Molecular Plant • Volume 3 • Number 1 • Pages 2–20 • January 2010 REVIEW ARTICLE

Phenylpropanoid Biosynthesis
Thomas Vogt1
Leibniz-Institute of Plant Biochemistry, Department of Secondary Metabolism, Weinberg 3, D-06120 Halle (Saale), Germany

ABSTRACT The general phenylpropanoid metabolism generates an enormous array of secondary metabolites based on
the few intermediates of the shikimate pathway as the core unit. The resulting hydroxycinnamic acids and esters are am-
plified in several cascades by a combination of reductases, oxygenases, and transferases to result in an organ and devel-
opmentally specific pattern of metabolites, characteristic for each plant species. During the last decade, methodology
driven targeted and non-targeted approaches in several plant species have enabled the identification of the participating
enzymes of this complex biosynthetic machinery, and revealed numerous genes, enzymes, and metabolites essential for
regulation and compartmentation. Considerable success in structural and computational biology, combined with the an-
alytical sensitivity to detect even trace compounds and smallest changes in the metabolite, transcript, or enzyme pattern,
has facilitated progress towards a comprehensive view of the plant response to its biotic and abiotic environment. Trans-
genic approaches have been used to reveal insights into an apparently redundant gene and enzyme pattern required for
functional integrity and plasticity of the various phenylpropanoid biosynthetic pathways. Nevertheless, the function and
impact of all members of a gene family remain to be completely established. This review aims to give an update on the various
facets of the general phenylpropanoid pathway, which is not only restricted to common lignin or flavonoid biosynthesis, but
feeds into a variety of other aromatic metabolites like coumarins, phenolic volatiles, or hydrolyzable tannins.

Key words: Phenylpropanoid; biosynthetic pathway; flavonoid; anthocyanin; tannin; coumarin; volatiles; lignin.

INTRODUCTION tases, and various superfamilies of transferases (Figure 1).

Phenylpropanoids contribute to all aspects of plant responses
towards biotic and abiotic stimuli. They are not only indicators
of plant stress responses upon variation of light or mineral
treatment, but are also key mediators of the plants resistance
towards pests (La Camera et al., 2004). They promote invasion
of new habitats (Bais et al., 2003) and provide the biochemical
resources for successful reproduction (Dudareva et al., 2004).
Phenylpropanoid-based polymers, like lignin, suberin, or con-
densed tannins, contribute substantially to the stability and
robustness of gymnosperms and angiosperms towards me-
chanical or environmental damage, like drought or wounding.
The magnificent diversity of phenylpropanoids, in principle,
is the result of efficient modification and amplification of
a very limited set of core structures, derived from the shikimate
pathway (Herrmann, 1995). Aromatic amino acid lyases link
the pool of phenylalanine and tyrosine to biosynthetic path-
ways sometimes downgraded as secondary metabolism,
which, however, turn out to be equivalently relevant to plant
survival as photosynthesis or the citrate cycle. A clear differen-
tiation between secondary and primary metabolites may seem
Some of these enzymes might exhibit overlapping specificities
in vitro, but their developmentally and spatially controlled ex-
pression specifically contributes to tissue and plant-specific
chemical phenotypes.
In the last decade, knockout mutations and RNAi-mediated
suppression in Arabidopsis and other model plants have facil-
itated the identification and functional characterization of
genes and enzymes (Alonso et al., 2003; Ossowski et al.,
2008). A plethora of mutants is available for candidate genes
central to the various branches of the phenylpropanoid path-
way. Identification of single loss-of-function mutants may not
be immediately accessible based on a clear phenotype such as
impaired growth or fertility (Briggs et al., 2006). In the absence
of any phenotype, high throughput screening of phenylpropa-
noid patterns by GC– or UPLC–MS may enable the correlation
of metabolite pattern with transcript profiles in an organ or
1
To whom correspondence should be addressed. E-mail tvogt@ipb-halle.de,
fax +49 345 5582 1509, tel. +49 345 5582 1530.

obsolute (Hartmann, 2007). The diversity and plasticity of the
resulting natural products, more specifically the phenylpropa-
noid profile, are then achieved by a set of enzymes organized
usually in superfamilies, like oxygenases, ligases, oxidoreduc-
ª The Author 2009. Published by the Molecular Plant Shanghai Editorial
Office in association with Oxford University Press on behalf of CSPP and
IPPE, SIBS, CAS.
doi: 10.1093/mp/ssp106, Advance Access publication 24 December 2009
Received 30 September 2009; accepted 19 November 2009

Figure 1. Schematic View of the Diversity of the Secondary Metab-
olite Formation.
tissue specificity manner (Matsuda et al., 2008). In some cases,
this approach may reveal the impact of a single phenylpropa-
noid pathway gene on other metabolic pathways (Rohde
et al., 2004). Metabolite profiling can be combined with immu-
nofluorescence or imaging techniques based on intrinsically
fluorescent proteins, like GFP, to follow also the expression
and interaction of participating enzymes down to the cellular
level (Dixit et al., 2006).
In Arabidopsis and poplar, nearly all genes required for phe-
nylpropanoid and lignin formation are encoded by small gene
families (Hamberger et al., 2007). Clustering of these sequen-
ces in cladograms suggests a variety of functions other than
the bona fide suggested roles of selected members of a gene
family based on sequence information. If genes of a single
pathway are compared, whole genome data indicate that in
poplar, bean, or alfalfa, the pattern of genes involved in fla-
vonoid hydroxylation appears more complex as compared to
Arabidopsis (Hamberger et al., 2007).
In recent years, various excellent reviews summarized the
current knowledge on structural genes involved in phenylpro-
panoid, specifically lignin and flavonoid formation, regulatory
transcription factors, hormonal control of the whole pathways
by jasmonate or auxin and evolution of pathway genes from
primary metabolism (Weisshaar and Jenkins, 1998; Pichersky
and Gang, 2005; D’Auria and Gershenzon, 2005; Ober, 2005;
Boudet, 2007). This report tries to emphasize that phenylpro-
panoid derived metabolites comprise and contribute to multi-
ple biosynthetic branches other than lignin and flavonoid
biosynthesis only (Figure 2). Some decisive steps in important
pathways were resolved only recently, such as 2#-hydroxylation
in coumarin biosynthesis (Kai et al., 2008). In a few cases, new
organ-specific pathways could be established in the last couple
of years based on functional transcriptomics (Alves-Ferreira
et al., 2007; Ehlting et al., 2008) including the formation of ta-
petum-specific trisacyl-polyamine conjugates of Arabidopsis
(Matsuno et al., 2009; Fellenberg et al., 2009). Other pathways,
Vogt d Phenylpropanoid Biosynthesis | 3
like sinapate ester formation in Arabidopsis, have been clari-
fied in detail in the last decade and may have direct practical
impacts on current breeding programs, resulting in consumer
or farmer benefits (Bhinu et al., 2009). Several new ideas and
insights into the complex organization of pathways are due to
the advancement of analytical, chemical, and biomolecular
tools, like whole genome tilling arrays and application of fluo-
rescent biosensors (Herbst et al., 2009). These techniques will
be a critical driving force towards understanding plant biology
(Yonekura-Sakakibara et al., 2008; Zeller et al., 2009). They
promote rapid annotation of genes, the structural and func-
tional characterization of enzymes, realize quantification of
even trace amounts of metabolites, and enable identifica-
tion of signal transduction pathways in living cells towards
a comprehensive view of the plant organism.
THE CENTRAL PHENYLPROPANOID
PATHWAY
The plant shikimate pathway is the entry to the biosynthesis of
phenylpropanoids. Its intracellular, plastidial location and
complex regulation have been investigated for more than a de-
cade (Schmid and Amrhein, 1995). Regulation of the entire
pathway in plants can be performed at multiple levels.
Transcriptional control was shown for 3-deoxy-D-arabinose-
heptulosonate (DHAP) synthase (Herrmann and Weaver,
1999), whereas arogenate and prephenate dehydratase are
inhibited by phenylalanine, one end-product of the pathway
(Yamada et al., 2008). The plastidal location of two elusive
steps of the shikimate pathway, arogenate dehydratase and
dehydrogenase, leading to phenylalanine and tyrosine biosyn-
thesis has recently been proven (Rippert et al., 2009). No recent
molecular evidence is available for the location of prephenate
aminotransferase, although its activity within a plastidal frac-
tion of Sorghum bicolor was shown (Siehl et al., 1986). There-
fore, a location of the complete pathway within plastids
appears plausible. It is noteworthy that genes of the shikimate
pathway in plants apparently do not originate from a single
prokaryotic ancestor of cyanobacterial origin, but are likely
derived from at least three different sources. A complex,
multi-step shuffling of loss and gain of function occurred dur-
ing phylogeny of this pathway, which might explain the mul-
tiple factors contributing to the genomic organization and
expression of the pathway genes in Arabidopsis and other
plants (Richards et al., 2006). With respect to its important con-
tribution to the carbon flow within the plant, the response of
individual genes of the shikimate pathway to variations in
light or nutrient content appears tightly regulated and more
complex than the transcriptional responses of the entry genes
of phenylpropanoid and flavonid biosynthesis, encoding phe-
nylalanine ammonia lyase (PAL) or chalcone synthase (CHS)
(Lillo et al., 2008).
PAL and tyrosine ammonia lyase (TAL) catalyze the non-
oxidative deamination of phenylalanine to trans-cinnamate
and direct the carbon flow from the shikimate pathway to
4 | Vogt d Phenylpropanoid Biosynthesis

