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218
Biosynthesis of flavonoids and effects of stress
Brenda Winkel-Shirley
The accumulation of red or purple flavonoids is a hallmark of
plant stress. Mounting evidence points to diverse physiological
functions for these compounds in the stress response.
Advances are also being made toward understanding how
plants control the types and amounts of flavonoids that are
produced in response to different cues.
Addresses
Department of Biology, Virginia Tech, Blacksburg, Virginia 24061-0406,
USA; e-mail: [email protected]
Current Opinion in Plant Biology 2002, 5:218–223
1369-5266/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Published online 22 March 2002
Abbreviations
CHI
chalcone isomerase
CHS
chalcone synthase
FLS
flavonol synthase
IAA
indole acetic acid
NPA
naphthylphthalamic acid
tt
transparent testa
UV
ultra-violet
Introduction
Flavonoids are ubiquitous plant secondary products that
are best known as the characteristic red, blue, and
purple anthocyanin pigments of plant tissues [1]. These
compounds serve essential functions in plant reproduction
by recruiting pollinators and seed dispersers. They are also
responsible for the beautiful display of fall color in many
plant species, which has recently been suggested to
protect leaf cells from photo-oxidative damage, thereby
enhancing the efficiency of nutrient retrieval during
senescence [2•]. In fact, flavonoids are a remarkably
diverse group of secondary products (Figure 1) with a vast
array of biological functions, including apparent roles in
stress protection. The flavonols may be among the most
important flavonoids in this regard; they are the most
ancient and widespread of the flavonoids, synthesized
even in mosses and ferns, and have a wide range of potent
physiological activities [3]. Progress continues to be made
in understanding the roles of flavonoids in stress protection,
as well as in defining the mechanisms that control the
amounts and varieties of flavonoids that are produced in
plants in response to diverse environmental cues [4].
Flavonoids and stress protection
The ultra-violet (UV)-absorbing characteristics of
flavonoids have long been considered to be evidence for
the role of flavonoids in UV protection. Indeed, flavanoids
are often present in the epidermal cell layers of leaves and
in tissues that are susceptible to UV light, such as pollen
and the apical meristem. The first direct evidence in
support of a role for flavanoids in UV protection came from
experiments with Arabidopsis mutants, which showed that
lesions in chalcone synthase (CHS) or chalcone isomerase
(CHI) resulted in UV-hypersensitive phenotypes [5].
Interestingly, the CHI mutants were the most sensitive to
UV light and showed a corresponding decrease in sinapate
esters. This evidence suggests that other phenolic
compounds may be at least as important as flavonoids in
UV protection, a hypothesis that has been supported by
subsequent studies [6,7]. However, work by Ryan et al.
[8,9] on petunia and Arabidopsis has provided new
evidence that UV light induces the synthesis of flavonols
with higher hydroxylation levels. Because hydroxylation
does not affect the UV-absorbing properties of these
compounds but does affect their antioxidant capacity, it
was suggested that flavonols may play as yet uncharacterized
roles in the UV stress response. A role for flavonoids in UV
protection is further supported by Bieza and Lois’ [10]
isolation of an Arabidopsis mutant that is tolerant of
extremely high UV-B levels. This line shows constitutively
high levels of a number of phenolics, including flavonoids,
and upregulation of the CHS gene. Clearly, much still
remains to be learned in this area, including how the
synthesis of specific flavonoids and other phenolics is
regulated in response to UV light, how flavonoids compare
to other phenolics in contributing to UV stress protection,
and whether there are roles for flavonoids in UV protection
beyond the absorption of UV radiation.
There is also new evidence that flavonoids play a role in
resistance to aluminum toxicity in maize [11]. Roots of
maize plants that were exposed to aluminum exuded high
levels of phenolics, and an aluminum-resistant variety
exuded a 15-fold higher level of flavonoids when pretreated
with silicon than when no such pre-treatment was applied.
