Plant phenolics: Recent advances on their biosynthesis, genetics, and ecophysiology

Abstract

Land-adapted plants appeared between about 480 and 360 million years ago in the mid-Palaeozoic era, originating from charophycean green algae. The successful adaptation to land of these prototypes of amphibious plants - when they emerged from an aquatic environment onto the land - was achieved largely by massive formation of “phenolic UV light screens”. In the course of evolution, plants have developed the ability to produce an enormous number of phenolic secondary metabolites, which are not required in the primary processes of growth and development but are of vital importance for their interaction with the environment, for their reproductive strategy and for their defense mechanisms.

Full text

Research article
Plant phenolics: Recent advances on their biosynthesis, genetics,
and ecophysiology
Véronique Cheynier
a
, Gilles Comte
b
, Kevin M. Davies
c
, Vincenzo Lattanzio
d
,
*
,
Stefan Martens
e
a
INRA, UMR1083 Sciences Pour l’oenologie, 2 place Viala, 34060 Montpellier Cedex 1, France
b
UMR 5557 CNRS-Université de Lyon, Ecologie Microbienne, USC INRA 1193-VetAgroSup, 6 rue Raphael Dubois, 69622 Villeurbanne Cedex, France
c
The New Zealand Institute for Plant & Food Research Limited, Private Bag 11-600, Palmerston North 4442, New Zealand
d
Dipartimento di Scienze Agrarie, degli Alimenti e dell’Ambiente, Via Napoli 25, 71100 Foggia, Italy
e
Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach (FEM), Via E. Mach 1,
38010 San Michele all’Adige, Italy
article info
Article history:
Received 30 January 2013
Accepted 10 May 2013
Available online xxx
Keywords:
Plant phenolics definition and classification
Acyltransferase
Glycosyltransferases
Biosynthesis regulation
Plant phenolics as underground and
aboveground signaling molecules
abstract
Land-adapted plants appeared between about 480 and 360 million years ago in the mid-Palaeozoic era,
originating from charophycean green algae. The successful adaptation to land of these prototypes of
amphibious plants ewhen they emerged from an aquatic environment onto the land ewas achieved
largely by massive formation of “phenolic UV light screens”. In the course of evolution, plants have
developed the ability to produce an enormous number of phenolic secondary metabolites, which are not
required in the primary processes of growth and development but are of vital importance for their
interaction with the environment, for their reproductive strategy and for their defense mechanisms.
From a biosynthetic point of view, beside methylation catalyzed by O-methyltransferases, acylation
and glycosylation of secondary metabolites, including phenylpropanoids and various derived phenolic
compounds, are fundamental chemical modifications. Such modified metabolites have altered polarity,
volatility, chemical stability in cells but also in solution, ability for interaction with other compounds
(co-pigmentation) and biological activity.
The control of the production of plant phenolics involves a matrix of potentially overlapping regulatory
signals. These include developmental signals, such as during lignification of new growth or the pro-
duction of anthocyanins during fruit and flower development, and environmental signals for protection
against abiotic and biotic stresses. For some of the key compounds, such as the flavonoids, there is now
an excellent understanding of the nature of those signals and how the signal transduction pathway
connects through to the activation of the phenolic biosynthetic genes.
Within the plant environment, different microorganisms can coexist that can establish various in-
teractions with the host plant and that are often the basis for the synthesis of specific phenolic me-
tabolites in response to these interactions. In the rhizosphere, increasing evidence suggests that root
specific chemicals (exudates) might initiate and manipulate biological and physical interactions between
roots and soil organisms. These interactions include signal traffic between roots of competing plants,
roots and soil microbes, and one-way signals that relate the nature of chemical and physical soil prop-
erties to the roots. Plant phenolics can also modulate essential physiological processes such as tran-
scriptional regulation and signal transduction. Some interesting effects of plant phenolics are also the
ones associated with the growth hormone auxin. An additional role for flavonoids in functional pollen
development has been observed. Finally, anthocyanins represent a class of flavonoids that provide the
orange, red and blue/purple colors to many plant tissues. According to the coevolution theory, red is a
signal of the status of the tree to insects that migrate to (or move among) the trees in autumn.
Ó2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
“We live in a chemical world, dominated by color, by scent and
by taste. Many animal phyla have developed a discriminatory acuity
*Corresponding author.
E-mail addresses: cheynier@supagro.inra.fr (V. Cheynier), gilles.comte@univ-
lyon1.fr (G. Comte), kevin.davies@plantandfood.co.nz (K.M. Davies), v.lattanzio@
unifg.it (V. Lattanzio), stefan.martens@fmach.it (S. Martens).
Contents lists available at SciVerse ScienceDirect
Plant Physiology and Biochemistry
journal homepage: www.elsevier.com/locate/plaphy
0981-9428/$ esee front matter Ó2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.plaphy.2013.05.009
Plant Physiology and Biochemistry xxx (2013) 1e20
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in these latter two senses which rivals their sight”..“For the
majority of living organisms chemical signals are the main means of
communication”..“The majority of chemical signals are complex.
They contain mixtures of many different compounds, the majority
of which are the so called secondary products”[1]. A typical char-
acteristic of plants and other sessile organisms is their capacity to
produce a vast and diverse array of the so-called secondary me-
tabolites, i.e. compounds present in specialized cells that are not
directly essential for basic photosynthetic or respiratory meta-
bolism, but are thought to be required for plant survival in the
environment. Phenolic compounds are the most widely distributed
secondary metabolites, ubiquitously present in the plant kingdom,
even if the type of compounds present varies according to the
phylum under consideration. Phenolics are uncommon in bacteria,
fungi and algae. Bryophytes are regular producers of phenolic
compounds, including polyphenols such as flavonoids, but it is in
the vascular plants that the full range of phenolics is found. Leaves
of vascular plants contain esters, amides and glycosides of
hydroxycinnamic acids, glycosylated flavonoids, especially flavones
and flavonols, and proanthocyanidins and their relatives. Lignin,
suberin and pollen sporopollenin are examples of phenolic con-
taining polymers [2e4].
As a general rule recently proposed by Quideau et al. [5], the
term ‘plant phenolics’should be strictly used to refer to secondary
natural metabolites arising biogenetically from the shikimate/
phenylpropanoid pathway, which directly provides phenyl-
propanoids (Fig. 1), or the ‘polyketide’acetate/malonate pathway,
which can produce simple phenols, or both of them. These path-
ways produce a bewildering array of monomeric and polymeric
structures (the term ‘polyphenols’defining those with more than
one phenolic ring) that fulfill a very broad range of physiological
roles in plants. Although the bulk of these compounds play cell wall
structural roles, plant tissues synthesize a vast array of non-
structural constituents that have various roles in plant growth
and survival. Thus, the expression “plant phenolics”embraces a
highly diverse group with an extremely large structural diversity:
tens of thousands of diverse structures have been identified, with
the number continually increasing [5,6].
Land-adapted plants appeared between about 480 and 360
million years ago in the mid-Palaeozoic era. From a simple plant
body consisting of only a few cells, land plants (embryophytes)
consisting of liverworts, hornworts, mosses and tracheophytes
originated from charophycean green algae. The ability to synthesize
phenolic compounds has been selected throughout the course of
evolution in different plant lineages when such compounds
addressed specific needs, thus permitting plants to cope with the
constantly changing environmental challenges over evolutionary
time. For example, the successful adaptation to land of some higher
members of the Charophyceae ewhich are regarded as prototypes
of amphibious plants that presumably preceded true land plants
when they emerged from an aquatic environment onto the land e
was achieved largely by massive formation of “phenolic UV light
screens”[7e10]. The ultraviolet part of the solar radiation is capable
of promoting the cleavage of chemical bonds. Hence the organic
compounds of the prebiotic phase and the primordial organisms
were viable only in aquatic medium, protected by the UV absorbing
oxygen of H
2
O. Thus, about half a billion years ago, when invading
humid environments of the land surface, possibly through
Fig. 1. Schematic of the major branch pathways of (poly)phenol biosynthesis. PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl:CoA-ligase;
HCT, hydroxycinnamoyl transferase; C3H, p-coumarate-3-hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase; ANS, anthocyanidin synthase; DFR, dihydroflavonol
reductase; FS, flavone synthase; FLS, flavonol synthase; F3H, flavanone 3-hydroxylase; IFS, isoflavone synthase; ANR, anthocyanidin reductase; LAR, leucoanthocyanidin reductase.
V. Cheynier et al. / Plant Physiology and Biochemistry xxx (2013) 1e202
Please cite this article in press as: V. Cheynier, et al., Plant phenolics: Recent advances on their biosynthesis, genetics, and ecophysiology, Plant
Physiology and Biochemistry (2013), http://dx.doi.org/10.1016/j.plaphy.2013.05.009

primitive members of the bryophytes, the plant phylum must have
solved the problem of the UV screen. The most conspicuous
chemical difference between aquatic protoctists and terrestrial
plants is the exploitation of the shikimate pathway. In all forms of
algae, this is limited to the production of phenylalanine and tyro-
sine, already incorporated in proteins since the primitive bacteria.
1
Only with the bryophytes, a post-tyrosine chemistry, based on
cinnamic acid, is initiated. Deamination of aromatic amino acids to
cinnamic acids may have occurred sporadically in other groups and
certainly occurs in fungi. Polyketides, compounds formed by the
condensation of acetyl-CoA as starter unit and malonyl-CoA for
chain extension, dominate the allelochemistry of aerobic bacteria
and of algae. The substitution in this process of the starter unit by
cinnamoyl-CoA in bryophytes led to flavones and flavonols. Flavo-
noids, such as chalcones, aurones, flavones and flavonols, absorb
UV light and should act as photoscreens in Bryophyta and in all
divisions of more modern terrestrial plants [11e13]. Apart from UV
radiation, there exist other phenomena in an aerial environment,
such as pests and pathogens, requiring special adaptation of plant
life. It is again polyphenol chemistry that conditions the adaptation
to these environmental challenges. For example, reductive devia-
tion from the biosynthetic route leading to anthocyanidins from
leucoanthocyanidins results in catechins. Polymerization of repre-
sentatives belonging to the two latter classes of flavonoid com-
pounds gives the so-called condensed tannins, important general
defense against virus, bacteria, fungi, insects and herbivores [14,15].
Several classes of phenolics have been categorized on the basis
of their basic skeletons: C
6
(simple phenols, benzoquinones), C
6
eC
1
(phenolic acids and aldehydes), C
6
eC
2
(acetophenones, phenyl-
acetic acids), C
6
eC
3
(hydroxycinnamic acids, coumarins, phenyl-
propanes, chromones), C
6
eC
4
(naphthoquinones), C
6
eC
1
eC
6
(xanthones), C
6
eC
2
eC
6
(stilbenes, anthraquinones), C
6
eC
3
eC
6
(flavonoids, isoflavonoids, neoflavonoids), (C
6
eC
3
eC
6
)
2,3
(bi-, tri-
flavonoids, proanthocyanidin dimers, trimers), (C
6
eC
3
)
2
(lignans,
neolignans), (C
6
eC
3
)
n
(lignins), (C
6
)
n
(catechol melanins, phlor-
otannins), (C
6
eC
3
eC
6
)
n
(condensed tannins). Low-molecular
weight phenolics occur universally in higher plants, some of them
are common in a variety of plant species and others are species
specific[2,16e18].
Arbutin (hydroquinone
b
-
D
-glucoside, I), is an example of simple
phenol found in leaves of various Vaccinium spp., such as blueberry,
cranberry, cowberry, and pear trees (Pyrus communis L., Rosaceae).
It is of sufficiently restricted occurrence to have been proposed as a
marker for adulteration of other juices by pear juice [19]. Phenolic
acids are usually present in the bound soluble form conjugated
with sugars or organic acids and are typically components of
complex structures such as lignins and hydrolyzable tannins [20].