CHO
H OH COOH
H OH + H2C
CH2O P O P

Erythose-4-Phosphat PEP

Gallotannins COOH COOH

+
Ellagitannins
HO OH O OH
OH OH

Gallic acid Dihydroshikimate

HO O O COOH COOH

COOH
CH3O

Coumarins HO OH O CH2
OH
Isochorismate
Shikimate

OH
HO O
HO O
COOH
COOH OH

OH O
OH OH O NH3+

Isoflavonoids Flavonoids Phenylalanine Salicylic acid

PAL

S CoA
OH OH COOH

O O
HO
OH
HO Cinnamate Benzoic acid
p-Coumaroyl CoA
OH
Stilbenes

OH

O glucose
HO O OH CH3O
O
OH

O CH3O HO
OH HO O
CH3O
Aurones
OH HO Phenylpropanoid esters
OH
Catechin Lignans Phenylpropenes
+
Cutin Acylated Polyamines
Lignin
Suberin
Sporopollein

Proanthocyanidins

Figure 2. Diversification of Phenylpropanoids Based on the General Phenylpropanoid Pathway.

The metabolites of the shikimate pathway and the central metabolite, 4-coumaroyl CoA, are shaded in gray.

the various branches of the general phenypropanoid metab-
olism. A crystal structure became available for a codon-opti-
mized, heterologously expressed enzyme from Petroselinum
crispum (Ritter and Schulz, 2004). The structural data are con-
sistent with the unusual deamination reaction, which is inde-
pendent of any co-factor. The required electrophilic prosthetic
group, 4-methylidene-imidazole-5-one, is auto-catalytically
formed. These data also provide evidence for the requirements
of antioxidative SH-protective reagent, like dithiothreitol
(DTT), for full enzyme activity in vitro, since an oxidized
DTT-derivative was detected in the electron density map of
the crystal, when DTT was present into the crystallization
buffer. Replacing the characteristic amino acid His89 of the ac-
tive site of TAL in bacteria by phenylalanine, a characteristic
feature of PALs, converts TAL to PAL and vice versa (Watts
et al., 2006; Louie et al., 2006). This is one rare example that
a single amino acid exchange leads to a drastic change in sub-
strate specificity. From an evolutionary perspective, plant and
microbial PALs and TALs are part of a superfamily of enzymes
from plants, funghi, and bacteria, and are likely derived from
a precursor of the widespread histidine ammonia lyase (HAL)
family in the histidine degradation pathway (Röther et al.,
2002). Both can be distinguished from HALs by an additional
N-terminal domain and by a more effective shielding of their
active sites from molecular oxygen.
Several copies of the PAL-genes are found in all plant spe-
cies, comprising four genes in Arabidopsis, to five in poplar
and nine in rice (Hamberger et al., 2007). The individual genes
may respond differentially to biotic and abiotic stressors and
their expression is developmentally and spatially controlled
(Bhuiyan et al., 2009; Lillo et al., 2008). In poplar, an organ-
specific expression of three of the PAL-genes has been sugges-
ted—all display a different function (Kao et al., 2002). Two
genes are expressed in lignifying tissues; only one is involved
in lignin formation. The second PAL-gene is specifically tar-
geted to condensed tannin formation. The third gene is asso-
ciated with flowering, although its functional role remains to
be established (Hamberger et al., 2007). In contrast to Arabi-
dopsis and poplar, in the Solanaceae, the entry step of the phe-
nylpropanoid pathway is represented by a remarkable set of
an estimated 20 putative PAL-genes, as shown for Lycopersicon
esculentum (Chang et al., 2008). However, only a single gene
appears to be strongly expressed in all tissues, whereas the
remaining genes appear effectively silenced. This underutiliza-
tion is peculiar and the precise genetic mechanisms of this
extensive gene silencing remain to be established.
The initial three steps of the pathway catalyzed by PAL, cin-
namate 4-hydroxylase, and 4-coumaroyl CoA-Ligase are man-
datory and provide the basis for all subsequent branches and
resulting metabolites. Subsequent steps downstream of PAL,
leading to phenylpropanoid monomers (with exception of cin-
namate 4-hydroxylase), are encoded by small gene families in
all species investigated so far (Hamberger et al., 2007). With
respect to its impact on the phenylpropanoid metabolism, it
seems surprising that among nearly 300 functional genes
Vogt d Phenylpropanoid Biosynthesis | 5
encoding cytochrome P450s in Arabidopsis, 4-hydroxylation
of trans-cinnamate to 4-coumarate is encoded by a single gene
only, At2g30490, encoding CYP73A5. As expected, mutations
in At2g30490 severely affect growth and development of the
plants and result in the accumulation of the quite unusual cin-
namoylmalate in leaves (Schilmiller et al., 2009).
The subsequent step, encoded by the small gene family of
4-CL ligases, channels the resulting aromatic CoA-esters to dif-
ferent biosynthetic pathways. 4-coumaroyl CoA probably rep-
resents the most important branchpoint within the central
phenylpropanoid biosynthesis in plants. It is either the direct
precursor for flavonoid or H-lignin biosynthesis, or is fed into
the production of increasingly methoxylated guaiacyl (G)- and
syringyl (S)-monolignols. A decisive step is initiated by the en-
zyme hydroxycinnamoyl CoA:shikimate/quinate hydroxycin-
namoyltransferase (HCT). HCT belongs to the large family of
BAHD-like acyltransferases (D’Auria, 2006) and may require
regulation at multiple levels. Its down-regulation results in
an increase in H-units, a depletion in S-units, and a significant
increase in caffeoylquinate esters in stems, with only minor
effects in leaves (Hoffmann et al., 2004). Silencing of the
SHT gene in tracheary elements forming Pinus radiata cell cul-
tures demonstrated the importance of this gene for lignin
quantity and quality, most notably a drastic shift towards
the H-type of monolignols (Wagner et al., 2007).
Only the shikimate and quinate esters, but not the free phe-
nylpropanoid acid or its CoA and glucose ester, are the sub-
strates of Cyp98A3, which catalyze the hydroxylation of the
meta-position of the hydroxycinnamoyl shikimate or quinate
esters, respectively (Schoch et al., 2001; Abdulrazzak et al.,
2006). Supported by reports of a high in vitro KM of HCT for
shikimate and quinate, two explanations for this fuzzy and ap-
parently complicated meta-hydroxylation appear most likely:
(1) effective protection and channeling of the reactive cate-
chol moiety for subsequent methylation by CCoAOMT and
(2) a versatile metabolic control for channeling of the shiki-
mate pathway intermediates into the biosynthesis of second-
ary metabolites only when photosynthesis is running well.
Under sub-optimal photosynthesis, when plants are stressed,
low concentration of shikimate pathway intermediates may
re-direct the entire phenylpropanoid pathway towards the
production of phytoalexins, volatiles, flavonoids, and antho-
cyanins and de novo synthesis of (defence?) proteins (Schoch
et al., 2006; Abdulrazzak et al., 2006). A sophisticated cross-
talk and effective transport system between photosynthetic
active parenchyma cells and the cambial cells or tracheary ele-
