S. cerevisiae Bio5 mutant partially complemented yeast growth in the
presence of KAPA but not DAPA, suggesting that this protein could be
involved in KAPA transport in plant cells (Claude Alban, unpublished
observation). Also, synthesis of biotin depends on the presence of AdoMet
in plant mitochondria. In plant cells, AdoMet is synthesized in the cytosol
and a specific carrier is thus required to ensure the import of AdoMet into the
mitochondrial matrix (Palmieri et al., 2006). Understanding how
these carriers operate is particularly challenging because they are key
elements to understand how different parts of the pathway are co-ordinately
regulated.
In conclusion, recent and forthcoming advances in plant biotin metabo-
lism comprehension will allow for more rational and directed efforts at
manipulation of biotin pathway by genetic engineering. Indeed, some of
the processes that involve biotin generate biochemicals that serve a broad
range of nutritional and industrial purposes. For example, plant storage oils,
the biosynthesis of which requires acetyl-CoA carboxylase, are a major
resource for both human and animal nutrition, and also for a number of
non-food uses including pharmaceutical, cosmetics, detergents and even
biofuels. Biotin itself is also added to many food, feed and cosmetic products,
but it is mainly produced in a chemical process. Thus, the production of
biotin in natural and particularly plant environments as compared to syn-
thetic chemistry might be advantageous, especially for meeting positive
public acceptance. Finally, we anticipate that the future elucidation of
the structure of the enzymes of the plant biotin synthesis pathway will
allow the design of new inhibitor families having herbicidal activities, affect-
ing plants in a specific manner and therefore having a lower impact on the
environment.
ACKNOWLEDGEMENTS
Dr. Olivier Bastien is gratefully acknowledged for his invaluable expertise in

phyllogenetic tree construction. I thank all my collaborators and students
who were involved in the plant biotin metabolism project. Finally, I would
like to thank Professor Roland Douce for his indefectible enthusiasm and the
exciting discussions we have had during the past 25 years.
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Metabolism of Folates in Plants
STÉPHANE RAVANEL,*,{,{,},1 ROLAND DOUCE*,{,{,} AND

FABRICE RÉBEILLÉ*,{,{,}
*Laboratoire de Physiologie Cellulaire et Végétale, CNRS, UMR5168,

F-38054 Grenoble, France
{
CEA, iRTSV, F-38054 Grenoble, France
{
INRA, UMR1200, F-38054 Grenoble, France
}
Université Joseph Fourier, F-38054 Grenoble, France
I. Folates Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
II. Biological Functions of Folates: C1-Metabolism and Beyond . . . . . . . . . . . . . 70
A. Generation of C1-Units ....................................................... 70
B. Interconversion of C1-Substituted Folates ................................. 72
C. Utilization of C1-Units ........................................................ 73
D. Other Functions of Folates ................................................... 75
III. Folate Synthesis, Turnover and Homeostasis in Plants. . . . . . . . . . . . . . . . . . . . . 77
A. Biosynthesis of THF ........................................................... 77
B. Catabolism and Salvage Pathways........................................... 82
C. Cellular Compartmentation and Transport of Folates ................... 84
D. Folates Distribution in Plant Organs and Tissues......................... 85
E. Control of Folates Homeostasis ............................................. 87
IV. Folate Synthesis in Other Autotrophs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
A. Species–Specific Differences in THF Biosynthesis......................... 88
B. Folate Biosynthesis as a Target for Therapies
Against Infectious Diseases ................................................... 91
V. Physiology of Folate in Human Health and Disease . . . . . . . . . . . . . . . . . . . . . . . 93
A. Metabolic and Clinical Manifestations of Folate Deficiency ............ 93
B. Dietary Sources of Folate and Intake Recommendations ............... 94
VI. Folate Biofortification in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
1
Corresponding author: E-mail: stephane.ravanel@cea.fr

68 STÉPHANE RAVANEL ET AL.
ABSTRACT
Tetrahydrofolate and its derivatives, collectively termed folates or vitamin B9, are
essential cofactors for one-carbon metabolism. They transport and donate C1-units
for the synthesis of pantothenate, purines, thymidylate, serine, glycine, methionine
and formylmethionyl-tRNA. Also, recent studies indicate that folates can act as
electron donors in major cellular processes. Plants and many microorganisms synthe-
size folates de novo through a complex metabolic route that is now fully elucidated. In
contrast, humans and other vertebrates lack a complete biosynthetic pathway and
thus need dietary folates, of which plants are major sources. Folate deficiency is
widespread in rich and developing countries and is associated with severe health
problems. Supplementation of foods with synthetic folic acid and biofortification is
an alternative strategy to fight folate deficiency. Encouraging pilot metabolic engi-
neering studies in plants enabled significant enhancement of folate contents. In
the next future, increasing our knowledge about the mechanisms controlling folates
homeostasis in plants will provide the keys towards efficient biofortification of
plant foods.
ABBREVIATIONS
AAH aromatic amino acid hydrolase

ADC aminodeoxychorismate
AdoMet S-adenosylmethionine
DFE dietary folate equivalent
DHFR dihydrofolate reductase
DHN dihydroneopterin
DHPS dihydropteroate synthase
FCL 5-formyl-THF cycloligase
FPGS folylpolyglutamate synthetase
FTHFS 10-formyl-THF synthetase
GDC glycine decarboxylase
GGH -glutamyl hydrolase
GTPCHI GTP-cyclohydrolase I
HPPK hydroxymethyldihydropterin pyrophosphokinase
MTHFC 5,10-methenyl-THF cyclohydrolase
MTHFD 5,10-methylene-THF dehydrogenase
MTHFR 5,10-methylene-THF reductase
NTD neural tube defect
pABA para-aminobenzoate
RDA recommended dietary allowance
SHMT serine hydroxymethyl transferase
THF tetrahydrofolate
TS thymidylate synthase
METABOLISM OF FOLATES IN PLANTS 69
I. FOLATES STRUCTURE
Folate(s) or vitamin B9 is a generic term for tetrahydrofolate (THF) and its

derivatives. Chemically, folates are tripartite molecules composed of a pterin,
a para-aminobenzoic acid (pABA) and a -linked glutamate residue (Fig. 1).
From this chemical architecture, there is a large diversity of related species
resulting from the oxidation state of the pterin ring, the differential substitu-
tion of single-carbon units on the pterin and/or pABA moieties, and/or the
length of the glutamyl side chain. Cellular folates occur as dihydro- or
tetrahydro-derivatives of pteroylglutamic acid but folic acid, the most oxi-
dized form, does not exist in nature to any significant extent. Its occurrence is
dependent on the chemical oxidation of reduced folates or on commercial
synthesis for use in supplements and in food fortification. Only THF ‘parti-
cipates’ in one-carbon (C1) metabolism by accepting and donating C1-units.
These C1-groups range in oxidation state from formyl (most oxidized) to
methyl (most reduced) and are attached at N-5 of the pterin moiety, N-10 of
the pABA moiety or bridged between the two (Fig. 1). Also, naturally
occurring folates are predominantly polyglutamylated and are therefore
termed folylpolyglutamates. The glutamyl side chain of folates (1–8 residues)
is somewhat unusual in that residues are -linked and not -linked as with
O COOH
R2 N COOH
O N H n
R1 10
N 9
HN 5
6
7
8
H 2N N N
H
Pterin p-aminobenzoate Glutamate
Folates R1 R2
5-formyl-THF CHO H
10-formyl-THF H CHO
5,10-methenyl-THF —
— CH+-
5,10-methylene-THF -CH2-
5-methyl-THF CH3 H
5-formimino-THF — NH
CH — H
Fig. 1. Structure of THF and its derivatives. Cellular folates are substituted at the
N-5 and/or N-10 positions by C1-units of different oxidation states and usually
contain 5–8 glutamate residues.
proteins. In all organisms, polyglutamylation is known to be essential for

three physiological roles (Shane, 1989). First, folylpolyglutamates are the
preferred coenzymes for most of the enzymes involved in C1 metabolism.
Second, the chain enhances folate stability by favouring binding to proteins,
bound folates being less sensitive to oxidative degradation (Rébeillé et al.,
1994). Third, polyglutamylation is the principal means by which folates are
retained within cells and subcellular compartments. Chain elongation
increases the anionic nature of folates coenzymes by providing -carboxyl
charges and decreases affinity for membrane carriers, thus impairing folates
diffusion through hydrophobic barriers.
II. BIOLOGICAL FUNCTIONS OF FOLATES:

C1-METABOLISM AND BEYOND
The N-5 and N-10 atoms of the THF cofactor are modified with C1-units at
the oxidation state of methanol (5-methyl-THF), formaldehyde (5,10-meth-
ylene-THF) and formate (10-formyl-THF, 5-formyl-THF and 5,10-methe-
nyl-THF). Serine, glycine and formate are the principal sources for C1 units,
the catabolism of these compounds resulting in the synthesis of 5,10-methy-
lene-THF and 10-formyl-THF. These folates are then enzymatically inter-
converted to other derivatives which serve a particular metabolic function:
5-methyl-THF is required for methionine synthesis, 5,10-methylene-THF is
required to convert uridylate (dUMP) to thymidylate (dTMP) and to pro-
duce pantothenate, whereas 10-formyl-THF supplies C-2 and C-8 for purine
ring biosynthesis and contributes to formylmethionyl-tRNA synthesis. The
overall organization of this complex metabolic network is generally con-
served between organisms, from microbes to human. However, depending
on species, tissues and developmental stages, C1 metabolism has been
adapted to meet specific metabolic requirements (Christensen and
MacKenzie, 2006; Nzila et al., 2005a,b; Ravanel et al., 2004a).
A. GENERATION OF C1-UNITS
The conversion of serine into glycine, a reaction catalysed by the pyridoxal

50 -phosphate-dependent enzyme serine hydroxymethyl transferase (SHMT)
and leading to 5,10-methylene-THF formation, is by far the main source of
C1 units in all organisms. Although the interconversion of serine and glycine
by SHMT is fully reversible, the equilibrium distribution of the substrates
indicates that glycine formation is favoured. In eukaryotes, SHMT is present
in the cytosol and the organelles, mitochondria and plastids, indicating that
serine can act as C1-unit donor in these compartments (Fig. 2; Appling, 1991;
Hanson and Roje, 2001; Tibbetts and Appling, 2010). In mitochondria, the
glycine decarboxylase (GDC) activity allows glycine to be used as an alter-
native C1-unit donor. GDC is a multi-enzyme complex that catalyses the
oxidative decarboxylation and deamination of glycine into CO2 and NH3
with the concomitant conversion of THF into 5,10-methylene-THF (Douce
et al., 2001). In mitochondria from photosynthetic tissues, the functions of
SHMT and GDC have been adapted to participate in photorespiration, a
complex pathway connected to photosynthesis in C3 plants (Douce and
Neuburger, 1999; Foyer et al., 2009). In leaf mitochondria, almost all 5,10-
methylene-THF formed upon glycine oxidation is used in serine synthesis for
recycling of ribulose-1,5-bisphosphate, a key intermediate of the Calvin
cycle. In mitochondria from non-photosynthetic tissues, the coupled action
of GDC and SHMT is also dedicated primarily to serine formation, which is
used then as a source of C1 units in the cytosol (Mouillon et al., 1999).
Formate is an alternative source of C1-units in eukaryotes. The synthesis
of 10-formyl-THF from formate and THF is catalysed by the
Fig. 2. Overview of C1-metabolism and its compartmentation in plant cells. The

enzymes involved in C1-metabolism are: (1) serine hydroxymethyltransferase; (2)
glycine decarboxylase; (3) 5,10-methylene-THF dehydrogenase; (4) 5,10-methenyl-
THF cyclohydrolase; (5) 10-formyl-THF synthetase; (6) 5,10-methylene-THF
reductase; (7) 5-formyl-THF cycloligase; (8) methionyl-tRNA formyltransferase; (9)
glycinamide ribonucleotide transformylase and aminoimidazole carboximide ribonu-
cleide transformylase; (10) thymidylate synthase; (11) methionine synthase; (12)
AdoMet synthetase; (13) cytosolic AdoMet-dependent methyltransferases; (14)
AdoHey hydrolase; (15) ketopantoate hydroxymethyltransferase; (16) 10-formyl-
THF deformylase. Ado, adenosine; AdoHey, S-adenosylhomocysteine; AdoMet,
S-adenosylmethionine; Hey, homocysteine; tetrahydrofolate (THF) and its deriva-
tives: CH3- (methyl), CH2- (methylene), CHþ- (methenyl), 5-CHO- and 10-CHO-
(formyl).
ATP-dependent enzyme 10-formyl-THF synthetase (FTHFS; Fig. 2). As this

reaction is reversible, it could be also considered as a route by which formate
exits C1 metabolism. The cytosolic and mitochondrial FTHFS isoforms
found in yeast and the cytosolic isoform from mammals are associated with
two other activities, 5,10-methylene-THF dehydrogenase (MTHFD) and
5,10-methenyl-THF cyclohydrolase (MTHFC), to form a trifunctional
enzyme called C1-THF synthase (Appling, 1991; Tibbetts and Appling,
2010). Mammals also have a monofunctional FTHFS and a bifunctional
MTHFD–MTHFC in mitochondria, which is similar to the arrangement
found in the cytosol, mitochondria and chloroplasts from higher plants
(Fig. 2; Christensen and MacKenzie, 2006; Hanson and Roje, 2001). In
plants, most of the formate is degraded to CO2 via a mitochondrial formate
dehydrogenase, but low amount can serve as a C1 donor (Gout et al., 2000;
Li et al., 2003; Prabhu et al., 1996).
In animals and some microorganisms, the catabolism of histidine is linked
to folate metabolism through the key intermediate 5-formimino-THF
(Fig. 1). The bifunctional enzyme glutamate formiminotransferase/formi-
mino-THF cyclodeaminase catalyses two consecutive reactions in this path-
way (Mao et al., 2004). In plants, little is known about histidine catabolism,
and the formiminotransferase/cyclodeaminase activity has never been
described.
B. INTERCONVERSION OF C1-SUBSTITUTED FOLATES
5,10-Methylene-THF, 5,10-methenyl-THF and 10-formyl-THF can be inter-

converted by the enzymes MTHFD and MTHFC. These activities are
reversible and are associated with the cytosol, mitochondria and plastids
(Hanson and Roje, 2001). Thus, the combination of SHMT, MTHFD and
MTHFC activities can supply each cell compartment with C1-substituted
folates required for nucleotides, formylmethionyl-tRNA or pantothenate
synthesis (Fig. 2).
Methyl-THF has no other known metabolic fate than methionine synthe-
sis. Methylene-THF reductase (MTHFR) serves a key role in C1 metabolism
by converting 5,10-methylene-THF to 5-methyl-THF. The NADPH-depen-
dent MTHFR from yeast and animals irreversibly directs the methyl group
of 5-methyl-THF to methylation of homocysteine (Roje et al., 2002a).
Because this reaction has the potential to deplete the cytosolic 5,10-methy-
lene-THF pool, the regulation of MTHFR is crucial for C1 metabolism. In
yeast and animal cells, methyl-group biogenesis is regulated in vivo by a
feedback-loop in which S-adenosylmethionine (AdoMet), a derivative of
methionine that is used for methylation reactions, inhibits MTHFR (Roje
et al., 2002a). Plant MTHFRs are cytosolic enzymes that differ from their
yeast and mammalian counterparts because they are NADH-dependent,
reversible and not regulated by AdoMet (Fig. 2; Roje et al., 1999). The
reversibility of the reaction is sufficient to control C1-fluxes into methyl-
group biogenesis and does not need a feedback inhibition by AdoMet.
5-formyl-THF is a ubiquitous member of biological folates but is the only
derivative that does not serve as a C1-unit donor. It is considered that
5-formyl-THF is a potential regulator of C1 metabolism because it is a potent
inhibitor of SHMT and several other folate-utilizing enzymes (Stover and
Schirch, 1993). 5-formyl-THF is formed during the irreversible hydrolysis of
5,10-methenyl-THF catalysed by a side reaction of SHMT in the presence of
glycine. 5-formyl-THF cycloligase (FCL, also referred to as 5,10-methenyl-
THF synthetase) is the only enzyme that uses 5-formyl-THF by catalysing an
ATP-dependent conversion to the metabolically active form 5,10-methenyl-
THF (Stover and Schirch, 1993). FCL is a cytosolic enzyme in yeast and
animals, whereas in plants, it is located in mitochondria (Fig. 2), a compart-
ment where the 5-CHO derivatives represent up to 50–70% of the folate pool
(Chan and Cossins, 2003; Orsomando et al., 2005; Roje et al., 2002b).
C. UTILIZATION OF C1-UNITS
The pool of C1-substituted folates forms the core of C1 metabolism, from

which single-carbon units are withdrawn by anabolic reactions. The synthesis
of methionine using 5-methyl-THF is the largest anabolic flux of C1-units in
many physiological situations. Indeed, in addition to protein synthesis,
methionine serves as a methyl-group donor through conversion to AdoMet,
a key biological methylating agent involved in dozens of methyltransferase
reactions with a wide variety of acceptor molecules (metabolites, nucleic
acids, proteins). In plants, AdoMet is also involved in the biogenesis of biotin
(vitamin B8; see chapter 2 in this volume) and the phytohormone ethylene
and have regulatory roles in the synthesis of aspartate-derived amino acids
(Curien et al., 2009; Ravanel et al., 1998). In all organisms, methionine is
produced from homocysteine and 5-methyl-THF through a reaction cata-
lysed by methionine synthase. Two types of methionine synthases are known;
their activities are dependent or independent on the presence of a cobalamin
(vitamin B12) cofactor. Cobalamin-dependent enzymes are found in animals,
bacteria and algae, whereas cobalamin-independent isoforms exist in bacte-
ria, fungi, algae and plants (Croft et al., 2005; Drummond and Matthews,
1993; Ravanel et al., 2004b). Methionine synthase, like AdoMet synthetase
and S-adenosylhomocysteine hydrolase, is present in the cytosol, where it is
involved in the regeneration of Met to ensure a rapid turnover of AdoMet
(a set of reactions referred to as the activated methyl cycle; Fig. 2). Thus,
AdoMet is synthesized exclusively in the cytosol and thereafter is transported
to other cell compartments to enable numerous methylation reactions or
regulatory roles (Bouvier et al., 2006). In vascular plants, methionine
synthase activity is also located in plastids to participate in de novo synthesis
of methionine (Fig. 2; Ravanel et al., 2004b).
Synthesis of the purine ring is a central metabolic function of all organ-
isms. The products AMP and GMP provide purine bases for DNA and
RNA, as well as for a number of essential coenzymes (NAD(P), FAD,
AdoMet, CoA and folates) and signalling molecules (cAMP). In plants,
nucleotides are also the precursors for purine alkaloids and the hormone
cytokinins. The pathways for synthesis and salvage of nucleotides in animals,
plants and microorganisms are similar (Boldt and Zrenner, 2003). Starting
from phosphoribosyl pyrophosphate, de novo synthesis of AMP and GMP is
a complex 14-step process involving two formylation reactions that depend
on 10-formyl-THF. The third reaction of the pathway is the formylation of
glycinamide ribonucleotide (GAR) into formyl-GAR, a reaction catalysed
by GAR transformylase. The bifunctional enzyme aminoimidazole carbox-
amide ribonucleotide (AICAR) transformylase/inosine monophosphate
(IMP) cyclohydrolase is responsible for catalysis of steps 9 and 10 in the
purine pathway, with formyl-AICAR as an intermediate. In plants, purine
ring synthesis is chloroplastic (Fig. 2), whereas it is located in the cytosol of
animal and fungal cells (Boldt and Zrenner, 2003; Christensen and
MacKenzie, 2006).
Besides its role during the assembly of the purine ring, 10-formyl-THF
plays an essential function as donor of formyl group during the synthesis of
formylmethionyl-tRNA. Thus, protein synthesis in the organelles, which is
initiated by this formylated tRNA, is tightly associated with C1 metabolism.
The synthesis of formylmethionyl-tRNA from methionyl-tRNA and
10-formyl-THF is catalysed by methionyl-tRNA transformylase, an enzyme
present in both mitochondria and chloroplasts (Fig. 2; Appling, 1991;
Cossins, 2000).
The synthesis of thymidylate is closely linked to C1 metabolism through
the enzyme thymidylate synthase (TS). TS catalyses the final step in de novo
synthesis of thymidylate, the reductive methylation of deoxy-uridine mono-
phosphate (dUMP or uridylate) to deoxy-thymidine monophosphate (dTMP
or thymidylate) with concomitant conversion of 5,10-methylene-THF to
dihydrofolate. This is the only reaction in C1 metabolism in which the folate
substrate is oxidized during C1-unit transfer, with the electrons being used to
reduce the C1-unit to the methyl level. It is thus necessary to regenerate the
fully reduced form of folate for a sustained synthesis of DNA. This reduction
of dihydrofolate into THF is achieved by dihydrofolate reductase (DHFR), a

ubiquitous enzyme that is also involved in de novo synthesis of THF in folate-
autotrophs (Fig. 3). TS and DHFR are monofunctional enzymes in most
species except in protozoan and plants where they exist as a bifunctional
enzyme (Blancquaert et al., 2010; Nzila et al., 2005a; Rébeillé et al., 2006). In
plants, the existence of multiple isoforms of the bifunctional DHFR–TS
suggests a multi-compartmented synthesis of thymidylate (Fig. 2).
Pantothenate is a water-soluble vitamin (B5) that is synthesized de novo
by plants and microorganisms but obtained through the diet by
animals (see chapter 5 in volume 1). Pantothenate is the precursor of the
40 -phosphopantetheine moiety of CoA and acyl-carrier protein, cofactors in
energy-yielding reactions including carbohydrate metabolism and fatty acid
synthesis (Coxon et al., 2005). In plants, CoA is also important in many
aspects of secondary metabolism, including lignin biosynthesis. The first
reaction in the four-step pantothenate synthesis pathway involves the trans-
fer of a hydroxymethyl group from 5,10-methylene-THF to -ketoisovale-
rate, generating ketopantoate. This reaction is catalysed by ketopantoate
hydroxymethyltransferase. The folate-dependent enzymes from plants and
yeast are located in mitochondria (Fig. 2), whereas the remaining steps of the
pathway are most likely present in the cytosol (Coxon et al., 2005; Ottenhof
et al., 2004).
D. OTHER FUNCTIONS OF FOLATES
Besides its fundamental role in C1-metabolism, folate has been recently

implicated in three biological processes in which it functions solely as an
electron donor. As mentioned previously, the redox properties of folates are
exploited in C1 metabolism through TS, where 5,10-methylene-THF acts
both as a C1-unit and electron donor.
Cryptochromes and DNA photolyases are related proteins identified in all
kingdoms of life with FAD as a common cofactor. Photolyases repair
cytotoxic and mutagenic DNA lesions that are formed during exposure of
DNA to UV-B and are thus essential for genome maintenance in many
species. Cryptochromes generally lack repair activity but act in photo-per-
ception and signal transduction of UV-A/blue light in a broad range of
organisms (Muller and Carell, 2009). In plants, cryptochromes control sev-
eral physiological and morphogenetic processes. The repair of DNA lesions
by photoexcited photolyases requires the catalytic fully reduced flavin cofac-
tor that injects an electron directly into the DNA lesion. Photoreduction of
the flavin cofactor was also observed in cryptochromes, and the protein
bearing the semireduced flavin species is considered as the signalling state
of the photoreceptor. Besides the flavin cofactor essential for enzymatic

activity, the majority of DNA photolyases and cryptochromes contain
polyglutamylated 5,10-methenyl-THF as a second chromophore. The folate
cofactor is involved in transfer of excitation energy to the catalytic flavin
cofactor. During photoreduction of FAD, a photobleaching of 5,10-methe-
nyl-THF is observed and corresponds to a reduction of the cofactor into
5,10-methylene-THF (Moldt et al., 2009). Thus, it was suggested that the
cryptochrome CRY3 from Arabidopsis thaliana could contribute to the
production of 5,10-methylene-THF through a unique light-dependent way.
The extent to which CRY3 contributes to 5,10-methylene-THF formation
in vivo remains unclear and this may occur only under very high irradiance
conditions (Moldt et al., 2009).
Aromatic amino acid hydroxylases (AAHs) are iron-dependent mono-
oxygenases that use a tetrahydropterin cofactor as electron donor and hydr-
oxylate the ring of an aromatic amino acid (Fitzpatrick, 2003). Animal
AAHs comprise three subfamilies that prefer phenylalanine, tyrosine or
tryptophan, while most bacterial AAHs are essentially specific for phenylal-
anine. The physiological AAH cofactor is tetrahydrobiopterin in
animals and tetrahydromonapterin in bacteria. AAH reactions convert the
tetrahydropterin cofactor to a 4a-carbinolamine, which is recycled by pterin-
4a-carbinolamine dehydratase and quinonoid dihydropterin reductase.
Non-flowering plants possess a unique folate-dependent AAH that is loca-
lized in chloroplasts (Pribat et al., 2010). Plant AAHs are phenylalanine
hydroxylases with 10-formyl-THF as the preferred cofactor in vitro and
in vivo. The use of folates as cofactors in vivo is unique for phenylalanine
hydroxylase or any other pterin-dependent enzyme. Following electron dona-
tion, the folate cofactor is recycled through pterin-4a-carbinolamine dehydra-
tase, which is also present in chloroplasts (Naponelli et al., 2008; Pribat et al.,
2010). The unique role of 10-formyl-THF as a net electron donor in a physio-
logical reaction is for instance limited to non-flowering plants. Indeed, AAH
genes have so far been found only in gymnosperms, mosses and algae, but it
would be premature to conclude that flowering plants lack AAHs because
genome sequences are available only for a small subset of angiosperm taxa.
The assembly of the iron/sulphur (Fe/S) cluster prosthetic group of many
enzymes requires a set of auxiliary proteins. The protein family classified as
COG0354 is structurally related to folate-dependent enzymes, and members
of this group were shown to be involved in synthesis or repair of Fe/S clusters
in yeast and Escherichia coli (Gelling et al., 2008; Waller et al., 2010c).
COG0354 proteins exist in all domains of life, and genetic evidence indicated
that those of bacteria, protists (Leishmania) and animals require THF to
function. It is likely that THF acts as an electron donor for the assembly or
repair of Fe/S clusters (Waller et al., 2010c). Two COG0354 proteins occur in
Arabidopsis and many other plants, the first related to those of -proteobac-
teria and predicted to be mitochondrial, the second related to those of
cyanobacteria and predicted to be plastidial (Waller et al., 2010b). The
subcellular distribution of the Arabidopsis proteins was validated and their
THF-dependent function was established. Moreover, an Arabidopsis T-DNA
insertion line of the mitochondrial COG0354-protein was pollen lethal,
whereas inactivation of the chloroplastic form was not. Together, these
data established that plant COG0354 proteins have a THF-dependent func-
tion in mitochondria and plastids, almost certainly related to Fe/S cluster
metabolism in these organelles (Waller et al., 2010b).
III. FOLATE SYNTHESIS, TURNOVER AND

HOMEOSTASIS IN PLANTS
A. BIOSYNTHESIS OF THF
Plants, fungi, most microbes and parasites of the Apicomplexa phylum have
the capacity to synthesize THF de novo (Blancquaert et al., 2010; Cossins and
Chen, 1997; Nzila et al., 2005a; Rébeillé et al., 2006). Humans and animals in
general do not have this capacity because almost all the enzymes required for
this complex metabolic route are absent. The synthetic pathway is nearly
identical in all folate-autotrophic organisms, and the main differences
between plants and other organisms will be discussed later (Section IV).
The plant THF-biosynthetic pathway is now completely elucidated (Fig. 3;
Blancquaert et al., 2010; Rébeillé et al., 2006). The plant enzymes possess
unique structural and biochemical properties and present a fascinating spa-
tial organization, in which three subcellular compartments participate
(Fig. 4). The pABA and pterin parts of THF are first synthesized in separate
routes originating from chorismate and GTP, respectively. These moieties
are then assembled, together with glutamate, to produce dihydrofolate,
which is then converted to folylpolyglutamates in two steps.
Para-aminobenzoate is synthesized from chorismate, a compound that is
also involved in the synthesis of aromatic amino acids and their derivatives.
The synthesis of pABA from chorismate occurs in two steps localized in
plastids (Figs. 3 and 4). First, the amination of chorismate yields 4-amino-4-
deoxychorismate (ADC), which is subsequently aromatized to pABA with
elimination of pyruvate. In bacteria, the synthesis of ADC is catalysed by
ADC synthase, a two-component enzyme in which the glutamine amido-
transferase protein PabA supplies an amino group to PabB, which catalyses
Gln Glu
HO COOH H2N COOH
Chorismate ADC
O O
CH2 CH2
1
HOOC HOOC
O
2
N Pyruvate
H2Pterin-PPi HN O P P
H 2N N N
H
H 2N COOH pABA
AMP
7
O ATP 8
H2Pterin N
OH
PPi
HN
H 2N N N H2Pteroate O HN
COOH
H COOH
Giycolaldehyde N
6
HN
ATP + H2N COOH
9
H 2N N N
H Glu
O OH
O COOH
N
H2Neopterin HN OH
N COOH
OH H2Folate O HN H
H2N N N
H N
Pi HN
NADPH
5 H2 N N N 10
H
PPi NADP
4 O COOH
O OH
COOH
H2Neopterin- N
THF-Glu1 O HN
N
H
HN O P P P H COOH
triphosphate HN N
OH
H2N N N ATP + H2N COOH
H H2N N N 11
H
Formate Glu
O
3
O COOH
GTP N H
HN COOH
THF-Glun O HN
N N
N H
H2N N O H O COOH n
O P P P N
HN
H2N N N
OH OH H
Fig. 3. THF biosynthesis in plants. The enzymes involved in the synthesis of THF
polyglutamate are: (1) aminodeoxychorismate (ADC) synthase; (2) ADC lyase; (3)
GTP-cyclohydrolase I; (4) dihydroneopterin triphosphate pyrophosphatase; (5) phos-
phatase (probably non-specific); (6) dihydroneopterin aldolase; (7) hydroxymethyldi-
hydropterin pyrophosphokinase; (8) dihydropteroate synthase; (9) 8, dihydrofolate
synthetase; (10) dihydrofolate reductase; (11) folylpolyglutamate synthetase.
Abbreviations: Gln, glutamine; Glu, glutamate; H2X, dihydro-pteridine derivatives;
H2Pterin, hydroxymethyldihydropterin; H2PterinPPi, hydroxymethyldihydropterin
pyrophosphate.
the amination reaction. In plants, both these reactions are catalysed by a

single protein that is a fusion of the PabA and PabB domains (Basset et al.,
2004a; Sahr et al., 2006). Each domain can operate independently but cou-
pling increases the catalytic efficiency of each reaction. The amination (ADC
synthase) activity is the limiting step of the bifunctional enzyme and is
feedback-inhibited by ADC (Camara et al., 2011). This feedback-loop sug-
gests that plant ADC synthase could be a potential regulatory step to control
flux partitioning of chorismate towards folate, tryptophan, tyrosine and
Plastid Mitochondrion
pABA-Glc
pABA
Chorismate
H2Pterin
1 8
pABA
2 7
pABA Glu
9
H2Pterin
C1-THF-Glun 10
6
THF
4+5 11 Glu
THF-Glun
3 THF-Glun
Glu 11 GTP
THF
C1-THF-Glun
THF
11 Glu
C1-THF-Glu1 THF-Glun C1-THF-Glun C1-THF-Glu1
12
C1-THF-Glu1 C1-THF-Glun
Vacuole
Fig. 4. Subcellular compartmentation of THF synthesis in plant cells. Enzymes

involved in THF synthesis are numbered as in Fig. 3. The enzyme -glutamyl
hydrolase (#12) that controls the glutamate tail length of folates is also shown.
Probable transport steps of folates and precursors are indicated by black circles
(carrier-mediated transports) and white squares (possibly simple diffusion).
C1-THF-Glun, C1 derivatives of folylpolyglutamates.
phenylalanine syntheses. The second step of pABA synthesis in bacteria and

plants is catalysed by the pyridoxal-phosphate-dependent enzyme ADC lyase
(PabC; Basset et al., 2004b). The pABA pool in different plant tissues is
mainly in an esterified form with glucose (pABA-Glc) that is formed in the
cytosol and is largely sequestered in vacuoles (Eudes et al., 2008). The
physiological role of pABA-Glc is not yet elucidated, it may be involved in
regulating pABA storage or may be the form in which pABA is trafficked
within plant cells (Quinlivan et al., 2003).
The conversion of GTP into hydroxymethyldihydropterin is a four-step
process that is presumably cytosolic, as none of the enzymes involved possess
obvious targeting signals (Figs. 3 and 4). The first reaction is catalysed by
GTP-cyclohydrolase I (GTPCHI) to form 7,8-dihydroneopterin (DHN) tri-
phosphate. GTPCHI is present in folate-synthesizing organisms and in
mammals where it is involved in the synthesis of other pteridines. Indeed, the

tetrahydrobiopterin cofactors for AAHs (see Section II.D) and NO synthases
and other pteridines that have key roles as chromophores or UV protectants
are derived from DHN triphosphate in animals (Werner-Felmayer et al.,
2002). Given that GTPCHI catalyses the first step in the synthesis of pterin
derivatives, the enzyme is considered to control fluxes into these pathways
(Basset et al., 2002). In support to this proposal, genetic engineering of the
GTPCHI step in transgenic plants led to important increase in pteridines
production (see Section VI for details). The triphosphate side chain of DHN
triphosphate is further removed in two steps to produce DHN. First, DHN
triphosphate pyrophosphatase specifically cleaves DHN to produce
DHN monophosphate (Klaus et al., 2005a). Second, the remaining phos-
phate is cleaved from DHN monophosphate by the action of a phosphatase.
This enzyme has not been yet identified in plants; it may be non-specific as in
E. coli. The last step of the pterin branch is catalysed by DHN aldolase that
cleaves the trihydroxypropyl side chain of DHN to yield hydroxymethyldi-
hydropterin (Goyer et al., 2004). The plant enzyme is encoded by a small
gene family and is monofunctional whereas the fungal activity is part of a
trifunctional enzyme (Guldener et al., 2004; see below).
The combination of hydroxymethyldihydropterin, pABA and glutamate
to produce dihydrofolate involves three reactions that are located within
mitochondria (Figs. 3 and 4). First, hydroxymethyldihydropterin is activated
into its pyrophosphorylated form through the operation of hydroxymethyl-
dihydropterin pyrophosphokinase (HPPK). Second, dihydropteroate is pro-
duced by condensation of pABA with the activated pterin in a reaction
catalysed by dihydropteroate synthase (DHPS). In plants, these two reac-
tions are catalysed by a bifunctional enzyme, whereas the activities are
carried by separate proteins in bacteria (Rébeillé et al., 1997). In plant
HPPK–DHPS, the DHPS reaction is feedback-inhibited by dihydropteroate,
dihydrofolate and THF-Glu1, suggesting that this domain could be a poten-
tial regulatory point of the mitochondrial branch of the folate pathway
(Mouillon et al., 2002). Arabidopsis is unique among higher plants with
sequenced genomes in having two genes coding HPPK–DHPS
(Storozhenko et al., 2007a). The first one encodes the mitochondrial isoform
involved in de novo synthesis of THF, while the second is highly expressed in
developing seeds and encodes a cytosolic enzyme whose function remains to
be established. The third mitochondrial step is the ATP-dependent attach-
ment of glutamate to the carboxyl moiety of pABA to form dihydrofolate. It
is catalysed by a monofunctional dihydrofolate synthetase (Ravanel et al.,
2001), an enzyme that is essential for plant development because a mutation
of this gene is embryo-lethal in Arabidopsis (Ishikawa et al., 2003). Before
entering C1 metabolism, dihydrofolate is reduced to THF and polyglutamy-

lated by the operation of two enzymes that are present in all kingdoms.
Dihydrofolate is reduced to THF-Glu1 by DHFR using NADPH as electron
donor (Fig. 3). In animals, fungi and bacteria, DHFR is a monofunctional
enzyme (Cossins and Chen, 1997; Schnell et al., 2004). In higher plants, the
activity is catalysed by the third bifunctional enzyme of the folate pathway,
which also carries a TS activity (Blancquaert et al., 2010; Rébeillé et al.,
2006). As a result, the DHFR domain of the enzyme has a dual function; it is
involved in the reduction of dihydrofolate monoglutamate originating from
de novo synthesis of folate or dihydrofolate polyglutamate resulting from the
oxidation of THF-Glun during TS activity (Neuburger et al., 1996). DHFR is
inhibited by the dihydrofolate analogue methotrexate, a molecule that is used
as an antifolate in chemotherapy and as a chemical tool to manipulate the
folate pool in plants (Loizeau et al., 2007, 2008; Prabhu et al., 1998; van
Wilder et al., 2009).
The polyglutamate tail of THF-Glun is formed by the sequential addition
of -linked glutamate residues to THF-Glu1, a reaction catalysed by folyl-
polyglutamate synthetase (FPGS). In all eukaryotes studied so far, FPGS
isoforms are found in each subcellular compartment containing folylpoly-
glutamates, indicating that these derivatives cannot cross membranes and
must be synthesized in situ. In plant cells, FPGS is present as three distinct
isoforms located in the cytosol, mitochondria and chloroplasts (Fig. 4;
Ravanel et al., 2001). Higher plants have two or more genes coding FPGS,
and in the dicot model Arabidopsis, each isoform is encoded by a separate
gene (Blancquaert et al., 2010). Recently, the functional importance of folate
polyglutamylation in C1-metabolism and plant development was assessed
through genetic studies (Mehrshahi et al., 2010; Srivastava et al., 2011).
Biochemical characterization of single and double FPGS loss-of-function
mutants in Arabidopsis established that the glutamylation step is essential
for organellar and whole-plant folate homeostasis. Also, these data were
consistent with a degree of redundancy in compartmentalized FPGS activity,
with targeting of one or more FPGS to multiple organelles, at least in
above-ground organs (Mehrshahi et al., 2010). In roots, the plastidial
FPGS isoform is essential for quiescent centre organization, cell division
and expansion during primary root development, and none of the other
FPGS isoenzymes can fulfil this role (Srivastava et al., 2011).
The glutamate tail of folate coenzymes can be shortened or removed by the
enzyme -glutamyl hydrolase (GGH). Plant GGHs share many common
features with the enzymes from mammals; they act on both folylpolygluta-
mates and the folate-breakdown product pABA polyglutamate and are
located in the vacuole (Fig. 4; Orsomando et al., 2005, 2006). GGHs play
an important role in governing glutamate tail length in vivo and plant folate
homeostasis. Indeed, a threefold overexpression of GGH caused an impor-
tant deglutamylation of folates and reduced the coenzyme pool by 40% in
Arabidopsis leaves and tomato fruits (Akhtar et al., 2010). Conversely, an
almost complete silencing of GGH expression in Arabidopsis led to an
increase in both tail length and folates content. Together, these data suggest
that folates can enter the vacuole as polyglutamates, may accumulate there
following binding to folate-binding proteins as yet non-identified, are hydro-
lysed by GGH and exit to the cytosol as monoglutamates (Fig. 4; Akhtar
et al., 2010).
B. CATABOLISM AND SALVAGE PATHWAYS
The physiological turnover rate of folates in plants is unknown but high

folate-breakdown rates (up to 10% per day) have been measured in plants
treated with inhibitors of THF synthesis (Orsomando et al., 2006; Prabhu
et al., 1998) or following harvesting (Scott et al., 2000). For comparison, the
breakdown products resulting from folate catabolism are excreted in the
urine of mammals with rates of 0.5% per day (Gregory and Quinlivan,
2002). Most natural reduced folates are labile compounds that undergo
oxidative degradation. Oxidation can occur by at least two distinct mechan-
isms that are essentially irreversible. First, the pterin ring of THF can be
sequentially oxidized to dihydrofolate and then to folic acid. Second, THF or
dihydrofolate can undergo an oxidative scission reaction at the C9 N10
bond (Fig. 5), giving a pterin aldehyde derivative and p-aminobenzoyl
(poly)glutamate (pABA-Glun). Such non-enzymatic cleavage is thought to
be the main way by which folates break down in all organisms, although
proteins, for example, ferritin, may sometimes facilitate the reaction in
animals (Suh et al., 2001). C1 substitution at N-5 or N-10 can alter the
reactivity of THF to oxidative degradation, 5-formyl-THF being the most
stable derivative. Also, folates are less labile when bound to proteins than
when free in solution (Suh et al., 2001).
Plants have the capacity to re-use breakdown products in THF synthesis
(Fig. 5; Orsomando et al., 2006). Recycling of the pABA moiety is initiated
by the hydrolysis of the polyglutamate chain of pABA-Glun to release free
pABA. This is a two-step process involving the vacuolar enzyme GGH to
cleave the -glutamyl peptide bond and a pABA-Glu hydrolase to remove
the last glutamate residue (Orsomando et al., 2005, 2006). The hydrolase is
predominantly vacuolar or cytosolic and may exist as various isoforms, but
the corresponding genes have not been yet identified (Bozzo et al., 2008).
Following these two reactions, pABA should be transported to mitochondria
H2Pterin pABA
O
N
HN OH
H2N COOH
H2N N N
H
O COOH
H COOH
N N
3 O HN H THF-Glun
H O COOH
HN
N * n
H 2N N N
H
pABA-GIun
O
O COOH
N CHO
HN H COOH
H 2N N N
H
H 2N N N O COOH n
H
H2Pterin-6-aldehyde
1
Glu
2
Pterin-6-aldehyde O COOH
H 2N N COOH
H
Glu
Pterin-6-carboxylate pABA-GIu
Fig. 5. Folate catabolism and salvage reactions. Chemical structures of the main
products of folates breakdown and salvage reactions are shown. Oxidation of THF-
Glun at the C9N10 bond (asterisk) generates pABA-Glun and H2Pterin-6-aldehyde.
Salvage reactions of these catabolic products involved the enzymes -glutamyl hydro-
lase (1), pABA-Glu hydrolase (2) and pterin aldehyde reductase (3). The resulting
hydroxymethyldihydropterin (H2Pterin) and pABA can enter THF biosynthesis at
the level of the bifunctional HPPK–DHPS enzyme (reactions 7 and 8, Fig. 3).
and combined with hydroxymethyldihydropterin-PPi by DHPS for dihy-

dropteroate synthesis (Fig. 5). The possibility of a salvage of pABA-Glu
through a direct incorporation into dihydrofolate by DHPS was found to be
physiologically improbable (Orsomando et al., 2006).
The catabolism of the pterin moiety of THF and dihydrofolate results in
the production of tetrahydro- and dihydropterin-6-aldehyde, which are fur-
ther oxidized to pterin-6-aldehyde and then pterin-6-carboxylate (Fig. 5;
Noiriel et al., 2007a,b). Salvage of dihydropterin-6-aldehyde consists in the
reduction of its aldehyde side chain into hydroxymethyldihydropterin and
involves a NADPH-dependent pterin aldehyde reductase (PTAR). Multiple
isoforms of PTAR seem to occur in plants. Their ability to reduce diverse
other aromatic and aliphatic aldehydes suggests that dihydropterin-6-alde-
hyde can be salvaged by a series of enzymes with broad specificity. PTAR
activity is mainly cytosolic in pea and up to 1500-fold higher than de novo

THF-synthesizing enzymes, suggesting that a rapid reduction of dihydrop-
terin-6-aldehyde is necessary to limit further oxidation (Noiriel et al., 2007b).
This is consistent with the observation that the pterin moiety can no longer
be salvaged when dihydropterin-6-aldehyde gets oxidized to pterin-6-alde-
hyde and pterin-6-carboxylate (Fig. 5; Noiriel et al., 2007a). Given the high-
rate of post-harvest folate degradation in fruits and vegetables, a better
understanding of pterin salvage is now requested to explore strategies to
improve folate preservation in crop plants.
C. CELLULAR COMPARTMENTATION AND TRANSPORT OF FOLATES
Plant folates are present in different subcellular compartments. The overall

distribution of total folates in photosynthetic pea leaves is 40% in mito-
chondria, 10% in chloroplasts, 20% in vacuoles and 30% in the cytosol
(Chan and Cossins, 2003; Jabrin et al., 2003; Orsomando et al., 2005). In the
organelles, folates are almost exclusively polyglutamylated, with the penta-
and hexa-glutamate species being the most abundant, but folate profiles are
different. Mitochondrial folates are dominated by 5-formyl-THF, which is
not directly involved in C1 transfer reactions (Fig. 2), and unsubstituted
THF, which probably results from de novo synthesis (Chan and Cossins,
2003; Orsomando et al., 2005). Chloroplasts are rich in 10-formyl-THF/5,10-
methenyl-THF and contain significant amount of 5-methyl-THF
(Orsomando et al., 2005), in accordance with the metabolic activity of these
organelles regarding purines and methionine synthesis (Fig. 2). In pea leaves
and in red beet roots, vacuoles contain almost exclusively 5-methyl-THF, of
which 50–75% is polyglutamylated (Orsomando et al., 2005). Because methi-
onine synthase is absent from vacuoles, these data suggest that 5-methyl-
THF is a potential storage form for folate in plant cells, a situation that is
conceivable as this derivative is quite stable to oxidative breakdown and is
readily converted to other folates.
The plant folate-biosynthetic pathway is split among the cytosol, plastids
and mitochondria (Fig. 4). This complex organization, together with the
presence of folates in different subcellular compartments, suggests a sophis-
ticated traffic of folate coenzymes and their biosynthetic intermediates
between the organelles via the cytosol. These intracellular transport steps
include pterin uptake into mitochondria, pABA export from plastids and
import into mitochondria, folates release from mitochondria and uptake into
plastids, and folates influx and efflux into vacuoles (Fig. 4). In addition, there
is evidence that folates, at least 5-formyl-THF and the antifolate
methotrexate, can be taken up by plant cells (Loizeau et al., 2007, 2008;

Prabhu et al., 1998), thus indicating a folate uptake system at the plasma
membrane.
Except for pABA that is a hydrophobic weak acid possibly transported by
simple diffusion (Quinlivan et al., 2003), all these transport steps are likely
mediated by specific membrane-integral proteins. To date, only three plant
folate carriers have been functionally characterized (Bedhomme et al., 2005;
Klaus et al., 2005b; Raichaudhuri et al., 2009). Two of these transporters are
located on the envelope of chloroplasts and belong to distinct families. The
first system is homolog to the mitochondrial folate transporter formerly
characterized in mammals (Bedhomme et al., 2005), whereas the second
belongs to the folate-biopterin transporter family originally described in
Leishmania, a parasitic protist that is heterotrophic for folates and pteridines
(Klaus et al., 2005b). Because Arabidopsis null mutants for these proteins are
not affected in growth and display modest changes in chloroplastic folates, it
is likely that these carriers have redundant functions although they can
exhibit different specificity/activity towards folate derivatives. In animals,
several multidrug resistance-associated proteins (MRP) belonging to the
ATP-binding cassette transporter superfamily catalyse a high-capacity and
low-affinity transport of methotrexate and physiological folates (Kruh and
Belinsky, 2003). In Arabidopsis, two MRPs located at the plasmalemma
(AtMRP4) or tonoplast (AtMRP1) membranes have been cloned, but their
physiological role in regulation of folate homeostasis remains to be estab-
lished (Klein et al., 2006). The vacuolar MRP protein AtMRP1 proved to be
competent for folic acid and methotrexate transport in vitro and contribute
to antifolate tolerance in planta (Raichaudhuri et al., 2009). It is suggested
therefore that AtMRP1 and its counterparts in other plant species have the
potential for importing folates into the vacuole.
D. FOLATES DISTRIBUTION IN PLANT ORGANS AND TISSUES
As detailed above, folates are present in the cytosol, mitochondria, chloro-

plasts and vacuoles of plant cells where the C1-derivatives are not equally
distributed. When considering whole-plant tissues or organs, folates are
largely dominated by the methyl (45–65%) and formyl (30–55%) derivatives,
the unsubstituted and methylene-bearing forms representing only 10–15% of
the total pool (Cossins, 2000). Although the 5-formyl-THF is not directly
involved in C1-transfer reaction, it represents 15–40% of the folate pool in
photosynthetic leaves and other plant organs, which is approximately five-
fold higher proportion than in animals and yeasts (Cossins, 2000). The
metabolic role of 5-formyl-THF is still not well defined in plants, but it
could serve as a regulatory factor of photorespiration through the inhibition

of mitochondrial SHMT. However, the near-normal growth of mutant
Arabidopsis plants accumulating high 5-formyl-THF levels suggests that
this derivative does not much affect fluxes through SHMT or any other
folate-dependent reaction (Goyer et al., 2005). In seeds and quiescent tissues,
5-formyl-THF could act as a storage form of folates, this derivative being the
most stable natural folate.
The total folate content greatly varies from one plant to another and with
the nature of the organ or tissue. Also, the folate pool fluctuates importantly
during the course of plant development, suggesting that folate synthesis and
turnover is tightly controlled and modulated as a function of the metabolic
requirements (Basset et al., 2004a; Cossins, 2000; Jabrin et al., 2003). In
developing pea seedlings, it was found that tissues with a reduced metabolic
activity such as cotyledons, roots and stems contain limited amount of
folates (Jabrin et al., 2003; Rébeillé et al., 2006). Similarly, tomato fruits
have low folate content and the pool gradually decreases during ripening
(Basset et al., 2004a). In contrast, folate synthesis and accumulation is
important in rapidly dividing tissues. In pea, the germination process,
which correlates with the transition from a quiescent to an active metabolic
state and a resumption of cell-cycle activity, is accompanied by an increase in
folate cofactors in embryos (Jabrin et al., 2003). Also, meristematic tissues of
the root apex contain fivefold more folate than the mature root, and Arabi-
dopsis cell-suspension cultures, which have a short generation time, show
very high folate content (Loizeau et al., 2007, 2008). These observations
suggest that proliferating tissues have a high-capacity to synthesize and
accumulate folate coenzymes to meet the demand for nucleotide synthesis
and high C1-metabolism activity. Green mature leaves are also characterized
by a high folate content that is triggered by the acquisition of photosynthesis
and thus is related to light (Jabrin et al., 2003). The relationship between
folate accumulation in leaves and photosynthesis is not yet fully understood.
Photorespiration involves two folates-dependent enzymes, GDC and SHMT
(Fig. 2), that accumulate within the mitochondria during greening (Douce
et al., 2001). Part of the folate synthesized in light might contribute to the
photorespiratory process but most of folate accumulates in the extra-orga-
nellar fraction (cytosol plus vacuoles) as 5-methyl-THF derivatives. There-
fore, it is likely that the high folate content in green leaves is associated with
an elevated activity of the methyl cycle to ensure a fast turnover of AdoMet
(Rébeillé et al., 2006). This assumption is supported by the threefold decrease
in chlorophyll synthesis, which depends on an AdoMet-dependent methyla-
tion step, in pea seedlings displaying a modest ( 25%) shortage in folates
(van Wilder et al., 2009).
E. CONTROL OF FOLATES HOMEOSTASIS
As depicted above, the pool of folates varies importantly in a developmental-

and organ-dependent manner to meet the fluctuating physiological demands
for C1 units. In these different situations, folates homeostasis should be
tightly controlled through biochemical, genetic and developmental mechan-
isms. These mechanisms should occur at the level of THF biosynthesis,
generation and interconversion of folates species, intracellular traffic, catab-
olism and salvage. To date, these mechanisms are still poorly understood.
The kinetic characterization of some enzymes involved in THF synthesis
indicated the existence of regulatory feedback-loops in vitro. Thus, ADC
synthase is feedback-inhibited by ADC (Camara et al., 2011), whereas DHPS
activity is controlled by dihydropteroate, dihydrofolate and THF-Glu1
(Mouillon et al., 2002). The physiological relevance of these feedback regu-
latory loops is difficult to predict because the pools of regulatory intermedi-
ates are presumably very low.
Regulation of THF synthesis at the gene level was investigated during the
young stages of pea development and during fruit/seed maturation stages.
High expression levels of the genes coding HPPK–DHPS and DHFR were
found in pea organs/tissues with high folate levels, that is, embryos, root
apices and developing green leaves (Jabrin et al., 2003). Also, the expression
levels of genes coding GTPCHI, ADC synthase and ADC lyase were found
to decline during tomato fruit ripening and wheat seed maturation, develop-
mental stages that are characterized by a decrease in folate content (Basset
et al., 2002, 2004a,b; McIntosh et al., 2008). Recently, the expression level of
all folate synthesis genes was investigated during tomato fruit development
(Waller et al., 2010a). Except for the above mentioned genes (GTPCHI, ADC
synthase and ADC lyase), none of the other genes involved in THF synthesis
followed a similar expression pattern. Together, these studies indicated a
developmental regulation of part of the genes involved in THF synthesis and
suggested the absence of a global coordinated control of the whole pathway
at the gene level.
Studies of plants or plant cells in which folate homeostasis has been
genetically or chemically modified provided new insights into the regulatory
mechanisms governing folate metabolism. Thus, a microarray analysis of
transgenic tomatoes overexpressing GTPCHI and ADC synthase genes indi-
cated a two- to eightfold increased expression of the downstream genes in the
pathway coding DHNA, ADC lyase and mitochondrial FPGS (Waller et al.,
2010a). These engineered tomatoes contained up to 15-fold more folates than
the wild-type fruits as well as increased levels (> 20-fold) of pteridines and
pABA (Diaz de la Garza et al., 2007). The following feedforward control of
the pathway was proposed to explain these data: the accumulation of pter-
idines and ADC could have induced DHNA and ADC lyase, respectively,
and the accumulation of folates, which are less extensively polyglutamylated
in transgenic lines than controls, may have induced mitochondrial FPGS
(Waller et al., 2010a). In another study, a genome-wide and metabolic
analysis of Arabidopsis cells treated with the antifolate methotrexate was
done to investigate the dynamic response of C1 metabolism to folate limita-
tion (Loizeau et al., 2008). This transcriptomic study indicated that the
steady-state expression of only one gene involved in THF synthesis was
modified by folate depletion. Because the two- to fivefold induction
concerned the gene coding cytosolic FPGS (Fig. 4), this unique response
suggested a regulatory loop to control the extent of folate glutamylation in
the cytosol rather than to increase folate production through de novo synthe-
sis. Also, important changes in the distribution of folate derivatives and
increased expression levels for transcripts-coding enzymes manipulating
C1-moieties in plastids were consistent with a re-orientation of C1-units
towards the synthesis of purine and thymidylate. These data suggested that
the metabolic priority of Arabidopsis cells in response to folate limitation was
to shuttle the available folate derivatives to the synthesis of nucleotides at the
expense of methylation reactions (Loizeau et al., 2008). The increased expres-
sion level of chloroplastic SHMT suggested a key role of this enzyme as a
switch to modulate nucleotide synthesis and methylation reactions, as previ-
ously proposed for the cytosolic SHMT in animal cells (Herbig et al., 2002).
After a prolonged period of folate starvation, the synthesis of AdoMet is
restored through a post-translational process that consists in the cleavage of
the N-terminal regulatory domain of the first enzyme specific for methionine
synthesis (Loizeau et al., 2007).
Together, these data illustrate that control of folate homeostasis and
dynamics of C1 metabolism involves multiple levels of regulation. These
mechanisms are only poorly known, and a more comprehensive view has to
emerge to help future biofortification efforts.
IV. FOLATE SYNTHESIS IN OTHER AUTOTROPHS
A. SPECIES–SPECIFIC DIFFERENCES IN THF BIOSYNTHESIS
The pathway leading to THF synthesis is roughly the same in all organisms
studied so far, indicating that it is highly conserved. The most striking
difference is found in the protozoa Plasmodium falciparum, a lower eukary-
ote belonging to the Apicomplexa phylum. Indeed, in such an organism, there
is no gene coding for DHNA (reaction 6 in Fig. 6). Thus, dihydropterin is not
synthesized from dihydroneopterin but directly from dihydroneopterin-tri-
phosphate through an original reaction catalysed by an atypical orthologue
of 6-pyruvoyltetrahydropterin synthase (reaction 60 in Fig. 6). This reaction
leads to the formation of two products, the predominant of which is the
substrate of HPPK, dihydropterin (Dittrich et al., 2008). Thus, the DHNA
step is bypassed in P. falciparum.
Other differences are mainly in terms of mono- or multifunctional
enzymes. Indeed, phylogenetic and biochemical studies revealed that several
steps of the pathway can be catalysed either by mono- or multifunctional
Fig. 6. Comparison of THF biosynthesis in various autotrophic organisms.