Picein (II), a C
6
eC
2
compound, which has been found as the main
component of spruce needles (Picea abies (L.) Karst.), also occurs in
Larix decidua Mill., Populus balsamifera L., and Salix spp. The general
plant metabolism of phenylpropanoids furnishes a series of
hydroxycinnamic acids such as chlorogenic acid (i.e. 5-O-caffeoyl-
quinic acid, Fig. 1), ()-chicoric acid (dicaffeoyltartaric acid, III),
rosmarinic acid (IV), and verbascoside (V). Coumarins, which are
also C
6
eC
3
derivatives, are benzo-
a
-pyrones (lactones) formally
derived from o-hydroxycinnamic acids by cyclization and ring
closure between the o-hydroxy and carboxyl groups. This group of
phenolic compounds can be found free in nature or in combined
form with sugars as heterosides and glycosides in many eudicoty-
ledon (eudicot) families, including the Apiaceae,Asteraceae,
Fabaceae,Moraceae,Rosaceae,Rubiaceae and Solanaceae [21].
Naphthoquinones, such as plumbagin (VI), represent a class of
quinone pigments widespread in nature. The most notable higher
plant families containing naphthoquinones are the Avicenniaceae,
Bignoniaceae,Boraginaceae,Droseraceae,Ebenaceae,Juglandaceae,
Nepenthaceae, and Plumbaginaceae. They are biosynthesized via a
variety of pathways including the polyketide pathway, the shiki-
mate/succinyl CoA combined pathway and the shikimate/mevalo-
nate pathway [22]. Xanthones are a class of plant phenolics
occurring in only a few higher plant families (Gentianaceae,Gutti-
ferae,Logoniaceae,Podostemaceae, and Polygalaceae), therefore they
have a high taxonomic value in such families. Mangiferin (VII) is
unique among the natural xanthones in having a much wider
natural occurrence than that of any of the others. This 2-C-gluco-
side of 1,3,6,7-tetrahydroxyxanthone was first found in the leaves
of Mangifera indica L. [23e25]. The members of the stilbene family
have the C
6
eC
2
eC
6
structure and are widely distributed in the Plant
Kingdom, although some structures are characteristic of particular
plant families. They are found in liverworts, in some ferns, in
gymnosperms and in many eudicot angiosperms, ranging from the
unsubstituted trans-stilbene from Alnus and Petiveria to the hex-
asubstituted combretastatin A-1 (VIII) from Combretum caffrum
(Eckl. & Zeyh.) Kuntze and also including various glycosylated and
acylated derivatives [26,27]. Flavonoids and their conjugates form a
very large group of natural products with more than 10,000
different identified structures [17,28,29]. They are found in many
plant tissues, both inside the cells and on the surfaces of different
plant organs. The chemical structures of this class of compounds
are based on a C
6
eC
3
eC
6
skeleton. Depending on the position of the
linkage of the aromatic ring to the benzopyrano (chromano) moi-
ety, this group of natural products may be divided into three clas-
ses: the flavonoids (2-phenylbenzopyrans, e.g. kaempferol,
apigenin, Fig. 1), isoflavonoids (3-phenylbenzopyrans, e.g. genis-
tein, Fig. 1), and the neoflavonoids (4-phenylbenzopyrans).
Together with the proanthocyanidins, the bi- and triflavonoids
constitute the two major classes of complex C
6
eC
3
eC
6
plant phe-
nolics. These compounds arise from the oxidative coupling of
various flavonoid structures and thus predominantly possess a
carbonyl group at C-4 or its equivalent in every constituent unit.
However, a multitude of compounds that do not arise via the
phenol oxidative coupling of monomeric flavonoid structures
possessing C-4 carbonyl functional groups are also classified as bi-
and triflavonoids [17,30]. Lignans and neolignans are a large and
varied group of plant phenolics produced by the oxidative dimer-
ization of two phenylpropanoid units, which occur in a wide range
of plant species. Lignans are phenylpropanoid dimers that are CeC
linked mostly through their C
3
-side chains (tail-to-tail such as
pinoresinol, Fig. 2). Neolignans, such as eusiderin, are phenyl-
propanoid dimers that are linked head-to-tail [31,32]. Lignin, the
essential structural polymer of wood and second only to cellulose
as the most abundant organic substance in plants, is found as an
integral cell wall constituent of all vascular plants. Gymnosperm
lignins are primarily derived from coniferyl alcohol, and to a lesser
extent, p-coumaryl alcohol, whereas angiosperm lignins contain
coniferyl and sinapyl alcohols in roughly equal proportions. The
plant tannins are phenolic compounds of relatively high molecular
weight that have the ability to complex with carbohydrates and
proteins. In higher plants, tannins consist of two major groups of
metabolites: the hydrolyzable tannins and proanthocyanidins also
known as condensed tannins. The latter are very abundant poly-
phenols in woody plants, but are less common in herbaceous plants
and often restricted to specific tissues (e.g. seed coat of Arabidopsis
thaliana L. or alfalfa). Hydrolyzable tannins have a more restricted
occurrence than proanthocyanidins, being found in only 15 of the
40 orders of eudicots [16e18]. More recently a third class of tannins,
1
Some brown algae synthesize phlorotannins, i.e. phloroglucinol polymers
arising from the acetate-malonate pathway, which may also have a role as UV
protectants.
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the phlorotannins, has been isolated from several genera of algae.
The phlorotannins are composed almost entirely of phloroglucinol
sub-units linked in some cases by CeC and in others by CeOeC
(aryl ether) bonds. Hydrolyzable tannins are cleaved by acids, bases
and in some cases by hydrolytic enzymes (tannase) into sugars
(usually
D
-glucose) or related polyols and a phenolic acid. In the
case of gallotannins, which are polygalloyl esters, this is gallic acid.
Ellagitannins can be defined in a narrow sense as hexahydrox-
ydiphenoyl esters of carbohydrates or cyclitols, while the definition
of ellagitannins in a wider sense also cover compounds derived
from further oxidative transformations. When the hexahydrox-
ydiphenoyl group is cleaved from the molecule, the parent acid
rapidly lactonizes to yield the dilactone ellagic acid (IX). The
proanthocyanidins are flavan-3-ol oligomers and polymers that
produce anthocyanidins by cleavage of their CeC interflavanic
bonds (or CeC and CeOeC in A-type proanthocyanidins) under
strongly acidic conditions. Proanthocyanidins can occur as poly-
mers of up to 50 units [18,33e36]. Although much less common,
flavano-ellagitannins also occur in some plants (e.g. acutissimin A
and B (XeXI) first isolated from Quercus acutissima Carruthers) [16].
Finally, melanins are pigments of high molecular weight formed by
the oxidative polymerization of phenolic compounds and usually
are dark brown or black. In general, they are conjugated polymers
of ortho-dihydroxyphenols. The more general classification of such
compounds contains three main types of such polymers: (i)
eumelanins (black or brown) that are produced in the course
of oxidation of tyrosine (and/or phenylalanine) to 3,4-
dihydroxyphenylalanine (DOPA) and dopaquinone, which
further undergoes cyclization to 5,6-dihydroxyindole or 5,6-
dihydroxyindole- 2-carboxylic acid; (ii) pheomelanins (yellow,
red or brown) that are initially synthesized just like eumelanins,
but DOPA undergoes cysteinylation, directly or by the mediation of
glutathione, then polymerizes; and (iii) allomelanins (black),
the most heterogenous group of polymers, which emerge
through oxidation/polymerization of dihydroxynaphthalene or
tetrahydroxynaphthalene, homogentisic acid,
g
-glutaminyl-4-
hydroxybenzene, 4-hydroxyphenylacetic acid, as well as of cate-
chols [37e39].
O
HO
HO
OH
O
OH
OH
HO
O
O
HO
COOH
OH
OH
HO
OH
OH
OH
O
O
O
HO
HO
OH
OH
O
HO
OH
O
O
O
OHO
HO O
O
O
H
O
H
O
O
OH
O
OH
O
O
OH
HO
HO
H3C
OH
OH
O
HO
HO
OH O
O
CH3
O
HO
HO
OH
OH
O
CH3
O
OH
OH
OCH3
H3CO
OCH3
H3CO
III III
IV
V
VI VII VIII
IX
HO
HO OH
OH
OHHO
CO CO
O
O
O
O
CO CO
OH
HO
HO
HO
HO OH HO OH
OH
OC
OH
O
OH
OH
HO
OH
OH
6
XX I
O
OH
HO OH
OH
HO
HO
HO OH
OH
OHHO
CO CO
O
O
O
O
CO CO
OH
HO
HO
HO
HO OH HO OH
OH
OC
OH
8
HO
HO
OH
OH
O
O
O
O
V. Cheynier et al. / Plant Physiology and Biochemistry xxx (2013) 1e204
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As far as physico-chemical properties of plant phenolics are
concerned, the interaction of the hydroxyl groups of phenolics with
the
p
-electrons of the benzene ring gives the molecules special
properties, most notably the ability to generate free radicals where
the radical is stabilized by delocalization. The formation of these
relatively long-lived radicals is able to modify radical-mediated
oxidation processes. Phenolics that possess two ortho-positioned
hydroxyl groups are very good antioxidants, though the down
side of this is their relative instability toward oxidation to quinones
during storage or processing of plant foods. These latter oxidative
reactions are involved in both browning phenomena in raw fruit
and vegetables during handling and storage and resistance mech-
anism of plant tissues against fungal pathogens, quinones being
transient intermediates that rapidly undergo condensation re-
actions, thus forming melanins. In addition, many phenolics chelate
metal ions, but tight binding requires ortho-hydroxyl groups such
as those found on catechol group. Other properties of biological
relevance also come from the presence of the phenolic hydroxyl
groups. Owing to the potential for electronic delocalization, the
phenolic groups are readily ionized and thus act as weak acids. The
other main feature of phenolic hydroxyl groups is that they are
good H donors in the formation of hydrogen bonds. Some of the
polymeric phenolics carry very large numbers of such donor
groups, with the result that complexes formed with other mole-
cules are extremely stable and tend to precipitate out. Phenolic
compounds are usually white solids, though the complex electronic
conjugation of some of the flavonoids results in a yellow color, or
even red in the anthocyanins. An intriguing feature of anthocyanins
is the way that their colors vary with time and with pH. The an-
thocyanins are red in acid solutions but the color decreases as the
pH is raised, because of the conversion of the red flavylium cation
form to colorless hemiketal and blue quinonoidal bases though
hydration and deprotonation reactions, respectively. Neutral and
alkaline solutions are violet or blue when freshly made, but fade
slowly. In vivo, there exist stabilizing mechanisms that favor colored
structures by preventing hydration of the flavylium cation. Four
possible stabilization mechanism have been classified: (i) self-
association of anthocyanins; (ii) intermolecular co-pigmentation
with other non-covalently bound colorless substances such as fla-
vonoids, alkaloids, nucleic acids and amino acids; (iii) intra-
molecular co-pigmentation with aromatic acyl groups (namely
hydroxycinnamic acids); and (iv) metal complex formation [40].
Finally, certain low-molecular-weight phenolics are however quite
volatile, and often possess characteristic aromas, such as vanillin,
eugenol, etc. Some phenolics are quite hydrophilic (e.g. hydrox-
ycinnamic acid glycosides, quinate esters, etc), but others can be
quite hydrophobic (e.g. polymethoxy flavonoids) [34,40e42].
2. Biosynthesis, genetics and metabolic engineering
2.1. Advances in understanding decorating steps in the biosynthesis
of polyphenols
Plants have developed the ability to produce an enormous
number of secondary metabolites that are not required in the pri-
mary processes of growth and development but are of vital
importance for their interaction with the environment. This ‘che-
modiversity’is particularly developed in land plants, which have to
face numerous environmental challenges, and in particular in
vascular plants that also need to maintain efficient transport of the
metabolites, structural rigidity and a fine regulation of homeostasis
[43]. Beside methylation catalyzed by O-methyltransferases, acyl-
ation and glycosylation of secondary metabolites, including phe-
nylpropanoids and various derived phenolic compounds, are
fundamental chemical modifications. Such modified metabolites
have altered polarity, volatility, chemical stability in cells and so-
lution, ability for interaction with other compounds (co-pigmen-
tation), and biological activity. Furthermore, these modifications
play pivotal roles in many of the important processes mentioned
above. The alteration of natural compounds with glycosyl and acyl
Fig. 2. Parent structures of lignane and neolignanes.