ments of the vascular bundles would, however, be required.
Caffeoyl quinate (chlorogenic acid) and caffeoyl phenyl lac-
tic acid (rosmarinic acid) are the predominant soluble phenyl-
propanoids in the Solanaceae and Lamiales, respectively.
Possible roles for both esters as defence compounds or as po-
tential antioxidants are discussed (Petersen et al., 2009). To ex-
plore the potential benefits as a nutraceutical antioxidant,
overproduction of chlorogenic acid was achieved in tomato ei-
ther by functional expression of a BAHD-like HCT (Niggeweg
6 | Vogt d Phenylpropanoid Biosynthesis
et al., 2004) or specifically in potato tubers by overexpressing
the StMtf1-MYB-transcription factor (Rommens et al., 2009).
Diversification of this small subset of phenylpropanoids like
coumaroyl- and caffeoyl CoA esters is achieved by an array of
subsequent oxygenases, reductases, and transferases and
results in the observed complexity and heterogeneity of plant
phenylpropanoid metabolite profiles.
LIGNIN
Besides cellulose, lignin is the most prominent polymer on
Earth. Lignin is nearly exclusively based on phenylpropanoid
units derived from the oxidative polymerization of hydroxycin-
namoyl alcohol derivatives. As early as 1960, the contribution
of the shikimate pathway, the reduction of ferulic acid to con-
iferyl alcohol, and the polymerization of monolignols had al-
ready been realized (Neish, 1960). The term lignocellulose
implies that the phenolic polymer can be cross-linked to the
cellulosic material by feruloyl-linkages, a characteristic feature
of grasses (Burr and Fry, 2009).
The various aspects of lignin and lignan formation have
been summarized in a recent review by Davin et al. (2008),
including a detailed biochemical evaluation of the require-
ments leading to controlled versus random coupling of
4-hydroxyphenyl (H)-, guaiacyl (G)-, and syringyl (S)-derived
monolignols. This review also illustrates progress and experi-
mental limitations in structural elucidation of the various
forms of lignin in monocots and dicots. Due to its economical
value for timber and biofuel formation, lignin biosynthesis
and manipulation has been a central research focus (Rubin,
2008; Li et al., 2008). Numerous efforts to manipulate the lig-
nin content have largely focused on structural and regulatory
genes leading the accumulation of gymnosperm and angio-
sperm lignin (Sederoff et al., 1999; Boudet et al., 2003;
Vanholme et al., 2008). The contribution of the corresponding
enzymes leading to different monomer composition and
resulting in various ratios of guaiacyl or syringyl units have
been summarized and the effects of the knockout mutations
of the corresponding genes have been analyzed in detail
(Anterola and Lewis, 2002; Dixon and Reddy, 2003; Vanholme
et al., 2008). The activation of phenylpropanoid precursors, by
the initial formation of coenzyme A-thioesters and the subse-
quent reduction in two successive steps by cinnamoyl CoA re-
ductase (CCR) and cinnamyol alcohol dehydrogenase (CAD), is
thoroughly documented (reviewed in Davin et al., 2008). Both
enzymes are considered the committed steps into monolignol,
lignan, and lignin biosynthesis. Arabidopsis knockout mutants
of the CCR1-gene show a reduced lignin formation, which can
be partly compensated for by CCR-2, but they also display an
enhanced pool of feruloyl malate at the expense of sinapoyl
malate accumulation (Mir Derikvand et al., 2008). This again
indicates that metabolic fluxes within a complex pathway
can be, at least in part, redirected towards a pool of soluble
metabolites by eliminating a single, central member of this
pathway, without otherwise affecting the essential, vital func-
tions of the plant. Partly due to the substrate specificity of
most 4CL-ligases earlier in the pathway, sinapoyl CoA is usually
considered not to be a direct precursor for lignin monomer
production—this may, however, depend on the species inves-
tigated (Hamada et al., 2004). Comparison of annuals and per-
ennials with respect to lignin biosynthesis has revealed an
apparent functional redundancy of most participating genes
and enzymes. Although CAD is considered a key enzymatic
step in monolignol biosynthesis, a detailed analysis of 17
CAD encoding genes in Arabidopsis revealed that most inser-
tion lines of individual CAD-genes only had a marginal effect
on the overall lignin composition (Kim et al., 2004). This sug-
gests at least some functional redundancy among these genes
in case of Arabidopsis and single CADs are not rate-limiting.
On the other hand, it is less likely that all of these genes encode
bona fide CADs and actually contribute to monolignol forma-
tion. A complex grid of temporal and organ-specific regulatory
mechanisms may exist to respond rapidly to various biotic and
abiotic stressors and ensure the growth and development un-
der adverse conditions. The development of functional
genomics and genetic mapping of several species like poplar,
pine, eucalyptus, and alfalfa has already enabled to decipher
the numerous possibilities of various metabolic routes towards
lignin monomer and oligomer formation, oxidative phenol
coupling, and analysis of its polymeric structure (Boerjan
et al., 2003; Bhalerao et al., 2003; Davin et al., 2008).
Remarkably, information on enzymatic reactions from con-
iferyl alcohol leading to the various types of lignans and neo-
lignans is limited. Data on the 8–8#-stereospecific coupling of
two coniferyl alcohol moieties resulting in the formation of
pinoresinol have been reported (Davin and Lewis, 2003).
The lignans, in contrast, received considerable interest based
on pharmacological properties, such as in the case of the cyto-
static and anti-cancerous, but also severely toxic, podophyllo-
toxin. (Enzymatically) controlled, regioselective 8–8’ coupling
of isoeugenol-derived lignans without an 9–9#-hydroxygroup
was recently demonstrated in roots of the Aristolochiaceae
Holystis reniformis upon feeding radiolabeled precursors
(Messiano et al., 2009). The identification of two pinoresinol
reductase genes, catalyzing specifically the formation of lari-
ciresinol from pinoresinol in Arabidopsis, may facilitate a more
thorough investigation of the enzymatic steps of this pathway
on a molecular level in a model organism (Nakatsubo et al.,
2008).
In addition to the entry enzymes into lignin biosynthesis,
hydroxylation and methylation steps by cytochrome
P450s and S-adenosyl-L-methionine (AdoMet)-dependent
O-methyltransferases essentially determine the contribution
of guaiacyl and syringyl monomers to the gymnosperm and
angiosperm pattern. Overexpression of ferulate-5 hydroxylase
(F5H), also termed coniferylaldehyde hydroxylase, leads nearly
exclusively to shift to syringyl-lignin in poplar, emphasizing the
critical role of this gene for the structure and physical proper-
ties of angiosperm lignin (Stewart et al., 2009), whereas knock-
down of the f5h gene reveals pleiotropic changes in the