These observations are certainly consistent with the
metal-binding activity of many flavonoids. Although some
researchers have argued that flavonoids are unlikely to be
effective in binding metals in an acid environment because
of competition from H+ ions, Kidd et al. [11] make the
interesting point that a pentahydroxyflavone, morin, is
routinely used to stain for aluminum in the root apoplast.
Further work is needed to corroborate their findings and to
extend them to other parts of the plant, including the cell
interior, and to other plant species.
After an interval of almost ten years, new evidence has
been uncovered linking flavonoids with control of the
polar transport of the plant growth regulator auxin. This
hormone may function in the stress response by helping to
control stomatal opening [12] and by allocating resources
under poor growth conditions [13]. Flavonoids have limited
structural similarity to auxin, but do resemble the synthetic
auxin transport inhibitor naphthylphthalamic acid (NPA;
Figure 2), which is believed to bind a regulatory protein
Biosynthesis of flavonoids and effects of stress Winkel-Shirley
219
Figure 1
3-Malonyl-CoA
+
4-Coumaroyl-CoA
CHS/CHR
CHS
Trihydroxychalone
Tetrahydroxychalcone
AS
CHI
Liquiritigenin
DFR
Aurones
Pigmentation
IFS
Naringenin
F3H
F3′H
F3′5′H
Eriodictyol
FS1/FS2
FS1/FS2
Flavones
Nodulation
Defense
3-OH-flavanones
(dihydroflavonols)
IFR
2′-hydroxy isoflavanone
Pigmentation
Defense
DFR
F3′H
IOMT
I2′H
3-Deoxyanthocyanidins
CHI
IFS
Isoflavones
Flavan-4-ols
AS
FLS
DFR
Flavonols
VR
RT, OMT,
UFGT
Pigmentation (Bee's
purple)
UV protection
Male fertility
Signaling
Flavonol glycosides
DMID
Isoflavonoids
Nodulation
Defense
LCR
Flavan-3,4,-diols
(leucoanthocyanidins)
Flavan-3-ols
LDOX
(ANS)
3-OH-Anthocyanidins
Proanthocyanidins
(condensed tannins)
OMT
Pigmentation
Defense
UFGT
RT
Anthocyanins
Pigmentation for recruitment of
pollinators and seed dispersers
Current Opinion in Plant Biology
Schematic of the flavonoid biosynthetic pathway. ANS, anthocyanidin
synthase; AS, aureusidin synthase; C4H, cinnamate-4-hydroxylase;
CHR, chalcone reductase; DFR, dihydroflavonol 4-reductase; DMID,
7,2′-dihydroxy, 4′-methoxyisoflavanol dehydratase; F3H, flavanone
3-hydroxylase; F3′H, flavonoid 3′ hydroxylase; F3′5′H, flavonoid
3′5′ hydroxylase; FSI/FS2, flavone synthase; I2′H, isoflavone
2′-hydroxylase; IFR, isoflavone reductase; IFS, isoflavone synthase;
IOMT, isoflavone O-methyltransferase; LCR, leucoanthocyanidin
reductase; LDOX, leucoanthocyanidin dioxygenase; OMT,
O-methyltransferase; PAL, phenylalanine ammonia-lyase; RT, rhamnosyl
transferase; UFGT, UDP flavonoid glucosyl transferase; VR, vestitone
reductase. The names of the major classes of flavonoid endproducts
are boxed. Some of the known functions of the compounds in each
class are indicated in italics.
that is associated with the auxin efflux carrier [14]. In
1988, Jacobs and Rubery [15] published evidence that
flavonoids, particularly apigenin and the flavonol
quercetin, could compete with NPA to perturb auxin
transport. Although two additional reports provided some
supporting evidence [16,17], this topic remained controversial. Studies of Arabidopsis flavonoid mutants have now
provided new support for a role for flavonoids in auxin
transport [14]. The transparent testa (tt) mutants, which are
deficient in CHS activity, have been shown to have elevated
auxin transport and altered growth phenotypes that are
consistent with this [18•]. These mutants also accumulate
more indole acetic acid (IAA) in the upper root than do
wildtype controls, and IAA appears to leak from the root
tip of these mutants [19]. Flavonoids have been shown to
accumulate in the cotyledonary node, the hypocotyl–root
transition zone, and the root tip of young Arabidopsis
seedlings [20]. Moreover, the flavonoid biosynthetic
machinery has been localized to the apical end of cortex
cells in the root elongation zone of these plants [21].