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moieties is catalyzed by acyltransferases (AT) and glycosyl-
transferases (GTs) and generates thousands of molecular variants
[44]. Both steps usually occur in a regiospecific reaction after the
completion of the biosynthesis of the respective aglycone [45].
Although a wide array of cDNAs encoding members of both
enzyme groups have been cloned from different plant species and
tissues, the catalytical function is only described for a minor part of
gene products emainly using recombinant enzymes in vitro [43].
Both classes of enzymes are characterized by catalytic versatility,
which normally makes functional predictions from primary
sequence alone unfeasible. For almost a decade, our knowledge on
the organization of the gene families has been limited to the model
plant A. thaliana (L.) Heynh, as the largest numbers of flavonoid-
related genes have been identified from this plant by the exten-
sive use of various “omics”based approaches [46]. Nowadays, the
availability of more and more plant genomes and protein crystal
structures present possibilities to obtain a broader view of the
families of these two important protein classes in terms of func-
tional evolution and the key structural elements determining
the specificities, particularly regiospecificities and catalytical
properties. Integrated approaches including metabolite profiling,
transcriptomics, sequence comparison, modeling, biochemical
characterization and the use of comparative genomics plus genetic
engineering can be successfully applied to identify substrate re-
quirements and activity of the large number of candidate genes
predicted in plant genomes [43,47,48].
2.1.1. Acyltransferases (AT)
Enzymatic acylation confers aliphatic and/or aromatic acyl
moieties onto the nucleophile (OHeor NHe) of acceptor molecules
with ester and amide bonds. Coumaroyl or sinapoyl groups are
usually involved in aromatic acylation, which leads to stabilization
of the molecule and intensifies the color of anthocyanins. Malo-
nylation is the typical aliphatic acylation and is important for sta-
bility of the molecules but also for water solubility and protection
upon degradation by enzymes [49]. Different types of acylations are
widespread in the secondary metabolism of plants. Predominantly,
N-orO-acylation is involved in forming and modifying phenolics
and polyphenolics but also alkaloids and terpenoids with various
ecophysiological roles [47,48,50].
The BAHD ATs, named according to the first characterized
members (BEAT, AHCT, HCBT, and DAT), comprise a large family of
plant specific monomeric acyl-CoA-utilizing and functionally diverse
enzymes, showing only 10e30% similarity at the amino acid level. A
genome wide analysis across five angiosperm taxa supported a
refined grouping of eight major clades for this family. Newly
discovered clade specific motifs should facilitate functional studies of
substrate and donor specificities among different BAHD enzymes.
These data strongly support the view that the expansion of BAHD
family in different lineages is linked to taxon-specific metabolic di-
versity [48]. Beside the alcohol acetyltransferases, the second iden-
tified functional subfamily within BAHD ATs is formed by
anthocyanin/flavonoid ATs, such as hydroxycinnamoyltransferases
and malonytransferases involved in modification of various poly-
phenols [50]. These proteins are assumed to be located to the cytosol,
since a respective transit peptide for translocation to other sub-
cellular sites has not been identified so far. Products synthesized
include modified anthocyanins, small green-leaf volatile esters,
lignin, suberin as well as defense compounds and phytoalexins [47].
Regarding substrate usage, some members have a more restricted
and others a wider preference, at least in in vitro experiments.
However, due to the versatility in their substrate specificities, the
prediction of function based only on structural information isa major
problem and therefore the great majority of BAHD ATs are unchar-
acterized so far as regards their substrates and products [49].
Recently, a novel class of ATs, serine carboxypeptidase-like
(SCPL) acyltransferases, was characterized. Proteins belonging to
this class facilitate transacylation reactions of various secondary
metabolites using energy-rich 1-O-
b
-glucose esters in the synthesis
[51]. These vacuolar proteins define an alternative cellular route of
transacylation spatially separated from the cytoplasmic enzymes of
the BAHD AT family. Recent efforts in cloning and characterization
led to the identification of diagnostic peptides for SCPL acyl-
transferases, enabling the detection of candidate genes in several
plant genomes. Detailed biochemical analysis of SCPL acyl-
transferases is strongly dependent on comprehensive heterologous
expression systems, efficient protein purification protocols, and the
supply of appropriate substrates. Obviously, SCPL ATs have evolved
from a hydrolytic ancestor by adapting functional elements of the
proteases such as the catalytic triad, oxyanion hole, and substrate
recognition H-bond network to their new function [52].
2.1.2. Glycosyltransferases (GT)
GTs are a ubiquitous group of enzymes that catalyze the transfer
of a sugar moiety to a wide range of acceptor molecules from an
activated donor molecule. Beside plant secondary metabolites also
sugars, lipids, proteins, nucleic acids, antibiotics and other small
molecules can serve as potential substrates for GT proteins [53]. The
glycosylation results in the formation of poly-glycosides, di-sac-
charides, and various glycosides of non-carbohydrate moieties. The
biosynthesis of such molecules is of great biological importance
due to the diverse functions that they carry out within the organism
and involves the action of hundreds of different, and more or less
selective, GTs [43,54]. GTs that typically transfer sugars onto small
molecules are classified into family 1 of a classification scheme that
currently includes 94 GT families (CAZy database; www.cazy.org;
April 2013). This family, often referred to as UDP glycosyl-
transferases (UGTs), is the largest enzyme family in the plant
kingdom and comprises highly divergent enzymes from plants,
animals, fungi, bacteria, and also viruses. In plants, UGTs utilize
UDP-activated sugars (e.g. glucose, galactose, arabinose) as the
major donor molecule and contain the well-conserved UDP-gly-
cosyltransferases-defining motif as a unifying feature, which is one
of the few regions of significant sequence similarity. The so-called
PSPG motif (Plant Secondary Product Glycosyltransferase) is a
carboxyl-terminal consensus sequence of 44 amino acid residues
and is thought to represent the nucleotide-diphosphate-sugar
binding site of the enzymes ([43] and refs therein). UGTs involved
in plant secondary metabolism often display broad substrate
specificity, at least in in vitro experiments, with recombinant pro-
teins recognizing a wide range of natural products as acceptor
molecules. This promiscuity could also contribute to the immense
skeletal variations of small molecules regarding their glycosylation
pattern. However, UGTs can also be selective and the substrate
specificities of some enzymes are defined by regiospecificor
regioselective features of the aglycones [55]. Consistent with the
structural diversity of plant secondary metabolites, a large number
of genes encoding UGTs are present in plant genomes, e.g. more
than 100 in A. thaliana, 180 in Vitis vinifera L. or 240 in Malus x
domestica Borkh. [43].
To date, hundreds of UGTgenes from model and non-model plants
including crops, ornamentals and medicinal plants have been cloned
and functionally characterized by in vitro and/or in vivo studies.
However, considering the number of UGTs present in a plantgenome,
the numberof functionallycharacterizedproteinsis still relativelylow
[43,53]. Furthermore, a number of studies indicated the fact that
drawingconclusions on the in vivo activity of the UGTs can be difficult
and in vitro activity studies can sometimes be misleading. For
instance, the A. thaliana enzyme UGT73C6 was characterized as a
UDP-glucose:flavonol-3-O-glycoside-7-O-glucosyltransferase from a
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Physiology and Biochemistry (2013), http://dx.doi.org/10.1016/j.plaphy.2013.05.009

T-DNA knock-out line lacking quercetin-3-O-rhamnoside-7-O-
glucoside and in vitro assays using the recombinant protein [56].The
authors found that the enzyme was also able to convert several
flavonoid aglycones, such as kaempferol, quercetin, apigenin and
genistein to glycosylated products. However, flavones (apigenin) and
isoflavones (genistein) are not naturally occurring metabolites in
A. thaliana.Recently,Husar et al. [57] reported UGT73C6 as an enzyme
that glucosylatesbrassinosteroids in A. thaliana.Another examplewas
given byWitte et al. [58] describing an invitro glycosylation p attern of
Hieracium pilosella L. UGTs that could not be attributed directly to
in vivo functions based on the metabolites described in plant tissues.
These examples highlight the basic problem scientists face when
approaching the study of the functions of these enzymes that in some
cases are selective and can display regio- and stereo-specificity but in
other cases can be very promiscuous, recognizing a range of sub-
strates and producing multiple products that may not befound in the
plant. The release of more and more plant genomes is nowadays
providing a massive amount of information regarding the genes and
the multigene families involved in secondary metabolites biosyn-
thesis. This knowledge can be used for different purposes, such as the
understanding of the molecular adaptations occurred during plant
evolution, the elucidation of new or not well known metabolic
pathways and the identification of genes that could be exploited for
biotechnological applications. Some recent examples are given in
more detail in the following section.
Two novel UGTs from A. thaliana involved in anthocyanin
biosynthesis were identified by transcriptome coexpression anal-
ysis and independent component analysis. Knock out mutants with
a drastically reduced anthocyanin content and enzyme assays with
the respective recombinant proteins revealed that UGT79B1 en-
codes for an anthocyanin 3-O-glucoside:2
00
-O-xylosyltransferase.
The second candidate, UGT84A2, is known to encode sinapic
acid:UDP-glycosyltransferase and knock-out contribution of
UGT84A2 in sinapoylation of anthocyanins is postulated [46]. The
synthesis of cyanidin 3-O-xylosyl-galactoside in red-fleshed kiwi-
fruit (Actinidia chinensis Planch.) involves the action of two UGTs. In
afirst step F3GT1 is responsible for the formation of cyanidin 3-O-
galactoside, as clearly demonstrated by recombinant enzyme as-
says and RNAi experiments. The recombinant F3GGT1 was able to
use this product as a substrate of the transfer of UDP-xylose to the
galactose moiety. Apparently the first glycosylation step serves as
the key step for anthocyanin accumulation in the red-fleshed fruit
of A. chinensis [59]. 3-Deoxyanthocyanins have only been identified
in some plant species but are the main flower pigments in Ges-
neriaceae and Bignoniaceae, providing a bright red or orange-red
color. A highly specific5-O-glucosyltransferase was cloned from
Sinningia cardinalis (Lehm.) H. E. Moore, which accumulates 3-
deoxyanthocyanins in its flowers. The recombinant protein only
transferred the glucosyl moiety from UDP-glucose to the 3-
deoxyanthocyanidins apigeninidin and luteolinidin and would
not accept 3-hydroxyanthocyanidins or flavones, flavonols, and
flavanones [60]. The effect of glycosylation pattern on flower color
was demonstrated in blue-flowered Veronica persica Poiret. The
predominant and highly glycosylated anthocyanin is accompanied
by apigenin 7-O-(2-O-glucuronosyl)-glucuronide, which results in a
bathochromic shift toward blue. Two UGTs were isolated by reverse
genetics and shown to encode an anthocyanin 3-O-glucoside-2
00
-O-
glucosyltransferase and flavonoid 7-O-glucuronosyltransferase,
respectively. The preferred expression of both genes and the
obvious bathochromic effect of the flavone led to the suggestion
that they are involved in the bluish coloration in flowers [61].
Grapevine (V. vinifera) is one of the most important fruit crops
worldwide and has been cultivated since ancient times. In the
recently published genome, 181 putative UGTs were identified [43]
but only a few of them were functionally characterized. Two of
them, VvGT5 and VvGT6, were described as UDP-glucuronic
acid:flavonol-3-O-glucuronosyltransferase (GAT) and bifunctional
UDP-glucose/UDP-galactose:flavonol-3-O-glucosyltransferase/gal-
actosyltransferase, respectively. The genes share high sequence
similarity, which led to the suggestion that these genes are a result
of a gene duplication and subsequent neofunctionalization due to
minor amino acid modifications. The gene products contribute to
the structural diversification of flavonols described in grapes [45].