Arabidopsis metabolome (Huang et al., 2009). Decoration of
the various hydroxylated monolignol precursors in Arabidopsis
is performed either by cation-dependent CCoAOMT1, re-
quired for the 3-O-methylation of caffeoyl-CoA, or by a cation-
independent caffeic acid OMT (COMT), which is involved in
the formation of sinapoyl residues and S-lignin from the
5-hydroxyferuloyl derivatives (Davin et al., 2008). The role of
CCoAOMT1, originally considered to be only associated with
monolignol formation, has changed considerably in the last
couple of years (Do et al., 2007; Kai et al., 2008; Fellenberg
et al., 2009). Its involvement in various pathways, including
sinapoylmalate formation, scopoletin biosynthesis, and meth-
ylation of acylspermidine precursors, has proven its central
role in the modification of phenylpropanoids. Engineering
substrate promiscuous OMTs to act as monolignol 4-O-
methyltransferases and impaire oxidative radical coupling is
a novel approach to modify lignin composition (Bhuiya and
Liu, 2009). The key issue, if lignin formation proceeds via a met-
abolic grid or by precise channeling of individual precursors
through metabolons as proposed for other pathways in plant
secondary metabolism, remains to be resolved (Humphreys
and Chapple, 2002; Jørgensen et al., 2005; Conrado et al.,
2008). Based on current data, a clear decision favoring either
one of both possibilities cannot be made. However, one might
assume that precise channeling would be a superior mode of
control. Additional work is also required on the transport of
monolignols prior to assembly. Two possibilities are discussed,
either golgi-mediated vesicle transport (Samuels et al., 2002)
or specific translocation of monolignols by unknown (ATP
binding cassette?) transporters suggested for xylem formation
in angio- and gymnosperms (Ehlting et al., 2005; Kaneda et al.,
2008).
Since the basic principles in lignin and lignocellulose forma-
tion may be similar in dicots and monocots, molecular work on
fast-growing crops with high biomass, severe drought resis-
tance, or low fertilizer requirements, like Miscanthus spec.,
should be encouraged to grow lignin and lignocellulose-
producing plants under extreme climatic conditions
(Dohleman and Long, 2009) and enable developing countries
to benefit from recent progress in biotechnology.
PHENYLPROPANOID ESTERS AND
AMIDES (ACYLATED POLYAMINES)
Arabidopsis has been an excellent tool to investigate the bio-
synthesis of a specific class of phenylpropanoids characteristic
for the Brassicaceae: the sinapic acid esters. Sinapoyl choline
(sinapine) accumulates to high levels in the seeds of Brassica
napus (rapeseed), and, besides tannins and glucosinolates,
can negatively affect its use as a protein supplement (Bhinu
et al., 2009). The reduction of sinapine by genetic engineering
of various biosynthetic enzymes was one of the driving forces
in the analysis of the pathway, but also led to important dis-
coveries with respect to structural information and evolution
of the participating enzymes. In contrast to lignin or flavonoid
Vogt d Phenylpropanoid Biosynthesis | 7
biosynthesis, where hydroxycinnamic acids are activated by
CoA-esters, in sinapic ester metabolism, 1-O-acyl-glucose esters
serve as the energy-rich metabolites during seed maturation
and germination (Mock and Strack, 1992). The corresponding
glucosyltransferases have been investigated in molecular de-
tail (Milkowski et al., 2000; Sinlapadech et al., 2007; Meissner
et al., 2008). Two transferases, sinapoylglucose:choline sina-
poyltransferase (SCT) and sinapoylglucose:malate sinapoyl-
transferase (SMT), are involved in the accumulation of
sinapine in the seed or sinapoylmalate in the seedlings, respec-
tively. Compared with the presumed ancestors, the serine car-
boxypeptidases (SCPs), the SMT from Arabidopsis, has
maintained the catalytic triad, Ser–His–Asp (Milkowski and
Strack, 2004; Stehle et al., 2009). A single Asp/Glu substitution
at the amino acid position close to the catalytic Ser enables
binding of 1-O-glucose (Figure 3). In the absence of L-malate,
a low esterase activity resulting in the formation of sinapic acid
can still be observed in this SCP-like protein, which is reminis-
cent of the hydrolytic ancestor. The drastic consequences of
only slight differences in the catalytic site can be perceived
as one of the driving forces resulting in a total pool of 51 genes
encoding SCPLs in Arabidopsis (Fraser et al., 2007). Even a small
subset of this reservoir of novel catalytic activities may already
be able to amplify existing metabolites and prove advanta-
geous to the plant under changing environmental conditions.
The existence of acylated polyamine conjugates has been
described for several decades and their widespread occur-
rence, predominantly in flowers and pollen grains of wind-
pollinated species, has been attributed to UV-protection, fer-
tility, as well as defence (Meurer et al., 1988; Kakkar and Rai,
1993; Walters, 2003). The rationale of their existence has been
Figure 3. Model Structure of the Active Site of the AtSMT Modified
from Stehle et al. (2006) with the Docking Interaction between the
Substrates Sinapoylglucose and L-malate (A) and the Mutated Pep-
tidase Motif Gly–Asp–Ser–Tyr–Ser (B).
The amino acids replaced by the residues of serine carboxypepti-
dases are marked in green. The bulky side chain of Glu at position
172 would restrict binding of the glucose moiety of sinapoylglucose
sterically. Therefore, the Glu to Asp substitution enables the acyl-
trasferases to accommodate 1-O-glucose esters as substrates and
is the hallmark of glucose ester-dependent acyltransferases. Resi-
dues forming the catalytic triad are colored in black. The substrates
sinapoylglucose and L-malate are colored in orange. Dotted lines
indicate hydrogen bonds.
8 | Vogt d Phenylpropanoid Biosynthesis
disputed since then. The recent identification of phenylpropa-
noid polyamine conjugates in different organs of Arabidopsis
wild-type and insertion lines by LC–MS analysis was accompa-
nied by simultaneous identification of specific genes and
enzymes in the acyl conjugate formation (Böttcher et al.,
2008; Matsuda et al., 2008; Luo et al., 2009). The rapid
annotation of several genes and enzymes involved in the
biosynthesis of these conjugates illustrates the combined
power of Arabidopsis knockout and knockdown insertion lines
with sensitive UPLC–MS analyses (Fellenberg et al., 2008;
Grienenberger et al., 2009). In the tapetum of Arabidopsis
flower buds, a new pathway for the biosynthesis of acylated
spermidine conjugates was established. Hydroxycinnamic
acids are transferred to spermidine by a BAHD-like spermidine
hydroxycinnamic acid transferase (SHT). Knockout mutation of
this gene results in anthers and pollen grains, completely de-
void of trisacyl conjugates (Grienenberger et al., 2009). Two
other enzymes critical for this pathway, a cytochrome P450
and a methyltransferase (Figure 4), were recruited from
enzymes in an established biosynthetic pathway to catalyze
the precise modifications of acylspermidine derivatives.
CYP98A3 hydroxylates quinate and shikimate esters of 4-
Figure 4. A Simplified Version of the Biosynthetic Pathway Result-
ing in Acylated Spermidine Conjugates in flower buds Is Based on
Recent Data by Fellenberg et al. (2008, 2009), Grienenberger et al.
(2009), and Matsuno et al. (2009).
The exact sequence of the initial hydroxylation and Werner meth-
ylation steps by CYP98A3 and CCoAOMT1, the subsequent hydrox-
ylation by Cyp98A9 and the transferase reaction by SHT is not
resolved yet. Sequential steps catalyzed by CYP98A9 and AtTSM1
are established. AtTSM1, localized exclusively in the tapetum,
may serve as a tapetal marker in young flower buds of Arabidopsis.
coumaric acid in lignin biosynthesis (Schoch et al., 2001).
The new, pathway-specific CYP98A8 and its paralog CYP98A9
belong to a monophyletic CYP98A clade and are duplications
of an ancestor of CYP98A3, which evolved rapidly via retropo-
sition and positive Darwinian selection (Matsuno et al., 2009).
Knockout mutants of CYP98A8 display a shift in the major ac-
ylated spermidine conjugate pattern, from 5-hydroxyferuloyl
to feruloyl conjugates. Likewise, AtTSM1, a cation-dependent
OMT with significant similarity to CCoAOMT1 in lignin biosyn-
thesis, catalyzes the final step in acyl spermidine conjugate for-
mation. The enzyme methylates a single 5-hydroxyferuloyl
moiety and yields N,N’-bis(5-hydroxyferuloyl)-N’’ sinapoyl sper-
midine (Fellenberg et al., 2008). CCoAOMT1 and potentially
CYP98A3 play additional roles earlier in methylation and
hydroxylation of the quinate/shikimate esters, respectively.
Corresponding knockouts of CCoAOMT1 also resulted in
a severe decrease in acyl-spermidine formation (Figure 4;
Fellenberg et al., 2009). This pathway convincingly demon-
strates that genetic mechanisms like retroposition and gene
duplication are also active in plant secondary metabolism
and may result in an accelerated establishment of organ-
specific new pathways from ancestral genes (Matsuno et al.,
2009). The reason for the exclusive appearance of the pathway
in the tapetum is not yet understood, since neither the overall
plant phenotype nor the plant fertility appears affected
(Grienenberger et al., 2009). Regulatory elements of the
pathway have up to now not been identified, although can-
didate MYB transcription factors are described that regulate
phenylpropanoid–polyamine conjugate accumulation in other
plants (Gális et al., 2006).
ANTHOCYANINS, AURONES,
(ISO)FLAVONOIDS, AND STILBENES
Due to ease of visualization and control of mutants and
genetic imbalances, anthocyanin biosynthesis was one of
the first branches of the general phenylpropanoid metabo-
lism, for which biosynthetic enzymes and corresponding tran-
scription factors were identified and has been reviewed
extensively (Allan et al., 2008). The key enzymatic step of
the whole pathway, the formation of anthocyanidins via
(2R-3R)-dihydroflavonols and oxidation of (2R, 3S, 4S)-
leucoanthocyanidins by anthocyanidin synthase (ANS) has
recently been questioned due the apparent flavonol syn-
thase activity of Arabidopsis ANS and the identification of
(+)-catechin as a substrate of Gerbera hybrida ANS (Wellmann
et al., 2006). In a monomeric state, anthocyanins never display
a blue color. Therefore, investigation of blue color formation
of anthocyanins either by pH-shift, metal complex formation,
modification of the core unit by additional hydroxylation,
methylation, or acylation and subsequent molecular stacking
has been a major focus of anthocyanin research in the last de-
cade (Yoshida et al., 2009). This research was partly driven by
the cut-flower industry to develop blue varieties of those flow-
ers, which naturally do not exhibit this phenotype, like roses or