Enzymes involved in THF synthesis are numbered as in Fig. 3. Main differences
between species are the following: reaction 1 is catalysed by a single bifunctional
enzyme in eukaryotes, whereas two proteins participate in E. coli; reaction 60 in
P. falciparum is catalysed by 6-pyruvoyltetrahydropterin synthase; reactions 6, 7
and 8 are catalysed by mono-, bi- or trifunctional enzymes; enzyme 9 in E. coli and
P. falciparum possesses DHFS and FPGS activities; DHFR (enzyme 10) and thymi-
dylate synthase (TS) activities are part of a bifunctional protein in plants and api-
complexan parasites. Enzymes in yellow are present in the cytosol; in green, in
plastids; in red, in mitochondria. Subcellular localization is predictive for most
P. falciparum and S. cerevisiae enzymes. In P. falciparum, the gene coding ADC
lyase (reaction 6) has not been yet identified. Pictograms indicate the two steps
(DHPS and DHFR) targeted by antifolate drugs.
proteins. Multifunctional proteins are preferentially found in eukaryotes

(Fig. 6), but they are not, however, restricted to these organisms. For exam-
ple, most bacteria contain two separate proteins to catalyse the first reaction
required for pABA synthesis (Fig. 6), but various prokaryotic groups
(including actinobacteria, - and -proteobacteria, cyanobacteria) display
a bifunctional glutamine amido-transferase/ADC synthase, a situation also
found in all the eukaryotes studied so far (Camara et al., 2011). Likewise, all
eukaryotes display either bifunctional or trifunctional enzymes for the syn-
thesis of dihydropteroate (Fig. 6), a situation contrasting with most prokar-
yotes, although few bacteria (those of the Rickettsiella and the Chlamidya
groups) contain a bifunctional HPPK–DHPS (Storozhenko et al., 2007a).
Another significant difference concerns the addition of the glutamate moiety
to the folate molecule. Indeed the polyglutamate tail of folates is synthesized
in two steps: the first glutamate is attached to dihydropteroate and then the
others are added to THF (Fig. 3). In many bacteria, the same protein
catalyses these two reactions, whereas in plants and fungi, two specific
enzymes are involved. Unusually for eukaryotes, P. falciparum and other
members of the Apicomplexa phylum such as Toxoplasma gondii express
DHFS and FPGS as a single protein (Wang et al., 2010). Lastly, it must be
remembered that DHFR exists as a monofunctional enzyme or a bifunction-
al protein bearing also a TS activity in the C-terminal part of the protein. It is
interesting to note that DHFR and DHFR–TS are present in two different
taxonomic groups and possibly represent a maker of evolution. Indeed, a trait
shared by all organisms originating from a bikont (a eukaryotic cell with two
flagella) is the presence of DHFR–TS, whereas the monofunctional form of
DHFR is present in organisms originating from a unikont (a eukaryotic cell
with one flagellum). Thus, separate DHFR and TS genes are present in animals,
fungi and amoeba, whereas a fusion of these two genes is found in higher
and lower plants and numerous parasitic protozoa, including those of the
Apicomplexa and Trypanosoma genus (Stechmann and Cavalier-Smith, 2003).
The advantages gained by the fusion of prokaryotic genes to form these
multifunctional enzymes are still a matter of debate. In the case of DHFR–
TS, this association presents clear benefits, as an electrostatic channelling of
dihydrofolate between the TS and DHFR domains, avoiding the diffusion of
the cofactor in the bulk medium, has been clearly demonstrated (Knighton
et al., 1994). However, for the other multifunctional enzymes of the pathway,
the gains resulting from domain fusion are not so obvious. The case of the
bifunctional HPPK–DHPS has been studied in some detail (Mouillon et al.,
2002). The kinetic data strongly suggest that the intermediate dihydropterin
pyrophosphate is not channelled between the two domains but rather
released into the external medium. However, the close vicinity of the two
catalytic sites is probably, by itself, an advantage as it would limit diffusion
of the intermediate.
As previously mentioned, the plant THF-biosynthetic pathway is split
between the cytosol, plastids and mitochondria (Fig. 4). The subcellular
distribution of the pathway is less clear in the other autotrophic eukaryotes.
In yeast, experimental evidences support the presence of two enzymes in
mitochondria. The first one is the trifunctional enzyme DHNA–HPPK–
DHPS (Fig. 6) involved in the conversion of dihydroneopterin into dihy-
dropteroate, which is associated with mitochondrial membranes (Guldener
et al., 2004). In baker yeast, the MET7 gene encodes both the cytoplasmic
and mitochondrial forms of the FPGS enzyme (DeSouza et al., 2000). The
remaining enzymes of the pathway are predicted to be located in the cytosol.
In Apicomplexa, the subcellular location of THF biosynthesis is probably
cytosolic. Indeed, sequence analysis of the different proteins does not predict
any targeting towards either mitochondria or apicoplasts (a vestigial, non-
photosynthetic plastid found in most parasites belonging to this group).
Experimental evidences are now required to validate the location of the
pathway in parasites, as it was done for plants.
B. FOLATE BIOSYNTHESIS AS A TARGET FOR THERAPIES

AGAINST INFECTIOUS DISEASES
Rapidly dividing cells such as bacteria, parasites, embryonic or tumour cells

rely heavily on the availability of folates. Thus, blocking de novo folate
biosynthesis or folate regeneration leads to the arrest of cell division and
eventually to cell death. This feature has been exploited to cure microbial or
parasitic infections, and the development of antifolate drugs blocking the
regeneration of THF from dihydrofolate has been proven effective against
cancer cells proliferation. Biosynthesis of THF is mainly inhibited by two
groups of compounds (Fig. 7). The first group is represented by sulphona-
mides that are structural analogues of pABA and competitive inhibitors of
DHPS, blocking the condensation of the pABA moiety with the pterin ring.
The second group includes inhibitors of DHFR, often mimicking a pterin or a
pyrimidine structure and blocking the reduction of dihydrofolate into THF,
which is the active form in transport of C1 units. Inhibitors of DHFR are
commonly used as therapeutic agents for cancer (Bertino, 2009), whereas a
combination of these two types of inhibitors are most often used in clinical
treatments against parasites such as P. falciparum or T. gondii (Nzila, 2006;
Wang et al., 2004). Unfortunately, the use of these drugs is compromised by
the emergence of resistances, which occurred mainly by point mutations of
Fig. 7. Chemical structures of drugs inhibiting dihydropteroate synthase and

dihydrofolate reductase activities. Inhibitors of dihydropteroate synthase (DHPS;
reaction 8 in Fig. 6) are pABA analogues and belong to the sulphonamide family.
Asulam is used as an herbicide, whereas sulfanilamide, sulfadiazine and dapsone are
used as antibacterial drugs and, in combination with pyrimethamine, to treat toxo-
plasmosis or malaria. Inhibitors of dihydrofolate reductase (DHFR; reaction 10 in
Fig. 6) mimic pteridine or pyrimidine structures and are used as therapeutic agents for
cancer (methotrexate), as antibiotics (trimethoprim), and, synergistically with sulpho-
namides, in treatments against parasites of the Apicomplexa phylum (pyrimethamine,
cycloguanil). Note that methotrexate is also used to manipulate the folate
and AdoMet pools in plants (Loizeau et al., 2007, 2008; Prabhu et al., 1998; van
Wilder et al., 2009).
genes coding for the target enzymes (Triglia et al., 1997; Vinayak et al., 2010;
Wongsrichanalai et al., 2002). The established efficacy of folate metabolism as
a clinical target is, however, strongly stimulating to identify new molecules
efficient against other enzymes of the THF-biosynthetic pathway (Nzila et al.,
2005b; Rattanachuen et al., 2009; Wang et al., 2010). Because several steps
present in plants are similar to those found in Apicomplexa (see Fig. 6), it is
likely that the plant enzymes could serve as models to develop new inhibitors
of folate synthesis active against the proliferation of these parasites.
V. PHYSIOLOGY OF FOLATE IN HUMAN

HEALTH AND DISEASE
A. METABOLIC AND CLINICAL MANIFESTATIONS OF FOLATE DEFICIENCY
Folate deficiency is one of the most prevalent vitamin deficiencies worldwide.

It may be due to several factors including a limited diet, an impaired absorp-
tion and pharmacological treatments. Also, pregnant women are at risk of
folate deficiency because pregnancy significantly increases the folate require-
ment, especially during period of rapid foetal growth. It is generally accepted
that folate levels below 300–330 nmol/L in erythrocytes are considered to be
suggestive of risk and may be symptomatic of folate deficiency. Measure-
ments of plasma concentrations of homocysteine are also used as an indica-
tor of folate status (see below). This criterion is, however, not sufficient to
establish folate deficiency because vitamin B12 or vitamin B6 nutritional
status, as well as other factors, may also affect homocysteine concentration
(Stover, 2004). In the case of folate deficiency, all the reactions in C1 metab-
olism will be compromised to varying degrees, leading to the modification of
substrates/products pools that may have negative consequences. Folate is
attracting considerable interest as having an established role in the preven-
tion of neural tube defects (NTDs) and possible preventive roles against
cardiovascular diseases, certain cancers and neuropsychiatric disorders.
Upon folate deficiency, the inefficient re-methylation of homocysteine to
methionine is associated with increased homocysteine levels in blood. Epide-
miological evidences indicate that elevated plasma homocysteine concentra-
tion (> 14 mol/L) is an independent risk factor for cardiovascular disease
and stroke (Stover, 2004). Increased plasma homocysteine content may also
be a risk factor for neurodegenerative disorders, including Alzheimer’s and
Parkinson’s diseases (Mattson and Shea, 2003). The impairment of methio-
nine synthesis upon folate deficiency also results in insufficient amounts of
AdoMet available for methylation reactions. These are required for the
biosynthesis of many important products and for methylation of DNA and
histones, which are important epigenetic determinants in gene expression.
DNA hypomethylation is an early and consistent event in carcinogenesis and
is associated with genomic instability and increased mutations (Choi and
Mason, 2000). Another consequence of folate deficiency is a decrease of
dTMP synthesis due to limiting supply of 5,10-methylene-THF to TS. Mod-
ification of the intracellular dUMP/dTMP balance results in a higher incor-
poration of dUTP into DNA, which generates point mutations, single- and
double-strand DNA breaks and ultimately chromosomal breakage. These
events related to DNA structure, stability and transcriptional regulation are
likely to underlay a whole range of cancers. In particular, a relationship

between folate status and colorectal, cervical and breast carcinomas has
been observed in several studies (Choi and Mason, 2000).
Besides its role in dTMP synthesis, folate also supports nucleic acid
synthesis through the biogenesis of purine ring. Thus, there is general im-
pairment of cell division upon folate deficiency, which is more obvious in
tissues with rapid turnover, such as the haematopoietic system. The specific
type of anaemia associated with folate deficiency (megaloblastic anaemia) is
characterized by the accumulation in the bone marrow of large, abnormal,
nucleated precursor cells of erythrocytes. Folate deficiency also affects the
intestinal epithelium, where impaired DNA synthesis causes megaloblastosis
of enterocytes.
Finally, there is general consensus that reduced maternal folate status is
associated with an increased risk of NTDs (Geisel, 2003). Two of the most
common serious birth defects of the brain and spine are spina bifida and
anencephaly. Although the mechanism by which adequate folate intake
reduces risk during the crucial developmental phase of the embryonic neural
tube is unknown, public-health campaigns in many countries recommend
periconceptional supplementation of synthetic folic acid to reduce the risk
of NTDs.
B. DIETARY SOURCES OF FOLATE AND INTAKE RECOMMENDATIONS
The Food and Nutrition Board of the US National Academy of Sciences

Institute reviewed the evidence of folate intake, status and health for all age
groups (Food and Nutritional Board, 1998). Accordingly, folate require-
ments have been defined as the intakes required for maintenance of normal
C1-transfer reactions, as estimated by measuring red blood cell folate con-
centration (Bailey and Gregory, 1999). This exhaustive review led to calcula-
tions of an estimated average requirement and a subsequent estimation of the
recommended dietary allowances (RDAs). This definition agrees with the
recommended nutrient intakes edited by the Food and Agriculture Organi-
zation of the United Nations and the World Health Organization. For male
and female adults > 19 years of age, the folate RDA is 400 g dietary folate
equivalents (DFE)/day. DFE is defined as the quantity of naturally occurring
food folates (g) plus 1.7 times the quantity of synthetic folic acid (g) added
in the diet, folic acid being assumed to be 1.7-fold more bioavailable than
natural folates (Bailey and Gregory, 1999). For pregnant women, the RDA is
600 g to deal with the increased requirements for folate that are associated
with the rapid rate of maternal and foetal cellular growth and development
during pregnancy.
Folate contents have been determined for a wide variety of foods, includ-
ing raw, processed and cooked foodstuffs. With the exception of liver, which
is the major storage organ for folate, meat, poultry, and fishery products
generally contain small amount of folate (5–60 g/100 g portion). Also, folate
content in diary products is often low, for example, total folate in cow’s milk
is in the range 5–10 g/100 g. Many foods derived from plants are particu-
larly rich in folate; they include green leafy vegetables, legumes and certain
fruits (Table I). As mentioned above, the amount of folate in plant foods
depends primarily on the species and the nature of the tissue. The contribu-
tion of different food sources to the total dietary folate intake is influenced by
numerous parameters including bioavailability, stability throughout storage,
processing and cooking, and dietary habits (Scott et al., 2000). Various
dietary surveys in Northern America and Western Europe countries indicate
that plant foods are by far the main contributors to the folate intake in
adults. Thus, about 35–40% of dietary folate is provided by vegetables
(including potatoes) and fruits, and about one-third by cereal/grain products
(Scott et al., 2000).
TABLE I
Folate Content of Selected Plant Foods
Food Folate (g/100 g)a

Vegetables
Spinach (raw/cooked) 193/146
Lettuce, different cultivars, raw 29–136
Cauliflower (raw/cooked) 57/44
Carrot (raw/cooked) 19/14
Potatoes (baked) 9
Fruits
Avocado 35–88
Oranges (orange juice) 30 (30)
Tomatoes 15
Apples, apricots 3–8
Legumes
Lentils, mature seeds (raw/cooked) 479/181
Beans, yellow, mature seeds (raw/cooked) 389/81
Cereals
Wild rice (raw/cooked) 95/26
Rice, white, long-grain (raw/cooked) 8/3
Wheat, hard white, raw 38
a
The amount of dietary folate equivalents provided by different foods is
given per 100 g portion. Data were adapted from the US Department of
Agriculture, Agricultural Research Service, National Nutrient Database for
Standard Reference, release 23, 2010 (www.ars.usda.gov/ba/bhnrc/ndl).
These types of surveys also indicated that typical folate intakes are subop-
timal in rich countries. For example, the average total intakes of folate
among adults ranged from 168 to 326 g/day in several European countries,
that is, 20 to 60% below the recommended 400 g/day level (De Bree et al.,
1997). As a consequence, the Food and Drug Administration published
regulations requiring the addition of synthetic folic acid to cereal-derived
foods (fortification). The main motivation behind mandatory fortification
was to decrease the occurrence of NTDs. Effective from 1998, mandatory
folate fortification has clearly improved folate status with increased folate
levels by two- to threefolds in serum and by 38% in red blood cells, and
a decrease in total homocysteine concentration by 7%. More importantly,
the incidence of NTDs was reduced by 20–53% since the onset of
fortification in North America (De Wals et al., 2007; Eichholzer et al.,
2006). Although considering these beneficial effects, folate fortification
remains a controversial issue in the European Union as important intakes
of folic acid might mask the diagnosis of vitamin B12 deficiency, principally
in elderly people, allowing neurological complications to progress
undiagnosed.
VI. FOLATE BIOFORTIFICATION IN PLANTS
Although folate fortification and supplementation (capsule intake) programs

proved efficient to a certain extent in developed countries, these are probably
not the most effective options for a lot of developing countries due to
unstable political and economical situations. Enhancement of folate level in
crops by plant breeding or biotechnology (biofortification) provides a ratio-
nal alternative, or at least complementary solution, in addressing folate
malnutrition (Bekaert et al., 2008; Blancquaert et al., 2010; Rébeillé et al.,
2006; Storozhenko et al., 2005). Conventional breeding combined with the
recent developments in plant molecular genetics and genomics is a powerful
tool for the development of plant cultivars with useful traits. Examination of
the natural variability in folate levels in wheat genotypes, strawberries and
potato cultivars indicated about twofold changes (Goyer and Navarre, 2007;
Piironen et al., 2008; Stralsjo et al., 2003). Although these variations are
rather modest, they provide a basis for breeding program.
Recent progress in our understanding of folate metabolism in plants led to
the development of metabolic engineering strategies. Conceptually, there are
several potential means to improve folate content in plants through engineer-
ing, including overexpression of limiting steps in THF synthesis or salvage
(push strategy) or favouring the stabilization/sequestration of folates, for

example, through production of a folate-binding protein (pull strategy;
Bekaert et al., 2008; Blancquaert et al., 2010; Rébeillé et al., 2006;
Storozhenko et al., 2005). Although counterintuitive, an overall folate
increase can negatively affect plant metabolism and development because
of important changes in folates homeostasis. Thus, blocking 5-formyl-THF
utilization in Arabidopsis plants knocked out in the gene coding FCL (reac-
tion 7, Fig. 2) raised the overall folates pool by twofold. As a result of the
eightfold increase in mitochondrial 5-formyl-THF, these mutants displayed,
however, a marked impairment of photorespiration, reduced growth rate and
delayed flowering (Goyer et al., 2005). Other studies indicated that engineer-
ing of THF biosynthesis can result in more important enrichment in folates
without detrimental effects on growth. Several studies indicated that the
simultaneous enhancement of both the pterin and pABA branches of THF
synthesis is required to achieve a high level of folate accumulation. In a first
series of experiments, overexpression of a GTPCHI gene of plant or non-
plant origin led to very important production of pterins (up to 1000-fold the
wild-type level) in leaves of Arabidopsis (Hossain et al., 2004) or lettuce
(Nunes et al., 2009), tomato fruits (Diaz de la Garza et al., 2004) and grains
from rice (Storozhenko et al., 2007b) or maize (Naqvi et al., 2009). The
overall folate content was generally increased two to four times in these
engineered plants, but the excessive production of pterins may be undesirable
for human health. Evidence that the synthesis of pABA was a limiting factor
for folate accumulation in these plants was obtained (Diaz de la Garza et al.,
2004) and the pABA branch was subsequently engineered, alone or in
combination with GTPCHI. Overexpression of the ADC synthase gene
alone resulted in tomato fruits with 20-fold more pABA than controls but
unchanged levels of folates (Diaz de la Garza et al., 2007), whereas rice grains
contained about 50-fold more pABA and, surprisingly, significantly less
folates than wild-type (Storozhenko et al., 2007b). In the double GTPCHI/
ADC synthase transgenic tomato lines, fruits accumulated up to 25-fold more
folate than wild-type plants. Thus, these tomatoes contained enough folate
(840 g/100 g) to provide the RDA for a pregnant woman in a standard
serving portion (Diaz de la Garza et al., 2007). Folates accumulated up to
100-fold in rice grains bearing the two transgenes, providing four times the
adult RDA in just 100 g of polished raw grains (Storozhenko et al., 2007b).
Although other activities of the THF-biosynthetic pathway and/or transpor-
ters still constrain folate accumulation in these transgenic plants, the success
of this two-gene engineering strategy opens the door for folate biofortifica-
tion in a wide range of agricultural crops.
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Vitamin C: The Metabolism and Functions of
Ascorbic Acid in Plants
NICHOLAS SMIRNOFF1
Biosciences, College of Life and Environmental Sciences,

University of Exeter, Exeter EX4 4QD, United Kingdom
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
II. Ascorbate Biosynthesis: The D-Mannose/L-Galactose (Man/L-Gal)
Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
A. The Phosphomannose Isomerase Misconception........................ 114
B. Phosphomannose Mutase ................................................... 115
C. GDP-mannose Pyrophosphorylase ........................................ 115
D. GDP-mannose-3,5-epimerase............................................... 116
E. GDP-L-galactose Phosphorylase/Guanylyltransferase .................. 117
F. L-Galactose 1-P Phosphatase ............................................... 118
G. L-Galactose Dehydrogenase ................................................ 120
H. L-Galactono-1,4-lactone Dehydrogenase ................................. 120
III. Are There Multiple Pathways for Ascorbate Biosynthesis?. . . . . . . . . . . . . . . 123
A. Ascorbate Biosynthesis from D-GalUA ................................... 125
B. Ascorbate Biosynthesis from myo-inositol and D-GlcUA .............. 126
C. Ascorbate Biosynthesis from L-GulL via GDP-Mannose .............. 127
IV. The Control of Ascorbate Biosynthesis and Pathway Engineering . . . . . . . 127
V. Ascorbate Catabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
VI. Ascorbate Transport and Subcellular Compartmentation. . . . . . . . . . . . . . . . 134
VII. Ascorbate Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
VIII. The Redox Reactions of Ascorbate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
A. APX—An Enzyme That Does Exactly What It Says on the Tin ..... 139
B. Monodehydroascorbate Reductase ........................................ 142
1
Corresponding author: E-mail: n.smirnoff@exeter.ac.uk

108 NICHOLAS SMIRNOFF
C. Dehydroascorbate Reductase............................................... 143

D. Ascorbate Oxidase—An Enigmatic Enzyme ............................. 145
IX. The Functions of Ascorbate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
A. The Ascorbate–GSH (Foyer–Halliwell–Asada) Cycle .................. 148
B. Photosynthesis and Photoprotection ...................................... 149
C. Environmental Stress and Pathogens...................................... 153
D. Growth and Signalling: Cell Division and Cell Expansion ............ 154
X. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
ABSTRACT
It is widely accepted that the predominant ascorbate biosynthesis pathway in green
plants is via GDP-mannose and L-galactose. D-galacturonic, D-glucuronic acid and
GDP-L-gulose could be minor ascorbate precursors, but there is no definitive
evidence. Arabidopsis thaliana mutants lacking ascorbate cannot grow, but it is
not known which function is critical: control of reactive oxygen or the proposed
roles in modulating cell expansion and division. Ascorbate is transported in the
phloem, and glucose conjugates occur in the phloem of the Cucurbitaceae. Ascor-
bate or dehydroascorbate transporters have not been identified at the molecular
level. Pathways from ascorbate to oxalate in the apoplast and tartrate in grape
berries have been identified. Ascorbate-deficient (vtc) mutants tend to be smaller,
more sensitive to abiotic stresses and more resistant to biotrophic pathogens. The
use of mutants and overexpression shows the importance of ascorbate peroxidase,
monodehydroascorbate reductase and dehydroascorbate reductase in reactive oxy-
gen defence and signalling. Ascorbate accumulation in Arabidopsis leaves is
increased by high light along with expression and activity of L-galactose phosphor-
ylase (VTC2), reflecting multiple roles in photosynthesis. These roles are modula-
tion of hydrogen peroxide and singlet oxygen, enzyme cofactor in the xanthophyll
cycle and, speculatively, a photosystem II electron donor during photoinhibition.
ABBREVIATIONS
Asc ascorbate (or ascorbic acid)

cAPX cytosolic ascorbate peroxidase
DHA(R) dehydroascorbate (reductase)
fd ferredoxin
GR glutathione reductase
GSH glutathione
LOO lipid peroxyl radical
LOOH lipid hydroperoxide
MDHA(R) monodehydroascorbate (reductase)
OEC oxygen evolving complex
PET photosynthetic electron transport
sAPX stromal ascorbate peroxidase
VITAMIN C 109
tAPX thylakoid ascorbate peroxidase

Toc -tocopherol
Toc -tocopheroxyl radical
VDE violaxanthin de-epoxidase
Viol violaxanthin
Zea zeaxanthin
I. INTRODUCTION
Ascorbic acid (vitamin C) is synthesised by animals and plants but is appar-

ently absent from prokaryotes. The definition of ascorbic acid as vitamin C
arises from the dietary requirement of humans, who do not express the last
enzyme in the pathway (L-gulonolactone oxidase). A number of other ani-
mals, including primates, guinea pigs, fruit bats and teleost fish, also lack this
enzyme (Smirnoff, 2001; Smirnoff and Gatzek, 2004). Without further inves-
tigation, it is not known for certain if all the groups of protists synthesise
ascorbate, although it is present in photosynthetic protists and trypanosomes
(Grun and Loewus, 1984; Helsper et al., 1982; Ishikawa et al., 2006b;
Wilkinson et al., 2005). Fungi synthesise D-erythroascorbate, a 5-carbon
analogue of ascorbate, and a large proportion of the total pool is present
as glycosides (Baroja-Mazo et al., 2005; Keates et al., 1998; Okamura, 1998).
Most of the biological roles of ascorbate derive from its ability to act as a
reducing agent. Two types of biochemical activity are dependent on this
property. Firstly, it is an effective antioxidant and free radical scavenger.
Secondly, it is required for preventing over-oxidation of iron in 2-oxogluta-
rate-dependent dioxygenases (2-ODDs) (De Tullio, 2004). Ascorbate oxida-
tion is a two-step process, initially producing the monodehydroascorbate
(MDHA) radical. Further oxidation, or disproportionation of MDHA,
produces dehydroascorbate (DHA; Fig. 1A). Of key importance in the
effectiveness of ascorbate as an antioxidant and free radical scavenger is
the relatively high stability of MDHA (Buettner and Schafer, 2004). It has
a sufficiently long life that it can be regenerated back to ascorbate by mono-
dehydroascorbate reductase (MDHAR) rather than propagating the forma-
tion of more damaging radicals. Ascorbate can react readily with hydrogen
peroxide (catalysed by a specific ascorbate peroxidase (APX) in
plants and some other organisms), singlet oxygen and ozone. Reactions
that remove potentially damaging free radicals include interaction with
tocopheroxyl radicals and carotenoid radicals (Buettner and Schafer,
2004). The co-operation between tocochromanols, such as tocopherols
(vitamin E) and ascorbate, has been widely discussed (Li et al., 2003). The
role of ascorbate as a ‘‘cofactor’’ for some 2-ODDs is related to their active
site Fe. These enzymes have a co-ordinated FeII that takes part in catalysis.
In some cases, iron becomes over-oxidised to FeIV in substrate-uncoupled
reactions leading to enzyme inactivation (Clifton et al., 2006). Ascorbate
prevents inactivation by reducing FeIV to FeII. It is therefore not strictly
speaking a cofactor but protects against over-oxidation. This importance of
this function is classically seen in the vitamin C deficiency disease scurvy, in
which impairment of collagen synthesis is the most obvious symptom. Prolyl
residues in collagen must be hydroxylated by the 2-ODD prolyl hydroxylase
for proper function in connective tissue. A wide range of other 2-ODDs may
Fig. 1. (Continued)
VITAMIN C 111
Fig. 1. The redox reactions of L-ascorbate. (A) Interconversions of ascorbate and

its main oxidation products. Two monodehydroascorbate (MDHA) radicals can
disproportionate producing ascorbate and dehydroascorbate (DHA). MDHA is
reduced to ascorbate by reduced ferredoxin and by pyridine nucleotide-linked
MDHA reductase. DHA is reduced to ascorbate by thiols, the reaction being cata-
lysed by a range of enzymes, particularly glutathione-dependent DHA reductase.
Numbers on the ascorbic acid structure indicate carbon atoms 1 and 6. (B) Ascorbate
oxidation by singlet oxygen (1O2). This reaction is unlikely to be of major significance
in the case of 1O2 produced in the hydrophobic environment of PSII during photo-
synthesis. (C) The reaction products of ascorbate with ozone are pH dependent.
Apoplastic ascorbate, which is proposed to provide defence against incoming
ozone, could follow both routes, as apoplastic pH can be close to 5.
also be protected by ascorbate, and it is routinely added to in vitro enzyme

assays. There are 119 predicted 2-ODDs in Arabidopsis thaliana (Arabidop-
sis) covering a wide range of functions, including synthesis of gibberellins,
abscisic acid and flavonoids as well as synthesis and degradation of glucosi-
nolates (Hedden, 1992; John et al., 2001; Kliebenstein et al., 2001). Another
hormone, ethylene, is synthesised by 1-aminocyclopropane-1-carboxylic-
acid oxidase. This enzyme has ascorbate as a co-substrate (Clifton et al.,
2006; Prescott and John, 1996). One of the best documented ascorbate-
dependent enzymes in plants is violaxanthin de-epoxidase (VDE). VDE
catalyses the conversion of violaxanthin to zeaxanthin in the thylakoid
lumen and is part of the photoprotective xanthophyll cycle (Eskling and
Akerlund, 1997). The reaction is limited in ascorbate-deficient vtc mutants
of A. thaliana (Muller-Moulé et al., 2003; Smirnoff, 2000a). It is not clear to
what extent pathways using 2-ODDs in plants are normally limited by
ascorbate supply in vivo, although there is evidence that synthesis of cell
wall extracellular matrix proteins, such as extensin, that contain hydroxy-
proline may be a significant sink for ascorbate (DeGara et al., 1991; De
Tullio et al., 1999). The ability of ascorbate to reduce transition metal ions
such as Fe3þ and Cu2þ has a potentially negative effect, as Fe2þ and Cuþ can
participate with hydrogen peroxide in the Fenton reaction to generate highly
reactive hydroxyl radicals (Halliwell and Gutteridge, 1999). This is the basis
of the pro-oxidant effect of exogenous ascorbate widely reported in the
mammalian cell culture literature (Halliwell and Whiteman, 2004). However,
in plants, Cu-mediated hydroxyl radical production in the apoplast has
been proposed as a mechanism that contributes to cell wall loosening (by
scission of polysaccharides) and cell expansion (see Section IX.D). This
review will cover recent developments in our understanding of the metabo-
lism and functions of ascorbate in plants. The levels and bioavailability of
ascorbate in fruit and vegetables have been well reviewed elsewhere (Davey
et al., 2000).
II. ASCORBATE BIOSYNTHESIS: THE D-MANNOSE/

L-GALACTOSE (MAN/L-GAL) PATHWAY
The biosynthetic pathway of ascorbate from GDP-mannose was proposed

byWheeler et al. (1998) (Fig. 2). This proposal was based on the discovery
that exogenous L-galactose (L-Gal) is rapidly converted to ascorbate using a
newly identified enzyme, L-galactose dehydrogenase (L-GalDH), which oxi-
dises C1 of L-Gal to L-galactono-1,4-lactone (L-GalL). L-GalL had been
identified a long time before as a potential ascorbate precursor in plants,
VITAMIN C 113
Fig. 2. The mannose/L-galactose ascorbate biosynthesis pathway. Enzymes: 1,

phosphomannose isomerase; 2, phosphomannose mutase; 3, GDP-mannose
pyrophosphorylase (VTC1 ¼ CYT1 and HSN1); 4, GDP-mannose-3,5-epimerase;
5, GDP-L-galactose phosphorylase/guanylytransferase (VTC2, VTC5); 6, L-galactose
1-P phosphatase (VTC4; IMPL2); 7, L-galactose dehydrogenase; 8, L-galactono-1,
4-lactone dehydrogenase.
although the source of L-GalL was not identified (Smirnoff et al., 2001). The
derivation of L-Gal and ascorbate from mannose-1 P and GDP-mannose was
confirmed by in vivo and in vitro 14C labelling experiments (Wheeler et al.,
1998). The initial genetic evidence for this pathway came from vtc1 (formerly
soz1), the first ascorbate-deficient Arabidopsis mutant to be characterised
(Conklin et al., 1996, 1997, 1999). The vtc1 mutant has a decreased ability
to convert glucose and mannose to ascorbate, and VTC1 encodes GDP-
mannose pyrophosphorylase (GMP). At the same time, it was found that
antisense suppression of GMP in potatoes decreased the ascorbate concen-
tration in their leaves (Keller et al., 1999). All eight steps of the D-mannose/
L-galactose (Smirnoff–Wheeler) pathway, starting from the central metabo-
lite fructose 6-P, have now been confirmed by genetic analysis of Arabidop-
sis. At the same time, biochemical evidence for the D-mannose/L-galactose
pathway in the heterotrophic green alga Prototheca moriformis was obtained
(Running et al., 2003).
A. THE PHOSPHOMANNOSE ISOMERASE MISCONCEPTION
At least two routes could be used to convert glucose or fructose phosphates

to phosphorylated mannose intermediates: conversion of fructose 6-P to
mannose 6-P catalysed by phosphomannose isomerase (PMI) and epimerisa-
tion of GDP-glucose to GDP-mannose by GDP-glucose 2-epimerase. Due to
suggestions in the older literature that plants lack, or have very limited, PMI
activity (Smirnoff and Wheeler, 2000), the view that an alternative route is
required to produce mannose-6 P from the hexose phosphate pool has
propagated in the literature (Wolucka and Van Montagu, 2007). However,
PMI activity is readily detected in Arabidopsis leaf extracts as well as a
variety of other species (Smirnoff and Wheeler, 2000). An elegant dual
labelling experiment using Arabidopsis cell cultures shows that GDP-Man
is derived from a pathway involving mannose phosphates and must involve
PMI (Sharples and Fry, 2007). The results of Sharples and Fry (2007) also
show that formation of GDP-Man from GDP-glucose, catalysed by GDP-
glucose 2-epimerase, is a very minor route in Arabidopsis cell cultures. Two
predicted PMI genes are present in Arabidopsis (PMI1/MEE31 MATER-
NAL EFFECT EMBRYO ARREST 31/At3g02570 and PMI2/At1g67070),
and expression of recombinant enzymes shows that both have PMI activity
(Maruta et al., 2008). RNAi suppression decreased ascorbate in the case of
PMI1 but not PMI2 (Maruta et al., 2008), providing strong evidence that
PMI1 is involved in ascorbate synthesis. Overall, labelling and molecular
genetic evidence therefore support the involvement of PMI in ascorbate
biosynthesis. An alternative pathway, termed the VTC2 cycle, producing
VITAMIN C 115
GDP-Man and GDP-glucose was proposed to explain the supposed lack of

M6P input to ascorbate biosynthesis (Laing et al., 2007; Wolucka and Van
Montagu, 2007). However, given the evidence for the existence of PMI and
its role in ascorbate synthesis, the VTC2 cycle does not have an exclusive role.
B. PHOSPHOMANNOSE MUTASE
Interconversion of M6P and M1P is catalysed by phosphomannose mutase

(PMM). The involvement of PMM in ascorbate biosynthesis is supported by
molecular genetic evidence. Expression of a recombinant predicted PMM
(At2g45790) in Arabidopsis shows that it has PMM (and phosphoglucose
mutase) activity (Qian et al., 2007). Expressing PMMs from a number of
other species including tobacco in a PMM-deficient Saccharomyces cerevisiae
strain rescued the cells. Virus-induced gene silencing of PMM in Nicotiana
benthamiana caused a decrease in ascorbate, while PMM overexpression in
N. benthamiana and Arabidopsis caused a modest increase in ascorbate.
Likewise, overexpression of acerola (Malpighia glabra) PMM in tobacco
caused an increase in ascorbate (Badejo et al., 2009). Further evidence for
the involvement of PMM was provided by map-based cloning of a tempera-
ture-sensitive Arabidopsis mutant affected in growth and cell death. The
plants had decreased PMM catalytic efficiency due to a point mutation and
decreased ascorbate (Hoeberichts et al., 2008). Transfer from 16 to 28 8C is
lethal, and this effect is associated with impaired protein glycosylation rather
than ascorbate deficiency because the plants could not be rescued by down-
stream ascorbate precursors. A T-DNA knockout mutation in PMM resulted
in an embryo lethal phenotype. Interestingly, both PMI1 and GMP (cyt1)
knockout mutations are embryo lethal (Lukowitz et al., 2001; Pagnussat et al.,
2005), suggesting that GDP-mannose synthesis using these three enzymes is
essential for embryo development. Although synthesis of mannose containing
hemicelluloses is possibly affected, the most likely cause of death is impaired
protein glycosylation, for which GDP-mannose is the mannosyl donor.
C. GDP-MANNOSE PYROPHOSPHORYLASE
GMP catalyses the formation of GDP-mannose from mannose 1-P and GTP.
Although reversible, the reaction in vivo is likely to favour GDP-mannose
synthesis because the other product, pyrophosphate, is rapidly hydrolysed by
inorganic pyrophosphatase. Map-based cloning of the ascorbate-deficient
mutant vtc1 (soz1) showed that there is a point mutation in At2g39770, a
predicted GMP. This was confirmed by decreased enzyme activity in the
mutant (Conklin et al., 1999). A second mutation in this gene (cyt1) is
predicted to knock out protein expression and is embryo lethal (Lukowitz

et al., 2001). Evidence supports impaired protein glycosylation due to lack of
GDP-Man as the cause of lethality, as is also found for PMI and PMM
mutants. There are two other genes in Arabidopsis (At4g30570 and
At4g30570) encoding proteins with high sequence similarity ( 80% identity)
to VTC1/CYT1. However, examination of Affymetrix microarray data shows
that both genes are generally expressed at very low level. At4g30570 is
expressed in developing pollen. Therefore, it seems that VTC1/CYT1 encodes
most of the GMP activity in Arabidopsis leaves. Similarly, antisense suppres-
sion of GMP in potato decreased ascorbate, accelerated leaf senescence and
decreased mannose in cell wall polysaccharides (Keller et al., 1999). Recently,
it has been found that vtc1 and an allelic GMP mutant (hsn1) are hypersensi-
tive to ammonium ions in the rooting medium (Barth et al., 2010; Li et al.,
2010a,b; Qin et al., 2008). Although ammonium is a common nitrogen source
for plants, it can be toxic at high concentration. This phenotype is independent
of ascorbate deficiency and may be caused by impaired protein glycosylation
that then affects ammonium fluxes across the plasma membrane (Li et al.,
2010a). It would be interesting to determine if PMI and PMM mutants are
similarly ammonium sensitive. The vtc1-1 mutation has been very widely used
in investigations of ascorbate function. The ascorbate-independent pheno-
types of altered protein glycosylation, cell wall polysaccharides and ammoni-
um hypersensitivity of GMP mutants suggest that care needs to be taken in
inferring anything about the functions of ascorbate from studies of this mutant
alone. GDP-mannose is also the precursor of L-fucose, another important
extracellular matrix sugar (Bonin et al., 2003).
D. GDP-MANNOSE-3,5-EPIMERASE
GDP-mannose-3,5-epimerase (GME) catalyses the double epimerisation of

GDP-mannose with the production of GDP-L-galactose. The enzyme was
first identified in the green alga Chlorella pyrenoidosa (Barber and Hebda,
1982) and was more recently purified and cloned from Arabidopsis (Wolucka
et al., 2001). Investigation of the properties of the native and recombinant
Arabidopsis GME showed that it is inhibited by GDP, GDP-glucose, GDP-
L-fucose, ascorbate and L-GalL, suggesting that it could be regulated by these
metabolites (Wolucka and Van Montagu, 2003). Most interestingly, they
showed that GDP-L-gulose is also produced as a minor product as a result
of an initial 500 -epimerisation. This is of significance in relation to the pres-
ence of enzymes able to oxidise L-gulonolactone to ascorbate (Section III.B).
A crystal structure of GME has been produced, and its reaction mechanism
has been studied in detail (Major et al., 2005). This reveals GME to be highly
VITAMIN C 117
unusual in carrying out oxidation, epimerisation and reduction in the same

active site. Genetic evidence to support a role for GME in ascorbate biosyn-
thesis has been provided by RNAi silencing of the two predicted GME genes
in tomato (Gilbert et al., 2009) and transient expression in tobacco (Bulley
et al., 2009). In the case of tomato RNAi suppression, the plants have
decreased ascorbate. However, the plants also showed additional phenotypes
related to alterations in cell wall polysaccharide composition (mannose and
galactose) that resulted in petiole fragility and altered fruit firmness. The
results are consistent with GDP-L-galactose being a precursor for L-Gal
containing polysaccharides in the cell wall. Significant amounts of L-Gal
occur in the cell wall (Baydoun and Fry, 1988; Roberts, 1971). A relatively
minor, but functionally important polysaccharide, rhamnogalacturonan II,
contains L-Gal in specific residues (Reuhs et al., 2004).
E. GDP-L-GALACTOSE PHOSPHORYLASE/GUANYLYLTRANSFERASE
The conversion of GDP-L-galactose to L-galactose 1-P is the first step in the

Man/L-Gal pathway that is dedicated to ascorbate synthesis. As far as is
known, the intermediates beyond this step only give rise to ascorbate or its
breakdown products (Fig. 2). An enzyme able to catalyse phosphorolytic
breakdown of GDP-L-galactose to L-Gal 1-P (GDP-L-galactose phosphory-
lase; GDP--L-Gal:orthophosphate guanylyltransferase) was identified in
pea seedling extracts (Dowdle et al., 2007; Ishikawa et al., 2006a). Following
from this, the gene encoding this enzyme activity in Arabidopsis was identi-
fied as VTC2 by three groups (Dowdle et al., 2007; Laing et al., 2007; Linster
et al., 2007, 2008). The ascorbate-deficient vtc2 mutant (Conklin et al., 2000)
was identified by map-based cloning as At4g26850 (Jander et al., 2002),
although its function was not identified at the time. A homologue in Arabi-
dopsis (VTC5/At5g55120) also encodes GDP-L-galactose phosphorylase
(Dowdle et al., 2007; Linster et al., 2008). Amongst the other common
sugar nucleotides, the enzyme has significant activity with GDP-L-fucose
(Linster et al., 2008). When enzyme kinetics are measured using direct assays
for substrates or products as opposed to enzyme-coupled assays, the Km
values for pea and Arabidopsis enzymes for GPD-L-Gal are 10 M
(Dowdle et al., 2007; Linster et al., 2008). There is disagreement about the
other substrates for VTC2 and VTC5. Laing et al. (2004) found that recom-
binant Arabidopsis VTC2 and Actinidia chinensis enzymes had much higher
guanylyltransferase activity with the 1-phosphates of mannose, glucose, D-
galactose and myo-inositol than with phosphate (Fig. 2). The products of this
reaction are L-Gal 1-P and the corresponding GDP sugar, while the products
of phosphorolysis are L-Gal 1-P and GDP. In contrast, Linster et al. (Linster
et al., 2008; Linster and Clarke, 2008) found very low guanylyltransferase
activity compared to phosphorylase activity. The reason for this discrepancy,
proposed to be caused by the use of enzyme coupled versus direct assays,
needs to be clarified because the extended VTC2 cycle depends on the
guanylyltransferase activity (Wolucka and Van Montagu, 2007). VTC2 and
VTC5 are members of the histidine triad superfamily of hydrolases, phos-
phorylases and transferases that act on nucleotide-containing substrates
(Brenner, 2002). The properties of GDP-L-galactose phosphorylase are
reviewed in more detail by Linster and Clarke (2008). Confocal microscopy
of an Arabidopsis VTC2::YFP fusion protein expressed in Arabidopsis
showed that the protein may be located in the nucleus as well as the cyto-
plasm. A putative nuclear localisation signal was found in the VTC2 amino
acid sequence (Muller-Moulé, 2008). This observation merits further investi-
gation and, if not artefactual, could suggest a regulatory role for VTC2.
Based on the proposed lack of PMI activity and the guanylyltranferase
activity of VTC2 with sugar 1-Ps reported by Laing et al. (2007), a ‘‘VTC2
cycle’’ in which VTC2 catalyses GDP-glucose, GDP-mannose and GDP-L-
fucose synthesis from sugar 1-Ps, while the mannose carbon skeleton is derived
from GDP-glucose by a 2-epimerase, was proposed (Laing et al., 2007;
Wolucka and Van Montagu, 2007). This interesting suggestion, however, is
based on the uncertain substrate specificity of VTC2 and is not in accord with
the labelling evidence that carbon skeletons for mannose are derived through
PMI and PMM and that PMI not only exists but also is required for normal
ascorbate production (see Section II.A). The importance of the VTC2 cycle
cannot be assessed until the substrate specificity of VTC2 is resolved.
The identification of VTC2/VTC5 as two genes encoding GDP-L-galactose
phosphorylase in Arabidopsis enabled the construction of a double mutant using
the vtc2-1 allele, in which a truncated message is predicted, and two independent
T-DNA insertion knockout mutants (vtc5-1 and vtc5-2). The double vtc2-1 vtc5
mutant seedlings ceased to grow after the cotyledons had expanded and eventu-
ally bleached. The seedlings could be rescued by feeding with ascorbate or
L-galactose (Dowdle et al., 2007). The properties of the double mutant show
that the Man/L-Gal pathway is essential for ascorbate biosynthesis in Arabidop-
sis seedlings and also show that ascorbate is essential for seedling growth.
F. L-GALACTOSE 1-P PHOSPHATASE
L-Gal 1-P is hydrolysed to produce L-Gal. Plants contain abundant sugar 1-P
phosphatase activity towards a range of sugars, but a phosphatase with high
specificity for L-Gal 1-P was purified from Actinidia deliciosa and Arabidop-
sis. Mass spectrometry of tryptic digests identified the Arabidopsis gene
VITAMIN C 119
At3g02870 that was annotated as myo-inositol 1-P phosphatase (Laing et al.,

2004). Genetic evidence for the role of At3g02870 in ascorbate biosynthesis
was provided by mapping the ascorbate-deficient vtc4-1 mutant to
At3g02870. This mutant has a P92L substitution in a conserved region
(Conklin et al., 2006). Two T-DNA insertion mutants of At3g02870 lacking
transcripts were identified. They contained similarly low ascorbate to vtc4-1
providing further evidence for the role of this gene in ascorbate biosynthesis
(Conklin et al., 2006). Another study showed that in vtc4 knockout mutants,
ascorbate and also myo-inositol are decreased by 25–30% (Torabinejad et al.,
2009). Recombinant VTC4 was effective at hydrolysing inositol 3-P as well as
the previously reported L-Gal 1-P and inositol 1-P (Torabinejad et al., 2009).
It can be concluded that VTC4 is bifunctional, having a role in inositol
metabolism as well as ascorbate metabolism. The VTC4 knockout mutants
were more sensitive to cold, NaCl and ABA, and it is possible that this
phenotype is due to impaired inositol phosphate signalling.
Significantly, the At3g028704/VTC4 KO mutants still contain 50–70% of
wild-type ascorbate and 50% of wild-type L-Gal 1-P phosphatase activity
(Conklin et al., 2006; Torabinejad et al., 2009). Given the evidence that the
Man/L-Gal pathway is the main contributor to ascorbate biosynthesis in
Arabidopsis, it is therefore likely that other phosphatases could contribute.
There are two Arabidopsis genes closely related to VTC4: IMPL1
(At1g31190) and IMPL2 (At4g39120). Recombinant IMPL2 has similar
activity with inositol 3-P, L-Gal 1-P and inositol 1-P, suggesting it could
also function in both ascorbate and inositol metabolism (Torabinejad et al.,
2009). Ascorbate and myo-inositol concentrations in IMPL1 and 2 mutants
have not yet been reported. Another L-Gal 1-P phosphatase candidate is a
purple acid phosphatase AtPAP15 (At3g07130). Overexpression in an acti-
vation-tagged line or by transformation with 35S::AtPAP15 increases ascor-
bate, while T-DNA insertion mutants have decreased ascorbate. The
recombinant enzyme has high activity not only with phytate but also with
inositol 1-P and a pH optimum of 4.6 (Zhang et al., 2008). The authors
proposed that expression of this enzyme affects ascorbate because myo-
inositol is an ascorbate precursor. However, there is not strong evidence
for this conclusion (Section III.B). Alternatively, AtPAP15 could act on
L-Gal 1-P. This possibility was not tested but, given the ability of VTC4
and IMPL2 to use inositol 1-P and L-Gal 1-P, it cannot be ruled out that this
enzyme contributes to ascorbate biosynthesis via the Man/L-Gal pathway. If
this were the case, its effectiveness despite having an acidic pH optimum
would need to be explained.
G. L-GALACTOSE DEHYDROGENASE
L-GalDH catalyses NADþ-dependent oxidation of L-Gal at C1 to produce L-

galactono-1,4-lactone (Gatzek et al., 2002). The capacity of the enzyme
seems to be relatively high, as exogenously supplied L-Gal and its reaction
product L-galactonolactone (L-GalL) are very rapidly converted to ascorbate
resulting in a large increase in ascorbate pool size (Davey et al., 1999;
Wheeler et al., 1998). Arabidopsis L-GalDH activity is encoded by
At4g33670. Evidence for its role in ascorbate biosynthesis is derived from
decreased ascorbate in plants where L-GalDH expression was decreased by
antisense suppression. The enzyme has high specificity for L-Gal (Arabidop-
sis Km 0.4 mM, Spinach Km 0.1 mM) and much lower Vmax and lower affinity
for L-gulose (Km 4 mM) and L-fucose (Km 56 mM) (Gatzek et al., 2002; Mieda
et al., 2004). Purified spinach L-GalDH is competitively inhibited by ascor-
bate (Mieda et al., 2004). The Ki value of 0.1 mM is well above the inferred
ascorbate concentration in the cytosol (Table I), suggesting that the enzyme
could be regulated by feedback inhibition.
H. L-GALACTONO-1,4-LACTONE DEHYDROGENASE
The last step in the Man/L-Gal pathway is the oxidation of L-GalL to ascorbate
(Mapson and Breslow, 1958). This reaction is catalysed by L-galactono-1,
4-lactone dehydrogenase (L-GalLDH), an FAD-linked enzyme of the vanil-
lyl-alcohol oxidase (VAO) flavoprotein family that uses cytochrome c as its
electron acceptor. Earlier investigations had shown L-GalLDH to be localised
in mitochondria in association with respiratory complex I (Millar et al., 2003).
L-GalLDH is encoded by one gene (At3g47930) in Arabidopsis. A T-DNA
insertion in this gene causes growth arrest after seed germination followed by
bleaching of the cotyledons (Pineau et al., 2008). Addition of ascorbate rescues
growth, but the plants are still stunted compared to wild type. Detailed
analysis of the respiratory complexes by electrophoresis revealed that respira-
tory complex I is missing in the ascorbate-rescued mutant plants, showing that
L-GalLDH is needed for complex 1 assembly as well as for ascorbate biosyn-
thesis. Reduction of L-GalLDH expression by RNAi in tomato also affected
growth, the authors noting that some lines were very severely affected
(Alhagdow et al., 2007). It is interesting that the total ascorbate concentration
in the less severely affected lines was not affected, although it was more
oxidised in the RNAi lines. Altered respiration in isolated mitochondria and
changed levels of TCA cycle intermediates suggested that mitochondrial
function was impaired. The symptoms observed by Alhagdow et al. could
be explained by impaired complex I assembly (Pineau et al., 2008), while
TABLE I
Ascorbate Concentrations (mM) in Leaf Cell Intracellular Compartments from Plants Grown Under Low or High Irradiance (Units:
mol photons m 2 s 1)
Irradiance Ascorbate concentration (mM)

Cytosol Chloroplasts Mitochondria Peroxisomes Nuclei Vacuoles
Arabidopsis 250 21 10 10 23 16 2
700 29 20 13 16 21 12
Barley 100 35 2 n.d. n.d. n.d. 0.6
500 61 10 n.d. n.d. n.d. 3
Arabidopsis concentrations were estimated from immunogold localisation with ascorbate antibodies using data in Zechmann et al. (2010). Barley data were
obtained by non-aqueous fractionation (Rautenkranz et al., 1994). n.d., not determined.
L-GalLDH activity is not decreased sufficiently to affect ascorbate biosynthe-

sis. The increased oxidation state of ascorbate in the RNAi lines is intriguing
and remains to be explained.
In all organisms that synthesise ascorbate, the last step involves oxidation
of an aldono-1,4-lactone. As noted above, plant L-GalLDH is a dehydroge-
nase and, where substrate specificity has been determined, it is highly specific
for L-GalL (Leferink et al., 2008; Ostergaard et al., 1997). Animals and
protists, unlike plants, use L-gulonolactone (L-GulL) as their ascorbate pre-
cursor. Fungi have a C5 analogue, D-erythroascorbate, synthesised from
D-arabinonolactone (Amako et al., 2006; Baroja-Mazo et al., 2005; Huh
et al., 1994). In contrast to the plant enzyme, the animal and fungal enzymes
are oxidases that generate hydrogen peroxide and are most likely not loca-
lised in the mitochondria. Rat L-GulL oxidase is microsomal (Smirnoff,
2001). Sequence analysis and site-directed mutagenesis have been used to
identify residues that determine the substrate specificity. Members of the
VAO flavoprotein family have a conserved proline or glycine in the vicinity
of FAD. Mutation of the corresponding residue (alanine) in Arabidopsis L-
GalLDH to glycine caused a 400-fold increase in reactivity with oxygen
along with H2O2 production but with little change to the ability to use
cytochrome c as electron acceptor (Leferink et al., 2009a,b,c). The authors
concluded that the alanine residue in the wild-type enzyme blocks the access
of oxygen to the reduced FAD. VAOs have a conserved glutamate–arginine
pair in the active site. Mutation of the glutamate to aspartate in Arabidopsis
L-GalLDH changed its substrate specificity, causing greater activity with L-
GulL (Leferink et al., 2009a,b,c). Despite very low activity of the wild-type
enzyme with L-GulL, Arabidopsis and other plants can convert exogenous L-
GulL to ascorbate, although to a considerably smaller extent than L-GalL
(Davey et al., 1999). The existence of L-GulL oxidases or dehydrogenases has
therefore been suspected. In addition to the well-characterised L-GalLDH,
Arabidopsis has genes encoding seven possible aldonolactone oxidases/dehy-
drogenases. Five of these have been expressed in tobacco cells and, of these,
three conferred increased ability to convert L-GulL to ascorbate (GulLO2/
At2g46750, GulLO3/At5g11540, GulLO5/At2g26740) (Maruta et al.,
2010a). This study provides the first evidence for genes encoding enzymes
able to utilise L-GulL in plants. Unfortunately, activity with L-GalL and
extent of oxidase activity was not reported. Given the potential redundancy
of these genes, at least a triple mutant will be needed to assess their contribu-
tion to ascorbate biosynthesis via either the Man/L-Gal pathway or the
proposed D-GlcUA pathway (Section III.B). An inspection of publicly avail-
able microarray data suggests that expression is very low in shoots compared
to roots. This would suggest that, whatever the function of these enzymes,
VITAMIN C 123
they are primarily active in root tissue. In contrast, L-GalLDH ribosome-

associated transcripts are expressed evenly across cell types, and transcript
abundance is remarkably unaffected by biotic and abiotic stress, light, hor-
mones and chemical treatments. Despite this observation, there is evidence
that L-GalLDH enzyme activity is higher in the light and higher in high light
acclimated leaves in terms of extractable activity and rate of conversion of
exogenous L-GalL to ascorbate (Bartoli et al., 2005; Dowdle et al., 2007;
Smirnoff, 2000a; Yabuta et al., 2007, 2008) (J. Dowdle and N. Smirnoff,
unpublished data). L-GalLDH loses activity during storage and after hydro-
gen peroxide treatment and is reactivated by dithiothreitol (Leferink et al.,
2009a,b,c). Mass spectrometry shows that a specific cysteine residue in the
cap domain of the active site is oxidised to sulfenic, sulfinic and sulfonic
states. The sulfenic acid state can be S-glutathionylated, causing enzyme
inactivation while protecting against further oxidation. Site-directed muta-
genesis of this cysteine not only removed sensitivity of the enzyme to oxida-
tion but also increased the Km for L-GalL (Leferink et al., 2009a,b,c). It is
tempting to speculate that cysteine oxidation and S-glutathionylation could
have a regulatory function in vivo and perhaps explain the rapid modulation
of L-GalLDH activity by light and during programmed cell death (Valenti
et al., 2007). While it is likely that L-GalLDH activity is present in excess
compared to flux through the ascorbate biosynthesis pathway, the ability to
inactivate it could provide a mechanism to switch off ascorbate biosynthesis
rapidly. There is evidence from inhibition of electron transport out of PSII by
DCMU and DBMIB that L-GalLDH activity could be controlled by signals
related to photosynthetic electron transport (PET) (Yabuta et al., 2007). As
DCMU and DBMIB should have opposite effects on the redox state of
plastoquinone (Pfannschmidt et al., 1999), the results suggest that some
other aspect of PET gives rise to the putative signal.
III. ARE THERE MULTIPLE PATHWAYS FOR