Ectopic overexpression of two R2R3MYB (myeloblastosis) tran-
scription factors involved in proanthocyanidin (PA) biosynthesis in
grape berries led to the identification of three differentially
expressed cytoplasmatic UGTs when compared to control lines. The
respective recombinant proteins were able to catalyze the synthesis
of 1-O-acyl glucose esters of various hydroxybenzoic and hydrox-
ycinnamic acids. Surprisingly, none of the proteins accepted fla-
vonoids or stilbenes as substrate. Transcripts were only detectable
in early stages of berry development in skins and seeds, leading to
the proposal that the enzymes could be involved in PA galloylation
using glucose esters as intermediates or the formation of hydrox-
ycinnamic esters in vivo [62]. In a similar approach, Pang et al. [63]
expressed the TRANSPARENT TESTA 2 (TT2) transcription factor (an
R2R3MYB) of A. thaliana in hairy roots of Medicago truncatula
Gaertn. and identified a UGT (UGT72L1) that has unique enzymatic
activity toward ()-epicatechin, resulting in the formation of the
respective 3
0
-O-glucoside. Since the expression pattern in devel-
oping seeds could be correlated with the presence of the product
and the accumulation of PAs it was postulated that UGT72L1 is
involved in PA biosynthesis or its regulation [63].
Flavanone glycosides are known to determine the bitterness that
is a flavor characteristic of some citrus fruits. The underlying
biosynthetic step is catalyzed by two rhamnosyltransferases that
utilize, beside others, flavanone 7-O-glucose as the main substrate.
However, only the flavanone 7-O-neohesperidosides (rhamnose-2-
glucose; e.g. neohesperidin and naringin) led to a bitter taste while
flavanone 7-O-rutinosides (rhamnose-6-glucose; e.g. hesperidin and
narirutin) are tasteless. Common to both compounds is the initial O-
glucosylation at position 7, which is catalyzed by a 7-O-glycosyl-
transferase. Subsequently, a rhamnosyltransferase is involved in the
second step. Accordingly, a 1,2-rhamnosyltransferase (1,2RhaT) was
cloned from bitter pummello(Citrus maxima Burm.) and the encoded
enzyme was found to specifically catalyze rhamnosylation of flava-
none- and flavone 7-O-glucosides at position 2 of the glucose moiety.
The counterpart from non-bitter oranges (Citrus sinensis (L.) Osbeck)
was recently identified as 1,6-rhamnosyltransferase (1,6RhaT). This
enzyme directs the biosynthesis to the tasteless flavanones and
seems to be a more promiscuous enzyme with regard to its substrate
specificity compared to 1,2RhaT, since rhamnosylation was found
with 3- or 7-O-glycosylated flavanones, flavones, flavonols and an-
thocyanins at position 6 of the glucose. The encoding genes appear to
be only distantly related and it is hypothesized that they originated
separately before specification of the citrus genus [64].
A new reaction mechanism for the sugar transfer was described
for two novel acyl-glucose-dependent anthocyanin glycosyl-
transferases (AAGTs) from Dianthus caryophyllus L. (carnation) and
Delphinium grandiflorum L. (delphinium). These enzymes were
shown to catalyze the 5- and 7-O-glucosylation of anthocyanidins,
respectively, which had been generally been thought to be cata-
lyzed by UGTs. Variation of petal color of carnations was observed
depending on the presence or absence of the glucosylation at the
position 5 [65]. Both enzymes were using various aromatic acyl-
glucose instead of UDP-glucose as sugar donor. Furthermore, they
showed a strict acceptor preference for anthocyanins, with no ac-
tivity detected with various other flavonoids and phenolic com-
pounds. Based on the obtained amino acid sequences, both
enzymes were classified as members of glycoside hydrolase family
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1 (GH1), which are usually known to act as beta-glycosidases. Re-
sidual GH activity was detectable with the recombinant protein but
was negligible compared to GT activity. GH1s are involved in
several fundamental processes such as plant defense, lignification,
hydrolysis of oligosaccharides and phytohormone regulation [66].
In contrast to UGTs most of the GH1 proteins seems to have puta-
tive transit peptide sequences that are necessary for localization in
the endoplasmic reticulum, peroxisomes, mitochondrion, plasma
membrane, and for the sequestration into vacuoles. Both proteins
reported by Matsuba et al. [66] contain a putative transit peptide
for localization in the vacuole, indicating that the two proteins may
function in noncytosolic compartments. Recently, the activity of an
AAGT was detected also in flowers of Agapanthus africanus (L.)
Hoffmanns, a monocot ornamental plant. The isolated cDNA was
designated as an AA7GT and the recombinant protein shows similar
biochemical properties as the Dianthus and Delphinium proteins
[67,68]. In parallel, a bioinformatics approach based on phyloge-
netic analysis led to the identification of up to eleven GH1 candi-
date genes from the genome of A. thaliana with unknown function.
Based on gene expression analysis only three were postulated as
functional AAGT candidates. These approaches give rise to the
assumption that AAGTs were derived from glucosidases early dur-
ing the evolution of angiosperm [68].
2.2. Regulation of polyphenol production
The control of the production of polyphenols involves a matrix
of potentially overlapping regulatory signals. These include devel-
opmental signals, such as during lignification of new growth or the
production of anthocyanins during fruit and flower development,
and environmental signals for protection against abiotic and biotic
stresses. For some polyphenols, such as the flavonoids, there is now
an excellent understanding of the nature of those signals and how
the signal transduction pathway connects through to the activation
of the biosynthetic genes. In this section of the review we provide
an update of recent advances in the understanding of the molecular
mechanisms for the regulation of polyphenol production. No
attempt is given to make a comprehensive review of the field, but
rather to highlight significant areas of new knowledge.
Studies across a range of systems, including flower develop-
ment, berry ripening and stress-induced vegetative pigmentation,
have found that the regulation of flavonoid production occurs
principally through changes in the transcription rate of the
biosynthetic genes. This is achieved through the action of tran-
scription factors (TFs). Several types of TF have been identified that
regulate flavonoid biosynthesis, with the general types of TF
involved apparently conserved across monocots and eudicots, with
some of these also now identified for gymnosperms [reviewed in
Refs. [69e71]]. Central to the direct regulation of anthocyanin and
proanthocyanidin (PA) biosynthetic genes are core ‘MBW’regula-
tion complexes, comprising of specific members of the R2R3MYB
and basic helix-loop-helix (bHLH) TF families in conjunction with a
WD-repeat (WDR; tryptophan-aspartic acid (W-D) dipeptide
repeat) protein. Variant MBW complexes can form from different
MYB and bHLH components, and these can have different target
genes and vary in their activation or repression actions. The WDR
protein is common to all the variant MBW complexes. The bHLH
component may also be common to MBW complexes targeting
different biosynthetic pathways. However, distinct R2R3MYBs are
involved in regulating the different branches of flavonoid produc-
tion, such as anthocyanins or PAs. Furthermore, production of 3-
deoxyflavonoids (in grasses) and flavonols is controlled by
R2R3MYBs that do not require a bHLH partner [72,73].
The anthocyanin-related R2R3MYB activators have been charac-
terized in detail for many different species [71,74]. They form
R2R3MYB sub-group 6 and are typically encoded by small gene
families, with individual members of the families defining specific
pigmentation patterns in the plant [75e77]. The PA-related
R2R3MYBs (sub-group 5) were first described for A. thaliana,with
the R2R3MYB TT2 interacting with TT8 (bHLH) and TTG1 (WDR) to
form the MBW complex [70,74]. Subsequently, the genes involved in
regulation of PA biosynthesis have been characterized for other spe-
cies such as grape, in which several PA-related R2R3-MYBs have been
identified [78e80], various legumes [81], and persimmon (Diospyros
kaki Thunb.). PAs are of significant agricultural importance in le-
gumes, for the control of protein digestion by ruminant animals in
pasture systems. R2R3MYBs regulating PA production have been
identified from the legumes Medicago and Lotus and used in genetic
modification approaches to increase PA levels in forage crops [81].A
significant recent breakthrough is the identification of an R2R3MYB
from rabbit’sfootclover(Trifolium arvense L.) that when over-
expressed in Medicago sativa L. or Trifolium repens L. induces foliar
PA accumulation at up to 1.8% dry weight [82]. In grape, PAs are major
influences on the sensory qualities of the resultant wine. Persimmon
offers an interesting study system, as PAs can accumulate to such
levels that they render the fruit highly astringent [83].
Outside of the anthocyanins, PAs and flavonols, there are only a
few polyphenol biosynthetic pathways for which TFs have been
characterized. Most notably, the regulation of lignin biosynthesis
has been extensively studied in A. thaliana, and to a lesser extent
the grasses [84,85].InA. thaliana, MYB85 and members of
R2R3MYB sub-group 3 directly promote lignin biosynthesis under
the control of upstream MYB and NAC family (named from No
apical meristem,A. thaliana transcription activation factor, and Cup-
shaped cotyledon) TFs. In addition, members of R2R3MYB sub-
group 4 repress biosynthesis of the precursors for the branch of
the phenylpropanoid pathwayleading to lignin and a range of other
compounds. For example, AtMYB4 is an active repressor controlling
sinapate ester biosynthesis in a UV-dependent manner [86], and
Petunia hybrida (Hook.) Vilm. MYB4 represses formation of phe-
nylpropanoid scent compounds in the petals [87]. There has been
recent progress on understanding the regulation of another branch
of the phenylpropanoid pathway, that producing the isoflavonoids.
Changes in TF gene expression associated with isoflavonoid pro-
duction were characterized for the Lotus japonicus L., and a group of
R2R3MYBs (sub-group 2) identified as candidate activators for the
necessary biosynthetic genes [88]. In conjunction with the induc-
tion of the sub-group 2 genes there was down-regulation of TFs
that regulate branches of the phenylpropanoid pathway that might
compete with isoflavonoid production, specifically those for pro-
duction of flavonols and PAs and the likely bHLH component of the
anthocyanin MBW complex.
It has been several years since the components of the MBW
complexes for anthocyanin or PA regulation were first identified,
and they are now well-characterized for several species with regard
to their protein structure and in planta function [69e71]. However,
there are still aspects of MBW function only partly understood.
Hierarchical activation of production of components of MBW
complexes by the same or closely related MBW complexes can
occur, such as the MBW complex PRODUCTION OF ANTHOCYANIN
PIGMENT1 (PAP1) (MYB75)-GL3-TTG1 in A. thaliana activating gene
expression of the bHLH TT8 that then forms a PAP1-TT8-TTG1
complex [89]. Also, the WDR role is not resolved fully, and some
aspects of WDR function can be compensated for by over-
production of the MYB or bHLH, as shown by partial complemen-
tation of the A. thaliana ttg1 phenotype [90,91]. Furthermore,
recently, several additional mechanisms and TFs have been iden-
tified that regulate flavonoid biosynthesis, most of which are
thought to act by affecting the MBW complex activity. Of particular
note are mechanisms that antagonize the action of the MBW
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activator complex. Mechanisms involving the degradation of TF
protein are part of the response pathway for the induction of an-
thocyanins in response to light in A. thaliana leaves and apple fruit.
In A. thaliana, the bZIP TF protein LONG HYPOCOTYL5 (HY5) is a key
coordinator of the light response. HY5 may promote flavonoid
biosynthesis through both direct binding at promoters of antho-
cyanin biosynthetic genes and the induction of MYB12, the direct
activator of flavonol biosynthesis. In the dark HY5 is tagged for
degradation by the ubiquitin E3 ligase CONSTITUTIVELY PHOTO-
MORPHOGENIC1 (COP1) complex, the central response mediator
downstream of the light receptors [92]. In apple fruit the key
anthocyanin R2R3MYB activator is degraded in the dark via the
COP1-mediated ubiquitin-dependent pathway [93].
