Plant Respiration and Internal Oxygen

Methods in
Molecular Biology 1670
Kapuganti Jagadis Gupta Editor
Plant Respiration
and Internal
Oxygen
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
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Plant Respiration and Internal
Oxygen
Methods and Protocols
Edited by
Kapuganti Jagadis Gupta

National Institute of Plant Genome Research, New Delhi, Delhi, India
Editor
Kapuganti Jagadis Gupta
National Institute of Plant Genome Research
New Delhi, Delhi, India
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-7291-3 ISBN 978-1-4939-7292-0 (eBook)
DOI 10.1007/978-1-4939-7292-0
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Preface
Respiration is a very important biochemical process. Aerobic respiration in plants, as in all

eukaryotes, involves the oxidation of organic substrates such as carbohydrates, lipids,
proteins, amino acids, and organic acids, leading to the production of ATP and the release
of CO2 and H2O. As side products, several reactive oxygen and nitrogen species are also
produced during respiration and they play important roles in cellular signaling. Respiratory
metabolism can also generate intermediates that are required for the biosynthesis of amino
acids, nucleic acids, fatty acids, and antioxidants. Respiration involves glycolysis, the pyru-
vate dehydrogenase complex, the TCA cycle, and the mitochondrial electron transport
chain. In glycolysis energy is released for the synthesis of ATP by the breakdown of glucose.
In the TCA cycle organic acids are oxidized releasing CO2 and generating NADH, FADH2,
and some ATP. Reoxidation of NADH and FADH2 by the mitochondrial electron transport
chain leads to the generation of a proton electrochemical gradient and the production of
ATP. In addition to the classic respiratory pathway, plants have various alternative respiratory
pathways that are regulated by stress and development. These alternative pathways are
important in preventing the buildup of harmful levels of reactive oxygen and nitrogen
species.
Since oxygen is the terminal electron acceptor in the respiratory electron transport
chain, its concentration plays a role in regulating respiration. Flooding/waterlogging
induces hypoxia in plants that leads to reduced energy production and even under normal
conditions various plant tissues, such as tubers and seeds, experience hypoxia. In order to
understand the regulation of respiration and internal oxygen, methods are required for the
analysis of respiration and this book describes a series of informative experimental
approaches for studying this all important process in plants.
The oxidation of carbohydrates is a central feature of respiratory metabolism, and two
chapters describe the complementary information that can be obtained from radiotracer
experiments. Kruger et al. describe an approach to determine and interpret the pattern of
oxidation of carbohydrates based on monitoring 14CO2 release during the metabolism of
exogenously supplied [1-14C]-, [2-14C]-, [3,4-14C]-, and [6-14C]glucose. The method is
exemplified by studies on Arabidopsis cell suspension cultures, but the protocol can be easily
adapted for the investigation of other plant materials. Similarly Obata et al. show how
combining radiotracer experiments with chemical fractionation can be used for the estima-
tion of respiratory fluxes.
Conventional oxygen (micro-) sensors assess oxygen concentration within a particular
region or across a transect of tissue but provide no information regarding its bidimensional
distribution. Four chapters present measurement approaches based on a novel imaging
technology, ViSisens, in which an optical sensor foil (a planar optode) is attached to the
surface of the sample. The sensor converts a fluorescent signal into an oxygen value. Gupta
and Kumari describe a method to measure respiration and internal oxygen noninvasively in
root tissues, while Rolletschek and Liebsch describe the application of the same technique to
determine oxygen gradients in seeds. Liebsch et al. describe a multisense approach that
allows oxygen to be monitored in several samples simultaneously, and this high-throughput
v
vi Preface
analysis of respiratory activity is likely to become an important component of many

biological investigations. Concluding this section, Pandey et al. describe the measurement
of respiration and internal oxygen in germinating Cicer arietinum L. seeds using an optical
microsensor.
Plants develop resistance to avirulent pathogens via a hypersensitive response in which
reactive oxygen and nitrogen species play a major role in localized cell death. Two chapters
describe complementary methods for studying oxygen dynamics and respiration during this
process. Pathak and Gupta describe how a microoxygen sensor can be used to make
respiratory measurements on intact plants undergoing the hypersensitive response, while
Kumari et al. show how the oxygen dynamics in Arabidopsis leaves infected with Pseudo-
monas bacteria can be analyzed using a ViSisens microscope.
The isolation of intact, physiologically active mitochondria is a recurring need in
respiratory research, and several chapters are devoted to this important topic. Pandey et al.
describe a protocol for the isolation of mitochondria for physiological and confocal studies,
while Viswakarma and Gupta describe a method for isolating metabolically active pea root
mitochondria that can be used for confocal and electron microscopic studies. Shaw et al.
describe methods for determining respiratory electron transport, along with techniques for
the in vivo determination of oxygen tension and the measurement of the respiratory
quotient. In addition, Kerbler and Taylor provide protocols for the isolation of mitochon-
dria from Arabidopsis, pea, Medicago, rice, and potato tubers, while Oliveira et al. provide
detailed information about the isolation of mitochondria from papaya, guava, tomato, and
strawberry. The activity of the TCA cycle enzymes is an important determinant of mito-
chondrial function under different conditions, and Omena-Garcia et al. describe the latest
methods for the determination of the TCA cycle enzyme activities in different plant tissues.
Calorespirometry is a technique that provides insight into metabolic adaptation and
acclimation to environmental conditions. The chapter by Arnholdt-Schmitt provides a
detailed overview on this. Arnholdt-Schmitt and Patil describe a calorespirometric method
to monitor respiration traits that can be used as novel markers for plant robustness under the
threat of climate change. The protocol provides a detailed procedure for using calorespiro-
metry as a rapid means for identifying differential effects of endophytes on the temperature
response and predicted biomass productivity in microalgae and plant holobionts.
Understanding the structure, function, and regulation of the alternative oxidase (AOX)
is a cornerstone in plant mitochondrial research, and several chapters are dedicated to this
topic. The oxygen isotope fractionation technique is the only available method for deter-
mining electron partitioning between the cytochrome and alternative pathways in vivo. Del-
Saz et al. explain the isotope fractionation technique and its associated calculations, together
with a detailed description of a protocol using dual-inlet isotope ratio mass spectrometry.
Ragonezi and Arnholdt-Schmitt describe a method based on laser-capture microdissection
for amplifying AOX genes in carrot, while Hélio Costa and Arnholdt-Schmitt describe a
bioinformatic approach for exploring the co-regulation of AOX gene family members
during growth and development. Arnholdt-Schmitt and Patil provide a protocol for identi-
fying plant AOX gene variants as functional markers for early growth regulation, while
Chapter 19 by Hélio Costa et al. provides a step-by-step protocol for classifying AOX
proteins in flowering plants. The chapter by Sunil and Raghavendra provides a protocol
for the isolation of protoplasts and a study on cytochrome and alternative pathways. The
chapter by McLamore et al. provides information about measuring spatial and temporal
oxygen flux near plant tissues using a self-referencing optrode.
Preface vii
I am extremely grateful to the scientists from eight countries who have contributed to
the methods described in this book. I extend my heartfelt gratitude to John Walker for his
guidance and I give my special thanks to Aprajita Kumari for formatting and Index prepara-
tion. Finally I thank my young son Rithwik for his patience during the preparation of this
book.
New Delhi, Delhi, India Kapuganti Jagadis Gupta

Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Assessing Metabolic Flux in Plants with Radiorespirometry . . . . . . . . . . . . . . . . . . 1

Nicholas J. Kruger, Shyam K. Masakapalli, and R. George Ratcliffe
2 Coupling Radiotracer Experiments with Chemical Fractionation
for the Estimation of Respiratory Fluxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Toshihiro Obata, Laise Rosado-Souza, and Alisdair R. Fernie
3 A Method for Imaging Oxygen Distribution and Respiration
at a Microscopic Level of Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Hardy Rolletschek and Gregor Liebsch
4 VisiSens Technique to Measure Internal Oxygen and Respiration
in Barley Roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Aprajita Kumari and Kapuganti Jagadis Gupta
5 MultiSense: A Multimodal Sensor Tool Enabling
the High-Throughput Analysis of Respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Peter Keil, Gregor Liebsch, Ljudmilla Borisjuk, and Hardy Rolletschek
6 Measurement of Respiration and Internal Oxygen in Germinating
Cicer arietinum L. Seeds Using Optic Microsensor . . . . . . . . . . . . . . . . . . . . . . . . . 57
Sonika Pandey, Aprajita Kumari, Chellapilla Bharadwaj,
and Kapuganti Jagadis Gupta
7 Using an Oxygen Microsensor to Measure Oxygen Dynamics
in Tomato Plants in Response to Pseudomonas syringae Infection . . . . . . . . . . . . . 63
Pradeep Kumar Pathak and Kapuganti Jagadis Gupta
8 Measurement of Oxygen Status in Arabidopsis Leaves
Undergoing the Hypersensitive Response During
Pseudomonas Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Aprajita Kumari, Gail M. Preston, and Kapuganti Jagadis Gupta
9 Isolation of Physiologically Active and Intact Mitochondria
from Chickpea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Sonika Pandey, Aprajita Kumari, and Kapuganti Jagadis Gupta
10 Isolation and Structural Studies of Mitochondria from Pea Roots . . . . . . . . . . . . . 87
Abhaypratap Vishwakarma and Kapuganti Jagadis Gupta
11 Mitochondrial Respiration and Oxygen Tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Daniel S. Shaw, Karlia Meitha, Michael J. Considine,
and Christine H. Foyer
12 Isolation of Mitochondria from Model and Crop Plants . . . . . . . . . . . . . . . . . . . . . 115
Sandra M. Kerbler and Nicolas L. Taylor
ix
x Contents
13 Procedures of Mitochondria Purification and Gene Expression

to Study Alternative Respiratory and Uncoupling
Pathways in Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Jurandi Gonçalves de Oliveira, Luis Miguel Mazorra Morales,
Gláucia Michelle Cosme Silva, Kátia Daniella da Cruz Saraiva,
Diederson Bortolino Santana, Clesivan Pereira dos Santos,
Marcos Goes Oliveira, and José Hélio Costa
14 Measurement of Tricarboxylic Acid Cycle Enzyme Activities in Plants . . . . . . . . . 167
Rebeca Patricia Omena-Garcia, Wagner L. Araújo,
Yves Gibon, Alisdair R. Fernie, and Adriano Nunes-Nesi
15 Respiration Traits as Novel Markers for Plant Robustness
Under the Threat of Climate Change: A Protocol for Validation. . . . . . . . . . . . . . 183
Birgit Arnholdt-Schmitt
16 Calorespirometry: A Novel Tool in Functional Hologenomics
to Select “Green” Holobionts for Biomass Production . . . . . . . . . . . . . . . . . . . . . . 193
Birgit Arnholdt-Schmitt and Vinod Kumar Patil
17 Measurements of Electron Partitioning Between Cytochrome
and Alternative Oxidase Pathways in Plant Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Nestor Fernandez Del-Saz, Miquel Ribas-Carbo,
Gabriel Martorell, Alisdair R. Fernie, and Igor Florez-Sarasa
18 A Driving Bioinformatics Approach to Explore Co-regulation
of AOX Gene Family Members During Growth and Development. . . . . . . . . . . . 219
José Hélio Costa and Birgit Arnholdt-Schmitt
19 A Step-by-Step Protocol for Classifying AOX Proteins
in Flowering Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
José Hélio Costa, Clesivan Pereira dos Santos,
Kátia Daniella da Cruz Saraiva, and Birgit Arnholdt-Schmitt
20 Studying Individual Plant AOX Gene Functionality in Early
Growth Regulation: A New Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
21 Laser Capture Microdissection for Amplification of Alternative
Oxidase (AOX) Genes in Target Tissues in Daucus carota L. . . . . . . . . . . . . . . . . . 245
Carla Ragonezi and Birgit Arnholdt-Schmitt
22 Measurement of Mitochondrial Respiration in Isolated
Protoplasts: Cytochrome and Alternative Pathways . . . . . . . . . . . . . . . . . . . . . . . . . 253
Bobba Sunil and Agepati S. Raghavendra
23 Measuring Spatial and Temporal Oxygen Flux Near Plant
Tissues Using a Self-Referencing Optrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Eric S. McLamore, D. Marshall Porterfield, and Yinglang Wan
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Contributors
WAGNER L. ARAÚJO Departamento de Biologia Vegetal, Universidade Federal de Viçosa,

Viçosa, Brazil
BIRGIT ARNHOLDT-SCHMITT Functional Cell Reprogramming and Organism Plasticity
(FunCrop), EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal;
Science and Technology Park Alentejo (PACT), Évora, Portugal; Functional Genomics and
Bioinformatics, Department of Biochemistry and Molecular Biology, Federal University of
Ceará, Fortaleza, Brazil
CHELLAPILLA BHARADWAJ Division of Genetics, Indian Agriculture Research Institute, New
Delhi, India
LJUDMILLA BORISJUK Department of Molecular Genetics, Leibniz Institute of Plant Genetics
and Crop Plant Research (IPK), Stadt Seeland OT Gatersleben, Germany
MICHAEL J. CONSIDINE Centre for Plant Science, School of Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, UK; The UWA Institute of Agriculture, and School
of Molecular Sciences, University of Western Australia, Perth, Australia; Department
of Agriculture and Food Western Australia, Perth, Australia
JOSÉ HÉLIO COSTA Functional Genomics and Bioinformatics, Department of Biochemistry
and Molecular Biology, Federal University of Ceara, Fortaleza, Ceara, Brazil
NESTOR FERNANDEZ DEL-SAZ Grup de Recerca en Biologia de les Plantes en Condicions
Mediterranies, Universitat de les Illes Balears, Palma de Mallorca, Spain
ALISDAIR R. FERNIE Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm,
Germany
IGOR FLOREZ-SARASA Max-Planck-Institut f€ ur Molekulare Pflanzenphysiologie, Potsdam-
Golm, Germany
CHRISTINE H. FOYER Centre for Plant Science, School of Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, UK; The UWA Institute of Agriculture, and School
of Molecular Sciences, University of Western Australia, Perth, Australia
YVES GIBON UMR 1332 Biologie du Fruit et Pathologie, INRA, Villenave d’Ornon, France
KAPUGANTI JAGADIS GUPTA National Institute of Plant Genome Research, New Delhi, India
PETER KEIL Department of Molecular Genetics, Leibniz Institute of Plant Genetics
and Crop Plant Research (IPK), Stadt Seeland OT Gatersleben, Germany
SANDRA M. KERBLER The University of Western Australia, Crawley, WA, Australia
NICHOLAS J. KRUGER Department of Plant Sciences, University of Oxford, Oxford, UK
APRAJITA KUMARI National Institute of Plant Genome Research, New Delhi, Delhi, India
GREGOR LIEBSCH PreSens Precision Sensing GmbH, Regensburg, Germany
GABRIEL MARTORELL Serveis Cientı́fico-Tècnics, Universitat de les Illes Balears, Palma de
Mallorca, Spain
SHYAM K. MASAKAPALLI School of Basic Sciences, Indian Institute of Technology Mandi,
Mandi, HP, India
ERIC S. MCLAMORE Agricultural and Biological Engineering, Institute of Food
and Agricultural Sciences, University of Florida, Gainesville, FL, USA
xi
xii Contributors
KARLIA MEITHA The UWA Institute of Agriculture, and School of Molecular Sciences,
University of Western Australia, Perth, Australia
LUIS MIGUEL MAZORRA MORALES Universidade Estadual do Norte Fluminense Darcy
Ribeiro, Campos dos Goytacazes, RJ, Brazil
ADRIANO NUNES-NESI Departamento de Biologia Vegetal, Universidade Federal de Viçosa,
Viçosa, Brazil
TOSHIHIRO OBATA Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm,
Germany; Department of Biochemistry, University of Nebraska Lincoln, Lincoln, NE, USA
JURANDI GONÇALVES DE OLIVEIRA Universidade Estadual do Norte Fluminense Darcy
MARCOS GOES OLIVEIRA Universidade Federal do Espirito Santo, São Mateus, ES, Brazil
REBECA PATRICIA OMENA-GARCIA Departamento de Biologia Vegetal, Universidade Federal
de Viçosa, Viçosa, Brazil
SONIKA PANDEY National Institute of Plant Genome Research, New Delhi, India
PRADEEP KUMAR PATHAK National Institute of Plant Genome Research, New Delhi, India
VINOD KUMAR PATIL Functional Cell Reprogramming and Organism Plasticity
(FunCrop), EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal
D. MARSHALL PORTERFIELD Bindley Bioscience Center, Physiological Sensing Facility,
Purdue University, West Lafayette, IN, USA; Department of Agricultural and Biological
Engineering, Purdue University, West Lafayette, IN, USA
GAIL M. PRESTON Biochemistry and Systems Biology, Department of Plant Sciences,
University of Oxford, Oxford, UK
AGEPATI S. RAGHAVENDRA Department of Plant Sciences, School of Life Sciences,
University of Hyderabad, Hyderabad, India
CARLA RAGONEZI Functional Cell Reprogramming and Organism Plasticity (FunCrop),
EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal
R. GEORGE RATCLIFFE Department of Plant Sciences, University of Oxford, Oxford, UK
MIQUEL RIBAS-CARBO Grup de Recerca en Biologia de les Plantes en Condicions
Mediterranies, Universitat de les Illes Balears, Palma de Mallorca, Spain
HARDY ROLLETSCHEK Department of Molecular Genetics, Leibniz Institute of Plant
Genetics and Crop Plant Research (IPK), Stadt Seeland OT Gatersleben, Germany
LAISE ROSADO-SOUZA Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm,
Germany
DIEDERSON BORTOLINO SANTANA Universidade Estadual do Norte Fluminense Darcy
CLESIVAN PEREIRA DOS SANTOS Functional Genomics and Bioinformatics, Department of
Biochemistry and Molecular Biology, Federal University of Ceara, Fortaleza, Ceara, Brazil
KÁTIA DANIELLA DA CRUZ SARAIVA Functional Genomics and Bioinformatics, Department
of Biochemistry and Molecular Biology, Federal University of Ceara, Fortaleza, Ceara,
Brazil
DANIEL S. SHAW Centre for Plant Science, School of Biology, Faculty of Biological Sciences,
University of Leeds, Leeds, UK
GLÁUCIA MICHELLE COSME SILVA Universidade Estadual do Norte Fluminense Darcy
BOBBA SUNIL Department of Plant Sciences, School of Life Sciences, University of
Hyderabad, Hyderabad, India
Contributors xiii
NICOLAS L. TAYLOR The University of Western Australia, Crawley, WA, Australia

ABHAYPRATAP VISHWAKARMA National Institute of Plant Genome Research, New Delhi,
India
YINGLANG WAN Department of Horticultural and Landscape Architecture, Purdue
University, West Lafayette, IN, USA; Weldon School of Biomedical Engineering, Purdue
University, West Lafayette, IN, USA; College of Biological Sciences and Biotechnology,
Beijing Forestry University, Beijing, China
Chapter 1
Assessing Metabolic Flux in Plants with Radiorespirometry

Nicholas J. Kruger, Shyam K. Masakapalli, and R. George Ratcliffe
Abstract
Carbohydrates are the dominant respiratory substrate in many plant cells. However, the route of carbohy-
drate oxidation varies depending on the relative cellular demands for energy, reductant, and precursors for
biosynthesis. During these processes individual substrate carbon atoms are differentially released as carbon
dioxide by specific reactions in the network, and this can be measured by monitoring the release of 14CO2
from a range of positionally labeled forms of [14C]glucose. Although the relative amounts of carbon dioxide
produced from different carbon positions do not allow precise determination of fluxes, they are indicative of
the route of carbohydrate utilization. Such information can be used to determine whether a comprehensive
metabolic flux analysis is merited, and also to facilitate independent verification of flux maps generated by
other techniques. This chapter describes an approach to determine and interpret the pattern of oxidation of
carbohydrates by monitoring 14CO2 release during metabolism of exogenously supplied [1-14C]-, [2-14C]-
, [3,4-14C]-, and [6-14C]glucose. The method is exemplified by studies on Arabidopsis cell suspension
cultures, but the protocol can be easily adapted for the investigation of other plant materials.
Key words [14C]Carbon dioxide production, Carbohydrate oxidation, Glycolysis, Oxidative pentose
phosphate pathway, Positionally labeled [14C]glucose, Respiration, Tricarboxylic acid cycle
1 Introduction
Radiorespirometry describes the determination of respiratory activ-

ity by monitoring the release of radioactivity from 14C-labeled
substrates supplied to intact tissues, and in particular the rate and
extent of conversion of positionally labeled substrate to 14CO2 [1].
This technique allows the fate of individual substrate carbon atoms
to be determined, and since specific carbon atoms are released by
particular reactions in the metabolic network, the pattern of 14CO2
release from different positions will reflect the pathways responsible
for catabolism (see Note 1). The preferred substrate for such studies
in plant tissues is often glucose reflecting both the availability of an
appropriate range of positionally labeled forms of [14C]glucose and
the dominance of carbohydrates as a respiratory substrate [2].
Early versions of this method were based on the assumption
that both C-1 and C-6 of supplied glucose are released as CO2 after
Kapuganti Jagadis Gupta (ed.), Plant Respiration and Internal Oxygen: Methods and Protocols, Methods in Molecular Biology,
vol. 1670, DOI 10.1007/978-1-4939-7292-0_1, © Springer Science+Business Media LLC 2017
1
2 Nicholas J. Kruger et al.
the conversion to pyruvate and entry into the tricarboxylic acid

(TCA) cycle, whereas only C-1 is released by the oxidative pentose
phosphate pathway [3]. Thus, the difference between the two was
attributed to the oxidative pentose phosphate pathway. The
method has subsequently been elaborated to include consideration
of other features of metabolism and to involve the use of additional
positional labels [4–6]. However, despite the increasing sophistica-
tion of the analysis [7–10], it is clear that radiorespirometry alone is
unlikely to permit a precise definition of flux through the central
metabolic network [11]. Ultimately, the large number of reactions
involved in the oxidation of the supplied substrate, the number of
reactions releasing CO2, and the multiple opportunities for the
rearrangement of carbon atoms within the backbones of the meta-
bolic intermediates combine to make it unlikely that the system can
be sufficiently constrained by measurements of a single output (see
Note 2). Nevertheless, the approach is useful for two reasons. First,
it provides information on relative fluxes through different sections
of the metabolic network and can thus reveal qualitative differences
in metabolism between tissues or treatments. Although precise
quantification of flux through individual reactions is problematic,
this qualitative information can be useful in identifying situations in
which a full steady-state 13C-metabolic flux analysis would be war-
ranted. Second, radiorespirometry offers independent verification
of comprehensive flux maps of central carbon metabolism gener-
ated by other approaches [12, 13].
This chapter describes the method used routinely to examine
and interpret the relative rates of CO2 production from different
carbon positions of glucose metabolized by an Arabidopsis cell
suspension culture. The technique involves monitoring the time-
course of 14CO2 released during the metabolism of positionally
labeled glucose (see Note 3). It is readily applied to cell suspensions
growing on different sugars [14] or cultures from different sources
[15], and is easily adapted for use with other plant tissues, including
potato tuber [16], barley root [17], wheat and pea leaves [18], and
Arum spadix [19].
2 Materials
1. Cell suspension culture: Cell line of Arabidopsis thaliana (L.)

Heynh. (ecotype Landsberg erecta) maintained in MS medium
supplemented with 166 mM glucose, and subcultured every 7
days by transferring 10 ml of cell suspension into 90 ml of fresh
MS medium containing glucose in a 250 ml conical flask (see
Note 4).
2. MS medium: Dissolve 4.3 g Murashige and Skoog basal salt
mixture salts (Sigma-Aldrich, M5524), 0.5 mg
Respiration of Positioinally Labelled [14C]glucose 3
naphthaleneacetic acid (50 μl from stock solution: 10 mg/ml in

ethanol), and 0.05 mg kinetin (50 μl from stock solution: 1 mg/
ml) in deionized water, set to pH 5.8 using 1 M KOH and adjust
to a final volume of 1 l (see Note 5).
3. Positionally labeled [14C]glucose stock solutions: 166 mM of
either [1-14C]glucose, [2-14C]glucose, [3,4-14C]glucose, or
[6-14C]glucose (specific activity 0.223-0.446 GBq/mol) dis-
solved in MS medium (see Note 6).
4. Alkali trap: 10% (w/v) KOH in deionized water made up fresh
on the day of use (see Note 7).
5. Liquid scintillation cocktail: Ultima Gold XR or OptiPhase
HiSafe 2 (both from Perkin Elmer) or equivalent (see Note 8).
6. Liquid scintillation counter: Beckman Coulter LS 6500, Hidex
300 SL, or similar.
3 Methods
The method describes the preferred approach involving analysis of

triplicate samples for each of four labeling strategies involving
different positionally labeled forms of glucose.
3.1 Determination 1. Transfer 3.9 ml MS medium containing 166 mM glucose to

of 14CO2 Release from each of 12 wide-necked 100 ml conical (Erlenmeyer) flasks (see
Positionally Labeled Note 9).
[14C]Glucose 2. To each of three flasks, add 0.1 ml 166 mM [1-14C]glucose
(total activity 3.7-7.4 kBq).
3. To a second group of three flasks, add 0.1 ml 166 mM [2-14C]
glucose.
4. To a third group of three flasks, add 0.1 ml 166 mM [3,4-14C]
glucose.
5. To the remaining group of three flasks, add 0.1 ml 166 mM
[6-14C]glucose.
6. Place the 12 flasks on an orbital shaking platform rotating at
80–100 rpm, sufficient to allow continuous mixing of the cell
suspension after addition (step 8).
7. Equilibrate the flasks and their contents at the desired incuba-
tion temperature, typically 22 C, for 15–30 min.
8. Add 1.0 ml 4-day-old (mid-log phase) cell suspension culture
to each flask at precisely timed intervals, typically every 30 s (see
Notes 10 and 11).
9. Immediately after the addition of the cell suspension culture,
seal each flask with a rubber bung modified to accommodate a
cup holding an uncapped microfuge tube containing the alkali
Fig. 1 Design of culture flask used for supplying [14C]glucose to plant samples.
The cell suspension or tissue is incubated in the medium at the bottom of the
flask. The alkali trap for capturing respired 14CO2 is contained within a microfuge
tube placed in the cup taped to a plastic rod protruding from the rubber bung
used to seal the flask
trap (0.5 ml 10% KOH) that is suspended in the flask above the
level of the incubation medium (Fig. 1).
10. At appropriate timed intervals, typically every 1 or 2 h, remove
the microfuge tube, and replace with another containing a
fresh 0.5 ml aliquot of 10% KOH (see Note 12).
11. Continue to sample 14CO2 release by replacing the microfuge
tube with a fresh alkali trap at regular intervals for up to 12 or
18 h and, if desired, take a final sample 24 h after the start of
the incubation with [14C]glucose.
12. Seal each microfuge tube immediately after the removal from
the incubation flask and store at 4 C prior to subsequent
analysis.
13. Ensure that the 0.5 ml KOH containing dissolved 14CO2 is
well mixed, and then transfer a representative aliquot, typically
0.45 ml, to a scintillation vial.
14. Add 2 ml (approximately 4 volumes) of liquid scintillation
cocktail to each vial, seal, and mix well before determining
the quantity of radioactivity present using a liquid scintillation
counter (see Note 13).
15. Determine the amount of radioactivity added to each flask by
transferring a known amount of each of the initial [14C]glucose
stock solutions, typically 1–5 μl, separately into triplicate scintil-
lation vials, and adjust the volume of each to 0.45 ml with water
before combining with 2 ml liquid scintillation cocktail and
treating in the same way as the experimental samples (step 14).
16. To assess the background level of scintillation generate tripli-
cate “alkali blank” samples by combining 0.45 ml of the 10%
KOH solution used in the experimental incubations with 2 ml

liquid scintillation cocktail and measure alongside the experi-
mental samples (step 14).
17. Prepare a separate triplicate set of scintillation vials each con-
taining 0.45 ml water to provide a “water blank.” Add 2 ml of
liquid scintillation cocktail to each of the scintillation vials, mix
thoroughly and count alongside the samples of [14C]glucose
stock solutions (step 15).
3.2 Analysis of 14CO2 1. Determine the background level of scintillation by calculating

Release from the mean of the rate of scintillation in the series of alkali blank
Positionally Labeled samples containing 10% KOH, and the series of water blank
[14C]Glucose samples.
2. Subtract the background level of scintillation of alkali blanks
from the rate of scintillation measured in each of the experi-
mental samples, and then adjust to correct for the proportion
of the alkaline trap sampled to determine the total amount of
radioactivity captured during the period of incubation.
3. Calculate the cumulative amount of radioactivity released for
each flask by summing the total amount of radioactivity cap-
tured in the alkali traps over successive sampling periods start-
ing from the beginning of the timed incubation.
4. Determine the amount of radioactivity supplied to each flask by
subtracting the background level of scintillation of the water
samples from the mean of the rate of scintillation in the aliquots
of the [14C]glucose stock solution assayed, and then adjusting
for the amounts of the stock solution used for measurement of
radioactivity relative to those added to each flask.
5. Calculate the yield of 14CO2 by expressing the amount of
radioactivity released (either cumulatively or during a specific
time interval) as a proportion of the radioactivity supplied to
the flask (Fig. 2).
6. Determine the relative yields of CO2 from different positions
within glucose by calculating the ratio of the yields of 14CO2
from [14C]glucose labeled in the following six combinations of
carbon positions: C-1/C-3,4; C-2/C-3,4; C-6/C-3,4; C-1/
C-6; C-2/C-1; and (C-1–C-6)/C-3,4 (Fig. 3; see Note 14).
3.3 Interpretation Analysis of the ratio of 14CO2 yields from different positionally
of Pattern of 14CO2 labeled forms of [14C]glucose is based on a consideration of the
Release from [14C] rearrangement of the carbon skeletons of intermediates in the
Glucose central metabolic network and the principal CO2-releasing reac-
tions (Table 1, Fig. 4).
If sugars are metabolized exclusively through glycolysis and the
tricarboxylic acid cycle, then as a result of the interconversion
catalyzed by triose phosphate isomerase, carbon atoms in positions
Fig. 2 Time course of 14CO2 release by metabolism of positionally labeled

glucose by Arabidopsis cell suspension cultures. Respired CO2 was captured
by an alkali trap which was replaced at hourly intervals for 8 h. Cumulative 14CO2
release is expressed as a proportion of radioactivity added to the cell culture, and
is presented as the mean SE of measurements from four replicate cultures for
each substrate. These results are taken from [20] and are representative of the
relative rates of 14CO2 release from different positionally labeled glucose by a
typical plant sample
1, 2, and 3 of the three-carbon phosphate esters in the second half

of glycolysis (triose phosphates, phosphoenolpyruvate, and pyru-
vate) are derived equally from positions 3 and 4, 2 and 5, and 1 and
6, respectively, of the original glucose substrate. Subsequently,
carbon atoms originally present in C-3 and C-4 of hexose are
released as CO2 in equal amounts when pyruvate is decarboxylated
to acetylCoA. In the first round of the TCA cycle, the two mole-
cules of CO2 generated by the reactions catalyzed by isocitrate
dehydrogenase and 2-oxoglutarate dehydrogenase are derived
from oxaloacetate and no carbon atoms are released from acetyl-
CoA entering the cycle, but in the second round equivalent
amounts of carbon from C-2 and C-5 of the original hexose
(which are now found in the carboxyl groups of oxaloacetate) are
released as CO2. In the third round, 50% of carbon originating
from C-1 and C-6 of hexose are released at the same steps, and in
each subsequent round a further 50% of the remaining C-1 and C-6
are released. Thus:
1. Carbon release from C-3 and C-4 will be equivalent, as will
release from C-2 and C-5, and release from C-1 and C-6.
2. Carbon will be released in the order C-3 (and C-4), C-2 (and
C-5), C-1 (and C-6).
3. Release from C-3,4 will indicate entry of pyruvate into the
TCA cycle (via pyruvate dehydrogenase).
However, C-1 of hexose is also released as CO2 during the
decarboxylation of 6-phosphogluconate to ribulose 5-
Fig. 3 Ratios of CO2 release from different carbon positions within glucose by Arabidopsis cell suspension
cultures. The cumulative values of 14CO2 release from cultures metabolizing different positionally labeled
glucose [20] were used to determine the relative yields of CO2 from different carbon positions within the
respiratory substrate. Data are the mean SE of measurements from four replicate cultures. The delay before
attaining a constant value for some ratios reflects differences in the time needed for the different pools of
metabolic intermediates that contribute to CO2 release to become labeled, and the larger error bars for the
earlier time points indicate the greater variation associated with estimating the comparatively small amounts
of radioactivity released in the initial stages of the incubations. The dashed line in each panel denotes the ratio
of CO2 yields predicted from the flux map of central carbon metabolism determined by steady-state 13C-
metabolic flux analysis for Arabidopsis cell cultures growing in MS medium [13]
Table 1
Summary of principal decarboxylation reactions in the central network of carbon metabolism in plant
cells as depicted in Fig. 4
Metabolic
process Enzyme Reaction
Oxidative 6-Phospho gluconate 6-Phosphogluconate þ NADP+ ! Ribulose 5-
pentose dehydrogenase phosphate þ NADPH þ CO2
phosphate [EC 1.1.1.44]
pathway
Tricarboxylic Pyruvate Pyruvate þ NAD+ þ CoASH ! AcetylCoA þ NADH þ CO2
acid cycle dehydrogenase
[EC 1.2.4.1]
Isocitrate Isocitrate þ NAD(P)+ ! 2-oxoglutarate þ NAD(P)H þ CO2
dehydrogenase
[EC 1.1.1.41/42]
2-oxoglutarate 2-oxo glutarate þ NAD+ þ CoASH ! Succinyl
dehydrogenase CoA þ NADH þ CO2
[EC 1.2.4.2]
Cataplerosis Malic enzyme Malate þ NAD(P)+ ! Pyruvate þ NAD(P)H þ CO2
[EC 1.1.1.38/
39/40]
Pentan UDPglucuronate UDPglucuronate ! UDPxylose þ CO2
synthesis decarboxylase
[EC 4.1.1.35]
phosphate in the oxidative pentose phosphate pathway, mean-

ing that:
4. Carbon will be released preferentially from C-1 relative to C-6
if the oxidative pentose phosphate pathway is active.
This idealized scheme is confounded by several complicating
factors:
1. First, pentan synthesis withdraws glucose 6-phosphate for the
conversion to UDPglucuronate and this releases C-6 during
the production of UDPxylose. This results in an overestimate
of the release of CO2 in the TCA cycle originating from C-6
and C-1, and will lead to an underestimate of flux through the
oxidative pentose phosphate pathway.
2. Second, there may be randomization between C-1 and C-6
within hexose phosphates due to two processes. One is recy-
cling between triose phosphates and hexose phosphates. Ran-
domization of C-1 and C-6 within the triose phosphate pool
and subsequent exchange with the hexose phosphate pool can
result in an underestimate of the release of CO2 from C-1 due
to the transfer to the C-6 position, and also an overestimate of
the release of CO2 from C-6 due to the transfer to the C-1
Fig. 4 Simplified scheme of the sources of carbon dioxide during carbohydrate

oxidation in plants. The major sites of CO2 release are indicated, and the principal
carbon position(s) within glucose from which CO2 is derived are highlighted in italics.
Carbon from positions 3 and 4 (C-3,4) is released as CO2 during the conversion of
pyruvate to acetylCoA, conversion of malate to pyruvate (after inversion following
interconversion with fumarate), or in the first turn of the TCA cycle if citrate is formed
from oxaloacetate produced by the anaplerotic reaction (catalyzed by PEP
carboxylase). Collectively, these three routes reflect entry of carbon into the TCA
cycle. Carbon from position 2 (C-2) is released during the second turn of the TCA
cycle following entry as acetylCoA, and carbons from positions 1 and 6 (C-1 and C-
6) are released in equivalent amounts in the third and subsequent turns through the
cycle. C-1 is additionally released through the formation of pentose phosphate via
the oxidative pentose phosphate pathway (as is C-2, secondarily, following the
regeneration of glucose 6-phosphate from fructose 6-phosphate produced by this
pathway). C-6 is also released during pentan synthesis that can be appreciable in
actively growing tissues. Note that only the quantitatively most important sites of
CO2 release are considered in this figure; a more comprehensive summary of
decarboxylation and carboxylation reactions within the central metabolic network
is presented in [21]. Abbreviations are: AcCoA acetyl coenzyme A, Cit citrate, Fru6P
fructose 6-phosphate, Fum fumarate, Glc6P glucose 6-phosphate, Icit isocitrate,
Mal malate, OAA oxaloacetate, 2OG 2-oxoglutarate, Pen5P pentose 5-phosphates,
PEP phosphoenolpyruvate, Pyr pyruvate, SucCoA succinyl coenzyme A, TrioseP
triose phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate)
position. The other process contributing to randomization

between C-1 and C-6 is a series of exchange reactions catalyzed
by the reversible nonoxidative steps of the pentose phosphate
pathway which transfer atoms originally present in C-1 to the
C-6 position of fructose 6-phosphate, leading to an underesti-
mate of the release of C-1. Both these considerations will lead
to underestimation of flux through the oxidative pentose phos-
phate pathway.
3. Third, fructose 6-phosphate produced by the oxidative pentose
phosphate pathway may be converted back to glucose 6-
phosphate and then re-enter the oxidative pentose phosphate
pathway. In this circumstance, the molecule will be labeled with
the atom originally at C-2 which is now in the C-1 position.
This means that some of the carbon released by the oxidative
pentose phosphate pathway will originate from C-2, thereby
underestimating release from C-1 and, again, underestimating
flux through this pathway.
4. Fourth, withdrawal of TCA cycle intermediates for storage or
biosynthesis will necessitate replenishment of the pools of
organic acids by the production of oxaloacetate from phospho-
enolpyruvate through the anaplerotic reaction catalyzed by
phosphoenolpyruvate carboxylase. This allows entry of inter-
mediates into the TCA cycle without the concomitant loss of
C-3 and C-4 normally associated with the formation of acet-
ylCoA from pyruvate. This means that the release of CO2 from
C-3,4 may underestimate the combined flux through glycolysis
and the oxidative pentose phosphate pathway. However, the
extent of this underestimation will be decreased by subsequent
metabolism of oxaloacetate through the TCA cycle in which C-
3,4 will be released as CO2 in the reactions catalyzed by iso-
citrate dehydrogenase and 2-oxoglutarate dehydrogenase in
the initial turn of the cycle.
5. Finally, additional rounds of recycling or exchange through the
reactions described above will result in yet greater rearrange-
ment of the label within the carbon skeletons of the intermedi-
ates in the metabolic network. This will further obscure the
immediate source of any 14CO2 that is released, confounding
precise analysis of the measurements.
It is clear that there is no simple relationship between the
relative rates of release of CO2 from individual positions within
the original glucose and flux through a specific sequence of reac-
tions. Nevertheless, the pattern of CO2 release from different posi-
tions is determined by the fluxes through the metabolic network,
and any changes in the ratios of 14CO2 yields between different
treatments will reflect a difference in the flux distribution.
Specifically, increases in the values of particular ratios are likely to be

indicative of the following changes in flux:
1. C-1/C-3,4: an increase in the proportion of carbon entering
the TCA cycle that has passed through the oxidative pentose
phosphate pathway, and/or a decrease in the proportion of
pyruvate/acetylCoA entering the TCA cycle that is oxidized
to CO2.
2. C-2/C-3,4: an increase in extent of recycling of hexose phos-
phates through the oxidative pentose phosphate pathway, and/
or increase in the proportion of pyruvate/acetylCoA entering
the TCA cycle that is completely oxidized to CO2 (i.e., a
decrease in the withdrawal of TCA cycle intermediates for
biosynthesis).
3. C-6/C-3,4: an increase in the proportion of pyruvate/acetyl-
CoA entering the TCA cycle that is oxidized to CO2 (i.e., a
decrease in the withdrawal of TCA cycle intermediates for
biosynthesis), and/or an increase in the proportion of hexose
phosphates that are used for pentan biosynthesis.
4. C-1/C-6: an increase in the proportion of hexose phosphate
metabolized via the oxidative pentose phosphate pathway,
and/or a decrease in the proportion of pyruvate/acetylCoA
entering the TCA cycle that is oxidized to CO2.
5. C-2/C-1: an increase in the extent of recycling of hexose
phosphates through the oxidative pentose phosphate pathway,
and/or an increase in the extent of withdrawal of TCA cycle
intermediates for biosynthesis.
6. (C-1–C-6)/C-3,4: an increase in flux through the oxidative
pentose phosphate pathway and/or decrease in pentan synthe-
sis, relative to entry of pyruvate/acetylCoA into the TCA cycle.
Thus, none of these ratios of 14CO2 yields defines a unique
metabolic process. However, changes in the ratios will reflect varia-
tion in flux through the metabolic network and may provide the
incentive for a more comprehensive interrogation of the system by
13
C-metabolic flux analysis.
4 Notes
1. This approach complements the analysis of the fate of [U-14C]

substrate described in Chapter 2, which estimates the fluxes to
major respiratory products but does not determine the path-
ways by which they are produced. In principle, the two meth-
ods can be amalgamated and the fate of carbon atoms in
biosynthesis of cellular constituents and respiratory products
other than CO2 can be traced by combining the metabolism of
positionally labeled [14C]substrates with the fractionation

techniques described in Chapter 2. Quantifying the specific
yield of [14C]products in addition to CO2 may improve dis-
crimination between alternative routes of metabolism (e.g., ref.
[18]). However, this combined approach is seldom used and
has largely been superseded by the 13C-metabolic flux analysis
that can typically provide more comprehensive and robust
estimates of fluxes throughout the central metabolic network.
2. Although the equivalent techniques based on the fractional 13C
abundance of the carbon dioxide produced by cells supplied
with positionally labeled [13C]glucose have been reported to
allow the determination of net fluxes within the central meta-
bolic network [22], this requires knowledge of the fluxes to
biomass components and other metabolic end-products to
provide additional constraints in what would otherwise consti-
tute an under-determined system [23].
3. The technique described in this chapter is not designed to
achieve quantitative capture of respired 14CO2, which would
require acidification of the cells and incubation medium. How-
ever, the method does trap a representative proportion of the
14
CO2 released from [14C]glucose. Thus, the ratio of label
captured from identical samples incubated with differentially
labeled sources provides a convenient and robust measure of
the relative rates of oxidation of the different positional carbon
atoms within the supplied respiratory substrate [20].
4. This method can be adapted for suspension cultures growing
in media containing a carbon source other than exogenous
glucose. We recommend that such cultures are incubated in
the normal growth medium (including the regular carbon
source) supplemented with 0.3 mM positionally labeled [14C]
glucose. To achieve this, the positionally labeled stock solutions
should contain 15 mM [14C]glucose (specific activity
2.47–4.93 GBq/mol) and be added as 0.1 ml aliquots in
Subheading 3.1, step 2 to step 5.
The method can also be used to study metabolism of
respiratory substrate in excised organs or tissues, which may
be sliced to improve the uptake of [14C]glucose. Typically, the
plant material is incubated in a weak buffered solution, such as
K-Mes (pH 5.6). The incubation medium may be supplemen-
ted with an exogenous respiratory substrate (particularly if the
tissue contains limited endogenous carbohydrate reserves). If
glucose is not used as the exogenous substrate, the positionally
labeled [14C]glucose should be supplied at a concentration of
0.3 mM, as above [16].
5. To restrict caramelization of sugars during sterilization when
preparing MS medium supplemented with 3% (w/v) glucose,
make up double-strength MS medium and a 6% glucose solu-

tion. Autoclave each solution separately, and then combine in
equal volumes after cooling.
6. L-[14C]glucose labeled in various positions may be obtained
from PerkinElmer (http://www.perkinelmer.com/) and Amer-
ican Radiolabeled Chemicals (http://www.arcincusa.com/).
Typically, these compounds are supplied at a very high specific
activity (1.85–2.22 TBq/mol) and should be diluted to the
desired specific activity using an appropriate concentration of
unlabeled glucose—normally the amount of glucose contribu-
ted by the labeled source is negligible (<0.025%).
7. Hyamine hydroxide and similar organic bases traditionally used
to absorb CO2 in standard manometry experiments should be
avoided. Such bases have a high vapor pressure and can inhibit
metabolic processes by alkalinization of the incubation medium
[24].
8. The choice of liquid scintillation cocktail can affect the effi-
ciency and reliability of counting. Ultima Gold and OptiPhase
HiSafe 2 are high-performance, high flash point, general-
purpose scintillation cocktails that are compatible with alkaline
samples.
9. Use a set of identical flasks for these incubations since their
precise size and shape may influence the proportion of respired
14
CO2 that is released from the medium and captured in the
alkali trap (see Note 3).
10. Ideally, each of the three flasks used to monitor metabolism for
any specific positionally labeled [14C]glucose should be an
independent biological replicate, yet incubations involving dif-
ferent positionally labeled [14C]glucose should be identical
(except for the position of 14C within the supplied substrate).
To satisfy these constraints, one of the replicates for each
positionally labeled [14C]substrate is taken from each of three
separate cell cultures (or tissue samples taken from three sepa-
rate plants). This generates three series of measurements of
14
CO2 release from specific carbon atoms with glucose, with
each series derived from a discrete biological sample. This
arrangement of samples into three distinct groups assists
subsequent statistical analysis (see Note 14).
11. The precise volume of cell suspension culture used in the series
of incubations can be varied depending on the requirements of
the study, provided that the volume of medium is adjusted
accordingly. Thus, for example, to examine the pathways of
carbohydrate oxidation in cells under conditions that reflect
the metabolic steady-state of mid-log phase growth [13],
4.9 ml of 4-day-old Arabidposis cell suspension culture
(grown in MS medium supplemented with unlabeled glucose
at an initial concentration of 166 mM) was combined with

0.1 ml positionally labeled [14C]glucose (total activity
3.7 kBq, specific activity 1.11–2.07 TBq/mol) in MS medium
without any supplementary glucose. The incubations were
initiated at precisely timed intervals, and then processed as
described in Subheading 3.1, step 9 onward.
12. Typically, only a small proportion of [14C]glucose is metabo-
lized during this time course, and if necessary the frequency of
replacement of the alkali trap and the total incubation time can
be adjusted to ensure that this condition is met. This avoids
complications in the interpretation of the pattern of 14CO2
release arising from depletion of substrate. However, while
using a labeled compound that differs from the major respira-
tory substrate (derived from either the medium or endogenous
reserves), be aware that the labeled compound may not meta-
bolized in the same way as the major respiratory substrate. As a
result, the pattern of 14CO2 release may not reflect the oxida-
tion of the principal substrate, although the impact of this
effect is likely to be limited if both respiratory sources are
carbohydrates that directly enter a common pool of hexose
phosphates. Additionally, if using excised tissues or organs, be
aware of the potential depletion of endogenous respiratory
reserves resulting from detachment from a source tissue [25],
and the possible distortion of the normal pathways of carbohy-
drate oxidation due to wound-induced respiration [26].
13. For samples containing strong base, such as the alkali trap, the
vials may need to be stored in the dark for 12–18 h after the
addition of scintillation cocktail to decrease the extent of
chemiluminescence before measuring in the scintillation
counter.
14. The ratio of the yields of 14CO2 from different positions within
glucose should be calculated separately for each of the three
cultures used in the replicate incubations for each positionally
labeled [14C]glucose (see Note 10). The results may then be
presented as the mean SE of the precise ratios from the three
biological replicates rather than determining the ratio of yields
determined from the mean SE of the values for 14CO2
release from each positionally labeled source of [14C]glucose.
(This latter approach effectively considers each flask as an inde-
pendent sample and will normally lead to larger estimates of SE
and thus decrease the power of the subsequent statistical anal-
ysis). The significance of any difference in the pattern of 14CO2
release between cell types or treatments can be assessed by
MANOVA of the ratios of 14CO2 yields, noting that these
values should be log transformed prior to the analysis since
they are expressed on a ratio scale [27].
Acknowledgments
SKM acknowledges financial support from the University of

Oxford (Clarendon Scholarship), Exeter College (Mr Krishna
Pathak Scholarship), and a UK Overseas Research Student award.
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Chapter 2
Coupling Radiotracer Experiments with Chemical

Fractionation for the Estimation of Respiratory Fluxes
Toshihiro Obata, Laise Rosado-Souza, and Alisdair R. Fernie
Abstract
Carbohydrates catabolized via respiratory processes are not only used for energy production but also for
biosynthesis of cellular components including soluble molecules (sugars, amino acids, organic acids, and
their derivatives) and insoluble macromolecules (proteins, starch, and cell wall). Radiotracer experiments
using 14C-labeled glucose provide a global picture of the fate of respired carbon in the metabolic network.
This method is based on a chemical fractionation of biomolecules in 14C-glucose fed plant materials and the
subsequent determination of radioactivity in each fraction. Metabolic flux into each fraction can be
estimated from the specific activity of the hexose phosphate pool. Here, we describe the procedure for
glucose metabolism in potato tuber but similar protocols can be adopted for various plant organs and
substrates.
Key words Respiration, 14C-radiotracer analysis, Fractionation, Metabolic flux
1 Introduction
The primary function of cellular respiratory metabolism is energy

production. However, it also provides precursors for other meta-
bolic pathways. Carbon backbones of all 20 proteinogenic amino
acids are originated from 3-phosphoglycerate, phosphorenolpyru-
vate, pyruvate, 2-oxoglutarate, and oxaloacetate that are intermedi-
ates of the glycolysis or the tricarboxylic acid (TCA) cycle (Fig. 1)
[1]. Glucose 1-phosphate is used for the synthesis of sucrose and
macromolecules including starch and cellulose (Fig. 1) [2, 3].
Thus, the distribution of carbon in catabolized carbohydrates
depends on the genetic, developmental, and environmental con-
texts and is informative to gain an insight into the cellular metabolic
status. Radiotracer experiments using 14C-labeled respiratory sub-
strates are appropriate to gain a broad overview of the flux of
carbon within the metabolic network, while a stable isotope tracer
experiment coupled with nuclear magnetic resonance spectroscopy
or mass spectrometry is suitable for analyses of metabolic flux in
17
18 Toshihiro Obata et al.
Glucose
Starch Hexose-P pool
Sucrose Gluc-1P Gluc-6P
CO2
Cell wall Fruc-6P
(cellulose)
Fruc-1,6BP
Fructose DHAP GAP OPPP
3PG Glycolysis
Pyruvate CO2
Protein Amino acids
OAA
TCA cycle
2OG
Organic acids
Fig. 1 Glucose metabolism in plant tissues. Glucose is mainly catabolized via glycolysis, oxidative pentose
phosphate pathway (OPPP), and tricarboxylic acid (TCA) cycle. The metabolite classes analyzed by the
methods described in this chapter are shown in red. The reactions including multiple steps are shown as
arrowheads with dotted line. P phosphate, Gluc glucose, Fruc fructose, BP bisphosphate, DHAP dihydroxyac-
etone phosphate, GAP glyceraldehyde-3-phosphate, 3PG 3-phosphoglycerate, OAA oxaloacetate, 2OG 2-
oxoglutarate
specific metabolites or metabolic pathways [4]. Nevertheless, the

radiotracer method can also be applied for the analyses of some
specific metabolites by the combination of further separation meth-
ods such as thin-layer chromatography [5, 6] or by treatments with
specific enzymes as described in this chapter.
This radiotracer method is based on the fractionation of cellular
molecules using simple ion exchange chromatography and
subsequent determination of radioactivity in each fraction. Plant
material is fed with a 14C-labeled substrate within a sealed flask and
the radioactivity evolved as 14CO2 is captured as described in
Chapter 1 to determine the amount of carbon released as CO2.
Metabolites are extracted from the plant tissue in ethanol and
separated into their respective compound classes by ion-exchange
chromatography based on their ionization state in a neutral solvent.
In general, amino acids and organic acids (including hexose phos-
phates) are positively and negatively charged at neutral pH, respec-
tively, while sugars are essentially uncharged. The neutral fraction
containing sugars is further analyzed following enzymatic conver-
sion of specific major sugars into charged molecules in order to
determine the 14C-distribution into glucose, fructose, and sucrose.
14
C-incorporation into sugar phosphates is similarly determined by
the acid phosphatase treatment of acidic fraction. Specific enzy-
matic digestion and subsequent fractionation allows the quantifica-
tion of 14C-incorporation into protein, starch, and cell wall
Coupling Radiotracer Experiments with Chemical Fractionation for the. . . 19
Plant tissue
Ethanolic extraction
Total soluble
Insoluble fraction
fraction
Amyloglucosidade
Acid phosphatase
and pronase
treatment
treatment
AP cationic/basic fraction Soluble cationic/basic Insoluble cationic/basic

(amino acids) fraction (amino acids) fraction (proteins)
Soluble anionic/acidic
AP anionic/acidic fraction Insoluble anionic/acidic
fraction (organic acids,
(organic acids) fraction + pellet (cell wall)
hexose phosphates)
AP neutral fraction (sugars, Soluble neutral fraction Insoluble neutral fraction

hexose phosphates) (sugars) (starch)
Hexokinase treatment GOX treatment
HK anionic/acidic fraction GOX anionic/acidic

(glucose, fructose) fraction (glucose)
HK neutral fraction GOX neutral fraction

(sucrose) (sucrose, fructose)
Fig. 2 Flow chart showing the chemical fractionation procedure
included in the ethanol insoluble pellet. The radioactivity in each

fraction is measured using a liquid scintillation counter and used for
the calculation of carbon redistribution into each compound type.
The overall workflow of the fractionation procedure is depicted in
Fig. 2. Metabolic flux into each compound class can be calculated
by using carbon redistribution and the specific activity of the hexose
phosphate pool. The pathways of glucose catabolism are depicted
in Fig. 1. It should be noted that radioactivity in all carbon contain-
ing molecules derived from glucose is determined by this method,
rendering the analysis of total uptake and metabolism possible.
Here, we describe a procedure for 14C-glucose feeding to a
potato tuber [7–9]. However, this technique is applicable for
various plant materials and has been adopted for Arabidopsis seed-
lings [10, 11], tobacco callus [5], tobacco leaves [12], tomato
leaves [13], tomato fruits [14, 15], carrot storage tissue [16], and
spadix of Arum (Arum maculatum) [17]. It is also possible to apply
a similar procedure for an analysis of carbon redistribution from
other substrates such as sucrose, pyruvate, and acetate. However,
for the purpose of understanding the major pathways of the oxida-
tion, glucose is by far the most used.
2 Materials
2.1 [U-14C]-Glucose 1. Developing potato tubers were rapidly removed from 10-week-
Feeding old plants.
2. 10 mM MES-KOH (pH 6.5).
3. 100 mM [U-14C]-glucose (1.4 MBq mmol 1) (see Note 1).
4. 10% (w/v) NaOH.
5. Liquid nitrogen.
6. Cork borer with 10 mm in diameter.
7. Razor blades.
8. Orbital shaker.
9. 100 mL Erlenmeyer flasks with wide opening (for each
sample).
10. 1.5 mL tubes with a hole on the lid (e.g., Safe-Lock Micro-
centrifuge Tubes, Eppendorf).
11. Toothpicks.
12. Parafilm.
13. Liquid nitrogen.
2.2 Ethanol 1. Ball mill (e.g., Mixer Mill MM 400, Retsch).

Extraction 2. Bench-top microcentrifuge.
3. Thermomixer. Thermostable water bath can also be used
although it requires occasional mixing of solution every few
hours.
4. Vacuum concentrator (e.g., SpeedVac concentrator, Thermo
Fischer Scientific). An air flow sample concentrator can also be
used.
5. Glass tubes with 10 mL volume.
6. Screw cap tubes with 2.0 mL volume.
7. 20, 50, and 80% (v/v) ethanol solutions in deionized water.
8. Balance.
2.3 Chemical 1. Liquid scintillation counter (e.g., LS6500, Beckman Coulter).

Fractionation of the 2. Scintillation cocktail. Rotiszint® eco plus (Roth) is recom-
Ethanol Soluble mended due to its low chemoluminescence.
Fraction
3. Cationic resin AG 50 W-X8, 200–400 mesh, in hydrogen form
(Bio-Rad Laboratories).
4. Anionic resin AG 1-X2, 200–400 mesh, in acetate form (Bio-
Rad Laboratories).
5. Magnetic stirrer.
6. Ammonium hydroxide solution: 1 M NH4OH in water.
7. Formic acid solution: 4 M CH2O2 in water.
8. Hexokinase mix: 8 U mL 1 of hexokinase (E.C. 2.7.1.1 from
Saccharomyces cerevisiae, Roche) in 100 mM Tris–HCl
(pH 8.0), 26.6 mM MgCl2, 6 mM adenosine triphosphate
(ATP).
9. GOX mix: 2 U mL 1 glucose oxidase (E.C. 1.1.3.4 from
Aspergillus niger, Roche) and 64 U mL 1 peroxidase (E.C.
1.11.1.7 from Horse-radish, Roche) in 200 mM potassium
phosphate buffer (pH 6.0).
10. Acid phosphatase mix: 20 U mL 1 acid phosphatase (E.C.
3.1.3.2, grade II, from potato, Roche) in 10 mM MES-KOH
(pH 6.0).
2.4 Chemical 1. Amyloglucosidase mix: 5 U mL 1 amyloglucosidase (EC

Fractionation of the 3.2.1.3 from Aspergillus nidulans, Roche) in 0.4 M sodium
Ethanol Insoluble acetate (pH 5.5).
Fraction 2. Pronase mix: 10 U mL 1 pronase (E.C. 3.4.24.4 from Strepto-
myces griseus, Roche) in 0.5 M Tris–HCl (pH 7.8).
2.5 Determination of 1. Glucose-6-phosphate standard solutions: 0, 0.4, 1.0, 2.0, 4.0,

Hexose Phosphate 10, 25, and 100 μM of Glucose-6-phosphate (Roche) in water.
Pool Size 2. Reaction Mix A: 50 mM Tricine KOH (pH 9.0), 2.5 mM
MgCl2, 0.1 mM NADP+, 0.1 mM glucose-1,6-bisphosphate
(Sigma), 2.5 U mL 1 glucose-6-phosphate dehydrogenase (E.
C. 1.1.1.49, grade II from yeast, Roche), 1.0 U mL 1 phos-
phoglucose isomerase (E.C. 5.3.1.9 from yeast, Roche), 1.0 U
mL 1 phosphoglucomutase (E.C. 5.4.2.2, from rabbit muscle,
Roche).
3. Neutralization solution: mix HCl solution with 1.0 M Tricine
KOH (pH 9.0) and dilute by water to gain 0.5 M HCl and
0.2 M Tricine. The pH of the solution should not be adjusted
after mixing.
4. Reaction Mix B: 300 mM Tricine KOH (pH 9.0), 12 mM
EDTA, 9 mM glucose-6-phosphate (Roche), 100 U mL 1
glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49, grade I
from yeast, Roche), 1.8 mM thiazolyl blue tetrazolium bro-

mide (MTT, Sigma), 0.3 mM phenazine methosulfate (PMS,
Sigma). MTT and PMS stock solutions should be prepared
daily and protected from strong light. These stock solutions
should be mixed with other solutions just before use.
5. Adhesive microplate sealer (e.g., Aluminium Foil Tape 425,
3 M).
6. Dry oven.
7. Microplate reader that can read absorbance at 570 nm.
3 Methods
3.1 [U-14C]-Glucose Radiolabled glucose is fed to the potato tuber discs. 14CO2 evolved
Feeding from respiration is captured by NaOH trap to measure the amount
of 14C released as CO2.
1. Take a 10 mm diameter longitudinal core of potato tuber by a
cork borer. Slice the core into 1 mm thick discs and wash three
times with fresh 10 mM MES-KOH (pH 6.5) (see Note 2).
2. Preincubate 8 discs per sample in a 100 mL flask containing
5 mL of 10 mM MES-KOH (pH 6.5) for 30 min under the
experimental condition.
3. Add 100 μL of 100 mM [U-14C]-glucose solution into each
flask to gain a glucose concentration of 2 mM to start labeling.
A 1.5 mL tube with 0.5 mL of 10% NaOH solution is sus-
pended in the flask by a toothpick put through the hole on the
lid of the tube. The top of the flask is tightly sealed with
Parafilm (Fig. 3a).
4. Incubate the flask for 2 h at room temperature with rotation at
90 rpm.
Fig. 3 Pictures of experimental setup. (a) A labeling flask with CO2 trap. (b) Columns with resins. (c) A column
stack. (d) A spin filtration column
5. Store NaOH solution in the tubes as CO2 fraction.

6. Wash tuber discs three times with water and drain excess water
drops on the surface. Put two discs into four 2 mL tubes and
record the exact weight of materials (see Note 3). Freeze the
tubes in liquid nitrogen and store at 80 C.
7. Mix CO2 fraction with 4 mL of scintillation cocktail and vortex
vigorously. Determine the radioactivity by liquid scintillation
counting. The values in disintegration per minute (dpm) cor-
respond to the radioactivity released as CO2.
3.2 Ethanol Cellular metabolites are extracted from frozen material by ethanol
Extraction containing solutions. Major water soluble small molecules are
extracted into the soluble fraction and the insoluble precipitate
contains macromolecules including starch, protein, and cell wall.
1. Grind the frozen materials to a fine powder by a ball mill.
2. Add 1 mL of 80% (v/v) ethanol to the frozen material. Close
the tubes and incubate for 10 min at 95 C with shaking at
400 rpm.
3. Spin the tubes briefly to remove the condensed liquid and
residual insoluble material from the tube cap. Collect the
supernatant to a 10 mL glass tube.
4. Add 1 mL of 50% (v/v) ethanol to the pellet. Close the tubes
and incubate for 10 min at 95 C with shaking at 400 rpm.
5. Spin the tubes briefly and add the supernatant into the same
10 mL glass tube used in step 3.
6. Add 1 mL of 20% (v/v) ethanol to the pellet. Close the tubes
and incubate for 10 min at 95 C with shaking at 400 rpm.
7. Spin the tubes briefly and add the supernatant into the same
10 mL glass tube used in step 3.
8. Add 1 mL of H2O to the pellet. Close the tubes and incubate
for 10 min at 95 C with shaking at 400 rpm.
9. Keep the tubes containing pellets at 20 C for the analysis of
insoluble fraction in Subheading 3.4.
10. Dry down the combined ethanolic and water supernatant in
10 mL glass tubes (~4 mL) by vacuum concentrator overnight.
3.3 Chemical Ethanol soluble metabolites are fractionated into groups by ion-
Fractionation of the exchange chromatography based on their charge in a neutral solu-
Ethanol Soluble tion. Radioactivity in total hexoses, glucose and total hexose phos-
Fraction phates are determined by calculating the changes in distribution of
radioactivity into fractions by treatments with hexokinase, glucose
oxidase, and acid phosphatase, respectively.
1. Resuspend the dried pellet from the ethanolic and water super-
natant in appropriate amount of H2O, by vortexing and
shaking at 1400 rpm on a thermomixer at room temperature

until the pellets are completely dissolved. Combine the solu-
tion from all aliquots derived from a sample and adjust the
volume to 2 mL with H2O (soluble fraction). Take 50 μL
from each sample, add 4 mL of scintillation cocktail, and vortex
vigorously (see Note 4). Determine the radioactivity by liquid
scintillation counting. The values in disintegration per minute
(dpm) correspond to the assimilation in the total soluble
fraction containing sugars, organics acids, and amino acids.
2. Prepare resins. Weigh out each resin (1 g per sample) into
separate glass beakers (the resin should not occupy more than
20% of the volume). Wash the resins in an excess of water. Let
the resins settle and pour off the water. Repeat until the super-
natant is colorless. Stir for 30 min and let the resins settle.
Remove the supernatant without losing the resin (see Note 5).
3. Prepare empty columns. Place a small piece of Miracloth on the
tip of 1 mL pipette tips (Fig. 3b).
4. Resuspend resins in an equal volume of water. Mix the resin by
a magnetic stirrer and fill the columns with 0.5 mL aliquots of
resins and allow water to drain from the column. The resin bed
height should be approx. 1.5 cm (Fig. 3b). Wash the columns
twice with 1 mL of H2O. The columns should be prepared
immediately before use to avoid disturbing and drying resins.
5. Stack the column with cationic resin above the column with
anionic resin in a suitable rack, so that the flow drops directly
from one column to the next. Place a small vial below the
columns (Fig. 3c).
6. Apply 750 μL of the soluble fraction to the cationic column.
Let the sample run into the resins. Remaining soluble fraction
can be stored at 20 C.
7. Collect the outflow in small scintillation vials and elute by
passing 1 mL of H2O three times. This corresponds to the
soluble neutral fraction and can be stored at 20 C.
8. Transfer the cationic column to a new large scintillation vial
and elute by passing 1 mL of 1 M NH4OH three times. This
corresponds to the soluble cationic/basic fraction and con-
tains amino acids. Add 10 mL of scintillation cocktail to the
outflow and determine the radioactivity.
9. Transfer the anionic column to a new large scintillation vial and
elute by passing three times 1 mL of 4 M formic acid. This
corresponds to the soluble anionic/acidic fraction and con-
tains organic acids and hexoses phosphates. Add 10 mL of
scintillation cocktail to the outflow and determine the
radioactivity.
10. Treat a part of soluble neutral fraction with hexokinase.