220
Physiology and metabolism
Figure 2
O
O
OH
NH2
N
H
COOH
OH
+3
H2C
NH2
Tryptophan
Phenylalanine
COSCoA
Malonyl-CoA
OH
OH
N
H
O
HO
OH
O
OH
OH
IAA
Structures of representative auxins and
flavonols, and their aromatic amino acid
precursors. IAA, the major plant auxin, is
derived from tryptophan, whereas flavonoids,
including flavonols such as quercetin, are
synthesized from phenylalanine and malonylCoA. NPA is a synthetic inhibitor of the auxin
efflux carrier that is believed to interact with an
associated regulatory protein rather than
competing directly for auxin binding;
flavonoids such as quercetin may have a
similar effect.
H
O
Quercetin (a flavonol)
H
N
O
O
OH
NPA
Current Opinion in Plant Biology
Future studies will determine whether changes in the
synthesis or deposition of specific flavonoids within the cell
may act to change the rate or direction of auxin transport.
Studies of these types raise the long-standing question of
how the subcellular distribution of flavonoid compounds is
determined. Researchers are continuing to gain insights
into how flavonoids are partitioned into the vacuole, most
recently through analysis of the tt12 mutant of Arabidopsis
[22•]. The TT12 gene encodes a protein with similarity to
members of the MATE (multidrug and toxin extrusion)
family of transporters, which appear to control the vacuolar
sequestration of flavonoids in the seed-coat endothelium.
In maize, the bronze2 locus has been found to encode a
glutathione transporter that is a key player in vacuolar
uptake of flavonoids [23]. These findings suggest that
different plant species may use different mechanisms to
distribute flavonoids among subcellular compartments or
that multiple mechanisms are used in individual species.
This is an area of flavonoid biology that is still poorly
understood and that is of significant current interest.
Transcriptional regulation of the flavonoid
pathway
One important avenue to understanding the role of
flavonoids in the stress response is to understand how the
expression of the biosynthetic pathway is regulated. This
system represents one of the oldest examples of coordinated
gene and enzyme regulation in response to environmental
and developmental factors [24]. A great deal has been
learned from studies in a variety of plant species, primarily
about transcriptional regulation, although evidence for
other types of control also exists. Recent advances in this
area include the isolation by genetic approaches of
transcription factors that are identified by the petunia
anthocyanin1 (an1) [25] and Arabidopsis tt8 regulatory
mutants [26]. Both transcription factors are new members
of the basic helix-loop-helix (bHLH) family, having
similarity to R1 in maize and DELILA (DEL) in snapdragon,
and are required for the activation of the dihydroflavonol
reductase gene as well as other genes. The Arabidopsis TT2
gene has now also been cloned by a genetic approach and
found to encode an R2R3 myb domain protein that
appears to function as a key regulator of proanthocyanidin
accumulation in developing seed [27•]. This gene could
prove to be a powerful tool for engineering proanthocyanidin
synthesis in seed crops [28]. An apparent complex of novel
regulatory proteins that may interact with CHS, as well as
with other flavonoid genes, was recently identified in
parsley using South-western and two-hybrid screening
[29]. Analysis of strawberry R2R3 myb homologs by their
overexpression in transgenic tobacco plants has uncovered
what appears to be a negative regulator of flavonoid gene
expression [30]. Gareth Jenkins’ group has come at this
from the opposite direction. They studied the expression
of the CHS gene to develop a model for the interaction
of the various light signalling pathways, in which
phytochrome B balances flux through the cryptochrome1
Biosynthesis of flavonoids and effects of stress Winkel-Shirley
and UV-B signalling pathways [31]. The data emerging
from these studies as a whole suggest that different plants
use somewhat different mechanisms to control the expression
of flavonoid genes, consistent with the diverse requirements
of flavonoids in different species.