carnations. During the process of developing a bluish tone, im-
portant contributions to anthocyanin biosynthesis have been
made in terms of structural and regulatory genes. It was shown
that roses, in contrast to most other plants, have a unique bi-
functional glucosyltransferase that catalyses the glycosylation
of both the 3- and the 5-OH group (Ogata et al., 2005). How-
ever, they are devoid of a 5#-hydroxylase activity, which partly
explains the lack of any natural bluish color. Expression of the
Viola spp. 3#, 5#-hydroxylase and functional replacement of
the endogenous rose dihydroflavonol reductase (DFR) for an
orthologous enzyme from Iris hollandica resulted in plants
that nearly exclusively accumulated delphinidin in the petals
and displayed a bluish, slightly purple tone (Katsumoto et al.,
2007). Myricetin, in addition to quercetin, was detected as
a co-pigment in the transgenic plants, indicating that the hy-
droxylase modifies both flavonols and anthocyanins. Further
modification by co-expression of an additional acyltransferase
did not result in the desired increase of acylated compounds
and intensified color formation (Katsumoto et al., 2007). To
further enhance color formation, additional methylation of
these B-ring hydroxyl groups may be required. Methylation
of vicinal dihydroxy groups of anthocyanins from a petunidin
to malvidin type was recently demonstrated to proceed by
a cation-dependent OMT in grapevine (Hugueney et al.,
2009). It further extends the biosynthetic niches of this type
of enzyme towards modification of chromogenic compounds
with an aromatic cis-diol structure.
Yellow flower colors are not necessarily due to carotinoid ac-
cumulation, as in sunflowers. In Anthirrhinum majus, the yel-
low aurones display a bright yellow color with an additional
fluorescence and are considered to help attract pollinators.
Ono and co-workers (2006) were able to produce ornamental
Torrenia hybrida flowers with bright yellow petals. This was
achieved by the combined down-regulation of the anthocyanin
pathway and the co-expression of two genes involved in aurone
formation, the vacuolar localized aureosidin synthase (AmSA1)
and a cytoplasmatic chalcone 4#-O-glucosyltransferase. Co-
epxression of the modifying UGT together with the AmSA1
was absolutely required, in order to enable transport of the
4#-O-glycosylated chalcones to the vacuoles, where enzymatic
conversion to the aurone 6-O-glucosides was performed (Ono
et al., 2006).
The complex structures of flavonoids and anthocyanins re-
quire precise annotation of the participating modifying and
decorating enzymes. Most acyl-, glucosyl-, and methyltrans-
ferases identified up to now exhibit overlapping or promiscu-
ous substrate specificities in vitro (Bowles et al., 2006). If
a potential gene function is not immediately apparent by
sequence analysis of a multigene family, comparison of its
gene expression with the expression profiles of known genes,
complementation of the metabolite pattern combined with
reverse genetics help to annotate a single member from large
superfamilies, like CYPs or UGTs. CYP98A8/A9 was correlated
to the phenylpropanoid polyamine conjugate biosynthesis
in the Arabidopsis tapetum (Ehlting et al., 2008; Matsuno
Vogt d Phenylpropanoid Biosynthesis | 9
et al., 2009). UGT89C1 was linked to the late steps in flavo-
nol biosynthesis based on transcript and metabolite
pattern and therefore correctly identified as Arabidopsis 7-
O-rhamnosyltransferase (7RhaT) (Yonekura-Sakakibara et al.,
2007).
Several other late steps in the formation of the flavonoid
skeleton have only been poorly investigated. B-ring hydroxyl-
ation at positions 6 and 8 of the flavonoid skeleton is limited to
a few plant families only, like the Asteraceae. For Chrysanthe-
mum segetum, Halbwirth and Stich (2006) identified a new
type of 8-monohydroxylase dependent on both NADPH and
FAD, and was, therefore, clearly different from classical cyto-
chrome P450s. Obviously, there is a lack of enzymatic and ge-
netic information for this as well as other exotic plants.
Scutellaria baicalensis is one of several prominent examples
used in traditional folk medicine. Its roots harbor flavonoids
with unusual hydroxylation, methylation, or glycosylation pat-
tern in the flavonoid A- and B-ring (Li-Weber, 2009). Exponen-
tially growing databases and sequence information may
enable the identification and annotation of the corresponding
genes and enzymes in due course, if we manage to prevent
these valuable resources from eradication.
Three other elusive modifications or decorations of the fla-
vonoid molecules have been recently established at the molec-
ular level. These modifications include the characterization
and identification of the first soluble, rare plant glucuronosyl-
transferase (GlurT) UGT94B1 from Bellis perennis (Sawada
et al., 2005). The key amino acid distinguishing this type of en-
zyme from the common glycosyltransferases was identified as
an arginine at position 25 outside of the conserved UDP–sugar
binding-motif, the PSPG-box (Osmani et al., 2008).
A second modification has been known to phytochemists
for more than 20 years, but the corresponding enzymatic
step has not been resolved: the C-glycosylation of the
flavonoid A-ring. Unequivocal annotation of flavone 6- and
8 C-glycosylating enzyme was recently achieved for
a C-glucosyltransferase (CGT) from rice (Brazier-Hicks et al.,
2009). A biochemical and molecular approach was used in
the case of the Oryza sativa CGT. This CGT could not be iden-
tified by a simple sequence similarity approach, since no CGT-
specific amino acid sequence motif of OsCGT1 was detected
and the most similar enzymes with 30% overall identity were
described as UGTs involved in monolignol formation in Arabi-
dopsis. The authors propose that C-glycosylation activity in
rice and wheat may proceed via a mechanism involving a 2-
hydroxyflavone intermediate, although this hydroxylation ac-
tivity requires further confirmation. The 2-hydroxyflavone
exists in equilibrium with its open chain form, which is pro-
posed as the substrate C-glucosyltransferase.
Last but not least, the cloning and characterization of enzy-
matically active naringenin 8-prenyltransferase pave the way
for subsequent investigations of this important, yet poorly
characterized class of membrane enzymes (Sasaki et al., 2008).
Regulation of flavonoid and anthocyanin formation by ba-
sic TTG/basic helix-loop-helix bHLH and MybR2R3-type
10 | Vogt d Phenylpropanoid Biosynthesis
transcription factors has been the topic of many reports, such
as Stracke et al. (2007), and several reviews (Broun, 2005; Allan
et al., 2008). The complex pattern of factors regulating antho-
cyanin formation has recently been extended by Matsui et al.
(2008) and Zhu et al. (2009). They discovered that single repeat
R3 Myb transcription factors may also repress anthocyanin for-
mation, probably by competing with R2R3-Mybs for the bind-
ing sites of bHLH proteins, thereby down-regulating structural
genes and counteracting, such as the Myb-like PAP1/2 en-
hancer of anthocyanin formation in Arabidopsis (Gonzales
et al., 2008). The bHLH-domain of the maize R-gene, partici-
pating in anthocyanin formation, links flavonoid formation
to histone modification by interacting with an EMSY-like fac-
tor, which is involved in chromatin repair (Hernandez et al.,
2007).
Identification of metabolite pattern and elucidation of the
structural genes and enzymes, deciphering the complex net-
work of hundreds of transcription factors and their hormonal
control (Gális et al., 2006) in Arabidopsis and other plant spe-
cies, will provide a tremendous challenge to understand phe-
nylpropanoid metabolism and its contribution to plant
biology. Non-targeted approaches correlating co-expression
data and metabolomics at the organ or tissue level can provide
solutions to unravel parts of this intriguing puzzle (Tohge
et al., 2005).
Terpenophenolics, stilbenes, and isoflavonoids are limited
to a few species only, but have attracted major interest due
to potential health-promoting affects on mammals (Stevens
and Page, 2004; Barnes and Prasain, 2005; Shen et al., 2009;
Dixon, 1999; Veitch, 2009). The biosynthesis of prenylchalcone
and other prenylated chalcones represent a deviation from the
standard flavonoid biosynthesis. Xanthohumol is a major me-
tabolite accumulating specifically in glandular trichomes of
hops, Humulus lupulus. By an EST-approach, Nagel et al.
(2008) could recently show that in the glandular trichomes,
naringenin chalcone is first prenylated and subsequently
methylated by a substrate-specific 6#-O-desmethylxanthohu-
mol OMT, which is responsible for the stabilization of the
resulting xanthohumol. This result provides the basis for fur-
ther investigations of this important pathway, leading to
the formation of a family of compounds with considerable
phytoestrogenic properties.
The biosynthesis of isoflavonoid and related pterocarpans in
model legumes like Medicago sativa and M. truncatula or Lo-
tus japonicus has been a major research focus for the last two
decades. The oxidative migration of the aryl ring from 2S-
liquiritigenin, the 5-deoxynaringenin formation catalyzed by
the cytochrome P450 isoflavone synthase and the subsequent
4#-O-methylation are the decisive branch points leading to the
formation of isoflavonoids. In contrast, stabilization of the iso-
flavanone by a 4#-OMT, a dehydratase and second cytochrome
P450, isoflavone 2#-hydroxylase, are required for the pathway
to proceed towards the biosynthesis of pterocarpan-type phy-
toalexins, like pisatin (Aoki et al., 2000). A promiscuous 4#-
isoflavonoid OMT with identical catalytic properties towards
the 4#- hydroxyl group of 2,7,4#-trihydroxyisoflavanone at
an initial step of the pathway as well as towards the 3-hydroxy
group of the pterocarpan 6a-hydroxymaackiain resulting in
the formation of pisatin has been identified and structurally
characterized (Liu et al., 2006). Careful analysis of both sub-
strates revealed their close to identical three-dimensional
structure. A single enzyme able to convert two metabolites
of the same pathway suggests that substrate promiscuity does
not only provide evolutionary advantages in response to
novel, exogenous phytoalexins, but may also be maintained
to serve those rare cases of metabolic serendipity. This situa-
tion is somewhat different from the methylation step in
acyl-spermidine formation in Arabidopsis flowers, where not
CCoAOMT1, but a similar cation-dependent OMT (AtTSM1)
with distinct properties was recruited to catalyze the final dec-
orating step of conjugate formation (Fellenberg et al., 2009).
As in case of the isoflavonoids, also the stilbenes like resver-
atrol and its glycoside piceid are of limited distribution in the
plant kingdom. Both have been shown to display numerous
biological activities in vitro, including antitumor activity and
induction of apoptosis (Pervaiz, 2004; Crozier et al., 2009).
Trans-resveratrol apparently retards the ageing process and
increases longevity in various organisms from yeast to insects
and rodents (Orallo, 2008). Since dietary polyphenolics usually
display low absorption and are chemically modified by the
small intestine, the contribution to human health in vivo will
require profound epidemiological studies (Crozier et al., 2009).
The relative ease to produce transgenic edible crops by intro-
ducing only a single gene, in this case stilbene synthase under
the control of a 35S-promoter, results in substantial levels of
stilbenes in tomato fruits and may encourage other scientists
to explore the proposed positive in vitro effects in controlled
experimental set ups in vivo (Giovinazzo et al., 2005; Schijlen
et al., 2006).
HYDROLYZABLE AND NON-
HYDROLYZABLE PROANTHOCYANIDINS
AND TANNINS
Proanthocyanidin biosynthesis branches off the flavonoid
pathway from either 2,3-cis or trans flavanols and is initiated
by anthocyanidin reductase or leucoanthocyanidin reductase,
respectively (Dixon et al., 2005; Tian et al., 2008). The resulting
epicatechin and catechin derivates can be oxidized to qui-
nones, which are polymerized. However, it is not clear whether
this polymerization of the soluble precursors proceeds enzy-
matically by a laccase type of enzyme (encoded by the TT10
gene in Arabidopsis) or non-enzymatically (Pourcel et al.,
2005; Dixon et al., 2005). Transport of precursors across
vacuoles membranes is facilitated by multidrug-extrusion
MATE-type transporters in Arabidopsis (encoded by the
TT12-locus) and Medicago truncatula (Marinova et al., 2007;
Zhao and Dixon, 2009). Similar to anthocyanin formation,
the deposition of proanthocyanidin oligomers in the seed coat
of Arabidopsis is regulated by the interaction of a series of