Hexokinase uses ATP to phosphorylate hexoses sugars into
their respective hexose phosphates, which allow them to be
negatively charged and trapped in the anionic/acidic fraction,
leaving sucrose as the major component of the neutral fraction.
Take 200 μL of the soluble neutral fraction into a new 2 mL
tube. Add 200 μL of hexokinase mix and incubate for 4 h at
25 C with shaking at 400 rpm. Stop the reaction by heating at
95 C for 5 min and spin down briefly. Apply the mixture to a
new set of cationic/anionic columns and fractionate into HK-
neutral, HK-cationic/basic, and HK-anionic/acidic frac-
tions and determine the radioactivity by liquid scintillation
counting as described above (steps 6–9).
11. Treat a part of soluble neutral fraction with glucose oxidase
(GOX). GOX catalyzes the specific oxidation of glucose to D-
glucono-1,5-lactone, which allows glucose to be negatively
charged and trapped in the anionic/acidic fraction, leaving
fructose and sucrose as the major components of the neutral
fraction. Take 200 μL of the soluble neutral fraction into a
new 2 mL tube. Add 200 μL of GOX mix and incubate at
25 C for 6 h with shaking at 400 rpm. Stop the reaction by
heating for 5 min at 95 C and spin down. Apply the mixture to
a new set of cationic/anionic columns and fractionate into
GOX-neutral, GOX-cationic/basic, and GOX-anionic/
acidic fractions and determine the radioactivity by liquid scin-
tillation counting as described above (steps 6–9).
12. Treat a part of total soluble fraction with acid phosphatase.
The acid phosphatase releases orthophosphate from hexose
phosphates, which remove the charge in hexose phosphate to
be in the neutral fraction. Take 150 μL of total soluble frac-
tion into a new 2 mL tube. Add 50 μL of acid phosphatase mix
and incubate at 37 C for 3 h with shaking at 400 rpm. Stop the
reaction by heating the sample for 3–4 min at 95 C and spin
down briefly. Apply the mixture to a new set of cationic/
anionic columns and fractionate into AP-neutral, AP-cat-
ionic/basic, and AP-anionic/acidic fractions and determine
the radioactivity by liquid scintillation counting as described
above (steps 6–9).
3.4 Chemical Ethanol insoluble precipitate is mainly composed of starch, protein,

Fractionation of the and cell wall. Starch and protein are hydrolyzed into glucose and
Ethanol Insoluble amino acids, respectively. Cell wall components stay insoluble by
Fraction this treatment. Radioactivity in these fractions can be determined
by the chemical fractionation similar to that described above.
1. Resuspend pellets derived from Subheading 3.2, step 9 into
appropriate amount of H2O by vortexing. Combine the
solution from all aliquots derived from a sample and adjust the
volume to 0.5 mL with H2O (insoluble fraction). Then grind
the pellets using a ball mill.
2. Treat the ethanol insoluble fraction with amyloglucosidase and
pronase. Take 200 μL of resuspended pellet into a new 1.5 mL
tube. Add 200 μL of amyloglucosidase mix and incubate over-
night at 37 C with shaking at 400 rpm. Then add 100 μL of
the pronase mix and incubate overnight at 37 C in a thermo-
mixer with shaking at 400 rpm. Stop the reaction by heating
the sample for 5 min at 95 C and spin down briefly.
3. Separate pellet from supernatant by filtration. Place a 0.5 mL
centrifugation tube with a small hole on the bottom into a
1.5 mL centrifuge tube and put a small piece of Miracloth on
the hole of a 0.5 mL tube (Fig. 3d). Apply all of the enzyme-
treated solutions onto the filtration column and centrifuge
briefly.
4. Apply the outflow to a new set of cationic/anionic columns and
fractionate into insoluble-neutral, insoluble-cationic/basic,
and insoluble-anionic/acidic fractions and determine the
radioactivity by liquid scintillation counting as described
above (Subheading 3.3, steps 6–9). The Miracloth containing
insoluble material is added into the insoluble-anionic/acidic
fraction prior to the radioactivity measurement. The radioac-
tivity in neutral, basic/cationic, and Miracloth/acidic/anionic
fractions corresponds to that in starch, protein, and cell wall,
respectively.
3.5 Determination of Absolute amount of hexose phosphate pool is spectrophotometri-

Hexose Phosphate cally determined by coupling enzyme reactions. The specific activity
Pool Size and Specific of the hexose phosphate pool is used for the estimation of meta-
Activity bolic fluxes in Subheading 3.6. It is estimated by dividing the
radioactivity present in hexose phosphate pool by the amount of
hexose phosphates. Fructose-6-phosphate (F6P) and glucose-1-
phosphate (G1P) are converted into glucose-6-phosphate (G6P)
by the reactions of phosphoglucose isomerase (PGI) and phospho-
glucomutase (PGM), respectively. Resulting G6P is used to gener-
ate equivalent amount of NADPH via G6P dehydrogenase
(G6PDH) reaction. The above reactions take place simultaneously
in the first reaction. Resulting NADPH is quantified by a sensitive
cycling assay [18] based on the reduction of phenazine methosul-
fate (PMS) by NADPH in the second reaction.
1. Take 5 μL aliquot of soluble fraction or G6P standard solution
with concentrations of 0, 0.4, 1.0, 2.0, 4.0, 10, 25, and
100 μM into a microtiter plate.
2. Add 75 μL of reaction mix A and incubate for 30 min at room
temperature.
3. Add 20 μL of 0.5 M KOH, seal the plate with microplate sealer,

and spin down.
4. Incubate the plate at 95 C for 5 min and cool the plate down
to room temperature.
5. Neutralize the sample by adding 20 μL of neutralization solu-
tion (see Note 6).
6. Add 50 μL of reaction mix B and monitor the absorbance at
570 nm till the slope becomes stable (usually around 15 min).
7. The amount of hexose phosphate is calculated from the reac-
tion velocity using a standard curve drawn from the slopes of
reactions with G6P standard.
8. Specific activity of the hexose phosphates is calculated by divid-
ing the radioactivity in hexose phosphate with amount of hex-
ose phosphate (dpm nmol 1).
3.6 Calculation of The redistribution of radioactivity from fed 14C-glucose is calcu-

Label Redistribution lated as a percentage of the radioactivity determined in each fraction
and Absolute against the sum of metabolized radioactivity. Specific activity of
Metabolic Flux hexose phosphate pool is calculated from the pool size and radio-
activity of hexose phosphate. Since hexose phosphate is the major
source of carbon for carbohydrate biosynthesis (Fig. 1), the radio-
activity recovered in sucrose, starch, and cell wall will be derived
from the hexose phosphate pool. Thus, the amount of newly
synthesized molecules within the experimental period can be esti-
mated as hexose equivalent by dividing the radioactivity in those
fractions with specific activity of hexose phosphate. This value is
further divided by the amount of material used for extraction and
by the length of experimental period to calculate metabolic flux
(nmol 1 hexose equivalents g 1 fresh weight h 1). This calculation
is based also on the assumption of equal consumption of cellular
hexose phosphate pools for the synthesis of these molecules [19,
20]. It should be noted that the counts of the fractions must be
normalized to the amount in all starting material since only a part of
fractions are used for further fractionations.
1. The radioactivity respired as CO2 is the radioactivity detected
in CO2 trap.
2. The label in amino acids is calculated from the radioactivity in
the soluble cationic/basic fraction. Since the fraction derived
from 750 μL of 2.0 mL of soluble fraction, the value should be
divided by 0.375.
3. The label in organic acids is calculated from the radioactivity in
the AP anionic/acidic fraction. Since the fraction derived from
150 μL of 2.0 mL of soluble fraction, the value should be
divided by 0.075.
4. The label in hexose phosphate pool is calculated by subtracting

the radioactivity in soluble neutral fraction from that in AP
neutral fraction. Since soluble and AP fractions are derived
from 750 and 150 μL of soluble fraction, the values should
be divided by 0.375 and 0.075, respectively, prior to
subtraction.
5. The label in sucrose is calculated from the radioactivity in the
HK neutral fraction. Since the fraction derived from 200 μL of
3750 μL of soluble neutral fraction, the value should be divided
by 0.02.
6. The label in glucose is calculated from the radioactivity in the
GOX anionic/acidic fraction. Since the fraction derived from
200 μL of 3750 μL of soluble neutral fraction, the value should
be divided by 0.02.
7. The label in fructose is calculated by subtracting the radioactiv-
ity in HK neutral fraction from that in GOX neutral fraction.
Since both GOX and HK fractions are derived from 200 μL of
soluble neutral fraction, the values should be divided by 0.02.
8. The label in protein, starch, and cell wall is corresponding to
the radioactivity in insoluble cationic/basic, neutral, and
anionic/acidic fractions, respectively.
9. Total uptake is calculated as a sum of the radioactivity in all
fractions (i.e., amino acid, organic acid, hexose phosphate,
sucrose, glucose, fructose, protein, starch, cell wall, and
respired CO2 fractions). The metabolized radioactivity is the
sum without glucose fraction assuming ignorable flux of
gluconeogenesis.
10. The recovery of label by fractionation treatment is assessed by
calculating the ratio between radioactivity in 750 μL of soluble
fraction and sum of those in soluble cationic/basic, soluble
anionic/acidic, and soluble neutral fractions.
11. Redistribution of radioactivity in each fraction is calculated as a
percentage of radioactivity in the fraction against the sum of
metabolized radioactivity.
12. Metabolic fluxes into sucrose, starch, and cell wall are calcu-
lated by dividing radioactivity in the corresponding fractions
with specific activity in hexose phosphates (Subheading 3.5,
step 8), fresh weight of material, and incubation time (2 h).
4 Notes
1. Radiolabeled glucose can be purchased from providers such as

American Radiolabeled Chemicals and Perkin Elmer. The
radioactive glucose is diluted with non-labeled glucose to
gain appropriate concentration and specific activity (1.4 MBq

mmol 1). If a product with very high specific activity (like
10 GBq mmol 1) is used, the glucose concentration in the
radioactive solution is very low and can be ignored when
diluting the radioactivity.
2. Here, we describe a procedure for potato tubers but very
similar procedures can be applied for other plant tissues. For
example, three tobacco leaf discs with 10 mm diameter [21],
eight tomato pericarp discs with 10 mm diameter [14],
90–100 mg of tobacco callus [5], 7-day old Arabidopsis etio-
lated seedlings derived from 20 mg of seeds [10], 30–35 discs
of carrot storage tissue (10 mm diameter, 1 mm thickness)
[16], and 300 mg of sliced spadix of Arum have been used in
previous studies [17].
3. Use of 2 mL screwcapped microcentrifuge tubes is recom-
mended to avoid opening during ethanolic extraction steps.
4. Ensure complete mixing of the sample and the scintillation
cocktail.
5. Dowex 50 W, 100–200 mesh and Dowex 1, 100–200 mesh can
also be used as cationic and anionic resins, respectively. In this
case, resins must be treated differently. The cationic resin
Dowex 50 W is washed three times with excess 2 M HCl
followed by washes with H2O until the resin suspension
reaches pH 7.0. The anionic resin Dowex 1 is washed three
times with excess 1 M sodium acetate and subsequent washes
by 0.1 M acetic acid until the resin suspension reaches pH 3.0.
6. Check the pH of the solution after neutralization. The pH of
the solution should be around 9.0. The second reaction does
not work when the pH of the solution is out of the range.
Acknowledgment
This study was supported by DFG (OB438 to T.O.), the European

Commission’s Directorate General for Research within the 7th
Framework Program (FP7/2007-2013 grant no. 270089 [MUL-
TIBIOPRO] to T.O. and A.R.F.), Conselho Nacional de Desen-
volvimento Cientı́fico e Tecnológico (CNPq to L.R.S), and Max-
Planck Society (to A.R.F.). We thank Saleh Alseekh for useful
comments.
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Chapter 3
A Method for Imaging Oxygen Distribution and Respiration

at a Microscopic Level of Resolution
Hardy Rolletschek and Gregor Liebsch
Abstract
Conventional oxygen (micro-) sensors assess oxygen concentration within a particular region or across a
transect of tissue, but provide no information regarding its bidimensional distribution. Here, a novel
imaging technology is presented, in which an optical sensor foil (i.e., the planar optode) is attached to
the surface of the sample. The sensor converts a fluorescent signal into an oxygen value. Since each single
image captures an entire area of the sample surface, the system is able to deduce the distribution of oxygen
at a resolution level of few micrometers. It can be deployed to dynamically monitor oxygen consumption,
thereby providing a detailed respiration map at close to cellular resolution. Here, we demonstrate the
application of the imaging tool to developing plant seeds; the protocol is explained step by step and some
potential pitfalls are discussed.
Key words Respiration mapping, Planar oxygen sensor, Hypoxia, Seed, Optical sensor, Oxygen
imaging, Maize kernel
1 Introduction
The ability to precisely quantify the oxygen content within a partic-

ular living tissue and/or its respiratory activity is relevant to many
physiological studies in biology and life sciences. Conventional
measurement systems can at best provide mean values, which by
definition lack spatial resolution; they are therefore blind to any
compartmentalization that may be present in the sample. Needle-
type microsensors [1–3] partially address this limitation, since they
are able to quantify oxygen levels across a sample transect with a
resolution of several micrometers. These devices operate by driving
the microsensor into the subject in a series of timed steps, using an
(automated) micro-manipulator. In some cases, these measure-
ments are followed up by a destructive assay: the sample is dis-
sected/cut along the transect to relate the measured oxygen
concentrations with each of the structurally distinct zones within
the subject. The major limitation of the needle-type microsensor
31
32 Hardy Rolletschek and Gregor Liebsch
assay is that it is unable to image the bidimensional distribution of

oxygen within a living sample, and it is unsuited to deriving a
dynamic picture of oxygen distribution (i.e., to measure spatiotem-
poral pattern of respiration).
Recent progress has been made in the development of a novel
oxygen sensing approach able to deduce the bidimensional distri-
bution of oxygen concentration [4, 5]. The present chapter
describes a detailed procedure for obtaining a spatially resolved,
quantitative analysis of oxygen distribution and respiration in living
plant tissue. The assay is based on a compact fluorescence
ratiometric-based device, comprising an oxygen-sensitive foil and
a USB microscope. The sensor foil is laid over the sample surface,
and quantifies oxygen consumption (respiration) by detecting
changes in the emitted fluorescence signal. The level of spatial
resolution possible is sufficient to determine respiratory activity at
the cellular (microscopic) level. The necessary equipment has been
available since 2011, and has already been exploited in a number
ways, including the monitoring of respiration at the sediment/
water interface, activity screens of microbe colonies, and the track-
ing of biodeterioration of historical monuments [4, 6–10]. Here, it
has been deployed to map oxygen distribution and respiration in
a developing caryopsis (seed) of maize, the structure and physiol-
ogy of which induces, in vivo, zones of more or less severe
oxygen depletion (hypoxia); the significance of this physiological
state on seed development and crop yield has been reviewed else-
where [11, 12]. The procedure is explained step by step; potential
limitations and pitfalls of the oxygen imaging technology are also
discussed.
2 Materials
2.1 Equipment Three individual VisiSens systems (A1, A2, A3; PreSens GmbH,
Regensburg, Germany) have been optimized for the bidimensional
mapping of, respectively, oxygen, pH, and carbon dioxide. The A1
system (as applied here) consists of a detector unit (DU01), the
software VisiSens AnalytiCal-1 and the sensor foil SF-RPSu4. The
DU01 unit provides a USB interface to enable its connection to a
computer, a CMOS chip, a build-in excitation light source, and
optical filters specifically designed for oxygen detection. The SF-
RPSu4 foil can image oxygen distribution at the sample surface or
through a transparent medium at a concentration range of 0–100%
air saturation (¼ 20.95 vol % ¼ 283,03 μmol/L at 20 C and
atmospheric pressure of 1013 hPa).
After installing the software and connecting the detector unit
to a computer, the system is ready for use. For technical details
regarding camera settings and software, see ref. [13]; installation
instructions and various other resources are provided by the
Respiration Mapping by Planar Sensors 33
manufacturer, see http://www.presens.de/products/brochures/

category/imaging/brochure/imaging-solutions-biological-research.
html#tab-resources. In what follows, a procedure is described which
measures oxygen levels at the surface of a plant sample.
2.2 Reagents/ Two calibration standards need to be prepared: (1) oxygen-free

Calibration Standards water (Cal-0), and (2) air saturated water (Cal-100). To obtain
Cal-0, either dissolve 2 g sodium ascorbate (C6H7NaO6) in
100 mL 100 mM NaOH, or vigorously bubble nitrogen (or
another inert) gas through 50 mL distilled for ~20 min, or dissolve
1 g sodium sulfite (Na2SO3) in 100 mL distilled water. To obtain
Cal-100, vigorously bubble air through 50 mL distilled water for
~20 min, using an air-pump with a glass-frit (air stone). The solu-
tions are stable for at least a week if stored in a filled (to avoid an
oxygen-containing headspace) air-tight flask. All the above reagents
are available from Sigma-Aldrich (https://www.sigmaaldrich.com).
3 Methods
3.1 Focusing and Fix the detector unit to a stand if a long-term measurement is
Calibration intended. Insert the appropriate adapter tube into the top of the
detector unit. (Please note: there are tubes of different lengths
provided to suit the chosen field of view.) Place the green reference
foil plus the transparent length scale over the site where the sensor
foil will later be attached. Use the focusing ring on the detector unit
to ensure that the scale is clearly visible and a sharp image appears.
Capture a single image in order to allow the VisiSens software to
determine the mm/pixel resolution (this depends on the choice of
tube, see above).
Next, replace the reference foil and length scale with the sensor
foil (30 30 mm), making sure that the glossy red back (insensi-
tive) side of the foil (carrying a blue tag) faces the detector unit. To
ensure that the sensor is working correctly, and to enable quantifi-
cation, a calibration procedure is then applied based on the two
calibration standards. Cover the sensor foil with the Cal-100 stan-
dard, wait ~1 min and record a single image. Remove the liquid
and add the Cal-0 standard, wait ~1 min and record a second
image. Clean the sensor foil thoroughly with distilled water or
70% ethanol. Start the Evaluation Module in the VisiSens software
and load the two acquired images. The expectation is that the Cal-
0 standard will generate a high sensor response value (fluorescence
signal), and the Cal-100 standard a low one (note that low values
correspond to high oxygen concentrations)—compare Fig. 1a, b.
Use the Calibration sub-menu to perform the necessary calcula-
tions (see software manual), and establish a calibration curve
(Fig. 1c).
A C 2.4
2.2
2.0
Detector Response
1.8
1.6
1.4
B 1.2
1.0
0.8
0 20 40 60 80 100
oxygen (% air saturation)
Fig. 1 The calibration procedure. (a) Calibration images generated by the sensor exposed to two different
oxygen concentrations. (b) Typical two-point calibration plot generated by the VisiSens software; the plot is
used to convert the arbitrary units produced by the device into the oxygen concentration
A B C D 1.4
detector response (rel. units)

1.2
em 1
0.8
0.6
0.4
0.2
es
0
0 200 400
analysis time (sec)
Fig. 2 Oxygen and respiration mapping. (a) Hand-cut section of an immature maize caryopsis showing the
starchy endosperm (es) and the embryo (em). (b, c) Oxygen distribution across the cut surface of a maize
caryopsis (b) at the start of the experiment and (c) after ~4 min. (d) After selecting an region of interest (ROI;
rectangle marked in (c)), VisiSens software generates a plot showing the temporal changes in fluorescent
signal; this can be interpreted to give the localized rate of oxygen consumption within the selected ROI
3.2 Sample The following sections provide a working example of how the
Preparation and oxygen concentration in a developing maize caryopsis is imaged.
Sensor Attachment to The caryopsis is sliced into two using a razor blade (Fig. 2a).
the Sample Surface Because it is important to ensure close contact between the sensor
foil and the sample surface, a drop of water is placed on the sensor
foil, and the half caryopsis is laid over this. (Depending on the
nature of the sample, water can be substituted by hydrogel, per-
fluorodecaline, or buffer.) Avoid trapping any air bubbles between
sample surface and sensor foil; these should be visible on the live
image produced by the camera.
3.3 Single As soon as the sensor foil has been attached to the subject, record a
Measurements for single image: this provides the initial oxygen concentration across
Oxygen Mapping the sample (Fig. 2b). Respiration of the tissue will deplete the
oxygen present in the liquid medium lying between the sample
surface and the sensor foil, so the expectation is that the measured
oxygen concentration will fall over time, possibly in a nonuniform
manner across the surface. To record localized changes in oxygen
distribution, measurements can be repeated at appropriate intervals
(the length of which depends on the nature of the sample, see
below). Note that the derived oxygen concentration maps do not
necessarily correspond to the situation in vivo, either with respect
to the oxygen level or distribution, because cutting the maize
caryopsis allows the ingress of air to its interior, which necessarily
changes the local oxygen status. Moreover, dynamic changes in
oxygen concentration depend strongly on the balance between
consumption by the tissue and diffusion from outside (parameters
that also can vary for a number of reasons like temperature or air
pressure). Thus, the oxygen concentration and distribution
measured under these conditions reflect specifically the localized
consumption of oxygen.
3.4 Time Series To acquire accurate local oxygen consumption data (respiration
Measurements and mapping), a time series of oxygen images is needed; this is acquired
Data Evaluation for using the function “Time Drive Measurement.” For the maize
Respiration Mapping caryopsis, about 30 images are captured, at a rate of one every
~20 s (total analysis time ~ 7 min; the number and frequency of
the images required depend on how strongly the sample is respir-
ing; it can easily take hours if samples show minor respiratory
activity or just 1 min if activity is high).
Upon the completion of the experiment, start the Evaluation
Module in the VisiSens software and load the complete image
series. Compare the first and last images of the time series (function
“Side by Side”) to determine where there has been a significant
shift in the response: oxygen consumption is indicated by an
increase in the fluorescence signal. If no response is evident, repeat
the experiment using a longer incubation period and lower the
image capture rate. Once a sufficient shift in the fluorescence signal
has been achieved, mark the region of interest (ROI, Fig. 2c) and
calculate the average response value across the full set of uploaded
images. The resulting plot (Fig. 2d) expresses changes over time in
signal intensity in arbitrary units. If the calibration procedure has
been applied beforehand, the signal is converted into oxygen con-
centration. The plot illustrated in Fig. 2d shows that over the first
~200 s, there was a steady, linear increase in signal (¼ oxygen
consumption), but after this time, the signal was saturated. The
later phase corresponds to a situation where most of the oxygen
contained in the intercalary liquid layer between sensor foil and
subject had been consumed. The slope of the line during the linear
phase corresponds to the mean respiratory activity within the

selected ROI. By choosing a set of ROI’s across the surface, a
quantitative, bidimensional map of respiratory activity can be gen-
erated. Note that the unit of measurement is μmol oxygen per unit
area of the sample surface, which is not the conventionally used unit
for respiration (which is rather expressed on a per weight of tissue
basis).
Where the sample’s structure is required in better detail, it is
possible to overlay the native sample image over the fluorescence
image, but this requires first detaching the optical isolation layer
from the sensor foil (see Note 4). This procedure can generate
problems of interference from background signal emitted from
the subject, as exemplified in Fig. 2b: the fluorescence signal from
the maize embryo (arrowed) differs from that emitted by the
endosperm. As the image was taken at the beginning of the experi-
ment, the difference in signal between the embryo and endosperm
is unlikely to reflect a true difference in the rate of oxygen con-
sumption; more likely it involves tissue-specific interference. In
such cases, software such as ImageJ or MathLab needs to be
deployed to subtract the initial image from the subsequently
acquired ones.
Capturing the dynamic behavior of the sample enables the
spatially resolved tracking of respiration; various examples for appli-
cations in plant science are given by Tschiersch et al. (2012). While
this represents a unique feature of the planar sensor system, some
limitations are inherent in the underlying principle. The technique
provides information on oxygen distribution across the sample at a
given time point, but the measured local oxygen concentration
depends on a number of factors and does not necessarily reflect
the steady state concentration in vivo.
4 Notes
1. Various manuals, videos, and application notes are available as a

free download from http://www.presens.de/support/down
load-center/manuals.htmL
2. Carry out all the procedures at room temperature. If the exper-
iment requires a different temperature, first allow all the equip-
ment to pre-equilibrate to this temperature for at least 1 h.
Ensure that calibration constants are applied solely to experi-
ments performed under the same experimental (temperature)
conditions. As the detector unit itself has no integrated tem-
perature sensor, there is no possibility for temperature
compensation.
3. Ambient light can disturb oxygen imaging. For best results, do

not remove the optical isolation, and exclude ambient light as
effectively as possible.
4. The sensitive side of the sensor foil bears an optical isolation
layer that blocks ambient light and excludes interference from
the sample. While it is generally recommended to use the
sensor with this optical isolation enabled, it is possible to peel
off the optical isolation layer. This procedure increases the
spatial resolution of the oxygen image to ~25 μm, and also
allows the possibility of overlaying the fluorescence image with
a native color image (using the “Alpha Blending” function in
the software). Note that removing the optical isolation may
require the recalibration of the sensor foil; it can also introduce
interference due to pigmentation or background fluorescence
of the sample.
Acknowledgments
The author is grateful to H. Tschiersch (IPK-Gatersleben, Ger-

many) for helping with the establishment of the method and for
critical discussions.
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(2009) The methodology and significance of cence 2D in vivo imaging and monitoring of
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4. Tschiersch H, Liebsch G, Borisjuk L, Stangel- 045002
mayer A, Rolletschek H (2014) Imaging 8. Ochs CJ, Kasuya J, Pavesi A, Liebsch G (2014)
microbial culture O2 consumption : metabolic 2D-Visualisierung des zellul€aren Sauerstoffver-
activity of E. coli monitored inside the incuba- brauchs in Mikrofluidiksystemen. BIOspek-
tor with the VisiSens™ A1. Genet Eng Bio- trum 20(7):773–775
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gen.34.14.15 Sanza M (2014) Imaging of oxygen transmis-
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Chapter 4
VisiSens Technique to Measure Internal Oxygen

and Respiration in Barley Roots
Aprajita Kumari and Kapuganti Jagadis Gupta
Abstract
Respiration is vital for production of energy in plants where oxygen is essential for conducting respiration.
Internal oxygen levels in plants depend on respiratory rates. Tissues such as underground roots experience
hypoxia due to their isolation from atmospheric oxygen and their internal oxygen depends on tissue size and
local oxygen in soil and rhizosphere. Here, we used the ViSisens imaging technique, in which an optical
sensor foil (i.e., the planar optode) is attached surface of root. This sensor measures oxygen values from
fluorescence. Visisens microscope captures an entire area root surface and gives profile of internal oxygen.
Interestingly, this system is also able to measure respiratory rate by measuring oxygen levels in a vial that
contains root tissue.
Key words Respiration, Internal oxygen, ViSisens, Oxygen, Energy
1 Introduction
Plants need oxygen for aerobic respiration to generate energy.

Oxygen distribution in the tissue depends on tissue type, stage of
development, and their location. Flooding and waterlogging also
determines internal oxygen of tissue [1]. Root respiration contri-
butes a lot for entire plant development [2] because it determines
internal oxygen levels. Levels of internal oxygen determine meta-
bolic rate. Several lines of evidence suggest that energy required
pathways adapt to actual oxygen availability; therefore, it is essential
to monitor respiration and internal oxygen levels in plant tissues.
Oxygen homeostasis also depends on local oxygen levels [3]. Reac-
tive oxygen species (ROS) are produced as by-products of various
oxygen-dependent metabolic pathways. Several methods are avail-
able for measure of oxygen but each method has several advantages
and disadvantages [4]. In this method sensor foil is placed on a
sample, and any changes in oxygen concentrations can be calculated
using fluorescence signals [5]. The level of spatial resolution possi-
ble is sufficient to determine respiratory activity at the cellular
39
40 Aprajita Kumari and Kapuganti Jagadis Gupta
(microscopic) level. Previously, it was explored to measure oxygen

from various biological and chemical samples such as respiration at
the sediment/water interface [6] activity screens of microbe colo-
nies and measurement of respiration in non-symbiotic hemoglobin
over expressing barley roots [3]. Here, we used VisiSens micro-
scope to monitor oxygen concentrations and respiration from root
tissue of barley. This technique is useful for monitoring distribution
of oxygen in tissues and also for monitoring respiratory rates from
root slices.
2 Materials
2.1 Equipment for Visisens systems is called The A1 system, it contains three compo-
Oxygen and nents (a). a detector unit (DU01) with USB connector to connect
Respiratory to a computer, it also contains an in-built excitation light source
Measurement and optical fibers for oxygen detection (Fig. 1) (b). Software Visi-
Sens AnalytiCal-1 (c). the sensor foil SF-RPSu4.
The sensor foil has the ability to detect oxygen concentration
range from 0 to 100% air saturation. Thermo block should be set to
25 C.
2.2 Reagents/ Two types of calibration standards have to be prepared. (a). For
Calibration Standards establishing zero oxygen add 20 mg of sodium dithionate Na2S2O4
into 200 μL of distilled water and immediately place the drop of this
solution in the center of sensor foil and establish zero oxygen using
software. (b). For establishing 100% in air saturation, bubble
10 mL of distilled water with pure compressed air for 10 min and
then take drop of this air-saturated water and place on sensor foil
and record oxygen concentration and set as 100% using software.
2.3 Materials for 1. Barley seeds.

Plant Growth 2. Sodium hypochlorite 0.1%.
3. Conical flask.
4. Shaker.
5. Pipette and tips.
6. 15 mL Falcon tubes.
7. Hydroponic trays.
8. Air pump.
9. Needles.
10. Razor blades.
11. Plant growth chamber with controlled conditions such as tem-
perature, humidity, and light.
Hydroponic solution contains 1 mM NH4NO3, 250 μM
CaCl2, 100 μM FeEDTA, 1 mM MgSO4, 100 μM H3BO3,
Visisens to Measure Respiration and Internal Oxygen 41
Excitation light
Adapter
USB cable
Sample
Emission light
Sensor foil
Fig. 1 Visisens systems A1 system, contains three components A. a detector unit (DU01) with USB connector
to connect to a computer, it also contains an in-built excitation light source and optical fibers for oxygen
detection. The sensor foil has to be placed on a sample as indicated in the picture for internal oxygen
measurement
1.5 μM CuSO4, 50 μM KCl, 10 μM MnSO4, 0.1 μM

Na2MoO4, 100 μM Na2SiO3, 2 μM ZnSO4, 1 mM KH2PO4.
12. Blotting paper.
13. Petridish.
3 Methods
3.1 Plant Growth 1. Soak healthy barley seeds in autoclaved distilled water overnight.
2. Remove the water and incubate the seeds in 0.1% sodium
hypochlorite NaClO for a period of 10 min by continuous
shaking.
3. Remove NaClO solution and wash the seeds with autoclaved
distilled water for four times.
4. Place the seeds in water-soaked filter paper containing Petridish
for germination for 2 days.
5. Fill 0.5 hydroponic solution in hydroponic trays.
6. Place the lid of hydroponic trays.
7. Place germinate seeds on the holes of hydroponic trays.
8. Grow the plants for 2 weeks in green house.
9. Cut mid portion of the root for oxygen measurement (Fig. 2).
3.2 Calibration 1. Calibration fix the DU01 to a stand.

of Sensor Foil 2. Place the green reference foil plus the transparent length scale
over the site (in this case eppendort tube cap).
Fig. 2 Two week old Barley roots grown on hydroponics
3. Focus on image and record Pixel resolution.

4. Then replace the reference soil with sensor foil.
5. Place a drop of air-saturated water on the top of the sensor and
record image and set as Cal-100.
6. Wipe the sensor foil with soft tissue paper.
7. Place a drop of freshly prepared sodium dithionate Na2S2O4 as
described in Subheading 2.
8. Record image (see Note 1) and set as Cal-0.
9. Once both the images are achieved, using software set the
calibration using scale (Fig. 3).
3.3 Measurement of 1. Take detector until DU01 (Fig. 1) and clean the lens (see Note 2).
Internal Oxygen and 2. For single oxygen measurement hold the adapter with hand
Respiration and for respiratory measurements (long-term oxygen measure-
ment) fix the detector unit to a stand as shown in Fig. 4.
3. Place the manufacturer-supplied green reference foil plus the
transparent length scale over the site of oxygen detection and
O% OXYGEN
100% OXYGEN
Before calibration After calibration
Fig. 3 Calibration images of sensor foil with 0 and 100% oxygen in air saturation
Thermometer
Detection unit
Stand
Sample
Thermo block
Fig. 4 Setup for measurement of respiration from barley roots. All components are indicated in figure
take image for VisiSens software to determine the mm/pixel

resolution.
4. Next, remove reference foil and length scale.
5. Cut sensor foil according to the size of the tissue.
6. Place sensor foil on root and capture image to quantify single
internal oxygen concentration.
7. For the measurement of respiration place Eppendorf tube in
thermoblock to keep temperature constant.
8. Repeat Subheading 3.3 described for pixel detection.
9. Remove reference foil and stick sensor foil 0.5 0.5 on
Eppendorf tube lid.
10. Cut the roots from 3 week old barley plants grown on
hydroponics.
11. Wash roots with autoclaved distilled water four times.

12. Blot the roots on tissue and cut into 0.5 mm slices with a sharp
sterile razor blade.
13. Weigh root slices and place 0.5 g of roots in a 2 mL Eppendorf
tube (Fig. 2).
14. Add 1 mL of HEPES buffer in Eppendorf tube that was placed
(refer to point 7).
15. Close Eppendorf tube and start recoding image every 10 s (see
the setup in Fig. 4).
16. See example measurement of internal oxygen as Fig. 5.
17. See example graph as in Fig. 6.
Fig. 5 Image of internal oxygen levels from barley roots
120
100
(% in oxygen saturation)
Respiratory rate
80
60
40
20
0
0 10 20 30 40 50 60
Time (min)
Fig. 6 Respiratory rate of barley roots. 0.5 g of roots were placed in an eppendorf tube that contains 1 mL of
HEPES buffer and sensor foil was attached to the eppendorf tube and the oxygen image was recorded for every
5 min till 1 h
4 Notes
1. After placing sodium dithionate Na2S2O4, immediately take

the image to be sure that it is not oxygenated.
2. Clean the detector with lens, make sure there is no dust.
Acknowledgments
We thank PreSens Gmbh for providing instrument for the mea-

surement of oxygen, JGK thanks R.G Ratcliffe for laboratory facil-
ities. KJG research was supported by Marie Curie Intra European
Fellowship under FP7 framework. KJG and AK were supported by
Department of Biotechnology, Govt. of India. Work was performed
at University of Oxford and data analysis and manuscript prepara-
tion was done at NIPGR.
References
1. Bailey-Serres J, Voesenek LACJ (2008) Flood- 5. Tschiersch H, Liebsch G, Borisjuk L, Stangel-
ing stress: acclimations and genetic diversity. mayer A, Rolletschek H (2014) Imaging micro-
Annu Rev Plant Biol 59:313–333 bial culture O2 consumption: metabolic activity
2. Armstrong W, Strange ME, Cringle S, Beckett of E. coli monitored inside the incubator with
PM (1994) Microelectrode and modelling study the VisiSens™ A1. Genet Eng Biotechnol News
of oxygen distribution in roots. Ann Bot 34(14):30. doi:10.1089/gen.34.14.15
74:287–299 6. Dutta T, Rubol S (2013) Effect of Spatial Het-
3. Gupta KJ, Hebelstrup KH, Kruger NJ, Ratcliffe erogeneity on Rate of Sedimentary O2 Con-
RG (2014) Nitric oxide is required for homeo- sumption Reaction. In: Pardo-Igúzquiza E,
stasis of oxygen and reactive oxygen species in Guardiola-Albert C, Heredia J, Moreno-Merino
barley roots under aerobic conditions. Mol Plant L, Durán JJ, Vargas-Guzmán JA (eds) Mathe-
7:747–775 matics of Planet Earth, Proceedings of the 15th
4. Chaturvedi P, Taguchi M, Burrs SL, Hauser BA, Annual Conference of the International Associ-
Salim WW, Claussen JC, McLamore ES (2013) ation for Mathematical Geosciences. Springer
Emerging technologies for non-invasive quanti- Verlag, New York, pp 485–489. doi:10.1007/
fication of physiological oxygen transport in 978–3–642-32408-6_107
plants. Planta 238(3):599–614
Chapter 5
MultiSense: A Multimodal Sensor Tool Enabling

the High-Throughput Analysis of Respiration
Peter Keil, Gregor Liebsch, Ljudmilla Borisjuk, and Hardy Rolletschek
Abstract
The high-throughput analysis of respiratory activity has become an important component of many
biological investigations. Here, a technological platform, denoted the “MultiSense tool,” is described.
The tool enables the parallel monitoring of respiration in 100 samples over an extended time period, by
dynamically tracking the concentrations of oxygen (O2) and/or carbon dioxide (CO2) and/or pH within
an airtight vial. Its flexible design supports the quantification of respiration based on either oxygen
consumption or carbon dioxide release, thereby allowing for the determination of the physiologically
significant respiratory quotient (the ratio between the quantities of CO2 released and the O2 consumed).
It requires an LED light source to be mounted above the sample, together with a CCD camera system,
adjusted to enable the capture of analyte-specific wavelengths, and fluorescent sensor spots inserted into the
sample vial. Here, a demonstration is given of the use of the MultiSense tool to quantify respiration in
imbibing plant seeds, for which an appropriate step-by-step protocol is provided. The technology can be
easily adapted for a wide range of applications, including the monitoring of gas exchange in any kind of
liquid culture system (algae, embryo and tissue culture, cell suspensions, microbial cultures).
Key words Seed respiration, VisiSense TD, pH-monitoring, Planar oxygen sensor, Brassica napus
1 Introduction
Quantifying respiration is an important aspect of understanding the

process of germination and seed viability. The standard means of
achieving this is to mount an oxygen (O2) sensor within a sealed
incubation vessel, thereby enabling the measurement over time of
the O2 consumed by an individual seed. Such a setup is not easily
adaptable to a high-throughput scenario, as would be required to
screen a large germplasm collection. A number of first-generation
high-throughput devices have been based on the use of multiple
sensors. One such example, targeted at assessing seed viability, is
the Q2 device marketed by ASTEC (Sheridan, WY, USA), which
tracks a set of individual seeds laid out in a 24-, 48-, or 96-well plate
[1]. An alternative platform is the XF96 Extracellular Flux Analyzer
47
48 Peter Keil et al.
(Seahorse Bioscience, Billerica, MA, USA), which also works from a

96-well plate template and is sensitive down to 1 mg of biological
sample [2]. However, both these systems are restricted to the
detection of O2 and furthermore both their geometry and capacity
have been designed to accommodate small seeds, such as those
produced by species such as tobacco (Nicotiana tabacum), Arabi-
dopsis thaliana), and camelina (Camelina sativa); larger seeds
(which are the hallmark of most crop species) either do not fit the
format or would deplete the available O2 too rapidly, thereby
compromising the fidelity of the assay. In an alternative approach,
Xin et al. (2013) [3] attached microsensors to the seed coat surface
in order to quantify O2 influx, facilitating a real-time and noninva-
sive means of detecting seed viability. This experimental setup
would not be up-scalable without major modifications.
While the tracking of O2 consumption over time is highly
informative in the context of respiration, the quality of the data
could in principle be improved by including an assessment of CO2
release, as this gas is a direct by-product of respiration. In particular,
it would allow for the determination of the respiratory quotient, an
important physiological parameter, representing the quantity of
CO2 released per unit O2 consumed [4]. Tracking CO2 uptake is
a standard procedure in the analysis of photosynthesis, and is typi-
cally managed using an infrared gas analyzer. However, its quantifi-
cation in the liquid phase is less straightforward on account of its
pH-dependent solubility. To make quantification of CO2 concen-
trations accurate, one therefore needs to monitor also the pH of the
liquid (incubation buffer) which might change over time due to
release of acidic compounds or similar.
Here, the so-called MultiSense tool is described. The concept
was based on extant VisiSens TD equipment (PreSens Company,
Regensburg/Germany), and was intended to provide a means to
simultaneously monitor the concentrations of O2 and CO2 concen-
tration in a high-throughput manner. Its working principle is based
on the imaging of fluorescent planar sensor foils placed inside
individual, airtight vials. Quantification of the analytes’ concentra-
tions relies on the ratiometric principle, as described elsewhere
[5–7]. Depending on the type of sensor foil deployed, either
CO2, O2, or pH can be monitored, and with some modification,
even all three analytes can be monitored simultaneously. Here, a
detailed procedure is presented to enable the quantification of
respiratory activity of imbibing seeds. While the method is focused
on the high-throughput analysis of seed respiration, the device can
also be used to measure respiration in any other plant organ/tissue.
Notably, the device could equally be adapted to characterize the
bidirectional distribution of O2, CO2, and/or pH in the root
system [8], across the leaf or seed [2, 9] or for the spatial monitor-
ing of microbial activity [5, 10].
Multimodal Sensor Tool 49
2 Materials
2.1 Equipment The equipment consists of several components, which can be sup-
plied as a package from PreSens Precision Sensing GmbH (Regens-
burg, Germany; www.presens.de). The system comprises a cage to
house the camera, a series of LEDs, and several sample racks
(Fig. 1a, see also Note 1). We installed two separate imaging systems
(digital camera, lens, optical filters, and LED light source) for data
capture. One is responsible for detection of gaseous O2, based on the
appropriate excitation light source and optical excitation and emis-
sion filters for reading out the oxygen sensors; the other measures pH
and/or CO2 in the liquid phase, and is equipped with the respective
components for reading out pH and/or CO2 sensors. The system
also comprises so-called mode operation units which are essential to
run the system, and consists of a power supply, strobe controller
(LED flash control), function generator (synchronizes camera
and LED light sources), and an Ethernet switch (controls Ethernet
camera). The mode operation units are connected by several
cables namely BNC trigger cable, Ethernet cables, and power cables.
Fig. 1 Multisensor platform. (a) Overview. The cage (1) houses the two cameras (2, 3) and a sample tray (4).
Sensor foils are inserted into each glass vial (5) along with the sample (e.g., seed). The foils are excited by LED
light of a specific wavelength. Dynamic concentration changes in O2 and/or CO2 and/or pH are obtained from
emitted signals, captured by the camera system. (b–d) Each glass vial is made gastight by the seal ring (red),
and is equipped with sensor foils (arrowed) attached to the cap (or bottom) of vial
The equipment set is computer-controlled by the software package

“VisiSens VS” (PreSens). See also Note 2.
2.2 Reagents For O2 calibration, two standards need to be prepared: (1) O2-free
and Calibration tap water (O2-Cal-0) and (2) air (O2-Cal-100; 20.95% O2
Standards (8.69 mM) at 20 C and atmospheric pressure of 1013 hPa). To
prepare (1), either dissolve 1 g Na2SO3 in 100 mL distilled water or
vigorously bubble nitrogen or another inert gas through 100 mL
distilled water.
To calibrate pH, six standards over the range pH 6–7.5 are
required. Prepare stock solutions of both 2 M Na2HPO4 and 2 M
NaH2PO4 and mix them to form a phosphate buffer (the appropri-
ate proportions of the mixture are given in Table 1). Alternative
mixing ratios/buffers can be found in [11]. If necessary, make fine
adjustments to the pH using NaOH or HCl.
To calibrate CO2, four standards are prepared over the range of
1–25% (equivalent to 0.33–8.39 mM). Make a 0.2 M solution of 2-
(N-morpholino)ethanesulfonic acid (MES) and adjust pH to 6.37
using 1 M HCl (note: at this pH, the concentrations of CO2 and
HCO3 are equal). Add the appropriate quantity of NaHCO3 to
each standard vial (see Table 2), fill with MES buffer, and seal the
Table 1
Mixing ratios to be used to prepare pH standards. Values are given in %
(v/v) of 2 M solutions of Na2HPO4 and NaH2PO4
pH NaH2PO4 Na2HPO4
6.0 87.7 12.3
6.3 77.5 22.5
6.6 62.5 37.5
6.9 45.0 55.0
7.2 28.0 72.0
7.5 16.0 84.0
Table 2
The quantity of NaHCO3 and volume of 0.2 M MES required for preparing
the CO2 calibration series
NaHCO3 [mg] CO2 final [mM] MES buffer [mL]

0 0 24
5.04 1.25 24
10.08 2.5 24
20.16 5 24
vial. The CO2 calibration standards need to be measured as quickly

as possible as the solution is unstable. Thus it is important not to
allow the passage of more than 5 min between preparing the
calibration standard and measuring it. Alternatively, one can use
premixed gases with known (customized) CO2 concentrations
(http://www.airproducts.com/products/Gases.aspx).
2.3 Additional l 25 mL Screw-top glass vials: The caps include a silicone O-ring
Supplies and a glass septum, and can be purchased as item #9502-4906
from Latek Labortechnik & Analysen GmbH (Eppelheim, Ger-
many; www.latek.de).
l Sample racks, which can be ordered directly from PreSens
GmbH.
l Sensor foils for O2 (SP-RPSu4-NAU-D5-OIW or SP-RPSu4-
NAU-D5-SA; see Note 3), pH (SF-HP5R) (range pH 6–7.5),
and CO2 (SF-MT1R) (range 1–25%); all the above can be
ordered directly from PreSens GmbH; O2 sensors work in
both liquid and gaseous phases; sensors for CO2 and pH work
correctly only in liquid phase.
l Pipettors (100–1000 μL) with the appropriate tips.
3 Methods
3.1 Preparation 1. Check whether the system has been set up for the correct
of the Multisense analyte: the upper camera /violet LEDs are used for O2 detec-
System tion and the lower camera/blue LEDs for that of pH and CO2.
The cameras and LEDs need to be connected to, respectively,
the function generator and the LED strobe controller (adjust
the cables accordingly).
2. Switch on the devices in the following order:
l Power supply (first the power switch, then the output
switch; if necessary, rotate the CV adjuster to 20 V).
l Strobe controller.
l Function generator (first the main power switch at the rear,
then the power switch).
l Ethernet switch.
3.2 Sample 1. Insert the sensor foils into the glass vial: the one used for
Preparation measuring O2 should be positioned within the cap (Fig. 1b,
c), while the one used for measuring pH and CO2 should be
positioned at the bottom of the vial (Fig. 1d). Note that all foils
are provided with a self-adhesive layer.
2. Weigh the biological samples (dry mature seeds) and place into
a glass vial. Add 0.5–1 mL tap water or chosen buffer (see Notes
4–6). Screw on the cap to hand-tightness.
3. Prepare the calibration solutions as described above, pour an
aliquot into a glass vial, and screw on the cap to hand-tightness.
4. Position each vial in its allocated spot on the rack and insert the
racks into the system (Fig. 1a).
3.3 Initiating 1. Launch the “VisiSens VS” software and select from the “Cam-
Measurement era List” the appropriate camera (upper one for O2, lower one
for pH/CO2).
2. Enter “Measurement Mode,” and select the appropriate ana-
lyte button; then create a new Session (comments can be
included if desired).
3. Select “Default” settings and start a live preview by clicking
on “ ”.
(a) Select “camera settings”: 70 mA for all light sources and
30 ms (¼30,000 μs) for exposure time. See Note 3.
(b) Select “Save Current Settings,” and then “Close Live
Preview.”
4. Start the automatic recording procedure by selecting “ ” with
a user-selected time interval (>10 s).
5. To generate a snapshot image, select live preview “ ” and
“Snapshot.” See Note 7.
3.4 Terminating The system will terminate the recording procedure automatically
Measurement and when the selected time interval has expired.
Evaluating the Data
1. Press the IDL software analysis button “ ” or double-click on
any image on the left side of window.
2. Once the IDL software has launched, upload all the created
files to be evaluated by selecting “ ”; if more than one
image is to be analyzed, select “Series.”
3. Select the first image and enter the number of images to be
opened.
3.5 System To calibrate the system, select “ ” and click on the value of
Calibration column “Ratio.”
1. Select “ROI for Cal1.”
2. Select a pixel within the image of the vial containing the chosen
calibration solution (Cal1).
3. Define a region of interest (ROI) by a left click and hold of the
mouse and confirm the selection with a right click of the
mouse.
A Oxygen-Calibration B 2.0
pH-Calibration C Carbon dioxide-Calibration
4.0
3.0
Ratio R/G [-]
Ratio R/G [-]

1.5 R² = 0.9968 3.0
Ratio R/G [-]
2.0
1.0 2.0
1.0 0.5 R² = 0.9223
1.0
0.0 0.0 0.0

0 10 20 30 5 6 7 8 9 0 10 20
O2 [vol%] pH CO2 [vol%]
Fig. 2 Typical calibration curves. (a) The concentration of O2, (b) the pH, (c) the concentration of CO2. The
y-axis indicates the calculated ratio of red and green light emission for each calibration standard
Fig. 3 Fluorescence image (raw dataset) produced by the multisensor platform. The image shows 7 10 foils
(spots) corresponding to 70 glass vials, each containing ten imbibing B. napus seeds. Spot color (see color
scale on the right) corresponds to the O2 concentration obtained within each airtight glass vial. Images
captured at (a) the beginning of imbibition, (b) after 35 h
4. Repeat the procedure for each of the calibration standards and

select “Calibrate” when finished. Some typical for O2, CO2,
and pH calibration curves are shown in Fig. 2.
3.6 Typical The following describes an experiment involving the 70 accessions

Germination included in an oilseed rape (Brassica napus) diversity panel [12],
Experiment selected from the IPK Genebank collection. Ten mature seeds per
with Plant Seeds accession were placed within a single glass vial, 0.5 mL tap water
was added, and the vial was gently shaken to ensure the wetting of
all the seeds. The vials were sealed, placed in sample racks, and
positioned under the camera system. To continuously monitor the
O2 concentration inside the vials, images were captured at a rate of
one per hour over 35 h. The initial and final images are shown in
Fig. 3, where each spot represents one vial and the color intensity
reflects the O2 concentration. The analyte (O2) content was then
calculated for each vial by selecting “Z-Profile” “ ” in the Visi-
Sens software package. After selecting an ROI for each vial (see
above), the data were saved in a named folder. The resulting files
were exported into Excel (this step currently needs to be carried out
100 20
90
80 16
70
qs [%O2/gBM/h]
Oxygen [%]
60 12
50
40 8
30
20 4
10
0 0
0 10 20 30
Time [h]
Fig. 4 Typical output of a germination experiment. The O2 concentration (blue

circles) was measured in vials containing ten B. napus seeds. The specific O2
uptake rate (qs) is shown by orange circles
for each spot separately, but the next version of VisiSens software
will incorporate a facility to export multiple files in batch mode).
After specifying the time interval (one per hour), both the O2
conversion rate (rs) and the O2 uptake rate (qs) were calculated,
where rs ¼ (Δ analyte concentration [%])/(Δ time interval [h]) and
qs ¼ rs/biomass [g]. The best results were obtained by using a
moving average to calculate both parameters. Typical outputs for
both the O2 concentration and O2 qs during the course of imbibi-
tion are shown in Fig. 4. The O2 concentration declined slowly over
the first ~15 h, after which it declined more strongly in a linear
fashion (blue circles in Fig. 4). This behavior was mirrored by that
of qs (orange circles in Fig. 4). The rate of O2 consumption corre-
sponded well with published values [13].
4 Notes
1. Two different O2 sensor foils are available. The stated camera

settings (70 mA light intensity and 30 ms exposure time) have
been optimized for foils with optical enhancement layer (SP-
RPSu4-NAU-D5-OIW). The use of sensor foils without this
layer (SP-RPSu4-NAU-D5-SA) requires an adaptation of cam-
era settings. Always apply independent calibrations for these
two sensor types.
2. Any light reflections which may occur will only influence mea-
surement accuracy if they fall on the sensor foil. If this is a
problem, then one solution is to reduce the light intensity and
another is to reposition the LED lights. Note that reducing the
light intensity may result in those vials placed towards the end
of the rack receiving insufficient illumination. However, the
ratiometric principle ensures that heterogeneity in light inten-
sity should not affect the measurement.
3. Ensure that the sample vials are of a size appropriate for the size
of the seed to be analyzed. The 25 mL vials used for the B.
napus experiment were selected because the seed is small (dry
weight <10 mg) and their respiration rate was expected to be
low during the early phase of imbibition. The risk is that too
large a head space above the sample (the volume of gas in the
vial) would render O2 uptake during this phase lying below the
level of detection. For larger seeds (such as maize, pea), it is
feasible to use one seed per vial. Seeds expand during the
germination process, so when several seeds are present in a
single vial, they may end up affecting one another through
contact, which is undesirable.
4. To ensure successful imbibition, sufficient water must be
provided, but it is important to avoid submergence of the
seed, as this inhibits gas diffusion and can induce O2 depletion
(hypoxia). Care should be taken to provide each vial with the
same volume of water. If the seeds vary in weigh, it might be
better to adjust the amount of water to be the same on a per mg
DW basis. However, this will affect the size of gas headspace
and needs probably to be taken into account.
5. If microbial contamination is a potential problem, it is possible
to surface-sterilize the seed, following a standard protocol
using hypochlorite [14]; for example, immerse the plant mate-
rial in 1.0% sodium hypochlorite solution for 10–20 min. Care
must be taken to avoid any damage to the seed’s interior due to
phytotoxicity.
6. In its current form, we used sample racks for up to 100 samples
and glass vials of 25 mL volume. Depending on individual
needs, other racks for more samples and smaller vials can be
used, enabling a higher throughput, and better adjusted to
small sample volumes.
7. The “MultiSense” tool can be easily adapted to custom-specific
needs with respect to vial (sample) size, vial number, or type of
analyte detected. In particular, it can be used for measuring any
type of organisms growing in liquid media: in vitro tissue
culture in biomedical research [15] including cultivation of
tumor spheroids [16]; mycelium proliferation in liquid culture
and bacterial growth [17, 18]; in vitro culture of any plant
material like developing plant embryos [12]; and embryo res-
cue cultures [19].
Acknowledgements
This research was financially supported by the German Plant Phe-

notyping Network (DPPN).
References
1. Van Asbrouck J, Taridno P (2009) Using the 10. Tschiersch H, Liebsch G, Borisjuk L et al
single seed oxygen consumption measurement (2014) Imaging microbial culture O2 con-
as a method of determination of different seed sumption. Genet Eng Biotechnol News 34:30
quality parameters for commercial tomato 11. Stoll VS, Blanchard JS (2009) Buffers:
seed samples. Asian J Food Agro-Ind 2: principles and practice. Methods Enzymol
S88–S95 463:43–56
2. Sew YS, Ströher E, Holzmann C et al (2013) 12. Schwender J, Hebbelmann I, Heinzel N et al
Multiplex micro-respiratory measurements of (2015) Quantitative multilevel analysis of cen-
Arabidopsis tissues. New Phytol 200:922–932 tral metabolism in developing oilseeds of Bras-
3. Xin X, Wan Y, Wang W et al (2013) A real-time, sica napus during in vitro culture. Plant Physiol
non-invasive, micro-optrode technique for 168:828–848
detecting seed viability by using oxygen influx. 13. Borisjuk L, Neuberger T, Schwender J et al
Sci Rep 3:3057 (2013) Seed architecture shapes embryo
4. Millar A.H, Siedow J.N., Day D. (2015) Respi- metabolism in oilseed rape. Plant Cell
ration and photorespiration. In: B.B. Buchanan, 25:1625–1640
W. Gruissem, and R.L. Jones. Biochemistry and 14. Sauer DB, Burroughs R (1986) Disinfection of
molecular biology of plants, 2, John Wiley & seed surfaces with sodium hypochlorite. Phy-
Sons Ltd, New York pp. 610–655. topathology 76:745–749
5. Meier R, Schreml S, Wang X-D et al (2011) 15. Lin RZ, Chang HY (2008) Recent advances in
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pH in vivo using sensor films. Angew Chem ture for biomedical research. Biotechnol J 3
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6. Wang X-D, Meier RJ, Link M et al (2010) 16. S W, Doetsch J, Mueller-Klieser W, Kunz-
Photographing oxygen distribution. Angew Schughart LA (2000) Metabolic imaging in
Chem Int Ed 49:4907–4909 multicellular spheroids of oncogene-
7. Wang X-D, Meier RJ, Wolfbeis OS (2013) Fluo- transfected fibroblasts. J Histochem Cytochem
rescent pH-sensitive nanoparticles in an Agarose 48(4):509–522
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metabolism. Angew Chem Int Ed 52:406–409 (1995) Development peculiarities of
8. Blossfeld S, Schreiber CM, Liebsch G et al mycelial structures of Thielavia terrestris
(2013) Quantitative imaging of rhizosphere under batch conditions. Biopolym Cell 11
pH and CO2 dynamics with planar optodes. (3–4):73–81
Ann Bot 112:267–276 18. Chang ST, Miles PG (2004) Cultivation, nutri-
9. Rolletschek H., Liebsch G. (this issue). A tional value, medicinal effect and environmen-
method for imaging oxygen distribution and tal impact of mushrooms. CRC Press, Boca
respiration at a microscopic level of resolution. Raton, pp 73–77. ISBN 9780849310430
MiMB 19. Shen X, Gmitter FG Jr, Grosser JW (2011)
Immature embryo rescue and culture. Meth-
ods Mol Biol 710:75–92
Chapter 6
Measurement of Respiration and Internal Oxygen

in Germinating Cicer arietinum L. Seeds Using
Optic Microsensor
Sonika Pandey, Aprajita Kumari, Chellapilla Bharadwaj,
and Kapuganti Jagadis Gupta
Abstract
Internal oxygen concentrations vary in different tissues depending on tissue size, developmental stage, and
their location. Respiratory rate of tissue also determines internal oxygen levels. For studying various
signaling pathways it is essential to establish a correlation between respiration and internal oxygen. Seed
germination is associated with increase in respiration which can dictate the internal oxygen and subsequent
production of reactive oxygen species. Using optic oxygen microsensor we made an attempt to measure
respiratory rate and internal oxygen. We found that microsensor is able to sense internal oxygen and it is also
possible to measure oxygen levels in a close vial that contains seeds. Step-by-step protocol is described here
along with illustration.
Key words Respiration, Internal oxygen, Microsensor, Germination, Seed
1 Introduction
Bulky tissues and non-photosynthetic tissues often experience hyp-

oxia. Stress conditions such as flooding or water logging also impose
hypoxia to plant organs such as underground roots and germinating
seeds [1–3]. Two parameters contribute for hypoxic atmosphere in
these organs: (1) restricted capabilities of oxygen diffusions and (2)
high rates of cellular metabolism especially central metabolism [4].
These two processes lead to hypoxia and eventually that leads to
anoxia which can be detrimental due to energy crisis. Previous
studies revealed that various seeds have different internal oxygen
concentrations in a range from 2 to 10 μm [4]. In germinating seeds
photosynthetic oxygen helps to prevent seeds to become hypoxic
[5]. Recently it has been shown that tight control of respiration is
important for maintaining the internal oxygen concentration in
roots [4, 6]. Another important control of respiration is the free
57
58 Sonika Pandey et al.
radicals such as nitric oxide (NO) that are produced under hypoxia
and are able to inhibit respiration. For instance under hypoxia plants
produce a high amount of NO [7]. Oxygen homeostasis is deter-
mined by production of nitric oxide which is an inhibitor of cyto-
chrome c oxidase. Scavenging of nitric oxide leads to increase in
respiratory rate and drop in internal oxygen levels suggesting an
inverse correlation between respiration and internal oxygen [6].
Reactive oxygen species are by-products of metabolism, and if pro-
duced in excess they damage cell membrane and damage protein
and nucleic acids. Hypoxic stress leads to increase in ROS produc-
tion [8], and increased respiration can decrease internal oxygen and
increase ROS. In order to improve germination rates first one needs
to understand respiratory metabolism and oxygen concentrations.
Here we used C. arietinum as a model to determine respiration and
internal oxygen concentrations using oxygen microsensor. This is an
electrode-based sensor which functions on the basis of oxidation
and reduction via diffusion of oxygen.
2 Materials
1. Two different varieties of healthy C. arietinum seeds: (a) Desi

and (b) Kabuli.
2. 100 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesul-
fonic acid)).
3. 0.1% Mercuric chloride HgCl2.
4. TBR1025 single-channel free radical analyzer (World Precision
Instruments, Sarasota, Florida, USA).
5. Oxygen electrode.
6. Temperature sensor.
7. Nitrogen gas.
8. Air pump.
9. Syringe.
10. Rubber tube.
11. 10 mL G.C. vial with rubber stopper wrapped with aluminum
lid (Chromacol Ltd., UK).
13. Parafilm.
14. Cheese cloth.
15. Camera.
16. Shaker.
17. Autoclave.
18. Distilled water.
19. Beaker.
Measurement of Respiration and Internal Oxygen in. . . 59
3 Methods
3.1 Seed 1. Take healthy seeds (see Note 1) and incubate in 0.1% HgCl2 for
Germination a period of 10 min by shaking the seeds using a shaker rotator
at 50 rpm/min. Immediately after incubation, rinse the seeds
with autoclaved distilled water. Repeat the washing three to
four times.
2. Soak the sterilized seeds in autoclaved distilled water for 4 h.
3. Remove the seeds from water and wrap it in wet cheese cloth
(Fig. 1).
4. Keep the wrapped seeds in the dark overnight.
Fig. 1 Germination procedure for C. arietinum. (a) Desi variety on damp cheese cloth. (b) Kabuli variety on
damp cloth. (c) Desi or Kabuli seeds wrapped in cheese cloth for overnight incubation in the dark
3.2 Calibration of 1. Clean sensor with distilled water.

Oxygen Sensor: 2. Establish zero oxygen concentration by flushing pure nitrogen
Perform Calibration gas into 5 mL of water and insert oxygen sensor and using
Through the Following software establish zero oxygen.
Steps 3. For 100% oxygen concentration flush 5 mL of distilled water
with air for 10 min and insert sensor and save values as 100% in
software.
3.3 Measurement of Use TBR1025 single-channel oxygen sensor. The sensor carries a
Seed Respiration platinum electrode and a silver electrode (as reference). These two
are embedded in stainless steel sleeve. At the end of the sleeve a gas-
diffusible polymer membrane is fitted which allows only oxygen.
The available oxygen diffuses through the membrane which is then
reduced at the platinum cathode at 0.7 V. Diffusion of oxygen
leads to change in current and the magnitude of current is deter-
mined by the rate of diffusion. The amount of current generated is
proportional to the partial pressure of oxygen (Fig. 2).
Arrange the instrument setup as per Fig. 3a. For measurement
of respiration transfer approximately 3 g of imbibed seeds to 10 mL
vial using sterile forceps. Add 8 mL of 25 mM HEPES pH 7.2
buffer to the vial using pipette. Place the lid on the top of glass vial.
Seal the lid tightly using septa vial sealer. Pierce the lid using a
needle and flush with ambient air for 5 min. Remove the needle and
place the sensor carefully into the vial (refer to Note 2). Start
recording change in the output current. Example of measurement
result is shown in Fig. 4. Calculate the rate of oxygen consumption
by subtracting initial values (see Note 3) and final values.
Pair of electrodes (working & reference)
Probe handle
Gas permeable polymeric membrane
Electrode pair embedded in stainless steel sleeve

Cap for connecting sleeve to handle of probe
Fig. 2 Structure of oxygen sensor. Description of various components is indicated in the figure
Measurement of Respiration and Internal Oxygen in. . . 61
Fig. 3 (a) Setup for measurement of respiration from C. arietinum seeds. (b) Setup to measure internal oxygen
concentration in C. arietinum seed. Various components of setup are indicated in the figure
100
90
80
Oxygen concentration
(% in air saturation)
70
60
50
40
30
20
10
0
0 10 20 30 40 50 60
Time (min)
Fig. 4 Respiratory oxygen consumption from C. arietinum seeds. Approximately 3 g of seeds was placed in the
vial and oxygen consumption was recorded for a period of 60 min
3.4 Measurement of Using a micromanipulator calculate the approximate diameter of

Internal Oxygen seed. Connect oxygen sensor to micromanipulator. Using the scale
Concentration insert oxygen sensor steel part into seed where oxygen concentra-
tions are to be determined (see Note 4). Release sensor once the
approximate depth is reached. Record oxygen concentration imme-
diately. Example oxygen concentration is shown in Fig. 5.
4 Notes
1. Make sure that all seeds are approximately of same size and
healthy.
Fig. 5 Measurement of internal oxygen concentration. Red line indicates the

depth of seed. Yellow text indicates the depth where oxygen measurement was
taken and levels of oxygen (% of air saturation) in each depth are indicated in the
figure
2. Make sure that while inserting vial extreme care is taken to

ensure no damage to the sensor tip.
3. To avoid fluctuation try to omit initial 10-min values.
4. Insert oxygen sensor slowly into the seed with low pressure.
Acknowledgements
This work was supported by NIPGR short term research fellowship

to S.P., and IYBA to A.K. and K.J.G. from Department of
Biotechnology.
References
1. Armstrong W, Br€andle R, Jackson MB (1994) storage in developing soybean seeds. New Phy-
Mechanisms of flood tolerance in plants. Acta tol 167:777–786
Bot Neerl 43:307–358 6. Gupta KJ, Hebelstrup KH, Kruger NJ, Ratcliffe
2. Crawford R, Br€andle R (1996) Oxygen depriva- RG (2014) Nitric oxide is required for homeo-
tion stress in a changing environment. J Exp Bot stasis of oxygen and reactive oxygen species in
47:145–159 barley roots under aerobic conditions. Mol Plant
3. Drew MC (1997) Oxygen deficiency and root 7:747–750
metabolism: injury and acclimation under hyp- 7. Gupta KJ, Stoimenova M, Kaiser WM (2005) In
oxia and anoxia. Annu Rev Plant Physiol Plant higher plants, only root mitochondria, but not
Mol Biol 48:223–250 leaf mitochondria reduce nitrite to NO, in vitro
4. Borisjuk L, Macherel D, Benamar A, Wobus U, and in situ. J Exp Bot 56:2601–2609
Rolletschek H (2007) Low oxygen sensing and 8. Vergara R, Parada F, Rubio S, Pérez FJ (2012)
balancing in plant seeds – a role for nitric oxide. Hypoxia induces H2O2 production and activates
New Phytol 176:813–823 antioxidant defence system in grapevine buds
5. Rolletschek H, Radchuk R, Klukas C, Schreiber through mediation of H2O2 and ethylene. J
F, Wobus U, Borisjuk L (2005) Evidence of a Exp Bot 63:4123–4131
key role for photosynthetic oxygen release in oil
Chapter 7
Using an Oxygen Microsensor to Measure Oxygen Dynamics

in Tomato Plants in Response to Pseudomonas syringae
Infection
Pradeep Kumar Pathak and Kapuganti Jagadis Gupta
Abstract
Pathogen infection leads to induction of defense responses which includes modulation of gene expression
and changes in metabolism plants. Despite of extensive research little is information known about the role
of respiration and photosynthesis during pathogen infection in plants. Limited methods are available to
measure oxygen dynamics in response to pathogen infection. Here by using an oxygen microsensor we
measured oxygen changes in tomato plants infected with avirulent Pseudomonas syringae pv. tomato
DC3000. In this method plant is placed in a closed chamber and change in oxygen levels can be measured
by an oxygen sensor.
Key words Oxygen sensor, Pseudomonas, Respiration, Tomato, Metabolism
1 Introduction
Plant pathogens are ubiquitous in nature and studying their inter-

action with plants is very important in order to understand molec-
ular mechanism to generate resistant crops. Continuous interaction
of pathogens with plants lead plants to developed immunity against
most of the pathogens during evolution. Both innate and induced
defenses aid plants to develop resistance [1]. For example pre-
formed barriers such as specific structural component of cell and
metabolites help in primary defense against pathogen invasion [1,
2]. In contrast to preformed barriers, induced defense response is
triggered via pathogen-associated molecular pattern (PAMP) [3].
For instance elicitors secreted by pathogens are translocated into
the plant cell and these are recognized by receptors present in plant
cell [4, 5]. Despite of extensive research very little information is
known about the changes that take place in respiration and photo-
synthesis during pathogen attack. In response to pathogen attack
many changes take place in plants that include change in ion fluxes,
63
64 Pradeep Kumar Pathak and Kapuganti Jagadis Gupta
posttranslational modification of proteins, and generation of sig-

naling molecules such as salicylic acid, nitric oxide (NO), jasmonic
acid, and ethylene, and production of reactive oxygen species are
activated, which can aid in shift of metabolism toward the defense.
Plant resistance mechanisms also depend on nutrient conditions
and modulation of several metabolic processes such as photosyn-
thesis and respiration [6]. During pathogen attack plants alter their
metabolism toward synthesis of defense-related metabolites [7]. In
the previous studies it was found that genes involved in photosyn-
thesis and chloroplast biogenesis were downregulated in response
to pathogen attack [8–13].
During the pathogen attack plants resist themselves via devel-
opment of programmed cell death called hypersensitive response
(HR). Salicylic acid and nitric oxide (NO) are known to induce cell
death during HR development. NO that is produced during HR
can inhibit cytochrome c oxidase and can also induce alternative
oxidase [14].
Previously it was shown that photosynthesis is downregulated
while respiration rate is upregulated in response to pathogen infec-
tion. For instance Salvador et al. [15] used infrared gas analyzer to
measure respiration in attached leaf of tomato during Fusarium
oxysporum infection while Berger et al. [9] used Imaging-PAM to
measure photosynthesis rate in response to Pseudomonas syringae
and Botrytis cinerea infection. Here we used oxygen microsensor to
measure oxygen dynamics during 24 h early stages of infection with
Pseudomonas syringae pv. tomato DC3000. We found decreased
respiratory rate in response to early stage of Pseudomonas infection
for the development of HR. This method is rapid, sensitive, and
noninvasive in nature.
2 Materials and Equipment
1. King’s B medium (KB).

2. Weighing balance.
3. Autoclave.
4. Laminar airflow.
5. Pipetman.
6. Rifampicin.
7. P. syringae DC3000.
8. Culture tubes.
9. Conical flask.
10. Incubator (28 C).
11. Centrifuge.
12. Oak ridge tube.
Oxygen Dynamics in Tomato Plants 65
13. Cuvette.
14. Spectrophotometer.
15. MgCl2.
16. Pathogen infection room.
17. Tomato plants (4 weeks old).
18. 2 mL Syringe (without needle).
19. Nitrile gloves.
20. Chamber.
21. Beaker.
22. Oxygen sensor.
23. Parafilm tape.
24. Scissor.
25. Hoagland media.
26. Light source.
27. TBR1025 Instrument.
3 Methods
3.1 Plant Growth 1. Take healthy seeds of tomato (Solanum lycopersicum; cv PR)
and grow in Soilrite.
2. After 4–5 days of germination, transfer plantlets in plastic pots
(10 10 cm2) with composition of Soilrite and agro peat (1:1).
3. Grow the plants in phytotron with 16/8-h day/night photope-
riod along with day/night temperature regimes of
26 C/18 C.
4. Water the plants thrice in a week with half-strength Hoagland
media.
5. After 4 weeks transfer the plants in hydroponics supplemented
with Hoagland media and allow for acclimatization (2–3 h)
prior to pathogen infection.
3.2 Bacterial 1. For preparation of primary bacterial culture, inoculate single

Inoculation colony of P. syringae pv. tomato DC3000 in 3 mL of King’s B
liquid medium (KB) supplemented with antibiotic rifampicin
(50 mg/L) and keep overnight at 28 C in incubator shaker at
200 rpm speed.
2. Take 1% of primary culture and inoculate in 100 mL of fresh
media that is supplemented with the same antibiotic. Grow the
culture till OD600 and set OD to 0.6.
3. Thereafter, centrifuge the culture at 3000g for 10 min at 25 C.
4. Discard the supernatant, resuspend the pellet in 10 mM MgCl2

until OD600, and set the OD to 0.2.
5. Infiltrate tomato plant with pathogen suspension through
abaxial side of leaf using 2 mL syringe (without needle).
3.3 Measurement of 1. First calibrate the TBR1025 single-channel oxygen sensor. To

Oxygen Concentration establish 0 oxygen bubble 10 mL of distilled water with 100%
nitrogen gas and dip the sensor and record with instrument in
calibration settings. For establishing 100% oxygen dip the sen-
sor in air-saturated water and set the value in calibration set-
tings of software (see Note 1).
2. TBR1025 single-channel oxygen sensor is made up of platinum
electrode and a silver counter/reference electrode embedded
in a sleeve made up of stainless steel (see Note 2). At the end of
oxygen sensor, a permeable membrane is set that allows oxygen
to diffuse while inhibiting ions, fluid, and other particles to pass
through it. When oxygen diffuses through this permeable
membrane, it is reduced at platinum cathode resulting in gen-
eration of electric current which is measured as partial pressure
of oxygen (Fig. 1).
3. For measurement of oxygen concentration, use oxygen sensor
as shown in Fig. 1.
Fig. 1 Setup of TBR1025 single-channel oxygen sensor for measurement of respiration in tomato plant.
Various components for measurement of oxygen concentration are indicated in the figure
Fig. 2 An Illustration of oxygen-sensing equipment TBR1025 and sensor. Various components are indicated in
the illustration
4. Place the tomato plant in acrylic chamber and insert oxygen

microsensor into the chamber as shown in Fig. 2.
5. Seal the lid completely using parafilm and record the change in
oxygen level after every 5-min interval till 3 h as shown in Fig. 3
(see Notes 3 and 4).
200
Control Pathogen
180
Oxygen (% in air saturation)

160
140
120
100
80
60
40
20
0
0 20 40 60 80 100 120 140 160
Time (min)
Fig. 3 Change in oxygen concentration in tomato plants in response to pathogen

infection. The data presented here is from representative graph of two
independent experiments
4 Notes
1. Calibration is must before starting the oxygen measurements.