ASCORBATE BIOSYNTHESIS?
The evidence for the Man/L-Gal pathway described in the Section II is very
strong and indeed suggests it to be the only pathway able to support survival
of Arabidopsis seedlings. It is well established that mammals synthesise
ascorbate from D-GlcUA (Smirnoff, 2001) while some photosynthetic pro-
tists such as Euglena may use D-galacturonic acid (D-GalUA) (Ishikawa et al.,
2006b; Smirnoff et al., 2001). It has, however, been proposed that plants can
also synthesise ascorbate from myo-inositol/D-GlcUA, L-gulose or D-GalUA
(Fig. 3A) as well as via Man/L-Gal. In order to interpret the evidence for
VITAMIN C 125
these pathways, a key point to understand is that although external applica-

tion of uronic acid derivatives often increases ascorbate and label from
labelled uronic acids is incorporated into ascorbate, the labelling pattern is
not compatible with such pathways being quantitatively important (Fig. 3B)
(Loewus, 1999).
A. ASCORBATE BIOSYNTHESIS FROM D-GALUA
Exogenously supplied methyl ester of D-GalUA increases ascorbate concen-

tration in various tissues, including Arabidopsis cell cultures (Davey et al.,
1999; Loewus and Kelly, 1961). Free D-GalUA and D-GalUA methyl ester
are likely to be produced the breakdown of pectin and could therefore
provide a substrate for ascorbate synthesis. A gene whose expression corre-
lates with the increase in ascorbate during fruit ripening was cloned and the
recombinant enzyme shown to have NADPH-dependent D-GalUA reductase
activity (Agius et al., 2003). Its role in ascorbate biosynthesis was confirmed
by overexpression in Arabidopsis, which resulted in a several-fold increase in
foliar ascorbate. Presumably, the predicted L-galactonic acid product is
lactonised to the ascorbate precursor L-GalL. While it is clear that both
Fig. 3. (A) Possible ascorbate precursors in plants. Exogenous supply of the

compounds in boxes either increases ascorbate pool size (Davey et al., 1999) or the
radiolabelled compound is incorporated into ascorbate (Loewus, 1999). The intensity
of the grey shading indicates the relative efficiency of incorporation, which will be
affected both by pathway capacity and by ease of transport across the plasma
membrane. Reactions for which there is evidence for the involvement of specific
enzymes or genes are indicated by solid arrows, while other reactions are shown by
dotted arrows. Enzymes: 1, polygalacturonase; 2, methyl esterase; 3, D-galacturonic
acid reductase; 4, L-galactonolactone dehydrogenase; 5, L-gulonolactone oxidase/
dehydrogenase; 6, myo-inositol oxygenase; 7, L-galactose dehydrogenase; 8, GDP-
mannose-3,5-epimerase. (B) The labelling pattern of L-galactonolactone/ascorbate
from specifically labelled glucose depends on the prevailing pathway of ascorbate
biosynthesis. The experimental evidence shows that carbon atom 1 (C1) of glucose
(indicated by grey circles) becomes C1 of L-galactonolactone and ascorbate (Loewus,
1999). This labelling pattern is not compatible with uronic acids (e.g. galacturonic or
glucuronic acids) acting as intermediates but is compatible with ascorbate synthesis
via oxidation of L-galactose at C1. Nevertheless, incorporation of the uronic acid
precursors shown in (A), along with uncertainty introduced by label randomisation
via the triose phosphate pool and the increase in ascorbate caused by D-galacturonic
acid reductase overexpression (Agius et al., 2003), indicates that a small proportion of
ascorbate could be derived from uronic acids. It will not be possible to assess the
contribution of uronic acids until mutations specifically affecting these pathways are
identified. Straight chain structures, rather than the predominant ring structures, are
shown for simplicity. Carbon skeleton numbering follows the IUPAC rules for
carbohydrate nomenclature (Pure and Applied Chemistry, 1996, 68, 1919–2008) in
which the carbonyl group of aldose sugars and uronic acids are designated as C1,
while the carboxylic acid group of aldonic acids is C1.
feeding D-GalUA methyl ester and introducing D-GalUA reductase result in

increased ascorbate, there is currently no evidence for operation of the
pathway under normal conditions. Labelling studies strongly suggest that
ascorbate synthesis in a variety of tissues is unlikely to use uronic acid
intermediates (Loewus, 1999). Given the inconclusive evidence, further in-
vestigation is warranted. The critical experiment will be to identify D-GalUA
reductase homologues in Arabidopsis and then determine if ascorbate bio-
synthesis is affected if these genes are mutated. Arabidopsis has at least eight
proteins that are more than 40% identical to the strawberry D-GalUA reduc-
tase and that do not have identified functions. Unfortunately, this suggests
that functional redundancy could complicate genetic analysis. D-GalUA
methyl ester could be readily available in ripening fruit due to cell wall
breakdown. Labelling evidence shows that ascorbate is synthesised by the
Man/L-Gal pathway in blackcurrant (Hancock et al., 2007) and grape
(Melino et al., 2009a,b). However, the data cannot rule out a contribution
from D-GalUA. Interestingly, a second phase of ascorbate accumulation
during grape berry ripening corresponded with increased expression of a
gene homologous to strawberry D-GalUA reductase (Cruz-Rus et al., 2010;
Melino et al., 2009a,b). In blackcurrant fruit, D-GalUA had little effect on
ascorbate pool size (Hancock et al., 2007). An introgression line between
Solanum lycopersicum and Solanum pennellii (higher fruit ascorbate) retained
high ascorbate compared to the S. lycopersicum parent and also had
increased transcript levels of polygalacturonase and pectinesterase genes,
while Man/L-Gal pathway genes did not change (Di Matteo et al., 2010).
Surprisingly, the authors proposed this as evidence for ascorbate synthesis
from D-GalUA being the predominant pathway. Whatever, the predominant
pathway, gene expression does not provide the appropriate evidence. Indeed,
it is likely that molecular genetics would even reject the operation of glycolysis
or the Krebs cycle, as expression of their genes often do not follow pathway
flux. Both labelling and enzyme activity support the possibility that a number
of photosynthetic protists use the D-GalUA pathway (Grun and Loewus,
1984; Helsper et al., 1982; Ishikawa et al., 2006b; Shigeoka et al., 1979).
B. ASCORBATE BIOSYNTHESIS FROM MYO-INOSITOL AND D-GLCUA
Animals synthesise ascorbate from UDP-D-glucuronic acid via L-gulonolac-

tone (Smirnoff, 2001). Various lines of evidence suggest that the same path-
way could operate in plants (Fig. 3A). Exogenous D-glucuronic acid methyl
ester (which presumably is converted to D-glucuronic acid or D-glucurono-
lactone) and L-GulL increase the ascorbate content of Arabidopsis cell
cultures but a lot less effectively than methyl galacturonate (Davey et al.,
VITAMIN C 127
1999; Finkle et al., 1960). Apart from the recent identification of L-GulL
oxidases in Arabidopsis, the enzymes or genes needed to reduce D-glucuronic
acid or D-glucuronolactone to L-GulL have not been identified. In Arabidop-
sis, it could be one of the potential D-GalUA reductase homologues. myo-
Inositol is a potential source of D-glucuronic acid via the enzyme myo-inositol
oxygenase (MIOX). Overexpression of Miox4 (At4g26260) in Arabidopsis
was reported to increase foliar ascorbate (Lorence et al., 2004). However,
more recently, re-examination of the same transgenic plants found no change
in ascorbate but the expected decrease in myo-inositol (Endres and Tenhaken,
2009). The reason for this contradiction needs to be resolved, and at this
point, the role of myo-inositol in ascorbate biosynthesis is an open question.
C. ASCORBATE BIOSYNTHESIS FROM L-GulL VIA GDP-MANNOSE
The production of GDP-L-gulose by GME could provide substrate for a

pathway that is analogous to the L-Gal pathway (Fig. 3A; Wolucka and Van
Montagu, 2003). However, with the exception of some putative GulLO
enzymes (Maruta et al., 2010a), analogous enzymes have not been identified,
while GDP-L-galactose phosphorylase and L-GalDH have low affinity for the
L-gulose substrates (Gatzek et al., 2002; Linster et al., 2008). Interestingly,
overexpression of rat GulLO in Arabidopsis vtc mutants increases ascorbate
(Radzio et al., 2003), but the results are difficult to interpret because the
enzyme can use both L-GulL and L-GalL as substrate.
The evidence above suggests that plants have a higher potential to utilise
D-GalUA than D-glucuronic acid, and this can be boosted by overexpressing
D-galacturonate reductase. Labelling patterns show that the pathways are
minor but until genes encoding the proposed enzyme activities are identified
and knocked out, definitive evidence is lacking. Overexpression is clearly not
definitive evidence because pathways that are insignificant in wild-type plants
could be introduced or boosted. It is also important to back up molecular
genetic studies with metabolic analysis, which is often not done.
IV. THE CONTROL OF ASCORBATE BIOSYNTHESIS

AND PATHWAY ENGINEERING
Ascorbate concentration varies between tissues. Roots and other non-photo-

synthetic tissues tend to contain less ascorbate than leaves. For example, the
peel of apple has much higher ascorbate concentration than the flesh (Davey
et al., 2004; Li et al., 2008). Ascorbate concentration in fruits varies during
the ripening process and in tomato and apple is increased by high light
(Gautier et al., 2009; Li et al., 2009). Leaf ascorbate decreases during senes-
cence and usually increases on exposure to increased light intensity (Dowdle
et al., 2007; Smirnoff, 2000a) and low temperature (Schoner and Krause,
1990). There is a suggestion that ascorbate concentration is higher in meri-
stem cells (Cordoba-Pedregosa et al., 2003) and low in the quiescent centre
(QC) of the maize root meristem (Kerk and Feldman, 1995). Mature seeds
have little ascorbate, and after imbibition, it accumulates prior to germina-
tion (Arrigoni et al., 1992; Pallanca and Smirnoff, 2000). These observations
suggest that ascorbate concentration is regulated at a level appropriate to cell
type and environmental conditions. However, as will be seen from the
discussion below, we know very little about how the biosynthesis or break-
down is controlled.
The rate of biosynthesis depends on the amount of each enzyme and
kinetic properties in relation to substrate concentrations and other factors.
In many pathways of primary metabolism, the control of flux is shared
between enzymes, although strategically placed enzymes (e.g. if they are
irreversible or at branch points) may have complex regulatory behaviour
that is dependent on post-translational modification. In contrast, pathways
of secondary metabolism are often strongly controlled at the transcriptional
level. An example is the induction of anthocyanin synthesis by high light or
ABA in which transcripts of almost all biosynthesis genes increase under the
control of transcription factors (Vanderauwera et al., 2005). The role of
transcriptional regulation in ascorbate biosynthesis is unclear. A wide
range of studies have compared the transcript levels of various ascorbate
biosynthesis genes with ascorbate concentration. They have found various
degrees of correspondence between expression of one gene or another with
ascorbate pool size. Investigation of mutants has uncovered the genes
involved but has not been detailed enough to indicate the level of control.
Overexpressing biosynthesis genes has resulted in increased ascorbate in
some (PMM, GME, VTC1, VTC2, L-GalLDH) but usually not for others
(L-GalDH, L-GalLDH, PMI) (see Section II for references). In the most
illuminating example, transient expression of GME and VTC2 together in
tobacco leaves caused a bigger increase in ascorbate than each gene singly
(Bulley et al., 2009).
The very reproducible increase in ascorbate that occurs in Arabidopsis
leaves when plants are transferred from low light to high light and the rapid
decrease when the dark period is prolonged (Dowdle et al., 2007; Toledo
et al., 2003) provides a useful system to investigate ascorbate metabolism.
The only published attempt to measure enzyme activity, rather than tran-
script levels, of all the Man/L-Gal pathway genes compared plants acclima-
tising to low and moderate light intensity (Dowdle et al., 2007). This showed
VITAMIN C 129
that only GDP-L-galactose phosphorylase increased in activity ( 20-fold)

and L-GalLDH increased about twofold. L-GalLDH transcripts are not
highly light responsive in Arabidopsis, but there is evidence that its catalytic
activity rapidly increases in high light (Bartoli et al., 2006; Smirnoff, 2000a).
The recently discovered S-glutathionylation of GalLDH (Leferink et al.,
2009a,b,c) could provide a mechanism for rapid activation and inactivation.
However, VTC2 gene expression is strongly increased by light and decreases
on transfer to the dark (Dowdle et al., 2007; Muller-Moulé, 2008). Analysis
of Affymetrix transcriptome data shows that VTC2 and GME transcript
levels are most likely controlled by the circadian clock and GDP-L-galactose
phosphorylase activity peaks later in the day than the dawn peak in tran-
script VTC2 levels (Dowdle et al., 2007). In rice, high light increases GMP
transcripts and dark decreases L-GalLDH transcripts (Fukunaga et al.,
2010). Transient expression of promoter::luciferase constructs in Arabidop-
sis identified potential light response elements in the promoters. In relation to
light, results to date support roles for VTC2 transcription and light-induced
activation of L-GalLDH as control points of ascorbate biosynthesis. More
quantitative studies of pathway enzyme activity are needed. Presumably,
such studies are rare because the assays are difficult to perform and require
expensive substrates. Given the strong evidence that VTC2 transcription is
important in controlling ascorbate biosynthesis, promoter::luciferase fusions
of both genes have been constructed and expressed in Arabidopsis to visual-
ise changes in expression (T. Ishikawa, S. Shigeoka, M. Page and N. Smirn-
off, unpublished data). Preliminary analysis of these plants shows rapid
modulation of VTC2 gene expression by light–dark transitions. Pathway
control at the GDP-L-galactose phosphorylase step can be rationalised by
this being the first step in the pathway dedicated to ascorbate biosynthesis.
The importance of the reported nuclear localisation of VTC2 and the occur-
rence of post-translational modification remain to be determined.
A potentially powerful way to identify genes that control ascorbate accu-
mulation is to use natural variation and quantitative trait loci (QTL) analy-
sis. This has been used for tomato (Rousseaux et al., 2005; Stevens et al.,
2007; Zou et al., 2006), broccoli (V. Buchanan-Wollaston, personal commu-
nication) and Arabidopsis (M. Bennett, personal communication). In toma-
to, QTLs for ascorbate concentration were mapped to GME and MDHAR
(Rousseaux et al., 2005). AMR1 (Ascorbic acid Mannose pathway Regulator
1; At1g65770) is a predicted F-Box protein that is proposed to negatively
regulate ascorbate pool size that has been identified by activation tagging
(Zhang et al., 2009). Activation-tagged lines have decreased ascorbate, while
T-DNA insertion mutants (amr1-1 and amr1-2) have increased ascorbate.
AMR1 expression increases as leaves age and decreases in high light
conditions that decrease and increase ascorbate pool size, respectively. Acti-
vation-tagged and KO mutants had decreased and increased expression of all
Man/L-Gal pathway genes (except VTC5), respectively, the biggest effect
being on GME. These results suggest that AMR1 could regulate expression
of Man/L-Gal pathway genes, particularly in relation to light and leaf age
through proteasome-mediated degradation of a transcription factor. How-
ever, as the effect of mutation on other genes, particularly light- and senes-
cence-associated genes, is not known, it is not clear if the effect is specific or
indirect.
Jasmonates and wounding both impact ascorbate metabolism. Methyl
jasmonate (MeJA) treatment increases ascorbate content and its synthesis
from 14C-labelled mannose in Arabidopsis cell cultures (Wolucka et al.,
2005), and jasmonic acid (JA) and MeJA increase ascorbate in Arabidopsis
leaves (Sasaki-Sekimoto et al., 2005; Suza et al., 2010). A review of the
response of ascorbate to jasmonate shows that it usually increases ascorbate
in a range of species and tissues; however, ascorbate decreases in MeJA-
treated tomato leaves (Suza et al., 2010). A number of ascorbate-related
genes also respond to jasmonates including VTC1, VTC2, VTC5 in Arabi-
dopsis and GME and a possible GulLOX in tobacco leaves (Sasaki-
Sekimoto et al., 2005; Suza et al., 2010). Response to mechanical wounding
involves jasmonate signalling (Koo and Howe, 2009). Wounding Arabidop-
sis caused a small increase in ascorbate and a decrease in tomato (Suza et al.,
2010). This follows the same pattern as the jasmonate response, but interest-
ingly, neither response was abolished in JA mutants.
Engineering ascorbate biosynthesis has been reviewed recently (Ishikawa
et al., 2006a). Of the Man/L-Gal pathway enzymes, the biggest increases in
ascorbate have been produced by transient overexpression of GME and
GDP-L-Gal phosphorylase together (Bulley et al., 2009). The effects of over-
expressing the other enzymes are small and variable (Section II). It has
proved possible to increase ascorbate by overexpressing enzymes associated
with the uronic acid pathways. Examples are strawberry D-GalUA reductase
(Agius et al., 2003), rat L-GulL oxidase (Jain and Nessler, 2000; Radzio et al.,
2003), a purple acid phosphatase (which could also operate in the Man/L-Gal
pathway) (Zhang et al., 2008) and, with positive and negative results from
two different studies on the same plants, MIOX (Endres and Tenhaken,
2009; Lorence et al., 2004). The rationale for the success, or otherwise, of
these manipulations is discussed in Section III. An alternative engineering
approach is to increase the stability of ascorbate by boosting regeneration
capacity from MDHA and DHA by overexpressing MDHAR and DHAR.
This approach can increase ascorbate pool size by up to twofold (see Sec-
tions VIII.C and VIII.D).
VITAMIN C 131
While engineering plant ascorbate may have benefits for stress resistance
or post-harvest longevity, the benefit of improving nutritional value of crops
is probably marginal. However, engineering the plant pathway into microbes
for a one-step ascorbate manufacturing process could improve on current
industrial processes (Hancock and Viola, 2001, 2002; Running et al., 2004).
Expression of the plant Man/L-Gal enzymes in S. cerevisiae, along with
MDHAR to improve recycling, has introduced ascorbate synthesis and
accumulation into this fungus (Branduardi et al., 2007; Fossati et al., 2011;
Sauer et al., 2004).
V. ASCORBATE CATABOLISM
Ascorbate and DHA are catabolised in plants and give rise to a number of
end products, including L-threonate, oxalate and L-tartrate. Labelling studies
by Loewus and colleagues provided the first information on pathways of
ascorbate catabolism (Loewus, 1999). In plants that produce oxalate from
ascorbate, the carbon skeleton is cleaved between C2 and C3. This cleavage
gives rise to oxalate (from C1 and C2) and L-threonate (Fig. 4A). Ascorbate
appears to be the precursor of oxalate in a number of species (Horner et al.,
2000; Yang and Loewus, 1975). Microautoradiography of calcium oxalate
crystals forming in crystal idioblast cells (specialised oxalate-forming cells) of
Pistia stratiotes shows that the oxalate crystals are labelled by 1-14C-ascor-
bate and 1-14C-L-Gal. Oxalate is labelled by 1-14C-ascorbate in tomato, water
hyacinth, winged bean and water lily (Keates et al., 2000; Kostman et al.,
2001, 2007; Kostman and Koscher, 2003). Evidence from the isolation of
oxalate crystal-deficient mutants in Medicago truncatula suggests that ascor-
bate is an oxalate precursor in this species: the mutants contain less ascor-
bate, while ascorbate feeding increases production of oxalate (Nakata and
McConn, 2007a). A pathway for the formation of oxalate and threonate
from ascorbate in the apoplast of cultured rose cells has been proposed
(Green and Fry, 2005) (Fig. 4A). This involves a novel intermediate 4-O-
oxaly-L-threonate that is formed by a series of oxidations, reductions and
intramolecular rearrangement of DHA. Hydrolysis of 4-O-oxaly-L-threonate
gives rise to oxalate and L-threonate, and this reaction is catalysed by an
esterase activity or can occur non-enzymatically. A number of the reactions
in this pathway can potentially generate hydrogen peroxide (Green and Fry,
2005). It is currently not clear if 4-O-oxaly-L-threonate is also an intermediate
in the intracellular formation of calcium oxalate crystals (Keates et al., 2000;
Kostman et al., 2001, 2007). Some plants synthesise oxalate from glyoxylate
rather than ascorbate (Franceschi and Nakata, 2005). An example is rice (Xu
et al., 2006; Yu et al., 2010), where down-regulation of L-GalLDH decreased
ascorbate but not oxalate. However, exogenous ascorbate or L-GalL cause a
modest increase in oxalate in rice (Guo et al., 2005), suggesting that there
could be a limited capacity for oxalate production from ascorbate.
M. truncatula has two types of calcium oxalate crystals (raphide and
druse). Evidence from the oxalate-deficient mutants suggests that ascorbate
is the precursor for druse crystals but possibly not the raphides (Nakata and
McConn, 2007b).
Tartrate is of more limited occurrence in plants but is a determinant of
wine quality. The pathway of tartrate synthesis varies between species. In
grapes and other members of the Vitaceae, the carbon skeleton is cleaved
between C4 and C5. In Pelargonium (Geraniaceae), labelling evidence
Fig. 4. (Continued)
VITAMIN C 133
Fig. 4. Pathways of ascorbate catabolism. (A) Ascorbate breakdown in the apo-

plast via 4-O-oxalyl-L-threonic acid (Green and Fry, 2005) using a combination of
enzymatic and non-enzymatic steps. The esterase producing L-threonic acid has not
been characterised. (B) L-Tartrate synthesis by carbon skeleton cleavage between C4
and C5. Other than L-idonate dehydrogenase in grape (Vitis vinifera), the enzymes
involved have not been identified (DeBolt et al., 2006).
suggests tartrate is produced by a C2/C3 cleavage presumably giving rise to

oxalate and threonate, the latter being oxidised to tartrate (Loewus et al.,
1975; Wagner and Loewus, 1974). Until recently, there were no details about
the reactions and the enzymes that are involved in tartrate synthesis.
By searching for candidate grape (Vitis vinifera) genes that might catalyse
reactions appropriate to the proposed C4/C5 cleavage pathway and then
comparing their expression levels in tissues varying in tartaric acid content, a
candidate dehydrogenase gene was identified (DeBolt et al., 2006, 2007). The
gene could not be detected in Ampelopsis aconitifolia, a member of the
Vitaceae lacking tartrate. The recombinant protein has L-idonate dehydro-
genase (IDH) activity, forming 5-keto-D-gulonate (Fig. 4B). Given that grape
can convert exogenous L-idonate and 5-keto-D-gulonate to tartrate, this
enzyme is very likely to be involved. The other enzymes in the pathway
remain to be identified. Candidates for the last step, which requires oxidation
of an aldehyde to carboxylic acid, could be a hydrogen peroxide producing
aldehyde oxidase or a monooxygenase.
VI. ASCORBATE TRANSPORT AND

SUBCELLULAR COMPARTMENTATION
Ascorbate occurs in the phloem of all the species that have been investigated.
For example, it can be detected in phloem exudate collected from aphid
stylets and from the exudates collected from cucurbit fruits (Franceschi and
Tarlyn, 2002; Hancock et al., 2004). Interestingly, isolated vascular strands
of celery were able to synthesise ascorbate from labelled mannose and L-
galactose. Correspondingly, enzymes of the Man/L-Gal pathway with the
exception of L-GalLDH could be detected in phloem exudate (Hancock et al.,
2004). Feeding labelled ascorbate or its precursors shows that they accumu-
late in the vascular tissue of Arabidopsis, Medicago sativa and N. benthami-
ana (Franceschi and Tarlyn, 2002; Hancock et al., 2004). Further, labelled
ascorbate supplied to source leaves in Arabidopsis and M. sativa resulted in
appearance of label (still largely in ascorbate) in sink tissues such as buds,
root tips and developing seeds (Franceschi and Tarlyn, 2002). These results
clearly show that ascorbate is translocated in the phloem from source leaves
to carbohydrate sinks. In the case of apoplastic phloem loaders, there must
be transporters to load and unload ascorbate across the membranes. The
occurrence of 6-O-glucosyl-L-ascorbate in the symplastically loading cucur-
bits suggests that this could facilitate loading (Hancock et al., 2008). In the
case of apoplastic loading, ascorbate would be trapped due to ionisation at
the high phloem pH and could be stabilised through the high expression of
glutaredoxins and thioredoxins in the phloem. Phloem transport of ascorbate
could be significant in relation to the diet of phloem feeding insects such as
aphids.
VITAMIN C 135
Ascorbate seems to occur in all subcellular compartments including chlor-

oplasts (Anderson et al., 1983; Beck et al., 1983; Foyer and Lelandais, 1996;
Rautenkranz et al., 1994), mitochondria (Jimenez et al., 1997), peroxisomes
(Jimenez et al., 1997) and vacuoles (Rautenkranz et al., 1994). Ascorbate can
also be detected in apoplastic fluid where it is tends to have a much lower
concentration (< 1 mM). A significant proportion of the apoplastic pool is
DHA (Kollist et al., 2001; Pignocchi et al., 2006; Sanmartin et al., 2003) while
intracellular ascorbate in healthy tissue tends to be > 80% reduced. Recently,
immunogold localisation of ascorbate has been attempted in Arabidopsis
and tobacco leaves using an antibody raised against ascorbate coupled to
bovine serum albumin (Zechmann et al., 2010). Comparison of wild-type
Arabidopsis with ascorbate-deficient vtc1 and vtc2 mutants gave the expected
decrease in the density of gold particles detected with transmission electron
microscopy. The distribution of gold particles in different subcellular com-
partments and the total leaf concentration were used to estimate ascorbate
concentrations in various compartments (Table I). The highest concentra-
tions were in cytosol and peroxisomes and exposure to high light increased
ascorbate in cytosol, chloroplasts and vacuoles. Estimation of ascorbate in
various subcellular compartments from barley leaves, using non-aqueous
fractionation of organelles, shows a remarkably similar distribution and
response to high light in barley leaves (Table I). Reduction of acidic silver
nitrate at low temperature has been used as a histochemical detection method
for ascorbate and detects ascorbate in cell wall and cytosol but not in the
vacuoles of Cucurbita root cells (Liso et al., 2004). In summary, ascorbate
seems to be most highly concentrated in the cytosol (and perhaps peroxi-
somes), but it increases in chloroplasts in response to high light (including
appearance of gold particles in the thylakoid lumen), while the vacuole has a
low concentration and shows the biggest proportionate increase in high light.
One interesting feature of the immunogold detection of ascorbate was the
lack of gold particles in the cell wall—except in the cell walls and lumen of the
xylem vessels. Given that ascorbate is readily detected in apoplastic fluid, its
concentration may be below the detection limit of the immunogold method.
Alternatively, the methods for detecting apoplastic ascorbate need to be
critically reassessed.
Considering that ascorbate is produced by the mitochondria at complex 1,
it is very likely that carriers will be needed to transport it (or DHA) into other
organelles and in and out of the apoplast. Intracellular ascorbate will be
largely ionised (pKa ¼ 4.1; Buettner and Schafer, 2004), while a significant
proportion will be present as ascorbic acid in vacuoles and apoplast. Ascor-
bate and DHA transport have been reviewed by Horemans et al. (2000a,b);
so the results are summarised here, along with more recent information.
Ascorbate uptake by isolated chloroplasts is carrier facilitated with Km

values of 18–45 mM (Anderson et al., 1983; Beck et al., 1983; Foyer and
Lelandais, 1996). Uptake is increased by pre-loading with DHA and com-
petitively inhibited by DHA (Beck et al., 1983), suggesting that either an
exchange mechanism operates and/or that DHA is taken up was well as
ascorbate. Uptake across the thylakoid membranes is not carrier mediated
(Foyer and Lelandais, 1996). Ascorbate and DHA uptake is carrier mediated
in mitochondria (Km 36 and 6 mM, respectively) and faster for DHA (Szarka
et al., 2004). DHA uptake was decreased by glucose and genistein, suggesting
that it may share glucose uptake carriers, as occurs in mammals. Uptake of
DHA into isolated vacuoles is much faster than that of ascorbate, and uptake
kinetics of both are non-saturable indicating lack of carrier mediation
(Rautenkranz et al., 1994). There is no information about ascorbate
or DHA transport into peroxisomes. Uptake studies of ascorbate are ham-
pered by the rapid oxidation of ascorbate in the apoplast. When care is
taken to control oxidation of ascorbate, it appears that plant cells only
take up DHA, and this process is not mediated by glucose transporters
(Horemans et al., 2008). In Arabidopsis cell cultures, the Km is 40 M and
Vmax 99 mol min 1 g 1 fresh wt. The affinity is therefore much higher than
for intracellular ascorbate/DHA transporters and matches the concentration
of DHA measured in apoplastic fluid. Previous work with plasma membrane
vesicles had shown that DHA uptake is stimulated by pre-loading with
ascorbate (Horemans et al., 1998). Overall, it appears that there is a high-
affinity DHA uptake mechanism that is most likely coupled with ascorbate
efflux to the apoplast. Oxidative stresses, for example, ozone or hydrogen
peroxide (Luwe et al., 1993; Parsons and Fry, 2010), oxidise ascorbate to
DHA, giving rise to export of ascorbate as a result of DHA uptake. Pulsing
of ascorbate efflux from hydrogen peroxide-treated rose and Arabidopsis cell
cultures has been observed (Parsons and Fry, 2010). The wider occurrence
and significance of this response require further investigation. While ascor-
bate and DHA membrane transport must be important for distribution
between organelles, maintenance of the apoplastic ascorbate/DHA pool
and for phloem translocation, no transporter proteins or the genes encoding
them have been identified. In mammals, by contrast, high-affinity ascorbate
transporters are characterised (Wilson, 2005) and DHA uptake occurs
through the GLUT family of glucose carriers (Wilson, 2004). Recently, a
new family of sugar efflux transporters (SWEETs), conserved in animals and
plants, has been identified (Chen et al., 2010) Possibly, some of the 17
SWEET genes in Arabidopsis are DHA transporters. Identification of ascor-
bate/DHA transport mutants is likely to provide new insights into the func-
tions of ascorbate.
VITAMIN C 137
VII. ASCORBATE CONJUGATES
Ascorbate can form esters: for example ascorbate 2-sulphate occurs in brine
shrimp larvae (Bond et al., 1972). Substitutions on C2 stabilise ascorbate
against oxidation and are used (e.g. palmitoyl ascorbate) to supply ascorbate
in fish food. Ascorbate 2-sulphate and other organic acid esters have not been
reported in plants. However, glycosides have been described. Lycium bar-
barum fruit contains 2-O-glucosyl-L-ascorbate (Toyada-Ono et al., 2005),
and phloem sap from several species of Cucurbitaceae (e.g. Cucurbita and
Cucumis species) contains 6-O-glucosyl-L-ascorbate (Hancock et al., 2008).
In zucchini (Cucurbita pepo), there are approximately equal quantities of
ascorbate and its glucoside. As ascorbate is phloem translocated, Hancock
et al. speculate that the presence of 6-O-glucosyl-L-ascorbate in the phloem of
Cucurbitaceae could be related to their symplastic phloem-loading mecha-
nism. In contrast to apoplastic phloem loaders, symplastic phloem loaders
drive sugar uptake into phloem sap by using a polymer trap mechanism in
which sucrose is converted to raffinose series sugars. There is currently no
evidence for this proposal, and the occurrence of 6-O-glucosyl-L-ascorbate in
the phloem of a wide range of symplastic and apoplastic phloem loaders
needs to be investigated. Enzymes involved in the synthesis or hydrolysis of
glucosides of ascorbate have not been identified. More recently, attention has
been drawn to a wide range (33) of ascorbylated compounds that have been
identified in plant extracts (Kesinger and Stevens, 2009). These are mostly
formed in reactions where ascorbate acts as a nucleophile or DHA as an
electrophile. Ascorbigens are produced from indole glucosinolates (Wagner
and Rimbach, 2009). Some of these compounds have reported therapeutic
potential. Kesinger and Stevens note that a much larger number of ascorby-
lated compounds are likely to exist. The physiological roles of these com-
pounds and the extent to which they exist in vivo, or form during tissue
extraction, are unknown.
VIII. THE REDOX REACTIONS OF ASCORBATE
Ascorbate is an effective free radical scavenger (donor antioxidant) because

of two key properties. Firstly, it readily donates either one or two electrons or
hydrogen atoms to an oxidising species or free radical. Secondly, this func-
tion is aided by the relative stability of the resulting MDHA radical and the
existence of enzymes that reduce MDHA and DHA back to ascorbate
(Buettner, 1993; Buettner and Schafer, 2004). MDHA never reaches high
concentrations but can be detected in vivo by electron paramagnetic
resonance (EPR), particularly following oxidative stress. The redox reactions

of ascorbate are shown in Fig. 1A. MDHA disproportionates produce DHA
and ascorbate, or it can be reduced by pyridine nucleotide-dependent
MDHAR. DHA is rapidly reduced to ascorbate by thiols. This reaction is
catalysed by glutathione (GSH)-dependent dehydroascorbate reductases.
The resulting oxidised GSH (glutathione disulphide) is regenerated to GSH
by NADPH-dependent GSH reductase. The cycle of reactions in which
ascorbate is regenerated at the expense of NADPH is known as the
ascorbate–GSH cycle or the Foyer–Halliwell–Asada cycle (Foyer and
Noctor, 2011; Noctor and Foyer, 1998).
Some of the biologically relevant reactions of ascorbate are with tocopher-
oxyl, carotenoid, peroxyl and thilyl radicals, hydrogen peroxide, singlet
oxygen, ozone, Cu2þ and Fe3þ (Buettner, 1993; Buettner and Schafer,
2004). The reaction of ascorbate with singlet oxygen may be of interest
because the product is hydrogen peroxide (Fig. 1B). Therefore, ascorbate
could convert a very short-lived reactive oxygen species to a longer lived and
mobile species (Kramarenko et al., 2006). However, the main source of
singlet oxygen in plants is PSII (see later), and it is not clear if this would
be available to ascorbate which is most likely too hydrophilic to access the
site of production. Infiltration of leaves with a variety of photosensitising
agents that act in the chloroplast (e.g. aminolaevulinic acid, acifluorfen and
monuron) causes light-dependent loss of ascorbate, providing evidence that
ascorbate could react with singlet oxygen or its products in vivo (Gullner and
Dodge, 2000). The reaction of ascorbate with ozone is apparently pH depen-
dent. Above pH 5, the predominant reaction is the production of singlet
oxygen and DHA (Enami et al., 2008; Kanofsky and Sima, 1995). Ascorbate
reacts with the resulting singlet oxygen producing hydrogen peroxide and
DHA (Kramarenko et al., 2006) (Fig. 1B). Therefore, the interaction of
ascorbate and ozone gives rise to hydrogen peroxide. Ascorbic acid also
reacts with ozone below pH 5 with the production of ozonated compounds
(Fig. 1C), one of which then decays to threonic acid and a C2 fragment
(Enami et al., 2008). This is somewhat analogous to the oxidative mechanism
for threonate and oxalate production proposed by Green and Fry (2005) that
occurs at relatively low pH in the apoplast (Fig. 3A). Given that singlet
oxygen is produced by ozone-treated Sedum album leaves (Kanofsky and
Sima, 1995), this reaction may predominate over the production of ozonated
ascorbate derivatives. However, it is possible that some of the discrepancies
concerning the role of apoplastic ascorbate in protecting against ozone are
related to production of reactive ascorbate derivatives (Sandermann, 2008).
Ferric ions and, particularly, cupric ions are very readily reduced by ascor-
bate. Hydrogen peroxide is also produced in the presence of oxygen during
VITAMIN C 139
metal ion reduction (Dekker and Dickinson, 1940; Silverblatt et al., 1943)
which can subsequently react with the Cuþ or Fe2þ to produce highly
reactive hydroxyl radicals in the Fenton reaction. These reactions are the
basis of the well-publicised pro-oxidant activity of ascorbate. A key to
antioxidant defence is to ensure that redox active metals are not accessible.
Interestingly, cancer cells may be particularly sensitive to the pro-oxidant
effect of ascorbate (Chen et al., 2005), and a possible role in cell expansion is
discussed below.
Plants contain two enzymes that catalyse ascorbate oxidation: APX, which
has a well-characterised role in scavenging or controlling hydrogen peroxide
concentration, and ascorbate oxidase (AO), an enzyme that catalyses oxida-
tion of ascorbate by oxygen with the production of water. The physiological
role of this enzyme is obscure.
A. APX—AN ENZYME THAT DOES EXACTLY WHAT IT SAYS ON THE TIN
APXs are members of the class 1 family of heme peroxidases that catalyse the
reduction of hydrogen peroxide to water with concomitant oxidation of
ascorbate to MDHA (Asada, 1992; Ishikawa and Shigeoka, 2008; Shigeoka
et al., 2002). APX is present in green plants, red algae, Euglena and other
photosynthetic protists and trypanosomes (Ishikawa and Shigeoka, 2008;
Pitsch et al., 2010; Wilkinson et al., 2002). Plants contain multiple APX genes
which encode enzymes that are targeted to the cytosol, chloroplasts and
peroxisomes/glyoxysomes. Within the chloroplast, there is a soluble stromal
ascorbate peroxidase (sAPX) and a thylakoid ascorbate peroxidase (tAPX)
that is anchored to the thylakoid membrane near PSI (Ishikawa and
Shigeoka, 2008). In some species (e.g. Arabidopsis), sAPX and tAPX are
encoded by separate genes; in others, they are generated by alternative slicing
(Ishikawa and Shigeoka, 2008). APX also occurs in mitochondria and per-
oxisomes (Chew et al., 2003; Ishikawa and Shigeoka, 2008; Jimenez et al.,
1997). The properties, including the sensitivity of sAPX to inactivation by
hydrogen peroxide, and reaction mechanism of APX have been well studied
and will not be reviewed here (Asada, 1992; Ishikawa and Shigeoka, 2008).
Instead, the focus will be on the use of mutants and overexpression to probe
the role of APX.
Arabidopsis has nine APX genes of which APX1, APX2, sAPX and tAPX
have been studied in most detail. sAPX is also targeted to the mitochondrial
inter-membrane space (Chew et al., 2003). APX1 (At1g07890) encodes a
cytosolic APX whose expression is induced by high light and various oxida-
tive stresses (Asai et al., 2002, 2004; Davletova et al., 2005; Fourcroy et al.,
2004; Pnueli et al., 2003). The Zat12 transcription factor controls APX1
expression in response to hydrogen peroxide, paraquat, wounding and heat
shock (Rizhsky et al., 2004). A T-DNA KO mutant lacking APX1 expression
has been used to explore its physiological role (Davletova et al., 2005;
Koussevitzky et al., 2008; Pnueli et al., 2003). The mutant plants are smaller
than wild type and flower later but have also been reported to be unaffected
in development (Asai et al., 2004). The apx1 mutant has decreased photo-
synthesis rate and shows increased hydrogen peroxide and protein oxidation
when exposed to high light, along with decreased levels of Rubisco small
subunit and cytochrome f (Davletova et al., 2005). The results are consistent
with a role for cytosolic APX in providing protection against hydrogen
peroxide produced under high light. This protection assumes that hydrogen
peroxide must leak out of chloroplasts and also perhaps the peroxisomes.
This proposal is strengthened by the demonstration that about 5% of the
hydrogen peroxide produced by chloroplasts can escape and that its release
increases with light intensity (Mubarakshina et al., 2010). On the basis of
altered gene expression in apx1, it is suggested that various redox-related
signalling processes are affected (Davletova et al., 2005) and these are pre-
sumably related to events initiated by cytosolic hydrogen peroxide. A tobac-
co cytosolic APX mutant is more sensitive to paraquat and ozone and shows
enhanced hypersensitive cell death when challenged with Pseudomonas syr-
ingae pv. phaseolicola presumably because of its decreased ability to scavenge
cytosolic hydrogen peroxide (Mittler et al., 1999). APX2 is another presumed
cytosolic APX, whose expression levels are very low under normal condi-
tions. It is very rapidly induced in the leaf bundle sheath cells by high light in
a hydrogen peroxide and ABA-dependent manner as part of a process that is
required for longer-term acclimation to high light (Fryer et al., 2003; Galvez-
Valdivieso et al., 2009; Karpinski et al., 1997). The function of APX2 in the
bundle sheath cells during the high light response is not clear. However, given
that over the very early part of the response high light-induced hydrogen
peroxide is highest in the bundle sheath cells, it is possible that APX2 is
involved in modulating this burst in a cell-specific manner. Arabidopsis
knockout mutants of tAPX have been investigated, along with various
double mutants (apx1 sapx, apx1 taxp and sapx taxp). sapx and tapx mutants
have also been combined with the ascorbate-deficient vtc2-1 mutant
(Giacomelli et al., 2007). The overall conclusion is that in mature plants
sAPX is not as essential for controlling light-dependent hydrogen peroxide
production or for protection against high light as is tAPX (Giacomelli et al.,
2007; Kangasjarvi et al., 2008; Maruta et al., 2010b; Miller et al., 2007;
Tarantino et al., 2005) Similarly, in wheat, a mutant with reduced tAPX
activity is more susceptible to high light and exhibited decreased
VITAMIN C 141
photosynthetic capacity (Danna et al., 2003). The Arabidopsis apx1 taxp

double mutant showed much less high light-induced protein oxidation,
higher anthocyanin accumulation and greater basal thermotolerance than
the individual mutants providing a suggestion that chloroplast and cytosol
hydrogen peroxide signalling interact in a complex manner (Miller et al.,
2007). As expected, triple mutants lacking both sAPX and tAPX and having
low ascorbate (lacking VTC2) were highly susceptible to photo-oxidative
stress (Giacomelli et al., 2007). Transcriptomes of apx1 (Davletova et al.,
2005; Pnueli et al., 2003) and sapx tapx double mutants (Kangasjarvi et al.,
2008) have been carried out and provide a repository of data on the con-
sequences of APX deficiency.
Microbodies (peroxisomes and glyoxysomes) are organelles that harbour
hydrogen peroxide-producing oxidases, the most prominent being glycolate
oxidase in leaf peroxisomes, which is involved in photorespiration. The
related glyoxysomes are present in germinating oil seeds. The main source
of hydrogen peroxide in glyoxysomes is the acyl-CoA oxidase step of fatty
acid -oxidation. Microbodies are characterised by very high concentrations
of catalase, which is essential for removing hydrogen peroxide. However, the
presence of APX and ascorbate in peroxisomes, as well as other enzymes of
the ascorbate–GSH cycle (Jimenez et al., 1997; Zechmann et al., 2010),
suggests that some hydrogen peroxide could escape the attention of the
high capacity, but low affinity, catalase. In peas, all the APX activity in
isolated peroxisomes is attached to the outer side of the membrane
(Jimenez et al., 1997) to where it is trafficked by a subdomain of the endo-
plasmic reticulum (Mullen et al., 1999). In Arabidopsis, APX3 is trafficked to
the membrane with the aid of a chaperone protein ankyrin repeat-containing
protein 2A (AKR2A) (Shen et al., 2010). A knockout mutation of APX3 had
no effect on plant phenotype under the conditions tested, suggesting that its
function is redundant (Narendra et al., 2006).
Overexpression of various APX isozymes targeted to cytosol, stroma,
thylakoids and peroxisomes has produced plants that, at least under
laboratory conditions, are more tolerant variously to high light, paraquat
and temperature extremes (Gadjev et al., 2006; Hirooka et al., 2009; Kim
et al., 2010; Kwon et al., 2002; Laloi et al., 2007; Lee et al., 2010; Li et al.,
2010b; Mittler et al., 1999; Murgia et al., 2004; Sun et al., 2010; Yabuta
et al., 2002). In some cases, increasing the expression of APX along with
another antioxidant enzyme such as superoxide dismutase has a synergis-
tic effect on oxidative stress tolerance (Lee et al., 2010). It remains to be
seen if this transgenic approach, or selection for enhanced APX expression
by molecular breeding, could make a measurable improvement to crop
yield.
B. MONODEHYDROASCORBATE REDUCTASE
The earliest suggestions of the existence of enzymes able to reduce MDHA in

a pyridine nucleotide-dependent manner were made by Beevers (1954) and
Marre and Arrigoni (1958). MDHAR is an FAD-containing enzyme that
uses NAD(P)H as a reductant to reduce MDHA to ascorbate (Fig. 1A). The
catalytic cycle requires two MDHAs to be sequentially reduced by NAD(P)
H þ Hþ. It has a high affinity for MDHA ( 0.1 M) and is more active with
NADH than NADPH. Reduced ferredoxin reduces MDHA very rapidly, so
in the vicinity of the thylakoids, this reaction may out-compete MDHAR
activity (Asada, 1999). MDHAR is also able to reduce phenoxyl radicals
produced by the action of peroxidase on quercetin, ferulic acid, coniferyl
alcohol and chlorogenic acid, thereby inhibiting peroxidase activity
(Sakihama et al., 2000). A crystal structure of cucumber MDHAR is avail-
able (Sano et al., 2004). As previously noted, the MDHA radical is relatively
stable. However, the equilibrium constant for MDHA formation from DHA
and ascorbate is 5 10 9 at pH 6.4 (Buettner and Schafer, 2004), so at
equilibrium assuming 10 mM ascorbate and 1 mM DHA, MDHA concen-
tration would be 0.5 M, which is within the range of the Km of MDHAR.
MDHA can be detected by EPR spectroscopy in vivo. It is produced by
chloroplasts, and while near the detection limit in young leaves under non-
stressful conditions, it can be detected after inhibition of photosynthesis
application of stresses such as high light, UV-B radiation and infection
with Botrytis cinerea (Heber et al., 1996; Hideg et al., 1997; Miyake and
Asada, 1994; Muckenschnabel et al., 2002). Therefore, it is clear that condi-
tions which increase oxidative stress cause an increase MDHA concentra-
tion. EPR spectroscopy is an excellent non-destructive means of monitoring
the dynamics redox state of the ascorbate system, particularly to assess the
effects of altered MDHAR activity. Unfortunately, the equipment is not
readily available to most laboratories.
MDHAR isoforms are found in the cytosol, chloroplasts, mitochondrial
matrix, peroxisomes and plasma membrane (Berczi and Moller, 1998). The
moss Physcomitrella patens has three genes that encode proteins with
MDHAR activity and lack organelle-targeting sequences (Drew et al.,
2007; Lunde et al., 2006). In Arabidopsis, MDHAR is encoded by five
genes (Chew et al., 2003). One gene (At1g63940, AtMDAR6) produces
proteins that are alternatively targeted at the chloroplast (stroma) and mito-
chondrial matrix possibly because two different isoforms, differing in length
by 21 bp, are generated by alternative transcription start sites (Obara and
Fukuda, 2004). AtMDAR1 (matrix) and 4 (membrane) sort to the peroxi-
somes, while AtMDAR2 and 3 appear to be cytosolic (Lisenbee et al., 2005).
VITAMIN C 143
There have been two studies of MDHAR function using knockout mutants
of specific isoforms. AtMDAR4 mutants are unable to survive after germi-
nation unless grown on sucrose (Eastmond, 2007). As rescue with sucrose is
characteristic of mutants affected in the ability to use triacylglycerols, the
main seed reserve in Arabidopsis, lipid utilisation is affected. Eastmond
(2007) showed that the phenotype is caused by severe oxidative stress arising
from hydrogen peroxide generated by -oxidation. It is unclear why the
AtAPX3 knockout mutation is not similarly lethal (Narendra et al., 2006),
but it does suggest that regeneration of ascorbate from MDHA is more
important in this case than hydrogen peroxide removal. After photosynthetic
competence of the rescued seedlings is established, the plants grow normally,
showing that the loss of AtMDAR4 from peroxisomes is not problematic.
T-DNA insertion mutants of the putative cytosolic AtMDAR3 (and AtD-
HAR5) had relatively little effect on uninfected plant size but reduced the
growth response of the plants to the mutualistic endophyte fungus Pirifor-
mospora indica (Vadassery et al., 2009). The results suggest that the balance
between the partners depends on the control of redox state. MDHAR iso-
forms have been overexpressed in chloroplasts (Kavitha et al., 2010; Li et al.,
2010b) and cytosol (Eltayeb et al., 2007). These manipulations have variously
increased ascorbate or its reduction state and decreased damage caused by
salt, osmotic stress, chilling and methyl viologen. In support of a role of
MDHAR in recycling ascorbate, a QTL for tomato fruit ascorbate fine-
mapped to an MDHAR gene. Plants with higher MDHAR activity had
improved cold storage linked to a less oxidised fruit ascorbate pool
(Stevens et al., 2008).
C. DEHYDROASCORBATE REDUCTASE
DHA is readily reduced by GSH, particularly at pH 7 and above (Winkler

et al., 1994), and so regeneration of ascorbate from DHA is predicted to
occur at a significant rate in the cytosol, and very rapidly in the stroma in the
light, where pH is in the region of 8. Despite this, many organisms contain
DHAR enzymes that catalyse the reaction. This class of enzymes belongs to
the GSH transferase superfamily (Dixon et al., 2002). Soluble DHAR activi-
ty is found in chloroplasts (Shimaoka et al., 2000; Shimaoka et al., 2003),
mitochondrial matrix and peroxisomes (Jimenez et al., 1997). Arabidopsis
has three DHAR genes for which activity has been confirmed by analysis of
recombinant proteins (Dixon et al., 2002). These are predicted to be localised
in cytosol, chloroplast/mitochondria and peroxisomes. The localisation of
DHAR1/2 (At1g19750) in peroxisomes has been determined by mass
spectrometry and localisation of a YFP fusion protein (Reumann et al.,

2009). The role of DHAR in maintaining the ascorbate pool is shown by
an increase in total ascorbate concentration in leaves of DHAR overexpres-
sing plants that can be up to twofold higher than that in wild-type plants
(Chen et al., 2003), and an Arabidopsis mutant with decreased cytosolic
DHAR has decreased apoplastic ascorbate (Yoshida et al., 2006). This result
supports the proposal that apoplastic DHA is transported into the cytosol
and regenerated by the action of DHAR. In some cases, an increase in the
proportion of ascorbate to DHA is reported. However, in general, there is
not an obvious decrease in the measured DHA concentration, suggesting two
important points. Firstly, in general, the baseline concentration of DHA
measured in plant extracts is always much the same, so it is probably not
possible to draw conclusions from small changes in ascorbate redox state, as
oxidation is certain to occur during extraction. Secondly, it is likely that the
increase in ascorbate seen in DHAR overexpressing plants reflects decreased
degradation of DHA. Detailed physiological analysis of tobacco plants over-
expressing wheat DHAR in the cytosol and with suppressed DHAR activity
in both cytosol and chloroplast, due to overexpression of the tobacco gene,
has been carried out. This shows that low DHAR causes somewhat slower
growth and an increase in senescence rate, along with higher hydrogen
peroxide levels, lower photosynthesis rate, altered stomatal aperture and
ABA response and decreased capacity for non-photochemical quenching
(NPQ) (Chen and Gallie, 2004, 2005, 2006, 2008). The decreased NPQ is
reversed by ascorbate supplementation (Chen and Gallie, 2008) and is con-
sistent with the role of ascorbate in the xanthophyll cycle (Section IX.B).
Conversely, overexpression has relatively little effect on growth and photo-
synthesis (Chen and Gallie, 2006). DHAR overexpression in the cytosol
generally increases the resistance of plants to various short-term stresses
that presumably cause oxidative damage, such as ozone, paraquat, high
temperature, aluminium, drought, salt and osmotic stress (Chen and
Gallie, 2005; Eltayeb et al., 2006; Ushimaru et al., 2006; Wang et al., 2010;
Yin et al., 2010; Yoshida et al., 2006). In the case of ozone, this is related to
the decreased apoplastic ascorbate (Yoshida et al., 2006) and not to reduced
ozone access via the stomata (Chen and Gallie, 2005). DHARs have sequence
similarity to animal CLICs (intracellular chloride channels). When transient-
ly expressed in animal cells, a small proportion of Arabidopsis peroxisomal
DHAR1/5 is associated with the microsomal fraction and bestowed chloride
channel activity on the cells (Elter et al., 2007). Both DHAR1/5 and DHAR2
(and APX1) have been detected in the proteome of plasma membrane
(Marmagne et al., 2004) supporting a possible ion transport role in the
plant plasma membrane.
VITAMIN C 145
D. ASCORBATE OXIDASE—AN ENIGMATIC ENZYME
Plants have long been known to contain AO enzymes. These catalyse oxida-
tion of ascorbate by oxygen, with the production of MDHA and water with
fairly high specificity and affinity for ascorbate. AOs are glycosylated blue
multicopper oxidases of the cupredoxin superfamily (Messerschmidt and
Huber, 1990). Arabidopsis has three genes predicted to encode extracellular
AOs (At4g39830, At5g21100 and At5g21105). Other species also have multi-
ple AO genes (Al-Madhoun et al., 2003; Diallinas et al., 1997; Sanmartin
et al., 2007). Most of the apoplastic AO is probably ionically bound to the
cell wall, as it can be eluted by high ionic strength buffers while a small
proportion is soluble (perhaps in transit from the endomembrane system to
the apoplast). The crystal structure of Cucurbita (zucchini) AO has been
determined to 1.9 Å resolution (Messerschmidt et al., 1992, 1993). There
are two other groups of related Cu-containing (glyco)proteins: laccases,
which are o-diphenol or monophenol oxidases (Cai et al., 2006; Turlapati
et al., 2010), and SKU5-like proteins, some of which have functions in cell
growth and microtubule organisation (Sedbrook et al., 2002, 2004).
High AO activity is found in the QCs of maize and Cucurbita maxima
roots, along with correspondingly little deposition of silver granules pro-
duced by reduction of silver nitrate—a histochemical test for ascorbate (Kerk
and Feldman, 1995; Liso et al., 2004). The oxidation of ascorbate is sug-
gested to provide an oxidising environment that prevents cell division in the
QC (Jiang et al., 2003). As the stable product of ascorbate oxidation by AO is
DHA, it is interesting to note that cell division is inhibited by added DHA in
tobacco cell cultures (Potters et al., 2010). Additionally, overexpressing AO
in tobacco cells enhances auxin-induced cell expansion (Kato and Esaka,
2000), while added DHA increases auxin-induced cell expansion (Potters
et al., 2010). These experiments, along with a number of other observations
that correlate high AO activity with zones of rapid cell expansion and its
induction by auxin (Takahama and Oniki, 1994), support a role for AO in
cell expansion. Interestingly, tobacco seedlings overexpressing AO have a
decreased growth response to added NAA (Pignocchi et al., 2006). However,
antisense approaches to reducing AO activity in tobacco (Fotopoulos et al.,
2006; Pignocchi et al., 2003; Sanmartin et al., 2003; Yamamoto et al., 2005)
and a T-DNA knockout line of Arabidopsis At5g21100 (Yamamoto et al.,
2005) result, at best, in only small changes in growth and development under
normal environmental conditions. The overexpression experiments in tobac-
co were successful in increasing apoplastic AO activity and decreasing apo-
plastic ascorbate, so it is clear that this perturbation has a minor effect. The
antisense and T-DNA approaches to decreasing AO activity left 10% or more
residual AO activity, which could be accounted for by incomplete suppres-

sion of gene expression or redundancy. However, a very interesting observa-
tion is that AO overexpression in tobacco decreases stomatal aperture,
decreases water loss from detached leaves and increases ABA (Fotopoulos
et al., 2008), suggesting a role for apoplastic ascorbate or DHA ascorbate in
ABA synthesis.
If AO has a minimal role under favourable growth conditions, it is possible
that its major function may be in response to stress. AO gene transcripts are
induced by light and auxin (De Tullio et al., 2007; Esaka et al., 1992;
Pignocchi et al., 2006). Publicly available Arabidopsis microarray data
show that At4g39830 transcripts are increased by elicitors such as flagellin
and increased by inoculation with virulent P. syringae and in the non-vir-
ulant hrpA mutant. The expression pattern suggests that increased
At4g39830 expression is a basal response to bacterial infection that is subse-
quently suppressed by the virulent strain. In contrast, At5g21100 expression
is decreased by flagellin and by both strains of P. syringae (M. Grant and
N. Smirnoff, unpublished data). AO activity and protein accumulation are
increased by copper, although gene expression is little affected. This result
suggests that AO could be under translational control by copper supply
(Esaka et al., 1988, 1992). Drought stress increases AO activity substantially
in Arabidopsis leaves in the absence of increases in the transcript levels of the
three AO genes (Choon Kiat Lim and N. Smirnoff, unpublished data),
suggesting translational control could be common for this enzyme. In sup-
port of a role in stress responses, tobacco and Arabidopsis with decreased
AO expression are more tolerant of high salinity and oxidative stress (hydro-
gen peroxide, methyl viologen and ozone), while overexpressors were some-
what more sensitive (Fotopoulos et al., 2006; Sanmartin et al., 2003;
Yamamoto et al., 2005). Similarly, tobacco overexpressing AO was some-
what more susceptible to the necrotrophic fungal pathogen B. cinerea
(Fotopoulos et al., 2006) and P. syringae (Pignocchi et al., 2006). Although
the effects on pathogen responses in both experiments were small and exten-
sive analysis was not carried out, these results suggest that the redox state of
apoplastic ascorbate influences stress responses. De Tullio et al. (2007)
speculate that AO and perhaps other apoplastic oxidases could control tissue
oxygen concentration, a proposal that merits further investigation. Overall,
the evidence suggests roles for AO in growth and stress responses via its effect
on apoplastic ascorbate and perhaps hormone signalling (Kerk et al., 2000;
Pignocchi and Foyer, 2003). It seems that more complete suppression of AO
activity, for example, by production of double or triple mutants of the three
Arabidopsis genes, will be needed to resolve the roles of this enzyme more
fully.
VITAMIN C 147
IX. THE FUNCTIONS OF ASCORBATE
The use of the collection of ascorbate-deficient vtc mutants (Conklin and

Barth, 2004; Conklin et al., 2000) has not only provided evidence for the
Man/L-Gal biosynthesis pathway, but these mutants, particularly vtc1, have
also been used to probe the functions of ascorbate. The EMS-induced
mutants were selected either by an initial screen for ozone sensitivity or by
a high-throughput semi-quantitative ascorbate assay based on reduction of
nitroblue tetrazolium to a blue formazan precipitate by squashed leaves
(Conklin et al., 2000). The vtc1, vtc2-1 and vtc2-2 seedlings are quite similar
in size after germination (Colville and Smirnoff, 2008) but are smaller when
more mature, the effect being greatest in the mutants with the lowest ascor-
bate content (Conklin et al., 2000; Olmos et al., 2006; Veljovic-Jovanovic
et al., 2001). VTC2 and VTC5 encode homologues of GDP-L-Gal phosphor-
ylase, the first dedicated enzyme of the Man/L-Gal pathway. A double vtc2
vtc5 mutant shows growth arrest after emergence of the cotyledons. Rescue
by L-Gal or ascorbate shows that growth beyond initial germination requires
ascorbate (Dowdle et al., 2007). A knockout in the last enzyme of the
pathway, L-GalLDH, has an identical phenotype (Pineau et al., 2008). Fur-
ther study of these ascorbate null mutants is needed to find out if growth
arrest is related to oxidative stress, effects of ascorbate on growth and cell
division, failure of ascorbate-dependent dioxygenases or a combination of
these factors. Flowering and leaf senescence are accelerated in several vtc
mutants, irrespective of day length (Barth et al., 2004, 2006; Kotchoni et al.,
2009), although retarded flowering has also been reported in vtc1 and vtc2 in
a 10-h photoperiod (Pavet et al., 2005; Veljovic-Jovanovic et al., 2001). While
it is clear that growth and development are affected by ascorbate status,
caution must be exercised in interpretation of data from vtc1. This mutation
affects mannose metabolism, with effects consequent effects on protein
glycosylation (Lukowitz et al., 2001) and cell wall composition (Keller
et al., 1999), which could contribute to growth and stress response pheno-
types. It has been shown that vtc1 root growth is hypersensitive to ammoni-
um in an ascorbate-independent manner (Barth et al., 2010; Li et al., 2010a;
Qin et al., 2008). The vtc4 mutation compromises salt tolerance, most likely
through altered inositol phosphate metabolism (Torabinejad et al., 2009).
The nature of VTC3 is still under investigation, and preliminary evidence
suggests it is a protein kinase that could be involved in controlling ascorbate
metabolism (P. Conklin, personal communication). The vtc2 mutants are
therefore the most useful and, in an ideal investigation, the phenotypes would
also be confirmed in some of the other mutants. This view is supported by a
comparison of the transcriptomes of vtc1 and vtc2 seedlings (Data from
European Arabidopsis Stock Centre, NASCArrays-390, P. Muller-Moulé).