transcriptional regulators R2R3 MYB-type proteins (TT2),
bHLH-factors (TT8), and WD40-repeat proteins (TTG1), which
act synergistically. Overexpression of those transcription fac-
tors may create improved crop varieties with enhanced proan-
thocyanidin contents (Baudry et al., 2004; Mellway et al., 2009;
Terrier et al., 2009). The reported health-promoting activities
of 3-O-esterified proanthocyanidins like epigallocatechin-gal-
late (EGCG) (Rezai-Zadeh et al., 2005), a major metabolite in
green tea leaves, can be hampered by a low bioavailability
upon modification of the core structure by sulfatation, meth-
ylation, and glucuronylation (Henning et al., 2008).
Similar to the proanthocyanidins (non-hydrolyzable tan-
nins), the hydrolyzable tannins, like galloyl-glucose esters
and ellagitannins, characterized by oxidative coupling of
neighboring galloyl groups, display a strong tendency to scav-
enge radicals. Due to multiple aromatic hydroxyl groups, they
cross-link and precipitate enzymes, making them interesting
defence compounds specifically when present in high concen-
trations in oat or in sumac (Barbehenn et al., 2006; Gross,
2008). Remarkably little information is available on the biosyn-
thesis of gallic acid, which respresents the core structure in
gallo- and ellagitannin structures. Retrobiosynthetic 13C-
NMR-studies, by feeding 13C-labeled glucose to young leaves
of Rhus typhina and measurments of oxygen isotope abun-
dance, performed by Werner and colleagues (1997, 2004),
showed that gallic acid is derived from the shikimate pathway.
The formation via phenylalanine and hydroxylated cinnamic
acid derivatives could be ruled out by these experiments. Based
on these studies, it appears most likely that gallic acid is
formed via dehydrogenation of 5-dihydroshikimate, although
a dehydratation step via protocatechuic acid and subsequent
monooxygenation cannot be ruled out. Enzymatic evidence
for the existence of a specific cytochrome P450 monooxyge-
nase or a 2-oxoglutarate dependent dioxygenase as shown
in coumarin formation would help to resolve the contribution
of both possibilities. Clearly, a lot of research remains to be
done specifically on the hydrolyzable tannins, although enzy-
matic studies appear difficult due to the protein precipitant
properties of both, substrates and products.
C6–C1 METABOLITES
In contrast to the established biosynthesis of phenylpropa-
noids with a C6–C3 carbon skeleton, the formation of phe-
nolics with a C6–C1 skeleton, like benzoic acid (BA),
appears complex and is still fragmentary. Starting from
trans-cinnamate, two biosynthetic scenarios are proposed.
A b-oxidative pathway via CoA-activation and a 3-keto-acyl
CoA-thiolase was described for Petunia hybrida (Van
Moerkercke et al., 2009). An alternative non-oxidative path-
way, via benzoyl CoA and benzaldehyde, with a final step
catalyzed by a benzaldehyde dehydrogenase, was identified
in Anthirrhinum majus (Long et al., 2009). Additional routes,
specific for certain plants or specific organs, have also been
described (Abd El-Mawla and Beerhues, 2002; Ibdah et al.,
Vogt d Phenylpropanoid Biosynthesis | 11
2009). From the sum of data, it is evident that several path-
ways for benzoic acid biosynthesis may co-exist in a single
plant and relative fluxes from one or the other pathway
may be controlled by hormones or abiotic stressors. Feeding
deuterium-labeled phenylalanine showed that metabolic
fluxes of benzaldehyde formation of the non b-oxidative
pathway from cinnamate to benzaldehyde to benzoic acid
was about twice that of the b-oxidative pathway involving
benzoyl-CoA in P. hybrida petals (Boatright et al., 2004).
The 2-hydroxyl derivative of benzoic acid, salicylic acid (SA),
has long been known as a key component of plant innate immu-
nity (Lee and Raskin, 1999; Nawrath and Metreux, 1999) and also
plays a central role in systemic-acquired resistance (SAR) (Vlot
et al., 2008). Biochemical evidence for a direct pathway from
BA to SA was obtained first by the identification of benzoic acid
2-hydroxylase, a cytochrome P450 monooxygenase from to-
bacco (León et al., 1995), and was also based on an increase
of PAL-transcripts and activity in tobacco plants upon tobacco
mosaic virus (TMV) infection (Ogawa et al., 2006). However,
in contrast to the prokaryotic BA 2-hydroxylase encoded by
the NahG gene, there is no Arabidopsis insertion line indicating
a similar gene and no CYP-candidate genes have been anno-
tated yet. Transgenic tobacco plants overexpressing PAL also
did not show any significant change in the SA levels (Shadle
et al., 2003), suggesting that SA biosynthesis is not derived from
trans-cinnamate and benzoic acid. In contrast, experimental ev-
idence from the Arabidopsis sid2 mutant, encoding isochoris-
mate synthase 1 (ICS1) (Wildermuth et al., 2001; Strawn et al.,
2007), as well as virus-mediated RNA-silencing in N. benthami-
ana (Catinot et al., 2008) suggest that chorismate and ICS may be
central to and required for salicylic acid biosynthesis. The exact
contribution of the several pathways to SA bioynthesis may
thereforebespecies-specificandresearchmayrequireacompar-
ative investigation of several mono- and dicot families.
Modification of SA results in a significant increase in suscep-
tibility to pathogens (Vlot et al., 2008). Modification of salicylic
acid can be achieved either by methylation (Ross et al., 1999) or
by glycosylation, at the carboxy- or at the 2-hydroxy group, re-
spectively (Lee and Raskin, 1999; Lim et al., 2002). The meth-
yltransferase activity towards the carboxygroup of benzoic
acid and salicylic acid is part of the SABATH-family of enzymes
that are generally implicated in hormone homoeostasis or
phenylpropanoid volatile formation in plants (Zhao et al.,
2008; Barkman et al., 2007; Kapteyn et al., 2007). Methyl salic-
ylate has been identified as the critical mobile signal for SAR.
Its perception may include additional regulatory steps, such as
by a salicylic acid binding protein (SABP2), identified as a meth-
ylesterase (Forouhar et al., 2005; Zhao et al., 2009). Glycosyla-
tion of salicylic acid is much less investigated. A systematic
screen within Arabidopsis plant natural product UGTs revealed
only enzymes with activities towards the carboxyl group, but
none towards the 2-OH group (Lim et al., 2002). This suggests