2. Oxygen sensor should be handled very carefully because its
membrane is fragile.
3. Chamber should be properly sealed.
4. Temperature should be constant throughout the experiment.
Acknowledgement
KJG is grateful to Ramalingaswami reentry fellowship and IYBA

from DBT. PKP is the recipient of UGC-JRF.
References
1. Mysore KS, Ryu CM (2004) Nonhost resis- 5. Bonardi V, Dangl JL (2012) How complex are
tance: how much do we know? Trends Plant intracellular immune receptor signaling com-
Sci 9(2):97–104 plexes? Front Plant Sci 3:237
2. Senthil KM, Mysore KS (2013) Nonhost resis- 6. Gupta KJ, Brotman Y, Segu S, Zeier T, Zeier J,
tance against bacterial pathogens: retrospec- Persijn ST, Kaiser WM (2013) The form of
tives and prospects. Annu Rev Phytopathol nitrogen nutrition affects resistance against
51:407–427 Pseudomonas syringae pv. Phaseolicola in
3. Boller T, He SY (2009) Innate immunity tobacco. J Exp Bot 64(2):553–568
in plants: an arms race between pattern recog- 7. Scheideler M, Schlaich NL, Fellenberg K,
nition receptors in plants and effectors in Beissbarth T, Hauser NC, Vingron M, Hohei-
microbial pathogens. Science 324(5928): sel JD (2002) Monitoring the switch from
742–744 housekeeping to pathogen defense metabolism
4. Jones JD, Dangl JL (2006) The plant immune in Arabidopsis thaliana using cDNA arrays. J
system. Nature 444(7117):323–329 Biol Chem 277(12):10555–10561
8. Scholes JD, Rolfe SA (1996) Photosynthesis in 12. Denoux C, Galletti R, Mammarella N, Gopalan
localised regions of oat leaves infected with S, Werck D, De Lorenzo G, Dewdney J (2008)
crown rust (Puccinia coronata): quantitative Activation of defense response pathways by
imaging of chlorophyll fluorescence. Planta OGs and Flg22 elicitors in Arabidopsis seed-
199(4):573–582 lings. Mol Plant 1(3):423–445
9. Berger S, Papadopoulos M, Schreiber U, Kaiser 13. Bilgin DD, Zavala JA, Zhu JIN, Clough SJ,
W, Roitsch T (2004) Complex regulation of Ort DR, Delucia E (2010) Biotic stress globally
gene expression, photosynthesis and sugar down regulates photosynthesis genes. Plant
levels by pathogen infection in tomato. Physiol Cell Environ 33(10):1597–1613
Plant 122(4):419–428 14. LJ F, Shi K, Gu M, Zhou YH, Dong DK, Liang
10. Swarbrick PJ, Schulze-Lefert P, Scholes JD WS, Song FM, JQ Y (2010) Systemic induction
(2006) Metabolic consequences of susceptibil- and role of mitochondrial alternative oxidase
ity and resistance (race-specific and broad- and nitric oxide in a compatible tomato–to-
spectrum) in barley leaves challenged with bacco mosaic virus interaction. Mol Plant-
powdery mildew. Plant Cell Environ 29 Microbe Interact 23:39–48
(6):1061–1076 15. Nogués S, Cotxarrera L, Alegre L, Trillas MI
11. Truman W, Zabala MT, Grant M (2006) Type (2002) Limitations to photosynthesis in
III effectors orchestrate a complex interplay tomato leaves induced by Fusarium wilt. New
between transcriptional networks to modify Phytol 154(2):461–470
basal defence responses during pathogenesis
and resistance. Plant J 46(1):14–33
Chapter 8
Measurement of Oxygen Status in Arabidopsis Leaves

Undergoing the Hypersensitive Response During
Pseudomonas Infection
Aprajita Kumari, Gail M. Preston, and Kapuganti Jagadis Gupta
Abstract
When plants are infected with pathogens they defend themselves via various processes. Once such process is
the development of the hypersensitive response (HR), a kind of programmed cell death (PCD), in which
localized cell death takes place in order to prevent pathogen spread to other part of tissue. The Arabidopsis
and Pseudomonas syringae system is one of the best known examples to study the HR. Here we used the
VisiSens™ oxygen-imaging system to investigate oxygen distributions in Arabidopsis leaves infected with
an avirulent strain of P. syringae and undergoing the hypersensitive response. Using this method we
observed a change in oxygen status at 6 h post-infection and a drop in oxygen levels at 24 h after infection.
Key words Arabidopsis, Oxygen, VisiSens, Hypersensitive response, Programmed cell death
1 Introduction
When plants are infected with pathogens they adopt various defense
strategies to counter pathogen invasion. One well-known strategy
is the development of the hypersensitive response (HR), which
typically includes the onset of localized programmed cell death
[1]. The HR can activate defense responses both locally and sys-
temically. It is associated with induction of various genes and meta-
bolites and free radicals such as reactive oxygen and nitrogen species
[2, 3], including reactive oxygen intermediates (ROI) such as
superoxide (O2 ) and hydrogen peroxide (H2O2) [4].
Mitochondria are important organelles for respiratory metabo-
lism leading to production of ATP. Mitochondria produce various
reactive oxygen species such as superoxide (O2 ) and hydrogen
peroxide (H2O2) and reactive nitrogen species [5] such as nitric
oxide (NO) and peroxynitrite (ONOO ) [6]. Excess ROS is con-
trolled by a mitochondrial defense mechanism mediated by alterna-
tive oxidase (AOX) [6, 7]. Mitochondrial status also plays a role in
71
72 Aprajita Kumari et al.
the development of cell death and defense responses [8, 9]. During
the HR there is production of excess of ROS and NO. A reaction
between these two radicals leads to production of peroxynitrite
(ONOO ). NO can reversely bind to cytochrome c oxidase
(COX), leading to inhibition of mitochondrial respiration [10,
11]. Thus mitochondria are targets during plant infection and
disturbance of their ROS and RNS homeostasis leads to generation
of signals for reprogramming of various defense pathways [12].
The interaction of Arabidopsis and the bacterial pathogen Pseu-
domonas syringae is a well-established model system for studying
plant-pathogen interaction and disease development. Despite
extensive research very little is known about the oxygen distribu-
tions in infected leaves undergoing HR which is crucial for under-
standing plant disease development and resistance mechanisms.
The avirulent strain P. syringae pv. tomato DC3000 (pAvrB1)
(Pst) induces the HR in Arabidopsis ecotype Col-0. In this study
we used the VisiSens oxygen imaging system to monitor oxygen
distributions during infection and HR development process. In this
method optical sensor foil was attached to the surface of the leaf
and the signal read with the recorder. The recorded images were
used to quantify oxygen changes in the infected leaves.
2 Materials
2.1 Equipment for The VisiSens system is known as the A1 system and contains three
Oxygen and components: (a) A detector unit (DU01) with USB connector to
Respiratory connect to a computer. This also contains an in-built excitation
Measurement light source and optical fibers for oxygen detection (Fig. 1). (b)
Software VisiSens AnalytiCal-1 B. (c) The sensor foil SF-RPSu4.
The sensor foil has the ability to detect oxygen concentrations
ranging from 0 to 100% air saturation. The Thermo block (Eppen-
dorf) should be set to 25 C.
2.2 Reagents/ Two types of calibration standards have to be prepared. (a) For
Calibration Standards establishing zero oxygen add 20 mg of sodium dithionate
(Na2S2O4) into 200 μL of distilled water and immediately place
the drop of this solution in the center of the sensor foil and establish
zero oxygen using software. (b) For establishing 100% in air satu-
ration, bubble 10 mL of distilled water with pure compressed air
for 10 min and then take a drop of this air-saturated water and place
on the sensor foil and record oxygen concentration and set as 100%
using software.
2.3 Materials for 1. Arabidopsis seeds.

Plant Growth 2. Sodium hypochlorite 0.1%.
3. 2 ml Eppendorf tubes.
Measurement of Oxygen Status in Arabidopsis Leaves. . . 73
Fig. 1 (a) Setup for oxygen measurement on Arabidopsis leaves infected with Pseudomonas syringae pv.
tomato DC3000 (pAvrB1). (b) Recording the sensor response with the VisiSens detector unit
4. Shaker.
6. 15 ml Falcon tubes.
7. 500 ml Black pots and trays.
8. Air pump.
9. Needles.
10. Razor blades.
11. Plant growth chamber with controlled conditions such as tem-
perature, humidity, and light.
12. Soilrite and agro peat and vermiculate.
3 Methods
3.1 Plant Growth 1. Soak healthy Arabidopsis Col-0 seeds in autoclaved distilled
water for 20 min.
2. Remove the water and incubate the seeds in 0.1% sodium
hypochlorite NaClO and 0.05% Tween-20 detergent for a
period of 10 min by continuous shaking.
3. Remove the sterilization solution and wash the seeds with
autoclaved distilled water four times.
4. Keep the seeds at 4 C.
5. Prepare pots for plant growth.
6. Place 2–3 seeds in each pot and transfer these pots to the plant
growth chamber for growth and grow them for 4 weeks.
3.2 Calibration of 1. For calibration fix the detector unit (DU01) to a stand (see
Sensor Foil Note 1).
2. Place the green reference foil plus the transparent length scale
over the site on a plain surface.
3. Focus using coarse and fine adjustment knobs on the detector
unit and on and record Pixel resolution.
4. Then replace the reference foil with sensor foil.
5. Using a pipette place a drop of air-saturated water on the top of
the sensor and record the image and set as Cal-100.
6. Remove the water droplet on the sensor foil with soft tissue
paper.
7. Place a drop of 50–100 μl freshly prepared sodium dithionate
Na2S2O4 on the sensor foil and record the image as described
in materials (see Note 2).
8. Once both the images are achieved, using software set the
calibration using scale.
3.3 Bacterial Growth 1. For primary bacterial culture preparation take a loopful of
inoculum and streak the bacterium on the surface of a KB
agar plate to obtain single colonies using a sterile spreader.
Incubate the plate upside down in an incubator at 28 C.
2. Take a single colony from pure culture and inoculate 3 ml
King’s B liquid medium (KB) supplemented with antibiotic
rifampicin (50 mg/l) and incubate overnight at 28 C with
continuous shaking (180–200 rpm).
3. Take 1% of primary culture and inoculate in fresh media of
10 ml.
4. Thereafter, centrifuge the culture at 3000 g for 10 min at
25 C.
5. Discard the supernatant and resuspend the pellet in 10 mM
MgCl2 at OD600 0.2.
6. Prepare 20 ml of bacteria for inoculation for infiltration into
several plants.
3.3.1 Bacterial 1. Take a loopful of glycerol stock of Pseudomonas syringae pv.

Inoculation and DC3000 in KB medium-containing plates. The composition to
Measurement of Internal KB medium is Proteose Peptone 20 g/l, K2HPO4, 1.5 g/l,
Oxygen in Leaves (See agar 20 g/l, and MgCl2 1.5 g/l, pH 7.2.
Fig. 2a, b) 2. Centrifuge at 3000 g for 5 min at 4 C.
3. Discard the supernatant and add fresh MgCl2.
4. Measure the density of bacterium using spectrophotometer.
5. Dilute the bacteria to 0.005 OD using 10 mM MgCl2
Measurement of Oxygen Status in Arabidopsis Leaves. . . 75
0h 2h 6h 12 h 24 h MgCl2
B 200
180
Oxygen (% of air saturation)
160
140
120 Psp
100
MgCl2
80
60
40
20
0
0 1 3 6 16 24
Time (hours after post inoculation)
Fig. 2 (a) Images of leaves infiltrated with Pseudomonas syringae pv. tomato DC3000 (pAvrB1) taken with
VisiSens™ at different time points. Right image is from MgCl2 treatment control. (b) Oxygen content (% air
sat.) in Arabidopsis leaves infiltrated with Pseudomonas or 10 mM MgCl2 at different times post-inoculation.
Images are representatives of three independent biological replicates
6. Using a 2 ml needle-less sterile syringe infiltrate the leaves from

the abaxial side of leaf.
7. Place the sensor foil on the infiltrated leaf.
8. Record the image using VisiSens software and repeat the mea-
surement at 2, 6, 12, and 24 h.
4 Notes
1. Clean the detector with lens tissue prior to use to make sure
that there is no dust.
2. After placing sodium dithionate (Na2S2O4) on the film capture
the image immediately to be sure that it is not oxygenated.
Acknowledgement
We thank PreSens Gmbh for providing instrument for measure-

ment of oxygen. J.G.K. thank Gail Preston for laboratory facilities
to conduct the experiment. K.J.G. research was supported by Marie
Curie Intra European Fellowship under FP7 framework. K.J.G. and
A.K. were supported by the Department of Biotechnology, Govt.
of India. Work was performed at the University of Oxford and data
analysis and manuscript preparation were done at NIPGR.
References
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dative stress: electron transport, NADPH turn- tions. Mol Plant 7:747–750
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species. Annu Rev Plant Physiol Plant Mol Evidence of mitochondrial involvement in the
Biol 52:561–591 transduction of signals required for the induc-
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Chapter 9
Isolation of Physiologically Active and Intact Mitochondria

from Chickpea
Sonika Pandey, Aprajita Kumari, and Kapuganti Jagadis Gupta
Abstract
Chickpea is an important leguminous crop that belongs to Fabaceae family, highly valued for its nutritious
seeds. Seeds contain reserve food for the developing embryos. Mitochondria are crucial organelle for
generation of chemical energy in the form of ATP which is required for achieving metabolically active
state; therefore, investigating mitochondrial function and respiration rate is crucial for exploring various
metabolic and physio-biochemical changes that occur during seed germination. Here we describe a method
for isolation of mitochondria from germinating seeds of two chickpea varieties, i.e., Desi and Kabuli.
Structure of Mitotracker-stained isolated mitochondria was observed by confocal microscopy and respira-
tion rate was measured using an oxygen microsensor.
Key words Chickpea, Confocal, Germination, Mitochondria, Oxygen sensor, Respiration rate
1 Introduction
Chickpea (Cicer arietinum L.) belongs to Fabaceae family and is

the third most important pulse, cultivated in semiarid regions of
Asia and Africa. Among all countries, India is the world leader in
chickpea production, covering approximately 8.3 million hectares
of land that accounts for 0.33 million tons of seed production [1].
Chickpea gets nitrogen via association with rhizobacteria [2]. Seeds
of Chickpea are consumed in diets for their protein, carbohydrate,
and essential amino acid contents [3]. In addition, these seeds are
also rich source of minerals (calcium, phosphorous, iron, and zinc),
β-carotene, and crude fibers [4]. Based on phenotypic appearances
and geographical distribution chickpea is characterized into two
groups: Desi and Kabuli. Desi variety grows as small bush bearing
small leaflets and small dark-colored angular seeds [5]. However,
Kabuli plant is grown mainly in the Mediterranean region, and
forms white flowers that later produce large, yellowish colored
oval seeds.
77
During seed germination, rapid mobilization of storage

reserves occurs that initiates growth and morphological alterations
and also results in changes in physiological, biochemical, and meta-
bolic activity [6]. Seed germination is totally an energy-dependent
phenomenon mediated by generation of ATP by mitochondria.
Plant mitochondria is a complex double-membrane organelle
and is associated with biosynthesis (ATP/ADP) via generation of
proton motive force [7]. Out of many factors oxygen is an essential
component for mitochondria to produce ATP and speed germina-
tion associated with increased respiration rate, which is accompa-
nied by the mitochondrial biogenesis [8]. Therefore studying
mitochondrial function using germinating seed as a model system
can be useful for exploring the mitochondrial biogenesis and
energy metabolism during seed germination process. In this
work, we have described a protocol for isolating metabolically
active mitochondria from mature germinating seeds of two differ-
ent chickpea varieties, i.e., Desi and Kabuli. The isolated mitochon-
dria were further used for confocal imaging and studying the
change in respiration rate using an oxygen microsensor.
2 Materials
2.1 Plant Material Germinated chickpea seeds 50 g (see Note 1).
2.2 Reagents 1. Buffers: Prepare the following buffers 1 day before.

(a) Extraction buffer: Prepare 500 ml buffer for extraction of
mitochondria by dissolving different components in
double-distilled water [HEPES (100 mM, pH 7.2), sucrose
(0.3 M), skim milk (0.1%), polyvinylpyrrolidone (0.6%),
EDTA (1 mM), L-cysteine (4 mM), KH2PO4 (5 mM),
and MgCl2 (2 mM)]. Store buffer at 4 C (see Note 2).
(b) Suspension buffer: Prepare 100 ml suspension buffer that
comprises various components dissolved in double-
distilled water [HEPES (20 mM, pH 7.2), sucrose
(0.3 M), EDTA (1 mM), MgCl2 (2 mM), KH2PO4
(0.5 mM)]. Store buffer at 4 C (see Note 2).
2. Percoll gradient: Prepare by mixing varying percentage of Per-
coll (60, 45, 28, and 5%) with suspension buffer of final volume
20 ml. Discontinuous Percoll gradient should be prepared
freshly into 50 ml Oakridge tube, starting from bottom to
top: 6 ml of 60%, 8 ml of each 45%, 28% and 5%, respectively,
using a 10 ml syringe (Fig. 1i) (see Note 3). Prepared discon-
tinuous Percoll gradient must be chilled on ice before using.
3. Bradford reagent.
4. MitoTracker Red: Dissolve lyophilized powder in high-quality
DMSO. Store the solution in the dark at 20 C.
Isolation of Physiologically Active and Intact Mitochondria from Chickpea 79
Fig. 1 Illustration of mitochondria isolated from geminated seeds of chickpea. (a, b) Germinated seeds of Desi
and Kabuli, (c) balancing of extract, (d) homogenized extract of Desi and Kabuli seeds, (e) centrifuge used for
mitochondria isolation, (f) pellet showing debris, (g) centrifugation of supernatant from previous step, (h)
mitochondrial pellet along with impurities and other cellular debris, (i) preparation of Percoll gradient, and (j)
mitochondrial fraction in the interface between 45 and 28% Percoll gradient
5. Nicotinamide adenine dinucleotide (NADH): Prepare 0.5 mM

NADH by dissolving the powder in Milli-Q water and store in
aliquots at 20 C.
2.3 Equipment Centrifuge tubes 1 l, 50 ml, and 1.5 ml.

Plastic beakers.
Scissors.
Electric grinder.
Spatula.
Measuring cylinder.
Nitrile gloves.
Tissue paper.
Sandwich of miracloth and surgical cotton.
Syringe (10 ml).
Cut tips (0.2 and 1 ml).
Micropipettes.
Magnetic pellet.
Magnetic stirrer.
Marker.
Ice flakes.
Camera.
Centrifuge machine (Sorvall, Lynx 6000).
MitoTracker Red (Molecular Probes, Life Technologies, USA).
Amber Eppendorf tube (1.5 ml).

Eppendorf tube stand.
Glass slides (GEM).
Glass coverslips (0.16 mm thickness; Blue Star).
Oxygen microsensor.
Temperature sensor.
Nitrogen gas.
Air pump.
Parafilm.
HPLC vial (Agilent).
Needle.
TBR1025 single-channel free radical analyzer (World Precision
Instruments, Sarasota, FL, USA).
Confocal microscope (Leica TCS SP5 X).
Shaker.
3 Procedure
3.1 Germination 1. Take chickpea seeds in a Falcon tube and incubate them in 0.1%
of Chickpea Seeds HgCl2 for a period of 10 min for sterilization. During steriliza-
tion shake the falcon tube containing seeds. After that, rinse
seeds 3–4 times with autoclaved water to remove HgCl2
completely.
2. Soak the sterilized seeds for imbibition in sterile water for 4 h
(see Note 4).
3. After imbibition, take the seeds from water and cover and tie it
in wet miracloth.
4. Keep the wrapped seeds in dark incubation for germination,
i.e., emergence of radicle (Fig. 1a, b).
5. Record the weight of germinated seeds before starting mito-
chondrial isolation.
3.2 Steps for 1. Take germinated seeds, and rinse in distilled water. All the
Isolation of procedure of mitochondria isolation should be carried out at
Mitochondria 4 C.
2. Homogenize 50 g of seeds in 50 ml of prechilled extraction
buffer in cooled electric grinder. This step should be repeated
ten times in short spin so that seeds can be homogenized
completely and uniformly. Make final volume up to 200 ml
(see Note 5).
3. Squeeze the homogenate through single layer of cotton sand-

wiched between double-layered miracloth to separate liquid
part from the solid tissue. Collect the filtrate in 1 ml centrifuge
bottle (Fig. 1d).
4. Centrifuge the filtrate at low spin at 500 g for 10 min at 4 C.
Transfer the supernatant into fresh centrifuge bottle (1 l)
carefully.
5. Again centrifuge the supernatant at 2000 g for 15 min and
collect the supernatant.
6. Centrifuge the supernatant at 12,000 g for an hour at 4 C.
Discard the supernatant slowly as the pellet formed at the
bottom contains mitochondrial fractions along with other cel-
lular impurities.
7. Dissolve the pellet in 2–4 ml suspension buffer and load it
carefully into prechilled Percoll gradient prepared in centrifuge
tube (50 ml) (Fig. 1i). Centrifuge at 17,000 g for an hour at
4 C.
8. Mitochondrial fraction can be seen dispersed as whitish creamy
layer in zone of 45 and 28% Percoll gradient (Fig. 1j). Carefully
collect the mitochondrial fraction using 1 ml cut tip and trans-
fer into 50 ml centrifuge tube and add 5 ml suspension buffer.
9. Again centrifuge at 17,000 g for 10 min at 4 C. This step is
required to remove the content of Percoll gradient. Discard the
supernatant and gently dissolve the pellet in 1 ml suspension
buffer.
10. Prepare the aliquots in 1.5 ml Eppendorf tube, freeze in liquid
nitrogen, and store at 20 C for further experiments.
11. Stepwise illustration (Fig. 1a–j) and schematic representation
(Fig. 2) of mitochondrial isolation from germinated seeds of
chickpea are shown.
3.3 Estimation of 1. First prepare stock of bovine albumin serum (BSA) protein
Mitochondrial Protein 0.1 μg/μl in double-distilled water.
Concentration 2. Prepare a standard curve of BSA protein 0–10 μg using Brad-
ford reagent and record the absorbance at 595 nm.
3. Take 10 μl of unknown protein sample and mix well with 990 μl
of Bradford reagent and keep it in dark incubation for 10 min.
Record the absorbance at 595 nm.
4. Now measure the unknown protein sample concentration
obtained from BSA standard curve.
3.4 Imaging 1. Mix gently 50 μl of freshly isolated mitochondria with suspen-

Mitochondria in sion buffer.
Confocal Microscopy 2. Add 500 nM MitoTracker Red dye into mitochondrial suspen-
sion and keep it in dark incubation for 10 min at room
temperature.
Sterilize seeds in 0.1% HgCl2 and keep it in miracloth in dark for germination
Record weight of germinated seeds and homogenize in electric grinder along with extraction
buffer at 4°C
Filter extract into centrifuge bottle through a sandwich of miracloth and surgical cotton
Centrifuge filtered extract at 500×g for 10 min at 4°C and transfer supernatant into fresh tube.
Again centrifuge at 2,000×g for 15 min
Collect supernatant, centrifuge at 12,000xg for an hour. Pellet contains mitochondrial fraction
along with other cellular impurities, can be purified using discontinuous Percoll gradient
Discard supernatant and dissolve pellet in 2-4 ml of suspension buffer and load into Percoll
gradient. Again centrifuge at 17,000xg for an hour at 4°C
Collect mitochondrial fraction (lies between 45% and 28% Peroll gradient) using 1 ml cut tip,
transfer into 50 ml Oakridge tube
Again centrifuge at 17,000xg for 10 min at 4°C. Dissolve pellet in 1 ml suspension buffer and
store at 4°C. Use isolated mitochondria for confocal and respiration measurement
Fig. 2 Schematic representation of mitochondrial isolation from germinated seeds of chickpea
3. For imaging, add 3 μl of mitochondrial suspension onto a glass

slide and gently place coverslip over it.
4. Choose the appropriate excitation (488 nm) and emission
(500–530 nm) wavelengths for MitoTracker Red dye.
5. Adjust laser intensity of the images on a Zeiss imaging fluores-
cence microscope (60/1.0 numeric aperture [NA] oil objec-
tive) equipped with a Leica TCS SP5 X (Leica Microsystems)
inverted confocal laser scanning microscope and capture the
image (Fig. 3).
6. The acquisition software is LAS AF software (Leica
Microsystems).
3.5 Measurement of Respiration is measured using TBR1025 single-channel free radical

Respiration analyzer (World Precision Instruments, Sarasota, FL, USA) as
shown in Fig. 4 (see Pandey et al. Chapter 6 in this book).
3.5.1 Calibration of Before using the instrument for measurement, perform calibration
Oxygen Sensor of oxygen sensor as mentioned below:
1. Wash the sensor with distilled water.
2. Put distilled water (1 ml) into HPLC vial and flush nitrogen gas
for 5 min to maintain zero level of oxygen. Now insert oxygen
sensor and set zero level oxygen using software.
3. After that for setting concentration of oxygen 100%, flush air
using air pump into distilled water (1 ml) for 5 min. Dip sensor
and save values as 100% in software.
Fig. 3 Confocal imaging of mitochondria isolated from geminated seeds of chickpea
Fig. 4 Setup of oxygen microsensor instrument for mitochondrial respiratory rate measurement. Microsensor
is inserted inside HPLC vial containing isolated mitochondria and respiration was measured as shown in the
figure
3.5.2 Measurement of Take isolated mitochondria equivalent to 1 mg protein and make

Mitochondrial Respiration final volume to 1 ml with suspension buffer and put it in HPLC vial.
of Seed Put magnetic pellet inside HPLC vial and seal it with parafilm. Keep
it on magnetic stirrer for 5 min. Pierce the cap of HPLC vial at the
top using needle and flush with ambient air for 5 min. Remove the
needle and carefully insert the oxygen sensor into the vial (see Note
6). Record the change at 1min interval. After that add 0.5 mM
NADH, and record the rate of consumption at every 1-s interval as
shown in Fig. 4. Oxidation of NADH by chickpea mitochondria is
represented in Fig. 5.
105 Desi
Kabuli
100
Oxygen Concentration
(% in air saturation)
95
90
85
80
75
0 5 10 15 20 25 30 35 40 45 50
Time(s)
Fig. 5 Oxidation of NADH by chickpea mitochondria. 0.5 mM NADH was added to reaction mixture (625 μl
mitochondria þ 325 μl suspension buffer) and change in respiration was measured using oxygen microsensor
4 Notes
1. Healthy seeds must be selected for isolation of mitochondria.

2. All buffers must be prepared freshly and should be ice chilled
before using.
3. Percoll gradient must be prepared freshly and carefully during
gap of centrifugation step.
4. Avoid soaking of seeds for prolonged time in water as it will
affect germination.
5. Seeds must be homogenized completely.
6. Oxygen microsensor must be carefully inserted into the HPLC
vial to prevent breakage.
Acknowledgements
This work was supported by NIPGR short term research fellowship

to S.P., and IYBA and Ramalingaswami to K.J.G. from Department
of Biotechnology.
References
1. FAO (2003) http://www.fao.org. Production 3. Jukanti AK, Gaur PM, Gowda CLL, Chibbar
database RN (2012) Nutritional quality and health bene-
2. Carranca C, De Varennes A, Rolston D (1999) fits of chickpea (Cicer arietinum L.): a review. Br
Biological nitrogen fixation by fababean, pea and J Nutr 108:S11–S26
chickpea, under field conditions, estimated by 4. Pradhan S, Bandhiwal N, Shah N, Kant C, Gaur
the 15N isotope dilution technique. Eur J R, Bhatia S (2014) Global transcriptome analysis
Agron 10:49–56
of developing chickpea (Cicer arietinum L.) seed shape reveals differences in cellulose
seeds. Front Plant Sci 5:698 mutants. Acta Physiol Plant 36:1585–1592
5. van der Maesen LJG (1972) A monograph of 7. Bowsher CG, Tobin AK (2001) Compartmen-
the genus, with special references to chickpea tation of metabolism within mitochondria and
(Cicer arietinum L.). Its ecology and cultivation. plastids. J Exp Bot 52:513–527
Mededlingen Landbouw Hogeschool (Commu- 8. Logan DC, Millar AH, Sweetlove LJ, Hill SA,
nication Agricultural University), Wageningen Leaver CJ (2001) Mitochondrial biogenesis dur-
6. Martı́n JJ, Tocino Á, Ardanuy R, Juana G, Cer- ing germination in maize embryos. Plant Physiol
vantes E (2014) Dynamic analysis of Arabidopsis 125:662–672
Chapter 10
Isolation and Structural Studies of Mitochondria

from Pea Roots
Abhaypratap Vishwakarma and Kapuganti Jagadis Gupta
Abstract
For structural and respiratory studies, isolation of intact and active mitochondria is essential. Here, we
describe an isolation method which gave good yield and intact mitochondria from 2-week-old pea (Pisum
sativum) roots grown hydroponically under standard growth conditions. We used Percoll gradient centri-
fugation for this isolation procedure. The yield of purified mitochondria was 50 μg/g FW. Isolated
mitochondria maintained their structure which was observed by using MitoTracker green in confocal
microscope and scanning electron microscopy (SEM). Intact mitochondria are clearly visible in SCM
images. Taken together this isolation method can be used for physiological and microscopic studies on
mitochondria.
Key words Confocal microscope, Mitochondria, Percoll gradient, Pea
1 Introduction
Mitochondria are double-membrane-bound organelles and ubiq-

uitous throughout eukaryotic cells. They are known as power-
houses of the cell as they play a critical role in generation of
metabolic energy. Apart from ATP generation via proton pumping,
they have various alternative functions such as calcium homeostasis,
nitric oxide generation, generation of free radicals, vitamin biosyn-
thesis, redox regulation, and regulation of cell death thermogenesis
[1, 2]. These semi-independent organelles possess their own gen-
omes and transcription/translations systems [3, 4]. Several studies
have revealed the complexity and plasticity of mitochondria includ-
ing their changing size, shape (e.g., by elongation, shortening,
branching, buckling, swelling), and traveling of long distance on
cytoskeleton [5, 6]. Plant mitochondria differ significantly from
animal mitochondria, with specific electron transport components
and photorespiration process [7, 8]. Plant mitochondria exhibit a
remarkable plasticity such as (1) operation of the TCA cycle in a
noncyclic mode under hypoxia [9, 10]; (2) the presence of non-
87
88 Abhaypratap Vishwakarma and Kapuganti Jagadis Gupta
phosphorylating bypasses including the alternative oxidase (AOX),

uncoupling protein (UCP), and the external NAD(P)H dehydro-
genases [11–14]; and (3) substantial interaction of the respiratory
pathways with cytosol, plastid, and peroxisomal compartments [12,
15–17].
Experiments using complex I mutants of tobacco [18, 19] and
Arabidopsis [20, 21] revealed the importance of these organelles
maintaining the redox and carbon balances. In recent years, many
studies concentrated on non-phosphorylating bypass pathways of
mitochondria such as internal/external NAD(P)H dehydrogenases
[7], the electron transfer flavoprotein (ETF)/electron transfer flavo-
protein quinone oxidoreductase (ETFQO) [22, 23], glycerol-3-phos-
phate dehydrogenase [24, 25], AOX [26, 27], and UCPs [11, 14].
Any perturbations of the cellular energy status may result in a
reconfiguration of mitochondrial activities that affects the other
cellular compartments, including changes in nuclear gene expres-
sion and photosynthetic activity as a consequence of retrograde
signaling [28, 29]. Mitochondria continuously produce the reac-
tive oxygen species (ROS) as a by-product of respiration. Physio-
logical levels of ROS production help in retrograde signaling [30].
The ROS production further increases under circumstances of high
matrix NADH/NAD+ ratio or over-reduction of ubiquinone pool
due to inefficient oxidation of reducing equivalents [7].
This protocol illustrates a step-by-step procedure to obtain
functional mitochondria with high yield from pea root. The isola-
tion procedure is based on discontinuous Percoll gradient and
requires 2–3 h.
2 Materials
2.1 Plant Material 50 g of pea roots.
2.2 Chemicals Extraction buffer (100 mM HEPES pH 7.2, 0.3 M sucrose, 0.1%
and Reagents skim milk, 0.6% polyvinylpyrrolidone, 1 mM EDTA, 2 mM MgCl2,
4 mM L-cysteine, 5 mM KH2PO4).
Suspension buffer (20 mM HEPES pH 7.2, 0.3 M sucrose, 1 mM
EDTA, 2 mM MgCl2, 0.5 mM KH2PO4). Percoll (GE Health-
care), MitoTracker green (Molecular Probes, Invitrogen, USA).
Hydroponic nutrient solution (NS): 3 mM KNO3, 1 mM CaCl2,
1 mM MgSO4, 0.025 mM NaFe-EDTA, 1 mM K2HPO4, 1 mM
KH2PO4, and trace elements (15 μM H3BO3, 3 μM MnCl2.4H2O,
0.25 μM ZnSO47H2O, 0.1 μM CuSO45H2O and 0.04 μM
Na2MoO4) according to Johnson et al. [31].
Isolation of Mitochondria from Pea 89
2.3 Other Materials 1. Aluminum pin stub (Carl Zeiss).

2. Eppendorf tube (1.5 ml).
3. Carbon planchet (Carl Zeiss).
4. Camera (Nikon COOLPIX L840).
5. Centrifuge machine (Sorvall, Lynx 6000).
6. Centrifuge tubes 1 L.
7. Confocal microscope (Leica TCS SP5 X).
8. Cut tips (0.2 and 1 ml).
9. Disposable syringe (10 ml).
10. Eppendorf tube stand.
11. Glass coverslips (0.16 mm thickness; Blue Star).
12. Glass slides (GEM).
13. Ice flakes.
14. Magnetic stirrer.
15. Marker pen.
16. Measuring cylinder.
17. Micropipettes.
18. MitoTracker green (Molecular Probes, Life Technologies
USA).
19. Mixer grinder.
20. Mortar pestle.
21. Nail paint.
22. Nitrile gloves.
23. Oak Ridge tubes (50 ml; Tarsons) for gradient preparation.
24. Paintbrush.
25. Plastic beakers.
26. Plastic trays.
27. Sandwich of cheese cloth and surgical cotton.
28. Scanning electron microscope (EVO® LS10 Carl Ziess NTS
GmbH, Germany).
29. Scissor.
30. Spectrophotometer.
31. Spatula.
32. Surgical blade.
33. Tissue paper.
3 Methods
3.1 Germination 1. Select healthy pea seeds (Pisum sativum cv. Pusa pragati)
of Pea and sterilize them by incubating for 10 min in 4% NaOCl
(see Note 1).
2. Rinse the seeds with sterile water 4–5 times.
3. Place the seeds on wet filter paper for 48 h for germination.
3.2 Plant Growth 1. Place germinating seeds on hydroponic tray and then grow for
Conditions 2–3 weeks in 10 l hydroponic pots filled with nutrient solution
(refer to materials for composition of NS) (Fig. 1).
2. Initially, use 1/10 diluted NS and gradually increase strength
of NS to ¼, ½, ¾, to full strength for well adaptation to
hydroponics medium.
3. Put a needle diffuser in hydroponic tray and flush continuously
with ambient air using air pumps.
4. Place the plants in growth chamber with a light intensity of
50 μmol/m2/s, 16-h light and 8-h dark period cycle.
5. Maintain the day/night temperature regime of the chamber
25/20 C, respectively.
3.3 Mitochondrial 1. Prepare Percoll gradient (60, 45, 28, and 5%) (Table 1).
Isolation from Pea 2. Add discontinuous Percoll gradient into Oak Ridge tube from
Roots bottom to top, 6 ml of 60% and 8 ml of each 45%, 28% and 5%,
respectively, using a 10 ml syringe with a flow rate of 18 s/ml
(Fig. 2a) (see Note 2). Store the Percoll gradient on ice until
used.
3. Set the centrifuge at 4 C (Fig. 2b).
Fig. 1 Phenotype of pea plants grown in hydroponic medium. (a) One-day-old seedlings and (b) 15-day-old
plants showing hydroponically grown roots
Table 1
Composition of Percoll gradient
Percoll gradient (%) Percoll (ml) Suspension buffer (ml) Final volume (ml)
60 12 8 20
45 9 11 20
28 5.6 14.4 20
5 1 19 20
Fig. 2 Photographic representation of mitochondrial isolation steps. (a) Preparation of Percoll gradient; (b)
centrifuge used for mitochondrial isolation; (c) a small pellet shows the debris; (d) pellet derived from
12,000 g centrifugation (e) after loading the dissolved pellet on Percoll gradient; (f) a distinct layer
(interface between 45 and 28% Percoll gradient) indicates the mitochondrial fraction
4. Dissect the roots with sharp scissor (see Note 3), place them
into small beaker, and subsequently wash thrice with Milli-Q
water.
5. Chop the roots into small pieces (~1 cm) with the help of sharp
surgical blade.
6. Place the chopped roots in mixer grinder container, add

100–150 ml of extraction buffer, close the lid tightly, and
grind until it becomes fine liquid (approximately 10–12 short
spins).
7. Place four layers of cheese cloth sandwiched with cotton on the
top of 1 l centrifuge tube and pour the extract onto it.
8. Wait till most of the extract passes through the sandwich cheese
cloth, and squeeze the cheese cloth to make sure that maxi-
mum filtrate is obtained (see Note 4).
9. Centrifuge the filtrate at 1000 g for 15 min at 4 C (Fig. 2c).
Transfer supernatant into a new bottle.
10. Centrifuge the supernatant at 12,000 g for 40 min at 4 C
(Fig. 2d).
11. Discard the supernatant and resuspend the pellet in 2–4 ml of
suspension buffer using a paintbrush.
12. Crude pellet contains mitochondrial fraction along with impu-
rities of peroxisomes, microsomes which can be separated from
mitochondria via discontinuous Percoll gradient.
13. Transfer the crude mitochondria suspension into Percoll
gradient.
14. Centrifuge the Percoll gradient-loaded mitochondria at
17,000 g for 30 min at 4 C (Fig. 2e).
15. Collect the mitochondrial fraction (lies between 45 and 28%
Percoll gradient) using 1 ml cut tip, transfer into 2 ml Eppen-
dorf tube, and store on ice (Fig. 2f).
16. Centrifuge the Percoll-separated mitochondria at 17,000 g
for 10 min at 4 C to remove the excess of Percoll from
mitochondrial fraction.
17. Discard the supernatant and dissolve the pellet into 1–2 ml of
suspension buffer and store at 4 C.
3.4 Protein 1. Prepare bovine albumin serum (BSA) protein stock 0.1 μg/μl
Estimation in Milli-Q water.
2. Make a standard curve of 0–10 μg BSA protein using Bradford
reagent. Read the absorbance at 595 nm.
3. Take 10 μl of unknown protein sample and mix with 990 μl of
Bradford reagent and incubate for 10 min at room temperature
in the dark. Read the absorbance at 595 nm.
4. Calculate the concentration of unknown protein sample from
BSA standard curve.
3.5 Confocal 1. Take 50 μl of freshly isolated mitochondria (0.7 mg protein/

Microscopy ml) suspended in buffer (20 mM HEPES pH 7.2, 0.3 M
sucrose, 1 mM EDTA, 2 mM MgCl2, 0.5 mM KH2PO4).
Fig. 3 Confocal microscopy image represents the integrity of pea root

mitochondria using MitoTracker green. Image is a representative of two
independent experiments
2. To check the integrity of mitochondria, add 500 nM Mito-

Tracker green/red into mitochondrial suspension and incubate
for 10 min at room temperature under dark condition.
3. Place 3 μl of mitochondria onto a glass slide and mount with
0.16 mm glass coverslip.
4. Use the recommended excitation (488 nm) and emission
(500–530 nm) wavelengths for MitoTracker green/red and
set the microscope filters.
5. Capture the images (Fig. 3) at room temperature on a Zeiss
imaging fluorescence microscope (60/1.0 numeric aperture
[NA] oil objective) equipped with a Leica TCS SP5 X (Leica
Microsystems, Germany) inverted confocal laser scanning
microscope.
6. Image analysis is performed by the acquisition software, i.e.,
LAS AF software (Leica Microsystems).
3.6 Scanning 1. Take an aluminum pin stub and mount with carbon planchet.
Electron Microscopy 2. Add 5 μl of isolated mitochondrial suspension on carbon plan-
chet and spread uniformly.
3. Put the specimen on the stage of the chamber with a tempera-
ture of 25 C.
4. Set the vacuum pressure, i.e., 40 Pa, and voltage, i.e., 20 kV,
which are controlled by VPSE G3 and EHT detectors,
respectively.
5. Set the working distance (WD) of beam at 9.5 mm.
Fig. 4 Scanning electron microscopy of isolated root mitochondria. The scale

bar is 2 μM. Image is a representative of two independent experiments
6. Use 2 μM scale bar and 5450 zoom for mitochondrial visual-

ization in a scanning electron microscope (EVO® LS10 Carl
Zeiss NTS GmbH, Germany) (Fig. 4).
4 Notes
1. Make sure that all seeds and roots are healthy.

2. Before starting mitochondria isolation, make sure that all mate-
rials, buffers, and other accessories are ready.
3. Perform complete isolation procedure at 4 C.
4. Percoll gradient has to be prepared freshly and carefully.
Acknowledgements
A.P.V. is currently funded by NPDF program of SERB, DST, India.

We thank Aakanksha Wany for help in confocal microscopy.
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(2017) The conserved regulation of mitochon- ment of a glycerol-3-phosphate dehydrogenase
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eukaryotes to mammals. Biochim Biophys Act vides evidence of a mitochondrial glycerol-3-
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with photosynthetic carbon assimilation. mitochondrial respiratory pathway to maintain
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et al (2011) Regulation of respiration in plants: Sci 14(4):6805–6847
a role for alternative metabolic pathways. J 27. Fiorani F, Umbach AL, Siedow JN (2005) The
Plant Physiol 168(12):1434–1443 alternative oxidase of plant mitochondria is
17. Sunil B, Talla SK, Aswani V et al (2013) Optimi- involved in the acclimation of shoot growth at
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Chapter 11
Mitochondrial Respiration and Oxygen Tension

Daniel S. Shaw, Karlia Meitha, Michael J. Considine,
and Christine H. Foyer
Abstract
Measurements of respiration and oxygen tension in plant organs allow a precise understanding of mito-
chondrial capacity and function within the context of cellular oxygen metabolism. Here we describe
methods that can be routinely used for the isolation of intact mitochondria, and the determination of
respiratory electron transport, together with techniques for in vivo determination of oxygen tension and
measurement of respiration by both CO2 production and O2 consumption that enables calculation of the
respiratory quotient [CO2]/[O2].
Key words Respiration, Oxidative phosphorylation, In vivo, In vitro, Mitochondria, Oxygen tension,
Respiratory quotient (RQ), Respiratory control ratio (RCR), Gradient centrifugation
1 Introduction
Respiration is one of the most intensively studied and well-

characterized physiological processes in plants. Sophisticated and
accurate commercial instruments for the measurements of CO2 and
O2 exchange have been available since the middle of the last century
[1, 2]. However, it was only when relatively simple methods
became available for the determination of oxygen tension (partial
pressure of oxygen, pO2; ca. 21 kPa at standard atmosphere and
pressure) within plant organs that it was realized that many plant
dense organs have hypoxic regions [3]. The spatial regulation of
oxygen tensions and hence oxygen metabolism can be complex in
tissues such as the root [4], seed [5], or perennial bud [6]. Readers
interested in more high-throughput analyses, such as with the Sea-
horse XF (Agilent Technologies, Santa Clara, USA) or Q2 technol-
ogies (Astec Global, Sheridan, USA) can find relevant information
in references by Sew, Millar, and colleagues [7, 8].
Daniel S. Shaw and Karlia Meitha contributed equally in this chapter.
97
98 Daniel S. Shaw et al.
Intact plant mitochondria can be successfully isolated from

actively growing plant tissues and organs such as potato tubers,
pea shoots, and soybean cotyledons and roots [9–11] where mito-
chondria are relatively abundant and tissues are relatively free of
lignins, polysaccharides, and other cellular components. Here we
describe a method for isolating intact mitochondria from Arabi-
dopsis leaves, followed by purification by density gradient centrifu-
gation using Percoll and polyvinylpyrrolidone (PVP). The isolation
of intact mitochondria from plant tissues using density gradient
centrifugation has been a standard procedure over a decade [17].
The main features are the disruption of the cell structure so as to
release the contents while maintaining the integrity of the orga-
nelles, and then separating the intact mitochondria from other
organelles and cell debris by use of density gradient centrifugation.
The Percoll/PVP gradient serves to separate broken chloroplasts
and thylakoids from the intact mitochondria [9]. Methods to assess
the physical and functional integrity of the isolated mitochondria
are described here, as well as an assay to determine respiration
function and methods for the determination of respiration rates in
intact organs, such as the grapevine buds. We refer the reader to
other literature for commonly used assays to determine respiration
function, which vary according to the research question [12, 13].
These methods can be applied to measure O2 consumption, CO2
production, and profile internal pO2 from the same material within
a relatively short time, <20 min. This is useful because the physio-
logical state of plant organs changes with time, particularly once
they are detached from the plant.
The methods described to measure O2 consumption utilize a
Clark-type oxygen electrode [2, 14], which operates in a closed
system. Although measurement in a closed chamber has some
limitations, the electrodes are highly specific for O2. In addition,
commercially available O2 detection equipment that operates in an
open system, a differential fuel cell system, is still considerably
expensive. The oxygen electrode method assumes linearity and
hence prior testing should be used to establish the conditions
where changes in oxygen levels are linear, and to ensure that oxygen
depletion does not alter the rate of respiration (see Note 1).
Several methods are available for the sensitive internal pO2
detection, which predominantly involve sophisticated and expen-
sive equipment, such as the optical oxygen sensor system (chemilu-
minescent or fluorescent) or electron paramagnetic resonance
(EPR) oximetry. In a comparison of sensitivity in measuring oxygen
consumption by tumor cells, Clark-type oxygen electrodes were
shown to be more sensitive than MitoXpress fluorescent assays
but inferior to the EPR method [15].
The measurement of internal pO2 is often challenging in plant
dense organs, such as buds, roots, and seeds. The method described
here uses a Clark-type electrode that allows penetration into an
Mitochondrial Respiration and Oxygen Tension 99
aimed depth. The electrode is housed in a conical thin glass with a

tip diameter of 25 μm to minimize disruption, and in which the tip
size also allows extensive measurement. In our experience, the
25 μm electrode is sufficiently robust to penetrate layers of highly
lignified scales of a grapevine bud [6]. For more fleshy tissues,
electrodes as thin as 10 μm, or considerably wider for more hard-
ened tissues, are available (see, e.g., www.unisense.com/o2). A sta-
ble signal can be obtained using these electrodes within 1 s of the
start of the measuring period. Experiments measuring the internal
pO2 of seeds have been conducted by using O2-sensitive optical
glass sensors, which are more time efficient because they do not
require repetitious calibration or a pre-polarization period [16].
An additional consideration when using Clark-type electrodes
is the consumption of oxygen by redox reactions [15]. It is impor-
tant to ensure that this is negligible relative to the sensitivity of the
electrode. When measuring the respiration rate, this problem can
be considerably reduced by using a very small electrode and by
continuous stirring in a micro-respiration chamber to avoid a gra-
dient of O2. In spite of this limitation, Clark-type O2 electrodes are
highly reliable tools to determine and quantify internal pO2 and to
determine O2 consumption rate in dense and small plant tissues.
The measurement of respiration by both CO2 production and
O2 consumption enables calculation of the respiratory quotient
[CO2]/[O2], which provides important information about respi-
ratory control and substrate use. Hence we also present a method
to detect CO2 evolution in an open system by using an infrared gas
analyzer (IRGA). This high-resolution, rapid response method
requires rigorous environmental control of gas system exchange
[2]. Some important considerations when measuring CO2 produc-
tion by an IRGA and O2 by a micro-respiration system are as
follows:
1. The Clark-type electrode differs from most conventional
IRGAs in being a closed system (see Note 5).
2. The presence of light must be considered. For experiments
seeking to exclude the effect of photosynthesis, light must be
completely excluded from the chamber.
3. To seal any wound that could occur from detaching the tissue
or organ from the rest of the plant as this may become an
unwanted source of gas exchange activity. This can be simply
achieved by mounting the sample on a block of agar.
4. To use a measuring chamber with a minimal volume, as this will
greatly aid fast detection of the minute O2 depletion or CO2
evolution.
O2 is very sensitive to temperature fluctuation. The methods
described below should ideally therefore be performed in a
controlled-temperature room.
2 Materials
2.1 Density Gradient 1. Light gradient fraction: 28% Percoll (GE Healthcare, Little
for Isolation of Intact Chalfont, UK), 0.3 M sucrose, 10 mM 2-(2-[hydroxy-1,1-bis
Mitochondria (hydroxymethyl)ethyl]amino)ethane sulfonic acid (TES), 0.1%
(w/v) bovine serum albumin (BSA). Adjust pH to 7.5 with
NaOH.
2. Heavy gradient fraction: 28% Percoll, 4.4% PVP-40, 0.3 M
sucrose, 10 mM TES, 0.1% (w/v) BSA. Adjust pH to 7.5
with NaOH.
3. Gradient former (e.g., Model 485 Gradient Former from Bio-
Rad Laboratories Ltd., Hemel Hempstead, UK; optional).
4. Peristaltic pump, pump-head, and silicone tubing (e.g., #WZ-
07522-30, #WZ-77200-62, and #WZ-95802-02, respectively,
from Cole-Parmer Instrument Co. Ltd., London, UK;
optional).
2.2 Isolation 1. Powder-free gloves.

of Mitochondria 2. Miracloth (Merck Biosciences, Nottingham, UK): Ensure that
they are completely clean and free from detergent (see Note 2).
3. Buchner funnel.
4. Large mortar and pestle.
5. Cell extraction medium: 0.3 M Sucrose, 25 mM sodium pyro-
phosphate (Na4P2O7), 1% (w/v) BSA, 1% (w/v) PVP-40,
10 mM KH2PO4, 2 mM EGTA. Adjust pH to 7.5 with hydro-
chloric acid (HCl). If prepared before the day of use, store at
4 C. Add sodium L-ascorbate to 18 mM and cysteine to
20 mM on the day of use.
6. 50 mL Centrifuge tubes.
7. Centrifuge with fixed-angle rotor (Beckman JA-20 or
equivalent).
8. Mitochondria wash medium: 0.3 M Sucrose, 10 mM TES,
0.1% BSA. Adjust pH to 7.5 with NaOH. If prepared before
the day of use, store at 4 C.
9. Fine-art paintbrush.
10. 28% Percoll, 0–4.4% PVP-40 gradient.
11. 2.5 or 5 mL plastic pipette OR autopipette with a cut tip—this is
essential, as conventional pipette tips will shear mitochondria.
2.3 Determination 1. Isolated intact mitochondria.

of Mitochondrial 2. Clark-type oxygen electrode, such as those manufactured
Integrity, i.e., by Hansatech (Kings Lynn, UK) or Rank Brothers (Cam-
Percentage Intactness bridge, UK).
3. Mitochondrial reaction medium: 0.3 M Mannitol, 10 mM

TES-KOH pH 7.5, 3 mM MgSO4, 10 mM NaCl, 5 mM
KH2PO4, 0.1% (w/v) BSA.
4. Stock solutions for cytochrome-c oxidase (COX) assay: 0.5 M
Na-ascorbate; 5 mM cytochrome-c; 10% (v/v) Triton X-100
(see Note 3).
2.4 Respiratory 1. Stock solutions of respiratory substrates: 1 M Succinate,

Control, Non- 500 mM pyruvate, 100 mM nicotinamide adenine dinucleotide
phosphorylating Leak (NADH), 50 mM adenosine 50 -diphosphate (ADP), and
State, and Maximum 50 mM malate. All to be made up in 500 mM TES-KOH,
Uncoupled Flux in pH 7.5.
Isolated Mitochondria 2. 10 mM ADP made up in 500 mM TES-KOH pH 7.5.
3. 5 mg/mL Oligomycin made up in 500 mM TES-KOH
pH 7.5.
4. 10 mM Carbonyl cyanide-4-(trifluoromethoxy)phenylhydra-
zone (FCCP) made up in 500 mM TES-KOH pH 7.5.
2.5 O2 Consumption 1. Agarose 0.8%.

in Fresh Plant Material 2. Calibration chamber for micro-respiration sensors and O2
(See Note 1) microsensors.
3. Scalpel blades and handle.
4. Balance.
5. Pycnometer to measure sample volume.
6. 100% Nitrogen (N) gas.
7. Air pump.
2.6 Measuring O2 1. Instruments of the micro-respiration system (see Notes 4

Consumption in Fresh and 5): Micro-respiration sensors and chambers, rack for the
Plant Material sensors and chambers, solid glass cylinder which fits the micro-
respiration chambers, glass ring, nylon mesh, glass-coated stir-
ring magnet, stirrer controller, signal amplifier, acquisition
software for data logging and calculations.
2. Vaseline or industrial grease.
2.7 Measuring CO2 1. Instruments of the IRGA system (see Note 6): IRGA console
Production in Fresh and hand piece, respiration chamber, relevant cables, and
Plant Material tubings.
2. Small soda charger (8 g).
3. Soda lime.
4. Desiccant (Drierite, Ohio, USA).
2.8 Measuring 1. Instruments of micro-profiling system (see Notes 6 and 5): O2

Internal pO2 Profile in microsensor, signal amplifier, motorized micromanipulator,
Intact Plant Material calibration chamber, and acquisition software for data logging.
2. Stereoscopic microscope.
3. Solid blocks for plant material stage.
3 Methods
3.1 Preparation of 1. The formation of this gradient requires a gradient maker, which
Density Gradients in its simplest form consists of two cylindrical chambers
connected by a small pipe at their base, a tap to meter outflow
from these chambers, and an outflow pipe from one chamber
(see Fig. 1). Alternatively one can prepare the gradient manually
using pipettes which is not described here. Two solutions are
prepared: a heavy gradient fraction consisting of 28% (v/v)
Percoll and 4.4% (v/v) PVP-40 from a 20% (w/v) stock made
up in buffer, and a light gradient fraction made up of 28% v/v
Percoll in buffer.
2. Pour the light gradient solution (17.5 mL) into the chamber
with no outflow and pour the heavy gradient solution
(17.5 mL) into the chamber with the outflow.
3. Place a magnetic stir bar in the chamber with the outflow, and
rapidly stir the solution. The outflow pipe should be secured
against the inside of a 50 mL centrifuge tube held at a 45 and
on ice.
4. Use a peristaltic pump to pull the solution through the pipes
and into the 50 mL centrifuge tubes. Allow heavy gradient
solution to run until half dispensed. Open connection valve
between chambers, allowing solutions to mix. Allow both
heavy gradient and light gradient solutions to dispense until
the chambers are empty (see Note 7).
Fig. 1 Diagram of the density gradient maker used to prepare gradients for
purification of isolated organelle fractions
3.2 Isolation of Intact 1. Where possible conduct all steps, but especially steps 4–6, in a
Mitochondria [17] 4 C cold room. This greatly aids the fraction of intact
mitochondria.
2. Ensure that all plasticware and glassware are free from deter-
gent (see Note 2). Perform all procedures at 4 C. Solutions,
plasticware, and glassware should be prechilled. Centrifugation
should be performed using a refrigerated centrifuge at 4 C.
3. Harvest seedlings (~50 g), place in a Buchner funnel, and wash
with sterile distilled water (see Note 8).
4. Place washed seedlings in a clean mortar, cover with 300 mL
cell extraction medium, and homogenize tissue with a pestle.
5. Filter extract through two layers of Miracloth to remove starch,
cell debris, and unbroken cells. Lightly wring the Miracloth to
aid filtration.
6. Transfer filtrate to several 50 mL centrifuge tubes and centri-
fuge at 2450 g for 5 min. Transfer supernatant into fresh
centrifuge tubes, taking care not to transfer any of the pellet.
7. Centrifuge the supernatant from step 6 at 17,500 g for
20 min. Use slow braking. Aspirate and discard the supernatant.
8. Add 1 mL mitochondria wash medium to the pellets and gently
resuspend using a small paintbrush. Transfer the resuspended
pellets to two 50 mL centrifuge tubes and add mitochondria
wash medium to each to 40 mL.
9. Repeat steps 7 and 8.
10. Resuspend the final pellets in 1 mL mitochondria wash
medium using a paintbrush as before. This is the organelle
suspension.
11. Using a disposable pipette or autopipette with the tip cut,
carefully transfer the two organelle suspensions onto the sur-
face of two 28% Percoll, 0–4.4% PVP-40 gradients. Centrifuge
at 40,000 g for 40 min. Disengage the centrifuge brake as
rapid deceleration can disturb the contents of the gradient.
12. Towards the bottom of the gradient, mitochondria will form a
white/pale brown band and the thylakoids will form a dark
green band in the upper fraction of the gradient (Fig. 2).
Aspirate and discard the upper fraction of the gradient. Care-
fully transfer the mitochondria band to a fresh 50 mL centri-
fuge tubes.
13. Add mitochondria wash medium to the mitochondria to a total
volume of 40 mL and centrifuge at 31,000 g for 15 min.
Aspirate and discard most of the supernatant, leaving ~3 mL in
the bottom of the tube and ensuring that the pellet is not
disturbed.
Fig. 2 Example of a Percoll gradient after centrifugation showing the position of the intact mitochondria
250 y = -0.0298x + 237.36 Mitochondria
240 Ascorbate
Cytochrome c
230
Triton X
y = -0.0524x + 240.08
220
nmol O2 in chamber
y = -0.0494x + 240.32
210
y = -0.3823x + 321.46
200
190
180
170
160
150
0 60 120 180 240 300 360 420 480
Time (Seconds)
Fig. 3 Example of trace showing the changes in oxygen uptake after the addition of ascorbate and cytochrome
to intact mitochondria, and then the detergent, Triton, to disrupt the membranes, allowing access of the
substrate to cytochrome c oxidase with subsequent detection of enzyme activity by oxygen uptake
14. Repeat step 13 and aspirate the resultant supernatant. Finally,

add 0.5 mL mitochondria wash medium and resuspend gently
with a small paintbrush. This solution contains the isolated
mitochondria (Fig. 3).
3.3 Determination of 1. Fill the chamber of a Clark-type oxygen electrode with 2 mL

Mitochondrial mitochondrial reaction buffer and warm to 25 C.
Integrity, i.e., 2. Calibrate the oxygen electrode by aerating the buffer and
Percentage Intactness setting this value as 100%, and by adding sodium hydrosulfite
and setting this value as 0%.
3. Once calibrated, drain the chamber, flush with water, and fill
the chamber once more with 2 mL mitochondrial reaction
buffer.
4. Determine mitochondrial protein, according to Lowry [18].
5. Add 500 μg of mitochondria (in a volume of 10–40 μL) to the
chamber and close it.
6. Respiration rates are calculated by measuring oxygen saturation
of buffer and dividing by time.
7. Once a base respiration rate is established add 40 μL of 0.5 M
ascorbate using a 50 μL Hamilton syringe to reduce endoge-
nous cytochrome c (rate a).
8. Two minutes later 20 μL 5 mM cytochrome c is added (rate b).
9. Two minutes later add 10 μL 10% Triton X-100 to solubilize
mitochondrial membranes (rate c).
10. The following equation was used to calculate COX activity:
rate c rate a
11. The following equation was used to calculate the proportion of

intact mitochondria:
rate b rate a
100 100
rate c rate a
3.4 Respiratory 1. Fill the chamber of the oxygen electrode with 2 mL mitochon-
Control and Electron drial reaction buffer and warm to 25 C.
Transport Rates 2. Add 500 μg of mitochondria (in a volume of 10–40 μL) to the
chamber and close it.
3. Determine the background rate of oxygen consumption (rate
a).
4. Add 10 μL of each of the respiratory substrates and determine
the rate of oxygen consumption (rate b).
5. Add 10 μL 10 mM ADP. The rate of oxygen consumption
should increase.
6. After a few minutes, the ADP will be depleted, and the rate of
oxygen consumption will be reduced to (rate c). The respira-
tory control ratio is given by
rate c rate a
rate b rate a
7. Add oligomycin to a concentration of μg/ml. The rate of

oxygen consumption should reduce (rate d). Max Leak is
given by
rate c rate d
8. Add titrations of 10 mM FCCP in steps of 0.1 μL

corresponding to a step increase in the final concentration of
0.5 μM FCCP every 2 min. Maximum uncoupled flux (capacity
of the electron transfer system, ETS; rate e) once the rate of
oxygen consumption no longer increases with titrations of
FCCP. Max ETS is given by
rate d
rate e
3.5 O2 Consumption 1. Connect and assemble the instruments of micro-respiration