There are some genes in common that are differentially expressed compared
to wild type. However, each strain also has a distinct set of differentially
expressed genes that could be related to the specific mutation or could be due
to insufficient back-crossing to remove other mutations. An important ob-
servation is that plant phenotype, transcriptome analysis and other gene
expression studies suggest that vtc mutants grown under benign laboratory
conditions in the dim light typical of most controlled environment growth
chambers show little or no sign of oxidative stress. In particular, there is not a
wholesale up-regulation of other genes in the antioxidant network (Pastori
et al., 2003). The effect of altering ascorbate pool size is therefore rather
different to altering APX activity, as reviewed in the Section VIII.A. How-
ever, as will be shown below, the effects of ascorbate deficiency become more
obvious at high light intensity or under stress.
A. THE ASCORBATE–GSH (FOYER–HALLIWELL–ASADA) CYCLE
The ascorbate–GSH cycle comprises a system to regenerate ascorbate from

MDHA and DHA using GSH and reduced pyridine nucleotides (Fig. 5). The
enzymes involved (MDHAR, DHAR and GR) are found in the cytosol,
chloroplasts, mitochondria and peroxisomes (see above). The evidence for
the operation of this cycle is reviewed by Foyer and Noctor (2011), and
mathematical models have been constructed to probe the function in silico
(Polle, 2001; Valero et al., 2009). The cycle is involved in ascorbate-depen-
dent hydrogen peroxide scavenging using APX as well as other processes that
oxidise ascorbate. The importance of the cycle in both protection against
oxidative stress and in controlling hydrogen peroxide-related signalling is
shown by the effects of mutations or overexpression of APX, MDHAR and
DHAR (see above). As GSH and its redox state are signals that effect gene
expression (Foyer and Noctor, 2011), the rate of ascorbate oxidation and
regeneration can affect GSH concentration and redox state and thereby
modulate GSH-dependent signalling (Fig. 6). GSH (e.g. via GSH peroxi-
dase) is also able to react with ROS and lipid hydroperoxides. A link between
the thiol system and ascorbate is also apparent with chloroplast 2-cys perox-
iredoxin, whose expression is affected by ascorbate status—being higher in
vtc1 (Baier et al., 2000). A promoter::luciferase construct showed that 2-cys
peroxiredoxin expression is repressed by ascorbate in a light-dependent
manner (Shaikhali and Baier, 2010).
VITAMIN C 149
Fig. 5. The interaction of ascorbate and the ascorbate–glutathione cycle with

photosynthesis. Hydrogen peroxide, produced by oxygen photoreduction by PSI, is
removed by APX, a thylakoid bound isoform (tAPX) being particularly important.
MDHA (using NADPH or reduced ferredoxin) and DHA (in the ascorbate–
glutathione cycle) are reduced back to ascorbate. This process is termed the water–
water cycle and may account for 10% of electron flow. Singlet oxygen (1O2)
produced in PSII causes lipid peroxidation. Ascorbate aids -tocopherol and plasto-
chromanol-8 in scavenging lipid peroxyl radicals by recycling tocopheroxyl radicals.
High light induces synthesis of the carotenoid zeaxanthin from violaxanthin and
antheraxanthin using the ascorbate-dependent enzyme (VDE). Zeaxanthin contri-
butes to the process of non-photochemical quenching (NPQ), which dissipates excess
excitation energy as heat. VDE is localised on the lumenal side of the thylakoid
membrane. Lumenal ascorbate can donate electrons to PSII, a process that may be
important in preventing its photo-oxidation when the OEC is inactivated by UV-B or
high temperature. Ascorbate could therefore contribute to linear electron transport
and generate a trans-thylakoid proton gradient via the Q cycle. Lumenal ascorbate
may also donate electrons to PSI in bundle sheath cells of C4 plants and aid the
priming of cyclic electron transport. Key questions are how ascorbate and DHA are
transported across the thylakoid membrane and the physiological significance of
ascorbate as an electron donor. The thylakoid cytb/f and ATP synthase complexes
have been omitted from the scheme for the sake of clarity.
B. PHOTOSYNTHESIS AND PHOTOPROTECTION
In most plants, ascorbate concentration in leaves is light dependent and it

interacts with photosynthesis in a number of ways (Smirnoff, 2000b). Firstly,
it removes ROS as part of the ascorbate–GSH cycle. Secondly, it aids
photoprotection via the xanthophyll cycle. Thirdly, and more speculatively,
Fig. 6. A simplified scheme indicating the possible role of ascorbate in modulating

signalling processes initiated in response to high light intensity and excess excitation
energy. Within the chloroplast, hydrogen peroxide is produced in a light-dependent
manner and removed by tAPX and sAPX. The resulting DHA, and hydrogen perox-
ide itself, can then affect the thiol redox state (GSH, GSH peroxidase, 2-cys PRX,
thioredoxin, etc.). Oxidised thiols and/or hydrogen peroxide will inhibit gene expres-
sion and affect the activity thioredoxin-dependent Calvin–Benson cycle enzymes.
Hydrogen peroxide also leaves the chloroplast and influences nuclear gene expression.
Levels of cytosolic peroxide are modulated by cAPX, whose expression and activity
are highly responsive to light. Singlet oxygen (1O2), produced in PSII, oxidises lipids,
thereby generating a compound that mediates signalling to the nucleus ultimately
affecting the expression of a distinct set of genes. Ascorbate most likely modulates this
signal by aiding the lipid pexoxyl radical scavenging activity of -tocopherol and
plastochromanol-8. Chloroplast gene expression may be inhibited by oxidised thiols
and hydrogen peroxide—a possible contribution to photoinhibitory damage as a
result of decreased replacement rate of damaged photosystem proteins (e.g. D1).
Finally, the redox state of the electron transport system (e.g. plastoquinone) affects
VITAMIN C 151
it could under some circumstances act as an electron donor to the PET

system (Fig. 5). The involvement of ROS in photosynthesis has been exten-
sively reviewed (Asada, 1999; Foyer and Shigeoka, 2011). Superoxide and
hydrogen peroxide are produced by oxygen photoreduction (Mehler reac-
tion) at PSI in the ‘water–water cycle’ where electrons from the oxygen-
evolving complex (OEC) reduce oxygen at PSI forming hydrogen peroxide,
which is then reduced to water by the chloroplast ascorbate–GSH cycle and
associated reactions. Evidence from tAPX mutants reviewed above shows
the importance of this enzyme in scavenging peroxide. Further, the MDHA
produced by this reaction can be directly reduced by ferredoxin, providing a
further sink for excess electrons. While the importance of the Mehler reaction
as a protective mechanism against oxidative stress is debated (Ort and Baker,
2002), it seems that around 10% of electron flow reduces oxygen and that the
thylakoid/stromal ascorbate–GSH cycle has a role in controlling hydrogen
peroxide levels in the stroma. It is, however, intriguing that gene expression
studies and analysis of mutants suggest that expression of the cytosolic APX
and ascorbate–GSH cycle isoforms are much more light responsive than the
chloroplast isoforms. It has been suggested that hydrogen peroxide leakage
from the chloroplasts constitutes a signal (Davletova et al., 2005; Galvez-
Valdivieso et al., 2009) that is sensed and controlled in the cytosol (Fig. 6).
Consistent with this is the demonstration that chloroplasts release about 5%
of their hydrogen peroxide, the amount increasing with light intensity
(Mubarakshina et al., 2010). The subsequent increase in cytosolic and chlor-
oplastic and cytosolic ascorbate and cytosolic APX may then dampen the
signal as part of longer-term acclimation (Fig. 6). Singlet oxygen is produced
by PSII and scavenged by the lipophilic antioxidant tocochromanols (-
tocopherol and plastochromanol-8) either directly or via lipid peroxyl radi-
cals (Gruszka et al., 2008; Kruk et al., 2005; Mene-Saffrane et al., 2010;
Yadav et al., 2010). Photosensitisers that act in the chloroplast cause light-
dependent ascorbate loss (Gullner and Dodge, 2000), suggesting ascorbate is
involved in removing singlet oxygen directly (unlikely, because it is formed in
hydrophobic domain of the thylakoids) or by regenerating tocopherol/
plasto-8 from their radicals. Excess singlet oxygen provides a signal, perhaps
state transitions via protein phosphorylation and chloroplast gene expression. During
high light acclimation, the increase in ascorbate and other ascorbate–glutathione
cycle components will dampen the initial signals allowing a new steady state to
establish. Other so-called retrograde signalling processes via GUN (genome
uncoupled) proteins are also involved in chloroplast to nucleus communication,
possibly through chlorophyll biosynthesis intermediates, during chloroplast develop-
ment and greening, but their role in high light acclimation is unclear.
derived from peroxidised lipids, that is distinct from that produced by

hydrogen peroxide (op den Camp et al., 2003). A singlet oxygen responsive
gene (BAP1) is up-regulated in vtc1 (Wormuth et al., 2006) and vtc3 (M. Page
and N. Smirnoff, unpublished data) providing evidence that singlet oxygen-
mediated signalling is quenched by ascorbate. Further, heat-induced auto-
luminescence, which is likely to result from singlet oxygen production, is
enhanced in vtc2 (Havaux, 2003). Mutants with decreased ascorbate, -
tocopherol and plastochromanol-8 will be needed to assess their interaction
in photoprotection.
Ascorbate can act as an electron donor to PSI and PSII in isolated
thylakoids. This process can be experimentally observed for PSII when the
OEC is inactivated (e.g. by heat or UV-B radiation) or, for PSI, when
electron transport from PSII is blocked (Ivanov et al., 2005; Mano et al.,
2004; Toth et al., 2007). Recently, evidence that ascorbate can donate elec-
trons to PSII in vivo has been obtained by comparing wild-type and ascor-
bate-deficient vtc2 (Toth et al., 2009). Under stressful conditions that
inactivate OEC, it is quite possible that ascorbate protects PSII from
photo-oxidation and could also support linear electron transport and forma-
tion of a trans-thylakoid proton gradient via the Q cycle. Therefore, an
ascorbate–ascorbate cycle could operate alongside the water–water cycle
(Fig. 5). Lumenal ascorbate is also required for the VDE reaction in the
photoprotective xanthophyll cycle in which zeaxanthin is synthesised from
violaxanthin. Zeaxanthin formation is stimulated in the high light when a
proton gradient builds up across the thylakoid (Murchie and Niyogi, 2011),
and it contributes to dissipation of excess excitation energy (NPQ). This
process is impaired in vtc mutants (Golan et al., 2006; Muller-Moulé et al.,
2002, 2003, 2004; Smirnoff, 2000a). Both electron donation to PSII and VDE
depend on ascorbate in the thylakoid lumen, so a key issue is the nature of
ascorbate transport into the lumen (protonated or anionic) and diffusion or
carrier mediated. Likewise, DHA must be transported back to the stroma for
reduction by GSH-dependent DHAR. It is possible that the high stromal
ascorbate concentrations and increased ascorbate accumulation in high light
are important for ensuring that ascorbate transport into the thylakoids is
sufficiently rapid. Clearly, more information on ascorbate and DHA trans-
port across the thylakoid membrane is needed. Very recently, it has been
proposed that damage to the OEC by UV-B and yellow light (rather than
photosynthetically active light) is the primary cause of photoinhibition by
high light (Takahashi and Badger, 2011; Takahashi et al., 2010). Under this
paradigm, the photoprotective effect of lumenal ascorbate by electron dona-
tion to PSII takes on a possible physiological significance. The exceptionally
high ascorbate reported in the chloroplasts of alpine species (where increased
VITAMIN C 153
UV-B is an environmental factor; Streb et al., 1997) could be explained by

increased frequency of UV-B damage to the OEC. In this context, it is also
interesting to note that UV-B damage to photosynthesis is increased in vtc1
(Gao and Zhang, 2008). A possible direct UV-B screening effect of ascorbate
cannot be overlooked, particularly at the high concentrations observed.
Although its absorption maximum (265 nm) is well into the UV-C region,
absorbance at 280 nm (the UV-B/C boundary) is 10% of that at 265 nm. The
mesophyll and bundle sheath cells of C4 plants have distinct photosynthetic
characteristics. The bundle sheath chloroplasts have limited PSII activity and
rely on cyclic electron transport to generate ATP. These chloroplasts have
the ability to use ascorbate as electron donor perhaps helping to prime cyclic
electron transport (Ivanov et al., 2002, 2005).
C. ENVIRONMENTAL STRESS AND PATHOGENS
Much of the evidence for a role of ascorbate in resistance to environmental

factors that generate oxidative stress has been covered in Section VIII.
A–VIII.C. Additionally, investigations of the various ascorbate-deficient
vtc mutants indicate increased sensitivity to high light, ozone UV-B radia-
tion, UV-C radiation, high temperature and high salinity (Conklin et al.,
1996; Filkowski et al., 2004; Gao and Zhang, 2008; Huang et al., 2005;
Larkindale et al., 2005; Muller-Moulé et al., 2004). In the cases where only
vtc1 was investigated, the caveats about non-ascorbate-dependent pheno-
types apply. Many studies have shown a general relationship between ozone
resistance and ascorbate. However, it is curious that the ozone sensitivity of
vtc mutants does not follow ascorbate concentration, even within an allelic
series. vtc2-1 and vtc2-2 have similar ascorbate concentrations, but only vtc2-
1 is ozone hypersensitive (Conklin et al., 2000). The vtc mutants are more
resistant to bacterial (P. syringae) and fungal (Peronospora parasitica) bio-
trophic pathogens (Barth et al., 2004; Mukherjee et al., 2010; Pavet et al.,
2005). Increased resistance is associated with higher expression and more
rapid induction of genes encoding pathogenesis-related proteins (Barth et al.,
2004; Colville and Smirnoff, 2008; Pavet et al., 2005) and higher salicylic acid
(SA) and SA glycoside concentrations (Barth et al., 2004; Mukherjee et al.,
2010). It seems likely that low ascorbate, perhaps via elevated hydrogen
peroxide, induces SA-mediated defences. In support of this proposal, double
mutants of vtc1 with the SA signalling mutants pad4, eds5 and npr1 are not
more resistant to P. syringae (Mukherjee et al., 2010).
D. GROWTH AND SIGNALLING: CELL DIVISION AND CELL EXPANSION
The various aspects of ascorbate metabolism reviewed above suggest roles

for ascorbate in modulating growth and signalling processes. Areas of sug-
gested involvement are in cell division, cell expansion and interaction with
hormone signalling. Cell division is inhibited by oxidative conditions, and
studies with cell cultures and root QCs are consistent with low ascorbate or
high DHA preventing cell division (Horemans et al., 2003; Innocenti et al.,
1990; Kerk and Feldman, 1995; Kerk et al., 2000; Liso et al., 1988, 2004;
Potters et al., 2002, 2004, 2010). It is tempting to speculate that arrested cell
division is the reason for growth arrest in seedlings that lack ascorbate
(Dowdle et al., 2007). The recent demonstration that GSH locates to the
nucleus during cell division reinforces a role for both these antioxidants in
cell division (Vivancos et al., 2010). In relation to hormone signalling,
hydrogen peroxide, generated by activation of NADPH oxidase in the
guard cell plasma membrane, mediates ABA-induced stomatal closure
(Kwak et al., 2003). Ascorbate in the guard cell cytosol or apoplast could
therefore modulate stomatal response to ABA, and possibly high light accli-
mated leaves with more ascorbate could maintain more open stomata. Over-
expression of DHAR in tobacco, which elevates ascorbate by improving
recycling, reduces ABA responsiveness of stomata and results in less rapid
response of stomata to water stress (Chen and Gallie, 2004). In support of
this proposal, oxidation of apoplastic ascorbate by AO overexpression
decreases stomatal aperture (Fotopoulos et al., 2008).
Ascorbate and AO could have roles in cell expansion (Kato and Esaka,
1999, 2000; Key, 1962; Lin and Varner, 1991; Takahama, 1996; Takahama
and Oniki, 1994). The possible mechanisms are interaction of DHA with cell
wall proteins, for example, by modifying lysine residues thereby preventing
Schiffs base formation between the reducing ends of polysaccharides and
proteins, and modifying arginine residues and decreasing cell wall calcium
via oxalate formation. The latter effect would decrease pectin cross-linking
(Lin and Varner, 1991). An alternative mechanism, for which there is some
experimental evidence, is that ascorbate or its oxidation products give rise to
hydroxyl radicals. Hydroxyl radicals could be produced from Cu2þ-mediated
ascorbate oxidation or from hydrogen peroxide produced during apoplastic
DHA degradation via 4-O-oxalyl threonate (Fry, 1998; Green and Fry,
2005). Hydroxyl radicals are highly reactive and cause scission of polysac-
charide glycosidic bonds, thereby causing cell wall loosening (Dumville and
Fry, 2003; Fry et al., 2001, 2002; Karkonen and Fry, 2006; Schweikert et al.,
2002). Such a process could contribute to tissue softening during ripening
and cell expansion. Auxin-induced growth is inhibited by hydroxyl radical
VITAMIN C 155
scavengers (Liszkay et al., 2003; Schopfer, 2001) providing evidence that this
mechanism could contribute to wall loosening and growth.
X. CONCLUSIONS
Ascorbate pervades all subcellular compartments and, along with other
antioxidants, is involved in controlling reactive oxygen and the associated
signalling events required for acclimation to environmental stress, particu-
larly in relation to photosynthesis. Over the past decade, the biosynthesis of
ascorbate by the Man/L-Gal pathway has been established as the primary
biosynthetic pathway in green plants. Arabidopsis seedlings blocked in this
pathway stop growing after germination. The way in which lack of ascorbate
causes growth arrest needs to be identified. Some key areas that require
investigation include how biosynthesis and turnover are controlled to main-
tain the correct ascorbate concentration, the identity of ascorbate and DHA
transporters, the role of ascorbate in cell expansion and possible roles of AO
in growth and stress resistance.
ACKNOWLEDGEMENTS
The Biotechnology and Biological Sciences Research Council provided

research funding. Apologies are due to colleagues whose work has not been
properly represented through misinterpretation or oversight.
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Vitamin E
DEAN DELLAPENNA1 AND LAURENT MÈNE-SAFFRANÉ
Department of Biochemistry and Molecular Biology, Michigan State

University, East Lansing, Michigan, USA
I.
A Brief History of Vitamin E Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
II.
Structure and Chemistry of Tocochromanols and Vitamin E . . . . . . . . . . . . 181
III.
Tocochromanol Distribution in Plant Tissues and Foods . . . . . . . . . . . . . . . . 185
IV.Vitamin E Requirement in Humans and Biological Functions . . . . . . . . . . . 185
V.The Tocochromanol Pathway in Photosynthetic Organisms . . . . . . . . . . . . . 188
VI.Biochemical Genomics Enabled the Cloning of Tocochromanol
Pathway Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
A. Synthesis of the Tocochromanol Aromatic Head Group .............. 191
B. Prenylation of HGA for Tocochromanol
and Plastoquinone Synthesis................................................ 193
C. An Alternate Route for the Phytyl-PP used in
Tocopherol Synthesis ........................................................ 195
D. The Methyltransferases of Tocochromanol Synthesis .................. 196
E. The Tocopherol Cyclase Enzyme .......................................... 197
VII. Engineering Multiple Steps of the Pathway and Application
to Agricultural Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
VIII. Potential for Breeding Plants with Improved Vitamin E Content . . . . . . . . 199
IX. Progress in Elucidating Tocochromanol Functions in
Photosynthetic Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
A. Tocochromanol Functions During Seed Desiccation, Storage and
Seedling Establishment ...................................................... 201
B. Tocochromanol Functions in Adult Plants............................... 210
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
1
Corresponding author: E-mail: dellapen@msu.edu

180 DEAN DELLAPENNA AND LAURENT MÈNE-SAFFRANÉ
ABSTRACT
Tocochromanols are a small group of natural products synthesized exclusively by
photosynthetic organisms and include the tocopherols and tocotrienols that are
vitamin E, an essential lipid-soluble antioxidant in the human diet. The major source
of vitamin E in the human diet is plant-derived products, which can vary by orders of
magnitude in tocochromanol content and composition and hence vitamin E activity.
In the past decade, tremendous progress has been made in our understanding of the
molecular genetics of tocochromanol synthesis in plants and cyanobacteria, and all
the biosynthetic genes of the core pathway have now been cloned and studied in
detail. Significant progress has been made in engineering tocochromanol content and
composition in plant tissues. Our understanding of tocochromanol function(s) during
the plant life cycle has been advanced by the isolation and study of pathway mutants
that accumulate specific intermediates or completely eliminate tocochromanols from
the organism. Tocochromanols are absolutely essential for limiting lipid oxidation
during seed desiccation, storage and germination, and the severe fitness impact of
tocochromanol deficiency at this stage of the plant life cycle makes it obvious why
tocochromanol synthesis has been conserved in all seed-bearing plants during evolu-
tion. However, the functions of tocochromanols in mature plant tissues are surpris-
ingly more limited than had long been assumed, especially in regard with plant stress.
I. A BRIEF HISTORY OF VITAMIN E RESEARCH
The first half of the twentieth century was a remarkable period for research
into animal nutrition that saw the identification and structural elucidation of
numerous specific organic and inorganic compounds in the diet (vitamins
and minerals, respectively) that were shown to be essential for optimal
growth and development in animals, including humans. Vitamin E is one
such group of compounds originally identified in 1922 as a factor from green
leafy vegetables essential for reproduction in rats (Evans and Bishop, 1922).
Vitamin E is only synthesized by photosynthetic organisms, and like so many
other essential nutrients in our diet, our primary source is from plants. The
compound with vitamin E activity was first purified from wheat germ oil in
1936 and given the name -tocopherol, derived from the Greek words for
childbirth (tokos) and to bring forth (phero) with the suffix ol being added to
indicate the presence of an alcohol function in the molecule (Evans et al.,
1936). The year 1938 saw the elucidation of the structure of naturally occur-
ring -tocopherol (Fernholz, 1938), which is a single (R,R,R)-stereoisomer,
and the first chemical synthesis of racemic dl--tocopherol, which is com-
posed of eight stereoisomers (Karrer et al., 1938).
In the intervening decades, since the discovery of vitamin E, a tremendous
body of research has been performed to understand various aspects of its
chemistry, uptake, transport and in vivo activities of vitamin E in animals
with over 29,000 papers on these topics in PubMed over the past four decades
VITAMIN E 181
TABLE I
Number of Chapters in PubMed with the Indicated Search Terms
Decade
Search criteria (title or abstract) 1970 1980 1990 2000 Total
Vitamin E or tocopherol* 1795 4248 9191 12,556 27,790
Vitamin E or tocopherol* 23 27 144 613 807
þ plant*
Vitamin E or tocopherol* 1 4 4 61 70
þ plant* þ biosynthesis*
Vitamin E or tocopherol* – 2 13 101 116
þ plant* þ function*
Vitamin E or tocopherol* – – 6 77 83
þ plant* þ Arabidopsis*
Decades are from January 1 to December 31 of each decade.
(summarized in Table I). While impressive progress has been made on all
research fronts during this time (for detailed reviews, see Brigelius-Flohe,
2006; Mustacich et al., 2007; Schneider, 2005; Traber and Sies, 1996), it is
also clear there is still much to learn about the function(s) of vitamin E in
animals (Andersen, 2001; Brigelius-Flohe, 2009; Clarke et al., 2008; Sen
et al., 2006; Traber et al., 2008; Usoro and Mousa, 2010; Wagner et al.,
2004). In contrast to the steady progress of vitamin E research in non-plant
systems, many of the advancements in our fundamental understanding of the
synthesis, molecular biology, genetics and function of vitamin E in photo-
synthetic organisms have been related recently (Table I). Indeed, of PubMed
citations since 1970 with plants and tocopherol/vitamin E in their titles/
abstracts, 75% were published in first decade of the twenty-first century.
Much of this recent progress can be directly linked to elucidation of the
tocopherol biosynthetic pathway in cyanobacteria and plants and the
corresponding availability and analysis of mutants and transgenics with
altered vitamin E levels and composition in these organisms.
II. STRUCTURE AND CHEMISTRY OF

TOCOCHROMANOLS AND VITAMIN E
The term tocochromanol is used to delineate a small group of natural

products that have in common a chroman-6-ol ring system (Fig. 1) and
includes compounds that are collectively termed vitamin E. The most abun-
dant tocochromanols in nature and in the human diet are the tocopherols,
which have a saturated hydrocarbon side chain derived from phytyl
HO
Chroman-6-ol
O
R1 Tocopherols
HO H CH3 H H CH3
CH3
CH3
R2 O CH3
CH3
R1 Tocotrienols
HO
CH3 CH3 CH3
CH3
R2 O CH3
CH3
Activity versus a -Tocopherol
a-TPP Antioxidant
Compound R1 R2 binding Vitamin E (in vitro)
a-Tocopherol CH3 CH3 100 100 100

a-Tocotrienol CH3 CH3 12.5 16 100
b-Tocopherol CH3 H 38 56 71
b-Tocotrienol CH3 H nd 5 71
g -Tocopherol H CH3 9 16 68
g -Tocotrienol H CH3 nd nm 68
d -Tocopherol H H 1.5 <1 28
d -Tocotrienol H H nd nm 28
Fig. 1. Structures and activities of tocopherols and tocotrienols. Key differences

in molecules are indicated by grey shading. The table shows the number and position
of ring methyl groups in -, -, - and -tocopherol and tocotrienols. The binding of
each tocochromanol to an -tocopherol transfer protein (-TTP; Hosomi et al., 1997;
Panagabko et al., 2003), the vitamin E activity determined by the rat resorption–
gestation assay (Leth and Sondergaard, 1977) and in vitro antioxidant activity are
expressed as percentages relative to (R,R,R)--tocopherol (Burton and Traber, 1990).
diphosphate, and the tocotrienols, which retain three double bonds on their
geranylgeranyl diphosphate (GGPP)-derived side chain. Four different toco-
pherols and tocotrienols are found in nature and are designated , , or
depending on the number and positions of methyl groups on the chroman-6-
ol ring system (Fig. 1). Tocopherols have three chiral centres with naturally
occurring tocopherols being of the R,R,R configuration, while chemically
VITAMIN E 183
synthesized tocopherols are racemic mixtures of eight stereoisomers. Toco-

trienols have but a single chiral centre that occurs exclusively as the d-isomer in
nature and as a racemic mixture in synthetic tocotrienols. The number and
positions of ring methylation, degree of side chain saturation and stereochem-
istry all strongly impact the retention of each compound by the human body
and hence the activity of each as vitamin E in the human diet. For example,
chemically synthesized -tocopherol, which is the most common form in
vitamin E supplements, contains eight stereoisomers (one of which is R,R,R)
that range in vitamin E activity from 21% to 100%, relative to the single,
naturally produced (R,R,R)--tocopherol stereoisomer (Eitenmiller, 1997).
From a chemical perspective, tocochromanols are potent lipid-soluble
antioxidants that interact with polyunsaturated acyl groups and protect
membrane lipids (especially polyunsaturated fatty acids, PUFAs) from oxi-
dative damage in both animals and photosynthetic organisms (Fig. 2). Pro-
tection of PUFA by tocochromanols is due to two mechanisms. The first
occurs by donation of a hydrogen atom from the tocochromanol ring hy-
droxyl to a highly reactive PUFA peroxy radical thereby converting it to the
much less reactive PUFA hydroperoxide and breaking the chain reaction of
radical-mediated lipid peroxidation (Fig. 2, upper panel). The resulting
tocochromanol radical is more stable and long lived than PUFA peroxy
radicals and can be recycled back to the corresponding tocochromanol by
reaction with another cellular reductant (presumed to be ascorbate, ubiqui-
nol, plastoquinone, etc.). This allows each tocochromanol molecule to par-
ticipate in an estimated 120 lipid peroxidation chain-breaking events before
being degraded (reviewed in Schneider, 2005). The second protective mecha-
nism is by tocochromanols physically or chemically quenching singlet oxygen
(1O2), a highly reactive species that can damage most biological molecules
including PUFAs (Fig. 2, lower panel). Physical quenching of singlet oxygen
occurs by a highly efficient charge transfer mechanism and thermal dissipa-
tion that returns oxygen to its ground state (3O2), without damaging the
tocochromanol. Tocochromanols can also chemically quench singlet oxygen
(and other reactive oxygen species) which results in opening of the chromanol
ring and production of the respective tocopherolquinone, which can also
participate in electron transfer reactions in both plants and animals (Kruk
et al., 2000; Lass and Sohal, 1998; Munne-Bosch et al., 2005; Siegel et al.,
1997). As photosystem II is a major source of singlet oxygen in plants, these
mechanisms for limiting the potential damage by singlet oxygen are likely to
play a more important role in plants than in animals (Gruszka et al., 2008;
Kruk et al., 2005; Triantaphylides and Havaux, 2009). A pathway for con-
version of tocopherolquinones back to the corresponding tocopherol is dis-
cussed in later sections (Kobayashi and DellaPenna, 2008).
PUFA
Enzymatic lipid oxidation
R
Prostaglandins (animals)
Thromboxanes (animals)
Oxylipins (plants)
Chain propagation
RH Alkyl radical
Conjugated diene
a-Tocopherol
O2
HO
OO
Proposed recycling by
ascorbate, UQ, PQ
O R
Lipid peroxyl radical
OOH
O
Lipid peroxide
O R
a-Tocopherol radical
OH
Malondialdehyde (MDA)
Lipid hydroxides
Phytoprostanes (plants)
Isoprostanes (animals)
Other bioactive products
Physical quenching Chemical quenching

g-Tocopherol
1O HO
2 Tocopherol
1O
2
O R
3O
2 Tocopherol HO
+heat R
OH
OH
O
a-Tocopherolquinone
Fig. 2. The role of tocochromanols in disrupting lipid peroxidation chain reac-

tions and in quenching singlet oxygen. Upper Panel: Peroxidation of polyunsaturated
fatty acids (PUFA) in lipids is initiated by hydrogen abstraction from a PUFA by a
free radical. The resulting alkyl radical rearranges to a conjugated diene radical that
reacts with oxygen to produce a lipid peroxy radical, which can attack another PUFA
and reinitiate the cycle. Tocochromanols (-tocopherol is shown) can donate a
hydrogen from the 6-OH group to form a lipid peroxide and the more stable toco-
chromanol radical (-tocopherol radical is shown). The tocochromanol radical is
recycled back to the corresponding tocochromanol by quenching with ascorbate or
VITAMIN E 185
III. TOCOCHROMANOL DISTRIBUTION IN PLANT

TISSUES AND FOODS
Plant tissues vary enormously in their tocochromanol content and composi-

tion and hence in their vitamin E activities (reviewed in Grusak and
DellaPenna, 1999). In general, photosynthetic tissues contain relatively low
levels of total tocochromanols (10–50 g/gfw in green, unstressed leaves) with
a high percentage being -tocopherol (the most active form of vitamin E).
Seeds contain much higher levels of total tocochromanols (300 to > 2000 g/g
oil; Grusak and DellaPenna, 1999; Hess, 1993; McLaughlin and Weihraugh,
1979; Table II). However, in most seed crops, including those from which the
major edible oils are derived, -tocopherol is often present only as a minor
component and -, - and -tocopherols and tocotrienols, which have lower
relative vitamin E activities, tend to predominate (Table II). Despite the low
proportion of -tocopherol, seed oils still represent the major source of
naturally derived dietary -tocopherol due to the large amount of vegetable
oils consumed in the average American diet and hence the genetic or trans-
genic alteration of tocochromanol profiles in such crops has high potential to
positively impact the vitamin E status of populations.
IV. VITAMIN E REQUIREMENT IN HUMANS AND

BIOLOGICAL FUNCTIONS
Like all essential nutrients, a minimum level of vitamin E is required in the

human diet to maintain optimal health. Vitamin E deficiency is normally only
observed in cases of severe malnutrition or in individuals with genetic defects
in the -tocopherol transfer protein (-TTP; described later in this section) or
conditions that affect fat absorption from the diet (Di Donato et al., 2010;
other antioxidants, though direct evidence for a particular compound having this role
in vivo is limited. The lipid peroxide can be reduced to a lipid hydroxide by a variety of
enzymes or converted (via free radical mechanisms) to a range of biologically active
compounds including dozens of species of phytoprostanes in plants or isoprostanes in
animals and malondialdehyde and other electrophiles by both organisms. An inde-
pendent different set of pathways and reactions exists in both plants and animals for
the enzymatic (vs. free radical) production of different classes and types of oxidized
lipids that also serve as biologically signalling molecules in both organisms. Lower
panel: Physical and chemical quenching of singlet oxygen by tocochromanols. Physi-
cal quenching of singlet oxygen by a charge transfer mechanism that converts singlet
oxygen to the triplet ground state without damaging the tocochromanol. Chemical
quenching results in conversion of the tocochromanol to the corresponding tocopher-
olquinone (-tocopherol and -tocopherol quinone are shown as examples).
TABLE II
Tocochromanol Levels and Compositions in Selected Plants and Oils (Adapted from
Grusak and DellaPenna, 1999)
Percentages of
Total tocopherols Percentage other tocochromanols
Plant and organ (g/gfw or /g oil) -tocopherol and major types
Arabidopsis leaf 10–20 90 10% -T
Arabidopsis seed 200–300 2 95% -T; 5% -T
Potato tubers 0.7 90 10% , -T
Lettuce leaf 7 55 45% -T
Spinach leaf 30 63 5% -T; 33% -T
Rice (white grains) 17 18 30% -T3; 30% -T3; 18% -T
Corn seed 60 10 75% -T; 15% -T3
Wheat germ oil 2700 47 25% -T; 10% -T; 7% -T3
Corn seed oil 1000 20 70% -T; 7% -T
Soybean seed oil 1200 7 70% -T; 22% -T
Sunflower seed oil 700 96 4% .-T
-T, -T, -T and -T are -, -, - and -tocopherols, respectively. -T3, -T3, -T3 and -T3
are -, -, - and -tocotrienols, respectively. Note that considerable genetic variation for both
levels and compositions exists for tocochromanols in plants and the figures given are averages
from the literature and not upper or lower limits.
Muller, 2010; Traber, 2007; Traber et al., 2008). Severe vitamin E deficiency
results in various neurological conditions including ataxia (impaired balance
and coordination), myopathy (muscle weakness) and damage to the retina of
the eye. Suboptimal dietary intake or plasma levels of vitamin E have been
associated with increased risk to cardiovascular disease, some cancers and
decreased immune function (Knekt et al., 1994; Kushi et al., 1996; Wright
et al., 2006). However, the results of large-scale vitamin E intervention trials
with at risk populations have been equivocal (Traber et al., 2008).
Vitamin E is unique among vitamins in that it is not a known cofactor for
any enzymatic reaction and there remain debates about how to objectively
select appropriate minimum dietary levels or levels for other potential bene-
ficial health effects (Blumberg, 1999; Horwitt, 2001; Maras et al., 2004;
Monsen, 2000; Traber, 2006). Thus, the biochemical and molecular mecha-
nism(s) responsible for vitamin E being an essential nutrient have been much
more challenging to delineate and define than for other vitamins (Azzi, 2007;
Jialal et al., 2001; Ricciarelli et al., 2002; Traber, 2001, 2010; Traber and
Atkinson, 2007; Traber et al., 2008; Zingg and Azzi, 2004). An attempt to
take all these issues into account was made in establishment of the most
recent dietary reference intake (DRI) for vitamin E being at 15 mg/day
(Monsen, 2000). Only a minority of the U.S. population actually achieves
this dietary intake level (Maras et al., 2004).
VITAMIN E 187
The -tocopherol content of food is especially important from a nutritional

perspective, as -tocopherol has by far the highest vitamin E activity of all
tocochromanols (e.g. compare -tocopherol and -tocotrienol in Fig. 1). As
long as a modest level of fat is present in the diet, all tocopherols and toco-
trienols are absorbed equally by the intestine and packaged into chylomicrons,
yet their vitamin E activities differ dramatically. Though a large number of
transporters are required for the distribution and maintenance of specific
tocochromanols in the body, a primary reason for the difference in vitamin
E activity of different tocochromanols is the preferential retention and distri-
bution of -tocopherol in animals by a hepatic -TTP. Indeed, -TTP-bind-
ing kinetics for different tocochromanols and stereoisomers correlate well
with the relative vitamin E activity of each and with the large variation in the
half life of different tocochromanols in blood plasma (Brigelius-Flohe, 2006;
Hosomi et al., 1997; Kaempf-Rotzoll et al., 2003; Mustacich et al., 2007;
Traber, 2007; Traber and Arai, 1999; Traber and Sies, 1996; Traber et al.,
2004). The importance of -TTP binding in determining vitamin E activity
and the selective retention of -tocopherol by the body is made clear from the
severe phenotypes of -TTP knockout mice and naturally occurring muta-
tions in the human -TTP protein that are associated with severe vitamin E
deficiency and peripheral neuropathy (Bomar et al., 2003; Gohil et al., 2004;
Schock et al., 2004; Terasawa et al., 2000; Yokota et al., 2001).
As mentioned earlier, because -tocopherol does not serve as an enzyme
cofactor, conclusively defining, the precise biochemical and molecular mechan-
isms of -tocopherol activity, and any additional roles for it and other toco-
chromanols in the diet, have remained challenging and are still the subject of
ongoing research and spirited debates (Azzi, 2007; Azzi et al., 2004; Brigelius-
Flohe, 2005, 2006, 2009; Brigelius-Flohe and Traber, 1999; Clarke et al., 2008;
Jiang et al., 2001; Matringe et al., 2008; Schneider, 2005; Sen et al., 2007; Traber
and Atkinson, 2007; Wagner et al., 2004; Zingg and Azzi, 2004). All tocochro-
manols can and likely do act as lipid-soluble antioxidants and lipid peroxy
radical scavengers in human tissues with the presence, levels, tissue distribution
and hence activity of any individual tocochromanol being determined by a
combination of ingestion levels, uptake, transport and degradation (Brigelius-
Flohe, 2006; Traber, 2007; Traber et al., 2004). While antioxidant activity is
likely to play a role in many tocochromanol functions in humans, over the past
two decades, numerous studies have also suggested non-antioxidant functions
as well. Data in support of non-antioxidant functions include genome wide
expression studies of -TTP knockout mice showing specific groups of genes
(e.g. for cholesterol homeostasis, cellular trafficking and vesicular transport)
that are affected by the near absence of tocochromanols in these mice (Gohil
et al., 2003, 2004; Oommen et al., 2007; Vasu et al., 2010). Unfortunately, the
exact mechanism(s) of this altered gene expression is still to be determined.

There are also numerous reports of activation or inhibition of various signalling
components (protein kinase C, lipoxygenases, phospholipase 2A, cyclooxygen-
ase, etc.) by tocopherols, though whether this is by direct enzyme interaction,
substrate level interaction or alteration in association of enzymes with mem-
branes is still undefined. Tocopherols certainly have been shown to affect
membrane fluidity and may partition into membrane rafts (Lemaire-Ewing
et al., 2010). Suffice to say that nearly 90 years since its discovery, a definitive
molecular basis for vitamin E (and tocochromanol) activities in animals is yet to
be determined. As the full scope of this issue is beyond this chapter, interested
readers are referred to recent reviews for discussions and debate of this issue
(Atkinson et al., 2008; Azzi, 2007; Azzi et al., 2000, 2004; Brigelius-Flohe, 2005,
2006, 2009; Brigelius-Flohe and Galli, 2010; Brigelius-Flohe and Traber, 1999;
Ricciarelli et al., 2002; Traber, 2004, 2006, 2010; Traber and Atkinson, 2007;
Traber et al., 2008; Zingg and Azzi, 2004).
V. THE TOCOCHROMANOL PATHWAY IN

PHOTOSYNTHETIC ORGANISMS
The majority of the tocochromanol biosynthetic pathway shown in Fig. 3

was elucidated from a series of elegant radiotracer studies using isolated
chloroplasts and cyanobacteria during the mid-1980s (Fiedler et al., 1982;
Soll, 1987; Soll and Schultz, 1979, 1980; Soll et al., 1980a,b, 1985). Through
these experiments, it was demonstrated that all the pathway activities shown,
with the exception of p-hydroxyphenylpyruvic acid dioxygenase (HPPD),
were localized to the chloroplast and often highly enriched in the plastid
envelope. Because all plant cells contain plastids, all cells of the plant have
the capacity to synthesize tocochromanols and thus, unlike animals, neither
are transport mechanisms between tissues and cells required nor have they
been reported in plants. Tocochromanols are present at relatively high levels
in all plastid membranes, but there have been reported low levels of toco-
chromanols in extraplastidic membranes on the basis of cell fractionation
studies (Caro and Puntarulo, 1996; Dilley and Crane, 1963; Rautenkranz
et al., 1994; Yamauchi and Matsushita, 1976), but the possibility of low-level
contamination with plastid membranes could not be excluded. The clear
exceptions to these concerns are studies of oil bodies, which are derived
from the endoplasmic reticulum. Oil bodies from sunflower and oat seed
can be isolated to high purity and, when analyzed for compositions, were
found to contain 20–40% of the total tocochromanols of the seed tissue (Fisk
et al., 2006; White et al., 2006). How tocochromanols may come to reside in
oil bodies is a topic explored in more detail later in this chapter.
VITAMIN E 189
CH3
CH3 CH3 HO
H
H
PPO 4 HPP
HO 3 GGPP CH2COCOOH
Phytol O2
VTE5 4 CH3 CH3

CO2 1 PDS1
H
3
PO 3
HO
Phytyl-P CH3 CH3 HGA CH3
H OH
5 PPO 3
CH2COOH PPO 9
H
Phytyl-PP Solanesyl-PP
CO2 + PPi CO2 + PPi
VTE2 2 6 PDS2
CH3 CH3 CH3
Solanesyl (derived) side chain

Phytyl (derived) side chain
HO H HO H
3 9
OH OH
MPBQ MSBQ
CH3 CH3
SAM SAM
7 VTE3 7 VTE3
CH3 CH3 CH3
HO H HO H
3 9
H3C OH OH
DMPBQ H3C PQ-9
CH3 CH3
8 VTE1 8 VTE1 8 VTE1
HO HO HO
CH3 CH3 CH3
H H H
O H3C O H3C O
3 3 8
CH3 CH3 CH3
CH3 CH3 CH3
d-Tocopherol g -Tocopherol PC-8
SAM SAM
9 VTE4 9 VTE4
CH3 CH3
HO HO
CH3 CH3
H H
O 3 H 3C O 3
CH3 CH3
CH3 CH3
b-Tocopherol a-Tocopherol
Fig. 3. Tocochromanol biosynthetic pathway in Arabidopsis. This representation

of the tocochromanol biosynthetic pathway highlights the tocopherol (phytyl-derived
side chain, green box) and plastochromanol-8 (solanesyl-derived side chain, red box)
branches of the pathway. The two routes that can produce phytyl-PP for tocopherol
synthesis are in the blue box. Tocotrienol synthesis is not shown but differs from the
pathway shown only in the addition of GGPP to HGA at reaction 2 by homogentisate
geranylgeranyl transferase to yield MGGBQ. All subsequent steps and enzymes use
the equivalent GGPP-derived intermediates to produce the four corresponding toco-
trienols. 1: HPP dioxygenase, 2: HGA phytyl transferase, 3: geranylgeranyl reductase,
4: phytol kinase, 5: phytylphosphate kinase, 6: HGA solanesyl transferase, 7: MPBQ/
MSBQ methyl transferase, 8: tocopherol cyclase, 9: -tocopherol methyl transferase.
DMPBQ, 2,3-dimethyl-6-phytyl-1,4-benzoquinol; GGPP, geranylgeranyldipho-
sphate; HGA, homogentisic acid; HPP, p-hydroxyphenylpyruvic acid; MPBQ,
2-methyl-6-phytyl-1,4-benzoquinol; MSBQ, 2-methyl-6-solanesyl-1,4-benzoquinol;
PC-8, plastochromanol-8; PQ-9, plastoquinol-9; SAM, S-adenosylmethionine. With
the exception of GGPP synthase, each reaction of the Arabidopsis tocochromanol
pathway is encoded by a single gene (in orange). Some pathway genes have been
The plant tocochromanol biosynthetic pathway utilizes cytosolic aromatic

amino metabolism for head group synthesis and what is now known to be the
plastidic deoxyxylulose 5-phosphate pathway for the synthesis of hydrophobic,
isoprenoid-derived tail groups. The first step in tocochromanol synthesis
involves the production of the aromatic head group, homogentisic acid
(HGA), from p-hydroxyphenylpyruvic acid (HPP) by the enzyme HPPD
(Garcia et al., 1997, 1999; Kleber-Janke and Krupinska, 1997; Norris et al.,
1995, 1998). This is a complex, irreversible enzymatic reaction catalyzing addi-
tion of two oxygen molecules, a decarboxylation and rearrangement of the side
chain of HPP. HGA is a substrate for prenylation with either phytyl diphos-
phate (phytyl-PP) or GGPP to yield the first committed intermediates in to-
copherol and tocotrienol synthesis, 2-methyl-6-phytylbenzoquinol (MPBQ)
and 2-methyl-6-geranylgeranylbenzoquinol (MGGBQ), respectively. MPBQ
and MGGBQ are substrates for either tocopherol cyclase or MPBQ methyl-
transferase (MPBQ MT). MPBQ MT adds a second methyl group to form
2,3-dimethyl-6-phytyl-1,4-benzoquinol (DMPBQ) from MPBQ or 2,3-dimeth-
yl-6-geranylgeranyl-1,4-benzoquinol (DMGGBQ) from MGGBQ. Tocopher-
ol cyclase converts MPBQ and DMPBQ to - and -tocopherols, respectively,
and the corresponding geranylgeranylated intermediates (MGGBQ and
DMGGB) to - and -tocotrienols. Finally, -tocopherol methyltransferase
(-TMT) adds a methyl group to C-6 of the chromanol ring converting - and -
tocopherols and tocotrienols to - and -tocopherols and tocotrienols,
respectively.
VI. BIOCHEMICAL GENOMICS ENABLED

THE CLONING OF TOCOCHROMANOL
PATHWAY ENZYMES
While the above enzymatic steps and reaction sequence for the tocochromanol
pathway were well defined by the mid-1980s, further molecular and genetic
advances remained limited by the recalcitrance of pathway enzymes to classical
identified independently by multiple groups and hence may have different historical
loci names in the literature than those shown (e.g. the VTE3 mutant locus is also
named HD, for high delta tocopherol, and APG1 for albino or pale green1). Similarly,
alleles for specific loci are not uniform in the literature and are sometimes duplicated.
A unified nomenclature for all published genes and mutants for both tocochromanol
biosynthetic genes and mutants has been published and is used in this figure and
throughout the text (Mène-Saffrané and DellaPenna, 2010). PDS1 and 2, phytoene
desaturase1 and 2; VTE1 to 5, vitamin E1 to 5. At least one biosynthetic mutant has
been characterized for all the cloned biosynthetic genes highlighted in orange.
VITAMIN E 191
biochemical purification and significant progress in this regard did not occur
until the late 1990s. The onset of high-throughput DNA sequencing and geno-
mics during this period allowed researchers to determine the genome sequences
of a variety of organisms, including plants and cyanobacteria, which set the
stage for accelerating the identification and functional analysis of genes of
relevance to nutritional quality in plants, including those needed to understand
and manipulate tocochromanol synthesis. By combining the rapidly growing
genome sequence databases, high-throughput genotyping, genome wide expres-
sion analyses and metabolite profiling, researchers can first develop a knowl-
edge base and identify genes for pathways in model systems and then efficiently
bridge the information and research into agricultural crops. Indeed, there are
numerous examples in this book of this approach being used to improve the
micronutrient content of world agricultural crops and thereby address the
underlying basis of micronutrient malnutrition, especially for developing
countries. The term ‘‘Nutritional Genomics’’ has been coined to describe such
work at the interface of plant metabolism, genomics and human nutrition
(DellaPenna, 1999).
During the past decade, our understanding of the molecular genetics of
tocochromanol synthesis has become increasingly sophisticated. Several
groups have targeted the tocochromanol pathway with approach described
above, focusing primarily on two complementary model systems, the cyano-
bacterium Synechocystis PCC6803 and Arabidopsis thaliana, such that all the
core pathway enzymes in Fig. 3 have been isolated and studied in detail
(Bergmuller et al., 2003; Cahoon et al., 2003; Cheng et al., 2003; Collakova
and DellaPenna, 2001, 2003a,b; Gilliland et al., 2006; Kanwischer et al.,
2005; Motohashi et al., 2003; Norris et al., 1995; Porfirova et al., 2002;
Sattler et al., 2003, 2004; Schledz et al., 2001; Shintani and DellaPenna,
1998; Shintani et al., 2002; Tsegaye et al., 2002; Valentin et al., 2006; Van
Eenennaam et al., 2003). It is important to note that with the exception of
MPBQ MT, the first methyl transferase of the pathway, the enzymes and
genes for tocochromanol synthesis in plants and cyanobacteria share signifi-
cant homology, consistent with the endosymbiotic origin of plastids, and this
has greatly facilitated the genomics-driven isolation of orthologues between
the two organism groups. This is likely to continue to be a recurring theme in
plant biochemistry in the coming years.
A. SYNTHESIS OF THE TOCOCHROMANOL AROMATIC HEAD GROUP
The first step of the plant tocochromanol biosynthetic pathway to be cloned

was HPPD, the product of the PhytoeneDesaturase1, PDS1, locus in Arabi-
dopsis (Norris et al., 1998; reaction 1 in Fig. 3). HPPD catalyzes synthesis of
HGA from p-hydroxyphenylpyruvate that is derived from tyrosine or pre-

phenate by the activity of tyrosine amino transferase (TAT) or prephenate
dehydrogenase, respectively (Fig. 4). Disruption of HPPD activity in the pds1
mutant of Arabidopsis demonstrated that it is necessary for synthesis of both
tocopherol and plastoquinone in plants, and hence the mutant is albino and
soil lethal. Interestingly, disruption of the Synechocystis HPPD orthologue
(slr0090) was not lethal and only impacted tocopherol synthesis, indicating
that plastoquinone synthesis in Synechocystis sp. PCC6803 is both HGA
independent and different from that in plants (Dahnhardt et al., 2002;
Schledz et al., 2001). The plastoquinone biosynthetic pathway in Synecho-
cystis remains to be elucidated. Given the location of HPPD in the tocochro-
manol pathway, it seemed a likely candidate for an activity regulating
pathway flux for headgroup synthesis. To test this hypothesis, HPPD was
overexpressed in Arabidopsis seed and leaves resulting in a 20-fold increase
in activity but only a 15% and 30% increase in seed and leaf tocopherols,
respectively (Tsegaye et al., 2002). Similarly, modest increases were reported
in seed tocochromanol levels when overexpressed enzyme was targeted to the
cytosol or plastid of tobacco (Falk et al., 2003, 2005). These data indicated
that though required, HPPD activity alone is not a significant limitation to
tocochromanol flux.
An alternative approach to engineering head group flux yielded greatly
increased levels of tocotrienols in seed and leaves of various plants. In plants,
the production of HPP, the substrate for HPPD, is tightly regulated by
(–)
PAT ADeH TAT