Several TFs with a repressive affect on flavonoid biosynthesis are
now known. The first repressor TF to be identified from plants was
the R2R3MYB mentioned earlier, AtMYB4. AtMYB4 production is
down-regulated by exposure to UV-B irradiation, which in turn re-
lieves the cinnamate-4-hydroxylase gene from AtMYB4-based sup-
pression, thus promoting production of sinapate esters for UV-B
protection [86]. A potential repressor for anthocyanin production
was identified soon after AtMYB4, FaMYB1 from strawberry [94].
Expression of FaMYB1 in tobacco [94] or Lotus corniculatus L. [95]
represses anthocyanin or PA biosynthesis, respectively. Both
AtMYB4 and FaMYB1 contain residues required for R2R3MYB
interaction withthe bHLH partners, and alsohave a domain in the C-
terminal that mediates active transcriptional repression, called the
Ethylene-responsive transcription factors (ERF)-associated Amphi-
philic Repression (EAR) domain. This suggests that they can form
MBW complexes that repress, rather than activate, target genes.
The EAR domain, which is plant-specific and has the core
sequence of LxLxL or DLNxxP, occurs in a wide range of TF families
in addition to the R2R3MYBs [96]. Alignment of confirmed and
candidate R2R3MYB-EAR repressors for flavonoid biosynthesis
from a range of species shows several conserved regions, with the
most striking being the (P/L)DLNL(E/D)L sequence that is the EAR
domain. The high conservation of the EAR domain makes for easier
identification of potential repressors for flavonoid biosynthesis
from among genomic sequence data. R2R3MYB-EAR repressors of
note identified recently include AtMYBL2 of A. thaliana [97,98] and
MYB27 of petunia [75,99]. AtMYBL2 and PhMYB27 likely have
similar roles in controlling vegetative pigmentation, being
expressed at high levels in leaves of shade-grown plants but down-
regulated when plants are exposed to light stress, concurrent with
the induction of the R2R3MYB and bHLH activators [75,97,100].
AtMYBL2 is unusual in that it appears to be a truncated protein. It is
sometimes termed a single repeat MYB but it actually has a partial
R2 domain in addition to an intact R3 domain. The EAR domain of
AtMYBL2 diverges from the LXLXL consensus, and Matsui et al. [98]
demonstrated that it requires a second sequence (TLLLFR) for active
repression. This domain occurs less frequently than the EAR
domain [96], but is well conserved among a clade of candidate
R2R3MYB repressors [[98] and the authors’analysis].
There are other members of the MYB family that also can have a
repressive affect on transcription ethe true single repeat MYBs of
the R3MYB type. The R3MYBs are much smaller than the
R2R3MYBs, containing the R3-region of the MYB DNA-binding
domain and the amino acid motif necessary to bind bHLH part-
ners but lacking active repression domains [101]. R3MYB repressors
were first identified as components of the regulatory system that
determines trichome and root hair patterning. So far, six R3MYBs
have been identified as being involved in this process in A. thaliana,
with TRIPTYCHON (TRY) and CAPRICE (CPC) being the best char-
acterized [70,102]. Transgenic over-expression studies have
revealed that in addition to regulating epidermal cell fates CPC has
a role in anthocyanin regulation [103,104]. A homolog has been
identified from petunia, MYBX, and evidence to date suggests its
primary role is in negatively regulating anthocyanin biosynthesis
[99,100]. Candidate R3MYBs for flavonoid regulation are also pre-
sent in the sequence databases for many other species, including
notably in that of grape (Fig. 3).
The R2R3MYB and R3MYB proteins probably have distinct
modes of action, involving active and competitive repression
respectively. The EAR (and TLLLFR) motif mediates active repres-
sion of target genes, with the protein forming part of a MBW
complex that binds to cis-elements within target promoters. The
EAR motif can actively inhibit transcription from the promoter by
directly preventing the basal transcription machinery from
assembling and/or recruiting proteins that modify chromatin into
an inactive form, such as via histone deacetylation [97]. Addition of
an EAR domain can even convert activator TFs to inhibitor proteins
[105]. The R3MYBs lack such domains for active transcriptional
repression. Rather, they may be competitive inhibitors, binding and
titrating out the bHLH component of the MBW complex. This is
supported by transient co-expression studies of CPC and the
A. thaliana activator R2R3MYB PAP1 [104]. An important aspect of
the function of R3MYBs in trichome patterning is their movement
between neighboring cells. The system of short-range activators
(R2R3MYB) and long-range repressors (the R3MYB) seen in control
of trichome formation meets key criteria for the Turing substrate
depletion/reaction diffusion models for pattern generation
[106,107]. The WDR may also move between cells [108]. This raises
the possibility that TF mobility may contribute to formation of the
complex anthocyanin pigmentation patterns seen in flowers, such
as spots and stripes [71]. It is known that localized expression of the
R2R3MYBs accounts for the activation of anthocyanin biosynthesis
in specific cells to generate venation [75,109] and possibly some
spot patterns [110,111], but the higher level mechanisms that define
these expression domains are not known.
Other TFs or post-transcriptional mechanisms involved in
repression of anthocyanin biosynthesis have been identified. In
Fig. 3. Phylogenetic tree of a selection of MYB transcription factors related to flavonoid
biosynthesis. R2R3MYBs with an activation acivity on the pathway for sub-group 6,
which contains AtMYB75 and is highlighted in red. R2R3MYBs with a repressive action
form a major clade of several branches, which contains AtMYB4 and AtMYBL2 and is
highlighted in black. The R3MYB repressor proteins form the third clade, highlighted in
blue. The scale bar represents number of substitutions per site and the numbers next
to the nodes are bootstrap values from 1000 replicates. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
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A. thaliana, the SQUAMOSA promoter binding protein (SBP)-box TF
SBP-LIKE (SPL9) may physically disrupt formation of the activator
MBW complex, with SPL itself the target of the general inhibitor
microRNA miR156 [112]. Similarly, the JASMONATE (JA)-ZIM
domain (JAZ) proteins may inhibit MBW complex formation in
A. thaliana by binding to the bHLH and R2R3MYB factors, with
jasmonic acid promoting the ubiquitin-mediated degradation of
the JAZ proteins [113]. So in both cases the inhibitor of the MBW
complex is itself the target of a separate inhibitory pathway.
Furthermore, the transcripts for the R2R3MYB anthocyanin-related
activators of A. thaliana (PAP1, PAP2, and MYB113) are the direct
targets of microRNA inhibition through a miR828 and TAS4-
siRNA81() system [114]. Another group of repressor TFs of note is
the LATERAL ORGAN BOUNDARY DOMAIN (LBD) family of three Zn-
finger TFs, which can suppress PAP1 production as part of the
response to nitrogen status in A. thaliana [115].
While many of the recent advances have come from the model
species, such as A. thaliana, petunia and Antirrhinum majus L., there
have also been significant advances in the depth of understanding of
regulation of phenylpropanoid biosynthesis in important horticul-
tural species, in particular grape and apple [74,116]. The completion
of the genome sequences of these two species has enabled more
extensive analysis of the phenylpropanoid-related TF families. The
R2R3MYBs regulating anthocyanin production in grape have been
extensively studi ed over the last decade [74], but more recently those
for the PA [78e80] and flavonol [117] pathway branches have been
identified, as well as the phenylpropanoid-related bHLHs and WDR
[74,118]. Lin-Wang et al. [119] characterized a large number of
anthocyanin-related R2R3MYB activators from Rosaceae species,
with a focus on apple, and also identified a range ofapple R2R3MYBs
containing the EAR domain that were able to inhibit anthocyanin
gene activation in a transient assay system [120].
The use of TFs as transgenes for modification of the biosynthesis
of polyphenolics is now well proven, with many different genes
shown to be effective and a large number of species successfully
targeted [74,81]. However, there are still issues to be resolved using
in TFs for pathway regulation. In particular, great variation is
observed in transgenic phenotype depending on the specific
transgene/host species combination. For example, in a given spe-
cies it may vary whether the R2R3MYB or bHLH of the flavonoid-
related MBW complex is more effective, or two R2R3MYBs from
the same gene family may generate dramatically different pheno-
types. These variations likely reflect differences in the activation
strength and specific target DNA motifs of the individual TF pro-
teins and/or hierarchies of regulation among the TFs [70]. Some TFs
may activate a wide range of target genes when over-expressed,
including in pathways for which they are not normally key regu-
latory factors. For example, AtMYB12 can induce production of both
flavonols and caffeoylquinic acids when over-expressed in tomato
[121]. For some target pathways, the correct combination of acti-
vator and repressor TFs may be required if the desired level of
phenolic production is to be achieved [88].
The understanding of the regulation of polyphenol production is
well advanced compared to the state of knowledge for other plant
secondary metabolite pathways. However, there are still many
areas in which progress is limited. In particular, there is little depth
of knowledge outside of the phenylpropanoid pathway, with in-
formation lacking for groups of compounds as significant as the
coumarins, anthraquinones, naphthoquinones, xanthones, and
hydrolyzable tannins.
3. Role in plants and ecosystems
In plant physiological ecology the term stress has general con-
notations rather than a precise definition. Stress could be defined as
any factor (biotic and abiotic) that modifies (positively or nega-
tively) plant functioning, growth or reproduction. Most plants un-
dergo some form of stress during the various stages of their life
cycle, which affects the performance and survival of individual
plants. Shade or excessive light, low/high temperature, nutrient
deficiency, drought stress, biological stresses of herbivory by
vertebrate and invertebrate grazers and diseases caused by fungi,
bacteria, and viruses, and competition among plants are various
examples of biotic and abiotic stresses. Almost every perturbation
of a plant community or ecosystem results in stress and so affects
the performance and survival of individual plants [122].
Plant activity at the cellular level can be classified as growth and
differentiation. Both growth and differentiation are resource
demanding, with precise requirements for light, water, minerals,
temperature, photoassimilates, growth regulators, the regulating
effects of neighboring cells, and other factors. Plant growth and
productivity are greatly affected by environmental stresses that
divert substantial amounts of substrates from primary to secondary
metabolism. It is assumed that there is a cost in the production of
defensive compounds that is reflected in decrease in the growth
rate, reproductive output, or competitive ability of the organisms
producing them. Assuming resources are limiting, species that
produce defensive metabolites should allocate fewer resources to
growth and reproduction, resulting in trade-offs between growth
and defense that regulate carbon fluxes between primary and
secondary metabolism, thus obtaining protective adaptation to
environmental stresses. Activation of plant defenses following
imposition of environmental stress involves complex reiterative
signal networks with extensive signal amplification and trade-offs
[123,124].
Accumulation of phenolic compounds in plant tissues is a
distinctive characteristic of plant stress. This accumulation is due to
an increased enzyme activity of phenylalanine ammonia lyase
(PAL), chalcone synthase (CHS), and other enzymes, activity of
phosphoenolpyruvate (PEP)-carboxylase also increases, suggesting
a shift from sucrose production to processes in support of defense
and repair. Plant phenolics confer various physiological functions
for plants to survive and to adapt to environmental disturbances
[125e127].
Within the plant’s environment different microorganisms can
coexist, such as bacteria, filamentous fungi, protists, that can
establish various interactions with the host plant. These in-
teractions are on a continuum from the parasitism to mutualism
and are often the basis for the synthesis of specific metabolites in
response to these interactions. Natural products interacting in
these interactions are classified into three major groups: phyto-
alexins, phytoanticipins and the signal molecules. Phytoalexins are
antimicrobial molecules of low-molecular weight synthesized and
accumulated in plants after exposure to microorganisms. The
phytoanticipins are constitutively synthesized in the plant in
anticipation of a pathogen attack.
Defensive phenolic compounds appear to contribute to a gen-
eral reduction of reactive oxygen species and therefore impact
cellular processes sensitive to redox effects. However, plant phe-
nolics also have been implicated in more direct interactions with
transport and signal transduction pathways. In this connection, the
small phenolic compound salicylic acid (SA) plays an important
regulatory role in multiple physiological processes including plant
immune response. Thus, when plants are subject to attack by mi-
crobial pathogens, they enable sophisticated innate immune sys-
tems by which they recognize signals from injured cells, and
respond by activating effective immune responses through SA
signaling cascade and interactions with other phytohormones
[128,129]. One well-documented example is the role of flavonoids
in modulation of auxin transport as well as localized auxin
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accumulations observed during nodulation. Perhaps the best-
studied example of flavonoid signaling is that of flavonoid media-
tion of interactions between the plants and other organisms in the
environment at both competitive (allelopathy/defense) and coop-
erative (mycorrhizal association) levels. Phenolic signaling within
the plants is not well documented. It has been suggested that intra-
and intercellular signaling involve enzyme activity and cellecell
communication [130e133].