Rate (See Note 4) system as directed in the manufacturer’s manuals (see Notes 9
and 10).
2. Calibrate the micro-respiration sensors by setting zero oxygen
reading in fully flushed water by 100% N gas, and atmospheric
O2 reading in continuously bubbled water by ambient air. This
step is performed in the calibration chamber.
3. Assemble a stirring magnet, glass ring, nylon mesh, and solid
glass cylinder (if necessary for very small samples) inside the
micro-respiration chamber (see Fig. 4).
4. Proceed to setting up CO2 and pO2 measurement systems.
5. When all measuring systems are ready, start detaching the plant
samples and immediately record the weight and volume.
6. Place the samples onto a thin layer of 0.8% agarose, then cut-
side to fit, and seal the wounding site (see Note 9).
7. Place the sample-on-agar inside the prepared micro-respiration
chamber, put on the rack, and pull down the sensor.
8. Remove all possible source of light.
9. Record O2 depletion in approximately 5–6 min (caution, see
Note 5); the software will ask to input sample and chamber
volumes prior to this.
10. Transfer the sample-on-agar into the CO2 production rate
measurement chamber.
11. Micro-respiration data output could be saved as an Excel sheet
to aid further calculations if required.
3.6 CO2 Evolution 1. Connect and assemble the instruments of IRGA system as
(See Note 4) directed in the manufacturer’s manuals (see Note 11).
Fig. 4 Diagrammatic representation of the micro-respiration chamber
2. Calibrate and inspect that all elements in the IRGA console

and hand piece are working well, and detailed instructions are
available in the manuals. Calibration involves the using of soda
lime and desiccant to set zero CO2 and H2O reading
subsequently.
3. Warm up the system and set the desired experimental condi-
tions, for example: airflow at 100 μmol/m2/s, CO2 concentra-
tion at 380 μmol/mol air, and 55–75% relative humidity. Allow
the system to stabilize in this condition for 10 min.
4. Place the sample-on-agar into the CO2 production rate mea-
surement chamber and log in sample weight.
5. Remove all possible light sources that directly irradiate the CO2
chamber.
6. On the display of IRGA console, pay attention to the “stable”
value and record the respiration rate when this value equals to
1. This suggests that the condition of humidity, CO2, and
airflow are in equilibrium and stable. Keep the fresh weight
(FW in grams) data handy to easily log them into the console
when prompted prior to recording the respiration rates.
7. Remove the sample-on-agar from the CO2 chamber and

immediately place on the sample stage of internal pO2 profiling
system.
8. Data output is also available as a spreadsheet and is readily
transferred from the IRGA console into computer.
3.7 The pO2 Micro- 1. Connect and assemble the instruments of internal pO2 micro-
Profile of Whole profiling system as directed in the manufacturer’s manuals (see
Tissues (See Notes 11 Note 9).
and 12) 2. Prepare a stage to place and hold the sample during internal
pO2 profiling.
3. Position O2 microsensor above the sample stage that there is
enough space for securely swapping the material between
profiling (see Note 9).
4. Establish the microscope to be able to visualize clearly O2
microsensor location relative to the sample (see Fig. 5). This
will greatly aid setting up the start point of each measurement
(e.g., 0 μm).
5. Prior to the measurement, calibrate O2 microsensor similarly as
the calibration for micro-respiration sensors.
Fig. 5 Diagrammatic representation of the use of a microscope to visualize the location of the O2 microsensor
within the tissue under analysis
6. Continue setting up the pO2 micro-profiling system and input

all required information according to the conditions of experi-
ment or instrument being used.
7. Determine the depth of measurement to conduct based on the
material size and purpose of experiment. Use a caliper to
precisely quantify the dimensions (length, width, height, or
diameter) of the material. For example, to measure internal
O2 concentration in a grapevine bud up to the meristem area
[6], diameter of the bud was firstly determined to determine
how deep the O2 microsensor was inserted from the surface.
8. Adjust all parameters to suit the experiment in the profile
settings page of the acquisition software. For instance, to mea-
sure internal O2 in a grapevine bud with 4 mm diameter the
settings would be as follows: start (0 μm), end measurement
(2000 μm), step size (25 μm), safe (1000 μm), sensor angle
(0 ), wait before measure (3 s), measure period (1 s), replicates
[3], delay between (0 s), and number of cycles [1]. See Note 13
for the explanation of key parameters.
9. Place and secure the sample on stage and position O2 micro-
sensor at step one of measurement or, e.g., 0 μm depth, and
then start measurement. The outer surface of the measured
material is commonly considered as the step one.
10. When the final step of measurement is done, save the data in an
Excel format from the software and carefully remove profiled
sample from the stage.
11. Store the plant material in a microtube and keep at 4 C for a
couple of days to visualize O2 microsensor trace inside the
sample. The browning of damaged tissue due to O2 microsen-
sor insertion will be visible against the lighter color of healthy/
undamaged surrounding tissues. However, for tissues with
natural dark color or a combination of dark and light color,
such as the grapevine bud, this could be achieved by preparing
microscope slides of 5 μm sections (see Ref. 6).
3.8 Calculation 1. The respiratory quotient is a dimensionless expression of respi-

of Respiration Rate ratory control, i.e., molar consumption CO2/molar consump-
and Interpretation of tion of O2 [19]. Therefore the units of expression of respiratory
Respiratory Quotient data must be equal.
2. The CO2 production data are acquired as μg(CO2)/g(FW)/
min, which is readily converted to a preferred dry weight
(DW) basis. For RQ, convert these data to mol(CO2)/g
(FW)/min.
3. The O2 consumption data are acquired as μmol/L of O2 con-
centration inside the micro-respiration chamber at the given
times (ms). Firstly, assess all data acquired to determine the linear
range of oxygen consumption (see Note 1). Set the “initial” time
point as the beginning of the linear range and the “final” time
point as the end of the linear range. Determine total O2 con-
sumed by subtracting the O2 concentration at the final point of
recording from the initial. Then, divide total O2 consumed by
the total time between two concentration points; here units are
μmol/L/s. Convert these data to mol(O2)/g(FW)/min.
4. RQ can then be calculated as the ratio between the number of
moles CO2 produced and O2 consumed [19]. This ratio is an
indicator of substrate types used in respiration and the follow-
ing energy usage to support biosynthesis. For example an RQ
of 1.0 suggests the use of glucose as the sole substrate of
respiration and is fully oxidized to CO2 and H2O, in the
absence of biosynthesis. An RQ lower than 1 may indicate the
use of substrates more reduced than glucose (e.g., lipid or
protein); RQ >1 may indicate the use of more oxidized sub-
strates (e.g., organic acids). However, these can be simplistic
assumptions, as the requirement of reducing power for cellular
processes other than primary metabolism may alter the rela-
tionship. However, typical relationships of RQ to substrate and
metabolic control have been demonstrated, e.g., for seed meta-
bolizing predominantly lipids vs. carbohydrates during germi-
nation [20].
3.9 Calculating pO2 Output data files present O2 concentration (μmol/L) at all given
Data and Statistical measurement depth. The data are usually ready to display after
Treatment measurements of soft organs, such as submerged root or germinat-
ing seeds. However, when measuring organs with alternate layers of
hard and soft tissues, e.g., the alternate layers of bracts and hairs in
grapevine buds, some false values or “noise” are sometimes
recorded too. These noises are caused by the tension to the micro-
sensor when penetrating hard tissues after the softer ones. The
generated vibration briefly influences electrical field inside the
microsensor, and is recognized by the single presence of abruptly
peaked reading. In this case, statistical analysis to visualize trends
without removing the genuine reading is preferable. Treating the
data as multivariate is suggested, in which trend visualization as
graphs could be easily generated, for example, by using latticeExtra
package [21] in R statistics software [22].
4 Notes
1. Prevent bubbles from forming in the gradient by stopping the

peristaltic pump once air bubbles are visible towards the end of
the outlet tube.
2. Ensure that all equipment is detergent free, as detergent will
damage the mitochondrial membranes. It is recommended that
plasticware and glassware are cleaned using hot water only and
are dedicated solely to mitochondrial isolations.
3. A common cause of poor extraction of intact mitochondria is a
low ratio of mitochondria extraction medium to the amount of
tissue used. Arabidopsis thaliana leaf cells are highly vacuo-
lated, and upon rupture, the cell volume is substantially
diluted. If an insufficient volume of extraction medium is
used, then the dilution of the osmoticum can lead to the
rupture of mitochondria.
4. Cytochrome c cannot traverse an intact outer mitochondrial
membrane. Therefore, if the outer membrane is intact, there
will be no activity when the membrane is intact and COX will
be 100% latent. Thus, by comparing COX activity in the
absence and presence of the detergent Triton X-100, the per-
centage intactness of the mitochondrial preparation can be
quantified.
5. Measurement of the rate of oxygen consumption in a closed
system assumes linearity between the initial and final time
points used for the calculations above. Thus the optimal
range should be determined prior to experimentation for
each treatment condition or tissue. This may require a short
period of stabilization but should not be delayed unduly; it is
imperative to establish the initial steady state rate of respiration,
as the rate will decline as oxygen becomes limiting and carbon
dioxide builds up in the chamber. This is particularly important
when seeking to measure the respiratory quotient.
6. For the O2 and CO2 gas exchange measurements, it is a must
to use fresh material and to be performed as soon as the sample
is detached as the physiological state of the tissues will change
gradually once they are separated from the rest of the plant.
The use of dry plant material for pO2 is possible but not ideal.
Dehydration often causes brittleness or constriction of the
tissue that could limit accessibility of the O2 microsensor.
When the tissue is too hard to penetrate, it is not impossible
to break the O2 microsensor. However, this pO2 profiling
method is not limited to use only in biological samples, mea-
surement could be performed in any penetrable material such
as in agar medium of plant tissue or bacterial culture, etc.
7. We use the micro-respiration and pO2 micro-profiling systems
from Unisense (Aarhus, Denmark). For the pO2 micro-
profiling, ensure to choose the best diameter size of O2 micro-
sensor outside tip that suits the dimensions of the sample. The
use of motorized micromanipulator is recommended for mea-
surement with fine step sizes of <100 μm.
8. We use an insect respiration chamber attached to a portable
IRGA (gas exchange) system from Li-COR (Lincoln, NB,
USA).
9. The signals of the micro-respiration sensors and O2 microsen-

sors are very small (1013 to 1010 A) and electrical fields may
interfere. It is recommended to switch off any unnecessary
electrical/mechanical equipment and avoid touching the sensor
or wires during measurements. Once connected to the electricity
current, the sensor signal (pA) usually takes a few moments to
stabilize for pre-polarization. Ensure to turn on the system at
least 2 h prior to calibration; this is crucial to remove all built-up
oxygen in the electrolyte when the sensor was not in use. Check
and repeat calibration frequently to ensure that all measurements
are calibrated to correct concentrations.
10. In addition to blocking O2 entry and CO2 exit through any cut
edges, the agar helps to retain sample moisture content during
measurement.
11. Pay extra attention to the fine cables around the hand
piece when removing swapping leaf to insect chamber. The
cables are easily trapped in between metal parts that may
break them.
12. Make sure that there is enough room between the stage and the
tip of O2 microsensor, for the plant material and operator hand
to move freely. This is crucial as often the O2 microsensor tip
was broken when the operator accidentally touched the sensor
when swapping the material in between profiling.
13. For the full explanation of all parameters please refer to the
manual of SensorTrace Profiling software. Here are some key
parameters of internal pO2 profiling in dense plant tissue:
(a) Start (μm): the first depth in which the measurement is to
be taken.
(b) End (μm): the final depth in which measurement is to be
taken. Both A and B are to be decided based on the
experiment purposes and dimensions.
(c) Step size (μm): the vertical step depths by which O2
microsensor is moved from start to end position.
(d) Wait before measure (s): after the O2 microsensor is
moved to a certain depth, it will wait for a period of
seconds before it starts measuring. This period is
important to get the sensor signal stabilized after the
movement. Adjust according to the character of the sam-
ple; harder sample may require longer waiting period.
(e) Measure period (s): duration of the measurement in
each position, with each measurement is an average
value over this period of time. When measuring in a
noisy environment, measuring in a longer period is
recommended.
References
1. Hunt S (2003) Measurements of photosynthe- 12. Gnaiger E (2014) Mitochondrial pathways and
sis and respiration in plants. Physiol Plant respiratory control. An introduction to oxphos
117:314–325 analysis. In: Mitochondr Physiol Network
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blood gas analysis. Iv. Leland Clark’s oxygen Innsbruck
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3. Geigenberger P (2003) Response of plant Ordered assembly of mitochondria during rice
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4. Konnerup D, Moir-Barnetson L, Pedersen O, import apparatus. Plant Mol Biol 60:201–223
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submergence tolerance in two species of stem- (1953) Continuous recording of blood oxygen
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status of the developing seed. New Phytol methods for measuring oxygen consumption
182:17–30 in tumor cells in vitro. Anal Biochem
6. Meitha K, Konnerup D, Colmer TD, Consi- 396:250–256
dine JA, Foyer CH, Considine MJ (2015) 16. Rolletschek H, Weber H, Borisjuk L (2003)
Spatio-temporal relief from hypoxia and pro- Energy status and its control on embryogenesis
duction of reactive oxygen species during bud of legumes. Embryo photosynthesis contri-
burst in grapevine (vitis vinifera). Ann Bot butes to oxygen supply and is coupled to bio-
116:703–711 synthetic fluxes. Plant Physiol 132:1196–1206
7. Sew YS, Stroher E, Holzmann C, Huang S, 17. Millar AH, Liddell A, Leaver CJ (2001) Isola-
Taylor NL, Jordana X, Millar AH (2013) Mul- tion and subfractionation of mitochondria
tiplex micro-respiratory measurements of ara- from plants. Methods Cell Biol 65:53–74
bidopsis tissues. New Phytol 200:922–932 18. Lowry OH, Rosebrough NJ, Farr AL, Randall
8. Sew YS, Millar AH, Stroeher E (2015) Micro- RJ (1951) Protein measurement with the folin
respiratory measurements in plants. Methods phenol reagent. J Biol Chem 193:265–275
Mol Biol 1305:187–196 19. Lambers H, Pons TL, Chapin FS (2008) The
9. Day DA, Neuburger M, Douce R (1985) respiratory quotient. Springer
Biochemical-characterization of chlorophyll- 20. Stiles W, Leach WC (1933) Researches on
free mitochondria from pea leaves. Aust J plant respiration. II.—variations in the respira-
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Chapter 12
Isolation of Mitochondria from Model and Crop Plants

Sandra M. Kerbler and Nicolas L. Taylor
Abstract
The ability to isolate intact and functional mitochondria has greatly deepened our understanding of
mitochondrial structure and function. With the advancement of molecular biology techniques and pro-
gression into omics-based research over recent decades, mitochondrial research has shifted from crop
species such as wheat, pea, and potato to genetically sequenced models such as Arabidopsis thaliana and
rice. Although there are many attributes that make model species particularly appealing for plant research,
they are often less than ideal for conducting biochemical investigations and as such, considerable modifica-
tion to mitochondrial isolation methods has been made.
As the cost of genome sequencing continues to decrease however, an increasing number of crop species
are now being sequenced and with these new resources it appears that the research community is turning
back toward crop research. In this chapter we present mitochondrial isolation methods using density
gradient centrifugation for both model species such as Arabidopsis thaliana, rice, and Medicago and crop
species including wheat, potato, and pea. In addition, we present a number of marker enzyme assays to
confirm mitochondrial purity as well as respiratory assays to determine outer membrane integrity and
respiratory function of isolated mitochondria.
Key words Mitochondrial isolation, Arabidopsis, Rice, Medicago, Potato, Wheat, Pea, Oxygen elec-
trode, Respiratory control ratio, ADP/O ratio
1 Introduction
For more than 50 years, the isolation of plant mitochondria has

deepened our understanding and knowledge of the intricate work-
ing of this important plant organelle [1]. In 1951, Millard, Bonner,
Axelrod, and Bandurski reported the first successful isolation of
mitochondria from plant tissues [2]. Using mitochondria isolated
from etiolated mung bean shoots, Millard et al. not only observed
“complete oxidation of pyruvate to CO2 and water,” but also
showed that this oxidation was tightly coupled with the phosphor-
ylation of ADP. Although mitochondrial isolation techniques have
become increasingly sophisticated over recent decades, with the
115
116 Sandra M. Kerbler and Nicolas L. Taylor
introduction of Percoll gradients to further purify organelles [3]

and the use of free flow electrophoresis [4], core principles such as
using isotonic sucrose solutions to homogenize plant tissue and
differential centrifugation to concentrate organelles remain the
same even today.
From the beginning of plant mitochondrial research, crop
plants such as cauliflower, potato, and beetroot were used for the
isolation of intact, functional mitochondria. As these large hetero-
trophic, homogeneous organs contain low concentrations of con-
taminating organelles (chloroplasts and peroxisomes), large
quantities of highly coupled mitochondria could be obtained,
which in turn fueled our understanding of mitochondrial proteins
and the biochemical processes they carry out. With the advent of
modern genetics however, mitochondrial research turned away
from these traditional plant species and instead focused on newly
sequenced model species such as Arabidopsis thaliana. There are
many features that make Arabidopsis particularly attractive for plant
research: high-quality genome sequences (including those of chlor-
oplasts and mitochondria), many single-gene knockout lines, tools
to produce overexpression and gene-edited lines, a short life cycle,
increasing data on its growth and development, and a large array of
natural variants or ecotypes; however, in terms of conducting bio-
chemical investigations, Arabidopsis is far from ideal. One of the
main problems with this species is that it is relatively small and thus
many hundreds, if not thousands, of plants are needed to produce
adequate amounts of biomass to isolate mitochondria effectively.
Furthermore, Arabidopsis has little non-photosynthetic tissue (root
and seed mass is relatively small compared to total plant mass);
therefore, mitochondria are generally isolated from heterogeneous
shoot tissues that are inherently more difficult to use due to thyla-
koid membrane contamination. To cope with these inherent diffi-
culties, mitochondrial isolation techniques have undergone
considerable adaptation to improve organelle isolation from this
model species [5].
As much as we have gained from Arabidopsis research, the cost
of genome sequencing continues to decrease, and with a greater
number of crop plants being sequenced, the research community
appears to be turning back toward crop research. In this chapter we
present mitochondrial isolation methods using density gradient
centrifugation for model species such as Arabidopsis thaliana
[6, 7], rice [8], and Medicago [9] as well as crop species such as
potato [10], wheat [6, 11], and pea [12]. We also provide a number
of marker enzyme assays to confirm mitochondrial purity and pro-
vide respiratory assays to determine outer membrane integrity and
respiratory function. Furthermore, common challenges and pitfalls
of intact mitochondrial isolation are discussed in detail.
Isolation of Mitochondria from Model and Crop Plants 117
2 Materials
2.1 Density 1. 2 Wash medium: 0.6 M Sucrose, 20 mM TES-KOH, pH 7.5,

Gradients 0.2% (w/v) BSA.
2.1.1 Density Gradients 2. 20% (w/v) PVP-40 solution.
for Arabidopsis Shoot 3. Percoll (GE Healthcare Life Sciences).
Mitochondria 4. Heavy gradient solution: 50% (v/v) 2 Wash medium, 28%
(v/v) Percoll, 22% (v/v) 20% PVP-40 solution.
5. Light gradient solution: 50% (v/v) 2 Wash medium, 28%
(v/v) Percoll.
6. Two-chamber gradient pourer, such as the SG 50 Gradient
Maker (Hoefer).
7. Peristaltic pump, such as the Econo Gradient Pump (Bio-Rad).
8. Magnetic stirrer and stirrer bar.
9. 18-G needle.
10. Ice bucket with 2 L of ice.
2.1.2 Density Gradients 1. 2 Wash medium: 0.6 M Sucrose, 20 mM TES-KOH, pH 7.5,

for Rice Mitochondria 0.2% (w/v) BSA.
2. 20% (w/v) PVP-40 solution.
3. Percoll (GE Healthcare Life Sciences).
4. Heavy gradient solution: 50% (v/v) 2 Wash medium, 28%
(v/v) Percoll.
6. 45% Percoll gradient solution: 50% (v/v) 2 Wash medium,
45% (v/v) Percoll.
Maker (Hoefer).
10. 18-G needle.
2.1.3 Density Gradients 1. 2 Wash buffer: 0.6 M Sucrose, 20 mM MOPS-KOH, pH 7.2.

for Medicago Mitochondria 2. 18% (v/v) Percoll solution.
3. 23% (v/v) Percoll solution.
5. 25 mL Syringe with an 18-G hypodermic needle.
6. Syringe holder.
2.1.4 Density Gradients 1. 2 Wash buffer: 0.6 M Mannitol, 20 mM TES-NaOH,

for Potato Mitochondria pH 7.5, 0.2% (w/v) BSA.
5. 25 mL Syringe with an 18-G hypodermic needle.
6. Syringe holder.

for Wheat Mitochondria 0.2% (w/v) BSA.
(v/v) Percoll.
Maker (Hoefer).
9. 18-G needle.

for Pea Mitochondria 0.2% (w/v) BSA.
(v/v) Percoll.
Maker (Hoefer).
9. 18-G needle.
2.2 Isolation of 1. Mortar and pestle (20 cm) or powered homogenizer (see
Mitochondria Note 1).
2.2.1 Isolation of 2. Plant tissue (see Note 2).
Mitochondria from Young 3. Grinding medium: 0.3 M Sucrose, 25 mM tetrasodium
Arabidopsis Shoots pyrophosphate, 2 mM EDTA, 10 mM KH2PO4, 1% (w/v)
(1–2 Weeks Old) PVP-40, 1% (w/v) BSA, 20 mM ascorbic acid (see Note 3),
5 mM cysteine (see Note 3), pH 7.5.
4. 1 Wash medium (with BSA): 0.3 M Sucrose, 10 mM
TES-KOH, 0.1% (w/v) BSA, pH 7.5.
5. 1 Wash medium (without BSA): 0.3 M Sucrose, 10 mM
TES-KOH, pH 7.5.
6. Acid-washed sand (if using mortar and pestle).
7. Funnel.
8. Miracloth.
9. 1 L Conical flask.
10. Soft artist paintbrush.
2.2.2 Isolation 1. Powered homogenizer (see Note 1).

of Mitochondria from Old 2. Plant tissue (see Note 2).
Arabidopsis Shoots
3. Grinding medium: 0.3 M Sucrose, 60 mM TES-KOH, 25 mM
(4–9 Weeks Old)
tetrasodium pyrophosphate, 2 mM EDTA, 10 mM KH2PO4,
1 mM glycine, 1% (w/v) PVP-40, 1% (w/v) BSA, 50 mM
ascorbic acid (see Note 3), 20 mM cysteine (see Note 3),
pH 8.0.
KH2PO4, 10 mM TES-KOH, 0.1% (w/v) BSA, pH 7.5.
TES-KOH, pH 7.5.
6. Funnel.
7. Miracloth.
2.2.3 Isolation 1. Mortar and pestle (20 cm).

of Mitochondria from Rice 2. Plant tissue (see Note 2).
Shoots/Coleoptiles
3. Grinding medium: 0.3 M Sucrose, 25 mM tetrasodium pyro-
phosphate, 2 mM EDTA, 10 mM KH2PO4, 1% (w/v) PVP-40,
1% (w/v) BSA, 20 mM ascorbic acid (see Note 3), pH 7.5.
4. 1 Wash medium (with BSA): 0.3 M sucrose, 10 mM
5. 1 Wash medium (without BSA): 0.3 M sucrose, 10 mM

TES-KOH, pH 7.5.
6. Funnel.
7. Miracloth.
2.2.4 Isolation 1. Waring blender (see Note 1).

of Mitochondria from 2. Plant tissue (see Note 2).
Medicago Cell Suspension
3. Grinding medium: 450 mM Sucrose, 15 mM MOPS-KOH,
Cultures
1.5 mM EGTA, 0.6% (w/v) PVP-40, 0.2% (w/v) BSA,
0.2 mM phenylmethylsulfonyl fluoride (PMSF), 14.3 mM
β-mercaptoethanol (see Note 3), pH 7.4.
4. Wash medium: 0.3 M Sucrose, 10 mM MOPS-KOH, 1 mM
EGTA, 0.2 mM PMSF, pH 7.2.
5. Vacuum filtration system.
6. Whatman paper.
7. Funnel.
8. Miracloth.
2.2.5 Isolation 1. Juice extractor (see Note 1).

of Mitochondria 2. Plant tissue (see Note 2).
from Potato Tubers
3. Grinding medium: 0.3 M Mannitol, 80 mM tetrasodium pyro-
phosphate, 2 mM EGTA, 10 mM KH2PO4, 1% (w/v) PVP-40,
1% (w/v) BSA, 25 mM cysteine (see Note 3), pH 8.0.
4. 1 Wash medium (with BSA): 0.3 M Mannitol, 10 mM
TES-NaOH, 0.1% (w/v) BSA, pH 7.5.
5. 1 Wash medium (without BSA): 0.3 M Mannitol, 10 mM
TES-NaOH, pH 7.5.
6. Funnel.
7. Miracloth.
9. Peeler.
10. Knife.
11. 500 mL Beaker.

Wheat Leaves and Shoots
1% (w/v) BSA, 20 mM ascorbic acid (see Note 3), 5 mM
cysteine (see Note 3), pH 7.5.
5. 1 Wash medium (without BSA): 0.3 M sucrose, 10 mM
TES-KOH, pH 7.5.
6. Funnel.
7. Miracloth.

Pea Shoots
1% (w/v) BSA, 20 mM ascorbic acid (see Note 3), pH 7.5.
TES-KOH, pH 7.5.
6. Funnel.
7. Miracloth.
2.3 Marker Enzyme 1. Spectrophotometer capable of measuring absorbance at

Assays to Assess 340 nm.
Mitochondrial Purity 2. Fumarase reaction mixture: 70 mM KH2PO4-NaOH, pH 7.7,
2.3.1 Fumarase 0.05% (v/v) triton X-100.
(Mitochondrial) 3. 1 M Malic acid.
2.3.2 Aconitase 1. Spectrophotometer capable of measuring absorbance at

(Mitochondrial) 340 nm.
2. Aconitase reaction mixture: 80 mM HEPES-NaOH, pH 7.5,
0.05% (v/v) triton X-100, 0.5 mM NADP, 0.1 M MnCl2, 2 U
NADP-isocitrate dehydrogenase (ICDH).
3. 0.1 M HEPES-NaOH pH 7.5.

4. 10% (v/v) triton X-100.
5. 20 mM NADP.
6. 0.5 M MnCl2.
7. 2000 U/mL NADP-isocitrate dehydrogenase (ICDH).
8. 0.2 M Aconitate.
2.3.3 1. Spectrophotometer capable of measuring absorbance at

Phosphoribulokinase 340 nm.
(Chloroplastic) 2. 2 Phosphoribulokinase reaction mixture: 200 mM Tris–HCl,
pH 7.8, 20 mM MgCl2, 40 mM KCl, 20 mM DTT.
3. 100 mM ATP in reaction buffer.
4. 100 mM Phospho(enol)pyruvic acid monosodium salt hydrate
in 1 reaction buffer.
5. 10 mM NADH in reaction buffer.
6. 350 U/mL Pyruvate kinase (rabbit muscle).
7. 500 U/mL Lactate dehydrogenase (Lactobacillus
leichmannii).
2.3.4 Catalase 1. Clark-type oxygen electrode, such as Oxytherm (Hansatech).

(Peroxisomal) 2. Catalase reaction mix: 0.3 M Sucrose, 5 mM KH2PO4, 10 mM
TES-KOH, pH 7.2, 10 mM NaCl, 2 mM MgSO4, 0.1% (w/v)
BSA.
3. Sodium hydrosulfite.
4. 1% (v/v) H2O2 solution.
2.4 Determination 1. Clark-type oxygen electrode, such as Oxygraph (Hansatech).

of Mitochondrial 2. Reaction buffer: 0.3 M Sucrose, 10 mM TES, 5 mM KH2PO4,
Membrane Integrity 10 mM NaCl, 2 mM MgSO4, 0.1% (w/v) BSA, pH 7.2.
and Respiratory
3. Stock solutions for cytochrome c oxidase assay: 0.5 M Sodium
Function L-ascorbate, 5 mM cytochrome c (bovine), 10% (v/v) triton
X-100.
4. Stock solutions of respiratory substrates: 500 mM Succinate,
100 mM NADH, 100 mM ATP, 100 mM ADP, 1 M pyruvate,
1 M malate, 100 mM NAD+, 50 mM thiamine pyrophosphate,
12 mM coenzyme A.
3 Methods
To maximize the quality and yield of mitochondrial isolations, a

number of general rules can be applied. To preserve the integrity of
isolated mitochondria, it is essential that all plasticware and
glassware are free from detergent (see Note 4). Materials and solu-
tions are best prepared a day in advance and stored together at 4 C.
Gradients should be prepared just before disruption of plant mate-
rial takes place and then stored on ice until required. During the
isolation procedure, all work should be done in a cold room and all
centrifugation should be done using a refrigerated centrifuge set at
4 C. Minimizing the time spent on any given step of the disruption
of plant material and differential centrifugation will increase both
yield and quality. Generally, more handling steps (centrifugation
spins, pellet resuspensions, and aspirations) have a negative effect
on mitochondrial yield, but often have a positive effect on the
quality of the preparation. Lastly, the use of plant material that is
low in starch content can have a beneficial effect on mitochondrial
integrity. Therefore, starting the isolation before lights on or soon
after is generally preferable to later in the day.
3.1 Preparation The number of gradients required for a given mitochondrial isola-
of Density Gradients tion depends on the amount of plant material used in the disruption
step. A general rule is to use one 35 mL gradient per 25 g FW of
disrupted material for photosynthetic tissues and one per 50 g FW
for non-photosynthetic tissues.
3.1.1 Density Gradients For isolation of mitochondria from Arabidopsis shoots, a single
for Arabidopsis Shoot linear 0–4.4% (w/v) polyvinylchloride (PVP-40) gradient in 28%
Mitochondria (v/v) Percoll is used. Preparation of this gradient requires a two-
chamber gradient pourer, which consists of a perspex block with
two cylindrical chambers linked by a valve that can be closed. Only
one of the chambers has an outflow. Typically, the gradient pourer
is placed on top of a magnetic stirrer and is connected to a peristaltic
pump by small-diameter (2–4 mm) silicon or Tygon® tubing. At
the end of the tubing an 18-G needle is placed, which is then
positioned at the top of a 50 mL centrifuge tube sitting on ice
angled at 45 . To produce consistent gradients, it is important that
the liquid flowing into the centrifuge tube does not drop directly
into the base of the tube, but rather flows down the side of the tube.
This can be easily achieved by securing the needle to the side of the
centrifuge tube with tape. The pump and the tube should also be
held below the level of the gradient pourer so that the solution will
flow by gravity.
1. Assemble apparatus as described above.
2. Close the inter-chamber valve of the gradient pourer, before
placing 17.5 mL of light gradient solution into the chamber
with no outflow.
3. Briefly open the inter-chamber valve to displace any air within
the connecting pipe and to ensure flow.
4. Place 17.5 mL of heavy gradient solution into the chamber
with the outflow.
5. Place a small magnetic stirring bar into the heavy solution and
stir the solution rapidly.
6. Start the peristaltic pump, allowing the heavy gradient solution
to flow into the centrifuge tube.
7. Once there is approximately 1 cm of the heavy solution in the
bottom of the tube, open the inter-chamber valve (you should
see the two solutions mix) and pour the solutions until gradient
formation is complete. The solutions in both chambers should
drop at the same rate.
3.1.2 Density Gradients The 28% (v/v) Percoll and 0–4.4% (w/v) PVP gradient pouring is
for Rice Mitochondria identical to density gradients for Arabidopsis shoot mitochondria (see
Subheading 3.1.1). For the 45% Percoll self-forming gradient, sim-
ply mix together gradient constituents and add to centrifuge tube.
3.1.3 Density Gradients For isolation of mitochondria from Medicago suspension culture
for Medicago Mitochondria cells, a step gradient consisting of 5 mL of 40% (v/v) Percoll over-
laid with 20 mL of 23% (v/v) Percoll and then 10 mL of 18% (v/v)
Percoll is used. All Percoll solutions are made using 2 wash buffer
to a final concentration of 1. Depending on the syringe holder
used, multiple syringes may be set up at once, allowing for a large
number of gradients to be poured relatively quickly. Once again, it
is highly important that the liquid flowing into the centrifuge tube
does not drop directly into the base of the tube, but rather flows
down the side, so that consistent gradients are produced.
1. Place clean 50 mL centrifuge tubes into an ice bucket and angle
at 45 .
2. Directly place 5 mL 40% (v/v) Percoll solution into the centri-
fuge tubes.
3. Place sterile syringes with 18-G needles attached into a syringe
holder. Remove plungers. Syringes should be placed directly
above the centrifuge tubes, such that the needles just touch the
inside lip of the centrifuge tubes.
4. Place 20 mL of 23% (v/v) Percoll solution into the syringes.
The solution should drip through the needle and flow down
the side of the centrifuge tube, forming a second layer above
the 40% (v/v) Percoll layer.
5. Once all of the 23% (v/v) Percoll solution has flown through
the syringe (you may have to use the plunger to push the last of
the solution through), place 10 mL of 18% (v/v) Percoll solu-
tion into the syringe. The solution should once again drip
through the needle and flow down the side of the tube to
form a third layer above the 23% (v/v) Percoll layer.
3.1.4 Density Gradients For isolation of mitochondria from potato, a step gradient consist-
for Potato Mitochondria ing of 8 mL of 50% (v/v) Percoll overlaid with 22 mL of 28% (v/v)
Percoll and then 5 mL of 20% (v/v) Percoll is used. All Percoll
solutions are made using 2 wash buffer to a final concentration of
1. Depending on the syringe holder used, multiple syringes may
be set up at once, allowing for a large number of gradients to be
poured relatively quickly. It is highly important that the liquid
flowing into the centrifuge tube does not drop directly into the
base of the tube, but rather flows down the side of the tube, so that
consistent gradients are produced.
1. Place clean 50 mL centrifuge tubes into an ice bucket and angle
at 45 .
2. Directly place 8 mL 50% (v/v) Percoll solution into the centri-
fuge tubes.
3. Place sterile syringes with 18-G needles attached into a syringe
holder. Remove plungers. Syringes should be placed directly
above the centrifuge tubes, such that the needles just touch the
inside lip of the centrifuge tubes.
4. Place 22 mL of 28% (v/v) Percoll solution into the syringes.
The solution should drip through the needle and flow down
the side of the centrifuge tube, forming a second layer above
the 50% (v/v) Percoll layer.
6. Once all of the 28% (v/v) Percoll solution has flown through
the syringe (you may have to use the plunger to push the last of
the solution through), place 5 mL of 20% (v/v) Percoll solu-
tion into the syringe. The solution should once again drip
through the needle and flow down the side of the tube to
form a third layer above the 28% (v/v) Percoll layer.
for Wheat Mitochondria identical to density gradients for Arabidopsis shoot mitochondria
(see Subheading 3.1.1).
for Pea Mitochondria identical to density gradients for Arabidopsis shoot mitochondria
(see Subheading 3.1.1).
3.2 Isolation The disruption technique may require optimization. Minimizing

of Mitochondria the time taken between the initial disruption of the tissue and
commencement of differential centrifugation is critical as concen-
trations of cellular proteases and damaging vacuolar compounds are
at their highest during this period.
3.2.1 Isolation of 1. Assemble the filter apparatus by placing the funnel into the
Mitochondria from Young neck of the conical flask and line with 2–4 layers of Miracloth.
Arabidopsis Shoots Wet the Miracloth with 100 mL of grinding medium and once
(1–2 Weeks Old) the liquid has run through remove from the conical flask.
2. For disruption of plant material by mortar and pestle: take 40 g

of plant tissue and add to the mortar with a small amount of
grinding medium and acid-washed sand. Start grinding, alter-
nating between small circles in the center of the mortar to
larger circles encompassing the periphery of the mortar.
Throughout the grinding process continue to add grinding
medium until 200 mL has been added to the mortar. Once
the plant material has been reduced to a fibrous grindate, pour
the contents of the mortar into the filter apparatus. This pro-
cess should take no longer than 2 min.
3. Gather the edges of the Miracloth and squeeze residual liquid
into the funnel. This can be quickly and safely achieved by
pressing the contents of the Miracloth against the side of the
funnel.
4. To obtain a greater mitochondrial yield, return residual plant
tissue from the Miracloth to the mortar, regrind for 30 s using
100 mL of grinding medium, and filter.
5. For disruption of plant material by powered homogenizer, cut
the plant tissue to be homogenized into 1 cm pieces. This can
be done with a sharp pair of scissors and directly into the
disruption vessel.
6. Add grinding medium to obtain a ratio of 5 mL buffer to 1 g
tissue.
7. Disruption speed is apparatus dependent: for Ultraturrax®
and Polytron® homogenizers use 50% full speed with a succes-
sion of 2-s bursts. Allowing the material to settle for approxi-
mately 5 s between bursts will enable greater disruption of the
tissue. We generally find that 3–5 bursts give an appropriate
amount of homogenization. Although further homogeniza-
tion may increase mitochondrial yield, this can also lead to a
decrease in mitochondrial quality, so it must be considered
carefully.
8. Pour contents of the disruption vessel into the filter apparatus.
Gather the edges of the Miracloth and squeeze residual liquid
funnel.
9. Distribute the filtrate (obtained from either mortar and pestle
or powered homogenizer) equally into 50 mL centrifuge tubes
and centrifuge at 2500 g for 5 min using a fixed-angle rotor
(Beckman-Coulter JA-25 or equivalent). Set centrifuge decel-
eration to slow.
10. Transfer the supernatant to fresh tubes, taking care not to
disturb the pellet, which is quite loose.
11. Centrifuge the supernatant at 20,000 g for 15 min with

centrifuge deceleration set to slow. Pour off the resulting
supernatant (pellet should be firm and well adhered to the
side of the tube) and discard.
12. Proceed to step 8 of Subheading 3.2.2 below.
3.2.2 Isolation 1. Assemble the filter apparatus by placing the funnel into the
of Mitochondria from Old neck of the conical flask and line with 2–4 layers of Miracloth.
Arabidopsis Shoots Wet the Miracloth with 100 mL of grinding medium and once
(4–9 Weeks Old) the liquid has run through remove from the conical flask.
disruption vessel.
tissue.
4. Disruption speed is apparatus dependent: for Ultraturrax® and
Polytron® homogenizers use 50% full speed with a succession
of 2-s bursts. Allowing the material to settle for approximately
5 s between bursts will enable greater disruption of the tissue.
We generally find that 3–5 bursts give an appropriate amount of
homogenization. Although further homogenization may
increase mitochondrial yield, this can also lead to a decrease
in mitochondrial quality, so it must be considered carefully.
funnel.
6. Distribute the filtrate equally into 50 mL centrifuge tubes and
centrifuge at 2500 g for 5 min using a fixed-angle rotor
eration to slow. Transfer the supernatant to fresh tubes, taking
care not to disturb the pellet, which is quite loose.
centrifuge deceleration set to slow. Pour off the resulting super-
natant (pellet should be firm and well adhered to the side of the
tube) and discard.
8. Resuspend the crude organellar pellet using a soft artist paint-
brush dipped in 1 wash medium (with BSA).
9. Transfer resuspended pellets to two fresh 50 mL centrifuge
tubes and add wash medium to each to 40 mL.
11. Resuspend the final pellets using a soft artist paintbrush dipped
in 1 wash medium (with BSA) as before. Add 1 mL 1 wash
medium (with BSA) to each tube and mix briefly by swirling.
You now have an organelle suspension. It is important to
complete these first steps and move onto density gradient
centrifugation as quickly as possible. Carefully pipette the
organelle suspensions onto the surface of two pre-prepared
28% Percoll and 0–4.4% PVP-40 gradients. Centrifuge in a
fixed-angle rotor at 40,000 g for 40 min with the centrifuge
brake off. It is important that the centrifuge brake is disen-
gaged at the end of the run as rapid deceleration can disturb the
gradient.
12. Mitochondria will form a pale yellow/brown band toward the
bottom of the gradient, while broken thylakoids and intact
chloroplasts will be apparent as dark and light green bands
(respectively) in the upper part of the gradient (Fig. 1). Aspi-
rate the upper part of the gradient to waste. Using a Pasteur
pipette, carefully transfer the mitochondrial band to a fresh
50 mL centrifuge tube, taking care not to collect the very
dense matter pooled at the very bottom of the tube.
13. Fill tubes with 1 wash medium (without BSA) and centrifuge
at 31,000 g for 10 min, with centrifuge deceleration set to
slow. Remove and discard the supernatant by aspiration. Take
care not to disturb the pellet as it will be very loose. Also, it is
not necessary to remove all of the supernatant—it is fine to
leave 3–4 mL at the bottom of the tube.
14. Repeat step 13 twice for a total of three washes. For the last
wash, combine all tubes into one and centrifuge with a 1
Fig. 1 Appearance of organellar bands on different density gradients following

centrifugation. (a) Linear 0–4.4% (w/v) PVP and 28% (v/v) Percoll gradient. (b)
45% (v/v) Percoll gradient. (c) 40/23/18% (v/v) Percoll step gradient. (d) 50/28/
20% (v/v) Percoll step gradient
wash medium balance. Remove and discard the supernatant by

aspiration. Add 0.5 mL of 1 wash medium (without BSA)
and gently resuspend the pellet with a soft paintbrush. These
are your isolated hydroponic Arabidopsis shoot mitochondria.
of Mitochondria from Rice neck of the conical flask and line with 2–4 layers of Miracloth.
Shoots/Coleoptiles Wet the Miracloth with 100 mL of grinding medium and once
the liquid has run through remove from the conical flask.
2. Cuts rice shoots or coleoptiles into 5–10 mm pieces using sharp
scissors. Take 100 g of plant tissue and add to the mortar with a
small amount of grinding medium and acid-washed sand. Start
grinding, alternating between small circles in the center of the
mortar to larger circles encompassing the periphery of the
mortar. Throughout the grinding process continue to add
grinding medium until 200 mL has been added to the mortar.
Once the plant material has been reduced to a fibrous grindate,
pour the contents of the mortar into the filter apparatus. This
process should take no longer than 2 min.
3. Gather the edges of the Miracloth and squeeze residual liquid
funnel.
4. Distribute the filtrate equally into 50 mL centrifuge tubes.
5. Centrifuge at 1000 g for 5 min using a fixed-angle rotor
centrifuge deceleration set to slow. Pour off the resulting super-
natant (pellet should be firm and well adhered to the side of the
tube) and discard.

gradient.
12. Fill the tube with 1 wash medium (with BSA) and divide into
six centrifuge tubes.
13. Centrifuge at 20,000 g for 15 min, with centrifuge decelera-
tion set to slow. Remove and discard the supernatant by aspi-
ration. Take care not to disturb the pellet as it will be very
loose. Also, it is not necessary to remove all of the
supernatant—it is fine to leave 3–4 mL at the bottom of the
tube.
14. Combine the six tubes into one and repeat step 13.
15. Remove and discard the supernatant by aspiration.
16. Add 1–2 mL of 1 wash medium (with BSA) and gently
resuspend the pellet with a soft paintbrush.
17. Carefully pipette the mitochondrial/peroxisome suspension
onto the surface of 45% (v/v) Percoll gradient. Centrifuge in
a fixed-angle rotor at 30,000 g for 30 min with the centri-
fuge brake off.
18. Mitochondria will form a pale yellow/brown band at the top of
the gradient, while peroxisomes will be apparent toward the
bottom of the gradient (Fig. 1). Using a Pasteur pipette,
carefully transfer the mitochondrial band to a fresh 50 mL
centrifuge tube.
19. Fill the tube with 1 wash medium (without BSA) and divide
into six centrifuge tubes.
tion set to slow. Remove and discard the supernatant by aspira-
tion. Take care not to disturb the pellet as it will be very loose.
Also, it is not necessary to remove all of the supernatant—it is
fine to leave 3–4 mL at the bottom of the tube.
22. Remove and discard the supernatant by aspiration. Add 0.5 mL
of 1 wash medium (without BSA) and gently resuspend the
pellet with a soft paintbrush. These are your isolated rice
shoot/coleoptile mitochondria.
of Mitochondria from neck of the conical flask and line with 2–4 layers of Miracloth.
Medicago Cell Suspension Wet the Miracloth with 100 mL of grinding medium and once
Cultures the liquid has run through remove from the conical flask.
2. Separate cells from culture medium by vacuum filtration using
Whatman filter paper. Weigh cells.
3. Transfer up to 150 g FW of cells to a 500 mL Waring blender
vessel. Add grinding medium (at least 2 volumes of extraction
medium per weight of cells (see Note 5)) and blend two times
for 30 s at maximum speed followed by one grinding step for
30 s at minimum speed, with 30-s breaks between grinding
steps to prevent excess foaming and heating.
4. Pour contents of the blender into the filter apparatus. Gather
the edges of the Miracloth and squeeze residual liquid into the
funnel. This can be quickly and safely achieved by pressing the
contents of the Miracloth against the side of the funnel.
5. Transfer filtrate to 250 mL centrifuge tubes and centrifuge at
2700 g for 5 min using a fixed-angle rotor (Beckman-
Coulter JA-14 or equivalent). Set centrifuge deceleration to
slow. Gently pour the resulting supernatant into fresh centri-
fuge tubes, taking care not to disturb the pellet, which is quite
loose.
6. Centrifuge the supernatant at 8300 g for 5 min with centri-
fuge deceleration set to slow. Once again, gently pour the
resulting supernatant into fresh centrifuge tubes, taking care
not to disturb the pellet, which is quite loose.
7. Centrifuge the resulting supernatant at 17,000 g for 10 min,
with centrifuge deceleration set to slow. Pour off and discard
the resulting supernatant (pellet should be firm and well
adhered to the side of the tube).
brush dipped in 1 wash medium. You now have an organelle
suspension. It is important to complete steps 1–7 and move
onto density gradient centrifugation as quickly as possible.
Carefully pipette the organelle suspensions onto the surface
of the 40/23/18% (v/v) Percoll step gradient, using a little
1 wash medium to rinse out each tube. The organelle suspen-
sion from up to ten cell cultures can be loaded onto a single
gradient. Centrifuge in an ultracentrifuge at 70,000 g for
90 min with the centrifuge brake off. It is important that the
centrifuge brake is disengaged at the end of the run as rapid
deceleration can disturb the gradient.
9. Mitochondria will form a pale yellow/brown band at the inter-
face of the 23 and 40% Percoll phases (Fig. 1). Peroxisomes will
form a band just underneath, but this is usually not visible.
Plastid membranes appear as a bright yellow band at the 18 and

23% Percoll interface. Aspirate the upper part of the gradient to
waste. Using a Pasteur pipette, carefully transfer the mitochon-
drial band to a fresh 50 mL centrifuge tube, taking care to avoid
the peroxisomal band below.
10. Fill the tube with 1 wash medium and centrifuge at
14,500 g for 10 min, with centrifuge deceleration set to
slow. Remove and discard the supernatant by aspiration. Take
care not to disturb the pellet as it will be very loose. Also, it is
not necessary to remove all of the supernatant—it is fine to
leave 3–4 mL at the bottom of the tube.
11. Repeat step 10. Remove and discard the supernatant by aspi-
ration (the pellet should be firm at this stage). Add 0.5 mL of
1 wash medium and gently resuspend the pellet with a soft
paintbrush. These are your isolated Medicago suspension cell
culture mitochondria.
of Mitochondria neck of the conical flask and line with 2–4 layers of Miracloth.
from Potato Tubers Wet the Miracloth with 100 mL of grinding medium and once
2. Peel and chop potatoes.
3. Using a juice extractor, grind potatoes into a 500 mL beaker
containing 100 mL of grinding medium. Continue grinding
with stirring until the volume is 500 mL and set aside for starch
to settle.
4. Pour contents of beaker into the filter apparatus, being careful
not to pour the large starch sediment at the bottom. Gather the
edges of the Miracloth and squeeze residual liquid into the
funnel. Do not squeeze the Miracloth too much to avoid
transferring excess starch.
5. Transfer filtrate to 250 mL centrifuge tubes and centrifuge at
1500 g for 5 min using a fixed-angle rotor (Beckman-Coulter
JA-14 or equivalent). Set centrifuge deceleration to slow. Gently
pour the resulting supernatant into fresh centrifuge tubes, taking
centrifuge deceleration set to slow. Pour off and discard the
resulting supernatant (pellet should be firm and well adhered to
the side of the tube).
organelle suspensions onto the surface of the 50/28/20%
Percoll step gradient. Centrifuge in a fixed-angle rotor at
40,000 g for 45 min with the centrifuge brake off. It is
important that the centrifuge brake is disengaged at the end
of the run as rapid deceleration can disturb the contents of the
gradient.
11. Mitochondria will form a pale yellow/brown band at the inter-
face of the 28 and 50% Percoll phases (Fig. 1). Plastid mem-
branes appear as a bright yellow band at the 18 and 23% Percoll
interface. Aspirate the upper part of the gradient to waste.
Using a Pasteur pipette, carefully transfer the mitochondrial
band to a fresh 50 mL centrifuge tube, taking care not to
collect the very dense matter pooled at the very bottom of
the tube.
12. Fill the tube with 1 wash medium (without BSA) and centri-
fuge at 15,000 g for 15 min, with centrifuge deceleration set
to slow. Remove and discard the supernatant by aspiration.
Take care not to disturb the pellet as it will be very loose.
13. Repeat step 12. Remove and discard the supernatant by aspi-
ration (the pellet should be firm at this stage). Add 0.5 mL of
1 wash medium and gently resuspend the pellet with a soft
paintbrush. These are your isolated potato mitochondria.
14. Additionally, 10–20 mg/mL potato mitochondria in 1 wash
medium (without BSA) can be supplemented with 5% (v/v)
dimethyl sulfoxide (DMSO), frozen in liquid nitrogen in
0.5 mL aliquots and stored at 80 C to maintain mitochon-
drial activity and coupling.
from Wheat Shoots Wet the Miracloth with 100 mL of grinding medium and once
disruption vessel.
tissue.

funnel.
gradient.
50 mL centrifuge tube, taking care not to collect the very dense

matter pooled at the very bottom of the tube.
13. Fill the tube with 1 wash medium (without BSA) and centri-
fuge at 31,000 g for 15 min, with centrifuge deceleration set
to slow. Remove and discard the supernatant by aspiration.
Take care not to disturb the pellet as it will be very loose.
14. Repeat step 13 twice for a total of three washes. Remove and
discard the supernatant by aspiration. Add 0.5 mL of 1 wash
medium (without BSA) and gently resuspend the pellet with a
soft paintbrush.
from Pea Shoots Wet the Miracloth with 100 mL of grinding medium and once
2. Cut off the pea leaves with a sharp pair of scissors and add them
directly into the disruption vessel.
tissue.
funnel.

gradient.
13. Fill the tube with 1 wash medium (without BSA) and divide
into six centrifuge tubes.
tion set to slow. Remove and discard the supernatant by aspi-
ration. Take care not to disturb the pellet as it will be very
loose. Also, it is not necessary to remove all of the
supernatant—it is fine to leave 3–4 mL at the bottom of the
tube.
16. Remove and discard the supernatant by aspiration. Add 0.5 mL
of 1 wash medium (without BSA) and gently resuspend the
pellet with a soft paintbrush.
3.3 Marker Enzyme During isolation of mitochondria most contamination occurs as co-
Assays to Assess purification of similarly dense chloroplasts and peroxisomes. By
Mitochondrial Purity profiling the activity of marker enzymes that are known to be
localized in other cellular compartments, we can determine the
purity of isolated mitochondria.
3.3.1 Fumarase 1. Add mitochondrial protein sample (10–100 μg protein) to

(Mitochondrial) 900 μL reaction master mix.
2. Add malate to start reaction to a final concentration of 50 mM.

3. Progression of the reaction is measured directly at 340 nM
(ε ¼ 2.55 mM1).
3.3.2 Aconitase 1. Add mitochondrial protein sample (10–100 μg protein) to

(Mitochondrial) 900 μL reaction master mix.
2. Add aconitate to start reaction to a final concentration of 8 mM
(see Note 6).
3. Progression of the reaction is measured as NADP reduction to
NADPH at 340 nM (ε ¼ 6.22 mM1).
3.3.3 Phosphoribulo- 1. Pipette 0.5 mL 2 reaction buffer and 5–20 μL of isolated

kinase (Chloroplastic) intact chloroplasts in a 1 mL cuvette.
2. 2 Reaction buffer 200 mM Tris–HCl (pH 7.8), 20 mM
MgCl2, 40 mM KCI, 20 mM DTT
3. Add 2 mM ATP, 2 mM phosphoenol pyruvate, 0.2 mM
NADH, 3.5 U/mL pyruvate kinase, and 5 U/mL lactate
dehydrogenase.
4. Add H2O to 975 μL and mix.
5. Monitor the A340 nm until constant.
6. Perform a baseline correction.
7. Add 25 μL 20 mM ribulose-5-phosphate (Ru5P) (final con-
centration 0.5 mM) to initiate the reaction.
8. Activities are assayed by following the oxidation of NADH at
340 nm (ε ¼ 6.22 mM1).
3.3.4 Catalase 1. Catalase activity can be measured in an oxygen electrode, by

(Peroxisomal) monitoring the production of oxygen after the addition of
H2O2.
2. Set up the electrode according to the manufacturer’s instruc-
tions using 50% (w/v) saturated KCl as an electrolyte and
calibrate between air-saturated water (253 nmol O2 mL1 at
25 C) and zero (established by adding a few crystals of sodium
hydrosulfite to the water) (see Note 7).
3. The assay should be conducted at 25 C. To 1 mL of mito-
chondrial reaction medium add 5–20 μL of mitochondrial
suspension (see Note 8).
4. Add 4 μL of 1% (v/v) H2O2 and monitor oxygen evolution.
Rates of oxygen evolution should be calculated as nmol O2
min1 mg1 protein. Typical rates for highly purified peroxi-
somes are ~14 μmol O2 min1 mg1 protein.
In addition to marker enzyme assays, purity of isolated mito-
chondria can be assessed by Western blotting of proteins of known
subcellular localization. For example, Rubisco can be used as a

chloroplast marker, KAT2 as a peroxisomal marker, and Porin
(VDAC1) as a mitochondrial marker. Although Western blotting
is still regarded as the “gold standard” for protein quantitation,
identification and absolute quantitation of proteins via mass spec-
trometry (MS) techniques such as selective reaction monitoring
(SRM) are becoming increasingly utilized. Although conceptually
similar to Western blotting, SRMs produce data of a higher quality
that can be easily verified by a fragment ion spectrum [13]. Fur-
thermore, for many of the performance characteristics underlying
each method, such as the limits of detection, linear dynamic range,
ability to multiplex, and reproducibility, MS-based methods now
outperform Western blotting [13]. To date however, only limited
series of SRM marker proteins and their transitions have been
compiled for Arabidopsis [14].
3.4 Determination Latency tests are commonly used to determine the proportion of
of Mitochondrial broken mitochondria in a sample. One test is the cytochrome c
Membrane Integrity oxidase assay, which relies on the impenetrable nature of added
cytochrome c to an intact outer mitochondrial membrane. Cyto-
chrome c oxidase (COX) is an inner membrane respiratory complex
(complex IV) and requires reduced cytochrome c in the intermem-
brane space as a substrate. As cytochrome c is too large to cross an
intact outer mitochondrial membrane, intact mitochondria incu-
bated in reduced cytochrome c should have a respiration rate close
to zero. Thus by comparing COX activity before and after addition
of nonionic detergents such as triton X-100, an estimation of outer
membrane integrity can be obtained.
1. Set up a Clark-type oxygen electrode according to the manu-
facturer’s instructions. Using saturated KCl (50% w/v) as elec-
trolyte, calibrate the electrode between air-saturated water
(253 nM O2 mL1 at 25 C) and zero (water with a small
quantity of sodium hydrosulfite) (see Note 7).
2. Resuspend 100 μg of mitochondria in 1 mL of reaction buffer
and place into the reaction chamber of the oxygen electrode. At
25 C, add the following respiratory substrates, waiting for a
linear rate of oxygen consumption to be established each time:
(a) 20 μL of 0.5 M sodium L-ascorbate (see Note 9).
(b) 10 μL of 5 mM cytochrome c.
(c) 5 μL of 10% (v/v) triton X-100.
COX activity is given by [rate (c) rate (a)]. Mitochondrial
outer membrane integrity is given by

rate ðb Þ rate ða Þ
1 100:
rate ðc Þ rate ðaÞ
3.5 Measurement The ratio between ADP-stimulated and ADP-exhausted respiration

of Mitochondrial is known as the respiratory control ratio (RCR) and indicates the
Respiratory Function tightness of coupling between the consumption of oxygen and the
phosphorylation of ADP. High RCRs (>2.5) imply a high capacity
for substrate oxidation and low proton leak and are an indication of
good-quality mitochondrial preparations.
1. Set up a Clark-type oxygen electrode according to the manu-
facturer’s instructions. Using saturated KCl (50% w/v) as elec-
trolyte, calibrate the electrode between air-saturated water
(253 nM O2/mL at 25 C) and zero (water with a small
quantity of sodium hydrosulfite) (see Note 7).
2. Resuspend 100 μg of mitochondria in 1 mL of reaction buffer
and place into the reaction chamber of the oxygen electrode. At
25 C, determine the background rate of oxygen consumption
(rate a).
3. Add one of the substrate combinations (all concentrations are
final):
(a) 1 mM NADH
(b) 5 mM Succinate, 0.5 mM ATP
(c) 10 mM Pyruvate, 10 mM malate, 2 mM NAD+, 0.2 mM
thiamine pyrophosphate, 12 μM coenzyme A and deter-
mine the rate of oxygen consumption. Add 1 μL of
100 mM ADP.
Measure the initial linear rate increase (rate b). When the ADP-
related oxygen consumption rate increase has expired, measure
this rate (rate c). RCR can be calculated by

rate ðc Þ rate ða Þ
RCR ¼
rate ðb Þ rate ða Þ
Additionally, ADP/O ratios express the relationship between

ATP synthesis and oxygen consumption during ADP-stimulated
respiration. It measures the efficiency of oxidative phosphorylation,
with higher values indicating that ADP phosphorylation is efficient
per unit of oxygen reduced. ADP/O ratios are calculated by divid-
ing the amount of ADP that was phosphorylated by the amount of
oxygen that was reduced to water. The moles of oxygen atoms (O)
is twice the moles of oxygen molecules, so if 50 nmol of O2 were
consumed, this would equate to 100 nmol of O consumed. Some-
times the transitions in and out of ADP-stimulated respiration are
not “sharp,” and so it can be difficult to precisely measure oxygen
concentrations at the beginning and end of this state. In these cases,
it is best to use “lines of best fit” over the top of the trace before,
after, and during ADP-stimulated respiration. Then, one can calcu-
late where these lines intersect to estimate where ADP-stimulated
respiration began and ended (this can be done electronically or with

paper and ruler). Maximum ADP/O ratios are usually about 2.5 for
complex I-linked substrates, while succinate produces ADP/O
ratios that are slightly lower at about 1.5.
4 Notes
1. Homogenizer types vary greatly; however most are applicable

to the disruption of plant cells. Our laboratory uses Waring-
type blenders, juice extractors, Kinematica™ Polytron®, Ika™
Ultra Turrax®, and Bullet-type blenders with various dispersing
attachments as well as mortar and pestles. In general, material
collected from plants less than 2 weeks old is better suited to
gentle homogenization techniques such as a mortar and pestle,
while larger, older, fibrous, and waxy tissues are better suited to
powered homogenization.
2. Common starting material for small-scale isolations (2–5 mg
mitochondrial protein) is 40–100 g FW of seedlings/leaves/
shoots. Large-scale preparations (100–300 mg mitochondrial
protein) can be made from 500 g FW suspension culture cells
or 5–10 kg potato tubers. Generally, younger plant tissues
contain greater concentrations of mitochondria and lower con-
centrations of interfering compounds such as phenolics. There-
fore, when selecting plants to isolate mitochondria from make
sure to select plant material that is healthy and not too old. The
following references may be used as guides for growing plants:
Arabidopsis [5, 15], rice [8] Medicago [9, 16], wheat [11], and
pea [12].
3. Reductants (such as ascorbic acid, cysteine, and β-mercap-
toethanol) should be added to the chilled grinding medium
immediately before use, correcting the pH after addition.
4. It is recommended that a dedicated stock of detergent-free
glass and plasticware be kept solely for mitochondrial isola-
tions. Buy new glass and plasticware for this purpose and
clean using hot water only.
5. There is a trade-off between preserving mitochondria by using
large volumes of grinding medium to dilute damaging com-
pounds and greater efficiency in tissue disruption by using
smaller volumes. For this reason, tissue disruption should be
done rapidly in a small volume to which additional grinding
medium is then added to minimize mitochondrial damage.
6. Aconitase activity can be determined through the measurement
of isocitrate production from citrate. Isocitrate production rate
is measured by the activity of an isocitrate- and NADP-
dependent enzyme. Aconitase contains an Fe-S center that is
readily damaged by H2O2-inhibiting activity of the protein,

and the protein itself has been shown to be degraded during
prolonged oxidative stress.
7. The performance of oxygen electrodes deteriorates over time
due to electrochemical deposition of chloride and oxide salts
on the silver anode. It is therefore necessary to periodically
clean the anode using aluminum oxide polishing paste.
8. It is important not to add too much mitochondrial suspension;
otherwise the rate of oxygen consumption will exceed the
response time of the oxygen electrode. It is therefore advisable
to test several different concentrations of mitochondrial sus-
pension and choose one in which the oxygen consumption rate
in the presence of substrate gives an appropriate rate.
9. Sodium L-ascorbate solutions oxidize rapidly and so should be
made up fresh on the day of use.
Acknowledgements
This work was supported by the ARC Centre of Excellence in Plant

Energy Biology (CE140100008) and N.L.T. as an Australian
Research Council Future Fellow (FT13010123). SMK is sup-
ported by an Australian Postgraduate Award and Grains research
and Development Corporation (GRDC) Graduate Research
Scholarship.
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electrophoresis for purification of plant mito- legume Medicago truncatula. Biochim Biophys
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5. Sweetlove LJ, Taylor NL, Leaver CJ (2007) 10. Millar AH, Knorpp C, Leaver CJ et al (1998)
Isolation of intact, functional mitochondria Plant mitochondrial pyruvate dehydrogenase
from the model plant Arabidopsis thaliana. complex: purification and identification of cat-
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Chapter 13
Procedures of Mitochondria Purification and Gene

Expression to Study Alternative Respiratory
and Uncoupling Pathways in Fruits
Jurandi Gonçalves de Oliveira, Luis Miguel Mazorra Morales,
Gláucia Michelle Cosme Silva, Kátia Daniella da Cruz Saraiva,
Diederson Bortolino Santana, Clesivan Pereira dos Santos,
Marcos Goes Oliveira, and José Hélio Costa
Abstract
We describe detailed procedures to get intact and well-coupled mitochondria from a variety of fruit species
such as papaya (Carica papaya), guava (Psidium guajava), tomato (Solanum lycopersicum), and strawberry
(Fragaria x ananassa) as well as the protocols to assess the capacities of AOX and UCP pathways in intact
fruit mitochondria. The procedures presented here were tested for the species mentioned above; their use
with other types of fruits must be tested for yield of intact and active mitochondria. This is possible from
individual adjustments. Strict care during extraction, including the use of osmotic protectants (i.e.,
mannitol/sucrose) and antioxidants (i.e., cysteine, ascorbate) at defined concentrations, are critical factors
to ensure mitochondrial integrity and to obtain higher yields. The mitochondria purified using the
discontinuous Percoll gradients described here can be used for the analysis of the capacity of alternative
respiration and uncoupling pathways in fruits. In addition, protocols for quantitative and semiquantitative
PCR applicable for the analysis of AOX and UCP gene expression in fruits are shown. Microarray and RNA-
seq data from public databases are also valuable for the analysis of AOX and UCP genes. In both cases
having the sequences of genes or cDNAs to be used in primer design or probe identification is necessary.
Key words Gene identification, Mitochondria isolation, Respiratory control ratio, RNA extraction,
Semiquantitative RT-PCR
1 Introduction
Mitochondria play an essential role in the regulation of bioenergetics

during fruit growth, ripening, and senescence. Fruit mitochondria
possess two energy-dissipating systems: the alternative oxidase
(AOX) and the uncoupling protein (UCP). To understand the
contribution of these pathways, mitochondria often need to be sepa-
rated from other cellular organelles (chloroplasts, peroxisomes, etc.)
143
144 Jurandi Gonçalves de Oliveira et al.
in an uncontaminated and functional form. The purification of

mitochondria from several fruit species to homogeneity poses
significant challenges due to the complexity of the chemical compo-
sition (abundance of phenols, sugars, etc.). The conditions for
isolation and purification in the discontinuous Percoll gradient
should be adjusted, depending on the type of fruit and even the
particular genotype of a fruit species. Because of these facts, it is
often the case that mitochondria are prepared under particular
extraction conditions.
With the advent of the genomic era, an impressive number of
genomes of angiosperm species have been sequenced and can be
used to retrieve the AOX and UCP genes to design primers or to
obtain probes applied in expression studies. These data are available
in public databases such as GenBank (NCBI) (http://www.ncbi.
nlm.nih.gov) and Phytozome (https://phytozome.jgi.doe.gov/
pz/portal.html). A recent in-depth search of GenBank using the
name “angiosperms” against the database “genome” (http://www.
ncbi.nlm.nih.gov/genome) returned 160 different species. In
addition, searches in phytozome databank showed 43 genomes
from angiosperm species. These available genomes include several
fruits species such as: Actinidia chinensis (kiwifruit), Ananas como-
sus (pineapple), Carica papaya (papaya), Citrus sinensis (orange),
Citrus clementina (clementine), Citrullus lanatus (watermelon),
Cucumis melo (melon), Cucumis sativus (cucumber), Fragaria
vesca (wild strawberry), Malus domestica (apple), Morus notabilis
(mulberry), Musa acuminata (banana), Prunus persica (peach),
Prunus mume (japanese apricot), Pyrus bretschneideri (pear), Sola-
num lycopersicum (tomato), Vitis vinifera (Grape), and Ziziphus
jujuba (jujube).
We describe detailed procedures to get intact and well-coupled
mitochondria from a diversity of fruit species such as papaya
(Carica papaya), guava (Psidium guajava), tomato (Solanum lyco-
persicum), and strawberry (Fragaria x ananassa) as well as the
protocols to assess the capacities of AOX and UCP pathways in
intact mitochondria. Some methods that could be applied to ana-
lyze the expression of AOX and UCP genes, such as quantitative
and semiquantitative PCR, microarrays, and RNA-seq, were also
presented.
2 Requirements
A. Extraction and purification of mitochondria and measure-

ments of capacity of alternative oxidase and uncoupling path-
ways in fruits
Procedures of Mitochondria Purification and Gene Expression to Study. . . 145
2.1 Buffers 1 M MOPS: dissolve 41.85 g MOPS in ultrapure water to a final

and Reagents volume of 200 mL.
2.1.1 Stock Solutions 0.1 M EDTA: dissolve 5.85 g EDTA in ultrapure water and com-
plete to a final volume of 200 mL.
1 M K2HPO4: dissolve 17.41 g K2HPO4 in a final volume of
100 mL of ultrapure water.
1 M KH2PO4: dissolve 13.61 g KH2PO4 in a final volume of
100 mL of ultrapure water.
0.1 M Phosphate buffer: combine 71.7 mL of 1 M K2HPO4 and
28.3 mL of 1 M KH2PO4. Adjust to pH 7.2 if needed.
1 M KCl: dissolve 7.45 g KCl in ultrapure water to a final volume of
100 mL.
1 M MgCl2: dissolve 9.52 g MgCl2 in ultrapure water to a final
volume of 100 mL.
1. Extraction buffer (1000 mL) used for papaya fruit (Golden
cultivar, a Solo-related papaya genotype)
50 mM MOPS (pH 7.4), 0.35 M mannitol, 3 mM EDTA, 0.4%
(w/v) PVP-25, 0.1% (w/v) defatted BSA, 8 mM
cysteine**
To make 1000 mL, combine 50 mL of 1 M MOPS, 63.77 g
mannitol, 30 mL of 0.1 M EDTA, 4 g PVP-25, 1 g
defatted BSA. Add 0.97 g cysteine (just before use) and
adjust the pH of solution to 7.4 using 1 M KOH. Com-
plete with ultrapure water to a final volume of 1000 mL.
**
(a) The use of 0.35 M mannitol was replaced by
0.6 M and 0.7 M sucrose in the Tainung01 and
UENF/Caliman01, respectively, increasing the
coupling of mitochondria in Formosa-related
papaya cultivars;
(b) For tomato mitochondria, use 0.4 M sucrose instead
of 0.35 M mannitol. In addition, add a higher EDTA
concentration (8 mM EDTA) and a lower cysteine
concentration (4 mM cysteine);
(c) For strawberry mitochondria, use 0.4 M sucrose
instead of 0.35 M mannitol.
2. Extraction buffer (1000 mL) used for guava fruit (Psidium
guajava L.) cultivars “Paluma” or “Cortibel”
100 mM MOPS (pH 7.4), 0.3 M sucrose, 3 mM EDTA,
0.4% (w/v) PVP-25, 0.1% (w/v) defatted BSA, 8 mM
cysteine.
sucrose, 30 mL of 0.1 M EDTA, 4 g PVP-25, 1 g defatted
BSA. Add 0.97 g cysteine (just before use) and adjust the
pH of solution to 7.4 using 1 M KOH. Complete with
ultrapure water to a final volume of 1000 mL.
3. Washing buffer (200 mL)
10 mM MOPS (pH 7.2), 0.35 M mannitol¥, 0.5 mM EDTA,
0.1% (w/v) defatted BSA.
mannitol, 1 mL of 0.1 M EDTA, 0.2 g defatted BSA.
Adjust pH to 7.2 with 1 M KOH and add ultrapure
water to a final volume of 200 mL.
¥
use 0.4 M sucrose instead of 0.35 M mannitol for tomato and
strawberry mitochondria.
2.2 Percoll Solutions 1. MOPS buffer solution (20 mL)

20 mM MOPS (pH 7.2), 0.6 M mannitol, 0.5% (w/v) defatted
BSA.
To make 20 mL, combine 0.4 mL of 1 M MOPS, 2.18 g
mannitol, 0.1 g defatted BSA. Adjust pH to 7.2 with 1 M
KOH and add ultrapure water to a final volume of 20 mL.
This solution is used to prepare Percoll gradients.
2. Percoll (13.5%) solution
10 mM MOPS, 13.5% (v/v) Percoll, 0.3 M mannitol, 0.25%
(w/v) defatted BSA.
To make 5 mL, combine 2.5 mL of MOPS buffer solution with
0.675 mL of Percoll and complete with 1.825 mL of
ultrapure water to a final volume of 5 mL.
3. Percoll (19%) solution
10 mM MOPS, 19% (v/v) Percoll, 0.3 M mannitol, 0.25%
(w/v) defatted BSA.
To make 10 mL, combine 5 mL of MOPS buffer solution with
1.9 mL of Percoll and complete with 3.1 mL of ultrapure
(w/v) defatted BSA.
To make 10 mL, combine 5 mL of MOPS buffer solution with
2.4 mL of Percoll and complete with 2.6 mL of ultrapure
(w/v) defatted BSA.
To make 5 mL, combine 2.5 mL of MOPS buffer solution with

1.75 mL of Percoll and complete with 0.75 mL of ultra-
pure water to a final volume of 5 mL.
6. Reaction buffer (50 mL)
10 mM MOPS (pH 7.2), 0.3 M mannitol#, 10 mM KPi,
10 mM KCl, 5 mM MgCl2, 0.5% (w/v) defatted BSA.
To make 50 mL, combine 0.5 mL of 1 M MOPS, 5 mL of
0.1 M phosphate buffer, 0.5 mL of 1 M KCl, 0.25 mL of
1 M MgCl2, and 0.25 g of defatted BSA. Adjust pH to 7.2
and add ultrapure water to a final volume of 50 mL.
#
use 0.4 M sucrose instead of 0.3 M mannitol for tomato and
strawberry mitochondria.
2.3 Stock Solutions 0.6 M L-Malic acid: To prepare 1 mL, dissolve 80.4 mg of malic
(Respiratory acid in 1 mL of ultrapure water.
Substrates, Inhibitors, 0.5 M L-Glutamic acid: To prepare 1 mL, dissolve 73.57 mg of
and Cofactors) glutamic acid in 1 mL of 1 M HCl.
0.1 M NADH dipotassium salt: To prepare 1 mL, dissolve
74.16 mg of NADH in 1 mL of 0.01 M NaOH solution.
Aliquot and store protected from light at 80 C.
0.5 M L-ascorbic acid: To make 100 μL, dissolve 8.8 mg of ascorbic
acid in 100 μL of ultrapure water. The solution should be
freshly made and kept protected from light.
1 M KCN: To prepare 100 μL, dissolve 6.51 mg of KCN in 100 μL
of ultrapure water.
50 mM n-propyl gallate: To make 1 mL, dissolve 10.6 mg of n-
propyl gallate in 50 μL of ethanol and then add 950 μL of
ultrapure water.
5 mM oligomycin: To make 1 mL, dissolve 3.958 mg of oligomycin
in 1 mL of ethanol. Aliquot and store frozen at 20 C.
50 mM propranolol hydrochloride: To make 1 mL, dissolve
14.79 mg of propranolol hydrochloride in 1 mL of ultrapure
water.
2 mM atractyloside potassium salt: To make 1 mL, dissolve 1.6 mg
of atractyloside potassium salt in 1 mL of ultrapure water.
100 mM dithiothreitol: To make 1 mL, dissolve 15.42 mg of
dithiothreitol in 1 mL of ultrapure water.
0.5 M pyruvate sodium salt: To make 100 μL, dissolve 11.0 mg of
sodium pyruvate in 100 μL of ultrapure water.
100 mM xanthine: To make 1 mL, dissolve 15.2 mg of xanthine in
1 mL of 1 M NaOH solution.
5 U xanthine oxidase from bovine milk: Dilute an ammonium

sulfate suspension of xanthine oxidase in 100 μL of ultrapure
water.
1 mM linoleic acid: To make 5 mL, dissolve 1.4 mg of linoleic acid
in 0.5 mL of 1% (v/v) Tween 20 and then add 0.1 M potassium
phosphate buffer to obtain a 5 mL final volume.
10% (v/v) Triton X-100: To make 1 mL, combine 100 μL of Triton
X-100 and 900 μL of ultrapure water.
50 mM ATP disodium salt hydrate (anhydrous): To make 1 mL,
dissolve 27.55 mg of ATP in 1 mL of ultrapure water.
50 mM ADP potassium salt hydrate: To make 1 mL, dissolve
25.06 mg of ADP in 1 mL of ultrapure water.
100 mM GTP sodium salt hydrate: To make 1 mL, dissolve
52.32 mg of GTP in 1 mL of ultrapure water. Aliquot and
store frozen at 20 C for up to 3 months.
5 mM cytochrome-c from bovine heart: To make 1 mL, weigh
61.63 mg of cytochrome c and dissolve in 1 mL of a 50 mM
phosphate buffer at pH 7.0. Aliquot and store frozen at
20 C.
100 mM CCCP (carbonyl cyanide m-chlorophenyl hydrazone): To
make 1 mL, weigh 20.4 mg of CCCP and dissolve in 1 mL of
acetone.
2.4 Gene 1. Total RNA extraction and reverse transcription