Prephenate Arogenate Tyrosine HPP
Engineered feedback insensitive TyrA
Tocopherols HPPD
HGA
Tocotrienols
Fig. 4. Pathway for synthesis of the aromatic head group of tocochromanols

leading from prephenate to homogentisic acid (HGA). Feedback inhibition of the
plant arogenate dehydrogenase (AdeH) by tyrosine is indicated by a dotted line. The
activity of the feedback-insensitive TyrA enzyme which converts prephenate directly
to HPP is indicated by a grey box and dashed line. HPP, p-hydroxyphenylpyruvate;
HPPD, HPP dioxygenase; PAT, prephenate amino transferase; TAT, tyrosine amino
transferase.
VITAMIN E 193
feedback inhibition of arogenate dehydrogenase by its product tyrosine

(Fig. 4). This allosteric regulation was bypassed by engineering a naturally
feedback-insensitive, bifunctional prephenate dehydratase from Saccharo-
myces cerevisiae, TyrA, for overexpression in tobacco (Karunanandaa
et al., 2005; Rippert et al., 2004). TyrA catalyzes HPP synthesis directly
from prephenate but had little impact on tocochromanol levels or content
when overexpressed in plants. However, coexpression of TyrA with Arabi-
dopsis HPPD yielded an eightfold increase in total leaf tocochromanol levels
due almost entirely to accumulation of various tocotrienols, which are nor-
mally only produced in tobacco seed (Rippert et al., 2004). Similar results
were obtained by seed-specific co-overexpression of TyrA and HPPD in
Arabidopsis, canola and soybean: two- to threefold increases of predomi-
nantly tocotrienols (Karunanandaa et al., 2005). These results suggest that
flux to HGA is indeed limiting for tocochromanol synthesis but that to
alleviate this bottleneck requires increases in both HPPD activity and flux
to HPP. However, it is still difficult to explain why co-overexpression of
TyrA and HPPD to yield HGA, which is a substrate for both tocopherol and
tocotrienol synthesis, specifically increases tocotrienols. One possibility is
that the high HGA level in transgenics specifically induces a GGPP-utilizing,
tocotrienol-producing homogentisate geranylgeranyl transferase (HGGT).
In this regard, it should be noted that Arabidopsis and soybean seed from
TyrA/HPPD overexpressing plants were black due to oxidative polymeriza-
tion of HGA (present at 60- and 800-fold higher levels, respectively, than
wild types; Karunanandaa et al., 2005), and perception of this as an oxidative
stress by the seed may also have contributed to induction of the pathway.
B. PRENYLATION OF HGA FOR TOCOCHROMANOL

AND PLASTOQUINONE SYNTHESIS
Homogentisate prenyl transferases catalyze the committed steps in tocochro-

manol (and plastoquinone) synthesis, producing tocopherols if phytylpyro-
phosphate (PDP) is the activated prenyl group, tocotrienols if
geranylgeranylpyrophosphate (GGPP) is the activated prenyl group and
plastoquinone if solanesyl pyrophosphate (SDP, C45) is the activated sub-
strate (see Fig. 3). Homogentisate phytyl transferase (HPT) catalyzes con-
densation of HGA and PDP (reaction 2 in Fig. 3) to form 2-methyl-6-phytyl-
benzoquinol (MPBQ), the committed intermediate of all tocopherols. HPT
was cloned utilizing whole genome information from Synechocystis sp.
PCC6803 and Arabidopsis (Collakova and DellaPenna, 2001; Savidge
et al., 2002; Schledz et al., 2001) based on the hypothesis that HPT should
show similarity to related cyanobacterial and plant prenyltransferases that
utilize similar prenyl-DPs as substrates, such as chlorophyll synthase (ChlG),

which attaches PDP or GGPP to chlorophyllide. Query of the Synechocystis
sp. PCC6803 genome database with the ChlG sequence identified several
candidate genes including one, slr1736, with 20% protein identity to
ChlG. Disruption of the slr1736 locus eliminated production of all tocopher-
ols and pathway intermediates in Synechocystis without affecting plastoqui-
none levels. An Arabidopsis orthologue (encoded by the VTE2 locus) was
isolated, and the bacterial and plant enzymes were expressed in E. coli and
assayed. VTE2 was shown to utilize phytyl-DP, while SLR1736 could utilize
both PDP and GGPP, an intriguing result given Synechocystis does not
accumulate tocotrienols (Collakova and DellaPenna, 2001). Mutation of
the VTE2 locus resulted in complete tocopherol deficiency in all tissues
demonstrating it is the only activity for the synthesis of tocopherols
in Arabidopsis (Sattler et al., 2004). VTE2 was subsequently shown to be a
limiting activity in unstressed Arabidopsis as overexpression of the
enzyme increased total tocopherol levels up to five- and twofold in leaves
and seeds, respectively (Collakova and DellaPenna, 2003a; Van Eenennaam
et al., 2003).
Isolation of VTE2 paralogs with 40–50% identity to Arabidopsis VTE2
from various monocots led to the identification of HGGT and demonstrated
the key role of the enzyme in determining the tocotrienol composition of a
tissue (Cahoon et al., 2003; Hunter and Cahoon, 2007). Overexpression of a
barley enzyme in Arabidopsis leaves and maize embryos increased tocotrie-
nols up to 15- and 6-fold of the total tocotrienol content without impacting
tocopherols. This result confirms that the other tocopherol biosynthetic
enzymes present in Arabidopsis, a species that does not synthesize tocotrie-
nols, are able to utilize tocotrienol intermediates as substrates, and also
suggests there is a separate pool of GGPP available for tocotrienol synthesis
that is regulated independently of the PDP pool needed for tocopherol
synthesis. While this experiment showed the tremendous possibility for
manipulating tocochromanol content, because the bulk of the increase in
these transgenic plants was -tocotrienol, which has low vitamin E activity
relative to -tocopherol, the vitamin E content of these transgenics was
increased less than 50% relative to wild type. A third class of homogentisate
prenyl transferase is that which transfers SDP to yield 2-methyl-6-solanesyl-
benzoquinol (MSBQ; reaction 6 in Fig. 3), the committed intermediate and
immediate precursor to plastoquinone-9 (PQ-9). The gene encoding HGA
solanesyl transferase (HST) has been identified through a bioinformatics
approach and was shown to complement the previously identified PQ-9-
deficient pds2 mutant (Norris et al., 1995; Tian et al., 2007; Venkatesh
et al., 2006). As described in later sections, PQ-9 is also a substrate for
VITAMIN E 195
tocopherol cyclase that yields a third class of tocochromanols in plants,

plastochromanol-8 (Mène-Saffrané and DellaPenna, 2010; Szymanska and
Kruk, 2010a,b; Zbierzak et al., 2010). Once the committed prenyl intermedi-
ates MPBQ, MGGBQ and MSBQ are formed, they can be subject to various
methylations and cyclizations to yield the full spectrum of tocochromanols
found in plants.
C. AN ALTERNATE ROUTE FOR THE PHYTYL-PP USED IN

TOCOPHEROL SYNTHESIS
It had long been assumed that the source of phytyl tail for tocopherol
synthesis was from the stepwise reduction of GGPP (C20) by a GGPP
reductase (Keller et al., 1998; reaction 3 in Fig. 3). That transgenic tobacco
and Synechocystis lines with decreased GGPP reductase activity exhibited
reduced tocopherol levels was consistent with this hypothesis (Havaux et al.,
2003; Shpilyov et al., 2005; Tanaka et al., 1999), though it could not be
excluded that increased ROS resulting from accumulation of geranylgerany-
lated chlorophyll compounds might also play a role in this decrease. Howev-
er, the identification of a novel Arabidopsis mutant that reduces leaf and seed
tocopherols by 80% and 65%, respectively, relative to wild type indicates the
majority of phytyl-DP for tocopherol synthesis in Arabidopsis results from
the reactivation and recycling of phytol, a by-product of chlorophyll degra-
dation (Valentin et al., 2006). The locus (VTE5; reaction 4 in Fig. 3) was
cloned and encodes a protein with similarity to yeast and Arabidopsis
dolichol kinase. When expressed and assayed in E. coli, the VTE5 protein
was shown to have CTP-dependent phytol kinase activity (Valentin et al.,
2006), a second kinase activity acts on the phytyl monophosphate produced
by VTE5 to yield phytyl-DP (Ischebeck et al., 2006; reaction 5 in Fig. 3). The
identification of VTE5 helps explain the inverse correlations between tocoph-
erol levels and chlorophyll degradation during natural and induced leaf
senescence (Rise et al., 1989) and developing canola seed (Goffman et al.,
1999). While the relative contributions of phytyl-DP from GGPP and the
VTE5-based recycling pathway to tocopherol synthesis have not been direct-
ly evaluated, this alternative source of phytyl-DP for tocopherol synthesis
may help explain some surprising pathway engineering results. Recall that
barley HGGT overexpression (Cahoon et al., 2003; Hunter and Cahoon,
2007) caused a large increase in tocotrienol in Arabidopsis without impacting
tocopherol levels. This result could be readily explained if the GGPP and
PDP for the two compound classes were derived from separate precursor
pools, with the majority of PDP for tocopherol synthesis coming from
activation of free phytol rather than reduction of GGPP.
D. THE METHYLTRANSFERASES OF TOCOCHROMANOL SYNTHESIS
The two methyltransferases of the tocochromanol pathway, MPBQ MT and

-TMT (reactions 7 and 9 in Fig. 3), do not appear to affect pathway flux, but
their activities and substrate specificities are essential for determining the
types of tocochromanols made, and hence the vitamin E activity of a given
tissue (reviewed in Clemente and Cahoon, 2009; DellaPenna, 2005a,b;
DellaPenna and Last, 2006; DellaPenna and Pogson, 2006; Hunter and
Cahoon, 2007; Mène-Saffrané and DellaPenna, 2010; Sattler et al., 2004).
Synechocystis -TMT (slr0089) was the first tocochromanol pathway methyl-
transferase to be identified, in part because Arabidopsis HPPD provided a
genomic stepping stone for the cloning and functional analysis (DellaPenna,
1999; DellaPenna and Pogson, 2006; Shintani and DellaPenna, 1998). Brief-
ly, a Synechocystis HPPD orthologue (slr0090) was identified using the
Arabidopsis HPPD sequence as query and found to be part of a 10-gene
operon. It was hypothesized that other tocopherol biosynthetic enzymes
might also be present in the operon and disruption of the adjacent slr0089
gene by homologous recombination resulted in loss of -tocopherol and
accumulation of -tocopherol, the anticipated phenotype for loss of -
TMT activity. Enzymatic analysis of SLR0089 and the Arabidopsis -TMT
orthologue (VTE4) from enzymes expressed in E. coli conclusively demon-
strated the activities and substrate specificities of the two enzymes. Arabi-
dopsis seed contain 95% -tocopherol and seed-specific overexpression of
VTE4 resulted in the near complete conversion of -tocopherol to -tocoph-
erol (an 80-fold increase in -tocopherol) and a ninefold increase in vitamin
E activity (Shintani and DellaPenna, 1998). These results have since been
extended to soybean where overexpression of both Arabidopsis -TMT and
MPBQ MT resulted in near complete conversion of seed -, - and -
tocopherols to -tocopherol with a nearly fivefold increase in the vitamin E
activity (Van Eenennaam et al., 2003). These combined experiments provide
a graphic example of the power of integrating studies from model systems
with agricultural crops and have inspired similar research in other crops
(Crowell et al., 2008; Tang et al., 2011; Yusuf and Sarin, 2007).
MPBQ MT was initially identified in Synechocystis based on sequence
similarity to -TMT. When the enzyme was expressed in E. coli, it was
found to use MPBQ and MSBQ but not - or -tocopherols as substrates
(Cheng et al., 2003; Shintani et al., 2002). Disruption of the sll0418 gene
nearly eliminated -tocopherol accumulation but had no effect on plastoqui-
none synthesis, again consistent with separate pathways for plastoquinone
and tocopherol synthesis in Synechocystis. Surprisingly, unlike all other
tocochromanol pathway steps, the SLL0418 protein sequence was not useful
VITAMIN E 197
for identifying a plant orthologue. Instead, two research groups used map-
based cloning approaches to isolate mutant alleles for Arabidopsis MPBQ
MT (the VTE3 locus; Cheng et al., 2003; Van Eenennaam et al., 2003). VTE3
has less than 20% amino acid identity to sll0418, but both proteins displayed
similar activities towards tocopherol and plastoquinone pathways substrates
(Cheng et al., 2003), suggesting convergent evolution for this step of the
pathway in cyanobacteria and plants. Based on available genomic data, it
appears that VTE3 arose from lateral gene transfer from an Archeabacteria
early during plant evolution (Cheng et al., 2003). Unlike the sll0418 mutant,
the phenotypes of various vte3 mutants showed that partial loss of function
affected both tocopherols and plastoquinone synthesis, while strong mutants
were lethal (Cheng et al., 2003; Motohashi et al., 2003; Van Eenennaam et al.,
2003). These data make it clear that there are no redundant activities for
MPBQ MT in Arabidopsis and that the enzyme is active towards both
intermediates in tocopherol, tocochromanol and plastoquinone biosynthesis
(MPBQ, MGGBQ and MSBQ, respectively). VTE3 and SLL0418 have
identical activities and substrate specificities in vitro but less than 20%
amino acid identity and represent a clear case of convergent evolution
(Cheng et al., 2003). Interestingly, the Chlamydomonas genome is unique
in containing orthologues for both SLL0418 and VTE3.
E. THE TOCOPHEROL CYCLASE ENZYME
The tocopherol cyclase (reaction 8 in Fig. 3) was first identified in Arabidop-

sis based on mutations that eliminated tocopherols but resulted in accumu-
lation of the pathway intermediate, DMPBQ, in leaves and seed (Porfirova
et al., 2002; Sattler et al., 2003). The mutated gene was isolated by chromo-
some walking and the encoded protein expressed in E. coli and its activities
characterized. An orthologue in Synechocystis, slr1737, was also identified
and found to be in a two ORF operon with the HPT gene (slr1736). Perhaps
even more surprising, a maize tocopherol cyclase orthologue, whose activity
was unknown at the time, had been previously cloned and studied 2 years
prior based on the negative impact of the mutated locus on carbon translo-
cation in maize leaves (Provencher et al., 2001). The maize gene was original-
ly designated SXD1 for sucrose export defective 1 and suggested tocopherols
have impacts beyond acting as lipid-soluble antioxidants in plants: in this
instance by somehow regulating carbon translocation from source tissues to
sink tissues. The phenotype resulting from RNAi of tocopherol cyclase
expression in potato was similar to maize sxd1, suggesting such functions
for tocopherols may be conserved in plants (Hofius et al., 2004).
Initial reports of overexpression of tocopherol cyclase in Arabidopsis

leaves produced results that were surprising and difficult to reconcile with
what was known about the pathway: leaf tocochromanols were increased
sevenfold due solely to -tocopherol, rather than -tocopherol, the major
tocochromanol normally found in Arabidopsis leaves (Kanwischer et al.,
2005). This was interpreted as a limitation in -TMT activity in the tocoph-
erol cyclase overexpressor (Kanwischer et al., 2005) which was puzzling as
overexpression of HPT and -TMT, singly and in combination, had demon-
strated -TMT activity only becomes limiting when plants are stressed
(Collakova and DellaPenna, 2003a,b; Shintani and DellaPenna, 1998).
Subsequent reports from other groups of tocopherol cyclase overexpression
singly or in combination with HPT and HPPD in transgenic rapeseed showed
increases in tocopherols and a 2.4-fold increase in plastochromanol-8, a
tocochromanol produced from cyclization of PQ-9, that co-migrates with
-tocopherol in many HPLC systems (Kumar et al., 2005; Raclaru et al.,
2006), including that used in Kanwischer et al., 2005. Recently, reassessment
of the biochemical status of the Arabidopsis tocopherol cyclase overexpr-
essors confirmed that the tocochromanol increase originally attributed to
-tocopherol is due almost entirely to increased plastochromanol-8
(Zbierzak et al., 2010).
VII. ENGINEERING MULTIPLE STEPS OF

THE PATHWAY AND APPLICATION
TO AGRICULTURAL CROPS
A small number of studies have utilized overexpression of multiple trans-
genes to modify both tocochromanol content and compositions in plants,
including agricultural crops. The consequences of overexpressing Arabidop-
sis HPT and -TMT on seed and leaf tocopherol content and composition
were described earlier. When HPT and -TMT overexpression lines were
crossed and double homozygotes selected, the two traits (increased total
tocopherol content and increased -tocopherol, respectively) were found to
be additive in the progeny: more total tocopherol was produced and virtually
all of it was converted to -tocopherol resulting in a 12-fold increase in the
vitamin E activity of Arabidopsis seed. Multiple tocopherol transgenes
expressed in Brassica napus also showed some degree of additive effects
(Raclaru et al., 2006). One of the best examples of the additive nature of
pathway enzymes was reported by Van Eenennaam et al. (2003) in which
coexpression of Arabidopsis -TMT and MPBQ MT in soybean seed
resulted in near complete conversion of -, - and -tocopherols to
VITAMIN E 199
-tocopherol. The resulting fivefold increase in vitamin E activity of the

transgenic soybean oil represents one of the clearest examples of Nutritional
Genomics being applied directly from a model plant (Arabidopsis) to an
agricultural crop (soybean). Finally, in a metabolic engineering tour de force,
Karunanandaa et al. (2005) utilized various combinations of the enzymes
described in this chapter (HPPD, TyrA, GGPP reductase and HPT) from
different biological sources with a variety of promoters in a systematic
approach to engineering the pathway in soybean leading to a 15-fold increase
in total tocochromanol levels from 320 ng/mg in WT seed to 4800 ng/mg in
the best combined transgenic, though 94% of this was as tocotrienols. None-
theless, crossing these lines to the -TMT and MPBQ MT soybean over-
expression lines resulted in a line with 11-fold higher vitamin E activity
than wild-type soybean.
VIII. POTENTIAL FOR BREEDING PLANTS WITH

IMPROVED VITAMIN E CONTENT
While transgenic approaches have clear potential to allow targeted altera-

tions in tocochromanol content and composition to affect the vitamin E
content of staple foods, the large body of knowledge about the genes,
pathway and proteins for tocochromanol synthesis in plants should allow
researchers to begin to connect genotype with phenotype and select from
natural variation present in crops for enhanced vitamin E content to improve
food and feed. Variation has been reported for tocopherol content and
composition in Arabidopsis, evening primrose, borage, Andean potato, oil-
seed rape, sunflower, maize, wheat, soybean and tomato (Almeida et al.,
2011; Andre et al., 2007; Chander et al., 2008; Gilliland et al., 2006; Goffman
and Galletti, 2001; Hass et al., 2006; Lampi et al., 2010; Lampi et al., 2008;
Li et al., 2010; Melendez-Martinez et al., 2010; Tang et al., 2006). In some
cases, QTL analyses have been performed for various tocochromanol
traits and, most recently, with the availability of tocochromanol pathway
biosynthetic genes, some QTL intervals have been associated with known
pathway genes as strong candidates for the QTL, most often MPBQ MT
and -TMT (Almeida et al., 2011; Chander et al., 2008; Hass et al., 2006;
Li et al., 2010).
Because of its fully sequenced genome and excellent molecular genetic
resources, a study of two QTL populations in Arabidopsis for tocochroma-
nol traits in seed was quite informative and, like the cloning and manipula-
tion of tocochromanol pathway genes, may provide a glimpse of what is to
come in the future for natural variation of tocochromanols in agricultural
crops (Gilliland et al., 2006). In this study, 14 QTL were identified in the two
populations, likely representing at least 12 loci. Less than half of these QTL
contained known tocochromanol or MEP (methyl-erythritol phosphate)
pathway biosynthetic genes as candidates in their intervals, and most impor-
tantly, several of the QTL with the largest explanation of a trait lacked
known candidate genes in their intervals. This suggests that although the
genes for the tocochromanol pathway in Fig. 3 are all now known, they
represent only half of the loci responsible for the natural variation of toco-
chromanol content and composition in Arabidopsis seed. Analysis of three
additional Arabidopsis populations by the author’s laboratory since this
publication suggests the percent of QTL for tocochromanol traits in Arabi-
dopsis that are explained by variation at biosynthetic loci is closer to 30%.
The rapid production of genome sequences for major agricultural crops,
high-throughput genotyping by sequencing of mapping progeny and culti-
vars combined with the development of association and nested association
mapping approaches in crops should greatly accelerate the identification and
utilization of genes and alleles responsible for natural variation of tocochro-
manols and other essential nutrients in food crops (Gore et al., 2009; Harjes
et al., 2008; Yu et al., 2006, 2008).
IX. PROGRESS IN ELUCIDATING TOCOCHROMANOL

FUNCTIONS IN PHOTOSYNTHETIC ORGANISMS
Like animal cells, there is no known enzyme in plants for which tocochro-
manols are a cofactor, and hence, mechanistic definition of the tocochroma-
nol functions in plants suffers from the same difficulties as animals. Plants,
especially model organisms like Arabidopsis, do have all the molecular and
genetic resources that the best animal systems have, and in addition, several
advantages. Unlike animals, plants produce tocochromanols in each cell
rather than consuming and transporting them throughout the body. Also,
unlike animals, complete tocopherol deficiency can be generated as a result of
mutating core pathway enzymes like HPT or tocopherol cyclase and this is
not lethal. Thus, plant researchers have several distinct advantages over their
animal counterparts: they can produce and utilize tocopherol-deficient plants
or plants in which only specific tocopherols can accumulate and use these to
assess the consequences of this absence or alteration and hence deduce more
directly the possible functions of tocochromanols. Moreover, additional
mutations affecting other antioxidants, ROS scavenging systems or other
biochemical pathways can be introduced into tocopherol mutant back-
grounds and any effects of these multiple genetic deficiencies assessed
VITAMIN E 201
in vivo. Finally, because of the fecundity of plants and the ease of generating
large mutant populations in homogenous genetic backgrounds, it is possible
to perform large genetic suppressor screens to provide unbiased genetic
analysis of tocochromanol function.
Like animals, tocopherols are thought to play an important role as lipid-
soluble antioxidants in plants. -Tocopherolquinone has been detected in a
number plant species over the past 30 years and identified as a tocopherol
oxidation product (Gruszka et al., 2008; Kruk and Strzalka, 1995; Kruk
et al., 2008; Threlfall and Whistance, 1977; Velasco et al., 2000). Unbiased
metabolite profiling of tocopherol oxidation products in Arabidopsis
(Kobayashi and DellaPenna, 2008) is consistent with -tocopherolquinone
being the most abundant oxidation product of -tocopherol in plants.
In response to high light treatment, wild-type plants accumulate only
-tocopherolquinone as the sole tocopherol oxidation product, whereas the
vte4 mutant (which contains only -tocopherol due to a defective -TMT
gene) accumulated only -tocopherolquinone. Introduction of high light
treated plants in darkness caused a reduction in the level of both compounds
with -tocopherolquinone levels in wild-type plants decreasing with much
faster kinetics than -tocopherolquinone in the vte4 mutant (T½ of 3 h vs.
> 12 h, respectively), suggesting an enzymatic process with higher specificity
for -tocopherolquinone. To address whether the -tocopherolquinone
produced was degraded or recycled back to the corresponding tocopherol,
14
C-labelled-tocopherolquinone was incubated with isolated chloroplasts.
Incubation with wild-type chloroplasts resulted in the formation of 14C-
labelled -tocopherol, while incubation with chloroplasts from the vte1-1
mutant, which is defective in tocopherol cyclase activity, led to accumulation
of 14C-labelled trimethylphytylbenzoquinone (TMPBQ), a substrate for TC.
These data conclusively demonstrated the existence of a plastid-based enzy-
matic mechanism that recycles the primary tocopherol oxidation product in
plants, -tocopherolquinone, back to -tocopherols (Fig. 5). This process is
similar to the reversible oxidation and reduction cycles of other well-studied
antioxidants in plants (e.g. ascorbate and glutathione).
A. TOCOCHROMANOL FUNCTIONS DURING SEED DESICCATION,

STORAGE AND SEEDLING ESTABLISHMENT
Seed are the plant tissues that generally contain the highest levels of toco-
chromanols by a wide margin (DellaPenna and Pogson, 2006; Grusak and
DellaPenna, 1999), and this trait is evolutionarily conserved among plant
species, suggesting an important function for tocochromanols in seed. The
first genetic evidence demonstrating an essential role for tocochromanols in
HO
HO O O
Chemical R 2e–/–H2O R
O R quenching Unknown O
O
dehydratase
a-Tocopherol a-TQ TMPBQ
VTE1
H
R: 3
Fig. 5. Proposed pathway for tocopherolquinone recycling in plants. -Toco-

pherolquinone (-TQ or its reduced equivalent) produced by chemical scavenging
of singlet oxygen or two electron oxidation by -tocopherol is dehydrated by an as yet
unidentified dehydratase enzyme(s) to produce trimethylphytylbenzoquinone
(TMPBQ, or its reduced equivalent) which is then a substrate for cyclization by
tocopherol cyclase (VTE1) to form -tocopherol (Stocker et al., 1996).
seed resulted from analysis of the germination levels of tocopherol-deficient

mutants (vte2-1, vte2-2 and vte1-1) subjected to an accelerating ageing treat-
ment (Sattler et al., 2004), which combines moderate heat (40 8C) and high
relative humidity (100% RH). In response to this treatment, germination
levels of vte2-1, vte2-2 and vte1-1 seed were drastically reduced by more than
95%, while those of WT (Col-0 and Ws ecotypes) were only reduced 20%.
Moreover, germination levels of untreated control vte seed were not signifi-
cantly different from WT demonstrating that tocochromanols play a role in
seed longevity.
In addition to this clear seed longevity phenotype, both vte2-1 and vte2-
2 mutants (but not vte1-1) exhibit a range of developmental phenotypes
during germination and seedling establishment (Sattler et al., 2004). The
vte2 mutants lack all tocopherols and all pathway intermediates (MPBQ
and DMPBQ) in both leaves and seed. Upon germination, vte2 mutants
exhibited a range of visible abnormalities including cotyledon defects,
bleached cotyledons and inhibition of root growth (Fig. 6; Sattler et al.,
2004) that was associated with enhanced lipid peroxidation as reflected by
a massive accumulation of fatty acid hydroperoxides and hydroxides, phy-
toprostanes and malondialdehyde (Sattler et al., 2004, 2006). Chiral HPLC
separation of lipid hydroxides isolated from vte2 seedlings showed that they
occur at extremely high levels and as a racemic mixture of almost equal
amounts of cis/trans and R/S isomers, the hallmark of free-radical-based
lipid oxidation (vs. enzymatic oxidation which generates only one type of
enantiomer; also refer to Fig. 2; Sattler et al., 2004). Gene-expression
profiling of vte2 seedlings with Affymetrix full genome chips showed that
VITAMIN E 203
A B
Individual siliques harvested when Seed harvested from fully dried

they first turn brown contain plants are a mixture of siliques
seed dried for 1–2 weeks. (and seed) dried for 2–10 weeks.
100% 10–30%
40–60%
30–50%
Fig. 6. Effect of seed harvesting methods on penetrance of the vte2 seedling

phenotype. (A) vte2 seed from siliques harvested individually 1–2 weeks after they
first turn brown (red triangles) produce seedlings (100%) without any visible pheno-
type or lipid oxidation (refer to ‘‘fresh seed’’ in Table III). (B) Seed harvested from
siliques of fully dried plants produce seedlings with variable developmental defects
ranging from no phenotype (10–30%) to seedlings with one (40–60%) or two (30–50%)
necrotic cotyledons (white triangles); bar ¼ 2 mm.
the accumulation of lipid peroxidation products correlated with a strong up-

regulation of numerous genes associated with biotic and abiotic oxidative
stresses, in particular pathogenesis (Fig. 7). Some of the lipid oxidation
products accumulated in vte2 seedlings (e.g. malondialdehyde and phyto-
prostanes) have previously been shown to be potent inducers of oxidative
stress-associated gene expression when applied exogenously to plant tissues
Biotic Abiotic
Pseudo.
Drought
Botrytis
Ozone
MeJA
Cold
vte2
vte1
5.0
3.0
2.0
Fold change
1.0
0.5
0.2
0.0
Fig. 7. Analysis of the expression levels of genes up-regulated in vte2 seedlings in

response to various treatments. Expression levels of genes up-regulated in 3-day-old
seedlings of the indicated treatments were retrieved from the public database Gene-
vestigator (Zimmermann et al., 2004) and compared to expression levels of 3-day-old
vte2-1 and vte1-1 seedlings. Up-regulated genes are in red, down-regulated are in blue
and unchanged levels are in yellow. MeJA, methyl jasmonate treatment; Pseudo,
avirulent Pseudomonas syringae treatment; Botrytis, Botrytis cinerea treatment.
(Farmer and Davoine, 2007; Thoma et al., 2003, 2004; Vollenweider et al.,
2000; Weber et al., 2004). The genetic removal of trienoic fatty acids from the
tocopherol-deficient vte2-1 background by introducing three mutated fatty
acid desaturases (fad3-2, fad7-1 and fad8) into the background reduced
malondialdehyde accumulation during germination by 75% and fully sup-
pressed the developmental defects observed in vte2 and the induction of the
oxidative stress marker gene glutathione-S-transferase1 (Fig. 8; Mène-
Saffrané et al., 2007). Collectively, these data demonstrate that essential
functions of tocopherols in planta include controlling non-enzymatic oxida-
tion of PUFAs during germination and early seedling growth that would
otherwise dramatically alter gene expression programmes and negatively
affect seedling establishment and growth.
VITAMIN E 205
Col-0 fad3/7/8
vte2 vte2fad3/7/8
Fig. 8. Genetic removal of trienoic fatty acids suppresses the vte2 seedling pheno-
type. Pictures show representative phenotypes of 12-day-old seedlings grown on ½
MS solid media not supplemented with sucrose at 12 h light/22 8C and 12 h dark/
18 8C. Seed were held quiescent (aged) at room temperature for 3 months prior to
sowing onto Petri dishes. fad3/7/8, fad3-2 fad7-1 fad8 triple fad mutant; bar ¼ 10 mm.
One intriguing aspect of vte2-1 was the wide variation in developmental

phenotypes exhibited by individual germinating seedlings from bulk seed
harvested from a single vte2 plant (Sattler et al., 2004; see Fig. 6B). While
most vte2 seedlings exhibit severe root growth inhibition and defects in
expansion of one or both cotyledons (white arrow heads Fig. 6B), approxi-
mately 10–30% of vte2 individuals are visually indistinguishable from WT,
indicating other factors combine with tocopherol deficiency to determine the
range of phenotypes observed. An important factor impacting the pene-
trance of the vte2 seedling phenotype is the extent of seed quiescence (ageing)
(Mène-Saffrané et al., 2010). When vte2-1 seeds are harvested ‘‘normally’’,
that is, from fully dry plants, the derived seedlings exhibit this variable
phenotype (Fig. 8B) but when siliques were individually harvested 1–2 weeks
after turning brown and these ‘‘fresh seed’’ are planted, they do not exhibit
any germination or lipid oxidation phenotype (Fig. 6A). When ‘‘fresh seed’’
are aged at room temperature for an additional 6 weeks, they uniformly
exhibited the most severe developmental defect shown in Fig. 6B (e.g. bottom
most panel) and a strong lipid oxidation phenotype (Mène-Saffrané et al.,
2010; Table III). This demonstrates that the vte2-1 seedling phenotype results
from the lack of tocopherols and is conditioned by the extent of seed
quiescence (ageing). The negative impact of extending seed quiescence on
vte2 is also clear from the enhancement of lipid oxidation after 60 days of
quiescence (Table IV) and also explains the range of germination phenotypes
in vte2-1 seed collected ‘‘normally’’ from dried plants. Since Arabidopsis
flowering is spread over a period of several weeks, seed collected ‘‘normally’’
from a dried plant are actually a mixture of individual siliques in which the
duration of seed quiescence varies by 6–8 weeks (Fig. 6B).
Despite being tocopherol deficient, the vte2-1 mutant is not completely
tocochromanol deficient as it contains another type of tocochromanol, plas-
tochromanol-8 (PC-8) at approximately 10% of total tocopherols (Mène-
Saffrané et al., 2010). PC-8 is synthesized by the VTE1-dependent cyclization
of PQ-9 (Mène-Saffrané et al., 2010; Zbierzak et al., 2010) and to assess any
role of PC-8 in vivo and the consequences of true total tocochromanol
deficiency in plants, PC-8 was genetically removed from the vte2-1 back-
ground by introducing the vte1-1 or vte1-2 mutations (Mène-Saffrané et al.,
2010). The resulting vte2-1vte1-1 and vte2-1vte1-2 double mutants lack all
tocopherols, pathway intermediates (MPBQ and DMPBQ) and PC-8 and are
thus truly tocochromanol deficient. Upon seedling establishment, both
vte2vte1 double mutants exhibited major developmental problems that
were much more severe than vte2 and characterized by necrotic cotyledons
that failed to expand and remained enclosed by the testa (Fig. 9A and C, red
triangles). Rarely, true leaves emerged between the necrotic cotyledons
(Fig. 9B, white triangle) for which development was drastically reduced.
This very strong seedling phenotype was associated with massive oxidation
of PUFAs, even when fresh vte2vte1 seed were used (Tables III and IV;
Mène-Saffrané et al., 2010).
To more precisely determine the developmental origin of lipid oxidation in
seed, lipid oxidation products were analyzed at various stages of seed devel-
opment, 15, 20, 30 and 60 days after pollination, which correspond to the end
of seed development, the end of desiccation and two different periods of
quiescence (ageing), respectively. Lipid oxidation is initiated during seed
desiccation (15–20 days after pollination) in both vte2vte1 double mutants,
while in the vte2-1 single mutant, it is initiated in quiescence between 30 and
TABLE III
Comparison of Root Growth, Lipid Hydroxide Accumulation and Eicosenoic Acid Metabolism in Arabidopsis ‘‘Fresh’’ and ‘‘Aged’’ Seed,
30 and 60 days After Pollination, Respectively (adapted from Mène-Saffrané et al., 2010)
Seedlings derived from ‘‘fresh seed’’

Seedlings derived from ‘‘fresh seed’’ aged 6 weeks at room temp.
Root LOH 20:1 Root LOH
length (mm) (pmol/nmol FA) (mol%) length (mm) (pmol/nmol FA) 20:1 (mol%)
Col-0 20.9 0.5 2.2 0.5 0.9 0.02 19.1 0.4 1.7 0.28 1.4 0.37
vte1-1 17.5 0.3 1.6 0.3 1.4 0.10 16.5 0.2 2.2 0.27 1.3 0.07
vte1-2 20.8 0.6 1.4 0.2 1.0 0.03 18.6 0.3 1.6 0.07 1.1 0.09
vte2-1 18.4 0.5 1.3 0.2 1.1 0.04 7.9 1.0 72.6 14.7** 22.4 0.54
vte2-1 vte1-1 1.6 0.2 75.8 14.8** 25.5 0.13 0.8 0.1 NA 22.9 0.15
vte2-1 vte1-2 1.6 0.3 64.8 20.9** 24.6 0.42 0.7 0.1 NA 22.7 0.28
Root length was quantified from 7-day-old seedlings grown vertically in vitro (average SEM; mm; n ¼ 40). Total lipid hydroxides were quantified by normal
phase HPLC from 3-day-old seedlings grown in vitro (average SEM; pmol/nmol FA; n ¼ 4). Eicosenoic acid metabolism was quantified by gas chromatog-
raphy-flame ionization detector from 8-day-old seedlings grown in vitro (average SEM; mol%; n ¼ 6). Asterisks represent significance levels using Student’s
t test of each genotype relative to Col of same age;
**, P < 0.01. FA, fatty acid; LOH, lipid hydroxides; 20:1, eicosenoic acid.
TABLE IV
Developmental Progression of Lipid Hydroxide Accumulation in Arabidopsis
Tocochromanol-Deficient Seed (adapted from Mène-Saffrané et al., 2010)
Total LOHs (pmol/nmol FA)

15 DAP 20 DAP 30 DAP 60 DAP
Col-0 2.37 0.14 2.81 0.31 0.07 0.01 0.20 0.04
vte1-1 2.62 0.07 2.83 0.23 0.19 0.01** 0.17 0.04
vte1-2 2.66 0.06 3.18 0.24 0.21 0.06* 0.19 0.03
vte2-1 2.76 0.23 3.43 0.29 0.29 0.03** 13.22 3.67**
vte2-1 vte1-1 2.33 0.24 11.39 0.86** 16.15 1.80** 50.97 5.62**
vte2-1 vte1-2 2.55 0.16 28.57 2.08** 26.12 5.54** 44.13 7.95**
Lipid hydroxides extracted from seed at various developmental stages were analyzed by normal
phase HPLC. Values are average SEM, pmol/nmol FA (n ¼ 5 or 6). 15 DAP, end of seed
maturation and initiation of seed desiccation; 20 DAP, end of seed desiccation and beginning of
quiescence; 30 and 60 DAP, quiescent seed; Asterisks represent significance levels using Student’s
t test of each genotype relative to Col of same age.
*, P < 0.05; **, P < 0.01; DAP, day after pollination; FA, fatty acid.
60 days after pollination. These results show that tocochromanols as a group

prevent oxidation of storage lipid during seed desiccation and quiescence and
that PC-8 is also a lipid-soluble antioxidant that can partially compensate for
the absence of tocopherols in vte2. These data are of particular interest
considering that PC-8 represents only 10% of the total tocochromanol levels
in WT seed, thus confirming the potent lipid-antioxidant activity of PC-
8 previously demonstrated in vitro (Olejnik et al., 1997). Collectively, the
data with vte2 and vte2vte1 mutants make clear why tocochromanol biosyn-
thesis is conserved in all extant seed-bearing plants and particularly abun-
dant in seed. One important function of seed that likely underlies the
evolutionary and ecological success of seed-bearing plants is their capacity
to maintain a viable desiccated embryo for extended periods, in particular, in
ecosystems with temporary non-permissive growth conditions (e.g. drought,
frost). Without tocochromanols, seed have severely compromised desicca-
tion tolerance, limited quiescence and defects in germination and early
growth of seedlings that would severely reduce the fitness of tocochroma-
nol-deficient mutants (Mène-Saffrané et al., 2010).
Despite also being tocopherol deficient, the Arabidopsis tocopherol cy-
clase vte1-1 mutant does not exhibit any of the severe developmental defects
of vte2 seedlings or massive accumulation of fatty acid hydroperoxides and
hydroxides and malondialdehyde (Sattler et al., 2004, 2006). This absence of
phenotype in vte1 mutant correlates with the accumulation of high levels of
the pathway intermediate DMPBQ, a redox active quinone that is apparently
capable of protecting PUFAs from non-enzymatic lipid oxidation in the
VITAMIN E 209
vte2-1 vte2-1
A Col-0 vte1-1 vte1-2 vte2-1 vte1-1 vte1-2
B C
Col-0 vte1-1 vte1-2
vte2-1vte1-1
vte2-1 vte2-1vte1-1 vte2-1vte1-2
vte2-1vte1-2
Fig. 9. Seedling phenotypes of tocochromanol-deficient vte1, vte2 and vte2vte1

single and double mutants. (A) Representative phenotypes of 2-week-old seedlings
derived from ‘‘aged’’ seed (60 days after pollination). Twelve seed were planted per
pot to assess the fitness of each genotype. The vte2vte1 double mutants that produce
true leaves and develop into mature plants (see Fig. 10) are circled; bar ¼ 20 mm. (B)
Phenotypes of 6-day-old seedlings deriving from ‘‘fresh’’ seed (30 days after pollina-
tion). With the exception of vte2vte1, all genotypes are indistinguishable. The white
triangle indicates the single vte2vte1 plant in the two images that developed first true
leaves. bar ¼ 1 mm. (C) Close up of vte2vte1 individuals from ‘‘aged’’ seed in panel (A)
that are not circled and will not develop further into adult plants showing their
‘‘stick’’ phenotype with undeveloped cotyledons that remain enclosed in the testa
(red triangle). On average, individuals with this ‘‘stick’’ phenotype represent 90% of
vte2vte1 seedlings derived from ‘‘aged’’ seed, bar ¼ 1 mm.
absence of tocopherols. The lipid-antioxidant property of DMPBQ was also

conclusively demonstrated with the vte2vte1 double mutant previously
described. vte1 mutants lack all tocopherols and PC-8, but accumulate
DMPBQ and do not exhibit any signs of lipid oxidation. Introduction of
the vte2 mutation into the vte1 background (in vte2vte1) also eliminates
DMPBQ from vte1, resulting in the severe developmental and lipid oxidation
phenotypes described earlier.
B. TOCOCHROMANOL FUNCTIONS IN ADULT PLANTS
Surprisingly, the few plants of tocochromanol-deficient Arabidopsis mutant

genotypes that survive germination and early seedling development (vte1
vte2 > vte2vte1 in terms of seedling survival; see Figs. 6 and 9) produce adult
plants that are virtually indistinguishable from wild type (chlorophyll and
carotenoid content, quantum yield of PSII, plant size, etc.) under standard
laboratory growth conditions of 120 mol photons m 2 s 1 (Fig. 10; Mène-
Saffrané et al., 2010; Maeda et al., 2006; Porfirova et al., 2002; Sattler et al.,
2004). These data indicate that unlike animals, in mature plants, tocochro-
manols are apparently dispensable under permissive conditions. Many of the
essential functions of tocochromanols that have been so strongly selected for
during the evolution of seed plants and which are so evident during seed
desiccation, storage and seedling germination of tocochromanol mutants
(Figs. 6–9; Tables III and IV) appear to either not be essential in mature
mutant plants or, more likely, are at least partially compensated for by the
myriad of other ROS defences present in mature photosynthetic tissues.
Attempts to demonstrate a role for tocochromanols in protecting mature
plants from lipid oxidation during severe stresses (a long-assumed role for
tocochromanols in plants) have been less convincing and consistent than for
seeds and seedlings and often variable between plant species. Exposure of
Arabidopsis vte1 and vte2 mutants to high light stress ranging from 850 to
1800 mol photons m 2 s 1 (2000 mol photons m 2 s 1 is full sunlight) did
not lead to obvious or significant differences in photoinhibition, photo-
bleaching or overall visible plant phenotypes relative to wild type (Havaux
Col-0 vte1-1 vte1-2
vte2-1 vte2-1vte1-1 vte2-1vte1-2
Fig. 10. Mature plant phenotype of Arabidopsis wild-type and tocochromanol-

deficient single and double mutants. Pictures show representative mature 8-week-old
plants of the various genotypes that grew beyond the seedling stage on soil with 12 h
light/22 8C and 12 h dark/18 8C.
VITAMIN E 211
et al., 2005; Maeda et al., 2006; Porfirova et al., 2002). It was only when high
light was combined with low temperature that differences were observed
(Havaux et al., 2005), though as described in later sections, because toco-
chromanol-deficient Arabidopsis mutants have a severe, light-independent
low-temperature phenotype, it is difficult to ascribe the reported results solely
to photooxidative stress. Similarly, the Synechocystis vte1 and vte2 knockout
mutants that eliminate tocochromanols (slr1737 and sar1736) were remark-
ably resistant to high light stress, it is only upon exposure to free PUFAs in
the media (18:3 and to a lesser extent 18:2) that they became more sensitive
than wild type to high light treatments (Maeda et al., 2005). Transgenic
tobacco overexpressing TyrA and HPPD (and therefore accumulating ele-
vated levels of tocotrienols) were found to be much less sensitive than wild
type to a combination of low temperature and high light and had significant-
ly higher levels of carotenoids and chlorophylls and lower levels of lipid
oxidation than wild type (Matringe et al., 2008). In contrast, overaccumula-
tion of tocopherols in transgenic Arabidopsis did not lead to alterations in
carotenoids or chlorophylls during high light stress relative to wild type
(Collakova and DellaPenna, 2003b). Finally, tobacco HPT RNAi lines in
which tocopherols were reduced to < 5% of wild-type levels showed a 25%
drop in photosynthetic electron transport in the absence of stress, while those
with > 5% of wild-type levels were indistinguishable from wild type (Abbasi
et al., 2007, 2009). These combined results indicate that tocochromanol-
deficient photosynthetic organisms are surprisingly resistant to high light
stress, suggesting a more limited and variable role for tocopherols in photo-
oxidative protection than had long been assumed.
When the Arabidopsis tocopherol cyclase gene was identified (Porfirova
et al., 2002; Sattler et al., 2003), it was found that the maize orthologue had
been cloned 2 years earlier, though the identity of the encoded maize protein
as tocopherol cyclase was not known at that time (Botha et al., 2000). The
maize mutant was originally designated sxd1 for sucrose export defective 1
and exhibits increased anthocyanin levels in leaves, reduction of growth,
deposition of callose in the phloem parenchyma, massive increases in soluble
sugar content and starch accumulation and decreased photoassimilate export
(Botha et al., 2000; Russin et al., 1996). In potato, suppression of tocopherol
synthesis by RNAi-mediated silencing of the tocopherol cyclase gene also
induces similar defects in photoassimilate export (Hofius et al., 2004). Curi-
ously, only those few transgenic potato lines with < 2% of wild-type toco-
chromanol levels displayed this phenotype, and unlike maize sxd1, where the
phenotype is constitutive, in transgenic potato, loss of photoassimilate trans-
port from source leaves is coincident with developmentally regulated vascu-
lar-specific deposition of callose and anthocyanin accumulation.
While the maize and potato tocopherol mutants both displayed a carbo-
hydrate accumulation phenotype at standard growth temperatures (e.g.
20–25 8C), it was puzzling that the orthologous Arabidopsis vte1 and vte2
mutants did not (Sattler et al., 2003, 2004). However, a carbohydrate accu-
mulation phenotype similar to maize sxd1 was rapidly and strongly induced
by non-freezing low-temperature treatment (7 8C) of adult vte2 mutants, and
to a lesser degree vte1 plants (Fig. 11; Maeda et al., 2006). The rapidity of this
induction and its reversion at permissive temperature allowed the time course
of biochemical, physiological and spatial changes associated with the pheno-
type to be dissected and studied in considerable detail in Arabidopsis, which
was not possible in the constitutive maize and potato mutants (Maeda and
DellaPenna, 2007; Maeda et al., 2006, 2008; Song et al., 2010).
The low-temperature phenotype of Arabidopsis vte2 and vte1 mutants was
coincident with the rapid and specific deposition of callose (as early as 6 h of
cold treatment) in the developing walls of phloem parenchyma transfer cells
adjacent to the companion cell–sieve tube complexes (Maeda et al., 2006).
Unlike the maize sxd1 mutant, plasmodesmata are not affected indicating
that defective transfer cell wall development at low temperature is
WT vte2 fad2 vte2fad2 fad6 vte2fad6
0 Days
at 7 ⬚C
14 Days
at 7 ⬚C
28 Days
at 7 ⬚C
Fig. 11. Phenotype of vte2 adult plants exposed to non-freezing low-temperature

(78C) and suppression of the vte2 cold-induced phenotype by the fad2 and fad6
mutations. Adult plants grown for 4 weeks at permissive temperature (228C, upper
panel) were transferred at 78C and representative individuals photographed 14 and 28
days after the beginning of the cold treatment (middle and lower panel, respectively).
Before the cold treatment (upper panel), none of the genotypes exhibit any obvious
phenotypic differences. However, vte2 individuals are smaller and accumulate
anthocyanins in response to the cold treatment. The vte2-cold induced phenotype is
suppressed totally by the fad2 mutation and partially by the fad6 mutation.
VITAMIN E 213
responsible for the phenotype. The resulting inhibition of photoassimilate

export subsequently leads to carbohydrate and anthocyanin accumulation
and eventually to growth inhibition of whole plants after extended low-
temperature treatment (Fig. 11). Given that tocopherols are lipid-soluble
antioxidants and the primary target of lipid oxidation, PUFAs, are abundant
in plastids, it seemed logical that lipid oxidation, especially in the plastid,
would be involved in the low-temperature phenotype. However, while lino-
lenic acid (18:3) levels and linoleic acid (18:2) levels were significantly de-
creased and increased, respectively, during low-temperature treatment, a
variety of approaches consistently failed to identify any differences in lipid
oxidation between the mutants and wild type during low-temperature treat-
ment (Maeda et al., 2006, 2008). Lipidomic analysis and pulse-chase labelling
of low-temperature-treated vte2 further confirmed the absence of lipid oxi-
dation and also demonstrated that these modifications to membrane lipid
composition occurred primarily on ER resident phosphatidylcholine (PC)
species or specific plastid galactolipid species with fatty acid compositions
that could be directly attributed to fatty acids/diacylglycerol imported into
the plastid from the ER (Maeda et al., 2006).
Genetic approaches to independently test the lipid oxidation hypothesis
provided further support that something much more interesting than lipid
oxidation was occurring in the low-temperature-treated tocopherol mutants.
A series of fatty acid desaturation (fad) mutations were introduced into the
vte2 background to block trienoic (18:3 and 16:3) fatty acid synthesis in the
ER or plastid (i.e. vte2 fad3 and vte2 fad7 fad8, respectively) or completely
eliminate it in both compartments (i.e. the vte2 fad3 fad7 fad8 quadruple
mutant). Surprisingly, none of these genotypes impacted the low-tempera-
ture-induced phenotypes of vte2 (Fig. 12; Maeda et al., 2008), a result that is in
sharp contrast with the complete suppression of the vte2 seedling phenotype
in the vte2 fad3 fad7 fad8 quadruple mutant (Fig. 8; Mène-Saffrané et al.,
2007) indicating the mechanism(s) for the seedling and low-temperature
phenotypes are quite different. These data also conclusively eliminated the
possibility that a host of trienoic fatty acid-derived signalling compounds (e.g.
JA, OPDA, dinor-OPDA and phytoprostanes) play a role in the initiation or
development of the tocopherol-deficient vte2 low-temperature phenotype.
In contrast, to these trienoic fatty acid mutations, the introduction of the
fad2 or fad6 mutations, which affect the conversion of monoenoic to dienoic
fatty acids in the ER and chloroplast, respectively, had dramatic impacts on
the vte2 low-temperature phenotypes: fad6 partially suppressed, while fad2
completely suppressed nearly all vte2 low-temperature phenotypes (Fig. 11).
In a complementary study using suppressor screening of EMS mutagenized
vte2, several loci whose mutation partially or totally alleviated the vte2 the
A B Col fad3fad7fad8
Col fad3fad7fad8
vte2 vte2fad3fad7fad8 vte2 vte2fad3fad7fad8
C D
400 Sucrose 20
Fructose
350 Glucose
16
Sugars (mmol/g FW)
300
% Exudation
250 12
200
8
150
100
4
50
0 0
Col
Col
vte2
fad3fad7fad8
vte2fad3fad7fad8
vte2
fad3fad7fad8
vte2fad3fad7fad8
Fig. 12. Trienoic fatty acid deficiency does not suppress the vte2-cold induced
phenotype. (A) Representative whole plant phenotypes of plants of the indicated
genotypes treated at 7 8C for 4 weeks. (B) Fluorescence microscopy of aniline
blue-stained leaves of the indicated genotypes treated at 7 8C for 7 days. Aniline
blue-positive fluorescence reveals callose deposition is not different in vte2 and
vte2fad3fad7fad8. (C) Leaf soluble sugars from plants of the indicated genotypes
treated at 7 8C for 2 weeks (average SEM, mol/g FW, n ¼ 4). (D) Leaf exudation
as a percentage of total 14CO2 by plants treated of the indicated genotypes at 7 8C for
1 week (average SEM, %, n ¼ 4).
low-temperature phenotype (Song et al., 2010). This included a novel allele of

fad2 and a new allele of trigalactosyldiacylglycerol1 (tgd1), a component of the
ER-to-plastid lipid ATP-binding cassette (ABC) transporter. Introduction of
mutations in the other two ABC transporter components, tgd2 and tgd3, as
well as in tgd4, an ER-localized protein involved in the transport process,
VITAMIN E 215
makes that there is an interaction of tocopherols with non-plastid (ER) lipid

metabolism that is required for low-temperature adaptation (Maeda et al.,
2008; Song et al., 2010).
The accumulated genetic and biochemical evidence from low-temperature-
treated vte2 and vte1 mutants (Maeda et al., 2006, 2008; Song et al., 2010)
makes a strong case that ER lipid metabolism is specifically and significantly
affected by the absence of tocopherols in the vte mutants (and presumably
also the presence of tocopherols in wild type), but it is challenging to envisage
how tocopherols, compounds that are clearly synthesized and presumably
localized in plastids, could affect ER processes. Large fluxes of lipids do
occur between the ER and plastids during plant lipid synthesis presumably
by the TDG pathway (Benning, 2009; Browse et al., 1986). Galactolipids,
which are normally restricted to plastids (Douce, 1974), are found in the
plasma membrane, mitochondria and tonoplasts during phosphate starva-
tion (Andersson et al., 2003) indicating that a yet to be discovered route exists
for export of these lipids from plastids to the endomembranes. Tocopherols
are also present outside the plastid at certain developmental stages, most
notably in ER-derived oil bodies where 20–40% of the total seed tocopherols
reside (Fisk et al., 2006; White et al., 2006) and are deposited there by an as
yet to be determined mechanism. Because all plant cells contain plastids, all
cells are capable of tocochromanol synthesis and thus, unlike animals, nei-
ther are transport mechanisms between tissues and cells required nor have
they been reported in plants. Movement of tocopherols between organelles
or at minimum interaction of biosynthetic enzymes, substrates and com-
pounds like tocopherols between organelles would be required for tocopher-
ols to exert an influence over ER lipid metabolism. A potential route for
transport of all lipid-soluble molecules from the plastid to the ER or to
ER-derived oil bodies is through ER:plastid contact sites known as plastid-
associated membranes or PLAMs (Andersson et al., 2007). Analogous mem-
brane contact sites in animals (Achleitner et al., 1999; Levine, 2004; Pichler
et al., 2001) provide a physical connection allowing specific enzymes, protein
complexes or compounds between the two organelles to interact directly.
That the low-temperature phenotype of Arabidopsis tocochromanol
mutants, and presumably maize and potato mutant as well, does not involve
lipid oxidation and in this regard is reminiscent of the non-antioxidant roles
described earlier for tocopherols in animal cells (Azzi, 2007; Azzi et al., 2004;
Brigelius-Flohe, 2006, 2009; Brigelius-Flohe and Traber, 1999; Zingg and
Azzi, 2004). However, like animal cells, a precise mechanistic understanding
of how tocopherols influence lipid metabolism during low-temperature treat-
ment of Arabidopsis, and at permissive temperatures in potato and maize, is
still lacking. However, the genetic suppression of the phenotype by
disruption of ER lipid desaturation or transport of lipids between the ER and

plastid, along with pulse-chase measurements of reduced incorporation of
label into 18:3 fatty acids specifically attached to PC and PE in the ER,
makes the system in Arabidopsis one of the most approachable for ultimately
describing the presumed non-antioxidant role of tocopherols in plants. In
this regard, it may be that animal and plant researchers studying the func-
tions of tocopherols may have more in common than they now realize and
increased dialogue and research interactions between the two communities is
warranted. The past two decades have witnessed truly amazing progress in
our understanding of plant tocochromanol synthesis, pathway manipulation
and functions and equally impressive progress in the role of these most
interesting compounds in non-photosynthetic systems. It is a near certainty
that the coming decades will bring into much sharper focus the activities of
these molecules in plants and animals alike.
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Vitamin K1 (Phylloquinone): Function, Enzymes and Genes
CHLOË VAN OOSTENDE, JOSHUA R. WIDHALM,