In the rhizosphere, a densely populated area in which plant
roots must compete with invading root systems of neighboring
plants and with other soil-borne organisms, including bacteria and
fungi, increasing evidence suggests that root specific chemicals
(exudates) might initiate and manipulate biological and physical
interactions between roots and soil organisms. These interactions
include signal traffic between roots of competing plants, roots and
soil microbes, and one-way signals that relate the nature of
chemical and physical soil properties to the roots [134e138].
3.1. Plant phenolics as underground signaling molecules
The plant interacts strongly with its biotic environment via the
synthesis of secondary, often diffusible metabolites. The root
exudation thus alters the physico-chemical properties of roots
surrounding soil which is called rhizosphere [139]. This environ-
ment is conducive to the survival of microorganisms around the
plants because most of the time the exudates include ions, en-
zymes, molecules rich in organic carbon (primary and secondary
metabolites) which is a source of nutrients for terrestrial organisms
[140]. Exudated secondary metabolites are often source of
chemotaxis to the selection of organisms (pathogens or commen-
sals) around the roots. This chemical environment is favorable to
the establishment of various interactions in particular with sym-
biotic microorganisms.
3.1.1. Rhizobium/legume symbiosis
One of the most described mutualistic symbioses is the
Rhizobium/legume symbiosis. This interaction is established pref-
erentially at the root level and allows the formation of specific
organs called nodules where the bacteroides are fixing atmo-
spheric nitrogen. This symbiosis is relatively recent in the evolu-
tion, and derives from a former mycorrhizal symbiosis that was
relatively common in the Plant Kingdom and allowed the plant to
obtain essential phosphated nutrients [141]. The Rhizobia colo-
nizing capacity was acquired by a symbiotic gene horizontal
transfer event 60 million years ago date of the appearance of le-
gumes in the living world [142].Rhizobium genes involved in
symbiosis are often localized in plasmids or chromosomal blocks
as is the case for Sinorhizobium meliloti, where the genes involved
in the synthesis of factors of nodulation (nod, dol, Noah) and
fixation of nitrogen (nif and nix) are located on the pSymA
plasmid [143]. In addition, it has been shown that the horizontal
transfer of these plasmids or these chromosomal genes is possible
between Rhizobia species [144].
Legumes via roots exude in the soil a variety of secondary me-
tabolites, including flavonoids, and in particular flavones and iso-
flavonoids, in the rhizosphere. These flavonoids, when they are
recognized (oxidation, position of the substituents, .) by the
nucleotide-binding and oligomerization domain D (NodD) re-
ceptors of the bacteria regulate the expression of other nod genes
(A, B, and C) and the production of factors Nod (lipo-chitin-oligo-
saccharide) (Table 1). Compatible bacteria are then able to move to
the flavonoid flow by chemotaxis, which implies that the host
specificity is settled before physical contact between the bacterium
and its host plant. Then Nod factors synthesized by the rhizobial are
specifically recognized by the host plant on the basis of their
structure (length and unsaturation of the acyl chain, number of
monomer of N-acetyl-glucosamine, substitutions.)[145].
The presence of flavonoids in the roots appears to be essential to
stimulate the synthesis of Nod factors in the infection tube [153].
The presence of Nod factors in the root would increase the
biosynthesis of kaempferol. The local presence of this flavonol in-
hibits the transportation of auxin which would facilitate cell divi-
sion and the initiation of the nodule.
The response of plants to nodulation factors in terms of sec-
ondary metabolites has been poorly studied. However there are
arguments supporting effect of Rhizobium/legume symbiosis, on
the modification of the synthesis of plant secondary metabolites.
Inoculation of Bradyrhizobium japonicum induced in soybean
changes of the expression of PAL, which is one of the major en-
zymes of the phenylpropanoid pathway (PHP), a major route in the
development of secondary metabolites, and of CHS, the first
enzyme of its flavonoid branch (Fig. 1)[154]. In addition, inocula-
tion of Nod factors on M. sativa cell suspensions can induce gene
encoding isoflavone reductase enzyme (IFR) [155]. This induction of
enzymes of the pathway suggests the possibility that the plant is
able to synthesize, in response to the perception of molecules re-
ported as the Nod factors or Rhizobiae, many secondary metabolites
and many cosubstrates (feruloyl CoA, sinapoyl CoA.). So poten-
tially, overexpression of the enzymes of the PHP pathway induced
by Nod factors can result in increased synthesis of phenolic com-
pounds and especially, flavonoids. Thus, it is possible that the
presence of Nod factors in the environment of the plant induces a
positive feedback by the plant for its infection by Rhizobia. More-
over, studies conducted on Arachis hypogaea L./Rhizobium spp.
symbiosis (Rhizobium isolated from peanut) were focused on the
observable differences between phenolic compounds synthesis in
inoculated plants versus non-inoculated ones [156]. The results of
this study show that in addition to the fact that the total phenolic
contents (
m
g/g of fresh matter) increases in inoculated plants, time-
dependent differences in qualitative and quantitative terms are
demonstrated. In inoculated condition, the plant specifically in-
creases the synthesis of phenolic acids such as trans and cis caffeic
acid, chlorogenic acid, p-coumaric acid, trans and cis ferulic acid,
vanillic acid. Other compounds see their synthesis not affected or
decreased in the presence of Rhizobium as p-hydroxybenzoic acid
and protocatechuic acid. Other authors [157,158] have conducted
studies on the compounds present in nodules formed by Rhizobium
on peanut and showed unambiguously that the total phenolic
content within the nodule is 1.6 times higher than that of non-
nodulated roots. Some authors have also shown that in alfalfa
plants inoculated with Rhizobium meliloti, three isoflavonoids
(medicarpin glucoside and aglycone and formononetin-7-O-(6
00
-O-
malonylglycoside)) were found while they were not present in non-
inoculated plants [152]. Among these compounds only the glyco-
side of formononetin is capable to induce Nod factors in R. meliloti.
In addition, a recent study conducted on exudates of common bean
(Phaseolus vulgaris L.) in non-inoculated and inoculated conditions
with a Rhizobium strain (Rhizobium tropici CIAT899) shows that the
flavonoid synthesis is strongly affected by the bacterium [159]. Four
days after inoculation, pinocembrin, apigenin and hesperetin are
no more detected in the plants exudates inoculated with the strain
of Rhizobium while apigenin is detected in non-inoculated plant
seedlings up to 7 days after inoculation. On the contrary, even if
morin is not present in the exudates of non-inoculated plant, it
occurs in plants inoculated with the strain of Rhizobium. Fourteen
days after inoculation, exudates from pea roots contain none of the
studied flavonoids. These studies demonstrate that the plant is very
reactive to its environment and that it is able to respond quickly to
changes in biotic ecosystem. A change in the synthesis of flavonoids
exuded by plant in inoculated condition is in favor of a regulation
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mechanism of the nodulation of plants in symbiosis with a Rhizo-
bium, to limit the rate of nodule formation.
As the synthesis of plant secondary metabolites is very reactive
because many compounds see increased or decreased concentra-
tion following inoculation, the question remains on the ecological
role of these metabolic changes. Plants may indeed by diffusion
(active or passive) excrete the phenolic compounds in the rhizo-
sphere and thus come into contact with the soil microorganisms.
Most of these compounds are often synthesized by the plant in
response to stress, whether biotic or abiotic [160]. Some Bradyrhi-
zobium strains have the ability to metabolize a large number of
phenolic acids such as 4-hydroxybenzoic acid, vanillic acid, caffeic
acid and coumaric acid, which belong to the most represented
simple phenols in the plant world [161]. Also it has been shown
that Rhizobium loti nodulating Lotus subbiflorus Lag. was able to use
ferulic and coumaric acid as carbon source. Syntheses of these
compounds by the host plant but also flavonoids induce a positive
chemotaxis in Rhizobium and furthermore allow Nod factors in-
duction [162]. The fact that the plant increases the synthesis of this
kind of compounds in the Rhizobium/legume symbiosis suggests
that these compounds can be used by the plant to select microor-
ganisms around roots and thus foster the bacteria that have
managed to adapt to these toxic molecules. On the other hand, the
specific modification of these phenolic compounds is probably due
to a coevolution mechanism between Rhizobia and their host
plants providing a selective advantage to the bacteria for the
implementation of new nodulations at the root-level.
3.1.2. Actinorhizal symbiosis
A second nitrogen root fixing symbiosis is the actinorhizal sym-
biosis. The latter associates soil filamentous bacteria, actinomycetes
of Frankia genus, to eudicot angiosperms, the actinorhizal plants.
These plants belong to eight families (Betulaceae,Casuarinaceae,
Coriariaceae,Datiscaceae,Elaeagnaceae,Myricaceae,Rhamnaceae and
Rosaceae) among which there are 25 genera and more than 200
species such as filao (Casuarina equistefolia L.), alder (Alnus sp), wild
olive (Elaeagnus angustifolia L.) and bog myrtle (Myrica gale L.).
Bacteria of Frankia genus belonging to the Frankiaceae family are
filamentous, microaerophilic or aerobic and for most of them ni-
trogen fixing microorganisms [163]. On the morphological point of
view, Frankia differentiate in several specialized structures: hyphes,
involved in the first steps of the symbiosis that can differentiate in
two other structures, sporangia and vesicles. Frankia strains can be
divided in 3 major clusters on the basis of the 16s sequences [164].
The different strains have host specificity abilities that allow to
distinguish compatible or incompatible strains to the symbiosis with
a plant species [165]. Like the Rhizobia/legume symbiosis, the bac-
teria can penetrate the roots of the host plant thanks to the Had
factor (Hair deforming) that is similar to Nod factor [166]. This Had
factor should be constitutive or inducible by the host plant through
the synthesis of original and rare dihydrochalcones (myrigalones)
like the nod factors areinducible by plant secondary metabolites and
especially flavonoids. As for the Rhizobia/legume symbiosis, only a
few studies have focusedon the impact of the symbiosis on the plant
host secondary metabolites. However, Hammad et al. [167] have
showed the activation of pal and chs plant genes during the symbi-
osis between Frankia and Alnus. The only study that focused on the
response of the host plant to the inoculation of compatible and
incompatible strains was realized on the Frankia/Myricaceae model
[168].TwoFrankia strains (Ea112 compatible with Morella cerifera L.