Identification, Liquid nitrogen.
Annotation, and
Mortar and pestle.
Expression in Fruits
RNase-free microcentrifuge tubes.
5 M NaCl (RNase-free).
Chloroform.
Isopropyl alcohol.
Ethanol (75%).
RNase-free water.
Concert Plant RNA Reagent (Invitrogen).
Commercial columns kit (RNeasy Plant Mini Kit—QIAGEN).
10 MOPs (3-(N-morpholino) propanesulfonic acid) buffer
solution—To prepare 1000 mL, weigh 41.69 g of MOPs
(200 mM final concentration) and 4.1 g of sodium acetate
(30 mM final concentration). Use 20 mL of 0.5 M sodium
EDTA (pH 8). Dissolve the MOPs and sodium acetate in
800 mL of deionized water and add 20 mL of sodium
EDTA and 1 mL of DEPC water. Adjust to pH 7 with
NaOH, complete the volume to 1000 mL, and store in

amber glass.
Agarose.
0.5 μg/mL Ethidium bromide.
Horizontal Gel Electrophoresis System.
2. Reverse transcription
30 ρmol/μL Oligo dT (18 mer) or randomic primers.
10 mM dNTP.
RNase-free water.
ImProm-II™ Reverse Transcription System (Promega).
Total RNA (1 μg).
3. Expression analyses by semiquantitative RT-PCR
Specific primers for each gene (20 ρmol/μL).
First strand cDNA (1 μL).
RNase-free H2O.
GoTaq® DNA Polymerase (Promega).
5 mM dNTP.
PCR instrument.
5 TBE (Tris-borate-EDTA).
To prepare 1000 mL, weigh 54 g of Tris-base and 27.5 g of
boric acid. Use 20 mL of 0.5 M Sodium EDTA (pH 8).
Dissolve the Tris-base and boric acid in 900 mL of deio-
nized water, add 20 mL of Sodium EDTA, and complete
the volume to 1000 mL. Store the solution in amber glass
and autoclave.
Agarose.
0.5 μg/mL Ethidium bromide.
Horizontal Gel Electrophoresis System.
4. Expression analyses by quantitative RT-PCR
Specific primers for each gene (6 ρmol/μL).
First strand cDNA (diluted 1:4).
RNase-free water.
Optical plate, cap strips or PCR tubes.
Optically clear adhesive film.
SYBR® Green PCR Master Mix (Applied Biosystems®).
3 Protocols
3.1 Extraction All steps should be performed at 4 C using precooled glassware or

and Purification plastic.
of Mitochondria
1. Precool centrifuge rotor at least 30 min before initiating mito-
and Measurements chondria isolation. Alternatively, this can be precooled in a
of Alternative Oxidase refrigerator overnight.
and Uncoupling
2. Surface fruits sterilize with 100 mL of sodium hypocloride
Pathway Capacity
[0.05% (v/v)], then wash with distilled water and allow to air
in Fruits
dry.
3.1.1 Isolation and 3. Peel fruit, remove seeds, and place the mesocarp pieces in cold
Purification of Mitochondria water for a few minutes. Weigh approximately 300 g of peeled
(Papaya Fruit, with papaya, or 50 g of tomato without seeds, or 150 g of
Adjustments to Tomato strawberry.
and Strawberry)
4. Quickly homogenize fruit pieces once using a blender (a Walita
R16720 domestic model is suitable) at maximum speed in
900 mL of extraction buffer (tissue extraction buffer ratio of
1:3). If necessary, use 1:4 ratio or higher to avoid forming the
homogenate into a gel after centrifugation. In the case of
tomato and strawberry, use a tissue extraction buffer ratio of
1:4.
5. Collect the homogenate (around 600 mL) and immediately
clarify the homogenate by filtering through four layers of gauze
and miracloth (Calbiochem, CA, USA).
6. Adjust the filtrate pH to 7.4 if needed.
7. Transfer about 300 mL filtrate to each of two 500 mL centri-
fuge tubes (Fig. 1), balance tubes, and centrifuge at low speed
(1500 g for 15 min at 4 C) in a precooled angle rotor (JA-10
rotor, Beckman Coulter, USA). In the cases of tomato and
strawberry mitochondria, centrifuge at 2000 g for 15 min
and 1000 g for 10 min, respectively.
8. Pour supernatants carefully into fresh 500 mL centrifuge tubes
(avoid disturbing pellets), balance tubes, and centrifuge at
higher speed (15,000 g for 15 min at 4 C).
9. Discard the supernatants gently (without transferring pellets).
In the cases of tomato and strawberry, centrifuge at 11,000 g
for 20 min and 10,000 g for 10 min, respectively.
10. Resuspend each pellet carefully in 1 mL of washing buffer
using a paint brush.
11. Pour 20 mL of the resuspended pellet into 45 mL centrifuge
tubes, balance tubes, and centrifuge at 1000 g for 8 min at
4 C in a precooled rotor (JA-25.50 rotor, Beckman Coulter,
USA). Centrifuge at 2500 g for 10 min and 1500 g for
10 min in the cases of tomato and strawberry mitochondria,
respectively.
homogenization
(A.1. step 4)
tissue fruits extraction buffer
(A.1. step 5)
homogenate
filtered
differential
centrifugation
1,500 g
15,000
1,000 g 10,000 g
9,000 g 10,000 g purified
12,000 g mitochondria
(A.1. step 22, 23)
transfer
supernatant
(A.1. step 7)
transfer pellet re-suspend the pellet in
(A.1. step 8) transfer transfer pellet
washing buffer
(A.1. step 19) (A.1. step 21)
supernatant transfer pellet transfer the
(A.1. step 11) (A.1. step 12) crude Percoll mithocondrial band
mitochondria gradient (A.1. step 16, 17)
(A.1. step 13) (A.1. step 15)
Fig. 1 Schematic diagram shows the process for extraction and purification of mitochondria from fruit pulp
tissue. The diagram presents the main steps described in protocol section (3.1.1.) with its respective
identification number in brackets as well as the speed of centrifugation
12. Pour about 20 mL of the supernatant into fresh 45 mL cen-

trifuges tubes, balance tubes, and centrifuge at 9000 g for
15 min at 4 C in a precooled rotor (JA-25.50 rotor, Beckman
Coulter, USA). Centrifuge at 10,000 g for 15 min and
10,000 g for 10 min in the cases of tomato and strawberry,
respectively.
13. Discard the supernatant and resuspend pellets in 1 mL of
washing buffer to obtain the crude mitochondria fraction
(Fig. 2).
14. Prepare a Percoll gradient in 80 mL centrifuge tubes using
Pasteur pipettes. Gently layer the first 5 mL of the 35% Percoll
solution on the bottom, then add 10 mL of the 24% Percoll
solution, 10 mL of a 19% Percoll solution, and finally 5 mL of
the 13.5% Percoll solution on the top. The final volume is
30 mL. Avoid mixing the bands.
15. Gently layer the crude fraction (1 mL) on the top of the 13.5%
Percoll gradient.
Fig. 2 Percoll gradient purification of papaya fruit. (a) Supernatant obtained after centrifuging filtrate (step 7);
(b) Pellet obtained after supernatant centrifugation (step 8); (c) Supernatant that resulted from the centrifuga-
tion of the resuspended pellet (step 11); (d) Pellet (crude mitochondria fraction) obtained after supernatant
centrifugation (step 12); (e) The crude fraction layered on the top of the 13.5% Percoll (step 15); (f)
Mitochondrial band at the 24–35% Percoll interface (step 17); (g) Pellet of mitochondria after Percoll gradient
purification
16. Balance tubes and centrifuge at slow speed up (12,000 g for

45 min at 4 C) using a precooled 45 mL angle rotor (JA-25.5
rotor, Beckman Coulter, USA).
17. Carefully recover the mitochondrial band from the 24–35%
Percoll interface using a Pasteur pipette.
18. Transfer the band to a fresh 45 mL tube.
19. Dilute the band tenfold with washing buffer, balance the tube,
and centrifuge at 10,000 g for 15 min at 4 C (JA-25.5 rotor,
Beckman Coulter, USA).
20. Carefully discard the supernatant and collect the pellet.
21. Wash the pellet again using 15–20 mL of washing buffer and
centrifuge at 10,000 g for 15 min at 4 C (JA-25.5 rotor,
22. Carefully remove the supernatant and resuspend the pellet in
1 mL of washing buffer stored on ice. The final pellet volume is
around 1.2 mL.
23. This protocol produces around 1–2 mg of mitochondrial pro-
tein, depending on the fruit ripening stage [1]. Fully ripe fruits
show lower yields.
3.1.2 Isolation All the steps should be performed at 4 C using precooled glassware
and Purification or plastic.
of Mitochondria
1. Precool centrifuge rotor for at least 30 min before initiating
(Guava Fruit)
mitochondria isolation. Alternatively, this can be precooled in a
refrigerator overnight.
2. Clean up guava fruits with sodium hypocloride [0.05% (v/v)],
wash with distilled water, and let fruit air dry.
3. Peel fruit, remove seeds, and quickly weigh about 200 g of

guava mesocarp and immediately place fruit pieces in chilled
homogenization buffer. This step is critical to avoid oxidation.
4. Quickly homogenize fruit pieces once using a blender (a
domestic Walita model R16720 is suitable) at maximum
speed in 300 mL of extraction buffer (ratio 1:1.5, tissue:extrac-
tion buffer).
5. Collect the homogenate (around 600 mL) and clarify the
homogenate immediately by filtering out through four layers
of gauze and Miracloth (Calbiochem, CA, USA).
6. Adjust the filtrate’s pH to 7.4 if needed.
7. Transfer about 300 mL filtrate to each of two 500 mL centri-
fuge tubes, balance tubes, and centrifuge at low speed
(1500 g for 15 min at 4 C) in a precooled angle rotor (JA-
10 rotor, Beckman Coulter, USA).
8. Pour supernatants carefully into fresh 500 mL centrifuges
tubes (avoid disturbing pellets), balance tubes, and centrifuge
at higher speed (15,000 g for 15 min at 4 C).
9. Discard the supernatants gently (without transferring pellets).
10. Carefully resuspend each pellet in 1 mL of washing buffer using
a paint brush.
11. Pour 20 mL of the resuspended pellet into 45 mL centrifuge
tubes, balance tubes, and centrifuge at 1000 g for 8 min at
4 C in a precooled rotor (JA-25.50 rotor, Beckman Coulter,
USA).
12. Pour about 20 mL of the supernatant into fresh 45 mL cen-
trifuges tubes, balance tubes, and centrifuge at 9000 g for
15 min at 4 C in a precooled rotor (JA-25.50 rotor, Beckman
Coulter, USA).
13. Discard the supernatant and resuspend pellets in 1 mL of
washing buffer to obtain the crude mitochondria fraction.
14. Prepare a Percoll gradient in 80 mL centrifuge tubes using
Pasteur pipettes. Gently layer the first 5 mL of the 35% Percoll
solution on the bottom, then add 10 mL of the 24% Percoll
solution, 10 mL of a 19% Percoll solution, and finally 5 mL of
the 13.5% Percoll solution on the top. The final volume is
30 mL. Avoid mixing the bands.
15. Gently layer the crude fraction (1 mL) on the top of the 13.5%
Percoll gradient.
16. Balance tubes and centrifuge at slow speed up (12,000 g for
45 min at 4 C) using a precooled 45 mL angle rotor (JA-25.5
rotor, Beckman Coulter, USA).
17. Carefully recover the mitochondrial band from the 24–35%
Percoll interface using a Pasteur pipette.
18. Transfer the band to a fresh 45 mL tube.

19. Dilute the band tenfold with washing buffer, balance the tube,
and centrifuge at 10,000 g for 15 min at 4 C (JA-25.5 rotor,
20. Carefully discard the supernatant and collect the pellet.
21. Wash the pellet again using 15–20 mL of washing buffer and
centrifuge at 10,000 g for 15 min at 4 C (JA-25.5 rotor,
22. Carefully remove the supernatant and resuspend the pellet in
1 mL of washing buffer stored on ice. The final pellet volume is
around 1.2 mL.
23. This protocol produces around 1–2 mg of mitochondrial pro-
tein, depending on the fruit ripening stage. Fully ripe fruits
show lower yields.
3.1.3 Test for All the steps should be performed at 25 C.

Mitochondrial Outer
1. Assemble the oxygen electrode (Oxytherm system, Hansatech)
Membrane (MOM) Integrity
and calibrate it 2 h before starting oxygen uptake measure-
ments. Follow the manual’s instructions.
2. Place 965–980 μL of reaction buffer into the incubation cham-
ber with continuous stirring. Use 600 μL of reaction buffer for
guava fruit mitochondria. Avoid air bubbles.
3. Add 10–20 μL of purified mitochondria (around 20–40 μg of
mitochondrial protein). In the case of guava mitochondria, add
75 μL of purified mitochondria (around 75 μg of mitochon-
drial protein).
4. Inject 20 μL of 0.5 M ascorbic acid (10 mM final concentra-
tion) using a 25 μL syringe and record the change in the slope
(O2 consumption rate) once it reaches linearity (Fig. 3). This
lasts around 10–20 s.
5. Inject 10 μL of 5 mM cytochrome c (50 μM final concentra-
tion) and record the slope.
6. Finally, inject 5 μL of 10% (v/v) Triton X-100 (0.05% final
concentration). A huge speed up of O2 uptake should take
place. Record the slope increase while the O2 consumption
stabilizes.
7. Calculate the rates of oxygen consumption (slopes).
8. The calculation of MOM integrity is the following:
MOM integrity ð%Þ

ðO2 consumption after adding cytochrome cÞ ðO2 consumption after adding ascorbateÞ
¼1 100
ð O2 consumption after adding Triton X‐100Þ ðO2 consumption after adding ascorbateÞ
9. This protocol should produce intact mitochondria with at least

70% of MOM integrity.
Fig. 3 Recording of oxygen uptake of papaya fruit mitochondria measured at 25 C using an oxygen electrode
(Oxytherm system, Hansatech). The slopes represent the oxygen uptake rates after adding specific com-
pounds. Graph was generated by the Oxygraph Plus software. (Courtesy of Hansatech Instruments Ltd.)
3.1.4 Test for Coupling 1. Assemble the equipment and calibrate the oxygen electrode 2 h
Efficiency (Respiration) before starting oxygen uptake measurements.
2. Place 965–980 μL of reaction medium into the incubation
chamber. Use 600 μL of reaction buffer for guava
mitochondria.
3. Add 5–20 μL of purified papaya mitochondria or 75 μL of
guava mitochondria.
4. Inject 2.4 μL of 50 mM ATP (120 μM final concentration).
5. Inject 10 μL of 0.1 M NADH (1 mM final concentration) and
then wait for linearity of the O2 consumption rate (steady
state).
6. Inject 2.4 μL of 50 mM ADP (120 nmol ADP). Use 6 μL of
50 mM ADP (300 nmol ADP) for guava mitochondria.
The O2 consumption speeds up, showing a linear increase
(state 3 respiration). It then decreases and reaches linearity
when all ADP is converted to ATP (state 4 respiration).
7. The assessment of the mitochondrial coupling efficiency is

determined by the respiratory control index (RCI) [1] as
follows:
Rate of O2 consumption after added ADP ðstate 3Þ
RCI ¼
Rate of O2 consumption in relaxed state ðstate 4Þ
3.1.5 Measurement 1. Place 915–930 μL of reaction medium into the incubation

of Alternative Oxidase chamber.
(AOX) Capacity in Purified 2. Add 5–20 μL of purified papaya mitochondria.
Mitochondria
3. Inject sequentially 2.4 μL of 50 mM ATP, 6.3 μL of 5 mM
oligomicin, 3.6 μL of 50 mM propranolol, 10 μL of 100 mM
DTT, and 2.5 μL of 0.5 M pyruvate.
4. Initiate the reaction by injecting 16.6 μL of 0.6 M malic acid
and 20 μL of 0.5 M glutamic acid. The O2 consumption speeds
up. Wait until it reaches the steady state.
5. Inject 2.4 μL of 50 mM n-propyl gallate (nPG) to inhibit the
AOX. The rate of oxygen uptake should drop. Wait it reaches a
linear decrease.
6. Inject 3.1 μL of 1 M KCN. The rate of oxygen uptake should
further drop to almost zero
7. The AOX capacity is determined following Ref. 1 as follows:
AOX capacity ¼ Rate of O2 consum . before add nPG Rate
of O2 consum . after add nPG and KCN
3.1.6 Measurement 1. Place 880–910 μL of reaction medium into the incubation

of Uncouple Protein (UCP) chamber.
Capacity in Purified 2. Inject sequentially 6.3 μL of 5 mM oligomicin, 3.6 μL of
Mitochondria 50 mM propranolol, 2.4 μL of 50 mM n-propyl gallate,
10 μL of 2 mM atractyloside, and 0.5 μL of xanthine 100 mM.
3. Add 20–50 μL of purified papaya mitochondria (40–100 μg of
mitochondrial protein). Note that this assay requires a greater
amount of mitochondria.
4. Inject 3 μL of 5 U xanthine oxidase (XO) to generate superox-
ide [1, 2] and 10 μL of 0.1 M NADH. The O2 consumption
rate increases. Wait until it reaches steady state.
5. Add 5 μL of 10% (w/v) defatted BSA and 10 μL of 1 mM
linoleic acid to induce UCP activity. Register the slope increase
(rate of oxygen uptake).
6. Inject 2.4 μL of 100 mM GTP. Register the slope decrease.
7. Inject 6 μL of CCCP and register oxygen consumption. The
rate of O2 consumption should increase quickly because CCCP
is a potent mitochondrial uncoupler.
8. The UCP capacity is determined following Ref. 1 as below:
UCP capacity ¼ Rate of O2 consum . after add linoleic acid

Rate of O2 consum . after add GTP
The protein content is determined following the method
described in Ref. 3.
3.2 Gene All AOX and UCP genes can be retrieved from fruit genomes using
Identification, BLAST (basic local alignment search tool) [4] searches according
Annotation, to the following steps:
and Expression 1. First, select suitable AOX and UCP sequences to be used as
in Fruits queries (protein or nucleotide sequences). In the case of angio-
3.2.1 Gene Identification sperm species, it is recommended to use sequences of plant
and Annotation models such as Oryza sativa (monocot) or Arabidopsis thali-
ana (eudicot).
2. Use AOX and UCP (nucleotide or protein) queries to search
for homolog sequences in the target genome database using
blastn or tblastn tools, respectively.
3. Next, use the corresponding retrieved gene sequences for
annotation (identification of exons/introns/promoters).
Compare the retrieved genomic sequence with homologous
mRNAs found in Refseq-RNA, ESTs (expressed sequence
tags), and TSA (transcriptome shotgun assembly) databases
using the blastn tool. The majority of AOX genes in angios-
perms are annotated by Ref. 5.
4. After, exclude manually the intron sequences from the anno-
tated gene to obtain the deduced cDNA sequence. Then,
translate the coding sequence of each cDNA into amino acid
sequence and identify the initiation (methionine, ATG) and
stop codons as well as -30 and -50 UTR regions.
5. Finally, perform a blastp search against protein databases to
check whether the deduced AOX and UCP proteins sequences
are complete.
3.2.2 Primer Design After obtaining the gene and deducing cDNA sequences, it is
possible to design specific primers to study the expression (RT-
PCR) of each gene member. It is important to highlight that the
same primer pair designed for quantitative real-time PCR (based on
syber green detection) can also be used for semiquantitative expres-
sion analyses. Several bioinformatic tools are available for primer
design. We recommend the use of PerlPrimer [6], which is available
online for Windows, Linux, Mac OS X and can be downloaded
from (http://perlprimer.sourceforge.net/download.html).
Specific primer pairs may be designed according to the main
rules:
1. The primers should have 17–28 nucleotides in length.
2. The proportion of GþC in each primer should vary from 40 to

60%.
3. Primers should have a GC clamp 30 (two of the last three bases
G or C).
4. The melting temperature (Tm) should vary from 55 to 80 C.
5. Amplicon size should vary from 80 to 200 pb.
6. Avoid the formation of stable secondary structures such as: self-
dimer, heterodimer, and hairpin of each primer pair.
In addition, the design of specific primers for multigene
families (AOX and UCP) first requires a multiple alignment of all
deduced cDNA sequences to detect non-conserved regions that
could be used to design specific primers to obtain the expression
profile of each gene member. In the case of a general expression
profile of a multigene family, it is more appropriate to design
degenerated primer pairs in conserved regions.
3.2.3 Total RNA The total RNA extraction method chosen is crucial to the accuracy
Extraction and Reverse of gene expression analyses such as semiquantitative polymerase
Transcription chain reaction (RT-PCR), quantitative real-time PCR (qRT-
PCR), microarrays, and RNA-seq. However, the success of the
extraction process depends on the extraction protocols applied to
the tissue.
Many commercial total RNA extraction kits are available,
providing fast RNA extraction with high purity. However, the
resultant total RNA are generally low in concentration due to
intense purification steps. The conventional extraction protocols
usually provide total RNA at high concentration and low purity. In
view of the difficulty of obtaining total RNA with reliable purity and
concentration in fruits, here we suggest a protocol that includes the
Concert Plant RNA Reagent in combination with RNA purification
in column kits.
1. First, pulverize the vegetal material with liquid nitrogen using
mortar and pestle. Then, add 750 μL of Concert Plant RNA
reagent extraction buffer.
2. Incubate the resulting mixture at room temperature for 5 min
and then centrifuge for 2 min at 12,000 g.
3. Add to the supernatant 100 μL of NaCl (5 M) and 300 μL of
chloroform. After, homogenize the mixture by successive
inversions, and centrifuge at 12,000 g (þ4 C) for 10 min.
This step is necessary to remove impurities.
4. Add an equal volume of isopropyl alcohol to the supernatant,
leave at room temperature for 10 min, then centrifuge at
12,000 g (þ4 C) for 10 min. Discard the supernatant
carefully keeping the pellet in the Eppendorf bottom.
5. Wash the pellet with 500 μL of 75% ethanol and centrifuge for
5 min at 12,000 g.
6. Elute the pellet containing the total RNA in 40 μL of RNase-
free water and then quantify the total RNA using spectropho-
tometer at 260 nm.
7. Treat the total RNA with DNase to eliminate genomic DNA
contamination as follows:
(a) First, calculate the volumes of buffer and DNase that
should be utilized. Example: For 5 μL of total RNA in a
concentration of 1 μg/μL, use 2 μL of RNase-free DNase
10 Reaction Buffer, 5 μL of RNase-free DNase (1 U/μ
L) and add nuclease-free water to a final volume of 20 μL;
(b) Incubate the solution at 37 C for 30 min.
8. The total RNA purification steps may be performed with a
commercial column (QIAGEN) extraction kit according to
the manufacturer’s instructions.
9. Perform the total RNA purification in columns. Initially, add
350 μL of RPL extraction reagent and 350 μL of ethanol to the
DNAse-treated RNA. After homogenization transfer the solu-
tion to the pink column and then centrifuge for 15 s at
11,000 g.
10. Add 350 μL of RW1 reagent and centrifuge at 11,000 g for
15 s. Then, repeat this step once.
11. Add 500 μL of RPE reagent and centrifuge for 15 s at
11,000 g. Repeat this step centrifuging for 2 min at
11,000 g. Finally, elute the total RNA using 40 μL of
RNase-free water.
12. Quantify the final concentration and purity of total RNA by
spectrophotometer at 260 nm. Then, check the non-
contamination by protein and polysaccharides verifying the
ratios 260/280 (optimal 1.8–2.0) and 260/230 (up to 2.0),
respectively.
13. Check the RNA integrity in agarose gel (1.5%) as follows:
(a) Weigh 0.375 g of agarose;
(b) Add it to a volume of 25 mL (2.5 mL of 10 MOPs
completed with 22.5 of autoclaved deionized water) and
then, heat the mixture until dissolve completely. After-
ward, place the comb slots correctly and then wait until
polymerization is complete (around 20 min).
(c) The run buffer should be prepared to a volume of 350 mL
containing 35 mL of 10 MOPs. The polymerized gel
must be placed in the electrophoresis tank containing the
run buffer.
(d) RNase-free water and 1.5 μL of bromophenol blue are

added to the total RNA samples (0.5 μg) for a resulting
volume of 5 μL. Next, apply the mixture in the well gel.
Then, stain the gel with an ethidium bromide solution
(0.5 μg/mL) and visualize with an appropriate gel imag-
ing system.
14. cDNA synthesis (Reverse transcription)
Reverse transcription should be carried out with the ImProm-
II™ Reverse Transcription System (Promega).
(a) Prepare a total volume of 19 μL of the mixture as follows:
1 μg of total RNA, 5 μL of 5 buffer, 2.4 μL of 25 mM
MgCl2, 3 μL of random hexamers or Oligo dT18
(20 ρmols/μL), 1 μL of 10 mM dNTPs, and RNase-free
water (variable according to total RNA concentration).
(b) Incubate the mixture obtained in step 14a at 65 C for
5 min and then on ice for at least 2 min.
(c) Add 1 μL of 160 U/μL Reverse transcriptase, then mix
and incubate the tubes at 42 C for 1 h and at 70 C for
15 min using the PCR instrument. Store the cDNA at
20 C until use for real-time PCR.
3.2.4 Expression The conventional PCR may be applied routinely to semiquantita-

Analyses by tively estimate the transcript levels. However, care must be taken to
Semiquantitative RT-PCR ensure reliable analysis. Some gene expression studies of AOX and
UCP have used semiquantitative PCR to quantify transcript levels
[1, 7, 8].
1. First, a PCR gradient must be performed with each primer pair
to obtain the best TM (melting temperature) and TA (anneal-
ing temperature), generally TA ¼ TM-5 C. The best TM and
TA are chosen according to the most intense bands visualized
in agarose gel (after PCR reaction) with or without reduced
backgrounds.
2. All total RNA samples must present similar concentration and
purity to ensure accurate gene expression profile.
3. Preliminary PCR reactions with variable cycle number must be
performed to certify that PCR amplification is not in the pla-
teau phase.
4. For RT-PCR, use 1 μL of cDNA, 15.9 μL of ultrapure water,
1 μL of (5 mM) dNTPs, 5 μL of buffer 5, 1 μL (20 ρmols/μ
L) of each primer pair (forward and reverse), and 0.1 μL of
(5 U/μL) GoTaq DNA Polymerase.
5. The standard PCR program used involves initial denaturation
at 95 C for 2 min, followed by cycles of denaturation at 95 C
for 0.5–1 min, annealing at 42 C to 65 C for 0.5–1 min, and
extension at 72 C for 1 min/kb. To finish, a final extension at
72 C is conducted for 5 min and held at 4 C. The PCR

program should be adjusted according to the amplicon size
and TA of each primer pair.
6. At least one stable expression gene must be used for the data
normalization.
7. The PCR products must be separated by electrophoresis in
agarose gel, stained with (0.5 μg/mL) ethidium bromide,
and visualized with an appropriate gel imaging system.
8. The image generated must be submitted to a suitable software
capable of measuring band density (pixels).
9. Finally, relative expression is obtained by the ratio between the
densities of the target cDNA bands compared to the reference
cDNA bands.
3.2.5 Expression Quantitative RT-PCR (qRT-PCR) is a powerful technique used to

Analyses by Quantitative measure gene expression because of its high sensitivity, specificity,
RT-PCR and accuracy. It comprises both relative and absolute quantification
methods. Relative quantification compares gene expression
between two distinct treatments and requires reference genes for
data normalization [9, 10]. Our group has used qRT-PCR to study
gene expression based on relative quantification in different tissues
of soybean [11, 12]. The qRT-PCR quantification is based on a
fluorescent label. During amplification, a fluorescent dye binds,
either directly or indirectly via a labeled hybridizing probe, to the
accumulating DNA molecules and fluorescence values are recorded
during each cycle of the amplification process [9, 13].
The relative expression of each AOX and UCP gene member
can be provided according to the following steps:
1. Primer pairs and cDNA samples must possess equivalent con-
centration and purity levels to ensure accurate gene expression
profiles.
2. A temperature gradient should be performed in order to obtain
the best TA for each primer pair as follows:
(a) The cDNA of all samples is synthetized as described in
B.3. Step 14 (Reverse transcription);
(b) Use 1 μL of pool cDNA (equal quantity of each cDNA
sample);
(c) Use 1 μL of each primer (forward and reverse) at 6 μM to
a final concentration of 300 nM;
(d) Use ultrapure water to complete 10 μL;
(e) Use 10 μL of master mix (SYBR Green);
(f) Select the temperature gradient in the quantitative PCR
instrument according to each primer pair.
3. Standard curves for each gene should be generated from ten-

fold serial dilutions of pooled cDNAs to determinate the ampli-
fication efficiencies for each primer pair. The corresponding
PCR amplification efficiencies (E) must be calculated according
to the equation E ¼ (1010/slope 1) 100 [14].
4. Use the PCR program indicated for SYBR® Green PCR Master
Mix (Applied Biosystems®) in temperature gradient and ampli-
fication efficiencies.
5. After determining the best TA and the amplification efficiencies
for each primer pair, the gene expression may be verified fol-
lowing the instructions of the Master Mix manufacturer and by
a suitable PCR reaction program in a real-time thermocycler.
(a) The cDNA (RT products) used in each reaction should be
diluted 1:4.
(b) The PCR reaction (in 96-well plates) should contain a
total volume of 20 μL which includes 4 μL of cDNA
(diluted 1:4), 10 μL of fluorescent molecules (SYBR
Green), 1 μL of each primer (forward and reverse) at
6 μM to a final concentration of 300 nM, and 4 μL of
ultrapure H2O.
(c) All the reactions should be performed in biological and
technical triplicates including a negative control (omitting
the cDNA template) for appropriate statistical analyses.
(d) The reaction should be performed in a real-time thermo-
cycler using the following program: 95 C for 10 min to
activate the hot-start Taq DNA polymerase followed by
40 cycles: (1) denaturation at 95 C for 15 s, (2) optimal
annealing temperature for each primer pair for 15 s, and
(3) extension at 60 C for 20 s.
(e) After the amplification, the melting curve must be
checked to verify the primer specificity.
6. At least four stable expression genes must be employed to data
normalization. Here, we suggest the GeNorm program (qBa-
sePLUS software) which calculates the gene expression stability
(M value) and the pairwise variation (V value). High M-values
indicate low gene expression stability and the V value deter-
mines the optimal gene combination required for normaliza-
tion [15].
7. The number of amplification cycles necessary for the flores-
cence (SYBR Green) to cross the baseline during the exponen-
tial reaction phase is denominated by Ct (Cycle threshold). The
Ct values are then imported to qBaseplus software (versão 2.4)
to provide relative gene expression.
3.2.6 In Silico Expression Before the advent of transcriptomic analyses, gene expression
Analyses Using Microarrays research was restricted to one or only several genes. However, it is
and RNA-Seq Data well known that at the cellular level genes interact among them-
selves participating in a complex expression and regulation network
[16]. The first transcriptomic approaches were conducted by a
microarray technique [17] that was widely used until some years
ago. More recently (after 2008), transcriptomic studies have
embraced a new method that emerged from the Next Generation
Sequencing [18] known as RNA-seq (sequencing of RNA).
Both transcriptomic technologies have accumulated a vast
quantity of information regarding the expression of almost all
genes in several species. Some databases have been created to
support the exponential increase of such information and to make
it publically available.
With regard to microarrays in fruits, data are scarce, comprising
only a few species such as citrus, grape, and tomato, available in
Plexdb database (http://www.plexdb.org/index.php?tmva¼0|10|
17|33|63|65|), and tomato and grape in Gene Expression Omnibus
(GEO), NCBI database. The following steps are necessary in order
to gain access to the information in the GEO database:
1. In the specialized Blast option (https://blast.ncbi.nlm.nih.
gov/Blast.cgi), select “Search sequences that have gene expres
sion profiles (GEO).”
2. Enter with AOX or UCP (protein or nucleotide query) select-
ing blastn, tblastn, or tblastx as well as the target organism to
detect the available probe sequence.
3. The expression profile is shown by first clicking the accession
number of the detected probe and then the graphic with the
expression data.
The microarray has some drawbacks due to the fact that this
technology is based on probe hybridization. These disadvantages
include the identification of transcripts only with an available probe.
In the case of AOX and UCP multigene families, the probes avail-
able frequently provide the expression profile of only one or a
reduced number of gene members.
RNA-seq technology has overcome these drawbacks and deep-
ened our understanding of fruit biology. The transcriptomic (RNA-
seq) data available are increasing exponentially and are available on
SRA (Sequence Read Archive) of the GeneBank (NCBI)]. To
evaluate the expression profile of each AOX and UCP gene mem-
ber, the following steps are suggested according to Ref. 12.
1. First all deduced cDNA or proteins must be aligned to identify
the nonconserved region in order to obtain at least two probe
sequences for each gene.
2. To confirm that the selected probes are specific for each gene
member, we recommend that a blast search be performed
against the studied genome (if available).
3. When the ORFs (Open Reading Frames) are highly conserved,
we recommend the use of the 30 and 50 UTR region, taking
advantage of nonconserved sequences to differentiate each
gene member.
4. Blast searches against each experiment deposited in SRA pro-
vide the number of mapped reads on each gene used to esti-
mate the gene expression.
5. Next the counted reads are normalized using the RPKM
(Reads Per Kilobase of transcript per Million of mapped
reads) method [19] according to the following equation:
RPKM ¼ (number of mapped reads 109)/(number of
reads in each database number of nucleotides of each probe).
References
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(2003) Superoxide stimulates a proton leak in tive PCR and the 2(-Delta Delta C(T))
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Chapter 14
Measurement of Tricarboxylic Acid Cycle Enzyme Activities

in Plants
Rebeca Patricia Omena-Garcia, Wagner L. Araújo, Yves Gibon,
Alisdair R. Fernie, and Adriano Nunes-Nesi
Abstract
Mitochondria are vital cytoplasmic organelle of eukaryotic cells responsible for oxidative energy metabolism
and the synthesis of intermediates utilized in various other metabolic pathways. The functions of mito-
chondrion are the oxidation of organic acids by the tricarboxylic acid (TCA) cycle and the synthesis of ATP
by the oxidative phosphorylation in the mitochondrial electron transport chain. The TCA cycle is com-
posed by a set of enzymes that are essential for optimal functioning of the primary carbon metabolism in
plants. The activity of each TCA cycle enzyme in plants may vary according to cell type, plant tissue, stage of
plant development, and the environment. Here, we describe current methods used for the determination of
the TCA cycle enzyme activities in different plant tissues.
Key words Activation assays, Mitochondrial enzyme activity, Tricarboxylic acid (TCA) cycle enzyme
assays
1 Introduction
The main role of the mitochondrial metabolism in the plant cell,

like in all eukaryotic cells, is the production of ATP, reducing
equivalents and metabolic intermediates for use in biosynthesis
elsewhere in the cell [1]. Moreover, plant mitochondrion partici-
pates in many other important cellular processes including meeting
the demand for carbon skeletons imposed by anabolic processes
such as amino acid and isoprenoid syntheses, carbon-nitrogen
interactions, photosynthetic optimization and plant cell redox
homeostasis, signaling, and biotic stress response [1]. In addition,
mitochondrion is intimately involved in the production of reactive
oxygen species and the processes of programmed cell death, flower
development, seed germination, and fruit ripening [2].
Inside the mitochondrion, the TCA cycle represents a sequence
of catabolic reactions that support ATP synthesis. Despite recent
research efforts to increase our knowledge on the roles of the TCA
167
168 Rebeca Patricia Omena-Garcia et al.
Fig. 1 Summary of the TCA cycle and its associated enzymes. TCA cycle enzymes are represented in violet
squares whereas associatedenzymes are represented in gray squares. The TCA cycle enzymes that undergo
regulation by thioredoxin (TRX) and thiamin pyrophosphate (TPP) are represented as follows: citrate synthase
(CS) activation by TRX is represented in green; and downregulation of succinate dehydrogenase (SDH) and
fumarase (FUM) is represented in red. TPP is an essential coenzyme (shown in blue) for the activity of pyruvate
dehydrogenase complex (PDH) and 2-oxoglutarate dehydrogenase (2-OGDH). Detailed reactions of individual
enzymes are given in the enzyme assays described below. Abbreviations: Oxaloacetate (OAA) and 2-
oxoglutarate (2-OG). Enzyme abbreviations are presented as in Table 1
cycle, the function of this metabolic pathway seems to be extremely

variable depending on tissue type and environmental conditions
[2]. Thus, the understanding of this cycle and its interactions
remains to be fully elucidated. The TCA cycle is comprised of a
set of eight enzymes, seven enzymes in the mitochondrial matrix
and one attached to the inner mitochondrial membrane, that cou-
ple the oxidation of pyruvate and malate (generated in the cytosol)
to CO2 with the generation of NADH and FADH2 for the oxida-
tion by the electron transport chain (Fig. 1) [3].
The activity of TCA cycle in plant tissues is subjected to a very
exquisite regulation since different steps of the pathway are
involved in distinct functions beyond the maintenance of the cycle
activity. It has been shown that TCA cycle may display a modular
structure, in which different steps of the cycle, according to the
plant tissue and environmental condition, have diverse functions
other than maintaining a cyclic flux [4]. It seems likely that the
metabolic balance between these roles strongly depends on the
physiological context within which the cycle is operating [4]. More-
over, it has recently demonstrated that some of the TCA cycle
Measurement of Tricarboxylic Acid Cycle Enzyme Activities in Plants 169
enzymes are regulated via thioredoxin (TRX) [5, 6] and thiamin

pyrophosphate (TPP) (Fig. 1) [7], providing a mechanism allowing
the coordination of mitochondrial function. TPP is the active form
of thiamin in plants and an essential coenzyme for the enzyme
complexes pyruvate dehydrogenase (PDH) and 2-oxoglutarate
dehydrogenase (2-OGDH) of the TCA cycle [7]. These enzyme
complexes are described as controlling steps of the TCA cycle [8,
9]. In addition TPP is limiting for the assembly of these complexes
[10, 11]. Thus, TPP is of great importance for the proper func-
tioning of the TCA cycle.
Since the activity of the TCA cycle enzymes is highly variable
between tissue types, developmental stages, and in response to
environmental conditions, measurement of activities of the
enzymes of this pathway can significantly improve our understand-
ing of the regulation of this pathway as well as the functioning of
the whole mitochondrial metabolism. In this chapter, relatively
simple enzymatic assays for the enzymes of TCA cycle and asso-
ciated enzymes (Table 1) as well as the activation by TRX [5, 6] and
TPP [7] are described in detail.
Table 1
List of enzymatic assays described here and related references
Reference Reference
Enzymes Abbreviation Regulation for regulation for enzyme assay
Pyruvate dehydrogenase PDH TPP [7] [12, 13]
Citrate synthase CS TRX [5] [14]
Aconitase ACO N.A. [14]
+
Isocitrate dehydrogenase-NAD IDH (NAD) N.A. [15]
Isocitrate dehydrogenase-NADP+ ICDH (NADP) N.A [14]
2-Oxoglutarate dehydrogenase 2-OGDH TPP [7] [8]
Succinyl-CoA ligase ScoAL N.A. [15]
a
Succinate dehydrogenase SDH TRX [6] [16]
(complex II)
Fumarase FUM TRXa [6] [14]
Malate dehydrogenase MDH N.A. [17]
+
Malic enzyme-NAD NAD-ME N.A. [18]
+
Malic enzyme-NADP NADP-ME N.A. [19]
N.A.: activation assay has not yet been described
a
Enzymes downregulated by TRX
2 Materials
2.1 Equipment 1. Plate reader UV-VIS spectrophotometer (wavelength from

190 to 700 nm).
2. Clear-bottom 96-well plates: Plastic plate (disposable) for
wavelengths from 380 to 780 nm (visible spectrum); quartz
plate (reusable) or plastic plate for wavelengths below 380 nm
(ultraviolet spectrum).
3. Micropipettes capable of dispensing 1–1000 μL of solution.
4. Plastic pipette tips of 10, 200, and 1000 μL.
5. Plastic tubes of 1.5 and 2.0 mL.
2.2 Suggested Stock Solutions for extraction buffer for total tissue extracts: 87% (v/v)
Solutions glycerol, 5% (w/v) bovine serum albumin (BSA), 10% (v/v) Triton
X-100, 500 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
2.2.1 Protein Extractions
acid (HEPES)/KOH pH 7.5, 100 mM MgCl2, 10 mM ethylene-
diamine tetraacetic acid (EDTA), 10 mM ethylene glycol tetraacetic
acid (EGTA), 10 mM benzamidine, 10 mM ε-aminocaproic acid,
100 mM phenylmethylsulfonyl fluoride (PMSF) in isopropanol,
2 mM leupeptin, and 500 mM dithiothreitol (DTT, see Note 1).
Solutions for extraction buffer and resuspended solutions for
mitochondrial enriched extract: 1 M Mannitol, 500 mM 3-
morpholinopropanesulfonic acid (MOPS)-KOH pH 7.5,
200 mM EDTA, 1% (w/v) BSA-fraction V, 1% (w/v) polyvinylpyr-
rolidone (PVP), and 40 mM cysteine.
2.2.2 PDH Assay Solutions for extraction buffer: 500 mM 2-{[2-Hydroxy-1,1-bis

(hydroxymethyl)ethyl]amino} ethanesulfonic acid (TES)-KOH
pH 7.2, 500 mM MgCl2, 10 mM Na2EDTA, 2% defatted BSA,
1 M mannitol, and 100 mM DTT.
Solutions for reaction buffer: 500 mM TES-NaOH pH 7.5,
1 M MgCl2, 50 mM β-nicotinamide adenine dinucleotide (NAD+),
100 mM lithium CoA; 100 mM thiamine pyrophosphate (TPP),
100 mM cysteine-HCl, and 30 mM sodium pyruvate. 1 mM TPP
for activation assay by TPP (see Note 2).
2.2.3 CS Assay Solutions for extraction buffer: 87% (v/v) Glycerol, 5% (w/v) BSA,
10% (v/v) Triton X-100, 500 mM HEPES/KOH, pH 7.5,
100 mM MgCl2, 10 mM EDTA, 10 mM EGTA, 10 mM benza-
midine, 10 mM ε-aminocaproic acid, 100 mM PMSF, 2 mM leu-
peptin, and 500 mM DTT.
Solutions for reaction buffer: 0.5 M Tricine buffer pH 8.5, 1 M
tricine buffer pH 9, 1.5 mM malate, 0.25 (v/v) Triton X-100,
20 mM acetyl-CoA, 25 mM NAD+, 100 Units/mL of malate
dehydrogenase in tricine pH 8.0, 10 mM MgCl2, 0.5 M NaOH,
0.5 M HCl in tricine buffer pH 9, 10 mM 2-(4,5-dimethyl-2-
thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT), 200 mM

EDTA, 50% ethanol, 4 mM phenazine ethosulfate (PES), 2000
units/mL alcohol dehydrogenase in tricine pH 9.0, 10 mM
MgCl2, and 1 mM β-nicotinamide adenine dinucleotide reduced
(NADH) (see Notes 1–3).
2.2.4 ACO Assay Solutions for extraction buffer: Stock solutions are prepared as
described for CS (see Subheading 2.2.3).
Solutions for reaction buffer: 1 M HEPES-NaOH pH 7.5,
100 mM β-nicotinamide adenine dinucleotide phosphate
(NADP+), 100 mM MnCl2, 2 units/mL of commercial NADP-
dependent isocitrate dehydrogenase from porcine heart, 10% (v/v)
Triton X100, and 400 mM aconitate (see Notes 2 and 3).
2.2.5 NAD+-Dependent Solutions for extraction buffer: Stock solutions are prepared as
IDH Assay described for CS (see Subheading 2.2.3).
Solutions for reaction buffer: 500 mM Tris(hydroxymethyl)
aminomethane (Tris)-HCl pH 7.6, 50 mM NAD+, 100 mM
MnCl2, 10% β-mercaptoethanol, and 450 mM isocitric acid
(see Note 2).
2.2.6 NADP+-Dependent Solutions for extraction buffer: Stock solutions are prepared as
IDH (ICDH) Assay described for CS (see Subheading 2.2.3).
Solutions for reaction buffer: 1 M Tricine-KOH pH 8.0; 1 M
tricine-KOH pH 9.0, 1 M MgCl2; 20 mM isocitric acid; 10 mM
NADP+, 20 mM EDTA, 10 mM phenazine methosulfate (PMS),
10 mM MTT, 30 mM glucose-6-phosphate (G6P), and 30
units/mL glucose-6-phosphate dehydrogenase (G6PDH) grade I
2.2.7 2-OGDH Assay Solutions for extraction buffer: Stock solutions are prepared as
Solutions for reaction buffer: 500 mM TES-NaOH pH 7.5,
10% (v/v) Triton X100, 1 M MgCl2, 50 mM NAD+, 100 mM
coenzyme A, 100 mM TPP, 100 mM cysteine, 100 mM adenine
monophosphate (AMP), 100 units/mL lipoamide dehydrogenase
from pig heart, and 30 mM 2-oxoglutarate (2-OG). For activation
assay by TPP addition of 10 mM TPP is suggested (see Note 3).
2.2.8 SCoAL Assay Solutions for extraction buffer: Stock solutions are prepared as
Solutions for reaction buffers:
Buffer 1: 1 M Tricine-KOH pH 8.0, 1 M MgCl2, 200 mM EDTA,
0.35 units/μL commercial glycerokinase, 1 M phosphate,
25 mM adenosine 50 -diphosphate (ADP), 1 mM 50 ,50 -diade-
nosinpentaphosphate, 87% (v/v) glycerol, and 1 mM succinyl
CoA.
Buffer 2: 1 M Tricine-KOH pH 8.0, 1 M MgCl2, 100 mM NADH,

500 units/mL commercial glycerol-3-phosphate oxidase
(GPOX), and 200 units/mL protein commercial glycerol-3-
phosphate dehydrogenase (GDH) (see Note 3).
2.2.9 SDH Assay Solutions for extraction buffer: 1 M mannitol, 500 mM MOPS-
KOH pH 7.5, 100 mM EDTA, 5% (w/v) BSA, 5% (w/v) PVP, and
20 mM cysteine.
Solutions for reaction buffer: 1 M Potassium phosphate buffer
pH 7.5, 300 mM succinate, 10 mM MTT, and 10 mM PMS
(see Note 1).
2.2.10 FUM Assay Solutions for extraction buffer: Stock solutions are prepared as
Solutions for reaction buffer: 1 M Buffer tricine-KOH pH 8.0,
1 M phosphate, 100 mM fumarate, 30 mM NAD+, 5000 units/mL
malate dehydrogenase in 200 mM tricine-KOH pH 8.0, 50 units/
mL citrate synthase in 200 mM tricine-KOH pH 8.0, 10 mM acetyl
CoA, 10%Triton X100, 0.5 M NaOH, 0.5 M HCl prepared in
200 mM tricine-KOH pH 8.0, 1000 units/mL alcohol dehydro-
genase in 200 mM tricine-KOH pH 9.0, 25 M ethanol; 200 mM
EDTA solution with pH 7.5 adjusted by adding KOH solution,
20 mM PES, 20 mM MTT, and 80 μM malate (see Note 3).
2.2.11 NAD+-Dependent Solutions for extraction buffer: Stock solutions are prepared as
MDH Assay described for CS (see Subheading 2.2.3).
Solutions for reaction buffer: 1 M TES buffer pH 7.2, 1 M
MgCl2, 10 mM β-NADH, 0.05% (v/v) Triton X100, and 50 mM
oxaloacetate.
2.2.12 NAD+-Dependent Solutions for extraction buffer: 1 M 2-(N-morpholino)ethanesul-

ME Assay fonic acid (MES)-NaOH pH 6.5, 500 mM MnCl2, 100 mM
EDTA, 500 mM 2-mercaptoethanol, 5% Triton X-100, 50% glyc-
erol, and 100 mM PMSF, and 100 mM DTT.
Solution for reaction buffers:
Buffer 1: 500 mM HEPES-NaOH pH 6.9, 100 mM NAD+,
100 mM CoA, 100 mM MgSO4, and 100 mM L-malate.
Buffer 2: 1 M HEPES-NaOH, pH 7.5, 100 mM β-NADH, and
50 units/mL of L-lactate dehydrogenase (see Note 3).
2.2.13 NADP+- Solutions for extraction buffer: 1 M Tris–HCl, pH 7.5, 500 mM

Dependent ME Assay MgCl2, 100 mM EDTA, 50% (v/v) glycerol, and 500 mM 2-
mercaptoethanol.
Solutions for reaction buffer: 1 M HEPES-NaOH pH 7.5,
100 mM MgSO4, 100 mM NADP+, and 100 mM L-malate.
3 Methods
3.1 General 1. The temperature assay and reads in spectrophotometer are

Procedures Used performed at 25 C.
in Enzyme Activity 2. The indicated spectrophotometers read between 0.01 and 2.0
Measurements absorbance units and the assay has been configured to work
within this zone.
3. The control assay, that does not contain any enzymatic extract
or containing denaturated protein extract, but have all the
solutions and substrates needed for initiating reactions, is
important to account for any drift in the baseline absorbance.
4. In general the final volume of reaction assay is 290 or 300 μL
because it is the maximum volume of plate. However, reaction
volumes can be scaled down to 100 μL or less.
5. Extraction procedure: Each assay uses between 20 and 50 mg
of fresh homogenized material to 500 μL of extraction buffer
(see Notes 4 and 5). 5–10 μL of total extract can be used for
reaction. When necessary, the extracts are further diluted or
desalted in the extraction buffer.
6. After addition of the enzyme extract and reaction mix, the reac-
tion is immediately monitored by measuring changes in absor-
bance or fluorescence in a microplate reader (see Note 6). The
rates of reactions are calculated as the decrease or increase in the
absorbance, which is expressed in mOD/min. VBase (values
obtained with the blanks, without enzyme extract) and VSat
(values obtained with enzyme extracts) assays are performed for
every sample in duplicate, and each plate includes at least four
standards and blanks (where reaction start is omitted). Samples
are randomly distributed to avoid local artifacts (see Note 7).
7. Enzymatic activity is usually expressed in nmoles per minute
per milligram of total protein (nmol/min/mg protein) or of
fresh weight (nmol/min/mg FW) (see Note 8).
3.2 Protein The activities of TCA cycle enzymes might be determined follow-
Extraction for Enzyme ing three different extraction procedures:
Assays
1. Total tissue extracts: For routine measurements, samples
corresponding to ~50 mg FW (leaf, root, or fruit tissues) are
collected, ground to a fine powder in liquid N2, and stored at
80 C (see Note 9). Aliquots of 10–20 mg FW are weighted at
very low temperature and 1% of insoluble PVP are added to
each sample. Then extraction buffer is added and the extraction
is performed by vigorous vortexing. The composition of the
extraction buffer is 20% (v/v) glycerol, 0.25% (w/v) BSA, 1%
(v/v) Triton X-100, 50 mM HEPES/KOH, pH 7.5, 10 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine,
1 mM ε-aminocaproic acid, 1 mM PMSF, 10 mM leupeptin,

and 1 mM DTT. PMSF is added just before extraction. DTT is
omitted when using peroxidase-based or MTT-based indicator
reactions. In all cases, 500–1000 μL of extraction buffer is
used, leading to an initial 25- to 50-fold (w/v) dilution.
When necessary, extracts are further diluted to generate appro-
priate dilutions for the different enzymes. This procedure has
been described in [14], except for the addition of 20% (v/v)
glycerol and 1% (v/v) Triton X-100. The assays described in
this chapter, at the exception of the assay for PDH and SDH,
are usually performed using this extraction procedure (see
Notes 10 and 11).
2. Mitochondrial enriched extract: The mitochondrial enriched
fraction is obtained by homogenizing 250 mg of plant tissue
(leaf, root, or fruit tissues) in prechilled extraction medium
containing 300 mM mannitol, 30 mM MOPS-KOH pH 7.5,
1 mM EDTA, 0.1% (w/v) BSA-fraction V, 0.1% (w/v) PVP,
and 4 mM cysteine. The homogenate is passed through three
layers of Miracloth (Calbiochem) and centrifuged at 1000 g
for 10 min. The supernatant is then transferred to new tubes
and centrifuged at 12,500 g for 20 min at 4 C, and the pellet
is resuspended using a soft paintbrush dipped in 300 mM
mannitol, 30 mM MOPS-KOH, pH 7.5, and 1 mM EDTA.
SDH activity is determined as described previously [16]
3. Purified mitochondrial preparation extracts: The mitochon-
drial isolation is performed as described in [20, 21].
3.3 Pyruvate The extraction and activity of PDH are performed as described
Dehydrogenase previously [12, 13].
1. Extraction: Purified mitochondrial preparations using two con-
secutive discontinuous Percoll gradients are used in the assay.
Mitochondrial preparations are stored at 4 C in 20 mM TES-
KOH pH 7.2, 2 mM MgCl2, 1 mM Na2EDTA, 0.1% defatted
BSA, 0.3 M mannitol, and 2 mM DTT. Mitochondria main-
tained at least 90% of their PDC for at least 4 days.
2. Reaction assay: The activity of PDH is determined by monitor-
ing the formation of NADH at 340 nm. The reaction medium
contains 75 mM TES-NaOH pH 7.5, 0.5 mM MgCl2, 2 mM
NAD+, 0.2 mM lithium CoA, 0.2 μM TPP, and 2.5 mM cyste-
ine-HCl.
3. Reaction start: The reaction is started by adding 1 mM sodium
pyruvate after at least 10 min of measurements.
4. Possible activation assay: For the activation of PDH, TPP

concentration in the reaction medium is increased from 0.2
to 10 μM [7].
3.4 Citrate Synthase The extraction and activity of CS are performed as described previ-
ously [14].
1. Extraction: Enzyme extracts are prepared as described in Sub-
heading 3.2, step 1.
2. Reaction assay: Protein extracts (2 μL), as well as NADH
standards prepared in the extraction buffer ranging from 0 to
200 μmol, are incubated in a medium containing 4 μL assay
mix (0.5 M tricine buffer pH 8.5, 1.5 mM malate, and 0.25 (v/
v) Triton X-100), 0.2 mM acetyl-CoA, 0.25 mM NAD+, and 5
units/mL of malate dehydrogenase. Incubate at 25 C for
40 min. The reaction is stopped with 250 mM of NaOH.
Incubate again at 95 C for 5 min. After neutralizing with
167 mM HCl prepared in tricine buffer pH 9, final concentra-
tions of 91.0 mM tricine buffer pH 9.0, 1.0 mM MTT,
7.3 mM EDTA, 1.0% ethanol, 0.18 mM PES, and 18 units/
mL alcohol dehydrogenase are added. The plate should be
protected from light. The absorbance at 570 nm can be
measured once the reaction is started by the addition of
0.36 M ethanol.
3. Reaction start: The reaction is started by the addition of acetyl-
CoA to a final concentration of 0 (blank) or 0.2 mM (maximal
activity).
4. Activation assay: It has been shown that CS is activated in vitro
by thioredoxin-dependent reduction [5]. The activation assay
is performed as described by Schmidtmann et al. [5]. The
protein is purified under non-disulfide-reducing conditions
and TRX-dependent reduction CS activity is measured with
TRX. The enzyme is incubated for 15 min at room temperature
in the presence of 35 μg recombinant E. coli TRX, 0.8 μg
recombinant E. coli thioredoxin-reductase (NTR), and
1.5 mM NADPH. After this procedure the activity can be
measured as described above.
3.5 Aconitase The extraction of ACO is performed as described previously [14].

Total ACO activity is determined according to [17].
2. Reaction assay: ACO is assayed spectrophotometrically by fol-
lowing the increase in absorbance at 340 nm. The reaction
mixture consists of 80 mM HEPES-NaOH pH 7.5, 0.5 mM
NADP+, 0.42 mM MnCl2, 0.2 units of NADP+-dependent
isocitrate dehydrogenase from porcine heart, and 0.05% (v/v)

Triton X100.
3. Start of reaction: The reaction is started by the addition of
aconitate to a final concentration of 8 mM.
4. Activation assay: To our knowledge no activation assay has
been reported for this enzyme yet.
3.6 Isocitrate The extraction of IDH is performed as described [14]. The activity
Dehydrogenase NAD+ of IDH is performed as described [15] with modifications.
Dependent 1. Extraction: Enzyme extracts are prepared as described in Sub-
2. Reaction assay: IDH is assayed by incubating crude extract or
isocitrate standards in a freshly prepared medium containing
40 mM Tris–HCl pH 7.6, 1.5 mM NAD+, 6.3 mM MnCl2,
0.05% β-mercaptoethanol, and 15 mM isocitrate. The absor-
bance is read at 340 nm.
3. Start of the reaction: The reaction is started by the addition of
isocitrate to a final concentration of 15 mM.
3.7 Isocitrate The extraction and activity of ICDH are performed as described
Dehydrogenase NADP+ previously [14].
Dependent
2. Reaction assay: ICDH is assayed incubating sample extracts, as
well as NADPH standards freshly prepared in the extraction
buffer, in concentration varying from 0 to 80 mM, in a medium
containing 100 mM tricine/KOH pH 8.0, 4 mM MgCl2, and
1 mM NADP+. The reaction is stopped with 20 mL of 0.5 M
NaOH and the plate is incubated at 95 C for 5 min. After
cooling down the plate, each reaction is neutralized with 10 μL
of HCl prepared in 0.2 M tricine buffer pH 9. NADPH is
measured in the presence of 3 units/mL G6PDH grade I,
100 mM tricine/KOH pH 9.0, 5 mM MgCl2, 4 mM EDTA,
0.1 mM PMS, 0.6 mM MTT, and 3 mM G6P. The absorbance
is read at 570 nm. The NADPH formed is then determined
using the NADP+-based cycling protocol.
3. Start of the reaction: The reaction is started by the addition of
isocitrate to a final concentration of 2 mM (maximal activity).
Blanks are also necessary for each sample using 0 mM of iso-
citrate (blank).
3.8 2-Oxoglutarate The extraction of 2-OGDH is performed as described previously

Dehydrogenase [17]. 2-OGDH activity is measured essentially following the pro-
tocol described previously [8].
2. Reaction assay: In brief, 2-OGDH activity is measured by
following the reduction of NAD+ at 340 nm. The reaction
medium contains 75 mM TES-KOH (pH 7.5), 0.05% (w/v)
Triton X100, 0.5 mM MgCl2, 2 mM NAD+, 0.12 mM lithium-
CoA, 0.2 mM TPP, 2.5 mM cysteine-HCl, 1 mM AMP, 1 mM
sodium-2-oxoglutarate, and 10 units/mL of lipoamide dehy-
drogenase. The enzyme is preincubated in the reaction
medium for 15 min. The initial linear part of the product
accumulation curves is used to calculate the reaction rates.
3. Reaction start: The reaction is started by adding 1 mM of 2-
oxoglutarate.
4. Activation assay: For the activation of 2-OGDH the concen-
tration of TPP is increased from 0.2 to 1 mM TPP [7].
3.9 Succinyl-CoA The extraction of SCoAL is performed as described previously [14].

Ligase SCoAL activity is measured following the protocol also described
previously [18].
2. Reaction assay: SCoAL is assayed in the forward direction by
measuring the production of ATP from ADP. Extracts, as well
as ATP standards ranging from 0 to 1 nmol, are freshly
prepared in the extraction buffer. They are incubated in a
microplate at 25 C, in a medium containing 100 mM tricine
buffer pH 8, 10 mM MgCl2, 100 μM EDTA, 1 unit/mL
glycerokinase, 10 mM phosphate, 2.5 mM ADP (ATP free),
100 μM 50 ,50 -diadenosinpentaphosphate, 120 mM glycerol,
0 (blank), or 100 μM (maximal activity) succinyl CoA. The
reaction volume is set to 50 μL. The reaction is stopped with
20 μL of 0.5 M HCl. After neutralization with 20 μL of NaOH
0.5 M, G3P is measured by adding GPOX, GDH, and NADH
to final concentrations of, respectively, 2 units/mL, 0.8 units/
mL, and 0.5 mM. The absorbance is read at 340 nm and at
30 C in a microplate reader until the rates are stable. The
reaction rates are calculated as the decrease of absorbance in
mOD/min.
3. Reaction start: The reaction is started by adding 100 μM of
succinyl CoA (maximal activity).
3.10 Succinate The extraction and activity of SDH are performed as described
Dehydrogenase previously [16].
2. Reaction assay: 50–100 mg Protein of a mitochondrial
enriched fraction is assayed for SDH activity by monitoring
the absorbance change of MTT in the presence of PMS, by
using the extinction coefficient of 17/mM/cm for MTT. The
measurements are performed spectrophotometrically at
570 nm. The reaction medium contains 50 mM potassium
phosphate buffer pH 7.4, 10 mM sodium succinate, 0.6 mM
MTT, and 2 mM PMS.
3. Reaction start: The reaction is started by adding 10 mM
sodium succinate.
4. Activation assay: The activation assay is performed as described
[6]. For the activation assay enzyme activities are measured in
mitochondrial extracts prepared as described in Subheadings
3.2, steps 2 or 3. The protein extracts are untreated (control)
or treated with TRXo1 (3 μg; 180 nM) or TRXh2 (3 μg;
210 nM) both reduced by NTRB (7.5 μg; 100 nM) and
NADPH (100 μM).
3.11 Fumarase The extraction and assay of FUM are performed as described
previously [14].
2. Reaction assay: FUM enzyme is assayed in the malate-forming
direction. Protein extracts (5 μL), as well as malate standards
prepared in the extraction buffer ranging from 0 to 1 nmol, are
incubated in a medium containing 100 mM tricine buffer
pH 8.0, 0.2 mM acetyl-CoA, 10 mM phosphate, 0.15 mM
NAD+, 0.05 (v/v) Triton X-100, 100 units/mL of malate
dehydrogenase, and 1 unit/mL of citrate synthase. Incubate
at 25 C for 20 min. The reaction is stopped with 20 μL of
NaOH 0.5 M.
The plate is incubated again at 95 C for 5 min. After cooling
down, neutralizing with 200 mM HCl prepared in 100 mM
tricine buffer pH 9, 50 μL of reaction mix containing 70 mM
tricine buffer pH 9.0, 0.7 mM MTT, 6 mM EDTA, 0.07 mM
PES, and 3.6 units/mL alcohol dehydrogenase are added. The
absorbance at 570 nm can be measured once the reaction is
started by the addition of 0.36 M ethanol.
3. Reaction start: The reaction is started by the addition of fuma-
rate to a final concentration of 0 (blank) or 10 mM (maximal
activity).
4. Possible activation assay: The activation assay is performed as

described [6]. For the activation assay enzyme activities are
measured in mitochondrial extracts prepared as described in
Subheading 3.2, steps 2 or 3. The protein extracts are
untreated (control) or treated with TRXo1 (3 μg; 180 nM)
or TRXh2 (3 μg; 210 nM) both reduced by NTRB (7.5 μg;
100 nM) and NADPH (100 μM).
3.12 Malate The extraction of MDH is performed as described previously [14].