FABIENNE FURT, ANNE-LISE DUCLUZEAU
AND GILLES J. BASSET1
Center for Plant Science Innovation, University of Nebraska-Lincoln,

Lincoln, Nebraska, USA
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
II. Structure and Chemistry of Vitamin K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
III. Biochemical Roles of Vitamin K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
A. Vertebrates..................................................................... 233
B. Plants and Cyanobacteria ................................................... 236
IV. Detection and Distribution of Phylloquinone in Plants . . . . . . . . . . . . . . . . . . 238
A. Detection....................................................................... 238
B. Tissular Distribution ......................................................... 238
C. Subcellular Distribution ..................................................... 238
V. Phylloquinone Biosynthesis in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
A. Early Work .................................................................... 240
B. Isochorismate Synthase/PHYLLO (Reactions 1–4) ..................... 242
C. OSB-CoA Ligase (Reaction 5) ............................................. 244
D. DHNA-CoA Synthase/DHNA-CoA Thioesterase (Reactions 6/7)... 244
E. DHNA Phytyl Transferase (Reaction 8).................................. 246
F. Demethylphylloquinone Methyltransferase (Reaction 9) .............. 246
G. Mutant Phenotype............................................................ 247
H. Subcellular Localization of Phylloquinone Biosynthetic Enzymes ... 247
VI. Evolution of Naphthoquinone Biosynthesis in Photosynthetic
Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
VII. Phylloquinone Turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
1
Corresponding author: E-mail: gbasset2@unl.edu

230 CHLOË VAN OOSTENDE ET AL.
VIII. Engineering of Phylloquinone in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

IX. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
ABSTRACT
Phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone) is a conjugated isoprenoid
that serves as a cardinal redox cofactor in plants and some cyanobacteria. In humans
and other mammals, it is required as a vitamin (vitamin K1) for blood coagulation
and bone metabolism. Until recently, the biosynthesis of phylloquinone in plants was
considered identical to that of menaquinone (vitamin K2) in facultative anaerobic
bacteria. It resulted that most of the plant research on phylloquinone focused histori-
cally on the study of its function, while very little was done on its metabolism per se.
There is today, tough, compelling evidence that plants have evolved an unprecedented
metabolic architecture to synthesize phylloquinone, including extraordinary events of
gene fusion, highly divergent enzymes and a separated compartmentalization in
chloroplasts and peroxisomes. Phylogenetic reconstructions also demonstrate that
the plant genes involved in the formation of phylloquinone display a high degree of
evolutionary chimerism owing to multiple events of horizontal gene transfer and gene
losses. Plant phylloquinone biosynthesis is also connected via shared intermediates to
the metabolism of salicylate, tocopherols, chlorophylls, and in some species to
anthraquinones.
I. INTRODUCTION
The discovery of vitamin K arose from the observation in the late 1920s and
early 1930s that chicks reared on a reconstituted ‘sterol-free’ diet developed a
hemorrhagic disease characterized by a severe impairment in blood coagulation
(Almquist and Stokstad, 1935; Dam, 1929, 1935; Dam and Schønheyder, 1934;
Holst and Halbrook, 1933; McFarlane et al., 1931; Schønheyder, 1935). The
lack of sterols or fat in the diet as the cause of the disease was quickly ruled out,
as haemorrhages still appeared in chicks receiving a daily supplement of choles-
terol and oil from cod-liver or flax seeds. Nor did it appear that the haemor-
rhages were caused by a lack of any of the known vitamins. Feeding experiments
with supplements obtained from various fractionation procedures showed,
however, that the protecting factor resembled vitamin E, being thermostable,
fat-soluble and non-saponifiable. (Almquist and Stokstad, 1935; Dam, 1935;
Dam and Schønheyder, 1934). The disease could be prevented or cured by
supplementing the chicks’ diet with various plant or animal products such as
fresh cabbage, dried alfalfa, tomatoes, hemp seeds, putrefied fish meal—but not
fresh—or hog liver fat (Almquist and Stokstad, 1935; Dam, 1935; Dam and
Schønheyder, 1934). Almquist and Stokstad (1935) at the College of Agriculture
of the University of California-Berkeley established early on that the green parts
VITAMIN K1 (PHYLLOQUINONE) 231
of the plant ingredients were the sources of the antihemorrhagic factor, and that
it was distinct from chlorophyll and xanthophyll. They also understood that, in
the case of putrefied fish meal, the antihemorrhagic factor originated from the
development of microorganisms (Almquist and Stokstad, 1935). Henrik Dam
at the Biochemical Institute of the University of Copenhagen recognized this
antihemorrhagic factor as a vitamin; he named it ‘vitamin K’, for K was the first
letter in the alphabet that had not been used to designate other vitamins, and
coincidently happened to correspond to the first letter in the word ‘koagulation’
as spelled in Scandinavian (Dam, 1935). A few years later, Edward A. Doisy’s
group at the Laboratory of Biological Chemistry from St. Louis University
School of Medicine purified vitamin K1 (phylloquinone) from alfalfa, deter-
mined its structure and achieved its chemical synthesis (Binkley et al., 1939;
MacCorquodale et al., 1939a,b; McKee et al., 1939). Shortly after, vitamin K2
(menaquinone) was isolated from putrefied fish meal and characterized (Doisy
et al., 1941). The 1943 Nobel Prize in Physiology or Medicine was co-awarded to
Henrik Dam ‘for his discovery of vitamin K’ and to Edward A. Doisy ‘for his
discovery of the chemical nature of vitamin K’. The award did not acknowledge
the pioneering work of Almquist and Stokstad, who co-discovered vitamin K
independently from Dam, or that of Schønheyder, who demonstrated that
vitamin K deficiency impaired blood coagulation. As for plants, one had to
wait until the mid-1980s to find out that phylloquinone participates in the
photosynthetic electron transfer chain and until the past couple of years to
discover that it doubles as an electron acceptor linked to the formation of
disulfide bridges in proteins.
Some readers may also be surprised to learn that until the middle of this
decade—and despite the cardinal role played by phylloquinone in photosyn-
thesis and human nutrition—not much was known about the biosynthesis of
this vitamin. As we will see later, plant biochemists were among the leaders in
the early studies of vitamin K biosynthesis. Unfortunately, as emerged a
general assumption that the biosynthesis of phylloquinone in photosynthetic
organisms was identical to that of menaquinone in facultative anaerobic
bacteria, research on the metabolism of vitamin K in plants virtually ceased
for decades. If it is indeed correct to view the individual steps of phylloqui-
none and menaquinone biosynthesis as similar, the most recent investiga-
tions showed that plants evolved an unprecedented architecture to synthesize
phylloquinone, including extraordinary events of gene fusion and horizontal
gene transfer, split of the pathway between plastids and peroxisomes and
multiple metabolic branch points that link the biosynthesis of phylloquinone
to that of salicylate, tocopherols and chlorophylls. The study of phylloqui-
none biosynthesis in cyanobacteria even led a couple of years ago to the
discovery of a ‘missing’ enzyme of the vitamin K biosynthetic pathway.
This review aims to summarize the current knowledge concerning

the function of vitamin K in vertebrates and oxygenic photosynthetic organ-
isms and its metabolism—emphasizing the most recent advances in under-
standing the phylloquinone biosynthetic pathway and its evolution
in plants—and to point out areas that are still obscure. We refer the reader
to the reviews of Sakuragi and Bryant (2006), Fromme and Grotjohann
(2006) and van der Est (2006) for a detailed coverage of the biosynthesis
and function of phylloquinone in cyanobacteria. We will nonetheless discuss
on occasion recent works concerning the biosynthesis of isoprenoid naphtho-
quinones in cyanobacteria and facultative anaerobic bacteria, as some of the
findings in these organisms could represent paradigms for plants.
II. STRUCTURE AND CHEMISTRY OF VITAMIN K

The term vitamin K encompasses a class of fat-soluble compounds formed
from a naphthoquinone ring attached to a poly-isoprenyl side chain of
variable length and saturation (Fig. 1A). Its main natural forms are vitamin
K1 (phylloquinone; 2-methyl-3-phytyl-1,4-naphthoquinone) that is found in
plants (Oostende et al., 2008), green algae (Lefebvre-Legendre et al., 2007)
and certain cyanobacteria (Collins and Jones, 1981) and vitamin K2 (mena-
quinones; 2-methyl-3-(all-trans-polyprenyl)-1,4-naphthoquinone) that is
found in most groups of archaea and bacteria (Collins and Jones, 1981),
the cyanobacterium Gloeobacter violaceus (Mimuro et al., 2005), red algae
(Yoshida et al., 2003) and diatoms (Ikeda et al., 2008). Vitamin K-synthesiz-
ing organisms appear to contain either phylloquinone or menaquinones but
not both. Phylloquinone has a partially unsaturated side chain formed of one
isopentenyl followed by three isopentyl units, while menaquinones have a
fully unsaturated side chain composed of 2–13 isopentenyl units (Fig. 1A).
Menaquinones are often designated as menaquinone-n (MK-n), where n
refers to the number of isopentenyl units in the side chain.
The naphthoquinone ring of vitamin K can exist at different levels of
oxidation, varying from epoxide (the most oxidized) to quinol (the most
reduced) through the intermediate quinone and semi-quinone (Fig. 1B).
The epoxide form is the product of an enzymatic reaction that so far
has been identified only in animal cells. Most of plant and cyanobacterial
phylloquinone is in the quinone form (Oostende et al., 2008; Widhalm et al.,
2009).
While in facultative anaerobic bacteria, menaquinones are often found
lacking the methyl group at position 2 of the naphthoquinone ring—for
instance, up to 90% of the menaquinone pool in aerobically grown
Fig. 1. (A) Structures of phylloquinone (vitamin K1) and menaquinones

(vitamin K2). The phytyl side chain of phylloquinone contains one isopentenyl unit
and three isopentyl units, while that of menaquinones are made exclusively of iso-
pentyl units. (B) Interconversion of the different redox forms of the naphthoquinone
ring. R: poly-isoprenyl moiety.
Escherichia coli is demethylated (Unden, 1988)—in plants and cyanobac-

teria, phylloquinone is virtually all in the methylated form [there is a single
report of the presence of trace levels of demethylphylloquinone in spinach
chloroplasts (McKenna et al., 1964)].
III. BIOCHEMICAL ROLES OF VITAMIN K
A. VERTEBRATES
The major known function of vitamin K in vertebrates is that of a cofactor

for the -carboxylation of specific glutamate residues, thus conferring strong
chelating properties to certain proteins whose activity depends on calcium
binding. Among such -carboxylglutamate (Gla)-containing proteins are
blood coagulation factors (prothrombin, factors VII, IX and X), proteins

that participate in bone metabolism (osteocalcin, Matrix Gla Protein) and
cell signalling (Gas6).
In order to fulfil its role as cofactor for the -carboxylase, vitamin K must
be in the quinol form. As a by-product of the -carboxylation, the bireduced
naphthalenoid ring of vitamin K is converted to the fully oxidized epoxide
form (Fig. 2). It is salvaged by an integral enzyme complex, named Vitamin
K epoxide reductase (VKOR), whose catalytic subunit (VKORC1; EC
1.1.4.1) reduces the epoxide back to quinone and the quinone back to quinol
(Chu et al., 2006; Fig. 2). This enzyme is the target of the vitamin K antago-
nist warfarin, used as an anticoagulant drug and rodenticide. Besides its role
as a cofactor, studies on mammalian cell cultures indicate that vitamin K acts
as a transcriptional regulator (Ichikawa et al., 2006) and as an antioxidant
(Li et al., 2003).
The lack of vitamin K results in non-functional Gla-containing proteins,
which, in turn, can lead to impaired blood clotting and bone mineralization.
Severe vitamin K deficiency, which can lead to easy bruising and bleeding, is,
however, rare in healthy adults because vitamin K is widespread in foods,
Fig. 2. Scheme of the vitamin K-dependent -carboxylation of glutamyl residues

in vertebrates. Vitamin K quinol is converted to an oxygenated intermediate that
abstracts a proton from the -carbon of the glutamyl residue of the carboxylase
substrate, followed by the addition of carbon dioxide. The concomitant oxidation
of vitamin K quinol into vitamin K epoxide, and its subsequent salvaging by the
enzyme complex vitamin K epoxide reductase (VKOR), is called the vitamin K cycle.
and the gut flora produces basal levels of menaquinones (Suttie, 1995). Only
individuals having chronicle hepatic and pancreatic disorders (Savage and
Lindenbaum, 1983), those receiving long-term antibiotic (Savage and
Lindenbaum, 1983; Shevchuk and Conly, 1990) or vitamin K antagonist
treatments (Bach et al., 1996), appear to be at risk of acute vitamin K
deficiency. Newborns, whose intestinal flora is not yet established, stand
apart and are naturally exposed to an increased risk of vitamin K deficien-
cy—often leading to dramatic haemorrhage of the central nervous system.
The risk is actually higher for infants who are exclusively breast-fed because
the human milk contains only traces of this vitamin (American Academy of
Pediatrics, 2003). It is therefore routine—and often mandatory—in many
countries to administer intramuscular or oral vitamin K at birth as a pro-
phylactic measure (American Academy of Pediatrics, 2003). The incidence of
unexpected bleeding during the first week of life in previously healthy neo-
nates ranges from 250 to 1700 per 100,000 births, and these numbers rise
to 4400–7200 per 100,000 births in infants 2–12 weeks of age who have
received no or inadequate vitamin K prophylaxis (American Academy of
Pediatrics, 2003).
The adequate intake values for vitamin K in the United States are current-
ly set at 120 g/day for men and 90 g/day for women (Food and Nutrition
Board, 2001). Specific levels have not yet been established in the European
Union, but the Committee on Medical Aspects of Food and Nutrition Policy
in the United Kingdom considered that an intake of 1 g/kg of body weight/
day is likely adequate for the proper carboxylation of blood coagulation
factors. In a typical western diet, phylloquinone is the main contributor of
vitamin K intake; about half of it comes from green leafy vegetables, fol-
lowed by soybean, olive, canola and cottonseed oils (Booth and Suttie, 1998).
American and British studies reported average values for dietary vitamin K
intake ranging from 60 to 70 g/day and suggested that one-half of the
populations investigated had vitamin K intakes below the present guidelines
(Vermeer et al., 2004). The impact on bone health of such suboptimal intakes
is currently debated. There is evidence that the level of circulating under-
carboxylated osteocalcin increases after menopause, and that it correlates
with an increased risk of hip fracture (Szulc et al., 1996). Some epidemiologi-
cal studies also reported that individuals with the highest vitamin K intakes
have lower risk of hip fracture than those with the lowest intakes (Booth
et al., 2000; Feskanich et al., 1999), but others found no correlations
(McLean et al., 2006; Rejnmark et al., 2006). None of these studies could
establish a relationship between vitamin K intake and bone mineral density.
Clinical trials indicated a possible increase in bone mineral density and bone
strength in postmenopausal women receiving vitamin K supplementation,
but the doses used were several orders of magnitude higher than those
commonly found in the diet (Iwamoto et al., 2001; Knapen et al., 2007).
B. PLANTS AND CYANOBACTERIA
Until recently, the sole firmly established function of phylloquinone in pho-

tosynthetic organisms was that of a light-dependent electron carrier—the A1
acceptor—in photosystem I (Brettel et al., 1987; Petersen et al., 1987;
Sigfridsson et al., 1995). The process is a one-electron transfer, that is, a
quinone/semi-quinone turnover—from chlorophyll a to the iron–sulphur
cluster of ferredoxin reductase (Boudreaux et al., 2001; Sigfridsson et al.,
1995; Fig. 3). There are two molecules of phylloquinone, called QKA and
QKB, that are bound to the PsaA and PsaB subunits, respectively, at the
stromal side of each photosystem I monomer (Ben-Shem et al., 2003;
Boudreaux et al., 2001; Jordan et al., 2001). Both molecules are active in
electron transport, but the transfer rate through QKB appears to be 50 times
higher than that through QKA (Guergova-Kuras et al., 2001). The reasons
for such a difference in kinetics between the two branches are not yet fully
understood (Fromme and Grotjohann, 2006).
Fig. 3. Scheme of the electron transfer in photosystem I and approximate mid-

point potentials of the cognate electron carriers in plants and cyanobacteria. Phyllo-
quinone is located at the A1 site of the PsaA and PsaB subunits of photosystem I to
serve as a one-electron carrier from chlorophyll aA0) to the Fe-S cluster (FX, FA/FB).
P700, photosystem I reaction center; P700*, excited photosystem I reaction center.
Reports that at least half of phylloquinone is not bound to photosystem I

(Gross et al., 2006; Lohmann et al., 2006), together with the detection of the
quinol form of phylloquinone in multiple plant species (Oostende et al., 2008)
and the cyanobacterium Synechocystis (Widhalm et al., 2009), have recently
indicated that in photosynthetic organisms, phylloquinone is probably
involved in redox reactions distinct from that of the one-electron transfer
in photosystem I. Further evidence for such an additional role came with the
discovery of phylogenetic relationships between mammalian VKOR and
predicted oxidoreductases in photosynthetic organisms. Indeed, although
there is no genomic or biochemical indication that the -carboxylation of
glutamic acid residues and the resulting generation of vitamin K epoxide
occur outside of the metazoan lineage, homology searches detect cyanobac-
terial and plant proteins that are similar to mammalian VKORC1. Remark-
ably, these VKORC1 homologues display a C-terminal fusion with a soluble
thioredoxin-like domain having the hallmarks of a protein disulfide isomer-
ase (Furt et al., 2010; Goodstadt and Ponting, 2004; Li et al., 2010; Singh
et al., 2008). In vitro assays demonstrated that the Synechococcus-fused
enzyme could couple the formation of disulfide bonds in an artificial protein
substrate to the reduction of phylloquinone (Li et al., 2010). Similarly, the
Arabidopsis orthologue—the At4g35760 gene product, which is localized in
plastids—was shown to catalyze the conversion of conjugated naphthoqui-
none species into their quinol forms using either dithiotreitol or its protein
disulfide isomerase moiety as electron donors. However, unlike mammalian
VKORC1, the plant and cyanobacterial enzymes lack phylloquinone epoxide
reductase activity and are resistant to warfarin (Furt et al., 2010). The
Arabidopsis enzyme also appears to be inactive on conjugated benzoqui-
nones such as plastoquinone and ubiquinone. Such a substrate stringency
might be unique to plants, for there is evidence that the recombinant Syne-
chococcus enzyme binds ubiquinone (Li et al., 2010), so as does the E. coli
DsbB protein (quinone oxidoreductase), which features similarities in se-
quence, structure and mode of action with VKORC1, and in fact uses
menaquinone or ubiquinone as oxidant molecules (Inaba et al., 2004;
Takahashi et al., 2004).
Along this line, it is noteworthy that in Synechocystis sp. PCC 6803, the
phenotype of the VKORC1-like (slr0565) knockout does not parallel that of
mutant strains lacking phylloquinone. Indeed, deletion of slr0565 causes
lethality or severe growth retardation depending on the presence or absence
of glucose in the culture medium, respectively (Singh et al., 2008), whereas in
similar conditions, Synechocystis mutants blocked in phylloquinone biosyn-
thesis display either no or moderate growth defects (see Section V.G). It is
therefore conceivable that in phylloquinone-deficient cyanobacteria, the
VKORC1-like enzyme functions using an alternate substrate, possibly plas-

toquinone. In plants, however, the lack of phylloquinone causes complete
loss of photoautotrophy (see Section V.G), and failure to obtain Arabidopsis
transgenics, whose VKORC1-like expression is deregulated, indicating that
this enzyme fulfils a core and vital role (Furt et al., 2010).
IV. DETECTION AND DISTRIBUTION OF

PHYLLOQUINONE IN PLANTS
A. DETECTION
Early methods for the determination of vitamin K in food or biological

extracts relied on a chick bioassay. Analyses were tedious, entailing large
extraction volumes—especially in samples of animal origin due to their
extremely low vitamin K content—and provided only semiquantitative
data (Dam and Schønheyder, 1936). Subsequent quantitative methods were
developed using thin-layer chromatography, gas chromatography and high-
performance liquid chromatography (HPLC); the latter coupled either to
fluorometric or to electrochemical detection (Davidson and Sadowski, 1997;
McCarthy et al., 1997). HPLC methods based on the reduction of the
naphthoquinone ring to its fluorescent quinol form prior to its detection by
fluorometry have proven to combine high sensitivity and selectivity and are
today the preferred applications for the routine quantification of vitamin K
in complex extracts (Booth and Sadowski, 1997; Davidson and Sadowski,
1997). On an additional technical note, let us mention that in green plant
tissues, phylloquinone is often sufficiently abundant to be detected and
quantified using HPLC–spectrophotometry (Fraser et al., 2000).
B. TISSULAR DISTRIBUTION
The level of phylloquinone varies greatly between different plant species and
tissues (Table I). Leaves usually have the highest levels, while most fruits,
tubers and seeds contain several-fold less. It is noteworthy that staple crops
(e.g. grains and tubercles) are among the poorest plant sources of
phylloquinone.
C. SUBCELLULAR DISTRIBUTION
At the subcellular level, plastids account for most if not all of the phylloquinone
content of plant tissues (Lohmann et al., 2006; Oostende et al., 2008). Subplas-
tidial fractionation experiments demonstrated that about a third of
TABLE I
Phylloquinone Content of Some Plant Species and Plant Food-Products
Phylloquinone (g/100 g)
A. thaliana (green leaf) 365(a)
Brassica oleracea (canola oil) 127(b)
Brassica oleracea (collard greens) 440(b)
Brassica oleracea (broccoli) 180(b)
Brassica oleracea (brussel sprouts) 177(b)
Brassica oleracea (cauliflower) 20(b)
Cicer arietinum (chickpeas) 9(c)
Daucus carota (tuber) 2.7(a)
Lactuca sativa (green leaf) 126(c)
Lactuca sativa (‘iceberg’ lettuce) 35(b)
Manihot esculenta (cassava) 1.9(c)
Olea europaea (olive oil) 55
Oryza sativa (grain) 0.1(c)
Oryza sativa (green leaf) 662(a)
Phaseolus vulgaris (dry bean) 5.6(c)
Phaseolus vulgaris (green beans) 33(b)
Solanum. lycopersicon (green leaf) 1217(a)
Solanum. lycopersicon (green fruit) 19(a)
Solanum. lycopersicon (red ripe fruit) 8(a)
Solanum tuberosum (tuber) 1.3(a)
Glycine max (soybean oil) 193(b)
Glycine max (‘Edamame’ seed) 31(c)
Triticum spp. (whole grain flour) 1.9(c)
Vicia faba (fava bean) 9(c)
Zea mays (grain) 0.3(c)
Zea mays (green leaf) 1514(a)
Zea mays (oil) 3(b)
Data are compiled from Oostende et al. (2008)(a); Booth and Suttie (1998)(b); USDA National
Nutrient Database for Standard Reference (http://www.nal.usda.gov/fnic/foodcomp/search/)(c).
Staple crops are shown in bold.
phylloquinone in Arabidopsis chloroplasts is deposited in plastoglobules, and

therefore, a significant amount of phylloquinone is not bound to photosystem I
(Lohmann et al., 2006). A similar conclusion was inferred from the observation
that Arabidopsis mutants containing less than a quarter of wild-type levels of
phylloquinone retained most of their photosystem I activity (Gross et al., 2006).
The enrichment of naphthoquinone oxidoreductase activities in plasma
membrane preparations of corn roots (Lüthje et al., 1998) and soybean
hypocotyls (Bridge et al., 2000; Schopfer et al., 2008) suggests that small
pools of phylloquinone may occur outside plastids. One of these studies
reported the direct detection of phylloquinone in the plasma membrane
(Lüthje et al., 1998), but the possibility of proplastid breakage (e.g. using
galactolipids as markers) was not investigated.
V. PHYLLOQUINONE BIOSYNTHESIS IN PLANTS
In essence, the phylloquinone biosynthetic pathway of plants consists of two

separated metabolic branches: one for the naphthoquinone ring and the
other for the phytyl moiety, which is also used for the biosyntheses of
tocopherols and chlorophylls. We focus hereafter on the enzymatic steps
leading to the formation of the naphthoquinone ring, as the biosynthesis of
the phytyl-diphosphate precursor from the methylerythritol-phosphate path-
way in plastids is not specific to phylloquinone and has been previously
covered (see, for instance, Lichtenthaler, 1999; Rohmer, 2003).
The biosynthesis of the naphthoquinone ring entails seven enzymatic steps.
The immediate precursor chorismate is first converted into isochorismate, to
which a succinyl side chain is added at the C2 position (Fig. 4). After
elimination of pyruvate and aromatization of the cyclohexadiene ring, the
succinyl chain is activated by ligation with CoA and then cyclized, yielding
1,4-dihydroxynaphthoyl-CoA (DHNA-CoA). The CoA moiety is then
removed, DHNA is conjugated to its phytyl partner and then methylated.
An alternative pathway, in which the naphthoquinone backbone originates
from a chorismate–inosine conjugate termed futalosine, was recently
described in some species of Deinococcus-Thermus, Actinobacteria and
E-Proteobacteria (Hiratsuka et al., 2008). There is currently no genomic or
biochemical evidence that this biosynthetic route occurs in phylloquinone-
synthesizing eukaryotes.
A. EARLY WORK
The basic architecture of isoprenyl naphthoquinone biosynthesis was estab-

lished in the 1970s and 1980s simultaneously in plants and facultative anaer-
obic bacteria using radiolabelling experiments. It quickly emerged from these
studies that the individual steps of the phylloquinone and menaquinone
biosynthetic pathways were virtually identical. In plants, shikimate was
identified as the precursor of the naphthoquinone moiety via the formation
of o-succinylbenzoate (OSB), OSB-CoA and DHNA (Dansette and Azerad,
1970; Heide et al., 1982; Hutson and Threlfall, 1980; Thomas and Threlfall,
1974), while the ring prenylation and methylation steps were found to use
phytyl-diphosphate and s-adenosylmethionine as substrates, respectively
(Gaudillière et al., 1984; Schultz et al., 1981). Interestingly, some of these
early works revealed that in Rubiaceae, OSB doubles as an intermediate in
the biosynthesis of anthraquinone species—of which still today not much is
Fig. 4. The biosynthesis pathway of phylloquinone. 1, isochorismate synthase;

2, SEPHCHC synthase; 3, SHCHC synthase; 4, OSB synthase; 5, OSB-CoA ligase;
6, DHNA-CoA synthase; 7, DHNA-CoA thioesterase; 8, DHNA prenyltransferase;
9, demethylphylloquinone methyltransferase. SAH, s-adenosylhomocysteine;
SAM, s-adenosylmethionine; SEPHCHC, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-
cyclohexene-1-carboxylic acid; SHCHC, (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohex-
adiene-1-carboxylic acid. EC numbers are indicated under each corresponding
reaction.
known—and suggested that anthraquinone and naphthoquinone biosynth-

eses intersect at the level of DHNA.
The plant genes of phylloquinone biosynthesis have been identified only in
the past decade; for most of them using the genomic and genetic resources of
Arabidopsis thaliana and homology searches with the bacterial men genes as
query. Table II lists the cognate Arabidopsis proteins with their orthologues
in Synechocystis sp. PCC 6803 and E. coli. Two steps (reactions 6 and 7,
Fig. 2) remain to be characterized: for the first one, the gene is predicted but
has not been functionally confirmed; for the second one, orthology appears
to be missing (see below).
TABLE II
Correspondence Between the Phylloquinone Biosynthesis Enzymes in Arabidopsis
and Synechocystis and Their Orthologues Involved in the Biosynthesis of
Menaquinone-8 in E. coli
Synechocystis sp.
A. thaliana PCC6803 E. coli
Isochorismate synthase At1g74710 (ICS1) Slr0817 MenF
At1g18870 (ICS2)
SEPHCHC synthase At1g68890 (PHYLLO) Sll0603 MenD
SHCHC synthase At1g68890 (PHYLLO) Slr1916 MenH
OSB synthase At1g68890 (PHYLLO) Sll0409 MenC
OSB-CoA ligase At1g30520 (AAE14) Slr0492 MenE
DHNA-CoA synthase At1g60550 (putative) Sll1127 MenB
DHNA-CoA thioesterase Unknown Slr0204 Unknown
DHNA phytyltransferase At1g60600 (ABC4) Slr1518 MenA
Demethylphylloquinone At1g23360 Sll1653 UbiE
methyltransferase
B. ISOCHORISMATE SYNTHASE/PHYLLO (REACTIONS 1–4)
Genetic approaches identified an Arabidopsis gene, termed PHYLLO

(At1g68890), that encodes an extraordinary protein composed of four mod-
ules homologous to the bacterial MenF (5.4.4.2), MenD (2.2.1.9), MenC
(4.2.1.113) and MenH (4.2.99.20) proteins, respectively (Fig. 5). The MenF
module lacks a complete chorismate binding domain, suggesting that it
cannot catalyze the conversion of chorismate into isochorismate (this
truncated domain appears nevertheless to be conserved from monocots to
dicots, pointing to a selective driving force for its maintenance. What this is,
however, is still an enigma). The Arabidopsis genome encodes in fact two
separated and catalytically active isochorismate synthases, ICS1 (At1g74710)
and ICS2 (At1g18870), that share about 80% identity (Garcion et al., 2008;
Strawn et al., 2007; Wildermuth et al., 2001). The ics1/ics2 double knockout
is devoid of phylloquinone (Garcion et al., 2008; Gross et al., 2006), thus
providing genetic evidence that PHYLLO is not sufficient for the de novo
synthesis of isochorismate, and that phylloquinone biosynthesis is dependent
upon a pool of isochorismate that is produced by separated isochorismate
synthases. As ICS1 bears most of the flux of isochorismate biosynthesis
(Garcion et al., 2008; Gross et al., 2006) and several plant genomes encode
for a single ICS copy, it is unclear if Arabidopsis ICS1 and ICS2 have
dedicated functions, or simply originate from recent duplication events and
are evolving separately. Whatever the case, isochorismate represents a meta-
bolic branch point where plant phylloquinone biosynthesis is likely to
Fig. 5. Arrangement of the functional domains of the Arabidopsis PHYLLO

protein and their approximate percentage of identity with their separated Men
orthologues in E. coli and with Arabidopsis isochorismate synthases 1 and
2 (AtICS1 and AtICS2). CTP, chloroplast transit peptide.
compete with that of the plastid-produced hormone, salicylate, which also

uses isochorismate as a precursor (Wildermuth et al., 2001). Confirming this
view, the constitutive expression in tobacco chloroplasts of a bacterial iso-
chorismate pyruvate lyase, which converts isochorismate to salicylate, was
shown to result in an increase in salicylate level at the expense of phylloqui-
none (Verberne et al., 2007). At the time of the identification of PHYLLO,
MenH was still thought to correspond to DHNA-CoA thioesterase. The
menC and menH fused modules were therefore viewed as encoding for
domains that catalyzed reactions two steps apart from each other. It was
hypothesized that such an arrangement could indicate the existence of physi-
cal associations between PHYLLO, OSB-CoA ligase and DHNA synthase
(Gross et al., 2006). It is now evident that the PHYLLO MenDCH modules
catalyze consecutive reactions that lead to the synthesis of OSB. This does
not actually rule out that PHYLLO interacts with other enzymes in the
pathway, particularly because the fused structure of PHYLLO itself is sug-
gestive of a metabolon where biosynthetic intermediates are channelled from
one catalytic domain to the other.
Homology searches point to the existence of clusters of menF, menD,
menC and menH orthologues in green algae, mosses, diatoms and rhodo-
phytes. The menF domain of such clusters, in contrast to that of flowering
plants, features a full chorismate binding domain and is a priori functional.
A Chlamydomonas reinhardtii cDNA corresponding to the menD orthologue
was shown to contain in-frame stop codons both upstream of the initiation
codon and at the end of the coding sequence, suggesting that in this species,
the menF, menD and menC modules are translated as separated polypeptides
(Lefebvre-Legendre et al., 2007). It remains therefore to establish if fused and
multifunctional enzymes equivalent to PHYLLO occur outside of the flower-
ing plant lineage.
C. OSB-COA LIGASE (REACTION 5)
OSB-CoA ligase (6.2.1.26) activates the carboxyl group on the succinyl side
chain of OSB by creating a high-energy bond with the pantetheine moiety of
CoA (Kolkmann and Leistner, 1987; Fig. 2). Plant OSB-CoA ligase was
identified as part of a general characterization effort of the CoA ligase family
in Arabidopsis (Kim et al., 2008). A putative CoA ligase termed AAE14 (acyl
activating enzyme 14; the product of gene At1g30520) was singled out as one
of the top coexpressors of some previously identified phylloquinone biosyn-
thetic genes (At1g60600, DHNA phytyl transferase; At1g23360, demethyl-
phylloquinone methyltransferase; At1g68890, PHYLLO) and of the
predicted DHNA-CoA synthase (At1g60550; see below). Direct evidence
for the involvement of AAE14 in phylloquinone biosynthesis came from
the isolation of three independent T-DNA mutant lines corresponding to
insertions in the first intron, and fourth and ninth exons of At1g30520,
respectively; all of which lacked phylloquinone (Kim et al., 2008). The
T-DNA mutants were also found to accumulate OSB and could be partially
rescued by exogenous applications of DHNA (Kim et al., 2008). Expression
of At1g30520 cDNA was shown to fully restore menaquinone biosynthesis in
the E. coli menE knockout, thus verifying that AAE14 bore OSB-CoA ligase
activity (Kim et al., 2008).
D. DHNA-COA SYNTHASE/DHNA-COA THIOESTERASE (REACTIONS 6/7)
DHNA-CoA synthase (4.1.3.36) catalyzes the cyclization of OSB-CoA

(Fig. 2). The enzyme, which belongs to the crotonase-fold family, is often
termed in the literature and numerous databases as DHNA synthase or
naphthoate synthase, but it is clear that its reaction product is DHNA-
CoA, not DHNA (Jiang et al., 2010; Truglio et al., 2003). The enzyme’s
substrate, OSB-CoA, is highly unstable at physiological pH and has been
shown to spontaneously decompose in vitro into the spirodilactone form of
OSB (Fig. 6) (Heide et al., 1982; Meganathan and Bentley, 1979). Should
such a decomposition occur in vivo, it is not known if or how OSB spirodi-
lactone is recycled.
Bacterial DHNA-CoA synthases appear to fall into two catalytic classes.
Type I enzymes use a bound bicarbonate anion as a catalytic base, while type
II enzymes use the side-chain carboxylate of one of their acidic residues
(Jiang et al., 2010). Type I enzymes are consequently deemed bicarbonate
dependent and their type II counterparts bicarbonate independent (Jiang
et al., 2010). Sequence comparisons and phylogenetic reconstructions indi-
cate that cyanobacterial DHNA-CoA synthase and its predicted Arabidopsis
Fig. 6. Hydrolysis and lactonization of OSB-CoA.
homologue belong to the type I category, suggesting that phylloquinone

biosynthesis is regulated by the intracellular level of bicarbonate.
The subsequent removal of CoA from DHNA, catalyzed by DHNA-CoA
thioesterase (3.1.2.-), has long puzzled the elucidation of the vitamin K
biosynthesis pathway. After being misattributed to DHNA-CoA synthase
(MenB), then to SHCHC synthase (MenH) in E. coli, it was later proposed
that the hydrolysis of DHNA-CoA, which like its OSB-CoA precursor
spontaneously decomposes at physiological pH, could be merely chemical
(Sakuragi and Bryant, 2006). But recent phylogenomics approaches in cya-
nobacteria detected putative CoA thioesterases, whose encoding genes were
arranged in clusters with known phylloquinone biosynthetic genes (Widhalm
et al., 2009). Deletion of the Synechocystis orthologue—gene slr0204—
resulted in a dramatic decrease of the phylloquinone content in the knockout
cells, thus verifying the existence of a functional linkage between the putative
CoA thioesterase and phylloquinone biosynthesis (Widhalm et al., 2009).
Further investigations demonstrated that the knockout mutant accumulated
DHNA-CoA and could be chemically rescued with DHNA, but not with
OSB, thus pointing to the location of the blockage in the pathway (Widhalm
et al., 2009). The purified recombinant Slr0204 was shown to catalyze the
hydrolysis of DHNA-CoA and to display absolute preference for this sub-
strate. It is thought that such a substrate stringency may reflect the presence
of OSB-CoA upstream in the pathway, and whose enzymatic hydrolysis
would create a futile cycle in the phylloquinone biosynthesis pathway
(Widhalm et al., 2009). Although the Synechocystis slr0204 knockout
completely lacked DHNA-CoA thioesterease activity, low levels of phyllo-
quinone could still be detected in this mutant revealing the occurrence of a
basal chemical hydrolysis of DHNA-CoA in vivo (Widhalm et al., 2009).
Such a background decomposition likely explains why DHNA-CoA
thioesterase did not show up in forward genetic screens aimed at identifying

men genes in bacteria, and one can therefore grasp through this illustrative
case the power of phylogenomics in predicting gene function based on the
detection of conserved physical associations of genes in genomes.
Except for the extremophilic rhodophytes Cyanidiales (Cyanidioschyzon
merolae and Cyanidium caldarium) and the cercozoan (Paulinella chromato-
phora), the plastid or chromatophore of which encode homologues of cya-
nobacterial DHNA-CoA thioesterase arranged in clusters with
phylloquinone biosynthetic genes (see Section VI), DHNA-CoA thioesterase
remains elusive in phylloquinone-synthesizing eukaryotes. Homology
searches do detect two pairs of Arabidopsis paralogs (At1g68260/
At1g68280, At1g35250/At1g35290) that share 17–28% of identity with Syne-
chocystis Slr0204, but these Arabidopsis genes have recently been shown to
encode orthologues of solanaceous methyl ketone synthases (Yu et al., 2010).
E. DHNA PHYTYL TRANSFERASE (REACTION 8)
DHNA phytyl transferase (2.5.1.-), an integral membrane protein, couples

the naphthoquinone ring to the phytyl side chain (Fig. 2). It was the first
enzyme specific to phylloquinone biosynthesis to be described in plants and
was initially identified in the Arabidopsis abc (aberrant chloroplast develop-
ment) T-DNA mutant series (Shimada et al., 2005). The cognate mutant-
designated abc4 was shown to correspond to an insertion in the ninth exon of
gene At1g60600 and to lack phylloquinone (Shimada et al., 2005). Function-
al assignment was based on homology with Synechocystis sp. PCC 6803
DHNA phytyl transferase, which shares 41% identity with the At1g60600
protein (Shimada et al., 2005).
F. DEMETHYLPHYLLOQUINONE METHYLTRANSFERASE (REACTION 9)
Demethylphylloquinone methyltransferase (2.1.1.-) catalyzes the methyla-

tion of 2-phytyl-1,4-naphthoquinone (demethylphylloquinone) and corre-
sponds to the last step of the phylloquinone biosynthetic pathway (Fig. 2).
Mining the Arabidopsis genome with Synechocystis demethylphylloquinone
methyltransferase—the E. coli UbiE homologue, which doubles in the bio-
synthesis of ubiquinone, hence its name (Lee et al., 1997)—as query detected
the product of gene At1g23360 as a likely orthologue (Lohmann et al., 2006).
Expression of the cognate cDNA fully rescued phylloquinone biosynthesis in
the Synechocystis demethylphylloquinone methyltransferase knockout
(Lohmann et al., 2006). In parallel, a T-DNA line corresponding to an
insertion in the seventh exon of At1g23360 was found to be devoid of
phylloquinone and to accumulate 2-phytyl-1,4-naphthoquinone, thus estab-

lishing definite evidence that this gene encodes the only demethylphylloqui-
none methyltransferase in Arabidopsis (Lohmann et al., 2006).
G. MUTANT PHENOTYPE
Arabidopsis lines corresponding to phyllo (the fused SEPHCHC synthase–

SHCHC synthase–OSB synthase), aae14 (OSB-CoA ligase) and abc4
(DHNA phytyl transferase) knockouts and to the double knockout ics1/
ics2 (isochorismate synthase 1 and 2) display loss of photoautotrophy and
are seedling lethal (Gross et al., 2006; Kim et al., 2008; Shimada et al., 2005).
A few pale-green leaves can be obtained from these mutants providing that
they are grown on a medium containing sucrose and under low illumination,
but even so the plants eventually stop developing. Analyses of the phyllo and
abc4 mutants showed that the lack of phylloquinone results in the disruption
of photosystem I assembly (Gross et al., 2006; Shimada et al., 2005). Plasto-
quinone level and photosystem II activity were also shown to be dramatically
reduced in the abc4 knockout, while photosystem II was found to be only
moderately affected in the phyllo mutant (Gross et al., 2006; Shimada et al.,
2005). In contrast, green algal and cyanobacterial mutants, which are
blocked in the formation of the naphthoquinone ring or its prenylation, are
able to recruit plastoquinone into the A1 site of photosystem I in place of
phylloquinone and—though being sensitive to high light intensity—can grow
photoautotrophically (Johnson et al., 2000; Lefebvre-Legendre et al., 2007).
The demethylphylloquinone methyltransferase knockout is the sole viable
phylloquinone-deficient mutant in plants. The reduction in photosynthetic
efficiency and number of photosystem I subunits observed in the cognate
Arabidopsis insertion line indicate nonetheless that the replacement of
phylloquinone by demethylphylloquinone is not fully functional (Lohmann
et al., 2006).
H. SUBCELLULAR LOCALIZATION OF PHYLLOQUINONE

BIOSYNTHETIC ENZYMES
Early radiolabelling experiments showed that the prenylation and methylation

steps of plant phylloquinone biosynthesis were associated with the chloroplast
membranes; the two activities appeared to be localized in separate subfractions:
the prenylation occurring in the chloroplast envelope and the methylation in
thylakoids (Gaudillière et al., 1984; Kaiping et al., 1984; Schultz et al., 1981).
Cloning of Arabidopsis DHNA phytyl transferase and demethylphylloquinone
methyl transferase later confirmed that both enzymes possess N-terminal
signalling peptides and are indeed targeted to plastids (Lohmann et al., 2006;
Shimada et al., 2005). Similar findings were obtained for isochorismate synthase
1 and 2 (Garcion et al., 2008; Strawn et al., 2007), PHYLLO (Gross et al., 2006)
and OSB-CoA ligase (Kim et al., 2008). Contrasting with this apparent all-
plastidial localization of the pathway, predicted DHNA-CoA synthases from
dicots and monocots have N-terminal extensions that contain a canonical
peroxisomal targeting signal type 2 (RLx5HL) and proteomic approaches
have identified the putative Arabidopsis enzyme and its spinach orthologue in
purified peroxisomes (Babujee et al., 2010; Reumann et al., 2007). Expression in
onion epidermal cells of the Arabidopsis protein fused at its C-terminal end to a
fluorescent reporter protein further verified that the resulting construct was
imported into peroxisomes (Babujee et al., 2010). The green alga C. reinhardtii
and moss P. patens orthologues, however, lack a peroxisomal targeting signal,
so as do their cyanidiale C. merolae and C. caldarium and cercozoan P. chro-
matophora counterparts, which are chloroplast or chromatophore encoded,
thus indicating that the targeting of DHNA-CoA synthase to peroxisome is
not ubiquitous in phylloquinone-synthesizing eukaryotes.
Interestingly, the preceding enzyme, OSB-CoA ligase, displays in most
monocotyledonous and dicotyledonous species a predicted peroxisomal target-
ing signal. In this case, it corresponds to a C-terminal tripeptide (SSL, SNL,
SRL or SKL depending on the species) that typifies a peroxisomal targeting
signal type 1 (Babujee et al., 2010). N-terminally fused fluorescent versions of
Arabidopsis OSB-CoA ligase (AAE14) or of its last 10 residues containing the
SSL signal were expressed in onion epidermal cells and confirmed here again
that the hybrid proteins were targeted to peroxisomes (Babujee et al., 2010).
These exciting observations imply that the activation of OSB and its cyclization
into DHNA-CoA occur in peroxisomes, thus requiring the shuttling of phyllo-
quinone biosynthetic precursors in and out of plastids and peroxisomes. One
should note, however, that transient expressions of C-terminally tagged fluo-
rescent versions of AAE14 or its first 120 residues in Arabidopsis leaf proto-
plasts and tobacco leaf mesophyll cells, respectively, have demonstrated that the
enzyme also bears a functional plastid targeting presequence and is targeted to
chloroplasts (Kim et al., 2008). The obvious bias of each of the aforementioned
fusion strategies is that the reporter protein conceals either the peroxisomal
targeting signal type 1 (C-terminal fusion) or the plastid targeting presequence
(N-terminal fusion). Although the current view is that AAE14 could be dual
targeted, direct identification using proteomics approaches and/or assays of
OSB-CoA ligase in purified peroxisomes and chloroplasts is needed to precisely
determine the subcellular localization of this enzyme.
As for the hydrolysis of DHNA-CoA, preliminary data from our labora-
tory indicated that although DHNA-CoA thioesterase was detectable in
whole extracts of pea and Arabidopsis leaves, it was absent in the

corresponding preparations of purified chloroplasts (unpublished results).
As previously suggested (Babujee et al., 2010), this makes the localization of
DHNA-CoA hydrolysis in peroxisomes all the more likely. Figure 7 sum-
marizes the current knowledge concerning the subcellular compartmentation
of the phylloquinone biosynthesis pathway in Arabidopsis.
Fig. 7. Subcellular localization of the phylloquinone biosynthetic enzymes in

Arabidopsis. Letters in brackets specify the type of experimental evidence: fusion to
a fluorescent reporter protein and transient expression in Arabidopsis leaf or leaf
protoplasts (a), onion epidermal cell (b), tobacco mesophyll cells or leaf protoplasts
(c); C-terminal fusion to V5-6xHis epitope and stable expression under the control of
native promoter in Arabidopsis transgenics, and immunolocalization (d); in vitro
import assay in chloroplasts purified from pea seedling (e); subcellular fractionation
and identification using mass sprectrometry (f). Dashed arrows indicate putative
transport steps between plastid and peroxisome, or the possible occurrence of
DHNA-CoA hydrolysis in peroxisome (reaction 7). Evidence from our laboratory
indicates that DHNA-CoA thioesterase activity is lacking in chloroplasts
(Widhalm J.R., unpublished data).
VI. EVOLUTION OF NAPHTHOQUINONE

BIOSYNTHESIS IN PHOTOSYNTHETIC EUKARYOTES
Isoprenoid naphthoquinones are evolutionarily the most ancient of all con-

jugated quinones, their biosynthesis in prokaryotes predating the rise of
atmospheric dioxygen level ca. 2.5 billion years ago (Schoepp-Cothenet
et al., 2009). Menaquinones have thus been detected in most prokaryotic
lineages (Collins and Jones, 1981), rhodophytes (Yoshida et al., 2003) and
diatoms (Ikeda et al., 2008). Phylloquinone in contrast appears to be restrict-
ed to some cyanobacterial species, green algae and plants (Collins and Jones,
1981; Lefebvre-Legendre et al., 2007; Oostende et al., 2008).
All the phylloquinone biosynthetic enzymes identified so far in photosyn-
thetic eukaryotes are nuclear encoded. However, the cyanidiale orthologues—
with the exception of the MenG orthologue (see below)— are plastid encoded,
indicating that the cognate genes have likely been retained from the former
cyanobacterial endosymbiont. While it might therefore seem that such genes
are merely of direct cyanobacterial descent, some phylogenetic studies suggest
that this is not so for the menF, menD, menC, menE and menB orthologues,
which are in fact more closely related to the chlorobi/-proteobacteria lineage
(Gross et al., 2008). To reconcile this surprising genealogy with the
fact that the corresponding enzymes are plastid encoded in cyanidiales, it
has been proposed that men genes originating from an organism of the
chlorobi/-proteobacteria descent have been captured through horizontal
gene transfer by the free living cyanobacterial progenitor of plastids, that is,
prior to its endosymbiosis, and have replaced their pre-existing cyanobacterial
counterparts (Gross et al., 2008). The remnant of this horizontal gene transfer
would now ‘survive’ as a men gene cluster in the plastid genome of modern-day
cyanidiales and in the nuclear genome of diatoms, green algae and plants—the
aforementioned tetramodular PHYLLO locus (Gross et al., 2008).
In contrast, the cyanidiales menA homologue—also part of such a men
gene cluster—would have been acquired through an independent horizontal
gene transfer with a prokaryotic donor, whose identity remains unclear
(Gross et al., 2008). So is the case for the menA homologue of diatoms,
which would define another event of horizontal gene transfer that occurred in
the nucleus (Gross et al., 2008). As for the nuclear-encoded menG homo-
logue, phylogenies suggest that it would originate from -proteobacteria in
both cyanidiales and diatoms (Gross et al., 2008). Plants and green algae
further complicate the picture, having menE homologues that would branch
from the -proteobacteria lineage and menA and menG homologues that
would be of cyanobacterial ancestry (Gross et al., 2008). In essence, accord-
ing to such phylogenetic reconstructions, the eukaryotic genes involved in the
formation of isoprenoid naphthoquinones display a high degree of evolu-

tionary chimerism that varies with the lineage considered, owing to multiple
and unrelated events of horizontal gene transfer and/or gene losses (summar-
ized in Fig. 8).
Fig. 8. Tentative scenario for the evolution of the men genes in photosynthetic
eukaryotes as proposed by Gross et al. (2008). Arrows symbolize gene transfers. Note
that menH homologues are not detected in the plastid genomes of cyanidiales. As for
menA, the cyanobacterial progenitor of plastids would have harboured two cognate
homologues: menA1, of cyanobacterial descent and menA2, acquired by HGT from
an unknown prokaryotic donor. Cyanidiales would have lost menA1 and retained
menA2; the opposite would have happened in plants and green algae. An alternative
explanation would be that menA1 was acquired from cyanobacteria by direct HGT in
the nucleus of the green algal ancestor. HGT, horizontal gene transfer.
One should, however, point that comparative genomics of modern cyano-

bacteria, cyanidiales and chlorobi/-proteobacteria question some parts of this
evolutionary model. For instance, the genomic organization of the men and
DHNA-CoA thioesterase homologues of certain present-day cyanobacteria
and of cyanidiales displays striking similarities that are not conserved in the
chlorobi/-proteobacteria lineage (Fig. 9). Although such a conservation might
be purely coincidental or driven by identical selective constraints (e.g. transcrip-
tional regulation), it could also point to an overlooked phylogenetic closeness
between the menaquinone biosynthetic genes of cyanidiales and some of their
homologues involved in phylloquinone biosynthesis in cyanobacteria.
Fig. 9. Organization of the phylloquinone/menaquinone biosynthetic gene clus-

ters in representative species of cyanobacteria/cercozoan, cyanidiales, -proteobac-
teria and chlorobi. The dashed frame highlights the conserved arrangement of the men
and DHNA-CoA thioesterase (THIO) homologues in cyanobacteria (N. punctiforme,
Nostoc punctiforme; P. marinus, Prochlorococcus marinus; S. sp. CC9605, Synechoc-
coccus sp. CC9605), cercozoan (Cerc.) (P. chromatophora, Paulinella chromatophora)
and cyanidiales (C. caldarium, Cyanidium caldarium; C. merolae, Cyanidioschyzon
merolae). The gene cluster of the cercozoan species P. chromatophora is located in a
plastid-like organelle called the chromatophore; the later is thought to originate from
a recent endosymbiosis of a cyanobacterium of the Prochlorococcus/Synechococcus
lineage. A. hydrophila, Aeromonas hydrophila; C. limicola, Chlorobium limicola;
C. ferrooxidans, Chlorobium ferrooxidans.
VII. PHYLLOQUINONE TURNOVER
Our knowledge of vitamin K metabolism is almost exclusively restricted to

mammals and is largely extrapolated from the data obtained with tocopher-
ols. Pharmacological studies have shown that phylloquinone is rapidly cat-
abolized into shortened side-chain carboxylic acids, which are excreted in
urine as water-soluble glucuronic acid conjugates (Harrington et al., 2007;
Landes et al., 2003). The enzymatic reactions that lead to the shortening of
the side chain are not known sensu stricto. Nevertheless, as tocopherols and
phylloquinone share the same phytyl side chain and in mammals the pro-
ducts of tocopherol catabolism come from !-hydroxylation and subsequent
-oxidation of the side chain, it is believed that phylloquinone follows a
similar catabolic route (Harrington et al., 2007; Landes et al., 2003).
Close to nothing is known about the catabolism of vitamin K in plants. One
study showed that non-physiological doses of phylloquinone can be fed to pea
stem sections, and that more than 90% of the incorporated vitamin could be
recovered after 18-h incubation (Gaunt and Stowe, 1967). The occurrence of
degradation products of phylloquinone in plants is not documented.
VIII. ENGINEERING OF PHYLLOQUINONE

IN PLANTS
There has not been so far any dedicated engineering of phylloquinone in

plants; the only data available are for tobacco transgenics engineered for
salicylic acid biosynthesis, and functional complementation experiments in
Arabidopsis. Thus, in tobacco, the overexpression of an E. coli isochorismate
synthase targeted to plastids led to a fourfold increase of phylloquinone
above wild-type levels (Verberne et al., 2007). In Arabidopsis, overexpression
of demethylphylloquinone methyltransferase or OSB-CoA ligase did not
change phylloquinone content compared to that of wild-type plants (Kim
et al., 2008; Lohmann et al., 2006).
IX. CONCLUDING REMARKS
Findings from the most recent studies of phylloquinone metabolism in plants

illustrate once more that the architecture of plant secondary metabolism is
hardly inferable from previous work in microorganisms. Witness the unprec-
edented and extraordinary multifunctional PHYLLO, the occurrence of
functional redundancies (isochorismate synthases), the apparent lack of
orthology (DHNA-CoA thioesterase) and the split of phylloquinone biosyn-

thesis between plastids and peroxisomes.
Besides the identification of the ‘missing’ DHNA-CoA thioesterase and
the characterization of DHNA-CoA synthase—especially with regards to its
possible regulation by carbonate—one of the priorities of the research on
phylloquinone biosynthesis in plants is now to determine the arrangement of
the plastid and peroxisomal branches that lead to the formation of the
naphthoquinone ring. One cannot indeed overemphasize that the cognate
transport steps between these two organelles are as determinant for the flux
of phylloquinone production as the biosynthetic enzymes themselves. It will
therefore need to be established which biosynthetic intermediates are trans-
ported, and if specific transporters are involved. Such future investigations
are predictably challenging owing to the very low abundance and high
instability of most of the naphthoquinone ring’s biosynthetic intermediates,
and to the difficulty inherently attached to the isolation of reasonably pure
plant organelles and to the functional study of integral proteins.
Another area to further explore is the integration of phylloquinone in the
metabolic network of plastids. As mentioned earlier, there is experimental
evidence in Arabidopsis and tobacco that the biosynthetic pathways of
phylloquinone and salicylic acid intersect through isochorismate. It is prob-
able that plants tightly regulate this metabolic node because salicylate is
massively produced in response to certain stresses, while phylloquinone is
absolutely needed as a redox cofactor. One fascinating hypothesis could be
that the flux of isochorismate usage towards phylloquinone biosynthesis
depends on a—yet-to-be demonstrated— physical association between iso-
chorismate synthase and the multifunctional PHYLLO, thus creating a
metabolon from chorismate up to OSB. Such a scenario could also explain
why flowering plants have maintained a truncated and catalytically inactive
MenF domain in PHYLLO; it could serve for instance as a recognition/
binding domain to assemble the metabolon.
Although species specific, the flux split at the level of DHNA between the
naphthoquinone and anthraquinone biosynthetic pathways is even more
enigmatic. Here, again one can expect that anthraquinone-producing species
must have evolved strategies to commit a steady flux of DHNA towards
phylloquinone biosynthesis.
Through phytyl-diphosphate as a common precursor of the isoprenyl
side chain, we also know that phylloquinone is connected to the metabolism
of tocopherols and chlorophyll. However, we cannot currently tell to
what extent a change in the biosynthesis flux of one of this compound will
impact the others. Answering this question is important for the basic under-
standing of the metabolic network of plastid isoprenoids as well as for
engineering purposes. For instance, an increase in phylloquinone level that

would occur at the expense of chlorophyll and/or tocopherols could nega-
tively impact photosynthesis or paradoxically decrease the nutritional value
of the derived plant products. The remark stands for the engineering of
tocopherol levels.
As for the fused VKORC1-PDI enzyme, the research problem is now to
identify the proteins whose folding is connected to the reduction of phyllo-
quinone in plastids. The fate of the formed quinol is also intriguing because
the phylloquinone/phylloquinol ratio appears to be remarkably stable in
plants and Synechocystis (Oostende et al., 2008; Widhalm et al., 2009).
A tacit conclusion is that phylloquinol is re-oxidized, and that these organ-
isms can sense the redox status of their phylloquinone pool.
ACKNOWLEDGEMENTS
G. J. B. and C. v. O. dedicate this chapter to the memory of Dr. Philippe