and ACN14a compatible with M. gale and M. cerifera) were inoculated
on the both host plants. Root extracts were realized 2 days and 14
Table 1
Different polyphenols inducing the nod gene expression. (SE: seed exudates, RE: root exudates)
Compounds Substitutions Origin microbial strain References
3457 3
0
4
0
5
0
Flavones
Luteolin OH OH OH OH Medicago (SE) R. meliloti [146]
Chrysoériol OH OH OCH
3
OH Medicago (SE) R. meliloti [146]
Apigenin OH OH OH [147]
4
0
,7-dihydroxyflavone OH OH Medicago (RE) R. leguminosarum bv. trifolii [148;149]
Flavonols
Myricetin OH OH OH OH OH OH [147]
Myricetin-O-Glu O-Glu OH OH OH OH OH Phaseolus vulgaris R. leguminosarum bv. phaseoli [150]
Quercetin OH OH OH OH OH [147]
QuercetineO-Glu O-Glu OH OH OH OH Phaseolus vulgaris R. leguminosarum bv. phaseoli [150]
Kaempferol OH OH OH OH [147]
Kaempferol-O-Glu O-Glu OH OH OH Phaseolus vulgaris R. leguminosarum bv. phaseoli [150]
Flavanones
Eriodictyol OH OH OH OH OH [147]
Naringenin OH OH OH Phaseolus vulgaris R. leguminosarum bv. phaseoli [151]
Hesperetin OH OH OH OCH
3
[147]
Liquiritigenin OH OH Medicago (SE) R. meliloti [148]
Anthocyanins
DelphinidineO-Glu O-Glu OH OH OH OH OH Phaseolus vulgaris R. leguminosarum bv. phaseoli [150]
Petunidin-O-Glu O-Glu OH OH OCH
3
OH OH Phaseolus vulgaris R. leguminosarum bv. phaseoli [150]
Malvidin-O-Glu O-Glu OH OH OCH
3
OH OCH
3
Phaseolus vulgaris R. leguminosarum bv. phaseoli [150]
Isoflavones
Genistein OH OH OH Phaseolus vulgaris R. leguminosarum bv. phaseoli [151]
Daidzein OH OH [147]
Formononetin-7-O-Glu O-Glu OCH
3
Medicago (RE) R. meliloti [148]
Formononetin-7-O-(6
00
-O-malonyl-Glu) Malonyl-Glu OCH
3
Medicago (RE) R. meliloti [152]
Substitutions
3457 2
0
4
0
5
0
Chalcones
4,4
0
-Dihydroxy-2
0
-methoxychalcone OH OCH
3
OH Medicago (RE) R. meliloti [148]
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days after inoculation and analyzed by LCeMS means. Results
showed indubitably quantitative variations of specific secondary
metabolites. The specificityof interaction seems here to be related to
the biosynthesis of specific phenylpropanoids and flavonoids. The
amount of a great number of hydroxycinnamic acids (HCA) is
modulated (increase or decrease depending of the inoculated bac-
terial strain). The decrease of HCA in the case of Frankia ACN14a/
M. gale could be related to the modification of the production of
coumarins or lignin. Actually, the modification of lignin biosynthesis
is a common mechanism allowing the plant to regulate the bacterial
penetration in its tissues [169]. This decrease of HCA biosynthesis
would allow facilitating the colonization of the host plant tissues by
Frankia. On the contrary, the incompatible strain EA112 induced an
increase of the HCA level limiting and/or preventing the symbiosis.
Furthermore, the differential synthesis of specificflavonoids, often
involved in plant defense mechanisms but also essential for symbi-
osis process in the case of Rhizobium/legume interaction, could be
the result of an evolutive co-adaptation mechanism of the partners
in the actinorhizal symbiosis.
3.1.3. Symbiosis involving plant growth promoting rhizobacteria
While in the most cases of mutualistic interactions, the symbi-
osis is characterized by the formation of specialized structures (as it
is the case for Rhizobium/legume and actinorhizal symbioses) with
the formation of nodules and pseudonodules, another kind of
bacteria is able to fix atmospheric nitrogen and interact through a
symbiosis with its host plant without forming specialized struc-
tures. These bacteria are called PGPR (Plant Growth Promoting
Rhizobacteria). PGPR are mainly represented by two genus, Pseu-
domonas and Azospirillum. Bacteria from the Pseudomonas genus
are biocontrol bacteria whereas bacteria from the Azospirillum
genus are phytostimulating bacteria. Pseudomonas bacteria are able
to inhibit some pathogen growth through several mechanisms like
siderophore synthesis (competition for iron availability), antibiotic
synthesis (e.g. 1,4-diacylphloroglucinol ¼DAPG). On the contrary,
Azospirillum bacteria are able to fix nitrogen (and transfer it under
bioavailable form for the plant) and synthesize phytohormones
(auxins) stimulating plant growth. These bacteria have a broad
spectrum of host plants from monocotyledons to eudicots even if
specificity is detectable in term of efficiency promotion of the root
and shoot growth. PGPR ecosystem is constituted by the host plant
root, some mucilages, plant exudates, soil around the roots, but also
other soil organisms. For wheat, as example, among exudated
compounds we can find many organic acids like fumarate or suc-
cinate, amino acids but also sugars like fructose and maltose and
oligosaccharides [170]. The first substrates utilized by Pseudomonas
PGPR are sugars, organic acids and amino acids. This explains the
chimiotactism to roots [171,172]. The recognition mechanism of the
bacteria by the host plant is still not elucidated but seems to involve
two bacterial molecules. Indeed, the flagellin (Flg22 peptide) that is
a subunit of the polar flagella of Pseudomonas is recognized by the
plant. The second molecule that allows the specific recognition of
bacteria is lipopolysaccharide (LPS). Plant roots colonization by
Azospirillum begins as for Pseudomonas by a positive chimiotactism
of bacteria to roots for the same reasons (carbon sources necessary
for growth and nitrogen fixation). Azospirillum also presents a
positive chimiotactism for sugars, amino acids and organic acids
that are exudated by the root tissues [173]. This chimiotactism
phenomenon allows the both partners to meet each other. The
following steps leading to symbiosis establishment are not well
known. The bacteria will adherate to the host plant root thanks to
the outer membrane, LPS, exopolysaccharides and proteins. For the
plant, mucilages, polysaccharides, and lectins are also involved in
the recognition [174]. Once recognized, bacteria colonize intercel-
lular spaces in the root cortex. The preferential adhesion areas
on the root correspond to the elongation zone root hairs [175].
The action mechanism of Pseudomonas is quite different from
Azospirillum. Actually, Pseudomonas PGPR is called “Biocontrol”due
to their ability to confer to the host plant a protection against
diverse pathogens. As example, the protection of Pseudomonas
fluorescens to the host plant is mainly related to the induction of
resistance mechanisms in the plant (Table 2).
However, some Pseudomonas strains possess the gene encoding
ACC deaminase (1-aminocyclopropane-1-carboxylate) that is
involved in the growth promotion of the host plant [186]. The action
mechanism of Azospirilla is different. Once the colonization realized,
the growth promotion is regulated by different mechanisms. These
bacteria are able to fix atmospheric nitrogen and to transfer it to the
plant under a bioavailable form. Moreover they also can synthesize
plant hormones like indole-3-acetic acid (IAA). Then an interaction
between plant and Azospirilla allows for the plant a better mineral
nutrition because its root system is more developed and allows a
more important resource mobilization [187]. Only a few data are
available on the impact of PGPR on the metabolism of host plant.
Besides induced systemic resistance (ISR) induction and other
compounds related to resistance mechanisms (jasmonate, ethyl-
ene.), inoculation by biocontrol PGPR can modify the metabolic
synthesis of the host plant. Some studies on chickpea (Cicer arieti-
num L.) showed that in inoculated conditions by P. fluorescens Pf4
and Pseudomonas aeruginosa Pag, there is an increase of total
phenolic content in leaves and roots three weeks after inoculation
and especially for ferulic acid [188]. Moreover, the inoculation of
betel (Piper betle L.) with Serratia marcescens Bizio 1823 NBRI1213
showed the modulation of the phenolic acid synthesis compared to
non-inoculated plants. The protocatechuic acid content decreased
of 50% and 40% for ellagic acid in leaves. In roots the synthesis of
these compounds is stopped. Furthermore, the plant specifically
synthesizes chlorogenic acid under inoculated conditions and the
biosynthesis of ferulate increases specifically in roots [189]. These
variations could be related to defense and plant protection by
biocontrol PGPR as these compounds have been largely described as
antifungal compounds that are able to induce ISR. Little is known
about the impact of phytostimulating PGPR on the host plant
metabolome. However, inoculation of Burkholderia phytofirmans
PsJN on vine generates an increase of total phenol content in all
plants. Moreover, this strain also induces an increase of free proline
in plant host tissues [190]. Another study showed that the presence
of Azospirillum brasilense Cd on P. vulgaris induces the synthesis of a
particular flavonoid, naringenin, at the fourth day after inoculation
[159]. PGPR inoculation impacts mainly phenylpropanoid de-
rivatives. However, it also seems that the interaction between plant
and PGPR affects also the synthesis of some amino acids. Most
phenolic acids are involved in plant defense mechanisms. So fer-
ulate at 250
m
g/ml is able to inhibit radial growth of the fungus
Sclerotium rolfsii, a chickpea pathogen [191]. Cinnamic acids
involved in the interactions between plants and PGPR can be found
Table 2
Determinants in Pseudomonas inducing ISR. Adapted from Bakker et al. [176].
Determinants Bacterial strain Host plant References
Lipopolysaccharides P. fluorescens WC374 Radish [177]
P. fluorescens WC417 Arabidopsis [178]
Radish [177]
Carnation [179]
Siderophores P. fluorescens CHA0 Tobacco [180]
P. fluorescens WC374 Radish [181]
Salicylic acid P. fluorescens P3 pchBA Tobacco [182]
DAPG P. fluorescens CHA0 Arabidopsis [183]
Tomato [184]
P. fluorescens Q2e87 Arabidopsis [185]
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in rhizosphere as these compounds are easily diffused. An increase
of the concentration of these compounds in rhizosphere conse-
quently modifies the physicochemical properties of the environing
soil around roots. The fact that PGPR are able to modify the syn-
thesis of phenols is in favor of a biocontrol supplementary mecha-
nism allowing the plant to fight against pathogens more efficiently
in coordination with other antimicrobial and siderophores.
The first steps of the mycorrhizal symbiosis imply the recogni-
tion of signal molecules between the two partners. The establish-
ment of the interaction begins for the arbuscular mycorrhizal fungi
(AMF) by the perception of signal compounds secreted by the plant
and specifically recognized by the fungus. Several studies are
dealing about the identification of these signals that are necessary
for the symbiosis. These compounds are secondary metabolites and
more precisely flavonoids. They are present in high concentrations
in roots and exudates of plants growing under lack in phosphate and
they can stimulate AMF growth in vitro at micromolar concentra-
tions. Moreover, flavonoids can stimulate mycorrhization and
flavonoid content is modified in mycorrhized roots. However, fla-
vonoids are not the only molecules that can be considered as signal
molecules. Actually, a study working on maize lines deficient for
CHS showed normal mycorrhization patterns [for review, see Ref.
[192]]. Strigolactones are also able to induce the germination of
spores and the ramification of fungal hyphaes [193]. However,
strigolactones are also known to stimulate seed germination of
phytoparasitic plants such as Striga and Orobanchae. Once the
mycorrhizal symbiosis established, the fungus induces great mod-
ifications of the plant metabolism. Several studies have focused on
the characterization of a yellow pigment specific to legume roots
infected by endophytic fungal parasites. Identified for the first time
in 1924 [194] this pigment has been characterized 70 years later as
mycoradicine, a compound derived from carotenoids. Since this,
several other carotenoid-derived compounds have been identified
in plants belonging to Poaceae and especially in the tribes Aveneae,
Oeantheae,Paniceae,Phalarideae, and Poeae [195]. The interaction
between plants and AMF doesn’t only modify the synthesis of
carotenoid derivatives. Other compounds and especially phenolics
are also influenced by the presence of the fungus. A study on the
mycorrhization of date palm showed a clear increase of phenolic
content when the plant is inoculated with Glomus. This increase
mainly concerns caffeoylshikimate isomers [196]. Otherwise,
studies on other plant species such as Begonia showed a significant
increase of several compounds classes in leaves as ortho-
dihydroxyphenols, flavonoids and more generally total soluble
phenols when the plant is inoculated with Glomus mossae [197].
Another study realized on purple Rudbeckia inoculated with Glomus
intraradices demonstrated that several cinnamate derivatives are
increased. This variation is significant only in roots. Thus cichoric
acid, caftaric acid, chlorogenic acid and 1,3-dicaffeoylquinic acid are
increased 1.5,1.7, 2.6 and 1.3 fold respectively. Furthermore the total
phenolic content is three times more important in inoculated leaves
[198]. Otherwise, mycorrhization usually induces phytoalexin syn-
thesis such as medicarpine (isoflavonoid) for M. truncatula.