Dehydrogenase MDH activity is determined as defined previously [17].
2. Reaction assay: MDH is assayed in the direction of malate
formation following the oxidation of NADH spectrophoto-
metrically at 340 nm. The reaction mixture contains 50 mM
TES buffer pH 7.2, 5 mM MgCl2, 0.2 mM NADH, and 0.05%
(v/v) Triton X100.
3. Reaction start: The reaction is started by the addition of oxalo-
acetate to a final concentration of 1 mM.
4. Possible activation assay: To our knowledge activation assay has
not been reported for this enzyme yet.
3.13 NAD+- The extraction and activity of NAD-ME are performed as described
Dependent Malic previously [22].
Enzyme
1. Extraction: The extraction buffer consists of 50 mM MES-
NaOH pH 6.5, 5 mM MnCl2, 1 mM EDTA, 10 mM 2-
mercaptoethanol, 0.05% Triton X-100, 20% glycerol, and
1 mm PMSF. The homogenates are clarified by centrifugation.
The supernatants are desalted using a Sephadex G-50 column
equilibrated with buffer containing 50 mM MES-NaOH,
pH 6.5, 5 mM MnCl2, 5 mM DTT, and 20% [v/v] glycerol
buffer and separated for activity measurements.
2. Reaction assay: NAD-ME activity is measured following the
production of pyruvate from malate. NAD-ME activity can be
measured in crude extracts from whole-plant tissues or from
isolated mitochondria. The measurements are made spectro-
photometrically at 340 nm. The reaction mixture contains
50 mM MES-NaOH, pH 6.5, 4 mM NAD+, 5 mM DTT;
10 mM MnCl2, and 10 units of commercial MDH. With this
assay there is a rapid but small increase of the A340 as the
reaction catalyzed by the MDH reached the equilibrium. After-
wards, the subsequent steady increase of A340 is attributable to
the decarboxylation of L-malate by the NAD-ME.
3. Reaction start: The reaction is started by the addition of

10 mM malate.
4. Possible activation assay: To our knowledge no activation assay
has been reported for this enzyme yet.
3.14 NADP+- The extraction and activity of NADP-ME are performed as

Dependent Malic described previously [19].
Enzyme
1. Extraction: Tissue material is ground in liquid N2 and the
resulting powder is suspended in 100 mM Tris–HCl, pH 7.5,
5 mM MgCl2, 2 mM EDTA, 10% (v/v) glycerol, and 10 mM
2-mercaptoethanol, in the presence of a protease inhibitor
cocktail. The homogenates are clarified by centrifugation and
the supernatants are separated for activity measurements.
2. Reaction assay: NADP-ME activity is measured following the
oxidation of malate producing pyruvate and NADPH which is
measured spectrophotometrically at 340 nm. The reaction mix-
ture contains 50 mM Tris–HCl, pH 7.0, 10 mM MgCl2, 0.5 mM
NADP+, and 10 mM L-malate in a final volume of 0.5 mL.
3. Reaction start: The reaction is started by the addition of the
10 mM L-malate.
4. Possible activation assay: To our knowledge no activation assay
has been reported for this enzyme yet.
4 Notes
1. The stock solutions of PMS, PES, MTT, and DTT should be

stored in the dark and freshly prepared.
2. The stock solutions of pyridine nucleotides (phosphate and/or
reduced) should be shock frozen and stored at 80 C.
3. The stock solutions of enzymes should be shock frozen before
storage at 80 C. Note that lactate dehydrogenase cannot be
frozen once in solution.
4. Samples should be harvested by shock freezing in liquid nitro-
gen, and then stored at 80 C until extraction.
5. Samples should be ground with great care, as powder granulo-
metry and homogeneity are essential to obtain reproducible
results.
6. The reaction is started after at least 5–10 min of measurements.
7. During the preparation of the microplates, sample extracts,
stock solutions, and reaction mix should be maintained on
ice. Then, microplates should be equilibrated at 25 C before
starting assays.
8. Since the activity is based on mass, fresh weight annotation

should be accurate.
9. The samples should not thaw during sample preparation until
addition of the extraction buffer.
10. Total tissue extract is appropriate for most of the TCA cycle
enzymes. For SDH, due to its low concentration in the extract,
mitochondrial preparations are recommended. Although not
tested due to ease of total extract, mitochondrial preparations
seem highly feasible for all mitochondrial enzymes described
here.
11. Extracts can be shock frozen and stored at 80 C for later use.
The presence of 20% glycerol in the extraction buffer protects
most enzymes from denaturation following freezing-thawing
cycles. However, it is recommended to check it for each
enzyme prior to storage.
Acknowledgements
Financial support was provided by Conselho Nacional de Desen-

volvimento Cientı́fico e Tecnológico (CNPq- to W.L.A.), Funda-
ção de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG),
and Max Planck Society to A.N.N. and W.L.A. Research fellowships
granted by CNPq to A.N.N. and W.L.A. as well as scholarship
granted by the Coordenação de Aperfeiçoamento de Pessoal de
Nı́vel Superior (CAPES) to R.P.O.G. are also gratefully
acknowledged.
Conflict of interest: The authors declare that they have no conflict of
interest.
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Chapter 15
Respiration Traits as Novel Markers for Plant Robustness

Under the Threat of Climate Change: A Protocol
for Validation
Birgit Arnholdt-Schmitt
Abstract
Respiration traits allow calculating temperature-dependent carbon use efficiency and prediction of growth
rates. This protocol aims (1) to enable validation of respiration traits as non-DNA biomarkers for breeding
on robust plants in support of sustainable and healthy plant production; (2) to provide an efficient, novel
way to identify and predict functionality of DNA-based markers (genes, polymorphisms, edited genes,
transgenes, genomes, and hologenomes), and (3) to directly help farmers select robust material appropriate
for a specified region. The protocol is based on applying isothermal calorespirometry and consists of four
steps: plant tissue preparation, calorespirometry measurements, data processing, and final validation
through massive field-based data.
The methodology can serve selection and improvement for a wide range of crops. Several of them are
currently being tested in the author’s lab. Among them are important cereals, such as wheat, barley, and rye,
and diverse vegetables. However, it is critical that the protocol for measuring respiration traits be well
adjusted to the plant species by considering deep knowledge on the specific physiology and functional cell
biology behind the final target trait for production. Here, Daucus carota L. is chosen as an advanced
example to demonstrate critical species-specific steps for protocol development. Carrot is an important
global vegetable that is grown worldwide and in all climate regions (moderate, subtropical, and tropical).
Recently, this species is also used in my lab as a model for studies on alternative oxidase (AOX) gene diversity
and evolutionary dynamics in interaction with endophytes.
Key words Calorespirometry, Predicting plant robustness, Yield stability, Temperature tolerance,
Climate change, Biomarker, Functional marker, Genomics, Hologenomics, Daucus carota L
1 Introduction
Calorespirometry was developed as a means for understanding

metabolic adaptation and acclimation to environmental conditions
[1]. Nevertheless, it was already early suggested that the method-
ology could be helpful in breeding programs to improve
temperature-dependent growth performance [2]. However,
despite this early insight, the concept has never been applied on
important crops in global breeding or for farmers in order to select
183
184 Birgit Arnholdt-Schmitt
the better material for their region. More recently, the basic con-
cept of this approach got significant support through new knowl-
edge on the significance of mitochondria and specifically on
alternative respiration for managing plants’ adaptive multi-stress
responses (reviewed in [3] and [4]) combined with deep phenotyp-
ing initiatives in plant breeding [5–7]. Alternative oxidase (AOX)
genes have been proposed as promising candidates for functional
marker development linked to temperature responses and growth
performance [8–11]. In carrot, applying the present protocol to
root meristems in a phenotyping assay with a small set of inbred
lines, it was possible to conclude that calorespirometry might be
used to identify genotype-specific optimum temperatures and low
temperature limits for root biomass growth [12]. Recently, it was
also found that early AOX expression during reprogramming of
quiescent carrot tap root tissue to cell division-based growth coin-
cided with a critical time point for biomass prediction measured by
calorespirometry [10].
Calorespirometry is capable of measuring temperature-depen-
dent metabolic heat rates and near-simultaneous CO2 production
rates in small amounts of growing plant tissue. CO2 production
rates can be obtained by capturing tissue-emitted CO2 as bicarbon-
ate through temporarily added NaOH in an exothermic reaction
(enthalpy change: ΔH ¼ 108.5 kJ/mol). This allows calculating
overall temperature-dependent substrate carbon conversion effi-
ciency and structural biomass formation rates. The calculation is
based on thermodynamic modeling explained by Hansen et al. [1,
13]. It takes into account an enthalpy balance model valid under
mainly aerobic conditions where the energy released by a respiring
tissue is equal to the sum of energy from catabolic reactions plus
that absorbed by anabolic reactions [1, 14, 15].
In carrot, yield stability depends crucially on the regulation of
the central root meristem that determines secondary growth of the
tap root, which is the harvest organ ([16] and references therein).
Shoot growth is not critically limiting root growth when plants are
growing under optimal abiotic and biotic conditions. However,
under difficult climate conditions due to extreme changes between
low and high temperatures and under weed pressure for competi-
tive plant growth, temperature-dependent rapid seedling and shoot
growth becomes critical for yield stability. Thus, a methodology
that is able to predict growth performance in both root and shoot
can serve as a valuable tool in carrot pre-breeding.
2 Materials
2.1 Plant Source Carrot seedlings and plants for shoot and secondary root sampling
Material (see Subheading 3.1): Genotypes should be selected from breeding
material or registered varieties with known environmental
responses across high numbers of locations and years.
Respiration Traits as Novel Markers 185
2.2 Equipment Calorespirometric measurements can be made in a model 4100

Multi-Cell Differential Scanning Calorimeter (TA Instruments,
USA). This instrument has four 1-mL ampoules, three for samples
and one ampoule acting as a reference. Heat emitted by material
inside the ampoules is measured by the equipment as heat rates.
The calorimeter is permanently connected to a water bath for
temperature control. A nitrogen container/bottle is connected to
the calorimeter in case temperatures will be measured below 15 C
in order to keep the atmosphere in the equipment dry (see Note 1).
2.3 Laboratory 1. Gloves, sharp laboratory knifes, and forceps are required for
Material carrot material sampling.
2. Tubes for preparing fresh 0.4 M NaOH solution (see Note 2).
3. Tips of plastic micro tubes are cut from normal tubes to a size
that allows placing them into the ampoules. They are used as
containers for 40 μL of 0.4 M NaOH solution for CO2 captur-
ing. Even when placed in a completely horizontal way, the liquid
will stay inside.
4. Laboratory nitrogen gas in a container/bottle connected to the
calorimeter.
3 Methods
3.1 Preparation Grow seeds that had been stored at cool temperatures (~4 C) in
of Carrot Samples pots with commercial substrate or into sandy soil (see Note 3).
from Root Tissue and Depending on maturity characteristics of the cultivar, sample carrot
Shoots plants from the moment when the root meristem is well developed,
meaning when root length growth is finalized and secondary root
3.1.1 Collecting Root growth is taking place (e.g., 3–4 months after seeding). Manually
Meristem from Tap Roots separate the tap root meristem circle as a layer of ten cells (thick-
ness) around the central xylem from xylem and secondary phloem
and cut it into smaller pieces in order to fit into the calorimeter
ampoules. Take samples of ca. 200 mg of meristem for each
measurement.
3.1.2 Collecting Shoot Sow seeds from selected genotypes on watered paper. In the seed-
Material ling stage, when both cotyledons are developed, transfer seedlings
to soil. When 5–10 leaves have emerged, sample leaf material
(see Note 4).
3.2 Calorespirometry Calorespirometry measurements on carrot samples follow the gen-

eral protocol given by Hansen et al. [1] and the specific protocols
3.2.1 Measurements
developed for carrot samples by Nogales et al. [12, 17]:
1. Place carrot root tissue or shoot samples into one of the three
ampoules for heat rate (Rq) measurements (see Subheading 2.2)
and run samples at a series of temperatures in isothermal mode
ranging normally between 5 and 40 C. Take endpoint measure-

ment values and subtract from each measured value a baseline
value (see Note 5) obtained from measurements with empty
ampoules. Temperature changes are run from less stressful to
more stressful conditions (see Note 6). For each temperature
change, program an equilibrium time of 600 s (s) before starting
measurements.
2. After the Rq signal has become stable during about 300 s, record
the first endpoint measurement value; normally this can be
obtained after around 35 min (see Note 7).
3. Open sample ampoules, introduce a vial of 0.4 M NaOH solu-
tion in order to capture emitted CO2 from the plant tissue at the
measurement temperature, and close the ampoule again. Record
the heat rate after it has become stable again for around 300 s
and take the second measurement value.
4. Remove the NaOH vial from the ampoules in order to measure
again the heat rate and take the third measurement endpoint
value when heat rates have become stable (see Note 8).
Perform measurements in repetitions in the same way with at
least three independent samples.
3.2.2 Data Processing 1. Calculate the mean of the measured heat rates (Rq) before and
(See Note 9) after adding NaOH (3.2.1 points 2 and 4) (unit: μW or μJ/s).
2. Subtract this mean heat rate from the measured Rq in the
presence of NaOH (unit: μJ/s) to obtain Rq that relates to
CO2 production (Subheading 3.2.1, step 3).
3. Calculate the rate of CO2 production (RCO2) by dividing the
mean Rq (Subheading 3.2.2, step 1) by the negative of the
enthalpy change, i.e., by 108.5 μJ/nmol (unit: nmol/s).
4. Calculate the calorespirometry ratio Rq/RCO2 (unit: μJ/nmol
or kJ/mol CO2) by using the Rq value obtained under Sub-
heading 3.2.2 step 2, and the value for the rate of CO2 produc-
tion obtained under Subheading 3.2.2, step 3 (see Note 10).
5. Calculate substrate carbon conversion efficiency (ε) in two steps
from calorespirometry ratios (Rq/RCO2) that are lower than
470 kJ/mol CO2 (see Subheading 3.2.2, step 4, and Note 10):
(a) Calculate a “factor x”: [Rq/RCO2–470]/30 (see Note
11).
(b) Calculate ε by dividing “factor x” (a) by its value plus 1 (no
unit) (see Note 12).
6. Calculate the rate of biomass formation (Rbiom): “factor x”
times RCO2 (see Subheading 3.2.2, steps 5a and 3) (no unit)
(see Note 13).
7. Analyze the temperature dependency of both values (ε and

Rbiom) by temperature response curves (x-axis: temperature;
y-axis: ε or Rbiom) and use these graphs as a basis to identify
differences between genotypes in terms of its stable or unstable
response across the tested temperatures (see Note 14).
8. Apply common statistics methodologies to identify significant
differences between genotypes at defined temperatures or for
the temperature response curve considering at least three inde-
pendent repetitions.
3.2.3 Validation 1. Validation depends on the association between ε and Rbiom of

roots and shoots with yield stability at a satisfactory level of
product quality and yield. Compare results achieved by this
protocol to already available large data from field experiments.
Data from many locations and different years can be obtained
from breeders or by variety registration catalogues. In case the
ranking of varieties or breeding material for robustness/yield
stability obtained from field trials could have been predicted by ε
and/or Rbiom, these respiration traits can be validated as useful
biomarkers for plant selection in carrot pre-breeding.
2. In case of a positive validation, the protocol can also be used to
select carrot varieties as a service to farmers. Local temperature
distribution curves, temperature-dependent climate changes,
predicted optimum temperature response, and temperature lim-
its for respiration and growth performances must be considered.
4 Notes
1. To avoid water condensation, the measuring chamber should

never be opened unless the temperature is above 15 C.
2. NaOH solution can be stored and used for short periods of
around 3 weeks depending also on how often and long it will
be opened during handling. For a new trial, fresh 0.4 M NaOH
should be prepared.
3. The exact conditions for carrot growth in pots (greenhouse or
growth chamber) are considered not important as long as all
genotypes for screening are grown in the same location under
comparable abiotic and biotic conditions.
4. The number of leaves taken for the measurement depends on
their size and the heat produced (see Subheading 2.2). Before
putting the leaves into the ampoules they are shortly dipped in
70% ethanol, washed twice with sterile aqua dest., and then
surface-dried by using a paper towel.
5. Baselines need to be taken for each ampoule, since the values
will differ depending on the ampoules and equipment
conditions.
Isothermal Curves minus/plus/minus NaOH

200
150
Heat Rate
100
50
0
4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000
measurement time (seconds)
Fig. 1 A typical aspect of isothermal curves minus/plus/minus NaOH
Table 1
Step-by-step data processing to obtain carbon use efficiency and rate of biomass formation
Example for data from carrot shoot (cv. Rotin): Minus NaOH (1) Plus NaOH (2) Minus NaOH (3)
Measured Rq values (Subheading 3.2.1) 83 108 80
Steps of data processing (Subheading 3.2.2)
1. 81.5
2. 26.5
3. 0.244
4. 333.69
5.a 4.54
5.b 0.82
6. 1.11
6. It is important to avoid beginning with stressed tissue that

might interfere with subsequent measurements. Before the
first measurement is started, it is also recommended to allow
an adaptation time of around half an hour at a non-stressful
temperature close to the first measurement temperature.
7. Stressed plant material that might show a rapid decrease in heat
rate will not give valid measurement values. On the contrary,
contamination by rapidly growing microorganisms might also
not result in valid measurement values because of increasing
heat rates.
8. A typical aspect of the isothermal curves minus/plus/minus
NaOH is given in Fig. 1.
9. An example for a step-by-step calculation is given in Table 1.
10. The ratio, Rq/RCO2, is inversely related to the efficiency by

which a plant system is using its available energy for biomass
growth. Heat released per mole consumed oxygen in living
material is called “oxycaloric equivalent” (e.g., [18]). Thorn-
ton’s rule states that this value is deduced from the oxidation
state of carbon and can be obtained by chemical reaction
through combustion of organic compounds. It reaches values
for ΔHO2 between 430 and 480 kJ/mol with an average
value of 455þ/15 kJ/mol. From carbohydrates every C-
mole releases 470 kJ/mol, a typical fatty acid provides 650 kJ/
mol, lipids 611 kJ/mol, and protein is expected to release
around 543 kJ per mole [1, 19]. The applied model assumes
carbohydrate as the main component in the growing biomass
and aerobic metabolism [1]. The amount of energy charac-
terizes the quantity of heat lost from the tissue per mole of
CO2 produced. Thus, it can be expressed by the ratio Rq/
RCO2. In growing tissue of plants, a measured value equal to
470 kJ/mol indicates under these assumptions that no growth
takes place, which is equal to zero substrate carbon conversion
efficiency. Values <470 kJ/mol indicate efficiencies >0 and
values higher than 470 kJ/mol indicate loss of energy for
biomass growth through the usage of reduced substances for
substrate oxidation. Thus, values for efficiency lie always
between >0 and <1.
11. This step can only be applied to calculate efficiency values >0,
meaning the ratio Rq/RCO2 needs to be <470 kJ/mol. Con-
sidering that the elemental composition and consequently heat
of combustion of all growing plant material are approximately
the same [1, 20, 21], it is assumed in the model that the change
of enthalpy (ΔHb) will be þ30 kJ mol-1 C. Then Rq/RCO2
can be set equal to 470–30 [ε/1-ε] [9]. Factor x ¼ [ε/1ε].
12. ε can be calculated from measured Rq/RCO2 values and the
calculated factor x:
ε ¼ x/(x þ 1).
13. According to the model, the rate of biomass formation
(Rbiom) can only be calculated when Rq/RCO2 is <470 kJ/
mol (see Note 8). Rbiom is equal to growth rate. Following
Hansen et al. [1, 13] respiration-driven growth of structural
biomass (Cbiomass) can be described as
Csubstrate þ NPK etc. þ xO2!εCbiomass þ (1ε) CO2.
Herein ε is the fraction of substrate carbon passing through
respiration and being used in anabolism to form structural
biomass. (1ε) is the fraction of substrate carbon lost as CO2.
By definition ε is called carbon use efficiency or substrate
carbon conversion efficiency. Rbiom/RCO2 is equal to ε/

(1ε); thus Rbiom is equal to the product of RCO2 times
“factor x.”
14. Small differences in ε will have high effects on growth over
time [13].
Acknowledgments
At first, the author wishes to thank Jagadis Gupta Kapuganti for his
invitation to make running protocols in my lab available to the
public via this valuable and respiration-focused collection. Special
thanks and my appreciation are going to Lee Hansen for his almost
daily availability as consultant and also collaborator in my lab to
support developing calorespirometry for application in plant breed-
ing. The author also wants to recognize the efforts of Amaia
Nogales, who was highly dedicated during her stay as postdoc
scientist at my Chair to become trained in calorespirometry and
to make the method working for carrot root material. The author
appreciates support from the Portuguese Foundation for Science
and Technology “Fundação para a Ciência e a Tecnologia” (FCT)
to establish this technology and is thankful to the University
of Évora for continuous invitations as Coordinating Investigator
since 2008 in order to prolong running of the established EU
Marie Curie Chair financed initially by the EC in the period from
2005 to 2008.
References
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from cell to ecosystem, Advances in photosyn- Schmitt B (2015) Functional marker develop-
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of DcAOX1 and DcAOX2a transcripts coin- carrot (Daucus carota L.) Eng Life Sci
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on DcAOX1 gene in carrot (Daucus carota CO2 on the development of musca domestica
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Chapter 16
Calorespirometry: A Novel Tool in Functional Hologenomics

to Select “Green” Holobionts for Biomass Production
Abstract
Endophytes can diversify temperature response and biomass production in plants and microalgae. Natural
and inoculated endophytes that modify growth performance are increasingly considered in research and
practical initiatives for sustainable agriculture. However, efficient, novel tools are required that are able to
support identification of differential effects of native endophyte populations and for pre-selection of
inocula.
This protocol gives instructions for applying calorespirometry as a rapid means for identifying differential
effects of endophytes on temperature response and predicted biomass productivity in microalgae and plant
holobionts. The protocol can help discriminating hologenomes, genes, and molecular neutral or functional
markers for microalgae strain and plant improvement. Here, we focus on the microalga Chlorella vulgaris
and associated microorganisms as an example for highlighting the methodology for its integration in
research and application.
Key words Functional genomics, Hologenomics, Respiration, Endophytes, Temperature response,

Biomass productivity, Plant holobionts, Microalgae, Chlorella vulgaris
1 Introduction
Knowledge on the relevance of endophytes for plant and micro-

algae growth improvement is increasing ([1] and references
therein). Functional marker development and gene as well as
genome editing are powerful instruments for targeting biodiversity
for eco-system and production system functionality. However,
break-through progress for closing the gap between functional
genomics/hologenomics and microalgal strain and plant improve-
ment depends strongly on appropriate novel concepts for screening
tool development. Successful tools should measure individual deep
phenotyping data that is centrally associated with adaptive growth
performance. Therefore, promising tool measures need to link to
energy metabolism, C-turnover rates, and epigenetics [2, 3].
193
194 Birgit Arnholdt-Schmitt and Vinod Kumar Patil
It is widely accepted that mitochondrial respiration in green

eukaryonts is critical for environmental signal perception, photo-
synthesis optimization, and final organism growth responses (e.g.,
[4–6]). Alternative oxidase (AOX) has been proposed and is now
broadly accepted as a potential source for powerful functional
marker candidates for multi-stress tolerance and yield stability
([7]; see also www.eu_chair.uevora.pt/ under publications and pre-
sentations). AOX/cytochrome oxidase (COX) activity ratio affects
carbon use efficiency that relates to efficient growth performance
[8, 9]. In green microalgae, biomass growth could be predicted by
calorimetric measures [10] and for plants calorespirometry was
developed as an emerging tool for rapid, predictive metabolic phe-
notyping in plant pre-breeding [11]. In mixotrophic or heterotro-
phic green microalgae cultures, in which media supplies a portion
or all of the carbohydrates and, in plant target tissues for breeding
especially, where photosynthesis is neither the driving nor the
limiting factor for growth, calorespirometry can become the tool
of choice for selection on superior growth performance.
Temperature is a major key factor for plant and microalgae
biomass production. Product quality linked to primary and/or
secondary metabolism is also influenced by temperature. Endo-
phytes are known to influence temperature response and overall
metabolism. Further, non-axenic microalgae production is normal
in commercial systems. Holobiont microalgae cultures can provide
higher stability to microalgae production systems (Martin Ecke
(GICON), personal communication). Thus, an efficient tool for
the selection on holobiont-dependent temperature response that is
based on respiration traits will also provide a means for combining
optimized biomass production with quality traits. In microalgae,
quality traits can relate to secondary metabolite compounds or lipid
production or other metabolic targets such as H2—production for
energy supply and, in plants, it is of interest to combine biomass
production with qualitative characteristics, such as the synthesis of
aromatic and/or medicinal compounds.
This protocol allows rapid screening for holobiont-dependent
predictive growth performance by calorespirometry. Calorespiro-
metry measures temperature-dependent metabolic heat rates and
near simultaneously CO2 production rates. CO2 production rates
can be obtained by capturing tissue-emitted CO2 as bicarbonate
through temporarily added NaOH in an exothermic reaction
(enthalpy change: ΔH ¼ 108.5 kJ/mol). This allows calculating
overall temperature-dependent substrate carbon conversion effi-
ciency and structural biomass formation rates. The calculation is
based on thermodynamic modeling explained in Hansen et al. [8,
9]. It takes into account an enthalpy balance model valid under
aerobic conditions where the energy released by a respiring tissue is
equal to the sum of energy from catabolic reactions plus that
absorbed by anabolic reactions [8, 12, 13]. This protocol is not
Calorespirometry: A Tool for Holobiont Selection 195
foreseen to allow quantitative analyses on metabolic pathways.

Instead, carbon use efficiency values obtained under aerobic con-
ditions are used here as relative, unitless biomarkers to rank geno-
types for selection.
2 Materials
2.1 Chlorella Strains of Chlorella vulgaris that contain endophytes or associated

vulgaris Strains microorganisms can be obtained from commercial industry or
germplasm banks.
2.2 Equipment 1. Autoclave and sterile working bench.

2. Incubators or growth chambers with integrated orbital shakers
that allow temperature and light/dark control.
3. Spectrophotometer that allows measuring in the cuvette mode
at 750 nm (see Note 1).
4. Calorimeter: Calorimetric measurements can be made in a
Multi-Cell Differential Scanning Calorimeter (TA Instru-
ments, USA). This instrument has four 1 mL ampoules, three
for samples and one ampoule acting as a reference. Heat emit-
ted by culture aliquots inside the ampoules is measured as heat
rates. The calorimeter is permanently connected to a water bath
for temperature control.
A nitrogen container/bottle is connected to the calorimeter in
case temperatures will be measured below 15 C to keep the
atmosphere in the equipment dry.
2.3 Laboratory 1. Gloves, glassware, petri dishes.

Material 2. Pipettes (500 and 1000 μL).
3. Cuvettes—1 mL (see Note 1).
4. Forceps to handle ampoules for calorimeter measurements.
2.4 Reagents/ Culture Chlorella vulgaris in BG 11 medium [14]. For medium

Buffers/Solutions/ preparation, the following nine stock solutions are needed:
Media
1. Sodium nitrate 15 g/L.
2.4.1 Culture Medium for 2. Dipotassium phosphate 2 g/L.
Chlorella vulgaris
3. Magnesium sulfate heptahydrate 3.75 g/L.
4. Calcium chloride dihydrate 1.8 g/L.
5. Citric acid 0.3 g/L.
6. Ammonium ferric citrate green 0.3 g/L.
7. Disodium EDTA 0.05 g/L.
8. Sodium carbonate 1 g/L.
9. Stock for trace elements:

Boric acid 2.8 g/L.
Manganese chloride tetrahydrate 1.81 g/L.
Zinc sulfate heptahydrate 0.22 g/L.
Sodium molybdate dihydrate 0.39 g/L.
Copper sulfate pentahydrate 0.08 g/L.
Cobalt nitrate hexahydrate 0.05 g/L.
To prepare the final BG11 medium mix the following:
#1 Stock solution 100 mL/L.
Make up the pH to 7.1 with 1 M NaOH or HCl.
Agar 15 g/L for solid medium.
Autoclave at 121 C for 15 min.
3 Methods
3.1 Microalgae Grow Chlorella vulgaris cultures in 100 mL Erlenmeyer flasks in

Culture 50 mL liquid BG 11 media at different temperatures in 16 h
photoperiod at 140 rpm. For controls free of associated microor-
ganisms, obtain isolated single cell colonies on solid BG 11
medium. Perform further propagation in increased volumes of
liquid culture. To maintain cultures, regular subcultures should
be done about every 10 days.
3.2 Growth For growth experimentation, start a subculture by inoculating

Experimentation 1000 μL as a standard into 50 mL BG 11 media. Growth should
be performed in 100 mL Erlenmeyer flasks in 16 h photoperiod at
140 rpm. Measure biomass production at defined time points after
inoculation by optical density, fresh weight, and/or dry weight.
3.3 OD Perform optical density measurements at 750 nm (see Subheading

Measurements for 2.2, item 3) at all critical time points for growth (lag phase, transfer
Growth Curves to linear growth phase, linear growth phase, at transmission to
stationary growth, stationary growth). Include control variants of
zero growth by suppressing growth during the experimental period
through a selected temperature. Identify appropriate strain-specific

temperatures for growth suppression by the help of calorimetry (see
Subheadings 2.2, item 4 and 3.4.1).
3.4 Calorimetric Take samples from zero growth controls and from microalgae
Measurements holobiont and single cell-derived colonies during the linear growth
phase determined by optical density measurements (see Subhead-
3.4.1 Measuring
ings 3.2 and 3.3). Pipette 500 μL of each sample under sterile
Temperature Response
conditions into the calorimeter ampoules (see Notes 2 and 3).
Measure heat rates (Rq—unit: μW or μJ/s) (see Subheading 2.2,
item 4) in isothermal mode at a range of temperatures (see Note 4).
3.4.2 Carbon Use To calculate carbon use efficiency (equal to substrate carbon con-
Efficiency and Biomass version efficiency) and to predict biomass formation rates, the
Formation Rate following steps have to be taken:
1. Calorimetric measurements:
(a) Take samples for genotype comparison at defined time
points during linear growth and measure in isothermal
mode under optimal temperature conditions. When the
Rq signal has become stable for about 300 s, take the first
of three measurement values (see Note 5).
(b) Open sample ampoules, introduce a vial with 40 μL 0.4 M
NaOH into the ampoule to capture emitted CO2 from the
microalgae cells, and close the ampoule. The heat rate
must be continuously recorded until it becomes stable
again for around 300 s, then take the second measurement
value.
(c) Remove the NaOH vial from the ampoules to measure
again the heat rate to obtain the third measurement value.
Perform measurements in repetitions in the same way
with at least three independent samples.
2. Data processing:
(a) Calculate the mean of the measured heat rates (Rq) before
and after adding NaOH (unit: μW or μJ/s).
(b) Subtract the mean heat rate (step 2a) from the measured
Rq in the presence of NaOH (unit: μJ/s).
(c) Calculate the rate of CO2 production (RCO2) by dividing
the mean Rq by the negative of the enthalpy change, i.e.,
by 108.5 μJ/nmol (unit: nmol/s).
(d) Calculate the calorespirometric ratio Rq/RCO2 (unit: μJ/
nmol or kJ/mol CO2) (see Note 6).
(e) Calculate substrate carbon conversion efficiency (ε) in two
steps from calorespirometric ratios (Rq/RCO2) that are
lower than 470 kJ/mol CO2 (see Note 6):
l Calculate “factor x”: [Rq/RCO2 470]/30 (see

Note 7).
l Calculate ε by dividing “factor x” (a) by its value plus 1
(no unit) (see Note 8).
(f) Calculate rate of biomass formation (Rbiom): “factor x”
times RCO2 (see under steps 2e and c) (no unit) (see Note 9).
4 Notes
1. A Nanodrop instrument with 1000 μL cuvettes can be used.

2. Samples are taken in a reproducible manner by shaking the
Erlenmeyer flask in a way that all cells are equally distributed
in suspension.
3. Ampoules are placed into the clean bench and are sterilized,
i.e., they are completely filled with 70% ETOH and kept for at
least 10–15 min.
4. Temperature response will typically be measured in the calo-
rimeter from 5 to 50 C to identify strain- and holobiont-
specific temperature response. Figure 1 shows, as an example,
temperature-dependent released heat rates of Chlorella vulgaris
at 17, 27, and 37 C. In the holobiont culture, bacteria were
Holobiont-dependent temperature response

250
200
Heat Rate
150
100 holobiont
single cell colony
50
0
0 10 20 30 40 50
Temperature (°C)
Culture at 27°C e Rbiom

Holobiont 0,4 0,60
Single cell colony- 0,4 0,21
derived culture
Fig. 1 Holobiont-dependent temperature response curve, carbon use efficiency (ε), and rate of biomass
formation (Rbiom)—Chlorella vulgaris. Holobiont and single cell colony-derived cultures show at 27 C the
same microalgae-determined relative carbon use efficiency values, but differ in the predicted rate of biomass
formation. Both ε and Rbiom can serve as unit-less relative marker indices (see Note 4)
visible under a microscope and it was possible to identify by

16S-based PCR analysis bacteria belonging to the group of
Proteobacteria, Firmicutes, Actinobacteria, Cyanobacteria,
and Chloroflexi. However, any treatment with bacteriocides
and efforts to separate bacteria and microalgae by other
means such as gradient separation were not successful (work
performed by Vera Valadas and Carla Ragonezi at the EU Marie
Curie Chair, unpublished). The single cell colony-derived cul-
ture was visibly free of bacteria or other microorganisms
checked by microscope analysis and through growth on Luria
Bertani (LB) agar plates.
5. Normally, this will be after about 35 min.
6. This ratio is inversely related to the efficiency by which a plant
system is using its available energy for biomass growth. Heat
released per mole consumed oxygen in living material is called
“oxycaloric equivalent” (e.g., [15]). The Thornton’s rule states
that this value is deduced from the oxidation state of carbon
and can be obtained by chemical reaction through combustion
of organic compounds. It reaches values for ΔHO2 between
430 and 480 kJ/mol with an average value of 455þ/
15 kJ/mol. From carbohydrates, every C-mole releases
470 kJ/mol, a typical fatty acid provides 650 kJ/mol, lipids
611 kJ/mol and protein is expected to release around 543 kJ/
mol [8, 16]. The applied model assumes carbohydrate as a main
component in the growing biomass and aerobic metabolism
[8]. The amount of energy characterizes the quantity of heat
lost from the tissue per mole of CO2 produced. Thus, it can be
expressed by the ratio Rq/RCO2. In growing tissues of plants,
a measured value equal to 470 kJ/mol indicates under these
assumptions that no growth takes place, which is equal to zero
substrate carbon conversion efficiency. Values <470 kJ/mol
indicate efficiencies >0 and values higher than 470 kJ/mol
indicate loss of energy for biomass growth through the usage
of reduced substances for substrate oxidation. Thus, values for
efficiency lie always between >0 and <1.
7. This step can only be applied to calculate efficiency values >0,
means the ratio Rq/RCO2 needs to be <470 kJ/mol. Consid-
ering that the elemental composition and consequently heat of
combustion of all growing plant material is approximately the
same, it is assumed in the model that the change of enthalpy
(ΔHb) will be þ30 kJ/mol C. Then Rq/RCO2 can be set
equal to: 470–30 [ε/1 ε] [9]. Factor x ¼ [ε/1 ε] (see also
in Arnholdt-Schmitt [17], chapter 15 in this book).
8. ε can be calculated from measured Rq/RCO2 values and the
calculated factor x.
ε ¼ x/x þ 1.
9. According to the model, the rate of biomass formation

(Rbiom) can only be calculated when Rq/RCO2 is <470 kJ/
mol (see Note 8). Rbiom is equal to growth rate. Following
Hansen et al. [1, 13] respiration-driven growth of structural
biomass (Cbiomass) can be described as:
C substrate þ NPK etc: þ xO2 ) εC biomass þ ð1 εÞCO2 ;
Herein, ε is the fraction of substrate carbon passing
through respiration and being used in anabolism to form struc-
tural biomass. (1 ε) is the fraction of substrate carbon lost as
CO2. By definition ε is called carbon use efficiency or substrate
carbon conversion efficiency. Rbiom/RCO2 is equal to ε/
(1 ε), thus Rbiom is equal to the product of RCO2 times
“factor x.”
Acknowledgments
B.A.S. appreciates the effort of the editor Jagadis Gupta Kapuganti

for this special edition related to respiration and for inviting sub-
mission to make running protocols in her lab available to the
public. The authors are grateful to Vera Valadas, who established
the Chlorella vulgaris growth experimental system in the lab of the
EU Marie Curie Chair and initiated analyses on the Chlorella
vulgaris holobiont cultures (see also Note 4). Vinod Kumar Patil
appreciates the scholarship provided by the Erasmus Mundus
FUSION Project, Postdoctoral Exchange Studies (2015–2016).
B.A.S. is thankful to the University of Évora for continuous invita-
tions as Coordinating Investigator since 2008 to prolong running
of the established EU Marie Curie Chair financed initially by the
EC in the period from 2005 to 2008.
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Chapter 17
Measurements of Electron Partitioning Between

Cytochrome and Alternative Oxidase Pathways
in Plant Tissues
Nestor Fernandez Del-Saz, Miquel Ribas-Carbo, Gabriel Martorell,
Alisdair R. Fernie, and Igor Florez-Sarasa
Abstract
Plant respiration is characterized by the existence of the alternative oxidase pathway (AOP) that competes
with cytochrome oxidase pathway (COP) for the electrons of the ubiquinone pool of the mitochondrial
electron transport chain, thus reducing ATP synthesis. The oxygen (O2) isotope fractionation technique is
the only available to determine the electron partitioning between the two pathways and their in vivo
activities in plant tissues. In this chapter, the basis of the O2 isotope fractionation technique and its derived
calculations are carefully explained together with a detailed description of the dual-inlet isotope ratio mass
spectrometry (DI-IRMS) system and the protocol developed at the University of Balearic Islands. The key
advantages of the DI-IRMS over other systems are highlighted as well as the potential problems of this
technique. Among these problems, those associated with leakage, diffusion, and inhibitor treatments are
noted and solutions to prevent, detect, and repair these problems are detailed.
Key words Oxygen isotope fractionation, Respiration, In vivo mitochondrial electron partitioning,
Cytochrome oxidase pathway, Alternative oxidase pathway
1 Introduction
Plant respiration is the combination of metabolic reactions where

reduced carbon compounds are oxidized to CO2 and H2O and the
energy released is used for the synthesis of reducing equivalents and
ATP. The synthesis of ATP is linked to mitochondrial electron
transport from respiratory substrates to terminal oxidases which
reduced oxygen to water. Therefore, while respiration can be
measured as CO2 production when the focus of interest is on
carbon budgets, measuring O2 consumption allows a more direct
assessment of the energetic efficiency of respiration.
Plants have a specialized mitochondrial electron transport chain
(mETC) with several proteins that allow alternative electron
203
204 Nestor Fernandez Del-Saz et al.
transport routes, thus modifying the energetic efficiency of respira-

tion (reviewed in [1, 2]). Among these energy bypass systems, the
alternative oxidase (AOX) couples the oxidation of the reduced
ubiquinone (UQ) to the reduction of oxygen to water without
proton translocation, thus dissipating energy as heat [3]. In addi-
tion, electron transport to AOX bypasses two of the three proton
pumping sites of the cytochrome oxidase pathway (COP) thus
considerably reducing ATP synthesis. Therefore, it is very impor-
tant to know to what extent the AOX is engaged and how is
regulated because of its major impact on the energetic efficiency
of plant respiration.
Past investigations on regulatory properties of AOX such as
redox [4] and allosteric activation by organic acids [5] indicated
that the AOX pathway (AOP) can operate under conditions when
the cytochrome oxidase pathway (COP) is not saturated. There-
fore, when the AOP is in its fully active state it can compete with the
COP for the electrons of the UQ pool [6]. Competition between
the COP and the AOP was demonstrated in isolated mitochondria
with measurements of the individual activities of both pathways by
using the oxygen isotope fractionation technique [7]. These lines of
experimental evidence supposed that approaches based on respira-
tory inhibitor titrations were no longer appropriate to measure the
in vivo activities of the COP and the AOP [8]. The basis of the
oxygen isotope fractionation technique resides in the differential
oxygen isotope fractionation by the cytochrome and the alternative
oxidases [9]. Both oxidases react preferentially with the 32O2 rather
than with the 34O2 because molecules that contain heavier isotopes
have chemical bonds that are more stable, and so take a greater
amount of energy to break [10]. However, the two oxidases use
different mechanisms to break the O2 molecule and this generates a
different isotope fractionation [11], being higher in AOX
(24–31‰) than in COX (18–20‰) (reviewed in Table 1 in [11]).
Thus, the oxygen isotope fractionation during respiration, i.e., by
examining the isotope fractionation of the substrate oxygen as it is
consumed in a closed and leak-tight cuvette, can be used to calcu-
late the partitioning of electrons between the two respiratory path-
ways (τ) in the absence of inhibitors ([11] and see Subheading 3.4).
Since the first oxygen isotope fractionation system [9], differ-
ent measurement systems have been developed for determining τ.
Ribas-Carbo et al. [11] reviewed the history of this technique as
well as the design advances. Since 2005, only two systems have
been newly developed including an off-line gas-phase system [12]
and an on-line gas- and liquid-phase systems [13]. While technical
problems on these new systems must be overcome after leakage
and/or fractionation corrections [12, 13], the precision and sensi-
tivity of the dual-inlet isotope ratio mass spectrometry (DI-IRMS)
system is still higher than any system developed (see Subheading
3.2). In addition, DI-IRMS has been the most frequently used
Electron Partitioning Between Cytochrome and Alternative Oxidase Pathways 205
system for determining mitochondrial electron partitioning

between COP and AOP in different tissues and species over the
last 10 years ([14, 15] and references therein). For all these reasons
and due to space limitation, only the gas-phase DI-IRMS system
developed at the University of Balearic Islands (UIB) is detailed in
this chapter with our focus being on in vivo determinations of AOP
and COP in plant tissues. Note that on-line liquid-phase systems
allow for in vitro studies in isolated mitochondria or enzymes while
off-line gas-phase systems allow for in vivo field measurements.
Readers are referred to Ribas-Carbo et al. [11] for the description
of previously developed systems, some of them still recently used
[16, 17], and to Kornfeld et al. [12] and Cheah et al. [13] for the
newly developed off-line and on-line systems, respectively.
2 Materials
2.1 Sample The air-sample collection system allows the sequential withdrawn
Collection System of air samples from the cuvette and its flux into the sample bellow
Coupled to the Dual- the dual-inlet isotope ratio mass spectrometer (DI-IRMS). The
Inlet Isotope Ratio system consists of a 3 mL and 10 cm2 stainless steel closed cuvette
Mass Spectrometer maintained at a constant temperature using a copper plate and a
serpentine around the cuvette with a temperature-controlled water
bath (Fig. 1). Typically, up to three leaf discs of 9.6 cm2 or 0.4 g of
fresh tissue can be fed into the cuvette; overfeeding of the cuvette
can cause O2 diffusion problems with its consequent impact on
fractionation (see Note 1). The respiration cuvette is equipped with
two inlets: one connected to the mass spectrometer sample bellow
through a 1 m long capillary tube (0.127 mm inside diameter), and
the other connected to a 1 mL air-tight syringe (Cromlab S.L.,
Barcelona, Spain) (Fig. 1). Throughout the experiment, the syringe
is used to both mix the air in the cuvette and maintain the cuvette at
constant pressure. There is a pneumatically controlled on-off
micro-needle valve at the capillary tube connecting the cuvette to
the mass spectrometer (Fig. 1). Finally, there is a liquid N2 trap
removing the H2O and CO2 from the cuvette-sampled air before
being fed into the mass spectrometer (Fig. 1). The removal of the
H2O must be ensured because it is crucial for a correct functioning
of the mass spectrometer (see Subheading 3.1). Also note that the
advantage of this system is its simplicity which diminishes the
possibilities for leaks, although they must be monitored conscien-
tiously (see Note 2).
2.2 Dual-Inlet The development of a system based on dual-inlet mass spectrome-

Isotope Ratio Mass try (DI-IRMS) improved the accuracy and sensitivity of the tech-
Spectrometer nique over previous on-line continuous flow systems [11]. The DI-
IRMS is generally considered to be the most precise method of
Fig. 1 Diagram of the gas-phase collection system coupled to dual-inlet isotope ratio mass spectrometer
(Delta XPlus, Thermo LCC, Bremen, Germany) developed at the Biology Department of the University of the
Balearic Islands
measuring the isotope ratios (see Subheading 3.2, steps 4–6 for
detailed explanations).
The DI-IRMS presents the following four independent systems
(Fig. 1):
1. System of bellows and pneumatic valves: this module is used to
collect the sample air, i.e., from the collection system described
above, and to allow simultaneous and automated measure-
ments of sample and reference gases (Fig. 1). Two inlets allow
the entry of air into two bellows with a capacity of 30 mL. One
of them is used as a reference and the other as a sample, and
both are able to be automatically compressed/decompressed;
the possibility to compress the gas captured into the bellows
allows dynamically increasing/decreasing the amount of gas
introduced into the ion source with the consequent increase/
decrease on the signal intensity detected for each generated ion
(i.e., the low signal intensity from a low amount of gas sampled
can be increased for about ten times after below compression
thus allowing a more precise measurement). There are 12
individual valves that can be opened/closed in order to fill up

either the reference bellow from standard air, or the sample
bellow from sample air from the cuvette; also, the valves system
allows to empty the bellows by connecting them to the rotatory
vacuum pump, i.e., once measurement is finished, the sample
bellow should be evacuated to prepare the system for the next
sampling. Moreover, the bellows are connected, via a capillary,
to a crimp that controls whether the sample or reference air
goes either into the mass spectrometer or to a waste line (i.e.,
ending into the turbo pump (Fig. 1)), via a “change-over valve
block” consisting of four valves. During the measurements,
two valves are always open and two closed to ensure one bellow
has an open path to the mass spectrometer, and the other to the
waste line.
2. Gas ionization system: the ionization chamber consists of an
ion source that ionizes the gas molecules throughout an elec-
tron beam, a positively charged ion repeller from the electron
beam, a magnetic field (disposed in parallel to the electron
beam flux) that allows the ions focusing, and an ion accelerator
that confers velocity to the ions by applying an electric potential
pulse before withdrawing the ionization chamber. The
obtained velocity of the ions will depend on the mass-to-charge
ratio.
3. System to separate ions on the basis of the mass-to-charge
ratio: this consists of a flight tube by which the ions beam is
directed by a magnetic field (or magnet) and separated into
multiple beams depending on their mass-to-charge ratios
(determined by their different isotopes composition), and Far-
aday cups that are collectors for that receive the impact of the
ions. On each impact, the ions are neutralized as well as the
electric potential, which reduction is proportional to the inten-
sity of the ions beam. The DI-IRMS system of the UIB has
eight Faraday cups thus allowing simultaneous detection of
14
N2, 32O2 and 34O2 among other gas molecules.
4. Data collection/monitoring system: it consists of a voltage/
frequency converter coupled to a software able to monitor the
ratio of the m/z 34/32 (18O2/16O2) and m/z 32/28 (O2/
N2) ratios of the sample gas. Isodat 3.0 (Thermo Fisher Scien-
tific, MA, USA) is the software for system control, data acqui-
sition, and data evaluation.
2.3 Chemicals for For many years, it was thought that electrons were only available to
End-Point the alternative oxidase pathway (AOP) when the cytochrome oxi-
Determinations dase pathway (COP) was either saturated or inhibited, and the
electron partitioning between the two respiratory pathways (τ)
was studied only by using specific inhibitors of the two pathways,
being the most used the potassium cyanide (KCN) for the COP,
and salicylhydroxamic acid (SHAM) for the AOP. While the O2

isotope fractionation technique can be used to determine τ in the
absence of inhibitors by measuring O2 isotope fractionation during
tissue respiration (Δn), the end-point fractionation values for each
pathway (Δc, COP discrimination; and Δa, AOP discrimination) are
still required for τ calculation (see Subheading 3.4). These end-
point values are obtained in independent experiments to those for
obtaining Δn and are fairly constant in each specific tissue of the
same species (see Note 6); though once Δc and Δa are obtained in
the tissue of interest, such values can be used for τ calculation in
different experiments (see Note 6 for special considerations). Typi-
cal concentrations used are 10 mM KCN and 25 mM SHAM (see
Subheading 3.3 and Note 4 for more details).
There are other inhibitors that can be used as an alternative to
KCN or SHAM, although they have not been extensively used for
the calculation of the respiratory end-points (Δc and Δa). Antimycin
A is one of the first known and most potent inhibitors of the
mitochondrial respiratory chain, and it is able to inhibit the flow
of electrons through COP [18, 19]. On the other hand, n-propyl
gallate has routinely been used to document the presence of a
cyanide-resistant respiratory pathway in a large number of tissues
[20]. Recently, ascofuranone, a ubiquinone analog isolated from
the pathogenic fungus Ascochyta viciae, has shown to be a potent
inhibitor of trypanosomal alternative oxidase [21]. Further studies
using the oxygen isotope fractionation technique are needed in
order to test the effectiveness and the appropriate concentration
of these alternative inhibitors.
3 Method
3.1 Preparatory Before starting an experiment different checks on the DI-IRMS

Checks system are recommended for its correct functioning. As for most
mass spectrometer systems, the vacuum pressure in the whole
system should be checked and kept at the levels indicated by man-
ufacturer’s recommendations; in case vacuum level is not achieved,
vacuum pumps may need repairing or replacing but also some leak
may be present and may require for technical assistance of the
manufacturer.
After checking correct vacuum levels on emptied system, the
background contaminants should also be checked to be under the
levels recommended by the manufacturers; these levels can vary
from lab to lab though adequate levels of contaminant for correct
functioning should be set and checked regularly. High water levels
is one of the main and typical problems and probably the most
difficult to solve (i.e., several days of vacuum-dry of the system may
be required to remove water contamination); water condensed
throughout the capillaries and ion source may react with the gas
samples thus causing undesired isotope fractionations; in the sys-

tem here described, special care must be taken when injecting gas
samples across the liquid N2 trap though (Fig. 1) ensuring com-
plete water removal (i.e., before starting the experiments, air sam-
ple tests can be introduced and m/z 18 can be monitored).
Once the system is ready to run samples, daily tests of machine
stability are strongly recommended before starting an experiment.
For this purpose, the two bellows can be filled up (see Subheading
3.2 for the procedure to inject the air samples into the bellows)
with the same standard gas; note that regular ambient air can be
used as standard gas and no special standard gas is needed to
determine the fractionation factor during respiration (see Subhead-
ing 3.4). Then, several (at least three are recommended) runs of six
cycle measurements (i.e., as for the “real” experiments, see Sub-
heading 3.2) can be started and two parameters should be checked:
(1) the isotope ratios m/z 34/32 (18O2/16O2) and m/z 32/28
(O2/N2) should be constant, i.e., standard deviations of the six
replicate cycles should be less than 0.1 and 0.05, respectively; (2)
the calculated isotope composition or delta (δ, calculation com-
monly used in stable isotopes studies which is usually given by the
software and indicates isotope ratio of the sample in relation to that
of the standard; for basic information on stable isotope analyses see
[10]) should be constant and around 0 because the same air is using
as reference and sample, thus indicating no fractionation due to
technical problems.
3.2 Protocol for 1. First, pre-evacuate the sample bellow (Fig. 1) and expand to its
Measuring Oxygen maximum volume (30 mL).
Isotope Fractionation 2. Close the vacuum line by closing valves n 2 and n 5, and open
in the Absence of the valves n 1, n 3, and n 4 that connect the sample bellow
Inhibitors and ion source with the external capillary of the collection
system (Fig. 1). No signal increase (i.e., m/z 32, O2) should
be detected, otherwise a leak is present which must be repaired
(see Note 2).
3. Open the pneumatically controlled micro-needle valve placed
next to the cuvette (Fig. 1) to let the air from the cuvette enter
into the bellow through the “sample” inlet of the DI-IRMS.
Keep this valve open until pressure in the sample bellow reaches
approx 2.5 mbars (a manometer is present in each bellow) and
then close it. With this pressure in the 30 mL bellow volume,
approximately 250 μL of air sample is fed into the sample
bellow. Note that before the air is introduced into the bellow,
the sample air passes through a liquid N2 trap to remove both
H2O and CO2.
4. Once the m/z 32 signal is stable, close valve n 3 to retain the
air sample inside the bellow and compress it to increase signal
intensity. This is a great advantage of the DI-IRMS because it
facilitates recording of a high signal (see Subheading 2.2, item
1) of a small amount of sampled air (i.e. 250 μL) thus allowing

not consuming more than 30–50% of the total O2 in the
cuvette at the end of the experiment. While signal (i.e., m/z
32) is stabilizing, open valve n 2 to connect the external
capillary to the rotatory vacuum pump to remove the remain-
ing sample so that the system can be prepared for the next
sample collection.
5. Once the m/z 32 signal is stable after bellow compression, the
mass spectrometer simultaneously measures the m/z 34/32
(18O2/16O2) and m/z 32/28 (O2/N2) ratios of the sample
gas. The sample gas is analyzed against standard air contained
in the other bellow (Fig. 1, reference bellow), previously filled
with standard air (see Subheading 3.1). Both sample and refer-
ence air can then enter ionization chamber under nearly iden-
tical conditions since the adjustable volume of the bellows
allows the equilibration of the air pressures in both bellows in
order to obtain the same signal intensity for the isotope mea-
surements (typically m/z 32 is adjusted and this can be done
automatically by the mass-spec software Isodat 3.0). This signal
adjustment avoids possible signal linearity problems derived
when very different signals are compared.
6. Once the signals of the two bellows are adjusted, a run of six
replicate cycles of each gas sample (sample and reference air) is
initiated (i.e., by starting a programed sequence of six cycles)
which takes approximately 10 min. The average values of the
isotope ratios of the six replicate cycles are taken as one single
point data in the plot for discrimination calculation (see Fig. 2,
Subheading 3.4).
7. After the run of six replicate cycles is finished, empty the bellow
by opening valve n 5 connecting the bellow volume to the
waste line of the vacuum rotatory pump. After evacuation,
expand the bellow to its maximum volume and start from
step 1 in this section. Note that a complete respiration experi-
ment usually consists of six data points with six cycles per point
thus requiring around 90 min. In case of a high respiration rate,
data points can be reduced to a minimum of five when the R2
of the discrimination plot is higher than 0.995 (see Fig. 2,
Subheading 3.4). Also, depending on the respiration rate of
the tissue under study replicate cycles can be increased thus
augmenting the sensitivity as needed and up to the limit of the
machine. This possibility of measuring the sample and reference
air as many times as necessary is one of the most important
advantages of the DI-IRMS system because it allows high preci-
sion and sensitive measurements and thus experiments to be
performed using plant material with very low respiration rates
and/or with low amount of tissue.
Fig. 2 Representative plots of the oxygen isotope fractionation during respiration

in the absence of inhibitors (Δn), in the presence of 25 mM SHAM (Δc), and in the
presence of 10 mM KCN (Δa). The three plots were obtained in three separate
respiration experiments in Florez-Sarasa et al. [22]. The fractionation factor (D)
was obtained from the slopes of the linear regressions between –ln f and ln(R/R0)
and then transformed to Δ values (see Subheading 3.4). The Δ values shown in
the figure legend are expressed in per mil units (‰). The r2 values of all
unconstrained linear regressions were higher than 0.995
3.3 Protocol for The same protocol described in Subheading 3.2 can be applied,
Measuring End-Point except that a previous inhibitor treatment is needed to obtain end-
Oxygen Isotope point oxygen isotope fractionation values (see Subheading 3.4 for
Fractionation the requirement of end-points values). In most organs, oxygen
isotope fractionation by the AOP (Δa) is easy to obtain, since
KCN penetrates tissues fairly easily; it can be applied by sandwich-
ing the tissue between medical wipes soaked in a concentration of
KCN ranging 1–16 mM, depending on the plant species and
incubation or inhibition periods [11]. In many cases, roots must
be submerged in solutions of KCN to obtain a complete inhibition
of COP. In recent years, full inhibition of the COP has been
obtained in leaves and roots of several species after 20–30 min
incubation with 10 mM KCN solutions [14, 15, 22–24]. The
alternative oxidase consistently gives Δa values between 24‰ and
27‰ in roots, while in leaves it is less variable, with values ranging
from 30 to 32‰ [11].
In many tissues the application of SHAM in order to obtain
oxygen isotope fractionation by the COP (Δc) is more problematic.
In some cases, the addition of this inhibitor leads to soaking of the
tissues, which can cause diffusion problems with subsequent frac-
tionation effects. Successful Δc measurements were however
obtained by incubation of tissue slices in solutions of 25 mM
SHAM diluted either in DMSO or in warmed water [14, 22, 25,
26]. In addition, the effectiveness of this inhibitor can be tested
using a Clark-type oxygen electrode (Rank Brothers, Cambridge,

England) in a solution containing 30 mM MES (pH 6.2) and
0.2 mM CaCl2, and by monitoring the oxygen consumption of
plant tissues that have previously been treated with KCN (see Note
4). In this sense, the application of increasing concentrations of
SHAM will decrease the cyanide-resistant respiration to some
extent in which the remaining respiratory rate is non-SHAM sensi-
tive, being defined as residual respiration (see Note 5). The
obtained values of Δc range between 16‰ and 20.8‰ in roots,
and 19.6‰ and 20.2‰ in leaves [11].
3.4 Calculations of Oxygen isotope fractionation is calculated using the fractionation

the Oxygen Isotope factor (D), which is derived from the slope of a linear regression
Fractionation and the through the origin of a plot of (ln R/R0) 1000 versus –ln f [9]:
Electron Partitioning D ð‰Þ ¼ ln ðR=R0 Þ= ln f :
where R is the 18O/16O ratio of the sample, R0 is the initial
18
O/16O ratio, and f is the fraction of remaining O2. With the
dual-inlet isotope ratio mass spectrometer (DI-IRMS) system,
masses 34 (18O16O) and 32 (16O16O) are used to measure the
oxygen-isotope 18O/16O ratios. On the other hand, masses 32
(16O2) and 28 (14N2) are used to calculate f and therefore total
oxygen uptake (Vt); note that mass 32 (16O2) is used as a good
approximation of total oxygen amount since natural abundance
levels of 18O are only 0.4% of the total oxygen, and N2 is an inert
nonreacting gas for the plant tissue. The values of m/z 34/32
(18O2/16O2) and m/z 32/28 (16O2/28N2) are obtained from a
standard and the sample air with DI-IMRS analysis with six repli-
cate cycles (see Subheading 3.2) for each of the six data points
regularly obtained for a complete plot to calculate D (Fig. 2). The
r2 values of all unconstrained linear regressions between ln f and
ln(R/R0), with a minimum of five data points, must be at least
0.995, considered minimally acceptable [27]. Note that D refers
to fractionation of the substrate of the reaction but then it is
transformed to the more common notation of “Δ” using the for-
mula [9]:
Δ ¼ D=1 ðD=1000Þ:
Once the Δ values are obtained, then the electron partitioning
to the AOP (τa) can be calculated as follows [9]:
τa ¼ Δn Δc =Δa Δc :
where Δn is the isotope fractionation in the absence of inhibitors,
and Δc and Δa are the so-called end-points corresponding to the
fractionation by the cytochrome and alternative oxidase pathways,
respectively (Fig. 2). These end-points are determined for each
experimental system using inhibitors of the COP and AOP (see
Subheading 3.3 for detailed explanations).
Finally, the individual in vivo activities of the COP (νcyt) and

AOP (νalt) are obtained by multiplying the total oxygen uptake rate
(Vt) and the electron partitioning to the AOP as follows:
νcyt ¼ V t ð1 τa Þ:
νalt ¼ V t τa :
4 Notes
1. Tissue diffusion problems: Poor diffusion through tissues is a

limitation to the present method. If diffusion limits the supply
of oxygen to the terminal oxidases, then the discrimination
values will be lower than those associated with respiration,
and will vary depending on the rate of respiration. This is a
problem with dense tissues such as stem and thick roots (dis-
cussed in [28]). Recently, in another dense tissue such florets of
Philodendron bipinnatifidum, this problem was overcome by
increasing the O2 partial pressure [16]. In leaves, diffusion is
less problematic, although in some cases it is recommended
cutting the leaf into slices to facilitate the oxygen diffusion.
Diffusion problems in leaves can be also observed when there is
an excess of tissue inside the cuvette, or in highly moisturized
tissues such as fine roots. The latter case can be observed in
roots that are washed to remove the excess of substrate after
being removed from the pot, or after the incubation period
using inhibitors. In both the cases, it is recommended to leave
to air dry before placing the sample inside the cuvette. Note
that KCN is highly volatile and its effect can be reverted (see
Note 4 for recommendations).
2. Leakage detection: The main problem of any isotope method of
this type is the possibility of contamination from outside air in
the form of leaks, which increases with every additional con-
nection. The connections of the system such as the syringe,
tight connectors of capillaries, and O-rings from inlet connec-
tors (Fig. 1) are the main points of air leaks. This contamination
affects Δn values, which can be lower than expected. In this
situation, it is recommended to check/replace these connec-
tors. Of course, the cuvette should also close tightly. If avail-
able, flushing argon around the connections allows testing for
leaks by monitoring the m/z 40 signal. Also, cuvette can be
filled with He or N2, tightly closed and a full experiment can be
performed (i.e., like a blank test); increasing O2 (m/z 32)
signals should not be detected over the course of such an
experiment. Alternatively, in order to detect whether the leak-
age problem is affecting discrimination values, it is recom-
mended to measure Δa value (i.e., tissue after KCN
incubation) as it has shown to be consistent in each tissue type

(see Note 6); also note that any leakage will decrease the
fractionation value obtained, thus an unusual (i.e., lower than
expected) fractionation value is easier to detect in high (i.e., Δa)
rather than in low (i.e., Δn or Δc) fractionation values.
3. Unusual O2 consumption rates: the length of a complete respi-
ration experiment depends on the respiration rate of the tissue
being studied and the experimental conditions applied. Sam-
ples with fast respiration rates can be measured by shortening
the air sampling time between experimental points. Neverthe-
less, faster respiratory rates can run out the available oxygen
inside the cuvette and the syringe (3 þ 1 mL), which can result
in unusual Δn values and respiratory rates. This problem can be
solved increasing the initial volume of oxygen to be respired by
pulling the plunger of the syringe. Another alternative is
decreasing the amount of plant material that is placed in the
cuvette and thus, reducing the consumption of available oxy-
gen to be respired. On the other hand, in the event of samples
with lower respiratory rates, the air sampling time between
experimental points must be extended as a minimum consump-
tion of oxygen is needed to obtain the minimum acceptable R2
values 0.995 of the linear regressions plots between ln(R/
R0) and –ln f (see Subheading 3.4). In this situation, the tissue
amount can be increased, however, care must be taken in order
not to cause diffusion problems (see Note 1), especially in
leaves. In the measuring system presented here, approximately
a maximum of three leaf discs of 9.6 cm2 or 0.4 g of fresh tissue
can be fed into the cuvette without causing diffusion problems.
4. Inhibitor effectiveness (partial inhibition and overexposure pro-
blems): Poor infiltration of the inhibitors can cause difficulties
in determining the two end-points, especially in dense or waxy
leaves. In such cases, cutting the leaf into two to four slices
before the incubation is recommended to facilitate the infiltra-
tion of the inhibitor. In KCN treatment experiments, a piece of
paper/wipe soaked in the KCN solution can be placed inside
the cuvette during the experiment to avoid the decrease of the
inhibitor concentration by cyanide evaporation. Vacuum infil-
tration/soaking of the inhibitor solutions is not recommended
because infiltration by itself can cause diffusion problems with
consequent discrimination effects (I. Florez-Sarasa, personal
communication). On the other hand, excessive periods of incu-
bation or concentration of the inhibitors can also cause pro-
blems in determining the end-points [29]. Such problems can
be detected when a very unusual fractionation value (usually
lower than expected) is observed together with highly changed
respiration rate after inhibitor treatment. In these situations, it
is recommended to test the effectiveness of the inhibitors using
a Clark-type oxygen electrode by monitoring the oxygen con-

sumption to establish the appropriate concentration of
inhibitor.
5. Residual respiration: The remained oxygen uptake that is
observed in the presence of inhibitors of both the cytochrome
(KCN) and alternative (SHAM) pathways is defined as residual
respiration. It has been reported to have an isotopic fraction-
ation between 19.6‰ and 21.0‰, much lower than Δa [9, 27].
Therefore, any significant residual respiration present in the
tissue would decrease Δa value, compared to isolated mitochon-
dria, since the latter do not present residual respiration. Ribas-
Carbo et al. [27] showed that Δa was similar in isolated mito-
chondria and intact tissues (30.9‰ and 31.5‰ respectively) of
green soybean (Glycine max) cotyledons. A similar result was
also observed in etiolated soybean cotyledons. These results
suggest that residual respiration may be an artifact that only
occurs in tissues in the presence of both inhibitors and which
does not interfere with the oxygen isotope fractionation mea-
surements [27].
6. Consistency of Δa and Δc among treatments, tissues, genotypes,
and species: the respiratory end-points are roughly constant in
tissues of different species and treatments, but not between
tissues or between green and nongreen tissues, especially Δa.
Ribas-Carbo et al. [27] observed that Δa in intact green coty-
ledons of soybean was high (31‰), and in roots and etiolated
cotyledons was low (25–26‰). However, Δc was demonstrated
to be constant in these tissues (19.5–21‰). These findings
were confirmed in mitochondria isolated from each of these
tissue types suggesting that the differential oxygen isotope
fractionation can be due to the existence of distinct AOX iso-
zymes that appear to be differentially expressed in various plant
tissues [30]; for instance, AOX2 and 3, later renamed as
AOX2a and 2b [31], were suggested to be responsible for the
high and low Δa values, respectively [32]. On the other hand,
the consistency of the end-points under a wide range of con-
ditions (e.g., reduction status of the ubiquinone pool, addition
of pyruvate and DTT), using different mitochondrial prepara-
tions, confirmed that these values can be used as standard
values for each pathway in subsequent experiments under dif-
ferent treatments [7]. In other words, the determination of
both end-points is generally not necessary for every experimen-
tal treatment that can or cannot affect the respiratory rate and/
or Δn value. Nevertheless, Δa changes were observed in AOX
mutated proteins [33] which indicate that dramatic changes on
expression of AOX different isoforms may have an impact on Δa
(discussed in [33]); however, similar Δa values were obtained in
plants with acute changes of wild-type AOX1a expression [23]
and also in mutated AOX1a overexpressors (I. Florez-Sarasa,

personal communication). Finally, recent genetic analysis of
AOX sequences revealed a high variability on the main AOX
isoform present in different plant species [34]. Therefore, it is
recommended to measure Δa and Δc values of species in which
O2 fractionation is measured for the first time, despite the
observed consistency of Δa and specially of Δc observed
among studied species [11, 15] and among different AOX-
altered expression genotypes [14, 23].
Acknowledgments
We would like to thank all the staff at the Serveis Cientifico-Tecnics

of the Universitat de les Illes Balears for their help while running
the experiments on the IRMS.
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Chapter 18
A Driving Bioinformatics Approach to Explore Co-regulation

of AOX Gene Family Members During Growth
and Development
José Hélio Costa and Birgit Arnholdt-Schmitt
Abstract
The alternative oxidase (AOX) gene family is a hot candidate for functional marker development that could
help plant breeding on yield stability through more robust plants based on multi-stress tolerance. However,
there is missing knowledge on the interplay between gene family members that might interfere with the
efficiency of marker development. It is common view that AOX1 and AOX2 have different physiological
roles. Nevertheless, both family member groups act in terms of molecular-biochemical function as “typical”
alternative oxidases and co-regulation of AOX1 and AOX2 had been reported. Although conserved
sequence differences had been identified, the basis for differential effects on physiology regulation is not
sufficiently explored.
This protocol gives instructions for a bioinformatics approach that supports discovering potential
interaction of AOX family members in regulating growth and development. It further provides a strategy
to elucidate the relevance of gene sequence diversity and copy number variation for final functionality in
target tissues and finally the whole plant. Thus, overall this protocol provides the means for efficiently
identifying plant AOX variants as functional marker candidates related to growth and development.
Key words AOX1, AOX2, AOX gene polymorphism, Gene copy number variation
1 Introduction
Bioinformatics approaches for studying “virtually” molecular-

physiology and exploring in silico the importance of genome orga-
nization for regulating growth and development [1] becomes now
more efficient with increasing data sets available from wet experi-
mentation. By making this protocol available to the public the
authors wish to stimulate research strategies that can progress
knowledge on the role of AOX in adaptive growth and develop-
ment regulation. It is regarded as a tool that should act through
interactive feedback circles with wet experimentation (see Note 1).
It starts with a scientific hypothesis, goes forward with a sophisti-
cated bioinformatics search for hypothesis validation that finally
219
220 José Hélio Costa and Birgit Arnholdt-Schmitt
requires “wet” confirmation. However, the knowledge retrieved by

the bioinformatics approach can expect ideally to enable a better
focused experimentation strategy.
This protocol is suitable to investigate in silico the relevance of
qualitative and quantitative AOX transcript variation from species
with transcriptomic data available in public databases. At this date
(September 2016), more than 44,000 experiments from several
plant species under different stages of growth and development,
tissues, and stress conditions are accessible in Sequence Read
Archive (SRA) database (NCBI). In order to use these data and
to identify AOX transcript variants related to physiology and dif-
ferentiation the data can be explored by taking the following steps:
(1) Determination of reference AOX sequences, (2) Annotation of
AOX genes in available genome, (3) Deduction of a reference
cDNA (mRNA) for each gene, (4) Transcript assembly and poly-
morphism detection, (5) Quantification of transcript variant
expression.
2 Materials
2.1 Equipment A computer with access to the internet.
2.2 Data Resources – Transcriptomic and genomic databases from plants available in
GenBank (NCBI).
– BLAST tool [2] available in GenBank (NCBI).
– CAP3: a Contig Assembly Program [3]. This program is available
on internet at mobyle (http://mobyle.pasteur.fr/cgi-bin/portal.
py?#forms::cap3) or PRABI-Doua (http://doua.prabi.fr/soft
ware/cap3) web servers. The CAP3 program is also available
upon request from Xiaoqiu Huang at xqhuang@cs.iastate.edu.
3 Methods
3.1 Reference AOX In the search for transcript variants, it is crucial to have the genes and
Sequences to Detect transcripts of AOX members available from the target species as
Transcript Variants reference. Therefore, the first concern is to know if all sequences of
AOX genes from a target species are present in some databases
[nucleotide collection (nr/nt), refseq_RNA] in GenBank. If this is
not the case, it is also possible to verify if its genome was sequenced
(http://www.ncbi.nlm.nih.gov/genome/?term¼streptophyta).
Thus, it would be promising to identify and annotate all AOX genes
from the target species exploring specific genomic databases. In the
case of genome inaccessibility, expressed AOX transcripts can also be
assembled from transcriptomic data using the CAP3 tool and ana-
lyses can be proceed directly as described under Subheading 3.4.
A Bioinformatics Approach to Explore Co-Regulated AOX Gene Family Actions 221
3.2 Annotation of At this date (September 2016), 184 plant genomes are available in
AOX Genes in Available GenBank. Thus, it is possible to identify and annotate all AOX
Genome genes for each available genome. Several online programs exist that
could be used in automatic gene annotation (detection of ORFs,
predict proteins and transcripts) such as ORFfinder (http://www.
ncbi.nlm.nih.gov/orffinder), Genscan (http://genes.mit.edu/
GENSCAN.html), NetStart (http://www.cbs.dtu.dk/services/
NetStart), etc. However, in general, the manual annotation gives
best quality and should preferably be applied to AOX, since it is
encoded by a small multigene family [4, 5].
Gene annotation from AOX genes retrieved from genome
consists in the identification of promoters, exons, introns, and
untranslated regions (UTRs). From the plant genomes available
in GenBank (refseq-genomics, NCBI genomes, and WGS data-
bases) the genes can be identified in a given species by using
BLAST and a plant AOX sequence as orientation (transcript or
protein). After BLAST search, it is important to delimitate
2000 bp upstream and downstream of the identified sequence
aiming to have the complete gene extension including promoters
and 30 UTRs. The gene annotation can be performed comparing
the identified sequence with AOX mRNA from the corresponding
or closely related species. Introns can be identified analyzing the
conserved splicing sites (GT-AG, GC-AG, AT-AC) [6] as well as
observing their absence in mRNA sequence.
3.3 Deduction of a In a third step, the deduced cDNA can be obtained from annotated
Reference cDNA genes (see Subheading 3.2) by removing introns and promoters.
(mRNA) for Each Gene
3.4 Transcript Deduced AOX cDNAs (see Subheading 3.3) and genes (see Sub-
Assembly and Variant heading 3.2) can be used in BLASTn searches to detect and retrieve
Detection the short sequences from transcriptomic data (RNA-seq) found in
SRA database (NCBI). This search must be performed using the
“megablast” which align only “highly similar sequences.” This
allows that only sequences belonging to the same gene/transcript
will be retrieved. The obtained SRA sequences can then be aligned
using the CAP3 assembly program in order to obtain contigs which
should represent the transcript variants. Thus, the transcript var-
iants can be identified comparing all contigs with the
corresponding deduced cDNA or gene using BLAST [GenBank
(NCBI)] to align two sequences.
As an example of Blast results, Fig. 1 shows a graphic revealing
the alignment of ten contigs derived from Daucus carota AOX1
(DcAOX1) for the gene (Fig. 1a) and for cDNA (Fig. 1b) (see
Note 2).
3.5 Quantification of Expression of transcript variants can be quantified by mapping all

Transcript Variant reads corresponding to each variation. The number of mapped
Expression reads can be normalized using the RPKM (Reads Per Kilobase of
a Color key for alignment scores

DcAOX1 Gene
<40 40-50 50-80 80-200 >=200 (5000 base pair)
Query
1 1000 2000 3000 4000 5000
1 7 8
3 6 9 10 Contigs
2 4
5
b Color key for alignment scores

<40 40-50 50-80 80-200 >=200 DcAOX1 cDNA
Query
1 400 800 1200 1600 2000
1
2 4 Contigs
3
5
Fig. 1 Graphics of Blast results aligning ten contigs (derived for Daucus carota L. transcriptomic data against
DcAOX1 gene/cDNA) with reference sequences DcAOX1 gene (a) and cDNA (b). Red or Pink rectangles indicate
possible exons, while lines between rectangles indicate possible introns in the gene (see Note 2)
7
6.22
6
5.34
4.96
5
4
RPKM
3.50
3.05 2.95
3
1 0.73
0.34 0.41
0 0 0
0
Lf1=leaves stage 1 Lf2=leaves stage 2 Lf3=leaves stage 3
DcAOX1.1 DcAOX1.2 DcAOX1.3 DcAOX1.4
Fig. 2 Gene expression estimation of carrot DcAOX1 transcript variants in different stages of leaf development.
Data in RPKM (Reads Per Kilobase of transcript per Million mapped reads)
transcript per Million of mapped reads) method [7] applying the

following equation: RPKM ¼ (number of mapped reads 109)/
(number of reads in each database number of nucleotides of each
mapped variation). This method was recently used by our group [8]
to study gene expression on RNA-seq data. Figure 2 indicates an
example of results that can be achieved by applying this protocol
A Bioinformatics Approach to Explore Co-Regulated AOX Gene Family Actions 223
4 Notes
1. It is important to highlight that this protocol is efficient to

detect all existing transcript variations in available data sets.
However, bioinformatics should be seen as an interactive com-
plementation to wet experimentation. It is very difficult to
reconstruct the original transcript variants due to the short
read sequences (50–150 bp) of RNA-seq data. Thus, experimen-
tal work is necessary to finally determine the original transcript
when more than one polymorphism exists distantly located in a
cDNA. Further, a more accurate quantification of transcript
variants should be performed by qPCR using specific primers.
2. Data of the Fig. 1 can be interpreted as follows: If all contigs
correspond to transcripts, at first glance, it could have ten tran-
script variations, however it is crucial to make some considera-
tions: with regard to Fig. 1a, all contigs support the presence of
until three introns in DcAOX1 gene, and intron 3 was retained
in the contig 1. Contigs 2 and 3 support transcripts with intron
2 retention while contigs 4 and 5 suggest transcripts without
intron 3 retention. Furthermore, possible differences in splicing
sites of intron 3 are supported by the existence of contigs 4 and 5
(same region). The contigs 6–10 seem transcripts belonging to
other neighbor gene (this can be certified by BLAST searches
with annotated sequences in GenBank). The comparison of all
contigs with the reference cDNA (Fig. 1b) reinforce some con-
clusions above, i.e., intron 3 retention in contig 1, similar to
reference cDNA; contigs 4 and 5 without intron 3 retention.
3. This protocol has some limitation when studying AOX mem-
bers (paralogous genes) with more than 90% identity. However,
it is known that the large majority of AOX members in studied
species have less than 90% of identity. In the case of very similar
genes, more specific tools such as TopHat 2 [9] and the Bowtie
2 Aligner [10] could be used to map reads and detect transcript
variants. Further, the Cufflinks package [11] could be employed
to calculate the number of mapped reads and to infer differential
gene expression.
Acknowledgments
J.H.C. is grateful to CAPES, CNPq, and FUNCAP for giving

financial support. B.A.S. appreciates having received support from
the European Commission (EC) and the Portuguese Foundation
for Science and Technology “Fundação para a Ciência e a Tecno-
logia” (FCT). B.A.S. is thankful to the University of Évora for
continuous invitations as Coordinating Investigator since 2008 in
order to prolong running of the established EU Marie Curie Chair
financed initially by the EC in the period from 2005 to 2008.
References
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4. Considine MJ, Holtzapffel RC, Day DA, Whe- development, stress and phytohormone treat-
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cut site (IVS4þ1A>G) of SEDL causes variable
Chapter 19
A Step-by-Step Protocol for Classifying AOX Proteins

in Flowering Plants
José Hélio Costa, Clesivan Pereira dos Santos,
Kátia Daniella da Cruz Saraiva, and Birgit Arnholdt-Schmitt
Abstract
The potential of alternative oxidase (AOX) genes to develop functional markers for plant breeding
programs has been emphasized. In this sense, it is essential to have a reliable classification system, which
could aid in the selection of candidate AOX genes from different species. In the case of angiosperms AOX, a
robust classification system is required because this enzyme is encoded by variable gene numbers (1–6
genes) with variable AOX subfamilies and subtypes. Thus, in this protocol, we present a detailed guideline
to application of a classification scheme of AOX based on specific amino acids and phylogeny. We believe
that this classification protocol provides an easier and practical way of classifying new angiosperm AOX
genes besides that it can help to standardize AOX gene names used in AOX research community.
Key words AOX1, AOX2, AOX subfamilies, Subtypes, Classification protocol
1 Introduction
Alternative oxidase (AOX) in flowering plants is of high interest for

plant stress research and functional marker development for molec-
ular plant breeding [1]. In angiosperms, the protein is encoded by
1–6 genes distributed in two subfamilies (AOX1 and AOX2) reveal-
ing diversity in patterns of gene duplication among plant species
[2]. This diversity challenges homogeneity in AOX denomination.
In the past, this was reflected by the lack of homologous expression
profiles when comparing predicted orthologous genes [3]. Addi-
tionally, we observed in databases that discrimination between
genes of AOX and PTOX (Plastid terminal oxidase) had created
difficulties in some cases [4]. Recently, our group advanced the
classification scheme for AOX in angiosperms by showing that
this enzyme could be distributed into four groups: AOX1a–c/1e;
AOX1d; AOX2a–c; and AOX2d [2]. However, despite this prog-
ress, misconceptions in AOX denomination are still a concern, an
225
226 José Hélio Costa et al.
observation that argues for a more user-friendly and accessible

approach for the scientific community involved in AOX research.
Here, we present a step-by-step protocol of the AOX classifica-
tion scheme that is mainly based on our recent publication [2]
using specific amino acids and phylogeny as a basis for nomination.
It aims to facilitate the classification process and to make it easily
more familiar to researchers and students working with this protein.
For this, we have classified AOX proteins retrieved from recently
released genomes of eudicots (Arabis alpina, Hibiscus syriacus,
Lupinus angustifolius) and one monocot (Zoysia japonica) species.
This example follows AOX classification of proteins belonging to
the four groups previously determined by Costa et al. [2].
Classification of AOX genes will improve not only depending
on the rapidly increasing knowledge about existing genes, but also
depending on actual evolution. Therefore, classification is a
dynamic process and the authors will regularly update classification
rules and procedures. In addition, extension of this scheme to other
plants besides angiosperms, and/or to other kingdoms such as
bacteria, fungi, and animals, where AOX have also been found
[5], will have to be considered. New updates can be requested
and discussed by contacting the first author of this chapter (helio.
costa@ufc.br). In complementation to this protocol, development
of an automated free tool is in progress that will be placed in near
future in the web to support further improvements in the AOX
classification procedure “in real time.”
2 Materials
2.1 Equipment A computer with access to the internet.
2.2 Data Resources – AOX proteins from angiosperms requiring classification.