that regiospecific SA-glycosylating enzymes share limited dis-
tribution in plants and that glycosylation is not the common
way of inactivating this hormone.
12 | Vogt d Phenylpropanoid Biosynthesis
PHENYLPROPENE VOLATILES
Benzenoid volatile formation in Petunia flowers has been
shown to be under strict diurnal and hormonal control,
and appears to follow the transcriptional control of central
shikimate pathway genes, like 5-enol-pyruvylshikimate-3-
phosphate synthase (EPSPS) (Schuurink et al., 2006). Methyl
salicylate and methyl benzoate are not the only phenolic
compounds emitted as volatiles by flowers. They are part of
a complex species-specific bouquet of compounds, including
terpenoids and phenylpropenes (Dudareva et al., 2004). These
volatiles are also either emitted as floral attractants to pollina-
tors, as defence compounds against microorganisms, insects,
and mammalian predators, or can display allelopathic effects
on other plants (Dudareva et al., 2004; Horiuchi et al., 2007).
For phenylpropenes from the Lamiales and simple catechol
derivatives, like orcinol from Chinese roses, several species
and organ-specific biosynthetic pathways have been eluci-
dated in the last decade (Scalliet et al., 2006; Koeduka
et al., 2006; Kapteyn et al., 2007). The biosynthesis of anti-
microbial phenylpropenes like eugenol, isoeugenol, or chavi-
col could be resolved due to the high production levels of
these compounds either in the corolla of Petunia hybrida
and the peltate glands of Ocymum basilikum. Both com-
pounds are formed via the general phenylpropanoid path-
way, including the conversion of coniferyl alcohol by a
BAHD-like coniferyl alcohol acetyltransferase and a subse-
quent NADPH-dependent reduction of the coniferyl acetate
(Dexter et al., 2007; Koeduka et al., 2006). The structure of
the key enzyme, eugenol synthase (EGS), a member of the
short chain dehydrogenases/reductases, catalyzing the reduc-
tive replacement of the propenyl side chain has been resolved
by X-ray crystallography (Louie et al., 2007). Methylation of the
4-hydroxy group of phenylpropenes is achieved by a specific
subfamily of methyltransferases. They may be either localized
specifically in the glandular trichomes as observed for sweet
basil or could be part of the phenylpropene pathway in the
leaves, as in the case of Pimpinella anisum (anise) (Koeduka
et al., 2009). As far as coniferylacetate-derived phenylpropenes
are concerned, a CCoAOMT1-like protein is again responsible
for the methylation of the 3-OH group, presumably at the
caffeoyl CoA level. Several levels of metabolic control at
the branch points of volatile emission are apparent when se-
lected basil lines were tested by a systems biology approach
combining transciptome, proteome, and volatile metabolite
profiles (Xie et al., 2008). With the exception of cinnamate
4-hydroxylase, all other transcript and protein levels of phenyl-
propanoid pathway genes correlate well with metabolite for-
mation. Nevertheless, additional control by post-translational
phosphorylation or ubiquintinylation is described for PAL and
chavicol OMT (CVOMT), respectively.
Engineering of transcription factors has not only been used
to control expression of flower color. Co-engineering scent
and flower color by a single transcription factor PAP1, known
to regulate anthocyanin formation, surprisingly also led to
a drastic increase in phenylpropanoid volatile formation when
expressed in Petunia flowers (Ben-Zvi et al., 2008). This fasci-
nating aspect of simultaneously enhancing flower color and
scent production illustrates only one part of the biotechnolog-
ical prospects this kind of research may lead into for potential
benefits to the consumer. Volatile emission from (genetically)
enhanced scent producers might be used to ‘prime’ and
thereby protect non-transgenic crops against pests (Dudareva
and Pichersky, 2008) and pave the way to an alternative way of
molecular farming approaches in an ongoing debate on the
sense and non-sense of transgenic crops.
COUMARINS
High expression levels in all organs, from seeds to roots to
flowers, suggested participation of CCoAOMT1 not only in lig-
nin biosynthesis, but in several biosynthetic pathways leading
to soluble phenylpropanoids (Do et al., 2007). Knockout of the
gene encoding CCoAOMT1 also led to a reduction in the cou-
marin scopoletin in Arabidopsis roots (Kai et al., 2008), sug-
gesting that feruloyl CoA is the phenylpropanoid-derived
precursor of the UV-fluorescent coumarin scopoletin. The de-
cisive step in coumarin biosynthesis in Arabidopsis roots was
verified by T-DNA insertion mutants (Kai et al., 2008). The
key ortho-oxygenation step is not performed by a CYP-like
monooxygenase, but by a position specific 2-oxoglutarate de-
pendent dioxygenase (F6#H1). This enzyme catalyzes the for-
mation of the 6#-O-hydroxylated trans-isomer from feruloyl
CoA, which undergoes isomerization of an enolate anion
and lactonization to form scopoletin (Kai et al., 2008; Figure 5).
The assumption that substrate specificities of orthologous
F6#H1-dioxygenase-like enzymes affect the substitution pat-
tern of coumarins justifies investigations of exotic plant species
containing unusual highly methoxylated coumarins, such as in
the roots of Pelargonium sidoites (Kolodziej, 2007). This might
lead to the detection of orthologous enzymes earlier in the
phenylpropanoid pathway, with novel substrate specificities,
like unusual CoA-ligases. The resulting coumarin aglycones
can be modified by a set of UDP-glucose-dependent glucosyl-
transferases to result, for example, in scopolin or esculin, the
most prominent soluble coumarins in plants. Alternatively, in
the Apiaceae, simple coumarins can be prenylated (Stanjek
et al., 1999) and after a series of subsequent cytochrome
P450-catalyzed reactions (Larbat et al., 2007), result in the for-
mation of furanocoumarins, generally considered as photo-
toxic defense compounds.
EXOTIC PHENYLPROPANOIDS
Several phenylpropanoid-derived metabolites are restricted
to a few species or families only. These include the
curcumins, benzophenones, biphenyls, or phenylphenale-
nones. Although of exotic distribution, some possess pharma-
cological and health-promoting effects, which make them an
interesting target for biosynthetic analyses (Bengmark et al.,