Raymond, whose mentoring and support have been seminal to their works.
Research in our laboratory is made possible in part by National Science
Foundation Grant MCB-0918258 to G. J. B. and by startup funds provided
by the Center for Plant Science Innovation and the Nebraska Tobacco
Settlement Biomedical Research Development Funds.
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AUTHOR INDEX
A Antonicelli, G.E., 247–248

Aasen, E.D., 191, 193–194, 196–197, Antoniw, J., 147–148, 153
198–199 Antony, G., 135–136
Abbasi, A.R., 210–211 Aono, M., 130, 139–141, 143–144
Abell, C., 75 Apel, K., 11–12, 22–23, 25–28, 141, 149–152
Abraham, Z., 24 Appel, J., 191–192
Abrams, G.D., 71–72, 80–81 Appling, D.R., 70–73, 74
Achleitner, G., 215 Aragao, F.J., 96–97
Adamantidis, A., 115 Arai, H., 182, 187
Adams, D., 129–130 Arendt, S., 240–241, 244
Adams-Phillips, L.C., 77–79, 86, 87 Arigoni, D., 18–20
Adler, L.N., 117–118, 127 Arita, M., 182, 187
Adrait, A., 49–51 Armijo, L., 139–141
Agalarov, R., 236 Arnal, N., 46–47, 54
Agius, F., 124, 125–126, 130 Aro, E.M., 139–141
Ahmed, N.J., 153 Aromaa, A., 185–186
Akerlund, H.E., 109–112 Arrigoni, O., 109–112, 127–128, 135, 142,
Akhtar, T.A., 81–82, 87 145–146, 154
Alam, M.T., 91–92 Arrigoni, R., 59–60
Alban, C., 40–42, 45, 46–47, 48–49, 52–53, Asada, K., 139, 142, 149–153, 183
54–58, 59–60 Asai, N., 139–141
Albanes, D., 185–186 Asami, T., 139–141, 149–152, 191, 196–197
Albersheim, P., 116–117 Asard, H., 135–136, 154, 239
Albin, R.L., 187 Asensi, A., 135, 145–146
Albrieux, C., 80–81, 86, 92 Asensi-Fabado, M.A., 12–13
Alcon, C., 143–144 Ashiuchi, M., 20–21
Alexeeva, M., 48–49 Ashurst, J.L., 75
Alexeev, D., 48–51 Asis, R., 199
Alhagdow, M., 116–117, 120–122 Aspinall, T.V., 91–92
Allen-Daniels, M.J., 118–119, 147–148 Atkinson, J., 182, 186, 187–188
Allen, J.F., 122–123 Atteia, A., 49–51
Allen, R.D., 141 Aubert, S., 70–72
Al-Madhoun, A.S., 145 Auldridge, M.E., 246
Almeida, J., 199 Aung, K., 143–144
Almquist, H.J., 230–231 Ausubel, F.M., 242–243
Alonso, J.M., 24–25 Avigliano, L., 145
Alvarez, M.E., 147–148, 153 Avila, C.A., 130
Alvarez, S., 76–77 Axiotis, S., 41–42, 45, 46–48, 53
Amako, K., 122–123, 143–144 Ayaki, M., 128–129
Ames, B.N., 187–188 Ayer, L.M., 54–58
Amrhein, N., 11–12, 15–20, 21–23, 24, Azerad, R., 240–241
25–28, 30 Azzi, A., 186, 187–188, 215–216
Anacleria, M., 125–126
Andersen, G., 191–192
Andersen, J.R., 180–181 B
Anderson, J.W., 135–136 Babujee, L., 247–249
Anderson, S.A., 234–235 Bach, A.U., 234–235
Andersson-Gunnerås, S., 24–25 Bacher, A., 79–80, 87, 91
Andersson, M.X., 215 Badawi, G.H., 142–144
Andre, C.M., 199 Badejo, A.A., 115
Antoniadis, A., 3–5 Badger, M.R., 139–141, 149–153
264 AUTHOR INDEX
Baek, K.H., 141 Bedo, Z., 96

Baier, M., 139, 148, 149–152 Beevers, H., 142
Bai, L., 199–200 Begley, T.P., 15–20, 29
Bailey, L.B., 94 Bekaert, S., 96–97
Bailey, L.M., 54–58 Belinsky, M.G., 85
Baker, N.R., 139–141, 149–152 Belitsky, B.R., 15–17, 18–20
Bakun, P.J., 186 Bellamy, W.D., 3
Baldet, P., 41–42, 45, 46–48, 52, 53, 120–122, Bellone, S., 154
129–130 Benavente, L.M., 24–25
Balestrini, R., 135, 145–146, 154 Ben-Israel, I., 246
Balk, J., 54 Benke, P.J., 187
Ballif, J., 145 Bennett, A., 129–130
Ball, L., 139–141 Bennett, M.J., 81–82, 96–97, 129–130
Bang, J.W., 141 Benning, C., 215
Banhegyi, G., 135–136 Ben-Shem, A., 236
Banks, J., 29 Benson, A.A., 71–72
Barber, G.A., 116–117 Bentley, R., 244
Barbier-Brygoo, H., 143–144 Bentsink, L., 191, 199–200
Barbosa, J.M., 20–21 Benzie, I.J.J., 109–112
Barendregt, A., 122–123, 128–129 Berczi, A., 142–143
Barghouthi, N.T., 153 Bergmuller, E., 191, 197, 198, 210–211
Barnes, J.D., 135, 145–146, 154 Berkovitch, F., 52–53
Barnwell, J.W., 91–92 Bermudez, L., 199
Baroja-Mazo, A., 109–112, 122–123 Bermudez, O.I., 186
Barone, A., 125–126 Bernard, S., 147–148
Barraclough, D., 117–119 Bernhardt, A., 18–20, 23–24, 25–27
Barrett, D.A., 81 Berry, A., 112–114
Barrette, T.R., 190, 191, 194–195 Bertino, J.R., 91–92
Barr, R., 239 Beverley, S.M., 76–77
Barth, C., 115–116, 147–148, 153 Beyer, P., 191–192, 193–194
Bartoli, C.G., 120–123, 128–129, 139–141 Bharani, N., 29
Basset, G.J.C., 77–80, 81–83, 85, 86, 87, Bhattacharya, D., 250–251
96–97, 232, 237–239, 244, 245–246, Bhattacharyya-Pakrasi, M., 237–238
247–248, 250, 253, 255 Bhuiyan, N.H., 145–146, 246
Bassie, L., 96–97 Bianchi, S., 185–186
Bastien, O., 73–74, 96–97 Bilski, P., 11–12, 15–17, 28
Baszis, S.R., 191, 192–195, 196–197, Binkley, S.B., 230–231
198–199 Birringer, M., 253
Batschauer, A., 75–76 Bishop, K., 180
Baumann, U., 142–143 Bitonti, M.B., 135, 145–146, 154
Baumgartner, E.R., 41 Bittl, R., 236
Bauw, G.C., 120, 122–123, 124, 125–127 Blaby, I.K., 76–77
Baxter, R.L., 48–51 Blagborough, A.M., 88–89
Baydoun, E.A.H., 116–117 Blancaflor, E.B., 81
Baymann, F., 250 Blanco, E., 59–60
Beachy, R., 96–97 Blancquaert, D., 74–75, 77, 80–81, 96–97
Beale, M.H., 81 Bligny, R., 71–72, 152–153
Beauparlant, S., 247 Block, M.A., 73–74, 80–81, 86, 92, 188
Beauvoit, B., 120–122 Bloom, A.J., 70–71
Becher, D., 79–80, 91 Bloom, R.E., 154
Bechtold, N., 24–25 Blumberg, B., 234
Beck, E., 135–136 Blumberg, J.B., 186
Becker, H., 195 Blundell, T.L., 75
Beckett, D., 40–41 Bock, R., 237, 238–239, 246–248, 253
Beckman, J.S., 180–181 Bodde, S., 154
Beckwith, J., 237 Boerjan, W., 116–117
Bedair, M., 76, 81 Bognar, A.L., 91
Bedhomme, M., 85 Bohnert, H.J., 72–73
Bedick, T.S., 153 Boldt, R., 74
AUTHOR INDEX 265
Bol, J.F., 242–243, 253 Brushett, D., 87

Bolognesi, M., 145 Brutnell, T.P., 199–200
Bolton, E.M., 183 Bryant, D.A., 191, 196–197, 210–211, 232,
Bolton-Smith, C., 235–236 245–246, 247
Bomar, J.M., 187 Brzezinski, P., 236
Bond, A.D., 137 Buchala, A., 242–243, 247–248
Bonfante, P., 135, 145–146, 154 Buchanan-Wollaston, V., 129–130
Bonin, C.P., 115–116 Buckler, E.S., 199–200
Bonjour, J.P., 44 Buehner, M., 10–11
Boody-Alter, E.L., 186 Buettner, G.R., 109–112, 135–136, 137–139,
Booth, S.L., 234, 235–236, 238, 239, 253 142
Bostick, R.M., 185–186 Bulley, S.M., 114–115, 116–119, 128, 130
Botella, J.R., 76 Buret, M., 129–130
Botella, M.A., 124, 125–126, 130 Burgner, J.W., 18–20
Botha, C.E.J., 211 Burkert, A., 135–136
Böttger, M., 239 Burla, B., 85
Bouchet, B., 116–117 Burlingame, R.P., 112–114
Boudreaux, B., 236 Burnett, J.R., 180–181, 187–188
Bourguignon, J., 54, 59–60, 70–71, 86 Burns, J.J., 25
Bouvier, F., 73–74, 195 Burns, K.E., 15–20
Bowden, E.F., 11–12 Burr, J.A., 183
Bowles, D.J., 77–79 Burton, G.W., 182, 187
Boycheva, S., 24, 30 Bush, D.F., 117–118
Boyd, D., 237 Bushman, E., 145
Bozzo, G.G., 77–79, 81–84, 85 Busto, F., 109–112, 122–123
Bracale, M., 139–141
Bramley, P.M., 199, 238
Branduardi, P., 131 C
Braun, S., 191–192 Caballero, J.L., 124, 125–126, 130
Breen, R.S., 49 Caffarri, S., 11–12, 14, 27–28
Brehelin, C., 194–195, 198, 206, 237, Cahoon, E.B., 191, 194–195, 196
238–239, 246–248, 253 Cahoon, E.D., 237, 238–239, 246–248, 253
Breitenbach, J., 96–97 Cai, X.N., 145
Brennan, R.M., 125–126 Camara, B., 73–74, 195, 240–241, 247–248
Brenner, C., 117–118, 127 Camara, D., 77–79, 87, 89–90
Breslow, E., 120–122 Campa, M., 139–141
Brettel, K., 236 Campopiano, D.J., 48–49
Breyer, I., 187–188 Cane, D.E., 29
Briat, J.F., 139–141 Capell, T., 96–97
Bridge, A., 239 Capron, A., 115
Briesen, I., 194–195, 198, 206 Card, D.J., 253
Brigelius-Flohé, R., 180–181, 187–188, Cardenas, M.L., 73–74
215–216, 253 Carell, T., 75–76
Briggs, S.P., 211 Caro, A., 188
Briggs, W.H., 199–200 Carrari, F., 59–60
Bringer-Meyer, S., 29 Carravieri, S., 141
Broe, K.E., 235–236 Carroll, K.L., 145
Brosch, M., 191–192 Carruthers, K., 130
Brosnan, J.T., 10–11 Carvalho, A., 188, 215
Brosnan, M.E., 10–11 Cassolato, P., 182
Brot, C., 235–236 Catinot, J., 242–243, 247–248
Brouwer, I.A., 96 Caubergs, R.J., 135–136, 154
Browse, J., 215, 244, 247–248, 253 Causse, M., 129–130, 142–143
Brugiere, S., 49–51 Cavalier-Smith, T., 89–90
Brunig, N., 13 Ceol, M., 232, 243, 247, 250
Brunisholz, R., 18–20 Chaerle, P., 96–97
Bruno, L., 135, 145–146, 154 Chakauya, E., 75
Bruno, R.S., 180–181, 187 Chakraborty, A., 187–188
Brunton, M., 49 Chander, S., 199
266 AUTHOR INDEX
Chang, S.C., 143–144 Codoba, F., 127–128

Chang, X.X., 153 Coffino, P., 6
Chan, S.Y., 73, 84 Cojocaru, M., 195
Chapple, A., 24–25 Colditz, G.A., 235–236
Chapuy, M.C., 235–236 Collakova, E., 85–86, 191, 193–194, 198,
Charles, P., 235–236 210–211
Chatson, K.B., 71–72, 80–81 Collins, M.D., 232, 250
Chatterjee, N.P., 49–51 Colussi, A.J., 138–139
Chatzopoulou, F., 145 Colville, L., 147–148, 153
Chaudhuri, B., 135–136 Combs, G.F. Jr., 43, 44
Chaykin, S., 25 Conklin, P.L., 25–27, 112–114, 115–116,
Chen, D.H., 72 117–119, 123–125, 147–148,
Chen, E., 49–51 152–153
Cheney, L.W., 230–231 Conly, J.M., 234–235
Cheng, Z., 191, 196–197 Conney, A.H., 25
Chen, H., 21–22, 25–28, 30, 235–236 Cook, D.R., 132–134
Chen, L.Q., 77, 135–136 Cooney, J., 114–115, 117–119
Chen, M., 244–245 Coppens, E., 115
Chen, Q., 138–139 Corbacho, A.M., 187
Chen, S., 85 Córdoba-Pedregosa, M.C., 239
Chen, W.S., 143–144, 187 Cordoba-Pedregosa, M.D., 127–128
Chen, Z., 143–144, 154 Cornish-Bowden, A., 73–74
Cherest, H., 80–81 Cornish, J.A., 29
Chermak, D., 135–136 Corpe, C.P., 138–139
Chetelat, A., 202–204 Correa da Silva, J.V., 199
Chetelat, R., 129–130 Cortes, D., 139–141
Chevone, B.I., 119, 126–127, 129–130 Cosper, M.M., 53
Chew, O., 139–141, 142–143 Cossins, E.A., 73, 74, 77, 80–81, 84,
Chia, J.M., 199–200 85–86
Chiang, E.-P., 87–88 Coughlan, S.J., 191, 194–195
Chiappetta, A., 135, 145–146, 154 Coutu, J., 139–141, 149–152
Chignell, C.F., 11–12, 15–17, 28 Coward, J.K., 89–90
Chitnis, P.R., 247 Cowman, A.F., 91–92
Chiu, W., 72 Coxon, K.M., 75
Choi, J.S., 46–47, 49–51 Crai, C.A., 3–5
Choi-Rhee, E., 53 Crane, F.L., 188, 232–233
Choi, S.W., 93–94 Creissen, G., 139–141
Cho, K.Y., 141 Crèvecoeur, M., 24, 30
Cho, S.J., 46–47, 49–51 Croft, K.D., 180–181, 187–188
Cho, W.K., 237, 238–239, 242–243, Cronan, J.E., 47–48, 53
247–248 Cross, C.E., 187–188
Christen, P., 6 Cross, R.H.M., 211
Christen, S., 187–188 Crowell, E.F., 196
Christensen, K.C., 117–118, 127 Crowley, M., 96
Christensen, K.E., 70, 71–72, 74 Cruz-Rus, E., 125–126
Christie, D.B., 116–117 Cupples, L.A., 235–236
Christou, P., 96–97 Curien, G., 73–74, 86, 87–88, 92
Chua, N.H., 22–23
Chudek, J.A., 134, 137
Chumnantana, R., 11–12 D
Chung, K.R., 11–12, 15–17 Dagan, T., 49–51
Chu, P.H., 234 Dahnhardt, D., 191–192
Ciraci, S., 135, 145–146, 154 Daicho, K., 143–144
Cirad, S., 146 Dairi, T., 240
Clarke, C.F., 246–247 Dale, M.A., 237–238
Clarke, M.W., 180–181, 187–188 Dam, H., 230–231, 238
Clarke, S.G., 117–118, 127 Danehower, D., 23–24, 27–28
Clemente, T., 196 Dangl, J.L., 154
Clifton, I.J., 109–112 Danna, C.H., 139–141
AUTHOR INDEX 267
Danon, A., 11–12, 120–122, 147–148, De Voss, J., 47–48

149–152 De Wals, P., 96
Dansette, P., 240–241 Dewdney, J., 242–243
Darvill, A.G., 116–117 De Wilde, L., 154
Da Silva, V., 76–77 Dey, S., 46–47
Daub, M.E., 11–12, 15–18, 21–24, 27–28, 30 D’Harlingue, A., 195, 240–241, 247–248
Daum, G., 215 Diallinas, G., 145
Davey, M.W., 109–112, 116–117, 120, Diaz de la Garza, R.I., 79–80, 81, 85–86,
122–123, 124, 125–128 87–88, 96–97
Davidson, K.W., 238 Di Cagno, R., 122–123
Davie, J.R., 54–58 Dickerman, A., 49–51
Davies, W.J., 139–141, 149–152 Dickinson, R.G., 138–139
Davin, L.B., 145 Di Donato, I., 185–186
Davis, B.G., 143–144 Dietrich, F.S., 47–48
Davis, E.J., 145 Dietz, K.J., 148, 149–152
Davis, J.M., 76 Diez, T., 45
Davletova, S., 139–141, 149–152 Dilley, R.A., 188
Davoine, C., 202–204, 213–215 Di Matteo, A., 125–126
Dawes, I.W., 76–77 Dimroth, P., 43–45
Dawson- Hughes, B., 235–236 Dinkins, R., 46–47
de Arriaga, D., 109–112, 122–123 Di Salvo, M., 29
Deaton, B., 3–5 Dittrich, S., 88–89
De Bock, M., 135–136 Dixon, D.P., 143–144
DeBolt, S., 132–134 Dixon, R.A., 194–195
De Bree, A., 96 Dodge, A.D., 138–139, 149–152
De Brouwer, V., 74–75, 80–81, 84–85, 86, 92, Doebley, J.F., 199–200
96–97 Doermann, P., 242–243, 247–248
de Cima, S., 109–112, 122–123 Doisy, E.A., 230–231
de Crecy-Lagard, V., 76 Dolezal, K., 24–25
DeGara, L., 109–112, 122–123, 127–128, 154 Donahue, J.L., 118–119, 147–148
de Godoy, F., 199 Dong, Y.P., 154
de Graaf, A.A., 29 Dong, Y.X., 15–17
Deighton, N., 142 Dormann, P., 191, 194–195, 197, 198, 206,
Dekker, A.O., 138–139 210–211, 237, 238–239, 246–248,
de la Garza, R.D., 73, 81–83, 84, 85 253
DellaPenna, D., 149–152, 183, 185, 186, Douce, R., 40–42, 45, 46–49, 52–53, 54–58,
190–195, 196–197, 198, 59–60, 69–72, 73–75, 77, 80–81, 86,
199–200, 201–204, 205–208, 87, 90–91, 92, 188, 215
210–211, 212–215 Douches, D.S., 196
Delledonne, M., 125–126 Dowdle, J., 117–119, 122–123, 127–129, 130,
Deller, S., 18–20 147–148, 154
Delmas, P.D., 235–236 Drennan, C.L., 52–53
Del Pozo, J.-C., 24 Drew, D.P., 142–143
del Rio, L.A., 135, 139, 141, 143–144 Drewke, C., 3–5, 18–20, 23–24, 25–27
del Valle, P., 109–112, 122–123 Dröge-Laser, W., 22–23
D’Emerico, S., 109–112 Drogoudi, P.D., 135, 145–146
Demol, H., 116–117 Drummond, J.T., 73–74
Denis, L., 54–58 Duan, M., 141
Denslow, S.A., 17–18, 21–23, 27–28 Dubald, M., 192–193
de Paepe, R., 120–122, 147–148 Duffe, P., 129–130
de Pinto, M.C., 122–123 Dugardeyn, J., 24–25
DeSouza, L., 91 Dumas, R., 54–58, 73–74, 77–79, 87,
Desrumaux, C., 187–188 89–90
De Steur, H., 74–75, 81 Dumville, J.C., 154–155
De Tullio, M.C., 109–112, 135, 145–146, Dunathan, H.C., 7–10
147–148, 154 Dunstan, H., 24–25
Deusch, O., 49–51 Durrett, T.P., 191, 193–194, 196–197,
Devaraj, S., 186 198–199
268 AUTHOR INDEX
Du, S., 29 F
Dutton, R.J., 237 Falk, J., 191–192, 237, 238–239, 242–243,
Duval, M., 45 247–248
Dyson, H.J., 80–81 Fall, R.R., 43–45
Fandeur, T., 91–92
Farese, R., 187–188
Farese, R.V. Jr., 187
E Farmer, E.E., 202–204, 213–215
Ealick, S.E., 17–20 Farrar, C.E., 53
Eastmond, P.J., 142–143 Farre, G., 96–97
Edelmann, M., 96 Faurobert, M., 116–117
Edison, A.S., 76–77 Favell, D., 109–112
Edwards, G.E., 152–153 Federico, A., 185–186
Edwards, R., 143–144 Feher, M., 187–188
Ehrenshaft, M., 11–12, 15–17, Feierabend, J., 152–153
22–23, 28 Feldman, L.J., 127–128, 145–146, 154
Ehrhardt, D.W., 145 Felix, G., 11–12
Ehrismann, D., 109–112 Feng, Y., 244
Eichholzer, M., 96 Fernandez, B., 96
Eiken, P., 235–236 Fernandez, L., 128–129
Einstein, J.R., 137 Fernholz, E., 180
Eirserich, J.P., 187–188 Fernie, A.R., 18–20, 23–24, 25–27, 30,
Eitenmiller, R.R., 181–183 59–60, 120–122
Eitinger, T., 14, 22 Ferrarini, A., 125–126
Eliot, A.C., 6, 7–10 Ferro, M., 143–144
Ellerbrock, B., 240–241, 247–248 Feskanich, D., 235–236
Ellis, B.E., 139–141 Feussner, I., 149–152
Elmadfa, I., 180–181, 187–188 Fiedler, E., 188
Elshire, R.J., 199–200 Filkowski, J., 153
Eltayeb, A.E., 142–144 Finamore, F.J., 137
Eltelib, H.A., 115 Finazziagro, A., 145
Elter, A., 143–144 Finazzi, G., 232, 243, 247, 250
Emerson, G.A., 180 Fincher, G.B., 142–143
Emerson, O.H., 180 Finkelstein, E., 187
Enami, S., 138–139 Finkelstein, Y., 3–5
Enders, D., 198 Finkle, B.J., 126–127
Endres, S., 126–127, 130 Fischer, M., 79–80, 87
Entcheva, P., 46–47 Fischer-Schliebs, E., 143–144
Epand, R.F., 187–188 Fisher, S.E., 145
Epand, R.M., 187–188 Fisk, I.D., 188, 215
Ephritikhine, G., 143–144 Fitzpatrick, P.F., 76
Eriksson, L.A., 12–13 Fitzpatrick, T.B., 11–12, 13, 15–20, 21–23,
Erkens, G.B., 14, 22 24, 25–28, 30
Ersoz, E.S., 199–200 Fletcher, J.M., 95, 109–112, 145–146
Esaka, M., 115, 128–129, 145–146, Flicker, K., 13, 15–18
154–155 Flint, D.H., 46–47, 52
Escalante, A.A., 91–92 Flipphi, M., 49–51
Escalettes, F., 53 Florentin, D., 53
Escobar, C., 139–141 Foley, A.L., 234–235
Eskling, M., 109–112 Folkers, K., 3
Espey, M.G., 138–139 Folsom, A.R., 185–186
Essen, L.O., 75–76 Fontecave, M., 53
Eudes, A., 14, 22, 77–79 Ford, C.M., 125–126, 132–134
Evans, H.M., 180 Forster, G., 15–18, 21–22, 25–27
Evans, J.A., 96 Fossati, T., 131
Evans, J.R., 152–153 Foster, S.J., 135, 145–146
Evers, D., 199 Fotopoulos, V., 145–146, 154
Evert, R.F., 211 Fouquet, R., 76
Eymery, F., 210–211 Fourcroy, P., 139–141
AUTHOR INDEX 269
Foyer, C.H., 70–71, 115, 120–123, 128–129, Garcia, I., 190

135–136, 137–138, 145–146, Garcia, V., 116–117, 120–122
147–148, 149–152, 153, 154 Garcion, C., 242–243, 247–248
Fraaije, M.W., 122–123, 128–129 Garin, J., 49–51, 80–81, 143–144
Franceschi, V.R., 131–132, 134 Gast, P., 236
Franck, C., 127–128 Gatzek, S., 109–112, 117–119, 120, 122–123,
Franck, F., 11–12, 14, 27–28 127–129, 147–148, 154
Frank, W., 76 Gaudilliére, J.-P., 240–241, 247–248
Franzmann, L.H., 46–47 Gaunt, J.K., 253
Fraser, P.D., 199, 238 Gaut, B.S., 199–200
Free, D.L., 194–195 Gautier, H., 127–128
Frei, B., 180–181 Gechev, T.S., 141
Frentzen, M., 198 Ge, F., 3–5
Freshour, G., 115–116 Gehrig, P., 18–20
Fretz, H., 202 Geiger, M., 197, 211
Frick, H., 202 Geisel, J., 94
Fridman, E., 246 Geisselbrecht, Y., 75–76
Friedrich, T., 250 Gelfand, M.S., 14, 22
Friedt, W., 198 Gelling, C., 76–77
Fritzsche, H., 180 Genard, M., 127–128
Frolow, F., 236 Gengenbacher, M., 13, 15–18
Fromme, P., 232, 236 George, S., 142–143
Frommer, W.B., 18–20, 23–24, 25–27 Gerbling, H., 46–48, 53
Frusciante, L., 125–126 Gershenzon, J., 109–112
Fryer, M.J., 139–141, 149–152 Giacomelli, L., 139–141
Fry, S.C., 114–115, 116–117, 131–132, 133, Giavalisco, P., 194–195, 198, 206
135–136, 138–139, 154–155 Gibson, K.J., 49–51, 52
Fujikawa, Y., 115, 128–129 Giglione, C., 24
Fujioka, Y., 143–144 Gilbert, L., 116–117, 120–122
Fujisawa, K., 146 Gillaspy, G.E., 118–119, 147–148
Fujita, K., 122–123 Gilliland, L.U., 191, 199–200
Fujiwara, T., 141 Gilot, C., 120, 122–123, 124, 125–127
Fukami, H., 137 Giovannoni, J.J., 77–79, 86, 87
Fukuda, H., 142–143 Gleave, A., 116–117, 128, 130
Fukui, H., 146 Glenn, J., 116–117
Fukunaga, K., 115, 128–129 Glick, B.R., 24–25
Furie, B.C., 234 Glushka, J.G., 116–117
Furihata, K., 240 Gobel, C., 149–152
Furt, F., 232, 237–238, 245–246, 255 Goda, K., 122–123
Godzdanker, R., 187–188
Goertzen, L.R., 20–21, 22
G Goffman, F.D., 195, 199, 201
Gadjev, I., 141 Goggin, F.L., 130
Gagnon, D.R., 235–236 Gogolewski, M., 206–208
Gaigg, B., 215 Gohil, K., 187–188
Gajda, R., 244 Goksör, M., 215
Gakiere, B., 73–74 Golan, T., 152–153
Galletti, S., 199 Golbeck, J.H., 76–77, 247
Gallie, D.R., 143–144, 154 Golderer, G., 79–80
Galli, F., 187–188 Goldschmidt, E.E., 195
Galvez-Valdivieso, G., 139–141, 149–152 Gomez, F., 128–129
Gambonnet, B., 77–79, 80–81, 84–85, 86, Gomez-Jimenez, M.C., 125–126
87–88, 89–90, 92 Gomez, T.A., 117–118
Ganzke, T.S., 191, 194–195 Gontero, B., 49–51
Gao, Q., 152–153 González, E., 23–24, 27–28
Garab, G., 152–153 Gonzalez-Jorge, S., 81
Garchery, C., 129–130, 142–143 Gonzalez-Lamothe, R., 124, 125–126, 130
Garcia-Carmona, F., 148 González-Reyes, J.A., 127–128, 239
270 AUTHOR INDEX
Gonzalez-Sanchez, M.I., 148 Guo, Z.F., 131–132, 244–245

Goodman, B.A., 142 Gupta, M., 202–204
Goodstadt, L., 237 Gurgui, C., 18–20, 23–24, 25–27
Goossens, A., 130 Gutteridge, J.M.C., 109–112
Gordon, H., 182 Gweon, H.S., 75
Gordon, S.L., 125–126 György, P., 3
Gore, M.A., 199–200 Gysin, R., 187–188, 215–216
Gotoh, T., 232
Goto, M., 146
Gottlieb, H.E., 195 H
Gouble, B., 116–117, 142–143 Haase, F.C., 54
Gout, E., 70–72 Haas, R., 76
Gouzd, Z.A., 115–116, 147–148 Hackert, M.L., 6
Goyer, A., 79–80, 85–86, 96–97 Hahn, M.G., 115–116
Graham, C.M., 22–23 Ha, H.T., 246–247
Graham, W.R. Jr., 230–231 Hah, Y.C., 122–123
Granatino, N., 109–112 Hajirezaei, M.R., 197, 210–211
Grandjean, O., 46–47, 54 Halbrook, E.R., 230–231
Grant, M., 146 Haldimann, P., 24–25
Gravel, R.A., 54–58 Halkier, B.A., 24–25
Gray, D.A., 188, 215 Hall, C., 47–48, 49–51
Gray, J.A., 49–51 Hall, G.E., 230–231
Green, B.J., 73, 82–83, 84 Halliwell, B., 109–112
Green, M.A., 131–132, 133, 138–139, Hall, J.D., 153
154–155 Hallmen, C., 3–5
Gregory, J.F. III., 3–5, 73, 76–80, 81–84, Hall, S.E., 191, 194–195
85–86, 87–88, 94, 96–97 Hamilton, R.L., 187
Grills, G.S., 199–200 Hancock, R.D., 125–126, 131, 134, 137
Grimaldi, S., 236 Hanes, J.W., 18–20
Grimm, B., 195 Hannan, M.T., 235–236
Grivet, C., 232, 243, 247, 250 Hannikainen, K., 139–141
Grolle, S., 29 Hanson, A.D., 14, 22, 70–73, 76–80, 81–84,
Grossemy, M., 54–58 85–86, 87–88, 96–97
Gross, J., 237, 238–239, 242–243, 247–248, Hanson, R.E., 47–48
250–251 Hansson, O., 236
Grotjohann, I., 232, 236 Han, Y., 120, 122–123, 124, 125–127, 199
Gruber, J., 198 Hao, M., 191, 192–194, 196–197, 198–199
Gruissem, W., 128, 204 Harjes, C.E., 199–200
Grun, M., 109–112, 125–126 Harms, E., 18–20
Grunwald, D., 80–81 Haroldsen, V., 145
Grusak, M.A., 185, 186, 201–202 Harrington, D.J., 238, 253
Gruszewski, H.A., 119, 129–130 Harris, S.A., 3
Gruszka, J., 149–152, 183, 201 Hartel, A., 143–144
Gruys, K.J., 191, 192–193, 198–199 Hartung, M.L., 135–136
Guan, X., 45 Hartung, W., 149–152
Guergova-Kuras, M., 236 Harwood, J.L., 45
Gu, F., 236 Hasegawa, E., 122–123
Gu, H., 85 Hass, C.G., 199
Guiamet, J.J., 122–123, 139–141 Hatch, M.D., 45
Guignard, C., 199 Haupt, S., 134
Guillon, F., 116–117 Hausman, J.F., 199
Guisez, Y., 135–136, 145–146, 154 Hausner, G., 22–23
Guldener, U., 79–80, 91 Haussmann, C., 79–80, 91
Gullner, G., 138–139, 149–152 Havaux, M., 11–12, 14, 27–28, 109–112,
Gunesekera, B.N., 118–119, 147–148 149–153, 183, 187–188, 195,
Gunsalus, I.C., 3 210–211
Guo, J.K., 153 Hawkins, N.D., 81
Guo, W.J., 135–136 Hayashi, H., 139–141
Guo, Y., 246 Haze, S., 47–48
AUTHOR INDEX 271
Healy, S., 54–58 Holcomb, W.F., 230–231

Heazlewood, J.L., 120–122 Hollander-Czytko, H., 149–152, 183
Hebbeln, P., 14, 22 Holland, J.B., 199–200
Hebda, P.A., 116–117 Holloway, D.E., 238
Heber, U., 135–136, 142 Holst, W.F., 230–231
Heck, A.J.R., 122–123, 128–129 Hopfgartner, G., 232, 243, 247, 250
Hedden, P., 109–112 Horemans, N., 135–136, 145–146, 154
Hegazi, A., 3–5 Horie-Inoue, K., 234
Hegemann, J.H., 79–80, 91 Horn, D., 109–112
Hegie, A., 139–141 Horne, D.W., 73
Heide, L., 240–241, 244 Horner, H.T., 131–132
Heliovaara, M., 185–186 Horwitt, M.K., 186
Hellmann, H., 5, 18–20, 23–24, 25–27 Hoser, D., 116–117, 128, 130
Helmreich, E.J.M., 10–11 Hosomi, A., 182, 187
Helms, G.L., 109–112 Hossain, T., 96–97
Helsper, J.P., 109–112, 125–126 Hostomska, Z., 90–91
Hemming, C., 191, 199–200 Hou, B.H., 135–136
Henderson, J.M., 13 Hou, C.M., 127–128
Hennig, L., 204 Howe, G.A., 130
Hennig, P., 210–211 Howell, P.L., 53
Henninger, M.D., 232–233 Howitt, C.L., 49–51
Henry, R.J., 87 Howland, E., 90–91
Herbig, A.K., 82 Hrastnik, C., 215
Herbig, K., 87–88 Hsieh, T.F., 190
Hermann, A.P., 235–236 Hsiung, Y., 29
Hermann, R.G., 237, 238–239, 242–243, Hsu, A.Y., 246–247
247–248 Huang, C.H., 153
Hernandez, H.L., 53 Huang, J.J., 109–112
Hernandez, I., 135, 145–146 Huang, T.Y., 234
Hernandez, J.A., 135, 139, 141, 143–144 Huang, W., 49–51
Hernandez, M., 182 Huber, R., 145
Herr, E.H., 139–141 Hugenholtz, J., 79–80, 96–97
Herrero, S., 30 Huh, W.K., 122–123
Hertel, B., 143–144 Hu, J.P., 143–144
Hess, J.L., 185 Hummel, S.G., 138–139
He, W.L., 153 Hung, K.F., 145
Heyno, E., 239 Hunter, S.C., 194–195, 196
He, Z.H., 131–132 Huntington, S., 139–141
Hideg, E., 142, 149–153 Hurwitz, B.L., 199–200
Hilby, C.L., 109–112, 125–126 Hutson, K.G., 240–241
Hillier, W., 139–141, 149–153 Huynh, B.H., 53
Hill, R.E., 29 Hwang, I.T., 46–47, 49–51
Hills, J., 87–88 Hyde, J.E., 70, 74–75, 88–90, 91–92
Himmeldirk, K., 29 Hymes, J., 43
Hirai, M.Y., 130
Hiratsuka, T., 240
Hirooka, S., 141 I
Hirsch-Hoffmann, M., 204 Ichikawa, T., 234
Hitz, W.D., 191, 194–195 Ichikawa, Y., 122–123, 127
Hobson, B., 187–188 Ichimura, S., 235–236
Hodges, S., 235–236 Ifuku, O., 47–48
Hoeberichts, F.A., 115 Igarashi, K., 187
Hofbauer, R., 3–5 Igarashi, O., 182, 187
Hoffmann, L., 199 Iijima, Y., 246
Hoffmann, M.R., 85, 138–139 Ikeda, K., 234
Hofius, D., 197, 210–211 Ikeda, Y., 232, 250
Hofmann, M., 135–136 Ikegami, I., 246, 247–248
Holaday, A.S., 141, 142–143 Illarionova, V., 79–80
Holbrook, D., 143–144 Imahori, Y., 128–129
272 AUTHOR INDEX
Imperio, R.M., 115–116, 147–148 Jiang, J.Z., 131–132, 191, 192–194, 196–197,
Inaba, A., 187 198–199
Inaba, K., 237 Jiang, K.N., 145–146, 154
Inada, N., 242–243, 247–248 Jiang, L.R., 131–132
Inanaga, S., 142–144 Jiang, M., 244–245
Ingala, L.R., 139–141 Jiang, Q., 187–188
Ink, S.L., 3–5 Jiang, X.H., 115–116, 147–148
Innocenti, A.M., 154 Jimenez, A., 135, 139, 141, 143–144
Inoue, H., 232 Jishage, K., 187
Inoue, K., 182, 187, 242–243, 247–248 Jitrapakdee, S., 43–45
Inoue, S., 234 Ji, X.M., 131–132
Inoue, Y., 143–144 Job, C., 45
Inze, D., 109–112, 115, 128, 130, 139–141 Job, D., 40–41, 45, 46–47, 54–58, 73–74
Ioki, M., 143–144 Johansen, I.E., 115
Isaac, G., 212–215 John, C.F., 109–112
Ischebeck, T., 195 John, P., 109–112
Ishikawa, J., 240 John, R.A., 6, 10–11
Ishikawa, T., 80–81, 109–112, 114–115, Johnson, C.S., 115
117–118, 120, 122–126, 127–129, Johnson, M.K., 53
130, 139–141, 147–148, 154 Johnson, T.W., 247
Isner, J.C., 73–74 Joliot, A., 236
Isupov, M., 118–119 Joliot, P., 236
Itoh, N., 240 Jones, A.D., 149–152, 205–208, 210, 247
Itoh, S., 232, 250 Jones, A.M., 139–141, 149–152
Itoh, Y., 232 Jones, C.M., 129–130
Ito, K., 237 Jones, D., 232, 250
Ito, T., 191, 196–197 Jones, J.D.G., 154
Ivanov, B.N., 139–141, 149–153 Jones, M.A., 112–114, 120
Ivanov, R.A., 54–58 Jong, Y.J., 25
Iwami, K., 14–15 Jonsson, M., 91–92
Iwamoto, J., 235–236 Joosten, H.J., 122–123, 128–129
Iwasa, N., 109–112, 123–126 Jordan, B.R., 109–112
Izumi, Y., 49 Jordan, P., 236
Jose, M.D.F., 122–123, 128–129
Jourdain, A., 80–81
J Joyard, J., 49–51, 143–144, 188
Jabrin, S., 73–75, 77, 80–81, 84, 86, 87–88, 92 Jürgens, G., 24–25
Jagendorf, A.T., 145–146 Just, D., 120–122
Jagerstad, M.I., 96
Jahn, O., 247–248
Jain, A.K., 130 K
Jain, S.K., 11–12 Kabir, H., 14–15
Jakoby, M., 22–23 Kaempf-Rotzoll, D.E., 187
Jameson, G.N., 53 Kagan, L., 109–112, 125–126
Janave, M.T., 72–73 Kainersdorfer, E., 215
Jander, G., 117–118 Kaini, R.R., 187–188
Jansen, M.A.K., 145–146, 154 Kaiping, S., 247–248
Janson, C.A., 90–91 Kajiwara, M., 47–48
Jarrett, J.T., 52–53 Kalkmann, D.C., 96–97
Jarvinen, R., 185–186 Kamada, H., 143–144
Jeltsch, J.M., 59–60 Kamal-Eldin, A., 180–181, 187–188
Jenns, A.E., 11–12, 15–17 Kaminaka, H., 142–144
Jensen, D., 117–119 Kan, C.C., 90–91
Jensen, P.K., 191, 192–193, 198–199 Kandianis, C.B., 199–200
Jeong, Y.J., 141 Kandlbinder, A., 149–152
Jeremic, V., 25 Kanellis, A.K., 109–112, 135, 145–146, 154
Jia, D., 54–58 Kangasjarvi, S., 139–141
Jia, J., 49–51 Kang, S.O., 122–123
Jialal, I., 186 Kang, Y.-N., 17–20, 142
AUTHOR INDEX 273
Kannan, K., 11–12 Kishimoto, R., 122–123

Kanofsky, J.R., 138–139 Kishore, G., 96–97
Kanwischer, M., 191, 194–195, 198, 206 Kisker, C., 244
Kaplan, F., 73 Kisu, Y., 146
Kappes, B., 13, 15–18 Kita, M., 15–17
Kariluoto, S., 96 Kitano, H., 80–81
Karkonen, A., 154–155 Kitaoka, S., 125–126
Karpinska, B., 139–141 Kittles, R.A., 187
Karpinski, S., 139–141 Kiyose, C., 182, 187
Karrer, P., 180 Klaus, S.M., 79–80, 85, 96–97
Karunanandaa, B., 191, 192–193, 194–195, Kleber-Janke, T., 190
198–199 Klein, H.W., 10–11
Kashino, Y., 232, 250 Klein, M., 85
Kästner, U., 3–5 Klessig, D.F., 147–148, 153
Kato, N., 145–146, 154–155 Kliebenstein, D.J., 109–112
Kato, T., 139–141 Klukas, O., 236
Katsuragi, T., 15–17 Knag, K., 20–21, 22
Kausch, A.P., 131–132 Knapen, M.H., 235–236
Kavitha, K., 142–143 Knapp, S.J., 199
Kawano, N., 142–144 Knekt, P., 185–186
Kaye, A.D., 3–5 Knight, M.R., 153
Kaye, A.M., 3–5 Knighton, D.R., 90–91
Keates, S.E., 109–112, 131–132 Knowles, J.R., 40–41
Keller, R., 112–114, 115–116, 147–148 Knox, J.P., 81
Keller, Y., 195 Kobayashi, M., 191, 196–197, 232
Kelly, J.M., 109–112, 139 Kobayashi, N., 183, 201
Kelly, S., 125–127 Koehler, G.J., 79–80, 91
Kemmerling, M., 188 Koga, N., 47–48
Kempinski, C.F., 147–148 Kögel, F., 41
Kempna, P., 187–188, 215–216 Kohlwein, S.D., 215
Kenk, B., 154–155 Koike, H., 232, 250
Kennedy, I.A., 29 Kojiro, C.L., 6
Keresztes, I., 18–20 Kojo, S., 137
Kerk, N.M., 127–128, 145–146, 154 Kolkmann, R., 240–241, 244
Kern, A.D., 6 Kolling, D., 247
Kernebeck, B., 191–192 Kollist, H., 135
Kershaw, N.J., 109–112 Komura, M., 232, 250
Kesinger, N.G., 137 Kondo, H., 15–17
Kessler, F., 194–195, 198, 206 Koo, A.J.K., 130
Kessler, K., 237, 238–239, 246–248, 253 Koornneef, M., 191, 199–200
Keulemans, J., 127–128 Kornyeyev, D., 141, 142–143
Key, J.L., 154–155 Koscher, J.R., 131–132
Khanna, S., 180–181, 187–188 Kossmann, J., 112–114, 115–116, 147–148
Kiddle, G., 115, 120–123, 135, 145–146, Kostman, T.A., 131–132
147–148, 153 Kotchoni, S.O., 147–148
Kiel, D.P., 235–236 Koussevitzky, S., 139–141
Kim, C.H., 149–152 Kovalchuk, I., 153
Kim, H.U., 244, 247–248, 253 Kovalchuk, O., 153
Kim, J.G., 135–136 Kozela, C., 24–25
Kim, J.S., 116–117, 141 Kramarenko, G.G., 138–139
Kim, K.-W., 145 Kramer, D.M., 152–153, 250
Kim, M.D., 141 Krasser, A., 215
Kim, S.T., 122–123 Krause, G.H., 127–128
Kim, Y.H., 141 Krauss, N., 236
King, C.G., 138–139 Kresovich, S., 199–200
King, J., 71–72, 80–81 Kress, W., 18–20
Kinsland, C.L., 15–17 Kricke, J., 79–80, 91
Kirsch, J., 6, 7–10 Krieger-Liszkay, A., 139–141, 149–152, 239
Kishimoto, J., 47–48 Krischke, M., 202–204
274 AUTHOR INDEX
Krishna, M.C., 138–139 la Veechia, F., 109–112

Kroj, T., 22–23 Lawson, K.A., 185–186
Kronzucker, H.J., 115–116, 147–148 Lawson, T., 139–141, 149–152
Kruh, G.D., 85 Layoune, O., 120–122, 147–148
Kruk, J., 149–152, 183, 194–195, 201 Lee, D.H., 46–47, 49–51
Krupinska, K., 190, 191–192, 201, 237, Lee, H.S., 141
238–239, 242–243, 247–248 Lee, L.-R., 87–88
Kruse, E., 195 Lee, M.J., 139–141
Ksas, B., 11–12, 14, 27–28, 187–188, Lee, N.S., 96
210–211 Lee, P.T., 246–247
Kubo, A., 139–141, 143–144 Lee, R.E., 46–47
Kubota, K., 146 Lee, Y.C., 25
Kuhn, I., 59–60 Lee, Y.P., 141
Kuhn, L., 49–51 Lefebvre-Legendre, L., 232, 243, 247, 250
Kulkarni, S., 130 Leferink, N.G.H., 122–123, 128–129
Kumar, G.K., 43–45, 54 Leistner, E., 3–5, 18–20, 23–24, 25–27,
Kumar, R., 198 240–241, 244
Kumar, S., 147–148, 153 Leklem, J.E., 14–15
Kunji, E.R., 85 Lelandais, M., 135–136
Kuppu, S., 141 Lemaire-Ewing, S., 187–188
Kuramitsu, S., 20–21 Lemke, R., 191, 197, 210–211
Kuroiwa, H., 141 Lemoine, Y., 59–60
Kuroiwa, T., 141 Lenne, C., 190
Kushi, L.H., 185–186 Leonard, S.W., 187–188, 199
Kushnir, S., 139–141 Leon-Del-Rio, A., 54–58
Kwak, J.M., 154 Leonhardt, N., 154
Kwak, S.S., 141 Lepisto, A., 139–141
Kwon, S.Y., 141 Lepkovsky, S., 3
Leskovac, V., 25
Leth, T., 182
L Leuendorf, J.E., 18–20, 23–24, 25–27
Laber, B., 29 Levering, C.K., 191, 193–194, 196–197,
Ladenstein, R., 145 198–199
Ladha, Z., 187 Levine, M., 135–136, 138–139
Lagrost, L., 187–188 Levine, T., 215
Lahnstein, J., 142–143 Levin, I.M., 117–118
Laing, W.A., 114–115, 116–119, 128, 130 Lewis, N.G., 145
Laloi, C., 11–12, 22–23, 25–28, 141, 149–152 Lewis, S.D., 6
Lalonde, S., 135–136 Lezhneva, L., 237, 238–239, 242–243,
Lambert, W.E., 74–75, 80–81, 84–85, 86, 247–248
87–88, 92, 96–97 Liang, D., 127–128
Lambrix, V.M., 109–112 Liang, H.J., 139–141, 149–152
Lam, H.-M., 29 Liang, M.X., 145
Lamodiére, E., 242–243, 247–248 Li, B.H., 115–116, 147–148
Lampi, A.M., 199 Li, B.S., 131–132
Landes, N., 253 Lichtenthaler, H.K., 240
Lane, J.M., 46–47 Lieberman, S., 13
Langebartels, C., 128 Liebler, D.C., 183
Lara-Nunez, A., 76–77, 81–82, 87 Lieutaud, C., 250
Larkindale, J., 153 Li, F., 115–116, 141, 142–143
La Rocca, N., 135, 145–146, 154 Li, H.X., 129–130, 199
Larondelle, Y., 199 Li, J., 199, 234
Larrimore, K.E., 147–148, 153 Li, L.J., 121, 135–136, 188
Larsson, K.E., 215 Liljenberg, C., 215
Lass, A., 183 Lill, R., 54, 76–77
Lassner, M.W., 193–194 Lim, E.K., 77–79
Last, R.L., 25–27, 112–114, 115–116, Lim, F., 43–45
117–118, 147–148, 153, 191, Lim, G., 11–12
196, 201–202 Lim, H.K., 46–47, 49–51
AUTHOR INDEX 275
Li, M.J., 127–128 Lu, S.Y., 131–132

Limka, N., 73–74 Lu, T., 20, 22, 27–28
Lim, P., 91–92 Lüthje, S., 239
Lim, Y., 187–188 Luttge, U., 143–144
Li, M.Y., 11–12, 15–17, 28 Lutz, C., 195
Lincoln, K., 191, 193–194, 196–197, 198–199 Luwe, M.W.F., 135–136
Lindenbaum, J., 234–235 Lu, Y.S., 131–132
Lindqvist, Y., 46–47, 49–51 Lye, L.F., 76
Lingard, M.J., 142–143 Lyon, G.D., 142
Ling, J., 143–144 Lyons, T., 135, 145–146
Lin, J.C., 234 Lytovchenko, A., 18–20, 23–24, 25–27
Linka, N., 143–144
Linkswiler, H., 13
Lin, L.S., 154–155 M
Linne, U., 75–76 Ma, C., 247–249
Lin, S., 47–48 MacCorquodale, D.W., 230–231
Linster, C.L., 117–118, 127 Macherel, D., 80–81
Linthorst, H.J.M., 242–243, 253 Macheroux, P., 13, 15–20
Lin, Y., 141, 142–143 Machida, C., 80–81
Li, Q., 115–116, 147–148 Machida, Y., 80–81
Li, R., 71–72 Machler, F., 121, 135–136, 188
Lisenbee, C.S., 141, 142–143 Machutta, C., 244
Liso, R., 109–112, 127–128, 135, 145–146, Macia, H., 148
154 MacKenzie, R.E., 70
Liszkay, A., 154–155 Mackerness, S.A.H., 109–112
Littlechild, J.A., 118–119 MacMillan, F., 236
Liu, E., 131–132 MacRae, E., 116–119, 128, 130
Liu, G., 199 Maeda, H., 191, 196–197, 210–211, 212–215
Liu, H., 199 Maeda, M., 137
Liu, X., 115 Ma, F.W., 127–128
Li, W., 199, 237 Magallanes-Lundback, M., 191, 199–200
Li, X., 109–112 Magliano, P., 49–51
Li, Y., 187 Majcherczyk, P., 202–204, 213–215
Lobreaux, S., 54 Major, L.L., 116–117
Locy, R.D., 20–21, 22 Malissiovas, A., 3–5
Loeffler, C., 202–204 Manandhar-Shrestha, K., 143–144
Loewus, F.A., 109–114, 123–127, 131–134 Mann, S., 49
Lohmann, A., 194–195, 198, 206, 237, Mano, J., 142, 152–153
238–239, 242–243, 246–248, 253 Manor, D., 182
Loizeau, K., 74–75, 76–77, 80–81, 84–85, 86, Man, T.K., 29
87–88, 92 Manzano, C., 24
Lokstein, H., 195 Mao, Y., 72
Lopez-Torrejon, G., 24 Mapson, L.W., 120–122
Lorence, A., 126–127, 129–130 Marahiel, M.A., 75–76
Lotierzo, M., 53 Maras, J.E., 186
Lowry, R.B., 96 Marceau, M., 6
Luan, L., 49–51 Marcovici-Mizrahi, D., 46–47
Lucas, W.J., 197 Marcus, S.E., 81
Lüder, F., 247–248 Markovic, J., 154
Ludtke, S.J., 72 Marmagne, A., 143–144
Ludwig, A., 59–60 Marquet, A., 48–49, 53
Luecke, H., 145 Marquis, N., 125–126
Lueder, F., 247–249 Marra, E., 122–123
Luhs, W., 198 Marre, E., 142
Lui, H., 80–81 Marr, S.K., 242–243, 247–248
Lukowitz, W., 115–116, 147–148 Marsh, K., 70, 74–75, 91–92
Lunde, C., 142–143 Martinoia, E., 85, 121, 135–136, 188
Lunn, J.E., 59–60 Martin, S.M., 138–139
Luomala, E.M., 139–141 Martin, W., 49–51
276 AUTHOR INDEX
Maruta, T., 114–115, 122–123, 127, 139–141 Mene-Saffrane, L., 149–152, 190–191,
Masi, A., 139–141 194–195, 196, 202–204, 205–208,
Mason, J.B., 93–94 210, 213–215
Masson, P.H., 145 Meng, Q.W., 115–116, 141, 142–143
Massot, C., 127–128 Meng, Y.L., 145–146, 199
Masuda, T., 130 Menting, J.G., 91–92
Masui, R., 20–21 Merrill, A.H., 13
Masumoto, I., 109–112, 123–126 Meshnick, S.R., 91–92
Mathis, P., 236 Messerschmidt, A., 145
Matringe, M., 187–188, 190, 192–193, Métraux, J.P., 242–243, 247–248
210–211 Meunier, P.J., 235–236
Matsumoto, F., 130 Meurer, J., 237, 238–239, 242–243, 247–248,
Matsushita, S., 188 250–251
Matsuyama, T., 139–141 Miao, L., 197
Mattanovich, D., 131 Mieda, T., 120, 122–123, 127
Mattevi, A., 122–123, 128–129 Miernyk, J.A., 141
Matthews, D.A., 90–91 Miersch, O., 11–12
Matthews, R.G., 73–74 Mikami, B., 142
Mattson, M.P., 93–94 Mikkelsen, M.D., 24–25
Matxain, J.M., 12–13 Millar, A.H., 120–122, 139–141, 142–143
Mauch, F., 121, 135, 141 Miller, G., 139–141
Maurer, W., 29 Miller, J.F., 199
Mauricio, I.L., 139 Miller, J.G., 154–155
Mayalagu, S., 115 Miller, L.T., 14–15
May, G.S., 11–12, 15–17 Milward, S.E., 152–153
May, J.M., 109–112 Mimuro, M., 232
Maynard, T.M., 109–112, 125–126 Minami, C., 232, 250
Mazurkiewicz, J., 17–20 Minkov, I.N., 141
McCarthy, E.A., 85 Mink, P.J., 185–186
McCarthy, P.T., 238 Mireau, H., 46–47, 54
McClelland, B.W., 137 Misumi, O., 141
McCollum, A.M., 91–92 Mitchell, J.B., 138–139
McConn, M.M., 131–132 Mitchell-Olds, T., 109–112
McDonald, M.K., 54–58 Mitchell, S.L., 88–89
McDonnell, L., 24–25 Mitsky, T.A., 193–194
McDonough, M.A., 109–112 Mitsuda, H., 14–15
McFarlane, W.D., 230–231 Mittenhuber, G., 15–17, 20
McGrath, J.M., 196 Mittler, R., 139–141, 149–152
McIntosh, L., 128–129 Mittova, V., 120–122
McIntosh, S.R., 87 Mixson-Hayden, T., 91–92
McKee, R.W., 230–231 Miyagawa, Y., 139
McKenna, M., 232–233 Miyake, C., 142, 143–144
McLafferty, F.W., 15–17 Miyaoka, H., 47–48
McLaughlin, P., 185 Miyashita, H., 232
McLean, R.R., 235–236 Miyashita, Y., 137
McMullen, M.D., 199–200 Mizusawa, H., 187
McNeil, S.D., 72–73 Moccand, C., 15–17, 18–20
McQuinn, R.P., 87 Moeder, W., 147–148, 153
McRae, D., 134 Mohammed, Z.M., 29
Meganathan, R., 244 Moldau, H., 135
Mehrshahi, P., 81–82, 96–97 Moldt, J., 75–76
Mehta, P.K., 6 Moller, I.M., 142–143
Meinke, D.W., 46–47, 49–51, 115–116, Monéger, R., 240–241, 247–248
147–148 Monsen, E.R., 186
Meinnel, T., 24 Mooney, S., 5
Melendez-Martinez, A.J., 199 Moore, M., 71–72
Melino, V.J., 125–126, 132–134 Moran, R.G., 85
Melzer, M., 197, 211 Mori, I.C., 154
Mendes, P., 126–127, 130 Morishima, I., 143–144
AUTHOR INDEX 277
Morishita, N., 142 Nakano, Y., 125–126