– A multiple sequence aligner as Clustal omega [6].
– List of specific amino acids for AOX protein classification (see
Fig. 1).
– Selection of AOX proteins previously classified by Costa et al. [2] to
be used as reference in phylogenetic classification of new proteins.
– Suitable tool to perform phylogenetic analyses as MEGA 7
program [7].
3 Methods
3.1 Identification 1. Select suitable AOX protein or nucleotide sequence to use as

and Annotation of New queries in BLAST search (see Note 1).
AOX Sequences 2. Use the selected AOX query sequence to search in public
in Public Databases databases for new AOX genes of the species of your interest
Classification of AOX Proteins in Flowering Plants 227
AA 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3
positi 1 2 2 2 2 4 6 6 6 6 6 6 7 7 7 7 7 8 8 2 3 3 3 3 4 5 7 7 9 9 1 2 3 4 4
on 2 2 4 6 9 2 1 2 4 6 7 8 0 3 5 6 8 0 1 9 0 2 3 8 1 7 7 8 2 5 7 0 8 2 3
AOX
type
AOX P n K n r L K S R P T D F r Y G R M M F M V a Y A g I H I V V V H R n
1a,b,c a d r k a s g a i H d
,e t p m l t Q
q s t l
g v m
s i y
f
m
AOX F V K H H L L l V T K
1d p M M v a T
(mon t i R s q
ocots) l s
a
AOX A V Q K Y H M H L I V N F Q
1d P M H i l i v
(eudic l i Q c r m
ots) g v
t
AOX r e P N M L K L R P T D F R Y G R M M M V L V Y l l i H I V I v H K e
2a,b,c k t k I v f v v v r
n a l v i i m a
t i c
v i
t
AOX R P M I M V I V v L K
2 p s l m a i n
mono t m v
cots t
AOX F L S Y E H H V I
2d S v k f
q
Fig. 1 Specific amino acids used to classify AOX proteins in angiosperms [2]. Red represents specific amino
acids to AOX1a–c/e; yellow shows specific amino acids to AOX2a–c; white represents conserved amino acids
to AOX1a–c/e or AOX2a–c at positions with specific amino acids to AOX1d or AOX2d; bright green represents
amino acids that distinguish monocots AOX1d from AOX1a–c/1e, while green illustrates amino acids that
differentiate eudicots AOX1d from AOX1a–c/1e; blue represents amino acids that distinguish AOX2d from
AOX2a–c. Capital letters represent the most predominant amino acid at a specific position
using BLAST tool (see Note 2). In order to demonstrate the

application of this protocol we proceeded with the annotation
of AOX sequences from three eudicot (Arabis alpina, Hibiscus
syriacus, Lupinus angustifolius) and one monocot (Zoysia
japonica) species (see Fig. 2).
3. Retrieve gene sequences and perform the annotation (identifi-
cation of exons/introns/UTRs/promoters). Compare the
retrieved genomic sequence with homologous mRNAs found
in Refseq-RNA (Reference RNA sequence), ESTs (Expressed
Sequence Tags), or TSA (Transcriptome Shotgun Assembly) in
GenBank (NCBI) databases using the BLAST tool. Use colors
to differentiate exon from intron region.
112 124 129 142 162

Aa_AOX-D ----DE----NSN-----STVTKDQNDSGSVAVPSYWGIETAKMKITRKDGSDWPWNCFMPWETYQADLSIDLKKHHIPKNIADKIAYRTVKLLR
La-AOX-L ----QE----DSE-----DKKKEVNNNNNNAVISSYWGITRP--KIQRDDGTEWPWNCFMPWDSYRADVSIDVTKHHEPKTIGDKFAFRTVKFLR
Hs_AOX-G ----KV----TSEREEKTEKSAAEETKGTEVASSSYWGISRP--KVTREDGTDWHWNCFMPWETYRSDLSIDLKKHHEPKNFMDKFAYRTVKILR
Hs_AOX-H ----KA----TTEREEKTEKSAAEETEGKEVVTSSYWGISRP--KVTREDGTDWPWNCFMPWETYRSDLSIDLKKHHEPKNFMDKFAYRTVKILR
Hs_AOX-K ----KA----PLEKKQHENSVAEDTKGK-DVVTSSYWGISRP--TITREDGTVWPWNCFMPWETYKADVSIDVKKHHMPQNFVDKLAYRTVKFLR
Hs_AOX-I ----KA----PLEKKERENSAAEDTKGE-DVVASSYWGISRP--TITREDGTVWPWNCFMPWETYKADVSIDVKKHHVPHNFVDKLAYRTVKFLR
Hs_AOX-J ----KE----SLEKKERENSAAEDTKGKGDVVASSYWGISRP--TITREDGTVWPWNCFMPWETYKADVSIDVKKHHEPQNFMDKLAYRTVKFLR
Aa_AOX-E ----------------NTTQPENPVRTVDGEVISTYWGIPP--AKITKPDGSAWKWNSFQPWDSYKPDVSIDITKHHKPSNLTDKFAYWTVQSLR
Zj-AOX-R TATAPP----PAAGSVGAVLPPAAGGEKEQKEAPSYWGVAP--TRVVKEDGTEWRWSCFRPWDTYEADLSIDLKKHHEPATLGDKVARWTVKAMR
Hs_AOX-F L---------------GGDEQQKEKQAKDAKGIVSYWGLEP--TRVSKEDGAPWKWTCFRPWEAYSADLSIDLKKHHEPMNLLDKIAYWTVKALR
La-AOX-M NGSTLAMSEKENKNDDVGSSFESVGGNKEENKVVSYWGVQP--PKVTKPDGTEWKWKCFRPWESYKADLSIDLKKHHAPVTFLDKMAYWTVKVLR
La-AOX-N NGSTLAMSEKENKSEVE---SGVAGGNKEEKKVISYWGVQP--SKVTKPDGTEWKWSCFRPWEAYKADISIDLQKHHEPVTFLDKMAYWTVKVLR
Aa_AOX-C GD--SEKTETNPKQVENESTGRDTGGNKGEKGIASYWGVEP--NKITKEDGSEW-WNCFRPWETYKADLTIDLKKHHVPKTFLDRLAYWTVKSLR
Aa_AOX-A FEKKTTE--NQSKKD--SSSGEADQGNKGEQLIVSYWGVKP--MKITKDDGTEWKWSCFRPWETYKADLTIDLKKHHLPSTLSDKLAYWTVKSLR
Aa_AOX-B LDVKTPTPETNPKKT--ENESTGGDANKGEKGINSYWGVEP--NKITKEDGTEWKWNCFRPWETYEADLKIDLKKHHVPTTFLDKLAYWTVKSLR
Zj-AOX-O TERPGGD-------TGEQKAAADG--QHNAKGIVSYWGIEP--RKLVKEDGTEWRWFCFRPWDTYRADTSIDMKKHHEPKALPDKLAYWLVKSLV
Zj-AOX-P TGAEHGR-------TGHQ--KPQG--EQTKKAIVSYWGINP--PKLVKEDGTEWRWACFRPWDTYKSDTSIDVKKHHEPTALPDKVAYWIVKSVR
Zj-AOX-Q --KKEAVRGA----DEKKEV--TKGSGEKEVVINSYWGIEK-TNKLIREDGTEWKWTCFRPWETYTADTSIDLTKHHKPKTMLDKIAYWTVKSLR
Zj-AOX-S ------SSSTAAAKAEVAEAAKDGELKGKEPVLSSYWGIPL--SKLVNKDGVEWKWSCFRPWETYTADTSIDLTKHHKPKVLLDKLAYWTVKSLR
Zj-AOX-T EGKEPTVSASEAKVEEVAEVAKDGELKGKEPALSSYWGIPP--SKLVNKDGVEWKWSCFRPWETYTPNTSIDLTRHHKPKVLLDKLAYWTVKSLR
166 178 229 241 257

Aa_AOX-D IPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHLKSIRRFEHSGGWIKALLEEAENERMHLMTMMELVKPKWYERLLVMLFQGIFFNSFFVCYV
La-AOX-L VLSDLYFKERYGCHAMMLETIAAVPGMVGGMLLHLKSLRKFQHTGGWIKALLEEAENERMHLMTMVELVKPSWHERLLVITAQGVFFNGFFVFYL
Hs_AOX-G IPTDIFFQRRYGCRAVMLETVAAVPGMVGGMLLHLKSLRKFKQSGGWIKALLEEAENERMHLMTMIELVQPKWYERLLVLTVQGVFFNAFFVLYL
Hs_AOX-H IPTDIFFQRRYGCRAVMLETVAAVPGMVGGMLLHLKSLRKFQQSGGWIKALLEEAENERMHLMTMIELVQPKWYERLLVLTVQGVFFNAFFVLYL
Hs_AOX-K VPMDMFFKKRYGCHAMMLETIAAVPGMVGGMLLHLKSLRKFQHGGGWIKALLEEAENERMHLMTMVELVQPKWYERLLVLAAQGVYFNAFFLVYV
Hs_AOX-I VPMDLFFEKRYGCHAMMLETIAAVPGMVGGMLLHLKSLRKFQHSGGWIKALLEEAENERMHLMTMVELVQPKWYERLLVLAAQGVYFNAFFLVYV
Hs_AOX-J VPMDLFFQKRYGCHAMMLETIAAVPGMVGGMLLHLKSLRKFQHSGGWIKALLEEAENERMHLMTMVELVQPKWYERLLVLAAQGVYFNAFFLVYV
Aa_AOX-E IPVNLFFQRKHMCHAMLLETVAAVPGMVGGMLLHLKSLRRFEHSGGWIKALLEEAENERMHLMTFIELSQPKWYERAIVFTVQGVFFNAYFMAYI
Zj-AOX-R WPTDLFFQRRYGCRAMMLETVAAVPGMVAGAVLHLRSLRRFEQSGGWIRALLEEAENERMHLMTFMEVSRPRWHERALVVAVQGVFLHAYLLAYL
Hs_AOX-F WPADLFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEHSGGWIKALLEEAENERMHLMTFMEVSDSRWYERALVFAVQGVFFNAYFLGYI
La-AOX-M YPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHFKSLRRFEQSGGWIKALLEEAENERMHLMTFMEVAKPKWYERALVISVQGVFFNAYFLGYL
La-AOX-N YPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEHSGGWIKALLEEAENERMHLMTFMEVAKPKWYERALVITVQGVFFNAYFLGYL
Aa_AOX-C LPTDLFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEQSGGWIKALLEEAENERMHLMTFMEVAKPKWYERALVITVQGVFFNAYFLGYV
Aa_AOX-A WPTDLFFQRRYGCRAMMLETVAAVPGMVGGMLVHCKSLRRFEQSGGWIKALLEEAENERMHLMTFMEVAKPNWYERALVIAVQGIFFNAYFLGYL
Aa_AOX-B LPTDLFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEQSGGWIKALLEEAENERMHLMTFMEVAKPKWYERALVITVQGVFFNAYFLGYV
Zj-AOX-O VPKQMLFQRRHASHALLLETVAAVPGMVGGMLLHLRSLRRFEHSGGWVRALLEEAENERMHLMTFMEVAQPRWWERALVLAAQGVYFNAYFVAYL
Zj-AOX-P GPMDLFFQRRHASHALLLETVAAVPGMVGGMLLHLRSLRRFEHSGGWIRALLEEAENERMHLMTFLEVTQPRWWERALVLAAQGVFFNAYFVGYL
Zj-AOX-Q FPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRRFEQSGGWIRALLEEAENERMHLMTFMEVAKPRWYERALVLAVQGVFFNAYFLGYI
Zj-AOX-S WPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRRFEPSGGWIRVLLEEAENERMHLMTFMEVAKPAWYERALVIFVQGVFFNAYFLGYL
Zj-AOX-T WPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRRFEHSGGWIRVLLEEAENERMHLMTFMEVAKPAWYERALVLAVQGIFFNAYFLGYL
277 295 317 342

Aa_AOX-D VSPRLAHRIVGYLEEEAIHSYTEFLKDIDNGKIENVAAPAIAIDYWRLPKDATLKDVVTVIRADEAHHRDVNHFASDIRNKGKELREAPAPIGYH
La-AOX-L LSPKTAHRFVGYLEEEAVISYTEHLNAIESGKVENVPAPAIAIDYWRLPKDATLKDVVTVIRADEAHHRDVNHFASDIHHQGKELKEAPAPVGYH
Hs_AOX-G LSPKLAHRIVGYLEEEAIHSYTEYLKDIESGRIENVPAPSIAIDYWRLPKDATLKDVITVIRADEAHHRDVNHFASDIHFQGKELREAPAPLGYH
Hs_AOX-H LSPKLAHRIVGYLEEEAIHSYTEYLKDIESGRIENVPAPSIAIDYWRLPKDATLKDVITVIRADEAHHRDVNHFASDIHFQGKELREAPAPLGYH
Hs_AOX-K TSPKLAHRFVGYLEEEAIISYTEYLEAIKSGEVENVPAPAIAIDYWRLPKDATLKDVITVIRADEAHHRDVNHFASDIQFQGKELREAPAPLGYH
Hs_AOX-I TSPKLAHRFVGYLEEEAIISYTEYLEAIKSGKVENVPAPAIAIDYWRLPKDATLKDVITVIRADEAHHRDVNHFASDIQFQGKELREAPAPLGYH
Hs_AOX-J TSPKLAHRFVGYLEEEAIISYTEYLEAIKSGEVENVPAPAIAIDYWRLPKDATLKDVITVIRADESHHRDVNHFASDIQFQGKELREAPAPLGYH
Aa_AOX-E ISPKLAHRITGYLEEEAVNSYTEFLEDIDAGKFENSPAPAIAIDYWRLPKDATLRDVVFVIRADEAHHRDINHYASDIQFQGRELKEAPAPVGYH
Zj-AOX-R LSPATAHRMVGYLEEEAVHSYTEFLRDIDAGKIEDVPAPAIAVDYWRLPPGATLRDVVEVVRADEAHHRDVNHYASDIHRQGHALREVAAPIGYH
Hs_AOX-F VSPKFAHRVVGYLEEEAIHSYTEFLKELDNGNIENVPAPPIAIDYWRLPPNSTLRDVVLAVRADEAHHRDVNHFASDIHYQGRQLKEAPAPLGYH
La-AOX-M LSPKFAHRMVGYLEEEAIHSYTEFLKELDKGTIENVPAPAIAIDYWQLPPNSTLRDVVMVVRADEAHHRDVNHFASDIHYQGRELREAAAPIGYH
La-AOX-N ISPKFAHRMVGYLEEEAIHSYTEFLKELDKGTIENVPAPAIAIDYWQVPPKSTLRDVVIVVRADEAHHRDVNHFASDIHYQGRELRDAAAPIGYH
Aa_AOX-C ISPKFAHRMVRYLEEEAIHSYTEFLKELDKGNIENVPAPAIAIDYWRLPADATLRDVVTVIRADEAHHRDVNHFASDIHYQGRELKEAPAPIGYH
Aa_AOX-A ISPKFAHRMVGYLEEEAIHSYTEFLKELDNGNIENVPAPAIAIDYWRLEANATLRDVVMVVRADEAHHRDVNHYASDIHYQGRELKEAPAPIGYH
Aa_AOX-B ISPKFAHRMVGYLEEEAIHSYTEFLKELDKGNIENVPAPAIAIDYWRLPADATLRDVVTVIRADEAHHRDVNHFASDIHYQGRELKEAPAPIGYH
Zj-AOX-O VSPKFAHRFVGYLEEEAVHSYTEYLKDLEAGLIENTPAPAIAIDYWRLPPDAKLKDVVAVVRADEAHHRDVNHFASDIHYQGIKLKDTPAPLGYH
Zj-AOX-P VSPKFAHRVVGYLEEEAVHSYTEYLKDLEAGIIENTPAPAIAIDYWRLPADAKLKDVVTVVRADEAHHRDVNHFASDIHYQGIKLKDTPAPLGYH
Zj-AOX-Q LSPKFAHRVVGYLEEEAIHSYTEFLKDLEAGKMENVPAPAIAIDYWRLPANATLKDVVTVVRADEAHHRDVNHFASDIHYQGMQLRESPAPIGYH
Zj-AOX-S MSPKFAHRVVGYLEEEAIHSYTEYLKDIEAGKIENVPAPSIAIDYWRLPPDATLKDVVTMVRADEAHHRDVNHFASDIHYQGLELKETPAPLEYH
Zj-AOX-T ISPKFAHRVVGYLEEEAIHSYTQYLKDIEAGKIENVPAPSIAIDYWRLPPDATLKDVVTMVRADEAHHRDVNHFASDIHYQGLELKETPAPLEYH
Fig. 2 Global alignment of AOX deduced proteins identified in Arabis alpina (Aa_AOX–A-E), Lupinus angusti-
folius (La_AOX–L-N), Hibiscus syriacus (Hs_AOX–F-K), Zoysia japonica (Zj_AOX–O-T) used as an example of
AOX classification in subfamilies and subtypes. The numbers indicate positions of specific amino acids to
AOX1a–c/e, AOX2a–c, AOX1d, and AOX2d subfamilies/subtypes according to Costa et al. [2]. Red represents
specific amino acids to AOX1a–c/e, while yellow shows specific amino acids to AOX2a–c; bright green
represents amino acids that distinguish monocots AOX1d from AOX1a–c/1e, while green illustrates amino
acids that differentiate eudicots AOX1d from AOX1a–c/1e; blue represents amino acids that distinguish AOX2d
from AOX2a–c
4. Remove manually the intron sequences from the annotated

gene to obtain the deduced cDNA.
5. Translate the cDNA coding sequence into amino acid sequence
using a translate tool (for instance expasy translate tool). Use
different colors to identify the initiation (methionine, ATG),
stop codons as well as 50 and 30 UTR (untranslated regions).
6. Compare the deduced protein with homologous sequences
available in protein databases to certify whether the sequence
was correctly annotated.
3.2 Classification 1. Rank all AOX proteins by provisional alphabetical naming. In

of AOX Proteins Using our example, the target AOX sequences (Figs. 2 and 3) were
Specific Amino Acids primarily named as follows: Arabis alpina AaAOX-A to E, Hibis-
cus syriacus HsAOX-F to K, Lupinus angustifolius LaAOX-L to
3.2.1 Classification N and Zoysia japonica ZjAOX-O to T (see Note 3).
of AOX in Subfamilies
2. Align all named AOX proteins using a multiple sequence
aligner (as Clustal omega). Use AOX1a from Arabidopsis thali-
ana as reference to determine the position of each amino acid.
3. Determine ‘specific amino acids’ in the alignment by analyzing
all positions available by help of Fig. 1. In Fig. 2 the ‘specific
amino acids’ and the according positions are given (see Note 4).
4. Classify the proteins firstly according to the subfamilies AOX1
or AOX2. Initially, use the 13 amino acid positions available in
Fig. 1 to differentiate AOX1 from AOX2.
5. Proceed with this examination, firstly checking the most reli-
able amino acids (position 229, 233 and 241) to distinguish
AOX1 from AOX2. Use different colors to highlight specific
amino acids of each subfamily. See Fig. 2: red represents specific
amino acids for AOX1 and yellow represents amino acid char-
acteristics of AOX2 subfamily.
6. Use secondary reliable amino acids (positions 112, 124, 129,
232, 342) to support your previous observation. Then, check
less reliable amino acids (positions 230, 257, 317, 320, 162
and 169) (see Fig. 2) (see Note 5).
7. Check all positions and classify your sequences as AOX1 or
AOX2. In the present example, AaAOX-A, B, C, E, HsAOX-
F, LaAOX-M and N as well as ZjAOX-O, P, Q, S and T were
classified as AOX1 while AaAOX-D, HsAOX-G, H, I, J, K and
LaAOX-L were classified as AOX2.
3.2.2 Classification 1. Check the 18 amino acid positions (122, 126, 142, 161, 164,
of AOX1 in AOX1a–c/1e 167, 168, 175, 176, 178, 180, 181, 230, 277, 278, 292,
or AOX1d Subtypes 295 and 343) available in Fig. 1 to differentiate AOX1d
(monocots and eudicots) from AOX1a–c/e.
Final Classification
Specific AA Phylogeny
99 AaAOX-A AOX1a-c/1e AaAOX1b

99 AtAOX1b
82 AtAOX1c
AtAOX1a
76 98 AaAOX-B AOX1a-c/1e AaAOX1a1
81 AaAOX-C AOX1a-c/1e AaAOX1a2
VuAOX1
60 LaAOX-M AOX1a-c/1e LaAOX1a
89
98 LaAOX-N AOX1a-c/1e LaAOX1b
86 HsAOX-F AOX1a-c/1e HsAOX1
100 GrAOX1
33 SpoAOX1a
100 ZjAOX-R AOX1a-c/1e ZjAOX1e
OsAOX1e
30 SpoAOX1e1
36 50 SpoAOX1e2
56 ZjAOX-P AOX1d ZjAOX1d1
100 OsAOX1d
ZjAOX-O AOX1d ZjAOX1d2
99 ZjAOX-Q AOX1a-c/1e ZjAOX1a
61
34 OsAOX1a
94 OsAOX1c
ZjAOX-S AOX1a-c/1e ZjAOX1c
65
100 ZjAOX-T AOX1a-c/1e ZjAOX1b
SpoAOX2
100 AaAOX-D AOX2a-c AaAOX2
AtAOX2
40 100 HsAOX-G AOX2a-c HsAOX2b
94 HsAOX-H AOX2a-c HsAOX2a
100 90 63 GrAOX2a
94 GrAOX2b
VuAOX2a
94 100 LaAOX-L AOX2d LaAOX2d
VuAOX2d
56 GrAOX2d
HsAOX-K AOX2d HsAOX2d1
74
100 HsAOX-I AOX2d HsAOX2d2
90 HsAOX-J AOX2d HsAOX2d3
AaAOX-E AOX1d AaAOX1d
100 AtAOX1d
100 Cgiga Aox0
Lanatina Aox0
CreAox0A
100 CreAox0B
10
Fig. 3 Classification of AOX deduced proteins identified in Arabis alpina (Aa_AOX–A-E), Lupinus angustifolius
(La_AOX–L-N), Hibiscus syriacus (Hs_AOX–F-K), Zoysia japonica (Zj_AOX–O-T) based on the phylogenetic
approach. AOX proteins from At (Arabidopsis thaliana), Gr (Gossypium raimondii), Os (Oryza sativa), and Spo
(Spirodela polyrhiza) previously classified as AOX1a–c/1e, AOX1d, AOX2a–c were used as reference. AOX
proteins from Crassostrea gigas (Cgiga), Lingula anatina (Lanatina), and Chlamydomonas reinhardtii (Cre)
were used as outgroup. Specific amino acid (AA) column represents the classification results obtained by
specific amino acids, while the phylogeny column shows the results based on phylogenetic analysis.
Percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000
replicates) is shown next to the branches
2. Start this examination checking the most reliable amino acids

(positions 175, 178 and 181) that distinguish AOX1d from
AOX1a–c/e. See the example in Fig. 2, bright green and green
represent specific amino acids for AOX1d.
3. Verify in Fig. 2 that proteins AaAOX-A, B and C, HsAOX-F,
LaAOX-M and N as well as ZjAOX-Q, S and T are proteins
belonging to the AOX1a–c/1e subtype while AaAOX-E,
ZjAOX-O, and P are representative of AOX1d.
4. Use amino acid positions (167 and 181) to verify whether the
sequence is AOX1d from monocot and positions (142, 161,
164, 168, 176, 230, 278, 292, and 338) for eudicot species (see
Note 6). In the present example, AaAOX-E is representative of
AOX1d from eudicots, while ZjAOX-O and P are representa-
tive of AOX1d from monocots.
3.2.3 Classification of 1. Check the nine amino acid positions (162, 166, 167, 170, 173,
AOX2 in AOX2a–c or AOX2d 178, 238, 277 and 278) available in Fig. 1 to distinguish
Subtypes AOX2d from AOX2a–c.
2. Differentiate AOX2d proteins from AOX2a–c primarily exam-
ining positions 167 and 178. Use positions 162, 166, and 170
to reinforce the correct AOX2 subtype nomination. In the
example of Fig. 2, blue represents specific amino acids for
AOX2d. AaAOX-D, HsAOX-G, H are proteins which belong
to the AOX2a–c subtype while La-AOX-L, HsAOX-K, I and J
are representative of AOX2d.
3. In the case of Fabales species, use other positions (173, 238,
277, and 278) (Fig. 1) to support AOX2d subtype nomination.
3.3 Final 1. Use a phylogenetic approach for protein sequences to support

Classification of AOX specific AOX classification into subfamilies and subtypes (see
Proteins Using Note 7).
Phylogenetic Analyses 2. Select reference AOX proteins previously classified that repre-
sent all existing subfamilies and subtypes (AOX1a–c/1e,
AOX1d, AOX2a–c or AOX2d) in angiosperms (see Note 8).
In the present example, Arabidopsis thaliana (Brassicales) was
used as reference to Arabis alpina (Brassicales), Gossypium
raimondii (Malvales) to Hibiscus syriacus (Malvales), Vigna
unguiculata (Fabales) to Lupinus angustifolius (Fabales) and
Oryza sativa (Poales) to Zoysia japonica (Poales) (see Note 9).
3. Use the provisional alphabetical ranked AOX proteins requir-
ing classification with the reference AOX chosen in Subheading
3.3, step 2 item to construct a phylogenetic tree using MEGA7
program [7] (see Note 10).
4. Proceed with the final classification identifying each pair of
orthologous genes formed from reference and target AOX
proteins and analyzing the phylogenetic tree.
5. Name the target AOX proteins properly according to the clas-

sification of your orthologous partner. Follow the example
shown in Fig. 3.
6. In the case of target sequences showing AOX duplication,
perform some additional adjusts. In the example in Fig. 3,
AaAOX-B and C, LaAOX M and N, ZjAOX-O and P,
ZjAOX-S and T, HsAOX-G and H as well as HsAOX-I, J and
K presented AOX duplications.
7. Denominate duplicated AOX sequences comparing the identi-
ties of duplicated target sequences with the orthologous refer-
ence AOX sequence using the Clustal omega [5].
8. In the case of AOX1a–c/1e or AOX2a–c subtypes (sequences
with high identity), denominate the target sequence with the
same letter of the orthologous reference AOX sequence (see
Note 11). Use the precedent or the next letter to denominate
the other (duplicated) target sequence. See the example in
Fig. 3: ZjAOX-S was classified as ZjAOXc, while ZjAOX-T as
ZjAOXb since these sequences presented 78.82% and 74.49%
of identities, respectively, with OsAOX1c.
9. In the case of AOX1d or AOX2d subtypes showing duplicated
target sequences, use numbers after the letter “d” (i.e., 1, 2, 3)
according to the identities of the target sequences with the
reference AOX sequence. For example, HsAOX-K, I and J
were respectively classified as HsAOX2d1, 2d2 and 2d3
because these sequences presented 84.89%, 84.68%, and
84.43% of identities, respectively, with GrAOX2d.
4 Notes
1. It is recommended to use Oryza sativa or Arabidopsis thaliana

proteins as references to retrieve new AOX genes from mono-
cot or eudicot species, respectively.
2. Data from genomic DNA and RNA increase exponentially for a
growing number of plant species with the advent of high-
throughput sequencing.
3. This provisional step is necessary to differentiate proteins from
each other while obtaining the reliable final classification.
4. Specific amino acids discriminate AOX proteins among each
other and therefore help to classify a protein as a member of a
specific AOX family. The presence of a single specific amino
acid is not enough to classify properly an AOX protein. For
correct classification, each subfamily and subtype generally
requires the presence of few or several specific amino acids.
5. Specific amino acids may appear in both subfamilies. Therefore,

care should be taken when using these positions.
6. No specific amino acid for AOX1d was detected at position
164, however, they were detected in other eight specific posi-
tions. It is important to clarify that variation at certain positions
can occur as highlighted by Costa et al. [2] without
compromising the correct AOX classification.
7. Phylogenetic analyses are especially required to differentiate
closely related sequences from AOX1a–c/e or AOX2a–c in
which specific amino acid approach is not effective.
8. Notice that for each case it will be necessary to use reference
AOX sequences from species closely related to the target spe-
cies. Similar gene number found in these species should be
considered.
9. The AOX multigene family of Spirodela polyrhiza was also
included in the analyses to help in the classification of AOX1e
proteins, since this species have two related proteins.
10. Phylogenetic analyses were inferred using the Neighbor-
Joining method [8] with bootstrap values (1000 replicates)
[9]. The evolutionary distances were computed using the num-
ber of differences method [10]. All positions containing gaps
and missing data were eliminated.
11. The orthologous reference AOX is the sequence presenting the
highest identity with the target sequence.
Acknowledgments
J.H.C., C.P.S., and K.D.C.S. are grateful to CAPES, CNPq, and

FUNCAP for giving financial support. B.A.S. appreciates having
received support from the European Commission (EC) and the
Portuguese Foundation for Science and Technology “Fundação
para a Ciência e a Tecnologia” (FCT). B.A.S. is thankful to the
University of Évora for continuous invitations as Coordinating
Investigator since 2008 in order to prolong running of the estab-
lished EU Marie Curie Chair financed initially by the EC in the
period from 2005 to 2008.
References
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Melo D (2006) AOX – a functional marker for onomic distribution and evolutionary history
efficient cell reprogramming under stress? of the enzyme in angiosperms. Mitochondrion
Trends Plant Sci 11:281–287. doi:10.1016/j. 19(Pt B):172–183. doi:10.1016/j.mito.2014.
tplants.2006.05.001 04.007
2. Costa JH, McDonald AE, Arnholdt-Schmitt B, 3. Thirkettle-Watts D, McCabe TC, Clifton R,
Fernandes de Melo D (2014) A classification Moore C, Finnegan PM, Day DA, Whelan J
(2003) Analysis of the alternative oxidase pro- using Clustal Omega. Mol Syst Biol 7:539.
moters from soybean. Plant Physiol doi:10.1038/msb.2011.75
133:1158–1169. doi:10.1104/pp.103. 7. Kumar S, Stecher G, Tamura K (2016)
028183 MEGA7: molecular evolutionary genetics anal-
4. Nobre T, Campos MD, Lucic-Mercy E, ysis version 7.0 for bigger datasets. Mol Biol
Arnholdt-Schmitt B (2016) Misannotation Evol 33:1870–1874. doi:10.1093/molbev/
awareness: a tale of two gene-groups. Front msw054
Plant Sci 7:868. doi:10.3389/fpls.2016. 8. Saitou N, Nei M (1987) The neighbor-joining
00868 method: a new method for reconstructing phy-
5. McDonald AE (2008) Alternative oxidase: an logenetic trees. Mol Biol Evol 4:406–425
inter-kingdom perspective on the function and 9. Felsenstein J (1985) Confidence limits on phy-
regulation of this broadly distributed ‘cyanide- logenies: an approach using the bootstrap.
resistant’ terminal oxidase. Funct Plant Biol Evolution 39:783–791. doi:10.2307/
35:535–552. doi:10.1071/FP08025 2408678
6. Sievers F, Wilm A, Dineen D, Gibson TJ, Kar- 10. Nei M, Kumar S (2000) Molecular evolution
plus K, Li W, Lopez R, McWilliam H, and phylogenetics. Oxford University Press,
Remmert M, Söding J, Thompson JD, Higgins New York
DG (2011) Fast, scalable generation of high-
quality protein multiple sequence alignments
Chapter 20
Studying Individual Plant AOX Gene Functionality in Early

Growth Regulation: A New Approach
Abstract
AOX1 and AOX2 genes are thought to play different physiological roles. Whereas AOX1 is typically
expected to associate to stress and growth responses, AOX2 was more often found to be linked to
development and housekeeping functions. However, this view is questioned by several adverse observa-
tions. For example, co-regulated expression for DcAOX1 and DcAOX2a genes was recently reported
during growth induction in carrot (Daucus carota L.). Early expression peaks for both genes during the
lag phase of growth coincided with a critical time point for biomass prediction, a result achieved by applying
calorespirometry. The effect of both AOX family member genes cannot easily be separated. However,
separate functional analysis is required in order to identify important gene-specific polymorphisms or
patterns of polymorphisms for functional marker development and its use in breeding. Specifically, a
methodology is missing that enables studying functional effects of individual genes or polymorphisms/
polymorphic patterns on early growth regulation.
This protocol aims to provide the means for identifying plant alternative oxidase (AOX) gene variants as
functional markers for early growth regulation. Prerequisite for applying this protocol is available Schizo-
saccharomyces pombe strains that were transformed with individual AOX genes following published proto-
cols from Anthony Moore’s group (Albury et al., J Biol Chem 271:17062–17066, 1996; Affourtit et al., J
Biol Chem 274:6212–6218, 1999). The novelty of the present protocol comes by modifying yeast cell
densities in a way that allows studying critical qualitative and quantitative effects of AOX gene variants
(isoenzymes or polymorphic genes) during the early phase of growth. Calorimetry is used as a novel tool to
confirm differences obtained by optical density measurements in early growth regulation by metabolic
phenotyping (released heat rates). This protocol enables discriminating between AOX genes that inhibit
growth and AOX genes that enhance growth under comparable conditions. It also allows studying
dependency of AOX gene effects on gene copy number. The protocol can also be combined with laser
microdissection of individual cells from target tissues for specified breeding traits.
Key words Early growth regulation, AOX gene variants, Gene copy number, Growth performance,
Heterologous expression, Schizosaccharomyces pombe, Calorimetric analysis
1 Introduction
The capacity for adaptive plant plasticity allows efficient growth

acclimation to rapidly changing environmental conditions. This
can be essential for abiotic as well as biotic stress tolerance [1].
235
Thus, for functional marker development it is important to under-

stand critical steps early in adaptive growth regulation [7]. This
protocol aims to help studies of the effects of AOX gene variants on
early adaptive growth regulation. It covers the following steps:
standardized yeast subculturing for growth experimentation, opti-
cal density measurements to obtain growth curves, and calorimetric
measurements for metabolic phenotyping.
In this protocol, yeast growth is induced through subculture at
high initial cell density levels to enable (1) measuring small
increases in cell division growth early during the lag phase and (2)
understanding the importance of quantitative differences in AOX
gene copy numbers per volume for growth performance. Yeast can
switch between anaerobic and aerobic metabolism. According to
Albury et al. [3] and Affourtit et al. [4] this can be controlled by
using modified media with 2% glucose supporting fermentation or
using glycerol (3%) and 0.1% glucose in order to support mito-
chondrial respiration. In this protocol, growth curves are obtained
at the latter conditions by simply measuring optical densities
(OD600nm). Calorimetry measures released heat from living organ-
isms. Heat generation in growing cells depends on aerobic and
anaerobic cell metabolism [5]. Therefore, AOX gene variant-
dependent heat production rate curves measured during growth
development at aerobic conditions can indicate metabolic changes
due to differential growth regulation and can be measured in real
time. The amount of heat produced and the dynamics of heat
release can vary between genotypes (e.g., [6] and references
herein). In this protocol, calorimetry is applied in two ways. (1)
Isothermal measures are taken at selected time points during early
and later growth under aerobic metabolism and (2) continuous
heat rate curves are measured under both aerobic and mainly
anaerobic conditions. (1) is applied to confirm differential results
between AOX gene variants achieved by OD measurements. (2) is
performed to study the effect of AOX gene variants in a continuous
mode under diverse metabolic conditions (aerobic and mainly
anaerobic metabolism). The protocol can also be combined with
laser microdissection of individual cells from target tissues for spe-
cified breeding traits (Lopes and Arnholdt-Schmitt (2017), chapter
21 in this book).
2 Materials
2.1 Yeast Strains Plant AOX gene-transformed Schizosaccharomyces pombe strains

(see Note 1).
2.2 Equipment 1. Incubator with orbital shaker that allows temperature and
light/dark control.
AOX Gene Functionality in Growth 237
2. Spectrophotometer that allows measuring in the cuvette mode

at 600 nm (see Note 2).
3. Calorimeter: Calorimetric measurements can be made in a
Multi-Cell Differential Scanning Calorimeter (TA Instru-
ments, USA). This instrument has four 1-mL ampoules, three
for samples and one ampoule acting as a reference. Heat emit-
ted by yeast culture aliquots inside the ampoules is measured as
heat rates. The calorimeter is permanently connected to a water
bath for temperature control.
2.3 Laboratory 1. Gloves.

Material 2. Pipettes (500 and 1000 μL).
3. Cuvettes—1 mL (see Note 3).
4. Forceps to handle ampoules for calorimetric measurements.
2.4 Reagents/ Yeast cultures are grown in PM media [8] with added adenine and
Buffers/Solutions/ uracil (named: PM-AU):
Media
1. Potassium hydrogen phthalate, 3.0 g/L.
2.4.1 Yeast Culture 2. Sodium hydrogen phosphate, 2.2 g/L.
Medium
3. Ammonium chloride, 5.0 g/L.
4. 3% Glycerol (30.0 g/L) and 0.1% D-glucose (1.0 g/L) or
alternatively, if mentioned (see Subheading 3.3.2, step 2), 2%
D-glucose (20.0 g/L).
5. Adenine hemisulfate, 75.0 mg/L.

6. Uracil, 75.0 mg/L.
7. 20 mL/L Stock solution of macroelements (50): Magnesium
chloride (MgCl2·6H20—53.5 g/L), calcium chloride
(CaCl2·2H2O—0.735 g/L), potassium chloride (50 g/L),
sodium sulfate (2 g/L).
8. 1 mL/L Stock solution of vitamins (1000): Sodium panto-
thenate (1 g/L), nicotinic acid (10 g/L), inositol (10 g/L),
biotin (10 mg/L).
9. 100 μL/L Microelements (10,000): Boric acid (5 g/L),
manganese sulfate (4 g/L), zinc sulfate (ZnSO4·7H2O—4 g/
L), ferric chloride (FeCl3·6H2O—2 g/L), molybdic acid
monohydrate (1.6 g/L), potassium iodide (1 g/L), copper
sulfate (CuSO4·5H2O—0.4 g/L), citric acid (10 g/L).
10. Thiamine (0.5 mM) is added, when AOX expression should be
suppressed as control.
11. 1 M NaOH (1 mL) (see Note 4).
12. Distilled water.
2.4.2 Test for Bacterial Aliquots of measured sample are tested for bacterial growth on
Contamination (See Luria Bertani (LB) medium:
Subheading 3.3.2, Step 3)
Sodium chloride 5 g/L.
Tryptone 10 g/L.
Yeast extract 5 g/L.
pH 7.2.
Agar 1.5% (15 g/L).
3 Methods
3.1 Growth 1. Yeast cultures are grown in 100 mL Erlenmeyer flasks in 50 mL

Experimentation liquid PM-AU media containing 2% glucose at 30 C in the dark
at 120 rpm until they reach stationary growth (see Note 5).
2. For experimentation, a subculture is started (see Subheading
3.1, step 1, and Note 5) with 1000 μL inoculum added to
50 mL PM-AU media as a standard containing 3% glycerol and
0.1% glucose plus/minus 0.5 mM thiamine. The culture is
performed in 100 mL Erlenmeyer flasks at 30 C in the dark
at 120 rpm for at least 65 h (see Note 6).
3. In order to study the effect of cell density the amount of
starting inoculum volume can be varied (see Note 7) (Fig. 1).
5
AOX1 - thiamine
4
OD 600nm
AOX2a - thiamine
3 AOX2b - thiamine
AOX1 + thiamine
2
AOX2a + thiamine
1 AOX2b + thiamine
0
0 20 40 60 80
growing time in hours
Fig. 1 Growth curves of yeast strains transformed with carrot AOX1, AOX2a, and AOX2b genes are shown as an
example for the methodology that were growing in the presence or absence of thiamine. The initial inoculum
volume was 1 mL/50 mL PM-AU medium with 3% glycerol and 0.1% glucose for all variants. Whereas the
control curves are close together and show intermediate growth, AOX1 suppresses growth while AOX2a/2b
variants show early enhanced growth
7
6
5
OD 600nm
4
3 0.5 mL + thiamine
2 1 mL + thiamine
1
0
0 50 100 150
Growing time (h)
Fig. 2 The effect of cell density on growth of yeast transformed with DcAOX1 is
shown for the control (plus thiamine ¼ no involvement of AOX): lower cell density
leads to a prolonged lag-phase and reduced final growth
3.2 OD 1. Optical density measurements are performed at 600 nm (see

Measurements Subheading 2.2, item 2) at critical time points for growth over
for Growth Curves at least 65 h (see Note 8).
2. The effect of cell density on AOX-dependent growth is studied
by varying the initial inoculum volume (e.g., 500 and 1000 μL
added to 50 mL medium) in the presence and absence of
thiamine (see Figs. 2 and 3).
3.3 Calorimetric 1. Samples are taken at selected time points of the OD-measured
Measurements growth curve (see Fig. 1) and heat rates (Rq—unit: μW or μJ/s)
for Metabolic (see Subheading 2.2, item 3) are measured in isothermal mode
Phenotyping at 30 C. 500 μL of yeast culture is pipetted under sterile
conditions into an ampoule of the calorimeter (see Notes 10
3.3.1 Isothermal and 11). Measurements can be performed at a range of tem-
Measurements peratures (see Note 12) (see Fig. 4).
3.3.2 Continuous Heat 1. 500 μL of freshly prepared yeast subculture (see Subheading
Rate Curves 3.1, step 2) is pipetted under sterile conditions into an
ampoule of the calorimeter (see Notes 11 and 13).
2. Heat rates are measured at 30 C continuously until the initial
values are reached again (see Note 14) (see Figs. 5 and 6).
3. An aliquot of measured samples is taken under sterile condi-
tions when heat rate curves are finished and checked for bacte-
rial growth on solid LB medium (see Subheading 2.4, item 2)
(see Note 15).
7
A
6
5
OD600nm
4
3 0.5 mL - thiamine
2 0.5 mL + thiamine
1
0
0 20 40 60 80
Growing time (h)
7
B
6
5
OD600nm
4
3 1 mL - thiamine
2 1 mL + thiamine
1
0
0 20 40 60 80
Growing time (h)
Fig. 3 (a, b) a/b show that the effects of AOX1 genes on growth regulation can be
dependent on cell densities (0.5 mL vs. 1 mL initial inoculum): at lower cell
density the lag phase of growth is shortened and growth is stimulated in the
absence of thiamine compared to the control (plus thiamine) (a); at higher cell
density level, similar to the control, growth starts early, but then growth is
strongly suppressed when AOX expression is not inhibited by thiamine (b) (see
Note 9)
4 Notes
1. S. pombe strains that contain vector constructs based on pREP1

with inserted coding sequences of AOX genes (transformation is
not part of this protocol, see published protocols in Ref. [3, 4]).
The thiamine-repressible nmt1 promotor allows suppressing
AOX expression as a control by adding thiamine. Yeast strains
can be stored on solid agar medium at 80 C and will be
initiated upon requirement on solid media subsequently trans-
ferred to liquid PM-AU media (see Subheading 3.1, step 2).
Temperature-dependent response at 20h A

40
35
30
25
Heat Rate
20 AOX1
15 AOX2a
10 AOX2b
5
0
0 10 20 30 40 50
Temperature
Temperature-dependent response at 54h

B
40
35
30
25
Heat Rate
20 AOX1
15 AOX2a
10 AOX2b
5
0
0 10 20 30 40 50
Temperature
Fig. 4 (a/b) This figure shows the added value of calorimetric measurements.
Heat rate measurements at selected time points of the growth curves (Fig. 1) can
confirm lower metabolic activity for the AOX1 variant (minus thiamine) in
comparison to the AOX2a and AOX2b variants at both selected time points of
20 h (a) and 54 h (b) (inoculum: 1 mL/50 mL). At 54 h calorimetric measurements
also confirm the results by decreased heat rates during reduced growth for
AOX1, while AOX2a/AOX2b show increased metabolic activity due to highly
active growth in the exponential phase (Fig. 1). Measurements were performed
at a range of temperatures. At 54 h (b) it can also be shown that the AOX1 variant
has already reduced metabolic capacity for growth at 43 C
2. A Nanodrop instrument can be used.

3. Measured volume 500 μL.
4. Adjustment to pH 5.5.
5. Cultures are regularly subcultured after around 10 days to keep
the material ready for starting growth experiments (see Sub-
heading 3.1, step 1).
Yeast culture in 3% glycerol and 0.1% glucose

160
140
120
100
Heat Rate
80
60 AOX1
40
20
0
-20 0 100000 200000 300000 400000 500000
Growing time (seconds)
Fig. 5 Continuous heat rate measurements can be used to confirm the low
metabolic activity during growth of the DcAOX1 variant in the absence of
thiamine (¼ AOX expressed). The graph is normalized for comparison to Fig. 6
Yeast cultures in 2% glucose

160
140
120
100
Heat Rate
80
AOX1
60
AOX2a
40
20
0
0 100000 200000 300000 400000 500000
Growing time (seconds)
Fig. 6 Continuous heat rate growth curves can also be used to demonstrate that
differential AOX functionality in growth regulation is related to mitochondrial
respiration. The figure shows earlier and rapid growth in the mostly anaerobic
mode at 2% glucose (see Fig. 5 for comparison). It can be seen that under
anaerobic conditions DcAOX1 and DcAOX2a do not differ in their effect on growth
(see Note 14)
6. It is important to have comparable cell densities as a starting

point. Starting with 1000 μL inoculum added to 50 mL will
give values of around 0.4–0.6 OD600nm in the starting media
(see Fig. 1).
7. Inoculum volume can be varied, e.g., to 500 μL instead of
1000 μL to highlight the importance of doubling gene copy
numbers. Other cell densities can be used to fine-tune the

observation.
8. Measurement time points for growth curves can be deduced
from Fig. 1.
9. Number of cells per volume in minus thiamine can be expected
to correspond to higher amounts of expressed AOX genes.
Thus, differential results between cell densities in minus thia-
mine could give indication on the importance of AOX copy
number variations in defined tissue volumes. For example, in
carrot the tap root meristem that is critical for yield produc-
tion/stability is restricted to a circled layer of only ten cells. The
amount of carrot AOX1 in this tissue can thus be critical.
However, this assumption that the yeast system can aid in
studying the importance of gene copy numbers for individual
genes requires still validation and the protocol can help in this.
10. Yeast samples are taken in a reproducible manner by shaking
the Erlenmeyer flask in a way that all cells are equally
distributed in suspension.
11. Ampoules are placed into the clean bench and are sterilized;
that is, they are completely filled with 70% ETOH and kept for
at least 10–15 min. This step is important especially when
continuous growth curves are reported over a long time,
since microorganism growth might rapidly influence heat rate
measurements. When continuous heat rates were measured, it
is recommended to check at the end of the run for bacterial
growth (see Subheading 2.4, item 2).
12. For isothermal measurements, endpoint values are considered
and a baseline value obtained from measurements with empty
ampoules is subtracted. Baseline values need to be measured
for each ampoule due to varying equipment and ampoule
conditions. As an option, measurements can be performed at
a spectrum of temperatures in order to confirm differences in
heat released by different AOX gene variants. Temperatures are
always started in a non-stressed mode for the material; for
example temperatures can be run from 30 to 17 C and then
going up from 17 to 43 C (Fig. 4). At each new temperature
step, an equilibrium time of 600 s is programmed before data
are collected.
13. PM-AU media with 3% glycerol/0.1% glucose is used in com-
parison to 2% glucose.
14. When stationary growth is achieved, heat rates go down to
initial values.
15. Colony growth is observed during several days.
Acknowledgements
At first, B.A.S. would like to appreciate the effort of the editor

Jagadis Gupta Kapuganti for this special edition related to respira-
tion and for inviting submission to make running protocols in her
lab available to the public. The authors thank Isabel Velada, Hélia
Cardoso, and Amaia Nogales for having established the yeast S.
pombe system at the Chair’s lab in Évora by help of Mary S. Albury
from the team of Anthony Moore’s group (University of Sussex,
UK) and for having provided carrot AOX gene-transformed yeast
variants (Velada et al. in preparation—research article). The idea of
using the yeast system for studying AOX gene variants/polymorph-
isms had been first developed within the frame of a bilateral coop-
eration running between the EU Marie Curie Chair team and
Anthony Moore’s group funded in Portugal by CRUP (“Concelho
de Reitores das Universidades Portuguesas”) and by the British
Council. Yeast transformation with carrot AOX genes was sup-
ported by Project AI/B-37/09 and Project EXCL/BEX-GMG/
0038/2012 coordinated at the EU Marie Curie Chair by Hélia
Cardoso. We also appreciate the consultancy of Amaia Nogales and
Lee Hansen for our first steps on applying calorimetry to yeast. To
Lee Hansen, B.A.S. is especially grateful for his permanently high
availability as consultant at the Chair for all theoretical and practical
questions related to calorimetry (see also Chapter 15 by Arnholdt-
Schmitt in this book). Vinod Kumar Patil appreciates the scholar-
ship provided by the Erasmus Mundus FUSION Project, Postdoc-
toral Exchange Studies (2015–2016).
References
1. Cardoso HG, Arnholdt-Schmitt B (2013) Func- alternative oxidase affects growth of the yeast
tional marker development across species in Schizosaccharomyces pombe. J Biol Chem
selected traits. In: L€
ubberstedt T, Varshney RK 274:6212–6218
(eds) Diagnostics in plant breeding. Springer, 5. Maskow T, Paufler S (2014) What does calorim-
Netherlands, pp 467–515. doi:10.1007/978- etry and thermodynamics of living cells tell us?
94-007-5687-8_21 Methods 76(2015):3–10
2. Campos M, Nogales A, Cardoso HG, Rajeev 6. Hansen LD, Thomas NR, Arnholdt-Schmitt B
Kumar S, Nobre T, Sathishkumar R, Arnholdt- (2009) Temperature responses of substrate car-
Schmitt B (2016) Stress-induced accumulation bon conversion efficiencies and growth rates of
of DcAOX1 and DcAOX2a transcripts coincides plant tissues. Physiol Plant 137:446–458
with critical time point for structural biomass 7. Ragonezi C, Arnholdt-Schmitt B (2017) Laser-
prediction in carrot primary cultures (Daucus capture microdissection for amplification of
carota L.) Front Genet 7:1. doi:10.3389/ alternative oxidase (AOX) genes in target tissues
fgene.2016.00001 in Daucus carota L. In: Kapuganti JG (ed) Plant
3. Albury MS, Dudley P, Watts FZ, Moore AL respiration and internal oxygen: methods and
(1996) Targeting the plant alternative oxidase protocols. Springer, New York
protein to Schizosaccharomyces pombe mito- 8. Mitchison JM (1970) Physiological and cytolog-
chondria confers cyanide-insensitive respiration. ical methods for Schizosaccharomyces pombe.
J Biol Chem 271:17062–17066 Methods Cell Physiol 4:131–165
4. Affourtit C, Albury MS, Krab K, Moore AL
(1999) Functional expression of the plant
Chapter 21
Laser Capture Microdissection for Amplification

of Alternative Oxidase (AOX) Genes in Target
Tissues in Daucus carota L.
Carla Ragonezi and Birgit Arnholdt-Schmitt
Abstract
Laser microdissection provides a useful method for isolating specific cell types from complex biological
samples for downstream applications. In contrast to the texture of mammalian cells, most plant tissues
exhibit a cell organization with hard, cellulose-containing cell walls, large vacuoles, and air spaces, thus
complicating tissue preparation and extraction of macromolecules such as DNA. In this study, we report a
method that allows tissue-specific gene amplification. An improved perception of genetic identity of the
entire plant can contribute to improved functional marker strategies. Alternative oxidase (AOX) has crucial
position for stress-induced responses/adaptation. Daucus carota sequence polymorphisms in AOX were
identified, however, never at tissue/cell level. This technology will support studying AOX gene sequences
in carrot organs/tissues/cells and specifically exploring differential polymorphisms in root meristem that
might be associated to adaptive growth upon all kind of stresses. Details on aspects of tissue preparation,
including fixation and embedding procedures, laser capture microdissection, DNA extraction, and amplifi-
cation, are provided. A combination of laser microdissection and polymerase chain reaction amplification
provides a powerful tool for the analysis of AOX gene amplification in methacarn-fixed paraffin-embedded
tissues.
Key words AOX gene amplification, Carrot, Laser microdissection, Methacarn-fixed tissues,
Plant tissue
1 Introduction
Methodologies for obtaining highest yield and best quality of

extracted DNA from microdissected tissues are well established
for animal and human tissues. In the case of plant tissues, chemical
fixation is the predominant method used for tissue preparation,
since it guarantees a high morphological integrity, especially when
fine structures and small tissue types have to be isolated. Owing to
the enormous differences in structure of plant tissues, no standard
protocol for tissue preparation exists. Existing protocols vary in the
choice of the chemical fixative, duration of fixation, and subsequent
245
246 Carla Ragonezi and Birgit Arnholdt-Schmitt
embedding procedure and it can be congruently summarized that

protocols have to be adapted and optimized for every tissue type.
Therefore, the isolation of specific tissue or cell types prior to a
molecular analysis, for example, is important to understand the
specification, differentiation, and function of these cells.
Carrot yield depends under normal growing conditions on the
activity of the central root meristem, not primarily on shoot growth
[1]. If crucial genes for meristem growth regulation show poly-
morphisms between diverse tissues/cells, then it will be important
to develop functional markers from the root meristem. AOX is a key
enzyme in alternative respiration. It is involved in cell homeostasis
[2], and adaptive growth and development upon all kinds of abiotic
and biotic stress ([3] and references herein, [4]). AOX has a central
upstream position in energy and carbohydrate metabolism and was
frequently reported to respond early to environmental signals.
AOX genes were highlighted as promising marker gene candidates
for general plant plasticity by regulating stress-tolerant, robust
plant behavior [4–6]. The carrot AOX gene family consists of the
three genes, DcAOX1, DcAOX2a, and DcAOX2b. Co-regulation
of carrot AOX1 and AOX2 genes has been reported for diverse
experimental systems, such as in plants, in primary cultures from
root explants during de novo growth induction [7, 8] and during
initiation of somatic embryogenesis [9, 10]. Recently, early
response of DcAOX1 and DcAOX2a has been demonstrated
upon chilling [8]. Response of both genes occurred previous to
high induction of a specific pathway gene, the antifreezing gene
AFP (2016). Early accumulation of DcAOX1 and DcAOX2a tran-
scripts in freshly inoculated primary cultures induced to callus
growth coincided with a critical time point in metabolism for
structural biomass prediction obtained by calorespirometry ([1,
8], for calorespirometry see [11]). Several reports are available on
the frequent occurrence of polymorphisms in functionally relevant
positions in both carrot AOX family genes [12–15].
In this protocol, we describe feasible methods allowing tissue-
specific transcriptome analysis in carrot tissues. Much information
about cell behavior and regulation can be revealed by examining
gene expression in cells which are grown as homogeneous cultures.
Sophisticated, controlled analyses of molecular changes in response
to different stimuli are possible. However, functional gene expres-
sion within individual cells and tissues in complex organisms can be
very different. Laser microdissection (LM) provides the opportu-
nity to bridge the gap for both requirements by enabling transcrip-
tome analyses at cell and tissue level [16]. A wide variety of methods
to fix and embed tissues prior to LM and downstream molecular
analyses have been developed for different plant species; however
few are published regarding tissue/cell analysis. This chapter
focuses on techniques for using LM and polymerase chain reaction
(PCR) analysis to evaluate gene expression in methacarn-fixed tis-
sue sections.
Laser Microdissection for AOX 247
2 Materials
2.1 Plant Material 1. Thin pieces (~3 mm—see Note 1) of root samples from D.
carota L. cv. Rotin.
2.2 Equipment 1. Hot bath.

2. Laser microdissection slides (frame slides with membrane type
for Leica instrument LMD6500).
3. Hot air oven.
4. Laser Microdissection System (Leica instrument LMD6500).
5. NanoDrop spectrophotometer.
6. Programmable thermocycler.
7. Microtome.
2.3 Laboratory 1. Double-distilled water and autoclaved water.

Material 2. Slides holder.
3. Nuclease-free PCR tubes and filtered pipette tips.
4. E.Z.N.A.® MicroElute® Genomic DNA Kit (Ômega).
5. TaqMan Polymerase.
6. dNTPs.
7. Primers specific for AOX1a gene amplification.
2.4 Reagents/ 1. Methacarn solution, methanol absolute, chloroform, and acetic

Solutions acid (6:3:1).
2. 70, 96, and 100% ethanol.
3. Paraffin.
4. Xylene.
5. Agarose.
3 Methods
In the following sections, methods are described for (1) pre-cutting

(histology), (2) laser microdissection cutting, and (3) post-cutting
(molecular analysis). An overview of the complete procedure is
given in Fig. 1.
3.1 Pre-cutting 1. Fixation: For this step, the pieces of root samples from D.
(Histology) carota L. cv. Rotin were fixed in 100 mL of methacarn in
covered glass flasks and stored at 4 C for at least 48 h (see
Note 2).
2. After fixation, samples were dehydrated in crescent concentra-
tion of ethanol (Table 1, see Note 3). All dehydration steps are
De-paraffinization
Carrot slices
LM cutting
Membrane slides
Fixation for LM cutting
DNA extraction with Kit
70% 96% 100% Xyl. Par.
Dehydration
Microtome cutting
PCR
1st bath 2nd bath Eletrophoresis

Infiltration Blocks formation
Fig. 1 Flowchart for the complete process enclosing pre-cutting (histology), laser microdissection cutting, and
post-cutting molecular analysis
Table 1
Dehydration and infiltration procedures
Reagent Time period

70% (v/v) Ethanol 4h
16 h (overnight)
1h
1h
Xylene 1h
1h
Paraffin 1 h (first bath)
1 h (second bath)
Table 2
De-paraffinization steps
Reagent Time period

Xylene 5 min
100% (v/v) Ethanol 1 min
done under the fume chamber and solutions maintained at

4 C (see Note 4).
3. For infiltration procedure, samples were placed in paraffin for
1 h (see Note 5), and for the second bath, the paraffin was
changed and samples were stayed also for 1 h (Table 1).
4. Paraffin-infiltrated samples were embedded in paraffin and
placed on plastic cassettes previously identified with a pencil
to do the blocks (see Note 6). The paraffin blocks were cut with
a microtome and section thickness was 10 μm. Cuts were
placed in a hot water bath at 45 C (see Note 7) and “caught”
with the proper membrane slides for LM cutting.
5. De-paraffinization was done with xylene and hydrated with
decreasing concentration of ethanol. All steps are done under
the fume chamber according to Table 2.
6. Let the slides air-dry and store in a proper box containing
desiccant.
3.2 Laser 1. Turn on all the components of the laser microdissection

Microdissection (LM) system.
Cutting 2. Log on to computer by clicking the user icon, and type in
“user” as the password. Click in the desktop “laser microdis-
section” icon to start the software.
3. Click on the rightmost “unload” button in the software toolbar
and place the tube in the tube holder into stage support with
caps facing upwards; there is only one way it will fit and click to
the loading window.
4. To load specimen slide put a slide on the stage, press the left-
hand “unload” button in the toolbar. The stage then moves to
a lower position for slide change. Withdraw specimen holder by
pulling towards yourself. Insert the slide onto specimen holder.
Click on “continue.”
5. Set the laser parameters according to the objectives: aperture,
speed, specimen balance, factory settings, and offset.
6. For cutting and collecting the area of interest—locate your area
of interest, select the cap, draw a shape around target area, and
press the “start cut” key (see Note 8).
7. Save image by selecting “save image as” in the “file” menu.