Taking into account the modifications noted on the biosynthesis
of secondary metabolites in mycorrhized plants, authors tried to
understand why the plant modifies the synthesis of specific me-
tabolites. They proposed different hypothesis to explain this phe-
nomena at the ecological level. Among the different classes that are
concerned by these modifications, phenolics were specially
focused. Actually, tannins are increased in presence of AMF. They
are known to be involved in different mechanisms related to the
resistance against microorganisms. The precipitation by tannins of
the enzymes secreted by necrotrophic phytopathogenic fungi is a
property that can contribute to resistance in planta. The biosyn-
thesis of tannins is in some cases induced by stress perception and
mediated by signaling mechanisms involving jasmonate or
ethylene. Among several possibilities, cinnamic acids can be
involved in the biosynthesis of lignin. Hydroxycinnamic acids such
as ferulate or para-coumarate are covalently linked to hemi-
cellulosic polymers in monocots and to pectic substances in eudi-
cots. They are involved in the formation of inter-chains bridges or
associated to lignin. The increase of cinnamic acids synthesis could
then lead to thickening of plant wall and thus limit the infection by
different cellulolytic organisms. Since few years, thanks to metab-
olomics, a great development of the study on secondary metabo-
lites occurs. This new approach is very promising because we can
consider that it allows bridging between genomics, transcriptomics
and proteomics to predict gene function [199]. This approach was
usually used to establish molecular footprints of species [200],to
analyze the impact of xenobiotics [201], to compare metabolic
content of genetically modified plants. However, one of the appli-
cations of metabolomics is also the study of developmental pro-
cesses like establishment of symbioses [202].
Due to the reactivity of secondary metabolites and their quick
turn over, a biotic interaction can induce deep modifications in
terms of synthesized metabolites. These are at the initiation of an
interaction through recognition mechanisms of these signaling
molecules by the different partners allowing thus a molecular
dialog.
3.2. Plant phenolics as aboveground signaling molecules
Plant phenolics can modulate essential physiological processes
such as transcriptional regulation, membrane permeability, signal
transduction, and vesicle trafficking. They induce or inhibit
oxidative bursts and affect the respiration and photosynthesis
rates. This means that, at the whole organism level, the processes
such as development, adaptation, symbiosis, diseases, and male
sterility can be better understood, being one of the main speci-
ficities of plants [203]. As far as the role of plant phenolics as in-
ternal physiological regulators or chemical messengers within the
intact plant is concerned, some interesting effects of plant phe-
nolics are the ones associated with the growth hormone auxin
(IAA). Monohydroxy B-ring flavonoids are suggested as cofactors
of peroxidase functioning as an IAA oxidase that destroys the
hormone, whereas dihydroxy B-ring forms act as inhibitors of the
IAA degrading activity [204,205]. Mono- and dihydroxy-flavonoids
are also implicated as inhibitors of IAA transport across the plasma
membrane by binding to a plasma membrane protein known as
the N-1-naphthylphthalamic acid (NPA) receptor. NPA is a syn-
thetic compound, which is believed to bind a regulatory protein
that is associated with the transmembrane efflux of IAA anions
mediated by carrier. Some flavonoids, such as quercetin, apigenin,
and kaempferol, do not directly compete with IAA but act through
their own receptor, the NPA receptor, in the plant cell plasma
membrane, thus blocking the polar auxin transport. These effects
upon auxin transport could influence plant architecture. Recent
studies have, in fact, affirmed the link between flavonoids and
plant architecture by showing that flavonoid-defective mutants
display a wide range of alterations to root and shoot development.
In this connection, it has been known for more than half a century
that auxin induces root formation. As far as the interactions be-
tween phytohormones and other bioactive molecules during the
commitment phase of root formation is concerned, it has been
observed that quercetin and the isoflavonoids formononetin and
genistein caused severe reduction in in vitro root formation in
model legume M. truncatula. This effect is related to an inhibition
of auxin transport and/or a regulation of the redox cell status
[206e210]. An additional role for flavonoids in functional pollen
development has been observed in petunia plants using antisense
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CHS and maize mutants lacking CHS. A lack of CHS activity has a
pleiotropic effect in petunia and maize mutants: pollen fertility as
well as flavonoid synthesis is disrupted, but the sterility could be
restored by adding micromolar quantities of the flavonol agly-
cones kaempferol or quercetin to mature pollen at pollination. The
flavonol-requirement for functional pollen occurs in monocots and
dicots as well as in angiosperms and gymnosperms, which sug-
gests that it might have arisen in an early ancestor of land plants
[211e214].
In general, plants are rooted and are unable to demonstrate
mobility. However, some plants are known to open their leaves in
the daytime and “sleep”at night with their leaves folded. This
circadian rhythmic leaf movement is known as nyctinasty and is
induced by the swelling and shrinking of motor cells in the pulvi-
nus, an organ located in the joint of the leaf. A flux of potassium
ions across the plasma membranes of the motor cells is followed by
a massive water flux, which results in the swelling and shrinking of
these cells. At the heart of such a mechanism is the regulation of the
opening and closing of the potassium channels involved in the
nyctinastic leaf movement, a process which is under metabolic
control. It has been found that nyctinastic plants have a pair of
endogenous bioactive substances that control the nyctinastic leaf
movement. One of these is a leaf-opening factor that “awakens”the
plant leaves, and the other is a leaf-closing factor that reverses this
process so that the plant leaves “sleep”. Five sets of leaf-closing and
-opening factors in five different nyctinastic plants have been
identified. All the leaf-opening factors have a common structural
feature, the p-coumaroyl moiety, and this result suggests that this
structural feature would be deeply involved in the common
mechanism for leaf-opening [215].
Anthocyanins represent a class of flavonoids providing the or-
ange, red and blue/purple colors familiar in many plant tissues.
These compounds are synthesized as visual cues, to attract
pollinators and other animals for seed dispersal, as well as mo-
lecular cues protecting plants from various stress conditions, and
are stored in the acidic vacuole of specialized cells [216]. Antho-
cyanins are also the pigments responsible for spectacular displays
of variable red to reddish-orange color in the leaves of deciduous
trees. Why do leaves turn red? The adaptive value of the autumn
colors of leaves is still a matter of controversy. Red may protect the
leaf from the damaging effects of light at low temperatures
(photo-inhibition and photo-oxidation), allowing a more efficient
resorption of nutrients, especially nitrogen (“photoprotection
theory”). Alternatively, red might be a warning signal of the status
of the tree (indicating high levels of defenses or low nutritional
capacity) to animals, particularly feeding insects like aphids
(“coevolution theory”). During winter, the combination of cold,
dry, and bright sunlight conditions can result in excess energy
capture relative to processing, photoinhibition of photosynthesis,
formation of reactive oxygen species (ROS), and greater photo-
oxidative damage. Red pigments are thought to alleviate these
stress factors by intercepting green sunlight (light attenuation),
and/or neutralizing ROS directly as antioxidants. On the other
hand, it has been suggested that red pigments reduce the leaf
damage either by making leaves less palatable or less visible to
animals lacking a red visual receptor (camouflage), or by signaling
a low leaf quality. According to the coevolution theory, red is a
signal of the status of the tree to insects that migrate to (or move
among) the trees in autumn. Migrating insects avoid red leaves
and colonize preferentially green leaves. Trees with red leaves
have better chemical defenses or a worst nutritional capacity that
induces a lower fitness in the insects. In this scenario, therefore,
color and preference coevolve in an arms race: autumn colors are
an adaptation of the trees to reduce their parasite load and insect
preference for green is an adaptation to find the most suitable host
trees [217e222].
Fig. 4. Relationship between proline cycle, oxidative steps of pentose phosphate pathway, and phenylpropanoid pathway (Redrawn from Ref. [127]).
V. Cheynier et al. / Plant Physiology and Biochemistry xxx (2013) 1e20 15
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4. Concluding remarks
Plant phenolics have a long history of scientific investigation
and represent the most abundant and the most widely represented
class of plant natural products. This class of secondary metabolites
have attracted some of the most distinguished of the Nobel-Prize-
winning organic chemists, including Emil Fischer, who studied
chemical substances used in tanning, Richard M. Willstätter, who in
1913 proposed the first chemical hypothesis concerning blue flower
color development, Robert Robinson (co-pigmentation theory),
Richard L.M. Synge (interested in interactions of tannins with
proteins), and Alexander R. Todd who worked from 1931 to 1934 on
anthocyanins and other coloring matters together with Sir Robert
Robinson. The continuing interest for polyphenols of scientists
involved with plant constituents is shown by the fact that both the
British and North American Phytochemical Societies originally
styled themselves ‘plant phenolics groups’. Earlier a new ‘plant
phenolics group’(now the “Groupe Polyphénols”) was established
in France (Narbonne 1970: the first meeting; Avignon-Montfavet
1971: the first International Conference on Polyphenols) [[223e
226], see also Ref. [5] for a fascinating history of (poly)phenol
chemistry]. Nowadays, investigations on plant polyphenols concern
many different scientific domains, ranging from chemistry and
physico-chemistry, biosynthesis, genetics and metabolic engineer-
ing, physiology and ecophysiology still to food technology and
nutrition and health.
When pioneering plant species spread out from the periphery of
their watery environments to occupy dry land, a challenging new
environmental niche, this important transformation was accom-
panied by several physiological adaptations, including the evolu-
tionary emergence of entirely new specialized metabolic pathways.
Much of the rich chemical diversity of the plant kingdom arises
from a limited number of chemical scaffolds (such as polyketide
structures), which are modified by specific types of chemical sub-
stitutions (hydroxylation, glycosylation, acylation, O-methylation
and so on) brought about by specific enzymes [227e230]. Signifi-
cant advances have been made concerning the biosynthesis,
regulation and genetic manipulation of plant phenolics. Therefore,
there are many opportunities to use this wealth of sequence in-
formation to accelerate progress toward a comprehensive under-
standing of the genetic mechanisms that control plant growth and
development and responses to the environment. In this context,
cells of eukaryotic organisms must not only respond to environ-
mental signals, they must also coordinate and organize the re-
sponses of partner cells. Determining the mechanisms that control
the information exchange between these cells is still a major
challenge for biology [130]. What about the role of plant phenolics
in the transduction pathway between perception of an environ-
mental stimulus and physiological response within the plant cell?
As previously stated, accumulation of phenolic compounds in plant
tissues is a distinctive characteristic of various environmental stress
that divert substantial amounts of substrates from primary meta-
bolism into secondary product formation and thus causes major
perturbations of the cellular homeostasis. At the same time strong
induction was observed for mRNAs encoding glucose 6-phosphate
dehydrogenase (carbohydrate metabolism, providing substrates for
the shikimate pathway) and 3-deoxyarabinoheptulosonate 7-
phosphate synthase (shikimate pathway, yielding phenylalanine).
In many plants, free proline also accumulates in response to a wide
range of biotic and abiotic stresses. It has been proposed that a
stress induced increase in the transfer of reducing equivalents into
proline synthesis (cytosolic) and degradation (mitochondrial) cycle
might enable sensitive regulation of cellular redox potential in
cytosol [127,231,232]. These experimental data suggest that envi-
ronmental stresses selectively induce all those primary as well as
secondary metabolic activities that are directly and indirectly
involved in the accumulation of phenolic compounds. A possible
sequence of biochemical reactions inside the cell, which transfer an
environmental signal from the outside of the cell into the plant cell,
thus producing a physiological response, could be envisaged (Fig. 4)
[127,233]. This signal transduction pathway postulates a link be-
tween primary and secondary metabolism that couples the accu-
mulation of the stress metabolite proline with the energy transfer
toward phenylpropanoid biosynthesis via the oxidative pentose
phosphate pathway. Under several conditions of stress, the plant is
forced to accumulate a large quantity of free proline. Its synthesis is
accompanied by the oxidation of NADPH. An increased NADP
þ
/
NADPH ratio is likely to enhance activity of the oxidative pentose
phosphate pathway providing precursors for phenolic biosynthesis
via the shikimic acid pathway.
Acknowledgments
Financial support by the Autonomous Province of Trento, Italy
(ADP 2012/13) is gratefully acknowledged (S. Martens).
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