Mori, T., 141 Nakao, M., 137
Morita, T., 20–21 Nakata, P.A., 131–132
Morley, S., 182 Naponelli, V., 76–79, 81–84, 85
Moroni, A., 143–144 Naqvi, S., 96–97
Morré, D.J., 239 Narang, M.A., 54–58
Morris, C.P., 43–45 Narendra, S., 141, 142–143
Morris, K., 109–112 Nater, M., 149–152
Morrow, J.D., 187 Naur, P., 24–25
Morse, A.M., 76 Navarre, D.A., 96
Mosekilde, L., 235–236 Navarrete, O., 80–81, 96–97
Moshiri, F., 191, 192–193, 198–199 Naydov, I.A., 139–141, 149–152
Motohashi, R., 191, 196–197 Néel, D., 187–188
Motoki, T., 120, 122–123, 141 Negis, Y., 187–188, 215–216
Mouillon, J.M., 70–71, 80–81, 87, 90–91 Nelson, K., 46–47
Mounet, F., 120–122 Nelson, M.J., 25
Mountjoy, K., 6 Nelson, N., 236
Mousa, S.A., 180–181 Nessler, C.L., 119, 126–127, 129–130
Mowla, S., 147–148, 153 Neuburger, M., 54, 69–71, 80–81, 86
Mubarakshina, M.M., 139–141, 149–152 Neuhaus, G., 191–192, 193–194
Muckenschnabel, I., 142 Neuwirth, M., 18–20
Mueller, M.J., 202–204 Newland, D., 187
Muhlenhoff, U., 54, 76–77 Ngo, Q.A., 115
Mujahed, N., 18–20, 23–24, 25–27 Nguyen, T.T., 246
Mukherjee, M., 147–148, 153 Nichols, B.P., 77–79, 86, 87
Mukhopadhyay, D., 24 Nickle, T.C., 115–116, 147–148
Mullen, R.T., 141 Nicolet, Y., 52–53
Muller, D.P., 185–186 Nielsen, D.M., 199–200
Muller, M., 75–76 Nikawa, J., 15–17
Muller-Moule, P., 109–112, 117–118, Nikolau, B.J., 45, 49–51, 54–58
128–129, 147–148, 152–153 Nilsson, A., 122–123
Müller, S., 13, 15–18 Nishida, K., 141
Mulliez, E., 53 Nishikawa, H., 109–112, 123–126
Mullineaux, P.M., 139–141, 149–152 Nishioka, H., 137
Munne-Bosch, S., 12–13, 183 Nitschke, W., 250
Munoz-Blanco, J., 124, 125–126, 130 Niu, J.K., 131–132
Munteanu, A., 187–188, 215–216 Niyogi, K.K., 109–112, 152–153
Muralla, R., 49–51 Noctor, G., 70–71, 137–138, 147–148
Murata, N., 143–144 Noel, J.P., 246
Murchie, E.H., 152–153 Nogala Kaucka, M., 206–208
Murgia, I., 139–141 Noiriel, A., 76, 82–84, 85
Murtif, V.L., 54 Noji, M., 130
Mustacich, D.J., 180–181, 187 Nonaka, H., 143–144
Muth, S., 91–92 Nonomura, A.R., 71–72
Mutterer, J., 73–74 Norris, S.R., 25–27, 112–114, 115–116,
117–118, 147–148, 153, 190,
191–192, 194–195
N Nudelman, A., 46–47
Nagano, S., 3–5 Nunes, A.C., 96–97
Nagata, N., 191, 196–197 Nunes-Nesi, A., 24, 30, 116–117,
Nagy, V., 152–153 120–122
Naismith, J.H., 116–117 Nurmi, T., 199
Naito, M., 143–144 Nushimura, S., 3–5
Nakagawa, T., 143–144 Nystuen, A., 187
Nakajima, N., 139–141, 143–144 Nzila, A., 70, 74–75, 77, 89–90, 91–92
Nakamura, A., 109–112, 122–126,
232, 250 O
Nakamura, S., 80–81 Obado, S.O., 139
Nakano, M., 20–21 Oba, K., 145–146
278 AUTHOR INDEX
Obara, K., 142–143 Page, M., 128–129, 149–152

Obayashi, T., 130 Pagnussat, G.C., 115
Oberbaum, M., 3–5 Pakrasi, H.B., 237–238
Obermueller-Jevic, U., 187 Pallanca, J.E., 112–114, 127–128
Ochsenbein, C., 149–152 Pallardo, F.V., 154
Oelmuller, R., 142–143 Palm, D., 10–11
Oertli, J.J., 121, 135–136, 188 Palmieri, F., 59–60
Oettmeier, W., 149–152, 183 Palmieri, L., 59–60
Ogasawara, N., 15–17 Panagabko, C., 182
Ogata, K., 49 Panopoulos, N.J., 145
Ogawa, D., 143–144 Parcy, F., 22–23
Ohlrogge, J.B., 45 Parida, A., 142–143
Ohno, C., 142 Park, N.J., 46–47, 49–51
Ohno, R., 246, 247–248 Parsons, H.T., 135–136
Ohto, M.A., 246, 247–248 Parsons, R., 182
Ohwaki, T., 152–153 Pasapula, V., 141, 142–143
Okamura, M., 109–112 Passarella, S., 122–123
Oka, T., 13 Pasternak, T., 145–146, 154
Oksanen, E., 135 Pastori, G.M., 122–123, 147–148
Olejnik, D., 206–208 Pastori, M., 187–188
Oliveira, M.A., 6 Patel, P.D., 187
Oliver, D.J., 139–141, 149–152 Pateraki, I., 135, 145–146
Ollagnier-de-Choudens, S., 53 Patton, D.A., 46–47
Ollilainen, V., 199 Paulowski, R.M., 29
Olmos, E., 147–148, 153 Pavet, V., 147–148, 153
Olsen, L.J., 143–144 Pawlak, A., 149–152, 183, 201
Onai, K., 246, 247–248 Pease, A.J., 29
O’Neill, M.A., 116–117 Peiffer, J.A., 199–200
Oniki, T., 145–146, 154–155 Pei, Z.M., 154
Oommen, S., 187–188 Pellny, T.K., 147–148, 154
Oostende, C.V., 232, 237–239, 250, 255 Peng, C.L., 131–132
op den Camp, R.G.L., 11–12, 149–152 Peng, M., 73, 82–83, 84, 85
Ordovas, J., 235–236 Peng, S., 131
Ormenese, S., 115 Peng, X.X., 131–132
O’Roark, E., 187–188 Pepin, R., 54–58, 190
Orselli, S.M., 143–144 Peracchi, A., 5
Orsomando, G., 73, 81–83, 84 Percudani, R., 5
Ort, D.R., 149–152 Perez-Cadahia, B., 54–58
Orth, C., 75–76 Perez Conesa, D., 96–97
Orvar, B.L., 139–141 Perilleux, C., 115
Osmani, A.H., 11–12, 15–17 Perkins, J.B., 49–51
Osmani, S.A., 11–12, 15–17 Perktold, A., 215
Ostergaard, J., 120, 122–123, 124, 125–127 Pero, J.G., 49–51
Osterman, A., 14, 22 Persiau, G., 116–117, 120, 122–123, 124,
Oster, U., 195 125–127
Otaiza, S.N., 199 Pers-Kamczyc, E., 141
Ottenhof, H.H., 75 Peter, C.I., 211
Otto, W., 116–117, 128, 130 Petersen, J., 236
Oufir, M., 199 Peterson, J.W., 234
Ouyang, B., 129–130 Petit, J., 116–117, 120–122
Oxborough, K., 139–141 Petruzzelli, R., 145
Peynot, P., 24
Pezzotti, M., 125–126
P Pfannschmidt, T., 122–123
Pacheco-Alvarez, D., 54–58 Phalip, V., 59–60
Paciolla, C., 109–112 Picciocchi, A., 52–53
Packer, L., 187–188 Pichersky, E., 246
Pagani, R., 131 Pichler, H., 215
Page, D., 116–117, 142–143 Pickard, A.L., 91–92
AUTHOR INDEX 279
Pietinen, P., 185–186 Quinlivan, E.P., 77–80, 81–83, 85–86, 87,

Pignocchi, C., 135, 145–146, 147–148 96–97
Piippo, M., 139–141 Quiocho, F.A., 72
Piironen, V., 96, 199 Qu, X.Q., 135–136
Pineau, B., 120–122, 147–148
Pinon, V., 48–49
Pinto, M.E.S., 238 R
Pitsch, N.T., 139 Raber, J., 187–188
Plett, J.M., 24–25 Raclaru, M., 198
Ploux, O., 48–49 Radzio, J.A., 127, 130
Plume, A.M., 76 Raeymaekers, T., 135–136
Pnueli, L., 139–141 Raichaudhuri, A., 85
Pogson, B.J., 196, 201–202 Rajani, S., 115
Poirier, Y., 49–51 Rambeck, W., 235–236
Pokorny, R., 75–76 Ramessar, K., 96–97
Pollard, M., 191 Ramos-Parra, P.A., 81
Polle, A., 148 Rapolu, M., 120, 122–123
Polyak, S.W., 54–58 Rapoport, T.A., 237
Ponting, C.P., 237 Rappaport, F., 232, 243, 247, 250
Pont, S.D.A., 125–126, 134, 137 Rasche, N., 247–248
Porfirova, S., 191, 194–195, 197, 198, 206, Raschke, M., 24, 30
210–211 Raschle, T., 13, 15–20, 21–22, 25–27
Porro, D., 131 Rascio, N., 109–112
Pospisil, P., 149–152 Rashotte, A.M., 20–21, 22
Post-Beittenmiller, D., 192–194, 198–199 Rassam, M., 116–117, 128, 130
Potters, G., 135–136, 145–146, 154 Rattanachuen, W., 91–92
Powell, A., 129–130 Rautenkranz, A.A.F., 121, 135–136, 188
Powell, J., 3–5 Ravanel, S., 48–49, 59–60, 70, 73–75, 76–79,
Prabhu, V., 71–72, 80–81, 82, 84–85, 92 80–83, 84–85, 86, 87–88, 90–91, 92,
Prasad, R., 142–143 96–97
Prasad, T.K., 45 Raymond, M.J., 118–119
Prathalingam, S.R., 109–112 Raymond, R.K., 72–73
Prescott, A.G., 109–112 Rea, P.A., 73, 82–83, 84, 85
Pressoir, G., 199–200 Reategui, R., 247
Pribat, A., 76 Rébeillé, F., 54, 59–60, 69–72, 73–75, 76, 77–
Prineas, R.J., 185–186 79, 80–83, 84–85, 86, 87–88, 89–91,
Provencher, L.M., 197 92, 95, 96–97
Przybyla, D., 149–152 Redding, K., 236
Pu, F., 127–128 Regan, S., 24–25
Pujadas-Salva, A.J., 201 Reichelt, M., 109–112
Puntarulo, S., 188 Reid, D.M., 22–23
Puthur, J.T., 152–153 Reiter, W.D., 115–116
Puttagunta, R., 187 Rejnmark, L., 235–236
Puyaubert, J., 54–58 Rendina, A.R., 49–51
Renou, J.P., 80–81, 84–85, 86, 92
Ren, W.W., 196
Q Renz, A., 112–114, 115–116, 147–148
Qian, W.Q., 115–116, 147–148 Reski, R., 76
Qiao, N., 186 Reuhs, B.L., 116–117
Qin, C., 115–116, 147–148 Reumann, S., 143–144, 247–249
Qin, H.J., 115 Reunanen, A., 185–186
Qi, Q., 191, 192–193, 198–199 Reuschhoff, E.E., 21–23, 27–28
Qiu, X.Y., 141 Rex, T.S., 143–144
Quadrado, M., 46–47, 54 Reymond, P., 202–204
Quadrana, L., 199 Rey, P., 187–188, 210–211
Quan, S., 143–144 Ricciarelli, R., 186, 187–188
Quemener, B., 116–117 Richefeu-Contesto, C., 77–79, 87, 89–90
Queval, G., 70–71 Richhardt, N., 76–77
280 AUTHOR INDEX
Riezman, H., 24 Russin, W.A., 211

Rimbach, G., 137 Ruttimann, A., 202
Ringier, B.H., 180 Ryan, T.J., 73, 82–83, 84
Rintamaki, E., 139–141
Ripoll, D.R., 139–141
Rippe, K., 17–20 S
Rippert, P., 73–74, 192–193 Sabar, R., 3–5
Ripp, K.G., 191, 194–195 Sacanell, C.J., 53
Rise, M., 195 Sacchettini, J.C., 46–47
Ristilä, M., 12–13 Sacco, A., 125–126
Rizhsky, L., 139–141, 149–152 Sacco, F., 139–141
Robert-Genthon, M., 73–74 Sadowski, J.A., 238
Robertson-Hoyt, J., 24–25 Sadre, R., 198
Roberts, R.M., 116–117 Saenger, W., 236
Robinson, A.L., 138–139 Sage, T.L., 210–211, 212–215
Robinson, J.K., 29 Sahm, H., 29
Robinson, K., 46–47 Sahr, T., 77–79
Robledo-Hernandez, A.L., 81 Said, H.M., 29
Rocca, J.R., 76–77 Sailland, A., 190
Rochaix, J.D., 232, 243, 247, 250 Saito, K., 130, 142
Rocheford, T.R., 199–200 Saji, H., 143–144
Rockett, H., 235–236 Sakai, A., 15–17
Rodgers, M., 190 Sakihama, Y., 142
Rodionova, I.A., 14, 22 Sakuragi, Y., 191, 196–197, 210–211, 232,
Rodionov, D.A., 14, 22 245–246
Rogers, W.O., 91–92 Sakurai, N., 130
Rohde, M., 43–45 Saldanha, S.A., 75
Rohmer, M., 240 Salomon, H., 180
Roitsch, T., 202–204 Samols, D., 54
Roje, S., 70–71, 72–73, Samuels, N., 3–5
77–80 Sanan, D., 187
Rolinski, S., 117–119, 122–123, Sanchez-Fernandez, R., 85
127–129, 147–148, 154 Sandelius, A.S., 215
Rolland, A., 190 Sandermann, H., 138–139
Rolland, N., 143–144 Sandmann, G., 96–97
Rombauts, S., 128 Sanekata, T., 142–144
Rosenberg, I., 96–97 Sanglard, D., 49–51
Rosenberg, P.A., 234 Sang, Y., 20–21, 22
Ros, G., 96–97 Sanmartin, M., 109–112, 135, 145–146
Ross, D., 183 Sano, H., 20–21
Rossi, A., 145 Sano, S., 142
Ross-Ibarra, J., 199–200 Sansuk, K., 242–243, 253
Rossi, M., 199 Santa-Maria, G.E., 139–141
Rosson, R.A., 131 Santoyo-Castelazo, A., 81
Rothan, C., 120–122, 129–130 Sanyal, I., 52
Rottnek, J.M., 194–195 Saracco, S.A., 25–27, 117–118, 147–148, 153
Rouet, M.A., 143–144 Sarin, N.B., 196
Rounsley, S.D., 117–118 Sasaki-Sekimoto, Y., 130
Rousseaux, M.C., 129–130 Satoh, K., 232, 250
Roux, C., 15–17, 18–20 Sato, K., 49
Roy, S., 180–181, 187–188 Sato, Y., 182, 187
Rozenberg, M., 139–141 Sattler, S.E., 191, 193–194, 196–197,
Rua, J., 109–112, 122–123 201–204, 205–206, 208–209,
Rubenstein, P.A., 10–11 210, 211, 212
Rubin, E.J., 46–47 Sauer, M., 131
Rudiger, W., 195 Sauer, N., 59–60
Ruffet, M.L., 52 Saur, A., 210–211
Rumeau, D., 11–12, 14, 27–28 Savage, D., 234–235
Running, J.A., 112–114, 131 Savidge, B., 191, 193–194
AUTHOR INDEX 281
Sawa, Y., 109–112, 123–126 Seppanen, R., 185–186

Sawyer, L., 48–51 Serino, S., 127–128
Sayer, B.G., 29 Sétif, P., 236
Schaap, P.J., 122–123, 128–129 Seto, H., 240
Schaefer, E.J., 235–236 Setta, N., 199
Schafer, A., 191–192 Sevilla, F., 135, 139, 141, 143–144
Schafer, F.Q., 109–112, 135–136, 137–139, Shachar-Hill, Y., 72–73, 77–79, 85–86
142 Shacter, E., 138–139
Schansker, G., 152–153 Shafer, J.A., 6
Scharf, S., 29 Shah, N.K., 91–92
Schauvinhold, I., 246 Shaikhali, J., 148
Scheibe, R., 149–152 Shane, B., 69–70, 87–88
Scheible, W.R., 145 Shang, P.F., 127–128
Schetter, A.L., 46–47 Sharkey, T.D., 211
Schiffmann, S., 79–80, 87 Sharples, S.C., 114–115
Schinzel, R., 10–11 Sha, W., 139–141
Schirch, V., 29, 73 Shearer, M.J., 235–236, 238, 253
Schlauch, K., 139–141, 149–152 Shea, T.B., 93–94
Schledz, M., 191–192, 193–194 Sheffield, V.C., 187
Schlereth, A., 24–25 Shellhammer, J., 46–47
Schmid, G.H., 183 Shen, G.X., 76–77, 141, 142–143, 247
Schmidt, F.S., 29 Shen, X., 190, 191–192
Schmitzberger, F., 75 Shen, Y., 91
Schneider, C., 180–181, 183, 187–188 Shestakov, S.V., 195
Schneider, G., 46–47, 49–51 Shevchuk, Y.M., 234–235
Schneider, T., 46–47 Shewmaker, C.K., 191, 193–194, 196–197,
Schnell, J.R., 80–81 198–199
Schock, B.C., 187–188 Shibahara, T., 142–144
Schoepp-Cothenet, B., 250 Shibata, D., 139–141
Schofield, C.J., 109–112 Shibata, H., 109–112, 123–126
Schoner, S., 127–128 Shibata, M., 246, 247–248
Schønheyder, F., 230–231, 238 Shigemizu, H., 142
Schopfer, P., 154–155, 239 Shigenaga, M.K., 187–188
Schorken, U., 29 Shigeoka, S., 109–112, 114–115, 120,
Schottler, M.A., 237, 238–239, 246–248, 253 122–126, 127, 128–129, 139–141,
Schroeder, J.I., 154 149–152
Schubert, K., 96–97 Shi, H., 20, 22, 27–28
Schulman, S., 237 Shikanai, T., 183
Schultz, G., 188, 240–241, 247–248 Shikano, T., 143–144
Schulz-Friedrich, R., 191–192 Shimada, H., 246, 247–248
Schunemann, N., 116–117, 128, 130 Shimaoka, T., 143–144
Schupp, N., 24–25 Shimohata, T.A., 122–123
Schurgers, L.J., 235–236 Shinozaki, K., 77–79, 86, 87, 191, 196–197
Schusseler, T., 198 Shinozaki, M., 237, 238–239, 242–243,
Schweikert, C., 154–155 247–248
Scimemi, C., 192–193 Shintani, D.K., 191, 196–197, 198
Scossa, A., 145 Shirano, Y., 139–141
Scott, D.L., 11–12 Shirley, N.J., 142–143
Scott, J., 82, 95 Shi, W.M., 115–116, 147–148
Scountzou, J., 3–5 Shi, W.P., 131–132
Sedbrook, J.C., 145 Shpilyov, A.V., 195
Seidler, A., 191 Shu, D.F., 141
Seki, M., 77–79, 86, 87, 237, 238–239, Shulaev, V., 139–141, 149–152
242–243, 247–248 Sibbald, B., 96
Selhub, J., 96–97 Sieben, C., 143–144
Sem, R., 91–92 Siegel, D., 183
Sen, C.K., 180–181, 187–188 Siemsen, T., 247–248
Seok, Y.J., 122–123 Sies, H., 180–181
Seong, E., 187 Sigfridsson, K., 236
282 AUTHOR INDEX
Silverblatt, E., 138–139 Stepanova, A.N., 24–25

Sima, P.D., 138–139 Stephens, S.B., 116–117
Sims, P.F.G., 70, 74–75, 88–90, 91–92 Stepusin, K., 29
Singer, S.R., 3–5 Stevens, J.F., 137
Singh, A.K., 237–238 Stevenson, B., 20, 22, 27–28
Singh, N.K., 20–21, 22 Stevens, R., 116–117, 127–128, 129–130,
Sinha, A.K., 202–204 142–143
Sinha, N., 197 Stilianou, E., 145
Sinha, R.K., 149–152 Stocker, A., 187–188, 202
Sinning, I., 13, 15–20 Stocklin, E., 235–236
Sirawaraporn, W., 91–92 Stok, J.E., 47–48
Siu, K.K., 53 Stokstad, E.L.R., 230–231
Sivasubramaniam, S., 22–23 Stolz, J., 14, 22, 59–60
Si, Y., 20–21, 22 Stolz, S., 202–204, 213–215
Sjoholm, I.M., 96 Storozhenko, S., 74–75, 77, 80–81, 89–90,
Slack, C.R., 215 96–97
Slattery, E.L., 187 Stover, P.J., 73, 82, 87–88,
Slattery, K., 139–141, 149–152 93–94
Slotbottom, D.J., 14, 22 Stowe, B.B., 253
Smekal, O., 49–51 Strain, J.J., 109–112
Smirnoff, N., 109–116, 117–119, 120, Stralsjo, L.M., 96
122–125, 126–129, 130, 139–141, Strasser, R.J., 152–153
146, 147–148, 149–153, 154 Strawn, M.A., 242–243, 247–248
Smith, A.G., 75 Streb, P., 152–153
Smith, A.M., 24–25 Streit, W.R., 46–48
Smith, J.L., 17–20 Strid, Å., 12–13
Smith, S.M., 24–25 Stridh, M.H., 215
Snell, E.E., 25 Strohmeier, M., 17–20
Soave, C., 139–141 Strominger, J.L., 10–11
Sohal, R.S., 183 Strzalka, K., 183, 201
Solinas, N., 131 Studart Guimaraes, C., 59–60
Soll, J., 188, 240–241, 247–248 Stumpe, M., 121, 135, 141
Solorzano-Vargas, R.S., 54–58 Stumpf, P.K., 45
Somerville, C.R., 115–116, 145, 147–148, Sueda, S., 15–17
215 Sugiura-Tomimori, N., 137
Sondergaard, H., 182 Suh, J.R., 82
Song, H.Y., 46–47, 49–51 Sumner, L.W., 76, 81
Song, W., 210–211, 212–215 Sundaresan, V., 115
Soni, P., 247–249 Sundberg, B., 24–25
Sonnewald, U., 197, 210–211 Sun, Q., 199–200
Soole, K.L., 125–126 Sun, W.H., 141
Sowinski, S.G., 199–200 Sun, Y.L., 115–116, 141, 142–143
Spenser, I.D., 29 Suormala, T., 41
Speziga, D., 18–20 Supplee, A., 191, 199–200
Sprenger, G.A., 29 Su, P.X., 153
Springer, F., 112–114, 115–116, 147–148 Surdin-Kerjan, Y., 80–81
Spycher, S., 187–188 Suttie, J.W., 234–236, 239
Srivastava, A.C., 81 Suza, W.P., 130
Stachowiak, M., 141 Suzuki, H., 130, 187
Staffieri, M., 187–188 Suzuki, K., 146
Stafford, D.W., 234 Suzuki, N., 139–141
Stanulović, M., 25 Swedberg, G., 91–92
Stapleton, A.E., 199–200 Sweeney, C., 49–51
Stechmann, A., 89–90 Switzenberg, R., 143–144
Steegers-Theunissen, R.P., 96 Sybesma, W., 79–80
Steele, H.P., 115–116, 147–148 Szarka, A., 135–136
Steffan, B., 202–204 Szewczyk, A., 11–12, 14, 27–28
Stehlik, D., 236 Szulc, P., 235–236
Stein, J.C., 191, 193–194, 196–197, 198–199 Szymanska, R., 194–195, 201
AUTHOR INDEX 283
T Tiedemann, J., 22–23

Tabata, S., 139–141 Tissot, G., 54–58
Tairou, F., 96 Titiz, O., 11–12, 15–18, 21–23, 25–28
Taji, T., 191, 196–197 Toledo, M.E.A., 128–129
Takabe, T., 145–146 Tomita, H., 187
Takahama, U., 135–136, 145–146, 154–155 Tommasi, F., 109–112, 127–128
Takahashi, S., 152–153 Toney, M.D., 7–10
Takahashi, Y.H., 237 Tonge, P.J., 244
Takamiya, K., 130, 246, 247–248 Tönnis, B., 41
Takanaga, H., 135–136 Tonz, T., 96
Takeda, T., 120, 122–123, 127, 139, 141, Torabinejad, J., 118–119, 145, 147–148
235–236 Torres, M.A., 154
Takeuchi, Y., 143–144 Toth, S.Z., 152–153
Taki, N., 130 Toyada-Ono, Y., 137
Tamaoki, M., 139–141, 143–144 Traber, M.G., 180–181, 182, 185–186,
Tambasco-Studart, M., 11–12, 15–20, 187–188, 199, 215–216
21–23, 25–28 Traktellis, A.C., 3–5
Tamoi, M., 114–115, 122–123, 127, 139–141 Traw, M.B., 153
Tanaka, K., 142–144 Trebst, A., 149–152, 183
Tanaka, R., 195 Trelease, R.N., 141, 142–143
Tanaka, Y., 145–146 Triantaphylidès, C., 11–12, 14, 27–28, 183
Tan, C.T., 22–23 Triglia, T., 91–92
Tang, K.X., 143–144, 196 Tripathi, S., 142–143
Tang, S., 199 Tropf, S., 191, 197, 210–211
Tang, Y.L., 81, 196 Trost, P., 154
Tan, H.Q., 131–132 Truglio, J.J., 244
Tani, Y., 15–17, 49 Truman, W., 139–141, 149–152
Tanouchi, A., 139–141 Tschiersch, H., 197, 210–211
Tarantino, D., 139–141 Tsegaye, Y., 191–192
Tardif, M., 49–51 Tse Sum Bui, B., 53
Tarlyn, N.M., 131–132, 134 Tsuchiya, T., 232
Taybi, T., 135, 145–146 Tsuji, H., 14–15
Taylor, A.M., 53 Tsuji, W., 143–144
Taylor, L.P., 187 Tucker, K.L., 186, 235–236
Taylor, M.C., 109–112 Tully, D.B., 13
Taylor, P.R., 185–186 Turlapati, P., 145
Taylor, S.V., 29
Taylor, W.S., 49–51
Teng, W., 199 U
Tenhaken, R., 126–127, 130 Uchida, M., 146
Terasawa, Y., 187–188 Uchihara, T., 187
Ter Beek, J., 14, 22 Uddin, M.I., 143–144
Teutloff, C., 236 Ueda, T., 182, 187
Tews, I., 13, 15–20, 21–22 Ueda, Y., 128–129
Thayer, S.A., 230–231 Ugalde, R.A., 139–141
Theis, K., 244 Ugulava, N.B., 53
Theodoulou, F.L., 120–123 Uh, S.H., 96
Thiel, G., 143–144 Unden, G., 232–233
Thoma, I., 202–204 Underwood, W., 135–136
Thomas, B., 109–112 Ushimaru, T., 143–144
Thomas, G., 240–241 Usoro, O.B., 180–181
Thorne, G.M., 191, 193–194, 196–197,
198–199
Thorneycroft, D., 24–25 V
Thornton, C.G., 54 Vacca, R.A., 122–123
Threlfall, D.R., 201, 240–241 Vadassery, J., 142–143
Thurnauer, M., 236 Vaeck, E., 115
Tian, L., 194–195 Valacchi, G., 187–188
Tibbetts, A.S., 70–72 Valenti, D., 122–123
284 AUTHOR INDEX
Valentin, H.E., 191, 193–195, 196–197, Verpoorte, R., 242–243, 253

198–199 Verrier, P.J., 147–148, 154
Valero, E., 148 Vestergaard, P., 235–236
Vallabhaneni, R., 199–200 Viaene, J., 74–75, 81
Valli, M., 131 Vicente-Carbajosa, J., 22–23
Valpuesta, V., 124, 125–126, 130 Vickers, T.J., 76–77
Van Allen, M.I., 96 Vidi, P.A., 194–195, 198, 206
Van Arsdell, S.W., 49–51 Vielreicher, M., 14, 22
van Bel, A.J.E., 211 Vierling, E., 153
van Berkel, W.J.H., 122–123, 128–129 Villacorta, L., 187–188, 215–216
Van Breusegem, F., 115, 128, 141 Villalba, J.M., 127–128, 239
Van Camp, W., 139–141 Vinayak, S., 91–92
Van de Cotte, B., 115 Viola, R., 125–126, 131, 134, 137
Vandekerckhove, J., 116–117 Virtamo, J., 185–186
Vandenabeele, S., 128 Visarius, T., 187–188, 215–216
van den Berg, W.A.M., 122–123, 128–129 Vivancos, P.D., 154
Van den Hof, M.C., 96 Vivera, S., 125–126
Vanderauwera, S., 128, 141 Volckaert, M., 80–81
van der Est, A., 232 Vollenweider, S., 202–204
van der Kooij, T.A., 191–192 Voll, L.M., 210–211
Van der Straeten, D., 24–25, 74–75, 77, Volpe, J.J., 234
80–81, 84–85, 86, 87–88, 92, Volrath, S., 46–47
96–97 Vroh Bi, I., 199–200
Vanderveer, P.J., 211 Vyas, M.N., 72
Van der Vliet, A., 187 Vyas, N.K., 72
Van Doorsselaere, J., 116–117
van Dorsselaer, A., 247–249
van Duijn, E., 122–123, 128–129 W
van Dusseldorp, M., 96 Wachi, Y., 47–48
Van Eenennaam, A.L., 191, 193–194, Waditee, R., 145–146
196–197, 198–199 Wagner, A.E., 137
Van Gestelen, P., 239 Wagner, B.L., 131–132
van het Hof, K.H., 96 Wagner, D., 149–152
van Lis, R., 49–51 Wagner, G., 132–134
Van Montagu, M.C., 109–112, 114–115, Wagner, K.H., 180–181, 187–188
116–118, 120, 122–123, 124, Wagner, S., 18–20, 23–24, 25–27
125–127 Walker, D.A., 135–136
Vanniasingham, V.M., 22–23 Walker, P.G., 125–126, 134, 137
Vannini, C., 139–141 Wallace, J.C., 43–45, 54–58
van Oostende, C., 232, 237, 244, 245–246, Waller, J.C., 76–79, 87–88
247–248, 253, 255 Wallner, S., 18–20
Vansuyt, G., 139–141 Walls, A.A., 17–18, 22–23
van Wijk, K.J., 139–141 Walter, P., 235–236
Van Wilder, V., 80–81, 86, 92 Wang, D.W., 115–116, 147–148
Vanzin, G.F., 115–116 Wang, H., 72–73, 234
Vapaavuori, E., 135 Wang, J., 141, 142–143
Varma, A., 142–143 Wang, L.Y., 115–116, 141, 142–143
Varner, J.E., 154–155 Wang, P., 88–90, 91–92
Vassiliev, I.R., 247 Wang, Q., 88–90, 91–92
Vasu, V.T., 187–188 Wang, S.W., 143–144
Velasco, L., 195, 201 Wang, W.F., 115–116, 147–148
Veljovic-Jovanovic, S.D., 147–148 Wang, Z.N., 143–144
Venkataraman, G., 142–143 Wan, J.T., 52–53
Venkataramani, S., 141, 142–143 Ward, E.R., 46–47
Venkatesh, T.V., 191, 194–195 Ward, J.L., 81
Venkatramesh, M., 192–193, 198–199 Ward, S.A., 70, 74–75, 91–92
Verberne, M.C., 242–243, 253 Ware, D.H., 199–200
Vermeer, C., 235–236 Warwick, N., 215
Verméglio, A., 250 Warzych, E., 11–12, 22–23, 25–28, 141
AUTHOR INDEX 285
Watanabe, M., 232, 250 Woggon, W.D., 202

Watanabe, T., 232, 250 Wolf, B., 43
Watt, R.M., 48–49 Wolf, E., 29
Webb, K., 117–118, 127 Wolucka, B.A., 114–115, 116–118, 127, 130
Weber, A.P.M., 73–74, 143–144 Wongsrichanalai, C., 91–92
Weber-Ban, E., 18–20 Wong, Y., 192–193, 198–199
Weber, H., 202–204 Wong, Y.H.H., 193–194
Weber, P., 235–236 Wood, H.G., 43–45, 54
Webster, S.P., 48–49 Wormuth, D., 149–152
Weck, M., 24–25 Wright, M.A., 114–115, 116–119, 128, 130
Weckwerth, W., 247–248 Wright, M.E., 185–186
Wegkamp, A., 79–80 Wright, P.E., 80–81
Weier, D., 198 Wu, G., 242–243
Weihrauch, J.C., 185 Wu, H.C., 20–21
Weinstein, S.J., 185–186 Wu, P., 115–116, 147–148
Weisshaar, B., 22–23 Wu, Q.Y., 115–116, 141, 142–143
Weiss, J.D., 192–194, 198–199 Wurtele, E.S., 45
Welsh, K.M., 90–91 Wurtzel, E.T., 199–200
Welti, R., 212–215 Wurtz, V., 247–249
Werner, E.R., 79–80 Wu, X., 199
Werner-Felmayer, G., 79–80 Wu, Y.J., 115–116, 145, 147–148, 185–186
Wernsdorfer, W.H., 91–92
Wertz, J., 237–238
Wheeler, G.L., 112–116, 118–119, 120, 127 X
Whelan, J., 139–141, 142–143 Xiang, Y., 15–17
Whistance, G.R., 201 Xiao, Y., 143–144
White, D.A., 188, 215 Xie, D.Y., 24–25
Whiteman, M., 109–112 Xie, L.F., 115
White, R., 77–79, 87 Xiong, L.M., 20, 21–22, 25–28, 30
Whitney, H.M., 75 Xu, H.W., 131–132
Widhalm, J.R., 232, 237–239, 245–246,
250, 255
Wiegert, T., 29 Y
Wienkoop, S., 247–248 Yabuta, Y., 109–112, 114–115, 120,
Wiese, M., 3–5 122–126, 127, 139–141
Wildermuth, M.C., 242–243, 247–248 Yadav, D.K., 149–152
Wilkerson, C.G., 143–144 Yagisawa, F., 141
Wilkinson, J.E., 145–146 Yagi, T., 3–5, 11–12, 20–21
Wilkinson, S.R., 109–112, 139 Yamaguchi-Shinozaki, K., 191, 196–197
Wille, C., 194–195, 198, 206 Yamamoto, A., 145–146
Willekens, H., 139–141 Yamamoto, T., 20–21
Willett, W.C., 235–236 Yamamoto, Y., 143–144
Williams, E.C., 234–235 Yamasaki, H., 142
Williams, E.H., 112–114, 115–116, 153 Yamasaki, M., 199–200
Williams, J., 234 Yamashita, H., 240
Williams, M., 199–200 Yamauchi, R., 188
Williamson, B., 142 Yamauchi, Y., 143–144
Williamson, J., 85–86 Yamawaki, K., 143–144
Wilson, C., 91–92 Yamori, W., 152–153
Wilson, J.X., 135–136 Yang, E., 29
Wilson, P.W., 235–236 Yang, G.Z., 131–132
Windeisen, V., 18–20 Yang, J.C., 131–134
Winkler, B.S., 143–144 Yang, K.S., 122–123
Winkler, M.E., 29 Yang, P.F., 143–144
Witsch, B., 139 Yang, S., 141
Wittenbach, V., 46–47 Yang, X.H., 115–116, 141, 142–143
Witthoft, C.M., 96 Yang, Y., 29, 89–90
Witt, H.T., 236 Yan, J.Q., 141, 199–200
Witt, S., 24, 30 Yasumoto, K., 14–15
286 AUTHOR INDEX
Ye, D., 115 Zarhloul, M.K., 198

Ye, N.H., 131–132 Zbierzak, A.M., 194–195, 198, 206
Ye, Z.B., 129–130 Zechmann, B., 121, 135, 141
Yin, L.N., 143–144 Zeeman, S.C., 24–25
Yocum, R., 49–51 Zein, F., 17–20
Yokochi, N., 3–5, 11–12 Zellnig, G., 215
Yokota, A., 143–144 Zempleni, J., 40–41, 54–58
Yokota, T., 187 Zhang, A.M., 115
Yonemitsu, M., 114–115 Zhang, C., 131–132
Yoon, H.J., 142 Zhang, G.F., 80–81, 86, 87–88, 92,
Yoshida, E., 232, 250 96–97
Yoshida, M., 141 Zhang, H., 141, 142–143
Yoshida, S., 143–144, 191, 196–197 Zhang, J.H., 129–130
Yoshida, Y., 141 Zhang, L.X., 143–144, 152–153, 196
Yoshikane, Y., 3–5 Zhang, M., 127–128
Yoshimura, K., 120, 122–123, 139–141 Zhang, W.Y., 119, 129–130
Yoshimura, M., 137 Zhang, Y., 17–20, 199
Yoshioka, Y., 80–81 Zhao, G., 29
Young, B.D., 117–118 Zhong, S.Q., 139–141, 149–152
Young, T.E., 143–144 Zhou, B.Y., 131–132
Yu, A., 80–81, 84–85, 86, 92 Zhu, C., 96–97
Yu, C.M., 115–116, 147–148 Zhu, G.H., 131–132
Yu, G., 246 Zhu, J.-K., 18–20, 22, 27–28
Yu, H.J., 115 Zhu, Z.H., 131–132
Yu, J.P., 128–129, 199–200 Ziegler, K., 154
Yu, L., 131–132 Ziemak, M.J., 72–73, 76–77, 79–80, 87
Yun, D.J., 141 Zimmermann, P., 128, 204
Yun, J., 24–25 Zimmermann, R., 96
Yusuf, M.A., 196 Zimmer, P., 96
Zimmer, S., 187–188
Zinchenko, V.V., 195
Z Zingg, J.M., 186, 187–188, 215–216
Zabeau, M., 115, 116–117 Zink, D.L., 109–112
Zablackis, E., 116–117 Zittermann, A., 235–236
Zalkin, H., 17–18 Zou, L.P., 129–130
Zamir, D., 142–143 Zrenner, R., 74
Zanor, M.I., 59–60 Zubieta, C., 242–243, 247–248
Zarhloul, K.M., 198 Zybailov, B., 247
SUBJECT INDEX
A Ascorbate-GSH cycle, 137–138, 148, 149

1-Aminocyclopropane-1-carboxylate (ACC) Ascorbic acid. See Vitamin C
precursor, ethylene biosynthesis, 27–28
synthase, 24–25
Ascorbate B
biosynthesis control and pathway Biosynthesis, phylloquinone
AMR1, 129–130 Arabidopsis, 242
fruits, 127–128 demethylphylloquinone
GME and GDP-L-Gal phosphorylase, methyltransferase, 246–247
130 DHNA-CoA
light intensity, 128–129 Cyanidiales, 246
methyl jasmonate (MeJA) treatment, hydrolysis and lactonization, 244, 245
130 Synechocystis, 245–246
rate, enzyme and kinetic properties, type I and type II enzymes, 244–245
128 DHNA phytyl transferase, 246
stress resistance/post-harvest enzyme subcellular localization
longevity, 131 Arabidopsis, 249
catabolism DHNA-CoA hydrolysis, 248–249
L-threonate, oxalate and L-tartrate, OSB-CoA ligase, 248
131–132 prenylation and methylation, 247–248
pathways, 133 gene identification, 241
tartrate, wine, 132–134 isochorismate synthase/PHYLLO
conjugates, 137 Arabidopsis ICS1 and ICS2,
functions 242–243
ascorbate-GSH cycle, 148 menF, menD, menC and menH, 243
cell division and expansion, 154–155 mutant phenotype, 247
environmental stress and pathogens, naphthoquinone ring, 240
153 OSB-CoA ligase, 244
photosynthesis and photoprotection, pathway, 240, 241
149–153 shikimate, 240–241
multiple pathways, biosynthesis, 107–179 Biosynthetic pathway, biotin
D-GalUA, 125–126 bacteria and plants, 46
L-GULL via GDP-mannose, 127 7,8-diaminopelargonic acid synthase-
Man/l-Gal, 123–125 dethiobiotin synthetase
myo-inositol and D-GlcUA, 126–127 AdoMet, 49
redox reactions bioA and bioD gene, 49–51
AO enzymes, 145–146 monocistronic BIO3–BIO1 transcript,
APXs, 139–141 49–51
dehydroascorbate reductase, 143–144 phylogenetic analysis, 50
MDHA and DHA, 137–138 TPTA, Arabidopsis, 49–51
monodehydroascorbate reductase, E. coli, 46–47
142–143 KAPA synthase, 48–49
oxidation, APX and AO, 139 pimeloyl-CoA origin, 47–48
singlet oxygen, 138–139 synthase
transport and subcellular AdoMet-dependent radical enzyme,
compartmentation 51–52
apoplastic fluid, 135 ADX1/ADR redox system and NFS1
intracellular, 135–136 protein, 54
phloem, 134 BIO2 protein, 52–53
uptake, DHA, 135–136 E. coli, 52
288 SUBJECT INDEX
Biotin enzymes, 71–72

Bio5p, 59–60 glycine formation, 70–71
biosynthetic pathway serine hydroxymethyl transferase
bacteria and plants, 46 (SHMT), 70–71
7,8-diaminopelargonic acid synthase- interconversion, C1-substituted folates
dethiobiotin synthetase, 49–51 FCL, 73
E. coli, 46–47 methylene-THF reductase (MTHFR),
KAPA synthase, 48–49 72–73
pimeloyl-CoA origin, 47–48
synthase, 51–54
deficiency, animal models, 41 E
description, 40–41 Enzyme commission (EC), 5, 6
distribution and nutritional aspects Escherichia coli (E. coli)
Arabidopsis, 41–42 biosynthetic pathway, 15–17
cycle in mammalian cells, 42 PL kinase, 20
dietary forms, 43 vitamin B6 biosynthesis, 15–17
forms, living cells, 41–42
humans, 43
metabolism, 59–60 F
protein biotinylation Folates
biotin-protein ligase (BPL), 54 biofortification
carboxylase activities, 54–58 agricultural crops, 96–97
[3H]-biotin, 54–58 metabolic engineering strategies,
HCS genes, 54–58 96–97
uORF-mediated translational control molecular genetics and genomics, 96
and HCS1, 57 catabolism and salvage pathways
proteins HPPK-DHPS enzyme, 82–83
food, 44 multiple isoforms, PTAR, 83–84
plant acetyl-CoA carboxylase, 43–45 physiological turnover rate, 82
structure, 41 cellular compartmentation
antifolate methotrexate, 84–85
multidrug resistance-associated
C proteins (MRP), 85
Cloning, tocochromanol pathway distribution, plant
aromatic head group synthesis metabolic requirements, 86
HPPD, 191–192 photorespiration, 85–86
TyrA/HPPD, 192–193 functions
molecular and genetic advances, 190–191 aromatic amino acid hydroxylases
phytyl-PP, tocopherol synthesis, 195 (AAHs), 76
plastoquinone synthesis, HGA genome maintenance, 75–76
prenylation redox properties, 75
Arabidopsis leaves and maize, 194–195 homeostasis
HPT and VTE2, 193–194 biofortification efforts, 88
PQ-9, 194–195 kinetic characterization, 87
Synechocystis PCC6803 and Arabidopsis microarray analysis, 87–88
thaliana, 191 THF synthesis regulation, 87
synthesis, methyltransferases physiology
MPBQ MT and g-TMT, 196 DFE, 94
SLL0418 and VTE3, 196–197 megaloblastic anaemia, 94
tocopherol cyclase enzyme plant foods, 95
HPT and g-TMT, 197 recommended dietary allowances
sxd1, 197 (RDAs), 94
C1-metabolism re-methylation, homocysteine, 93–94
C1-units utilization spina bifida and anencephaly, 94
AdoMet, 73–74 transcriptional regulation, 93–94
DNA synthesis, 74–75 vitamin deficiencies, 93
pantothenate, 75 species-specific differences
purine ring synthesis, 74 fusion, prokaryotic genes, 90–91
generation, C1-units multifunctional proteins, 89–90
SUBJECT INDEX 289
THF-biosynthetic pathway, 91 H
THF biosynthesis Holocarboxylase synthetase (HCS)
4-amino-4-deoxychorismate (ADC), Arabidopsis HCS1, 58
77–79 description, 54
dihydropteroate synthase (DHPS), HCS1 and HCS2 genes, 54–58
80–81
enzymes, 77, 78
folylpolyglutamate synthetase I
(FPGS), 81 Isoprenoids
g-glutamyl hydrolase (GGH), 81–82 naphthoquinones, 232, 250–251
GTP-cyclohydrolase I (GTPCHI), plastid, 254–255
79–80
subcellular compartmentation, 77, 79
synthetic pathway, 77 K
Folates metabolism, plants 7-Keto-8-aminopelargonic acid (KAPA)
biofortification, 96–97 synthase, 48–49
biological functions, 70–77
physiology, human health
dietary sources and intake M
recommendations, 94–96 Menaquinone
metabolic and clinical manifestations, anaerobic bacteria, 231, 232–233
93–94 cyanidiales, 252
structure E. coli, biosynthesis, 242
para-aminobenzoic acid (pABA), isolation, 230–231
69–70 menaquinone-n (MK-n), 232
polyglutamylation, 69–70 phylloquinone and, 240–241
THF and derivatives, 69 prokaryotic lineages, 250
synthesis, other autotrophs
DHPS and DHFR inhibitors,
91–92 N
species-specific differences, 88–91 Naphthoquinones
target, therapies against infectious HPLC methods, 238
diseases, 91–92 isoprenyl biosynthesis, 240–241
turnover and homeostasis oxidoreductase activities, plasma
biosynthesis, THF, 77–82 membrane, 239
catabolism and salvage pathways, photosynthetic eukaryotes, biosynthesis
82–84 chlorobi/g-proteobacteria
cellular compartmentation and lineage, 252
transport, 84–85 menaquinone, 250
distribution, plant organs and tissues, men genes, 250–251
85–86 nuclear and plastid encoded, 250
homeostasis control, 87–88 ring, vitamin K, 232
Foyer–Halliwell–Asada cycle, 137–138
Functions, ascorbate
ascorbate-GSH cycle, 148, 149 O
cell division and expansion OSB-CoA ligase, 244
and AO, 154–155
stomatal response, ABA, 154
environmental stress and pathogens, 153 P
photosynthesis and photoprotection Photosynthetic organisms, tocochromanols
interaction, 149–152 adult plants
PSI and PSII, 152–153 Arabidopsis wild-type phenotype, 210
‘water-water cycle’, 149–152 cyclase gene, 211
vtc mutants, 147–148 ER lipid metabolism, 215–216
maize and potato, 212
plasmodesmata, 212–213
G stress, lipid oxidation, 210–211
GTP-cyclohydrolase I (GTPCHI), 79–80 trienoic fatty acid, 213–215
vte2, 212
290 SUBJECT INDEX
Photosynthetic organisms, tocochromanols (cont.) PMP, 20–21

vte2vte1, 210 pyridine ring, 7–10
plants vs. animals, 200–201 role, 13
seed desiccation, storage and seedling Pyridoxamine (PM), 20, 25, 29
establishment Pyridoxine (PN)
ageing treatment, 201–202 plants, 14–15
DMPBQ, 208–209 and PM, 25, 27–28, 29
lipid hydroxide accumulation, 208 PN-glucoside form, 14–15
lipid oxidation, 206–208 SOS4, 20
plastochromanol-8 (PC-8), 206 vitamin, 3–5
root growth, lipid hydroxide and and Yjef_N oxidase domains, 20–21
eicosenoic acid, 207
trienoic fatty acids, vte2, 205–206
vte2-1 and vte2-2 mutants, 202–204 R
a-tocopherolquinone, 201 Reactive oxygen species (ROS)
Phylloquinone PDX1 and PDX2 transcription, 22–23
biosynthesis, plants vitamin B6, 11–12
Arabidopsis, 242 Redox reactions, ascorbate
demethylphylloquinone AO enzymes
methyltransferase, 246–247 description, 145
DHNA-CoA, 244–246 P. syringae, 146
DHNA phytyl transferase, 246 QC, 145–146
enzyme subcellular localization, APXs
247–249 genes, 139–141
gene identification, 241 isozymes overexpression, 141
isochorismate synthase/PHYLLO, microbodies, 141
242–243 sAPX and tAPX, 139
mutant phenotype, 247 dehydroascorbate reductase
naphthoquinone ring, 240 (DHAR)
OSB-CoA ligase, 244 chloroplast, 143–144
pathway, 240, 241 overexpression, cytosol, 143–144
shikimate, 240–241 MDHA and DHA, 137–138
detection, plants, 238 monodehydroascorbate reductase
engineering, 253 (MDHAR)
photosynthetic eukaryotes, AtMDAR4, 142–143
naphthoquinone biosynthesis description, 142
chlorobi/g-proteobacteria isoforms, 142–143
lineage, 252 oxidation, APX and AO, 139
menaquinone, 250 singlet oxygen, 138–139
men genes, 250–251 ROS. See Reactive oxygen species
nuclear and plastid encoded, 250
subcellular distribution
naphthoquinone oxidoreductase S
activities, 239 Serine hydroxymethyl transferase (SHMT),
plastids, 238–239 70–71
tissular distribution, 238, 239 Shikimate, 240–241
turnover, 253
PL reductase (PLR)
activity, 20–21 T
yeast, 20–21 Tocochromanols
Pyridoxal (PL) agricultural crops, 198–199
content, 25–27 biosynthetic pathway, photosynthetic
hydrogen atom subtraction, 12–13 organisms
kinase, 20 Arabidopsis, 188, 189
vitamin B6, 20 cytosolic aromatic amino metabolism,
Pyridoxal phosphate (PLP) 190
amino acids functioning, 6 chroman-6-ol ring system, 181–183
cofactor, 22, 24–25, 28 cloning, biochemical genomics
metabolic enzymes, 14
SUBJECT INDEX 291
aromatic head group synthesis, pathway engineering, nutritional

191–193 development, 30
molecular and genetic advances, regulation and turnover, biosynthesis
190–191 Arabidopsis, 22–23
phytyl-PP, tocopherol synthesis, 195 pentose and triose phosphate pool,
plastoquinone synthesis, HGA 24–25
prenylation, 193–195 post-transcriptional modification, 24
Synechocystis PCC6803 and sos4 and pdx1.3, 23–24
Arabidopsis thaliana, 191 role, antioxidant
synthesis, methyltransferases, 196–197 ROS, 11–12
tocopherol cyclase enzyme, 197–198 singlet oxygen, 12–13
function, photosynthetic organisms salvage pathways, biosynthesis
adult plants, 210–216 PDX3, 20–21
plants vs. animals, 200–201 sos4 mutant, 20
seed desiccation, storage and seedling Yjef_N domain, 20–21
establishment, 201–209 Vitamin B8. See Biotin
a-tocopherolquinone, 201 Vitamin C
lipid peroxidation chain reaction, 184 ascorbate (see Ascorbate)
plant tissues and foods, 185 definition, 109–112
PUFA, 183 D-mannose/l-galactose (MAN/l-Gal)
Trolox equivalent antioxidant capacity biosynthesis pathway
(TEAC) assay, 12–13 GDP-L-galactose phosphorylase/
guanylyltransfer, 117–118
GDP-mannose-3,5-epimerase (GME),
V 116–117
Vitamin B6 GDP-mannose pyrophosphorylase,
vs. autotrophic non-plant organisms, 29 115–116
cellular localization pathways L-GalDH, 112–114, 120
de novo biosynthesis, 21–22 L-GalLDH, 120–123
splice variants, 22 L-Gal 1-P, 118–119
cellular metabolism, 31 phosphomannose mutase (PMM), 115
degradation, 25 PMI activity, 114–115
de novo biosynthesis L-ascorbate redox reaction, 111
Arabidopsis, 18–20 2-ODDs, 109–112
Bacillus subtilis Pdx1/Pdx2 complex Vitamin E (see also Tocochromanols)
crystal structure, 18–20 breeding plants
E. coli, 15–17 QTL analyses, 199–200
pathways, 15–17 transgenic approaches, staple foods,
PDX1 and PDX2, 17–18 199
description, 2–3 chemistry, uptake, transport and in vivo
discovery, 3 activities, 180–181
distribution purification, 180
food content examples, 14–15 requirement humans and biological
intracellular and plant tissues, 14 functions
enzyme cofactor biochemical and molecular
activity-based classification, 5 mechanism, 186
mechanism, 7–11 deficiency, 185–186
predominant forms, 4, 5 tocochromanol, 187–188
structural classification, 6–7 a-tocopherol, 187
forms and derivatives, 3–5 tocochromanols (see Tocochromanols)
impact, physiology and development Vitamin H. See Biotin
mutations, 25–27 Vitamin K
PLP, 28 biosynthesis, 231
rsr4-1, 25–27 description, 230–231
sos4 plants, 27 function, 232
tocopherols, 27–28 plants and cyanobacteria
WT, pdx1.1 and pdx1.3 electron transfer, photosystem I,
growth, 25–27 236
importance, human health, 13 QKA and QKB, 236
292 SUBJECT INDEX
Vitamin K (cont.) intake values, US and UK, 235–236

VKORC1, Synechocystis, 237–238 VKOR, 234
structure and chemistry vitamin K1 (see Phylloquinone)
anaerobic bacteria, menaquinones, Vitamin K 2. See Menaquinone
232–233
and menaquinones, 232, 233
naphthoquione ring, 232 W
vertebrates Wild type (WT) growth, 25–27
deficiency, 234–235
g-carboxylation, 233–234

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Advances in

MICHEL DELSENY Laboratoire Génome et

First edition 2011

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Printed and bound in USA

CLAUDE ALBAN Laboratoire de Physiologie Cellulaire Végétale, CNRS,

VITAMINS: A PLANT AFFAIR

quite common in low-resource countries but also occur in developed

restricted to plants and microorganisms, a biochemical feature that can be

FABRICE RÉBEILLÉ AND ROLAND DOUCE

Series Editor (Volumes 35–44)

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Compartmentation of Proteins in the Protein Storage Vacuole:

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Aphids: Non-Persistent Transmission

Persistent Transmission of Luteoviruses by Aphids

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