8. For the shutdown procedure, simply do as follows: activate left-
hand “unload” slide and “unload” tube holders and remove
the slide and tube. Turn off laser, shut down computer, and
turn off microscope controller box.
3.3 Post-cutting 1. Follow the detailed instructions provided with the genomic
(Molecular Analysis) DNA kit, taking particular care to clean the work area. Also,
wear disposable gloves and proper laboratory coat.
2. The amount of DNA was measured using a NanoDrop spec-
trophotometer. Samples were amplified by PCR in the condi-
tion as described in Tables 3 and 4.
3. Primers used for amplification of AOX gene were previously
designed at the EU Marie Curie Chair [14]. DcAOX1 was
selected for establishing the protocol.
– For complete gene amplification: DcAOX1_24Fw/
DcAOX1_1021Rev.
Table 3
PCR mix used to amplify the samples
Mix Volume
10 TaqPolymerase Buffer with MgCl2 25 mM 2.5 μL
dNTPs (10 mM) 0.5 μL
Primer FW (10 μM) 1.0 μL
Primer Rev. (10 μM) 1.0 μL
TaqPolymerase 0.25 μL
DNA 1 μL
H2O 18.74 μL
Table 4
PCR conditions used to amplify the samples
Temperature Time
95 C 5 min

95 C 1 min
Primer dependent 1 min
72 C 2 min
72 C 10 min
Cycles 40
DcAOX1a_24Fw: AAAATAACAATGATGATGACACG.
DcAOX1a_1021Rev: CTCCACTTCAGTGATATCCAA.
– For Intron 1: DcEx1_ 263Fw/DcAOX1 _ex2_Rev.
DcEx1_263Fw: GGCCATGGGAGACGTACCAG.
DcAOX1a_ex2_Rev: AGGTCTTGATCCAACCTCCG.
4. PCR results (see Note 9) were analyzed by electrophoresis in
agarose gel (1.4%) and staining with ethidium bromide and
visualized in proper equipment.
4 Notes
1. Carrots were washed with tap water and cut with a scalpel.
2. The sample can stay in the fixation solution up to 3 weeks
without disturbing the histology protocol.
3. It is important to put the samples always in new ethanol solu-
tions when those are changed.
4. Other fixation solutions such as F.A.A. can be used for DNA
extraction.
5. The infiltration step must be done inside the hot air oven, so
that the paraffin stays liquid. Take care to be fast when chang-
ing the samples to the second bath.
6. Pencil is the best for cassette identification, since pen often is
erased.
7. The water should not exceed the temperature of 45 C; other-
wise the paraffin will melt.
8. You can use the specimen overview option to easily navigate
specific areas of your sample.
9. In case PCR results are not good enough, cloning of the same
samples is advised.
Acknowledgments
This work was supported through a Marie Curie Fellowship to C.R.

by the European Commission through the project AGRO-AMF-
AOX within the program Industry-Academia Partnerships and
Pathways (IAPP, FP7) coordinated by the EU Marie Curie Chair.
C.R. appreciates having been hosted during the 2-month fellowship
by Inoq GmbH (Germany). We acknowledge equipment funding
from the Programa Operacional Regional do Alentejo (InAlentejo)
Operation ALENT-07-0262-FEDER-001871 and also support
through National Funds by FCT—Foundation for Science and
Technology under the Project UID/AGR/00115/2013. This
work was supported by the program POPH-Operational Program

for Human Potential and “Fundo Europeu de Desenvolvimento
Regional” (FEDER) funds through the Operational Program for
Competitiveness Factors-COMPETE, and national funds through
FCT under the strategic project PEst-C/AGR/UI0115/2011 and
PEst-OE/AGR/UI0115/2014. We appreciate contribution on
primer design, and support on gene isolation and data analysis by
Amaia Nogales, Hélia Cardoso e Tânia Nobre. B.A.S. is thankful to
the University of Évora for continuous invitations as Coordinating
Investigator since 2008 in order to prolong running of the estab-
lished EU Marie Curie Chair financed initially by the EC in the
period from 2005 to 2008.
References
1. Nogales A, Muñoz-Sanhueza L, Hansen LD prediction in carrot primary cultures (Daucus

et al (2013) Calorespirometry as a tool for carota L.) Front Genet 7:1–17
studying temperature response in carrot (Dau- 9. Frederico AM, Campos MD, Cardoso HG et al
cus carota L.) Eng Life Sci 13:541–548 (2009) Alternative oxidase involvement in
2. Hansen LD, Church JN, Matheson S et al Daucus carota somatic embryogenesis. Physiol
(2002) Kinetics of plant growth and metabo- Plant 137:498–508
lism. Thermochim Acta 388:415–425 10. Zavattieri MA, Frederico AM, Lima M et al
3. Vanlerberghe GC (2013) Alternative oxidase: a (2010) Induction of somatic embryogenesis
mitochondrial respiratory pathway to maintain as an example of stress-related plant reactions.
metabolic and signaling homeostasis during Electron J Biotechnol 13:12–13
abiotic and biotic stress in plants. Int J Mol 11. Arnholdt-Schmitt B (2017) Respiration traits
Sci 14:6805–6847 as novel markers for plant robustness under the
4. Arnholdt-Schmitt B, Costa JH, Fernandes de threat of climate change – a protocol for vali-
Melo D (2006) AOX – a functional marker for dation. In: Walker JM, Gupta KJ (eds) Meth-
efficient cell reprogramming under stress? ods in molecular biology. Humana Press, New
Trends Plant Sci 11:281–287 York. ISSN: 1064-3745
5. Cardoso HG, Arnholdt-Schmitt B (2013) 12. Cardoso HG, Campos MD, Costa AR et al
Functional marker development across species (2009) Carrot alternative oxidase gene
in selected traits. In: L€
ubberstedt T, Varshney AOX2a demonstrates allelic and genotypic
RK (eds) Diagnostics in plant breeding, vol 21. polymorphisms in intron 3. Physiol Plant
Springer, Dordrecht, Netherlands, pp 137:592–608
467–515 13. Cardoso H, Campos MD, Nothnagel T et al
6. Arnholdt-Schmitt B, Hansen LD, Nogales A (2011) Polymorphisms in intron 1 of carrot
(2016) Calorespirometry, oxygen isotope anal- AOX2b-a useful tool to develop a functional
ysis and functional-marker-assisted selection marker? Plant Genet Resour 9:177–180
(‘CalOxy-FMAS’) for genotype screening: a 14. Nogales A, Nobre T, Cardoso HG et al (2016)
novel concept and tool kit for predicting stable Allelic variation on DcAOX1 gene in carrot
plant growth performance and functional (Daucus carota L.): an interesting simple
marker identification. Brief Funct Genomics sequence repeat in a highly variable intron.
15:10–15 Plant Gene 5:49–55
7. Campos MD, Cardoso HG, Linke B et al 15. Nobre T, Oliveira M, Arnholdt-Schmitt B
(2009) Differential expression and co- (2016) Wild carrot differentiation in Europe
regulation of carrot AOX genes (Daucus car- and selection at DcAOX1 gene? PLoS One
ota). Physiol Plant 137:578–591 11:e0164872
8. Campos MD, Nogales A, Cardoso HG et al 16. Day RC, Grossniklaus U, Macknight RC
(2016) Stress-induced accumulation of (2005) Be more specific! Laser-assisted micro-
DcAOX1 and DcAOX2a transcripts coincides dissection of plant cells. Trends Plant Sci
with critical time point for structural biomass 10:397–406
Chapter 22
Measurement of Mitochondrial Respiration in Isolated

Protoplasts: Cytochrome and Alternative Pathways
Bobba Sunil and Agepati S. Raghavendra
Abstract
The electron partitioning between COX and AOX pathways of mitochondria and their coordination is
necessary to meet the energy demands as well as to maintain optimized redox status in plants under varying
environmental conditions. The relative contribution of these two pathways to total respiration is an
important measure during a given stress condition. We describe in detail the procedure that allows the
measurement of the parameters of COX and AOX pathway of respiration in mesophyll protoplasts using
Clark-type O2 electrode. This chapter also lists the steps for rapid isolation procedure for mesophyll
protoplasts from pea leaves. The advantages and limitations of the use of metabolic inhibitors and the
protoplasts for measuring the respiration are also briefly discussed.
Key words Alternative oxidase (AOX) pathway, Alternative pathway engagement, Clark-type O2
electrode, Cytochrome oxidase (COX) pathway, Mesophyll cell protoplasts, Mitochondrial electron
transport, Mitochondrial inhibitors, Pisum sativum
1 Introduction
1.1 Respiration in The process of respiration involves the oxidation of substrates

Plant Cells through glycolysis and TCA cycle to produce NADH/FADH,
which are then utilized in mitochondrial oxidative electron trans-
port chain (ETC) to produce ATP. The mitochondrial ETC typi-
cally comprises of four large protein complexes (I, II, III, IV) that
drive the electron flow from NADH to oxygen while facilitating the
phosphorylation of ADP to ATP via ATP synthase (complex V) [1].
The oxidative electron transport of higher plants consists of
two different pathways—cytochrome oxidase (COX) pathway and
alternative oxidase pathway (AOX). The ATP production is mostly
accomplished by COX pathway. The AOX pathway is considered as
a sink for excess electrons and plays a prominent role in maintaining
the cellular redox balance [2, 3]. Apart from ATP generation,
respiration has several other functions in plants, such as optimiza-
tion of photosynthetic carbon assimilation, nitrogen assimilation,
253
254 Bobba Sunil and Agepati S. Raghavendra
reactive oxygen species homeostasis, and metabolite cycling needed

for stress acclimation [4–6]. The mitochondrial ETC pathways play
a prominent role as energy mediators and also help in the plant
adaptation to different stresses [7]. In plants, mitochondria con-
tribute to the metabolic flexibility and the coordination of both
COX and AOX pathways, which is necessary to cope up the fluctua-
tions in energy demands during diverse environmental conditions
[2, 8].
As the relative contribution of COX and AOX pathways to total
respiration is flexible and dependent on environment, it is quite
useful to measure the electron partitioning between the COX and
AOX pathways. This can be performed with the use of specific
inhibitors of the two pathways, such as Antimycin A, SHAM, or
KCN. After measuring the actual rates of oxygen uptake, formulae
can be used to differentiate the respiration via the two pathways.
This chapter describes in detail the principle and procedure for
determining the rates and proportion of alternative pathway,
using specific metabolic inhibitors, in protoplasts. The method of
protoplast isolation is also explained.
1.2 Methods to There are various methods that have been developed to measure
Measure Respiration in the rate of respiration based on “consumption of O2” or “produc-
Plants tion of CO2” [9, 10]. The choice of the method depends on the
experimental system, the exact purpose, and the ease of use.
The most popular methods use either the infrared gas analyzer
(IRGA) to measure the CO2 production or Clark-type O2 electrode
to monitor the O2 consumption. The IRGA can be used for CO2
measurement in both open and closed systems and has the advan-
tage of using leaves in vivo [11]. The O2 electrodes used in the
aqueous phase are excellent for studying photosynthesis and respi-
ration of isolated organelles/cells or suspensions, yet in vitro. Fur-
ther, the components of COX and AOX pathways can be easily
measured in the O2 electrode chamber, without disturbing the
experimental material. The details of theory, design, and use of
the O2 electrode for plants are described elsewhere [10, 12]. Typi-
cally, O2 accumulates in the chamber during photosynthesis (in the
presence of light) or is depleted from the chamber during respira-
tion (in the dark), when the sample is provided with a small polar-
izing voltage across the electrode. The loss of O2 from the sample
(consumption by tissue) is used as a measure of respiration rate by
the sample. The advantage of measuring O2 uptake over CO2 efflux
is that the gas being measured is not the gas being altered, avoiding
the need for instrument calibration [13].
Respiration can be measured by using radioactive or stable
isotopes. For example CO2 evolution using 14C (a radioisotope)
involves feeding the leaves with a substrate containing 14C and
measuring the CO2 efflux by a suitable monitor, e.g., beta-counter.
This method can also be coupled to measure the CO2 fixation by
Measurement of Respiration in Mesophyll Protoplasts 255
infrared gas analyzer (IRGA) simultaneously. The technique of

oxygen isotope fractionation (or discrimination) measures the
changes in the isotopic composition of O2, with varying masses
(18O/16O). This method is based on the observation that AOX and
COX discriminate to different extent against 18O, when reducing
oxygen to produce water. The cytochrome oxidase discriminates
less than the alternative oxidase, allowing the calculation of the
partitioning of electron flow between the two pathways [14, 15].
Fluorophore-based micro-oxygen sensors have been developed to
monitor oxygen levels inside plant seeds and leaves. Recent devel-
opments in the measurement of respiration include high-
throughput assay systems, by combining micro-respiratory tech-
nologies with multiplex assays. This allows real-time fluorimetric
detection of O2 consumption in live cells in a noninvasive way [16].
The contribution of cytochrome and alternative pathways to
total respiration is calculated from the titration curves of respiratory
rates against the concentrations of specific respiratory inhibitors of
mitochondrial electron transport chain. The extent of alternative
pathway can be quantified by measuring O2 uptake in the absence
and presence of KCN or Antimycin A (inhibitors of cytochrome
pathway). The subsequent respiration, which gets inhibited by
SHAM, reflects the capacity of alternative pathway [2, 17].
1.3 Protoplast Isolation of plant protoplasts was first reported more than five
System decades ago [18]. Since then, several isolation procedures have
been described that yield highly purified and functional protoplasts
from diverse organs, such as leaves, roots, petals, and guard cells
[19, 20]. Intact mesophyll protoplasts isolated from pea and Ara-
bidopsis leaves can respond to signals in a physiological manner
similar to the responses observed in leaves of whole plants
[21–23]. Protoplasts offer a versatile single cell-based experimental
system to evaluate a wide range of cellular processes, using physio-
logical, proteomic, and genomics approaches. The protoplasts find
use in tissue culture, somatic hybridization, plant transformation,
and metabolite production, besides high-resolution imaging and
subcellular localization [24–26]. Mesophyll protoplasts have been
routinely used to study photosynthesis, respiration, transient gene
expression, protein-protein interactions, circadian clock studies,
and signal transduction pathways [27–29].
Protoplasts allow free diffusion of O2 or CO2 due to the
absence of barriers like intercellular spaces and cell walls. Further,
they also allow the ease of manipulation of metabolism by exoge-
nous addition of substrates or metabolite inhibitors, whose effects
on metabolism can be observed within few minutes. For example
the intriguing concepts of intriguing interactions of chloroplast-
mitochondria and the phenomenon of light-enhanced dark respira-
tion (LEDR) were established by using protoplast system as a
tool [30].
The yield and intactness are important factors for protoplast

isolation. The favorable factors are short isolation time (to achieve
maximum yield) and maintenance of high metabolic integrity of
protoplasts. In order to obtain large quantities of pure/intact pro-
toplasts, it is necessary to optimize the conditions. The isolation
procedures need to be optimized, particularly, the osmotic
strength, concentration of digestive enzymes, reaction pH, and
time for each step during isolation. Short duration of digestion
leads to low protoplast yields whereas too long a time results in a
decrease in the viability and biological activity of the resulting
protoplasts [21, 22]. Further, the isolation procedure may lead to
the generation of free radicals (reactive oxygen species, ROS)
because of the stressful steps, such as peeling of leaves, enzymatic
digestion at acidic pH, and alternate periods of light and darkness.
Since the ROS are toxic to protoplasts, addition of sodium ascor-
bate, BSA, and EDTA during the isolation procedure is quite
effective for improving the protoplast quality.
1.4 Limitations Although the protoplasts seem to be quite good for metabolomic
of Protoplast System studies, they have certain limitations. Since protoplasts are quite
and the Biochemical fragile, their preparation and handling need to be mastered. To
Respiratory Inhibitors ensure consistency, the experiments with protoplasts need to be
repeated sufficient times. The protoplast system is not useful for the
studies related to cell differentiation (such as cell cycle regulation),
tissue-specific processes (such as root vs. leaf), intercellular signal-
ing, or long-term plant responses. Further discussion on limitations
of protoplast system can be found in the articles of [31, 32].
The use of chemical inhibitors has been an important approach
for analyzing the components of respiration. Mitochondrial respi-
ratory inhibitors are available for modulation of mitochondrial
function, which include electron transport chain inhibitors (e.g.,
Antimycin A, SHAM, KCN, Rotenone, n-propylgallate) or phos-
phorylation inhibitors (oligomycin) or uncouplers (CCCP/
FCCP). An ideal inhibitor/modulator is the one, which has a
high degree of specificity to the respective target site or enzyme.
However, most of the commonly used inhibitors have the disad-
vantage of being nonspecific, particularly at higher concentrations
[33–35]. Inhibitors are all toxic in nature, and therefore we have to
be very careful with the dosage (low or high) of the compound that
is being used. It is essential to determine the optimal concentration
of the inhibitor for each tissue or the plant species used. For
example SHAM or propylgallate used for assessing the role of
AOX may have nonspecific effects of these inhibitors in long-term
assays [36]. The solubility of the inhibitor compound also needs to
be considered before using. All the respiratory inhibitors may not
be soluble in water. The effect on respiration of alcohol or nonpolar
solvents used for stock solution needs to be ascertained.
In this manuscript, we have confined to the Clark-type O2

electrode-based respiratory measurements in protoplasts. The pro-
cedure is described for use with leaves of pea (Pisum sativum). It
can be used for preparing mesophyll protoplasts from the leaves of
also Arabidopsis thaliana with high rates of photosynthesis [21].
2 Materials
2.1 Plants 1. Seeds of garden pea (Pisum sativum L., cv. Arkel) (see Note 1).
2. Sodium hypochlorite (NaClO) 4% solution.
3. Plastic trays, potting soil, and farmyard manure.
4. Growth chamber or green house.
2.2 Protoplast 1. Two petri dishes (one of 15 mm and the other of 8 mm

Isolation diameter), forceps (bent forceps or normal), scalpel blade,
Pasteur pipette.
2. Water bath, balance, pH meter, round-bottom test tubes,
benchtop centrifuge.
3. Tungsten lamp as a light source during digestion of leaves.
4. Li-Cor quantum sensor (Li-Cor Instruments Ltd., USA) to
measure the photon flux.
5. The room temperature should be maintained at 25 1 C, so
as to optimize the function of protoplasts.
6. 60 μm sieve-sized nylon filter, cyclo-mixer, and
spectrophotometer.
2.3 Buffers and 1. Stocks of 2 M sorbitol, 100 mM MES buffer (2-N-morpho-

Solutions lino-ethanesulfonic acid-monohydrate), 100 mM HEPES
buffer (4-2-hydroxyethyl-1-piperazineethanesulfonic acid),
100 mM CaCl2·2H2O, 100 mM MgCl2, and 100 mM EDTA
prepared with Millipore water. Adjust the pH with potassium
hydroxide (KOH) to the desired values (MES pH 5.5 and 6.0,
HEPES pH 7.0 and 7.5).
2. Working solutions of pre-plasmolysis, digestion, suspension,
and reaction media are prepared from the above stock solutions
(Table 1).
3. Digestive enzymes: Cellulase Onozuka R-10 and Macerozyme
R-10 (Yakult Honsha Co. Ltd., Nishinomiya, Japan). Enzymes
are usually stored at 4 C and brought to room temperature,
before use.
4. Sodium ascorbate and bovine serum albumin (BSA) and 80%
(v/v) acetone.
Table 1
Media used for protoplast isolation, indicating the final concentrations of components
Pre-plasmolysis medium Sorbitol 0.3 M

CaCl2 1 mM
MES (pH 6.0) 10 mM
pH adjusted with 1 N KOH to 6.0
Digestion medium Sorbitol 0.4 M
CaCl2 1 mM
EDTA 0.25 mM
MES (pH 5.5) 10 mM
Components to be added just before use
Cellulase Onozuka R-10 2.0% (W/V)
Macerozyme R-10 0.2% (W/V)
BSA 0.25% (W/V)
Sodium ascorbate 10 mM
Washing medium Sorbitol 0.4 M
CaCl2 1 mM
MES (pH 6.0) 10 mM
Suspension medium Sorbitol 0.4 M
CaCl2 1 mM
MgCl2 0.5 mM
HEPES (pH 7.0) 10 mM
Reaction medium Sorbitol 0.4 M
CaCl2 1 mM
MgCl2 1 mM
HEPES (pH 7.5) 10 mM
2.4 Respiratory 1. Antimycin A (AA), salicylhydroxamic acid (SHAM), and car-

Measurements bonyl cyanide 3-chlorophenylhydrazone (CCCP) (from
Sigma, St. Louis, MO, USA); can be dissolved in ethanol.
Potassium cyanide (KCN) is soluble in water.
2. Clark-type O2 electrode (DW2, Hansatech Ltd., King’s Lynn,
UK, or equivalent).
3. Refrigerated circulatory water bath.
4. Black muslin cloth (thick one).
5. 35 mm Slide projector (with a halogen projection lamp, 24 V/
150 W, Philips, or Osram).
3 Methods
3.1 Plant Growth 1. Soak the seeds in water overnight, surface sterilize with 0.2%
(v/v) sodium hypochlorite solution for 30 min, and then wash
several times under running tap water. Germinate the seeds on
a moist blotting paper in the dark at 25 C (usually 3 days).
2. Transfer the germinating seedlings to plastic trays filled with
soil and farmyard manure (3:1). Water them daily in a green-
house or an environment-controlled chamber (average day/
night temperatures of 30 C/25 C and a natural photoperiod
of approximately 12 h).
3. Choose the second to fourth pair of fully unfolded leaves from
15- to 20-day-old plants for isolation of mesophyll protoplasts.
3.2 Isolation of 1. The protoplast preparation requires less than 90 min (as in our
Mesophyll Protoplasts hands and should be as much less as possible) [22, 27, 37]. A
typical protoplast preparation is shown in Fig. 1.
2. Gently peel off the abaxial (lower) epidermis of leaves with a
bent forceps and cut into small (ca. 1 cm2) pieces with a scalpel,
after discarding the midrib (see Note 2).
3. Float the leaf pieces on the pre-plasmolysis medium (Table 1)
with the peeled lower surface touching the medium in the petri
dish.
4. After 15 min, carefully remove the pre-plasmolysis medium
with a Pasteur pipette, leaving the leaf strips in petri dish. Add
Fig. 1 Photomicrograph of a typical mesophyll protoplast preparation from

leaves of pea (Pisum sativum). The protoplasts were suspended in the reaction
medium containing 0.4 M sorbitol (iso-osmoticum)
the digestion medium (Table 1) for enzymatic digestion (see

Note 3).
5. Use a tungsten lamp as a light source during digestion of leaves.
To avoid heat from the lamp, filter the light through water-
filled petri plate cover, leaving a small gap so as to avoid the
moisture.
6. The leaf pieces need to be digested for 25–30 min under a low
light of 100–150 μE/m2/s at 30 C [27, 37] (see Notes 4–6).
7. Gently remove the digestion medium with a Pasteur pipette
and add washing medium (Table 1) to the petri dish containing
the digested leaf pieces.
8. Swirl and tap gently the petri dish, so as to release the proto-
plasts into the washing medium, as evidenced by the green
color of the solution (see Note 7).
9. Filter the protoplast suspension through a 60 μm nylon filter
(pre-soaked in washing media) into a glass beaker along the
inner wall. Rinse again the petri dish with more washing media
and collect the suspension into the beaker (kept on ice) by
nylon filter (see Note 8).
10. Distribute the protoplast suspension into two or three 15 ml
tubes, depending on the volume collected. Centrifuge at
100 g for 5 min using a swinging bucket rotor in a tabletop
centrifuge (see Note 9).
11. Remove as much supernatant as possible and gently resuspend
the protoplast pellet, again in washing media. Wash the pellet
(by centrifugation at 100 g) again twice (for 4 and 3 min,
respectively) using the washing medium (Table 1). This step
would remove broken protoplasts while discarding the
supernatant.
12. Finally, resuspend the pelleted protoplasts once in the suspen-
sion medium (Table 1) and centrifuge at 100 g for 3 min (see
Note 10).
13. Suspend the final pellet in 0.5–1 ml of suspension medium,
depending on the yield of the pellet, mixed gently and left on
ice (see Note 11).
3.3 Purity/Intactness 1. The viability and intactness of protoplasts can be checked with
of Protoplasts neutral red and fluorescein diacetate (FDA) staining (10 μg/
ml). The purity of protoplast preparation should ideally be
above 80%.
3.4 Estimation of 1. Estimate the chlorophyll (Chl) in mesophyll protoplasts by

Chlorophyll extracting protoplasts into 80% (v/v) acetone [38].
2. Add an aliquot of 12.5 μl of protoplast suspension to 5 ml of
80% (v/v) acetone and mix well using a cyclo-mixer. Measure
the absorbance of acetone extract at 652 nm (A652—to deter-

mine chlorophyll) and 710 nm (A710—to correct for turbid-
ity), using a spectrophotometer.
3. Calculate the Chl concentration in the protoplast preparation
using the following formula:
Chl ðmg=ml of protoplast suspensionÞ ¼ ðA 652 A 710 Þ 11:11:
3.5 Monitoring 1. The rates of respiratory O2 uptake (in darkness for 5 min) of
Respiration: COX and the mesophyll protoplasts will be monitored polarographically
AOX Pathways using a Clark-type O2 electrode (DW2, Hansatech Ltd., King’s
Lynn, UK).
2. Calibrate the oxygen content in the electrode chamber at 25 C
with 1 ml of air-saturated water (assumed to contain 252
nmoles of oxygen/ml) and then using a pinch of sodium
dithionate, to get “zero” oxygen [12] (see Notes 12 and 13).
3. After calibration, thoroughly wash the electrode with distilled
water several times and then proceed to respiratory
measurements.
4. The mesophyll protoplasts equivalent to 10 μg Chl shall be
loaded to oxygraph chamber containing the reaction medium
(total volume 1 ml) (Table 1) and wait for oxygen consumption
rate to stabilize, approximately 2–3 min (see Note 14).
5. This reading will give the total respiration capacity of the pro-
toplasts in units of nmol O2/min/10 μg Chl. This can be
calculated to μmol O2/mg Chl/h.
6. The respiratory inhibitors Antimycin A (AA) or KCN (inhibi-
tor of COX pathway/complex III of mitochondria) and
SHAM (inhibitor of AOX pathway of mitochondria) should
be added to the reaction medium, at the required final concen-
trations, after the stable electrode signal during the experiment
(see Note 15).
7. The capacity of alternative pathway can be determined as the
cyanide (1 mM KCN or 1 mM Antimycin A) resistant respira-
tion that was sensitive to 5 mM SHAM [39].
8. The respiratory O2 uptake that was sensitive to either 1 mM
KCN or 1 mM Antimycin in the presence of both 1 μM CCCP
(an uncoupler) and 5 mM SHAM indicates the capacity of
COX pathway [40] (see Note 16).
9. The activities of both AOX and COX pathways can be deter-
mined by monitoring the respiratory O2 uptake in the presence
of SHAM and with/without AA (Fig. 2). The relative expres-
sion of these two pathways is calculated by using formulae [41]
as below:
Fig. 2 (a) A typical measurement of the sensitivity of respiration in pea mesophyll protoplasts to SHAM in the
absence and presence of 1 mM Antimycin A (AA). Respiratory rate in the absence of SHAM/Antimycin was
11 μmol/mg Chl/h, and 6.6 in the presence of Antimycin alone. (b) Plot of respiratory activity in the presence of
SHAM alone against that in the presence of both SHAM and AA. The slope of the line represents the
engagement of alternative pathway (ρ)
V SHAM V KCNþSHAM
% Cytochrome pathway activity : 100
VT
V T V SHAM
% Alternative pathway activity : 100
VT
% Residual respiration : 100 ð% cytochrome pathway activity
þ% alternative pathway activityÞ
10. The extent of engagement of alternative pathway (p) can be

determined by plotting the rate of respiration of mesophyll
protoplasts in the presence of SHAM alone (on Y-axis)
against those in the presence of both SHAM þ AA
(Table 2). The terms used are as follows:
Total respiration (VT): Rate of respiratory O2 uptake in the
absence of any inhibitor.
Capacity of COX pathway (VKCN): Rate of respiratory O2
uptake in the presence of both 1 μM CCCP and 5 mM
SHAM that were sensitive to 1 mM Antimycin (or KCN).
Capacity of AOX pathway (VSHAM): Rate of respiratory O2
uptake in the presence of 1 mM Antimycin A (or 1 mM
KCN) that was sensitive to SHAM (sequential addition of
Antimycin and SHAM).
Residual respiration (Vres): Rate of respiratory O2 uptake in
the presence of 1 μM CCCP þ 5 mM SHAM þ 1 mM
KCN.
Table 2
Relative proportion of the activities of the Cyt and alternative pathways of respiration in mesophyll
protoplasts of pea, calculated from the data of Fig. 2a (details in Subheading 3.5 of methods)
Activity/parameter Percentage (%)

Cyt pathway as % of total respiration 20.4
Alternative pathway as % of total respiration 66.6
Residual respiration 13.0
Engagement of AOX pathway (ρ) 1.28a
The fraction of the capacity of alternative pathway engagement (ρ) is determined as indicated in Fig. 2b
a
Represented as ratio of full capacity of AOX pathway
4 Notes
1. Any other suitable variety, such as “Azad” or “Bonneville” can

also be used.
2. The peeling of leaves should be done under water in a big petri
dish.
3. The enzyme solution should be prepared fresh and there
should be sufficient digestion media to cover the leaf strips.
About 10 ml of enzyme solution is sufficient for digesting 7–9
pairs of pea leaves.
4. The digestion time may depend on the type of leaf material and
the cultivar used.
5. Prolonged digestion time is stressful and might affect the
physiological responses of the protoplasts.
6. The color of the enzyme solution turning green indicates the
release of protoplasts.
7. Harvesting all the protoplasts from leaf pieces is not necessary.
8. The nylon filter will retain the leftover leaf material and debris.
9. Higher speed and longer duration of centrifugation may dam-
age the protoplasts.
10. The protoplasts should be kept on ice, between centrifugation
steps 10 and 12.
11. The protoplasts should be handled carefully throughout the
experiments.
12. Air-saturated water can be achieved by bubbling air through
water.
13. The O2 electrode should be cleaned periodically using alumi-
num oxide powder/paste.
14. The concentration of chlorophyll to be used for the study

should be selected after testing with several concentrations
and choose the optimum one to get a good response from
electrode.
15. The optimal concentrations of the inhibitors that are to be used
in the experiments need to be checked freshly with every batch
of chemical.
16. Since the adenylates determine the flux of electrons through
COX pathway, an uncoupler should be used.
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28. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis Padmasree K (2016) Alternative oxidase path-
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29. Confraria A, Baena-González E (2016) Using Front Plant Sci 7:68
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Chapter 23
Measuring Spatial and Temporal Oxygen Flux Near Plant

Tissues Using a Self-Referencing Optrode
Eric S. McLamore, D. Marshall Porterfield, and Yinglang Wan
Abstract
Self-referencing optrodic microsensing is a noninvasive method for measuring oxygen transport into/from
tissues. The sensing mechanism is based on fluorescence quenching by molecular oxygen at the tip of a
fiber-optic probe, and facilitates microscale spatial mapping and continuous monitoring at 100–350 mHz
sampling frequency. Over the last decade, this technique has been applied for plant tissues, including roots,
seeds, leaves, and flowers in both liquid and air. Here, we describe the operating principle of self-referencing
optrodic microsensing for the study of plant tissues with a specific focus on juvenile roots.
Key words Oxygen flux, Root physiology, Optrode, Self-referencing
1 Introduction
Real-time measurement of oxygen concentration, transport, and

respiration in living cells is crucial to solving fundamental physio-
logical questions related to development, metabolism, and stress
response. Dating back to the first oxygen sensor developed by
Leland Clark [1], there has been a significant ongoing effort to
measure oxygen with high temporal and spatial resolution in or
near living cells and tissues; see the seminal review by Wang and
Wolfbeis [2]. Early efforts were based on Clark’s polarographic
electrochemical technique, where a current is measured as a func-
tion of oxygen reduction versus a reference electrode and platinum
wire counter electrode [3]. Although electrochemical oxygen sen-
sors were transformative and paved the way for the field of modern
biosensing, the electrodes have limitations for direct oxygen sens-
ing. Namely, electrodes are susceptible to electromagnetic interfer-
ence, convective artifacts, and calibration drift, which create high
background noise and a requirement for frequent recalibration. It is
possible to partially alleviate convective artifacts by reducing the
diameter of an electrode and covering the electrode with a gas-
permeable membrane, but this in turn increases the susceptibility to
267
268 Eric S. McLamore et al.
electromagnetic noise. To overcome this, Kautsky and Hirsch [4]

developed the first optical sensors for measuring oxygen based on
adsorption of trypaflavin on silica gel beads. This pioneering
work led to the development of the first luminescent oxygen sensor
by Bergman [5] based on the chemical quenching of luminescence;
this premise was later used to develop the first fiber-optic O2
sensor [6].
Luminescent quenching is a non-emitting pathway for excited
valence electrons to return to ground state (Papkovsky and Dmi-
triev [7]; Wang and Wolfbeis [2]. Figure 1 shows the Jablonsky
diagram for quenching, which is a schematic representation that
illustrates the electronic states of a molecule and the transitions
between energy states. In the diagram, the energy of an incident
photon is transferred to the valence electron on a chromophore,
causing the electron to transition to a different eigenstate; from the
ground sate (So) to an excited state (S1). Each particular chromo-
phore has a finite difference in energy between the two eigenstates,
and therefore can only absorb specific wavelengths of incident light.
Once excited, valence electrons on chromophores undergo a small
loss of energy due to vibrational relaxation and internal conversion
(shown as curved arrow in Fig. 1), and the photon is then emitted
as the excited electron returns to ground state. The presence of
quenching chemicals inhibits photon emission due to overlap in
absorption and fluorescence spectrums (also shown as a curved
arrow in Fig. 1). Thus, quenching is a non-emitting mechanism
by which excited state electrons return to the ground state without
emission of a photon at a wavelength that corresponds to the
chromophore enantiostate, where long decay times correlate to
enhanced quenching efficiency; quenching is often accompanied
Vibration
S1
Fo τ o
= = 1 + K SV [Cq ]
Internal conversion
Excited F τ
State
where:
quenching F0 = Luminescent intensity in the presence of quencher [nrfu]
by O2 F= Luminescent intensity in the absence of quencher [nrfu]
Absorption
T = Luminescent lifetime in the presence of oxygen [sec]
T0 = Luminescent lifetime in the absence of oxygen [sec]
Ground State Ksv = Stern-Volmer constant [mM-1]
(So) [Cq ]=Quencher concentration [mM]
Fig. 1 Jablonsky diagram depicting energy state changes during fluorescence quenching. Energy states are
arranged vertically by energy, and grouped horizontally by spin multiplicity. Nonradiative transitions are
indicated by curved arrows, while radiative transitions are indicated by straight arrows. Vibrational ground
states (thick lines) and higher vibrational states (thin lines) are also shown. Quenching is a non-emitting
process which grounds excited valence electrons without emission of a photon at the expected wavelength
Biophysical Root Oxygen Flux 269
by the formation of singlet oxygen. The Stern–Volmer principle

describes relationship between quencher concentration and fluo-
rescence (Fig. 1). Dynamic quenching is a photophysical, rather
than a photochemical process; it is usually controlled by mutual
diffusion. Static quenching, on the other hand, involves formation
of a ground state complex between the fluorophore and quencher
with a nonfluorescent complex. Importantly, static quenching is a
temperature-dependent process.
Oxygen and aliphatic/aromatic amines are examples of efficient
quenchers. Oxygen has a unique triplet ground state that makes the
molecule an efficient quencher, and many O2 quenched dyes that
are available commercially. Sensors using metalloporphyrins as the
dye are among the most common, with molar absorbances up to
1.5 105 M1 cm1 at excitations in the Soret band
(390–420 nm). In this protocol we focus on use of the highly
photostable fluorine-substituted platinum porphyrin known as
PtTFPP (platinum tetrakis pentafluorophenyl porphyrin), which is
the most popular metalloporphyrin dye (Wang and Wolfbeis [2]).
Many techniques have been developed for measuring oxygen
near living tissues using PtTFPP; for details see reviews by Chatur-
vedi et al. [8], Papkovsky and Dmitriev [7], Wang and Wolfbeis [2].
Among these techniques, fiber-optic sensing (also known as optro-
dic sensing) offers the advantage of high spatial resolution (fiber
diameters as small as 5 μm), no need of exogenous reagents or
special lighting conditions, and ability to probe tissues without
making direct physical contact. One disadvantage of using micro-
sensors in the concentration domain is the relatively low signal
acquisition due to a small specific surface area of the fiber tip.
Work by Chatni et al. [9, 10] showed that this problem can be
overcome by including photocatalytic nanomaterials in the dye
membrane located on the fiber tip. Static quenching of PtTFPP
fluorescence by O2 is a simple microsensor technique and systems
can be constructed at low cost, but caution must be take to avoid
temperature artifacts.
For probing spatially and temporally variable regions near living
tissues, microfibers can be used in the self-referencing (SR) modal-
ity. SR is a microsensor technique that converts static microsensors
with an otherwise low signal-to-noise ratio into dynamic flux sen-
sors capable of filtering out signals not associated with biologically
active transport. The method uses a move-wait-measure technique
for detection of real time changes in differential analyte concentra-
tion (ΔC) over a fixed excursion distance (ΔX) and is based on
Fick’s first law of diffusion (Fig. 2). This is accomplished by simply
oscillating a single microsensor between two locations using
computer-controlled stepper motors. The SR technique uses
phase-sensitive detection to “self-correct” for signals produced by
ambient drift and noise (Fig. 2).
Soret LED
ΔC C1 − C2
J=D =
ΔX ΔX
PtTFPP O2
Tapered fiber optic
Dichroic
mirror
where:
Camera/Frame Grabber
J= Oxygen flux to/from specimen [mmol cm-2 sec-1],
ΔC= Oxygen concentration differential at surface [mM],
ΔX= Distance of microsensor oscillation [μm],
Zoom Microscope C1= Concentration of O2 at surface of specimen [mM], and
C2= Concentration of O2 at distance ΔX from surface of
3D Motion
specimen [mM].
Controller
P
Head Stage
Amp
Fig. 2 Schematic of SR optrodic sensing. (top) A Soret LED (XX nm) is used for excitation of the PtTFPP-TiO2
membrane in a polystyrene matrix on the tip of a 10 μm tapered optical fiber (inset shows oxygen quenching of
PtTFPP luminescence). (bottom) The micro-optrode is oscillated near the tissue surface using a 3D motion
controller, and differential concentration (ΔC) is recorded while oscillating the optrode over a fixed excursion
distance (ΔX); Fick’s first law of diffusion is used to calculate oxygen flux. Schematic of SR optrode system
courtesy of McLamore and Porterfield [28]
The SR technique has many advantage over microsensor arrays,

including higher signal-to-noise ratio, lower cost, and capability of
continuously monitoring specimens which are changing in morphol-
ogy during the experiment (i.e., elongating roots), among others. SR
was originally developed for electrochemical probing of ions [11–13]
and later oxygen [14]). Since these early discoveries, the technique
has since been extended to include biosensors for probing sugars
[15–19], hormones [20], and signaling molecules [21]. In 2006,
Porterfield et al. [22] developed the first SR micro-optrode, and the
Porterfield lab has since demonstrated this technique for monitoring
roots [23] and other medical and environmental applications [24,
25]. The methodology has since been adopted with the NMT system
and Xin et al. [26] demonstrated use of a SR micro-optrode for
screening seed viability. Here, we describe the operating system and
protocol for measuring O2 flux with a SR micro-optrode in plant
systems. There are many reviews describing the SR technique
[27–29], and commercial platforms include SIET (USA), MIFE™
(Australia), and NMT (China); Newman et al. [29] describe the
technical differences between these commercial systems.
2 Materials and Methods
2.1 Preparing Plant 1. Seeds of A. thaliana should be sterilized with 75% EtOH for
Tissues 30 s and 1.5% NaClO solution for 1 min, followed by rinsing
(five times) in sterilized H2O for 1 min. The seeds should be
placed on the surface of culture medium (1/2 Murashige and
Skoog medium with 1% sucrose and 0.4% phytagel) in trans-
parent petri dishes. The petri dishes are then incubated in
culture chambers for 4 days before the experiments (22 C,
12000lux, light/dark ¼ 16 h/8 h).
2. The MS salts mixture can be purchased from various compa-
nies, or prepared: major salt components are NH4NO3
(1.65 g/l), CaCl2·2H2O (0.44 g/l), MgSO4 (0.37 g/l),
KH2PO4 (0.17 g/l), KNO3 (1.9 g/l), and minor salt compo-
nents are H3BO3 (6.2 mg/l), CoCl2·6H2O (0.025 mg/l),
CuSO4·5H2O (0.025 mg/l), FeSO4·7H2O (27.8 mg/l),
MnSO4·4H2O (22.3 mg/l), KI (0.83 mg/l),
Na2MoO4·2H2O (0.25 mg/l), ZnSO4·7H2O (8.6 mg/l),
Na2EDTA·2H2O (37.2 mg/l).
3. Seeds of Z. mays or P. vulgaris are surface-sterilized as described
above, and then immersed into water for 2 h. After that, the
seeds are placed on moist filter paper and rolled into paper
cylinders. The cylinders are vertically inserted into water con-
tainers and covered by aluminum foil.
2.2 Micro Optrode 1. First, prepare the dye membrane: 96 mg of polystyrene beads
Fabrication should be vortex mixed with 1.15 g chloroform (Fisher Scien-
tific, Waltham, MA) for 30 min in a sealed glass vial. Titanium
dioxide (45 mg) and 5 mg of platinum tetrakis pentafluoro-
phenyl porphine (PtTFPP) (Frontier scientific, USA) should be
mixed and then vortex-mixed for 30 s. The solution is sealed
immediately to avoid evaporation of the chloroform.
2. Prepare the cladding material by mixing polystyrene (15% w/w)
in dichloromethane.
3. A standard waveguide connector (140 μm silica glass) should
be cut in half using a FBC-007 diamond blade fiber cleaver
(Corning, Inc. Corning, NY).
4. Cladding near the tip of the cleaved fiber should be stripped
and optical insulation under a dissecting microscope.
5. Cables should be placed in a horizontal laser puller (e.g., P-
2000 horizontal laser puller from Sutter instrument Co.,
Novato, CA) and tapered to produce a tip diameter of 5 μm
(see Fig. 3).
70
a) Chatni et al (2008) b)
60 WPI Optrode
Non shielded optrode
Measured phase angle Shielded optrode
50
40
30
20
10
0
0 20 40 60 80 100 120 10 μm
Oxygen concentration [%]
polystyrene cladding
c) PtTFPP membrane
tapered fiber
optic
Fig. 3 (a) Calibration of micro-optrode is linear between 0 and 21 kPa, but non linear above values of 21 kPa
(courtesy of McLamore et al. [23]). Generally, labs use a two point calibration curve in de-oxygenated media
and media at 21 kPa. Various cladding and shielding schemes described in McLamore et al. [23] do not alter
sensor calibration. (b) Photo of tapered fiber optic. (c) schematic of optrode assembly after tip is coated with
oxygen-sensitive membrane (PtTFPP + TiO2 + polystyrene)
6. The tip of the cladded fiber should be immersed in dichloro-

methane to expose the fiber (approximately 500 μm of fiber
should be exposed).
7. The fiber should then be dip coated in the PtTFPP membrane
with TiO2 microparticles dissolved in chloroform. The materi-
als for the membrane are: TiO2 microparticles (2 μm diameter),
PtTFPP, polystyrene (Sigma-Aldrich, Atlanta, GA), and
dichloromethane.
8. Next, exposed fibers should be re-cladded under a binocular
microscope using 15% (w/w) opaque polystyrene by carefully
immersing the fiber for 5 min (thickness will be approximately
100 μm).
9. Alternatively, commercial micro-optrodes may be obtained
from World Precision Instruments (Sarasota, FL; Cat. Number
501656). The fiber sensor is 140 μm long tapering to a sharp
sensor tip with a diameter of 50 μm housed inside a steel needle
Alternatively, the company Ocean Optics Sensors (USA) also
provides optical microsensors.
2.3 Fabrication of 1. Light-emitting diode (LED): A Soret LED (403–405 nm) is

Optical System used for excitation of the fluorescence dye on the tip of the
tapered fiber optic.
2. LED power: An amplifier can provide stable voltage signals to
the LED light source such as the SRS 530 (SRS, USA).
3. Laser coupler: A band-pass optical filter (Edmund Optics, USA)
prevents nonspecific light from reaching the fluorescent dye on
the optrode tip. The emitted fluorescent light from the optrode
is in the red color range, and thus a red color filter (Edmund
Optics, USA) was used in the light path to obtain pure fluores-
cence signals, designed and installed by Science Wares, Inc.
(USA).
4. Photomultiplier tube (PMT): The PMT transduces weak light
signals into measureable electric signals via the photoelectric
effect and electron secondary emission. A current-type photo-
multiplier consisting of a photoemissive cathode (sensitive
photon sensitivity) followed by an electron multiplier (in high
vacuum) and an electron collector (anode); for example Hama-
matsu (Japan) and Edmund Optics (USA) supply PMTs with
high signal to noise ratio.
5. PMT power: A high voltage power supply is integrated into the
system to for the PMT using products such as the optical signal
processor by YoungerUSA (YGOO-OSP).
6. Imaging device: Any kind of microscope is suitable, but
inverted microscopes are preferred. We used the Olympus
IMT2 microscope in our studies. An objective lens with 10
or 20 magnification is good for observation.
7. Temperature sensor: To account for thermodynamic effects, an
integrated thermocouple was used to provide input for the
digital signal processor (DSP)-based lifetime fluorometer (Pre-
sens, Regansburg, Germany).
2.4 Optrode 1. Prepare the calibration solution: 1⁄2 MS medium bubbled with
Calibration pure N2 (0% O2) or air (21% O2) in an Erlenmeyer flask, or
other kind of container with a narrow neck.
2. Probes should be calibrated in 1/4 MS medium (or growth
media of choice), and modulated emission amplitude and
phase angle shift should be recorded as described in Chatni
et al. [9, 10]. To calibrate, immerse the probe and temperature
sensor into calibration solution and wait for approximately 60 s
for the signal to stabilize; record phase angle and intensity with
constant frequency domain excitation. Rinse the probe with DI
and gently wick the excess solution from the tip with a Chem-
wipe (do not directly touch the tip as this could damage the
sensor membrane). Repeat in at least three calibration solutions
to obtain a calibration curve; alternatively, some researchers use
only de-oxygenated DI water and saturated (21 kPa) DI water

to construct a 2-point calibration curve, assuming linear behav-
ior between these two oxygen partial pressures.
2.5 Self-Referencing A custom or commercial SR system including camera/zoomscope,

System 3D motion controllers, vibration table, and data acquisition system
includes the following basic parameters (commercial systems vary
by region and are summarized).
1. Vibration isolation table (e.g., High Performance Newport
Research Series Plus Optical Table 50 80 1200 with sealed
1/400 20 threaded holes).
2. Commercial SR optrode system.
(a) In the USA, the commercial system is known as the Non-
invasive Scanning Microelectrode System (Applicable
Electronics, Inc., New Haven, CT). The system includes
a DC-coupled differential amplifier, low/high pass filters,
and video/data acquisition system. The preamplifier sig-
nal is capacitively coupled through a lock-in amplifier.
Temperature effects are corrected through the use of an
integrated thermocouple with a digital signal processor
(DSP)-based lifetime fluorometer (Presens, Regansburg,
Germany). Measured phase angle is transduced to an
analog signal via a digital signal processor (World Preci-
sion Instruments, Sarasota, FL) [28, 30, 31].
(b) In China, the commercial system is known as Noninvasive
Micro-test System (NMT) (BIO-IM, YoungerUSA, USA)
and is constructed using a three-dimensional stepper
motor (YGOO-LTS, YoungerUSA, USA) and a lock-in
amplifier (SR530, SRS, USA) [32, 33].
2.6 Software 1. The original software for the technique was developed by
Science Wares, Falmouth, MA) and is known as Automated
Scanning Electrode Technique (ASET). The software is used
for data acquisition (A/D) and control functions (D/A). An
A/D board with DC-coupled differential amplifier, low/high
pass filters, and video/data acquisition system can be obtained
through Applicable Electronics, Inc.
2. In China, the commercial software is known as imFLUX
(YoungerUSA). The software, controls several basic compo-
nents of this system by adjusting the power supply, imaging
with a digital camera, and managing the main component.
3. The following describes the construction of the measuring
system: A LED power supplier controls the voltage signal to a
blue LED (503–505 nm). A 20 microscope (Newport, USA)
objective focuses light from the LED onto the optical fiber,
which is coupled to a blue filter leading the excitation light to
the MicroTip—Fiber Optic Oxygen Sensor (World Precision

Instruments, USA). Fluorescence emission is conducted by the
fibers and split by the fiber coupler again. When the red fluo-
rescence signal reaches the PMT (Hamamatsu, Japan), the
signals are transformed into electrical signals and conducted
into a Lock-in-Amplifier (SRS 530). Then, the phase shift and
fluorescence intensities are recorded by the computer and ana-
lyzed by imFLUX (YoungerUSA, USA).
3 Measuring Root Biophysical Oxygen Flux
3.1 Positioning Micro 1. First, stepper motors should be positioned near the center of
Optrode Near Plant lead screw to avoid stripping of the leads during measurement.
Tissue and 2. The sample should be brought into focal field, and the probe
Background (Control) manually positioned approximately 2 mm from surface of the
Measurement tissue.
3. The stepper motors should then be used to position the
optrode as close the surface of the tissue as possible, without
contacting the sample. If necessary, magnification of the zoom-
scope should be increased to ensure the sensor is within 1–5 μm
of the tissue surface (Fig. 4a).
4. The optrode should be positioned at least 1 mm away from the
tissue surface using the computer-controlled stepper motors,
and a background (control) measurement taken for at least
1 min. Recommended oscillation parameters are ΔX ¼ 20 μm,
wait time at each position ¼ 0.5 s.
5. The sensor should be placed near the surface of the root
(approximately 200–500 μm from the root tip), and a measure-
ment taken using the same oscillation parameters in step 3,
above.
6. Note: temp control must be placed in same condition as sample.
3.2 Protocol for 1. First, a background measurement is taken approximately 1 mm

Spatial Profiling of from the root surface.
Plant Tissues 2. The sensor is positioned near the root tip, and the X, Y, Z
coordinates of the position are recorded. In software systems
such as ASET, this position can be used to “zero” the stepper
motors so that the tip is the spatial reference point.
3. Oxygen flux should be recorded for 2–5 min at the tip.
4. Next, the sensor should be positioned approximately 5 μm
from the root tip. Oxygen flux should be recorded for
2–5 min at this position.
a)
b) 30 c) 100
background
Oxygen flux [pmol cm sec ]

at DEZ
-1
Oxygen concentration [μM]
25
80
-2
20
60
15
at DEZ 40
10
5 20 background
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time [min] Time [min]
Fig. 4 (a) Photograph of micro-optrode near the surface of the DEZ of an A. thaliana root (inset shows the
working principle of oscillating the micro-optrode between two positions). (b) Example of oxygen concentra-
tion measured at background position (1 mm from root surface) and at the surface of a 4-day-old Z. mays root
near the DEZ in ¼ MS media at 20C. (c) Oxygen flux for data shown in panel b. Panels b and c are courtesy of
McLamore et al. [23]
5. This process should be repeated to create a spatial map of the

tissue surface. We recommend distances of 5, 10, 15, 20, 30,
40, 60, 80, 100, 150, 200, 300, 400, 500, 750, and 1000 μm
from the root tip (Fig. 5).

Temporal Profiling of from the root surface.
Plant Tissues 2. The sensor is positioned near a location of interest (e.g., the
DEZ determined in Subheading 3.4). Oxygen flux should be
recorded for at least 5 min.
3. A background measurement should again be taken after the
recording to ensure no signal drift artifacts (Fig. 6).
a) 300
Zea mays

-1
Bambusa vulgaris
250
-2
200
150
100
50
0
0 500 1000 1500 2000
Distance from tip [μm]
b) 120
Glycine max
-1
Phaeolus vulgaris
100
-2
80
60
40
20
0
0 500 1000 1500 2000
Distance from tip [μm]
Fig. 5 (a) Spatial profile of oxygen flux for 7-day-old monocots G. max and B. vulgaris roots. The location of the
DEZ is correlated with the highest oxygen uptake, which varies slightly for each of these monocots. The
characteristic “dip” in the oxygen uptake occurs near the tip for most monocots. (b) Oxygen flux for 7-day-old
dicots P. vulgaris and G. max. The location of the DEZ is correlated with the peak in oxygen uptake, and the
profile lacks the “dip” observed for monocots. Data for Z. mays, G. max, and P. vulgaris courtesy of McLamore
et al. [23]

Pharmacological from the root surface.
Experiments 2. The sensor is positioned near a location of interest (for exam-
ple, the root tip or DEZ). Oxygen flux should be recorded for
at least 5 min.
3. The oxygen flux should be continuously recorded during addi-
tion of the pharmacological agent/drug. The drug should be
carefully added to the dish at location at least 5 mm from the
tissue. A transfer pipette can be used to gently mix the drug
into solution (Fig. 7).
4. The experiment should be repeated while adding a blank con-
trol (e.g., ¼ MS media).
Fig. 6 Oxygen flux measured near the DEZ for 7-day-old G. max, P. vulgaris, and
Z. mays roots; a background measurement (1 mm from root surface) is also
shown for comparison; courtesy of McLamore et al. [23]
3.5 Data Analysis 1. Flux is calculated using Fick’s fist law of diffusion (Fig. 2). First,
concentration at each sensor position is calculated using the
calibration curve obtained in Subheading 3.2. Once concentra-
tion at each position is known, the excursion distance and the
diffusion coefficient (2.1 105 cm2 s1) can be used to direct
calculate the flux.
2. For filtering noise, smoothing algorithms such as a rolling
average (McLamore et al. [18, 30]) or differential drift artifact
[31] can be used.
a)
120
Glycine max

-1
100 Zea mays
-2
80
DEZ + 0.5 mM KCN
60
40
bg + 10 mM SHAM
20
0
0 10 20 30 40
Time [min]
b)
160
Glycine max
-1
140 Zea mays

120
-2
100
80
60
40
20
0
nd DE
Z N AM
gro
u KC SH
ck M
ba . 5m 0 mM
+0 +1
Fig. 7 (a) Effect of KCN and SHAM on oxygen flux on 7-day-old Z. mays and G.
max roots (recorded at the root DEZ). (b) Average oxygen flux for three replicate
roots following the protocol in panel a; courtesy of McLamore et al. [23]
3. Empirical models such as a simple harmonic oscillator can be

used to further analyze temporal patterns such as oscillatory
flux [19, 34].
3.6 Conclusions In conclusion, we describe here a real-time and noninvasive probe

system with highly sensitivity for detecting the oxygen flux rate in
plant systems, with a particular focus on roots. We describe detailed
experiments for characterizing temporal/spatial oxygen flux near
the root, and we also provide a protocol for conducting pharmaco-
logical studies. This simple method is a versatile tool for noninva-
sive measurement of oxygen transport.
4 Notes
1. It is critical that optrodes are calibrated before and after each

experiment and any calibration drift noted and corrected dur-
ing data analysis.
2. The main advantage of the technique is the ability to measure
directional oxygen flux in/out of a root with an extremely high
signal-to-noise ratio under a wide variety of conditions (e.g.,
lighting, temperature, salinity, pharmacology).
3. The main disadvantage is that the root must be submersed in
buffer, and experiments cannot be conducted in soils without
damaging the micro-optrode. Additionally, the technique is
temporally constrained and detaild spatial profiles of an intact
root can take multiple hours.
Acknowledgments
The authors thank the UF Opportunity Fund for supporting E.S.

McLamore, and the following for supporting Y. Wan: National
Basic Research Program of China (973 Program 2011CB809103,
2011CB944601), the CAS/SAFEA International Partnership Pro-
gram for Creative Research Teams (20090491019), the National
Natural Science Foundation of China (31000595, 30730009), the
Knowledge Innovation Program of the Chinese Academy of
Sciences (KJCX2-YW-L08, KSCX2-EW-J-1), and the China Post-
doctoral Science Foundation. We also thank Dr. Miguel Angel
Torres (University of North Carolina, USA.) for providing seeds
of atrbohD/F double mutant.
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INDEX
A Catabolism...................................................................1, 19
Catabolized................................................................17, 18
Abiotic .................................................184, 187, 235, 246 14
C-glucose ....................... 1, 3–5, 12–14, 18, 19, 22, 27
AcetylCoA .................................................................6, 8, 9 Chemical fixation .......................................................... 245
Adaptive growth..................................193, 219, 236, 246
Chemiluminescence ..................................................14, 98
Adenosine triphosphate (ATP)........................21, 25, 71, Chickpea ....................................................................77–84
78, 87, 122, 137, 139, 148, 155, 167, 177, 203, Chloroplasts....................................................64, 98, 116,
204, 253
122, 128, 130, 134, 136–138, 143, 255
Alternative oxidase (AOX)...............................64, 71, 88, Chromophore................................................................ 268
143, 184, 194, 219, 225, 246 Competitive plant growth ............................................ 184
Ammonium hydroxide.................................................... 21
Confocal ......................................... 78, 80–82, 89, 91, 93
Amplification ..................... 159, 161, 162, 245, 247, 250 Co-regulated expression ............................................... 246
Ampoules ...........................................185–187, 195, 197, Cotyledons .....................................................98, 185, 215
198, 237, 239, 243 14
C-radiotracer analysis................................................... 17
Amyloglucosidase......................................................21, 26
Analytes................................................48, 51–53, 55, 269 D
Angiosperms ............................... 144, 157, 225–227, 231
Anionic resin....................................................... 21, 24, 29 Daucus carota L. .................................................. 222, 245
Antimycin .......................... 208, 254–256, 258, 261, 262 Density gradient centrifugation .......................... 98, 116,
AOX gene variants ............................................... 236, 243 128, 130, 133, 134, 136
Arabidopsis thaliana ........................................2, 48, 111, De-paraffinization ......................................................... 249
116, 157, 230–232, 257, 271, 276 Desi ...............................................................58, 60, 77–79
Ascofuranone................................................................. 208 Dichloromethane ................................................. 271, 272
Automated Scanning Electrode Technique Disintegration............................................................22, 24
(ASET) ...................................................... 274, 275 DNA extraction............................................................. 251
B E
Binocular microscope ................................................... 272 Electron paramagnetic resonance (EPR) ....................... 98

Biodeterioration .............................................................. 32 Endosperm ................................................................34, 36
Bioenergetics ................................................................. 143 Enthalpy............................. 184, 186, 189, 194, 197, 199
Biosynthesis ..............................6, 8, 27, 78, 87, 110, 167
F
Biotic...........................................167, 184, 187, 235, 246
Bovine albumin serum (BSA)........................81, 91, 100, Fiber optic probe.................................268, 269, 273, 275
101, 117–122, 126, 128, 130, 132, 134, 136, Fluorescence quenching ............................................... 268
145–147, 170, 173, 174, 256–258 Fluorometer.......................................................... 273, 274
Bradford reagent ................................................ 78, 81, 91 Formic acid ................................................................21, 24
Fractionations...................................................24, 26, 29,
C 204–206, 208–215, 255
Calorespirometry........................ 183–187, 193–200, 246 Fructose 6-phosphate (F6P) ................................. 8, 9, 26
Carbohydrate oxidation....................................... 9, 13, 14 Functional markers.................................... 184, 193, 194,
Carbon dioxide production ................................... 12, 98, 225, 236, 246
99, 101, 106, 107, 109, 184, 186, 189, 194, 197,
G
199, 254
Carbonyl cyanide-4 ....................................................... 101 Gas-permeable membrane ............................................ 267
Caryopsis ............................................................ 32, 34, 35 Gene annotation ........................................................... 221
vol. 1670, DOI 10.1007/978-1-4939-7292-0, © Springer Science+Business Media LLC 2017
283
PLANT RESPIRATION AND INTERNAL OXYGEN
284 Index
Gene copy numbers ............................................. 236, 242 Molecular-biochemical.................................................. 219
Gene identification..............................148, 149, 157–164 Morphological alterations............................................... 78
Genome inaccessibility.................................................. 220 Murashige and Skoog ...............................................2, 271
Germination ................................. 41, 47, 53–55, 58–60,
65, 78, 80, 84, 90, 110, 167 N
Germplasm ............................................................. 47, 195 Nanodrop ............................................198, 241, 247, 250
Gluconeogenesis ............................................................. 28 Naphthaleneacetic acid ..................................................... 2
Glycolysis ................................................. 4, 8, 17, 18, 253
Gradient centrifugation .............................. 98, 116, 128, O
130, 133, 134, 136
Growth curves .....................................196, 236, 239, 243 Oligomycin ..........................................101, 106, 147, 256
Growth regulation .......................................235–243, 246 O2 microsensors ...................................... 63–68, 80, 101,
107–109, 111, 112, 269
H Optical glass sensors........................................................ 99
Optrodic microsensing ................................................. 270
Heterologous expression .............................................. 235 Orthologous genes .............................................. 225, 231
Hexokinase ......................................................... 21, 23, 25 Osmoticum........................................................... 111, 259
Hibiscus syriacus ..................................226–228, 230, 231 Overexpressors .............................................................. 215
High-throughput sequencing ...................................... 232 Oxaloacetate .............................................. 6, 8, 9, 17, 18,
Hologenomics ............................................................... 193 168, 172, 179
Homogeneity .............................................. 144, 180, 225 Oxidative phosphorylation ........................................... 139
Housekeeping................................................................ 235
2-Oxoglutarate dehydrogenase (2-OGDH) ..... 6, 8, 168,
HPLC ................................................................. 80, 82–84 169, 177
Hydroponics ................................. 40–43, 65, 88, 90, 129 Oxycaloric............................................................. 189, 199
Oxygen flux ................................................................... 267
I
Oxygen tension .......................................................97–112
Imbibition .......................................................... 53–55, 80
Isocitrate dehydrogenase ................................... 6, 8, 121, P
122, 169, 171, 175–177 Paralogous genes........................................................... 223
Isothermal.......................... 185, 188, 197, 236, 239, 243 Pentose phosphate pathway .....................................2, 8, 9
Percoll gradient ...................................78, 79, 81, 84, 88,
K
90–92, 94, 104, 116, 117, 128, 130, 144, 146,
Kabuli............................................................58, 60, 77–79 151–153, 174
Perennial bud .................................................................. 97
L Perfluorodecaline ............................................................ 34
Phenotyping ...............................184, 193, 194, 236, 239
Laser microdissection (LM) ................................ 245–251
Lignins ............................................................................. 98 Phosphoglucomutase ................................................21, 26
Liquid scintillation .................3–5, 13, 19, 21, 22, 24–26 3-Phosphoglycerate.............................................. 9, 17, 18
Phosphorenolpyruvate .................................................... 17
M Photocatalytic nanomaterials........................................ 269
Phylogenetic .................................................226, 230–233
Macromolecules ........................................................17, 22 Phytotoxicity ................................................................... 55
Magnetic field................................................................ 207 Plant plasticity ...................................................... 235, 246
Manometer .................................................................... 209 Platinum porphyrin....................................................... 269
Mass spectrometer ..............................205–208, 210, 211 Polarographic electrochemical...................................... 267
Metabolic flux.......................................... 1–14, 17, 26–28 Polymerase chain reaction (PCR) ..................... 144, 149,
Metalloporphyrins......................................................... 269 157–162, 199, 246, 247, 250, 251
Methacarn-fixed tissue .................................................. 246 Polymorphisms............................................ 220, 223, 246
Microarrays ..........................................144, 158, 163, 164 Polysaccharides ....................................................... 98, 159
MicroTip-Fiber Optic Oxygen Sensor......................... 275 Polystyrene ........................................................... 270–272
Misconceptions ............................................................. 225 Polyvinylpyrrolidone (PVP) .................................. 78, 88,
Mitochondrial respiration ................................72, 83, 97, 98, 100, 102, 103, 117–121, 123–125, 128, 134,
139, 140, 194, 208, 236, 253–264 136, 145, 170, 172–174
MitoTracker Red .......................................................79, 81 Positionally labeled .......................................... 1–7, 12–14
PLANT RESPIRATION AND INTERNAL OXYGEN
Index 285
Potassium cyanide ................................................ 206, 258 Stimuli............................................................................ 246
Potassium phosphate ............................21, 148, 172, 178 Streptomyces griseus .......................................................... 21
Predicting plant robustness .......................................... 183 Subtypes...............................................227, 228, 231, 232
Pronase ......................................................................21, 26
Proteinogenic .................................................................. 17 T
Pycnometer.................................................................... 101 TaqMan polymerase ...................................................... 247
Pyruvate .................................................2, 6, 8, 9, 17, 19, Thermocycler ....................................................... 162, 247
101, 115, 122, 137, 139, 147, 156, 168–170, Thermomixer...................................................... 20, 24, 26
174, 175, 179, 180, 215
Thin-layer chromatography............................................ 18
Thornton rule ...................................................... 189, 199
Q
Thylakoids ................................................... 98, 103, 116,
Quiescent....................................................................... 184 128, 130, 134, 136
Transcriptomic .............................................163, 220–222
R Tricarboxylic acid (TCA) cycle .............................. 2, 4, 6,
Radioactivity ............................... 1, 4, 6, 7, 18, 19, 22–29 8, 9, 17, 18, 87, 167–180, 253
Radiorespirometry .........................................1, 2, 8, 9, 14 Trypaflavin ..................................................................... 268
Radiotracer ................................................................17–29 Trypanosomal alternative oxidase ................................ 208
Respiration.................................... 14, 22, 31–34, 39–45,
U
47–55, 57–64, 66, 78, 82, 83, 88, 97, 138, 139,
155, 156, 183–190, 194, 203–205, 208–214, Ubiquinone ...........................................88, 204, 208, 215
246, 253, 262, 263, 267
Respiratory quotient ............................... 48, 99, 109, 110 V
Rhizobacteria................................................................... 77
Vacuum concentrator................................................20, 23
Vibrational states ........................................................... 268
S
Salicylhydroxamic acid ......................................... 206, 258 Y
Schizosaccharomyces pombe............................................. 236 Yeast culture medium ................................................... 237
Secondary growth ......................................................... 184 Yield stability ............................................... 184, 187, 194
SensorTrace Profiling software..................................... 112
Sequence Read Archive (SRA) ...........163, 164, 220, 221 Z
Spatiotemporal ................................................................ 32
Spectrophotometer ........................................65, 74, 121, Zeiss imaging fluorescence microscope ...................82, 93
159, 170, 173, 195, 237, 247, 250, 257, 261 Zoomscope ........................................................... 274, 275
Spirodela polyrhiza................................................ 230, 233 Zoysia japonica.....................................226–228, 230, 231

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Methods in

Molecular Biology 1670

Kapuganti Jagadis Gupta Editor

For further volumes:

Methods and Protocols

Kapuganti Jagadis Gupta

ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media LLC 2017

Printed on acid-free paper

This Humana Press imprint is published by Springer Nature

Respiration is a very important biochemical process. Aerobic respiration in plants, as in all

analysis of respiratory activity is likely to become an important component of many

New Delhi, Delhi, India Kapuganti Jagadis Gupta

1 Assessing Metabolic Flux in Plants with Radiorespirometry . . . . . . . . . . . . . . . . . . 1

13 Procedures of Mitochondria Purification and Gene Expression

WAGNER L. ARAÚJO  Departamento de Biologia Vegetal, Universidade Federal de Viçosa,

NICOLAS L. TAYLOR  The University of Western Australia, Crawley, WA, Australia

Assessing Metabolic Flux in Plants with Radiorespirometry

Radiorespirometry describes the determination of respiratory activ-

the conversion to pyruvate and entry into the tricarboxylic acid

1. Cell suspension culture: Cell line of Arabidopsis thaliana (L.)

naphthaleneacetic acid (50 μl from stock solution: 10 mg/ml in

The method describes the preferred approach involving analysis of

3.1 Determination 1. Transfer 3.9 ml MS medium containing 166 mM glucose to

KOH solution used in the experimental incubations with 2 ml

3.2 Analysis of 14CO2 1. Determine the background level of scintillation by calculating

Fig. 2 Time course of 14CO2 release by metabolism of positionally labeled

1, 2, and 3 of the three-carbon phosphate esters in the second half

phosphate in the oxidative pentose phosphate pathway, mean-

Fig. 4 Simplified scheme of the sources of carbon dioxide during carbohydrate

position. The other process contributing to randomization

Specifically, increases in the values of particular ratios are likely to be

1. This approach complements the analysis of the fate of [U-14C]

positionally labeled [14C]substrates with the fractionation

make up double-strength MS medium and a 6% glucose solu-

at an initial concentration of 166 mM) was combined with

SKM acknowledges financial support from the University of

Coupling Radiotracer Experiments with Chemical

Key words Respiration, 14C-radiotracer analysis, Fractionation, Metabolic flux

The primary function of cellular respiratory metabolism is energy

specific metabolites or metabolic pathways [4]. Nevertheless, the

AP cationic/basic fraction Soluble cationic/basic Insoluble cationic/basic

AP neutral fraction (sugars, Soluble neutral fraction Insoluble neutral fraction

Hexokinase treatment GOX treatment

HK anionic/acidic fraction GOX anionic/acidic

HK neutral fraction GOX neutral fraction

Fig. 2 Flow chart showing the chemical fractionation procedure

included in the ethanol insoluble pellet. The radioactivity in each

2.2 Ethanol 1. Ball mill (e.g., Mixer Mill MM 400, Retsch).

2.3 Chemical 1. Liquid scintillation counter (e.g., LS6500, Beckman Coulter).

2.4 Chemical 1. Amyloglucosidase mix: 5 U mL 1 amyloglucosidase (EC

2.5 Determination of 1. Glucose-6-phosphate standard solutions: 0, 0.4, 1.0, 2.0, 4.0,

from yeast, Roche), 1.8 mM thiazolyl blue tetrazolium bro-

5. Store NaOH solution in the tubes as CO2 fraction.

shaking at 1400 rpm on a thermomixer at room temperature

10. Treat a part of soluble neutral fraction with hexokinase.

3.4 Chemical Ethanol insoluble precipitate is mainly composed of starch, protein,

3.5 Determination of Absolute amount of hexose phosphate pool is spectrophotometri-

3. Add 20 μL of 0.5 M KOH, seal the plate with microplate sealer,

3.6 Calculation of The redistribution of radioactivity from fed 14C-glucose is calcu-

4. The label in hexose phosphate pool is calculated by subtracting

1. Radiolabeled glucose can be purchased from providers such as

gain appropriate concentration and specific activity (1.4 MBq

WAGNER L. ARAÚJO Departamento de Biologia Vegetal, Universidade Federal de Viçosa,

NICOLAS L. TAYLOR The University of Western Australia, Crawley, WA, Australia