O SCoA O SCoA
2-oxoglutarate
dependent
dioxygenase Rotation
Feruloyl-CoA
OH
6' F6’H1 6' H ical or molecular farming approaches.
MeO MeO
OH OH
O
O SCoA
MeO
O The contribution of the general phenylpropanoid metabolism
OH
SCoA
O O
MeO
CoAS OH
O
O tion of the contribution of the individual constituents of phe-
MeO
OH
Scopoletin
Figure 5. Recently Established Decisive Steps of Scopoletin Biosyn-
thesis, Slightly Modified According to Kai et al. (2008).
Starting from feruloyl–CoA, the 2-oxoglutarate dependent dioxy-
genase F6#H1 catalyzes the formation of the 6#-O-hydroxylated
trans-isomer from feruloyl CoA, which undergoes subsequent isom-
erization, rotation, and lactonization to form scopoletin and free
coenzyme A.
2009). The biosynthesis of curcumins with linear diarylhepta-
noid structure is not sufficiently clarified. Specifically, the
introduction of the oxygenation pattern, either before or after
the formation of the curcumin skeleton remains to be estab-
lished (Kita et al., 2008). That plant polyketide synthase type III
(PKS III), which catalyzes the formation of bisdemethoxycurcu-
min, from p-coumaroyl and malonyl CoA, was described orig-
inally from rice (Katsuyama et al., 2007). Its presence in the
actual curcumin producing Curcuma longa has most recently
been demonstrated and the corresponding enzymes have
been characterized (Katsuyama et al., 2009). Benzophenone-
synthase (BPS) and biphenyl-synthase (BIS) are specific PKS
III, use benzoyl CoA as a starter, and initiate the carbon skel-
eton formation of the benzophenones, xanthones, biphenyls,
and dibenzofurans (Beerhues and Liu, 2009). The compounds
are considered as phytoalexins and their occurrence is limited
to a few known plant taxa like the Hypericaceae (benzophe-
nones and xanthones) and the Maloideae (benzophenones
and biphenyls) (Beerhues and Liu, 2009). A new kind of type
III PKS was also suggested to be involved in the biosynthesis
of cyclic diarylheptanoids and phenylphenalenons from Musa-
ceae (Brand et al., 2006). The general phenylpropanoid path-
way is not different from the pathway from established
species. This was demonstrated for xanthone biosynthesis
from the pharmaceutically relevant Hypericum perforatum
(Franklin et al., 2009). Nevertheless, investigations of the ge-
Vogt d Phenylpropanoid Biosynthesis | 13
O SCoA netic resources and metabolic diversity of plants with exotic
phenylpropanoid-derived compounds are worth exploring
to detect enzymes with unusual specificities for biotechnolog-
O
MeO
OH
MIXED POLYMERS, SUBERIN, CUTIN,
AND SPOROPOLLENIN
O SCoA
towards the biosynthesis of mixed polymers is a challenging
task for structural biochemists and physiologists. The complex-
MeO ity of the pathways and robustness of cutin, suberin, or sporo-
OH
pollenin towards degradation requires harsh oxidative
chemical breakdown. Inherently, there is considerable chance
of artefact formation, which hampers the precise determina-
nylpropanoid metabolism to the structural integrity of these
polymers. These polymers essentially serve completely differ-
ent functions, as a water barrier for the sessile plant in case
of suberin and cutin, or as a highly protective and rigid scaffold
for the male gametophyte. Nevertheless, their complex and
variable, highly rigid structures are based on only two building
blocks, aliphatic and aromatic. The contribution of the latter is
apparently lower than in the case of lignin. The production of
suberin is limited to a few plant tissues, like the endodermis
with the casparian strip or the phellogen or cork cambium;
its principle components have been established (Bernards,
2002). As summarized in recent reviews by Pollard et al.
(2008) and Franke and Schreiber (2007), besides linear ali-
phatic, oxygenated long chain dicarboxylic acids and esters,
in Arabidopsis, feruloyl esters comprise a significant part of
the suberin structure. These are virtually absent in cutin. Poor
substrate availability and problematic detection of intermedi-
ates render any biochemical investigations and identification
of the participating enzymes of the phenylpropanoid pathway
difficult. This includes the acyltransferase(s) connecting feru-
loyl CoA to the aliphatic, oxygenated building blocks either
in or outside the symplast. A genomic approach towards su-
berin biosynthesis and cork differentiation in Quercus robur
(cork oak) stresses the major contribution of two gene families
in the assembly of this mixed polyester. Both the cytochrome
P450 and the BAHD-like HC-tranferases (Soler et al., 2007). The
exact mechanism, namely whether there is either coordinated
or random enzymatic linkage of the phenylpropanoid-derived
aromatic blocks and the aliphatic esters in the apoplast, either
catalyzed by peroxidases or by laccases, remains to be estab-
lished. Recent data demonstrate that a sterol glycosyltransfer-
ase determines efficient trafficking of cutin and suberin to the
Arabidopsis seed coat (De Bolt et al., 2009). Much less is known
about the regulatory networks affecting composition, trans-
port, and assembly of both polymers. Candidates of R2R3s
and WRKY type transcription factors have been identified,
but await further structural and functional characterization
(Soler et al., 2007).
14 | Vogt d Phenylpropanoid Biosynthesis
Sporopollenin, the major part of the pollen exine, appar-
ently contains similar building blocks compared to cutin and
suberin, namely phenylpropanoids and hydroxylated fatty
acids. In contrast to cutin and suberin this polymer is even more
resistant to chemical degradation, although advances in chem-
ical analysis have enabled the complete identification and sep-
aration of its constituents (Dominguez et al., 1999). Based on
labeling experiments, the presence of phenylpropanoid resi-
dues is generally accepted (Schulze-Osthoff and Wiermann,
1987). The mechanical strength and durability of the pollen
exine are probably due to a substantial amount of ether-
linkages between fatty acid and/or phenylpropanoids. Conclu-
sive data on the assembly and the exact contribution of
the phenylpropanoid conjugates are virtually non-existent.
Whether the variable phenylpropanoid pattern deposited as
soluble pollenkit serves as a fingerprint of individual units in-
corporated into the polymer is also unknown. Based on func-
tional characterization of Arabidopsis knockout mutants,
initial progress has been made recently at least on the aliphatic
components of the polymer. Tissue-specific and developmen-
tally controlled members of the cytochrome P450 family con-
trol the hydroxylation pattern of the aliphatic acids, with
CYP703 and CYP704 unequivocally involved in hydroxylation
of lauric acid and omega-hydroxylation of longer chain fatty
acids, respectively (Morant et al., 2007; Dobritsaa et al., 2009).
The coordinate expression of several genes of the general phe-
nylpropanoid metabolism with CYP703 indicates the tight as-
sociation of fatty acid biosynthesis and phenylpropanoid
biosynthesis structures in the establishment of the exine bar-
rier. The precise participation of phenylpropanoids in the pro-
posed ether and ester linkages remains to be established,
although it is already known that structures with vicinal dihy-
droxy groups are required to meet the current perceptions of
polymer organization (Morant et al., 2007). The functional
aspects of phenylpropanoids in sporopollenin and exine for-
mation besides the contribution to UV-light protection may re-
side in protection against various pathogens. Biosynthesis and
transport of aliphatic and aromatic building blocks are
performed by the tapetum in specific organelles, termed tape-
tosoms (Hsieh and Huang, 2007), whereas the factors contrib-
uting to transport and polymerization are still unknown.
CONCLUDING REMARKS
The diversity within the structural and regulatory genes of
several superfamilies of enzymes involved in secondary me-
tabolism are the direct consequence of a complex plant–
environment interaction mediated in part by soluble and
insoluble secondary metabolites, including phenylpropanoids.
The rapid progress in sequencing, structural elucidation, and
analytical tools will ultimately result in the desired systems bi-
ology approach, which may start with a few model plants and
crops, like Arabidopsis, alfalfa, poplar, and rice at the begin-
ning. A thorough understanding of the biosynthetic pathway
leading to the various branches of phenylpropanoid formation
and function should also consider in more detail the following
key aspects:
(1) A more detailed investigation of transport and shuttling of
metabolites across membranes and cells.
(2) Elucidation and identification of enzyme complexes and
metabolon formation to understand how several pathways
may co-exist but are independently regulated.
(3) A thorough understanding of the molecular mechanisms
resulting in the formation of unusual phenylpropanoids in me-
dicinal and exotic plants in a continuous search for new metab-
olites and new pathways.
New analytical and biophysical methods, such as the implemen-
tation and application of nanotechnology to solve biological
problems, are currently developed for new approaches towards
resolving a most complex scenario of biological systems (Borch
and Hamann, 2009; Zhong, 2009). Combining traditional meth-
ods with these new fascinating molecular and chemistry tools
will not only contribute to a better understanding of plant nat-
ural product and phenylpropanoid biosynthesis, but encourage
farmers and consumers to benefit from the diversity and plastic-
ity of plant natural product formation and modification.
FUNDING
Financial support by the Deutsche Forschungsgemeinschaft (VO
719/8-1) is gratefully acknowledged.
ACKNOWLEDGMENTS
I would like to thank Felix Stehle (IPB Halle, Germany) for the contri-
bution of Figure 3. I am also grateful to Christin Fellenberg, Dieter
Strack (both IPB Halle, Germany), Jörg Ziegler (University of Calgary,
Canada),andtwoanonymousreviewersforcriticalreadingoftheman-
uscript and many helpful suggestions. No conflict of interest declared.
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