Enzymology of flavonoid biosynthesis
Many, although not all, of the enzymes of flavonoid
biosynthesis are encoded by small gene families. The
functional significance of this redundancy has been the
subject of substantial interest over the years. Kimura et al.
[32•] reported recently that licorice (Glycyrrhiza echinata)
contains two CHI isozymes that can use both 6′-hydroxychalcone and 6′-deoxychalcone, and therefore are likely to
be involved in the legume-specific isoflavonoid pathway
(Figure 1). One of these genes is induced by elicitor treatment,
and is therefore proposed to function in the stress response
related to wounding. A third enzyme can use only
6′-hydroxychalcone and presumably functions in the
general flavonoid pathway. It will be interesting to discover
whether other legumes use similarly duplicated genes to
provide for the stress-inducible synthesis of isoflavonoids.
In non-legume plants, which do not synthesize isoflavonoid
phytoalexins, CHI generally appears to be encoded by a
single gene; although petunia does contain a second gene
with homology to CHI, the product does not appear to be
involved in the flavonoid pathway [33]. Work is also
underway to examine duplication in other genes of
flavonoid biosynthesis, such as flavonol synthase (FLS), the
only flavonoid enzyme in Arabidopsis that is encoded by a
gene family ([34]; B Winkel-Shirley, unpublished data).
Flavonols are among the most important flavonoids with
respect to biological activity, and the gene duplication of
FLS in Arabidopsis may provide a mechanism for controlling
the types and amounts of flavonols produced in different
tissues and in response to different environmental cues.
This past year has seen the first reported use of a flavonoid
enzyme to enhance the nutritional value of a vegetable crop
by increasing the content of flavonols, which have demonstrated health-promoting activities in animals [35]. The peel
of fruit from tomato plants expressing the petunia chi-a
gene, under control of the double-enhanced 35S promoter,
contained significantly higher levels of naringenin chalcone
and quercetin 3-trisaccharide, a flavonol glycoside [36].
Surprisingly, flavonoid accumulation was not altered in the
fruit flesh or leaves of these transformants. Nonetheless,
tomato paste produced from the transgenic fruits had a
21-fold higher flavonol concentration than paste from
comparable non-transformed tomatoes, presumably because
of leeching of flavonols from the peel into the paste.
221
three-dimensional structures of these enzymes by Joe Noel’s
group within the past few years [37,38•]. Most recently, this
group has combined site-specific mutagenesis with analysis
of X-ray crystal structures to correlate the size of the activesite cavity with the length of the polyketide chain produced
by CHS and related enzymes, providing insights into how
the synthesis of diverse polyketides may be engineered
[38•]. The CHI reaction has also been examined relative to
the spontaneous cyclization reaction of di- and tri-hydroxychalcones, showing that catalysis involves binding of the
ionized chalcone in a conformation that accelerates ring closure [39•]. In addition, some new information has emerged
on the organization of flavonoid enzymes within complexes
comprised of one or more enzymes. For example, a study
by Rick Dixon’s group has shown that metabolic channeling
in the isoflavonoid pathway controls the regiospecificity of
the alfalfa isoflavone O-methyltransferase reaction [40•].
As mentioned earlier, new information has also been
published on the subcellular localization of flavonoid
enzymes in Arabidopsis roots [21], and there is evidence that
this localization may change in response to wounding
(D Saslowsky, B Winkel-Shirley, unpublished data). At the
same time, complementation of Arabidopsis flavonoid
mutants using maize genes [41] suggests either that
enzyme interactions have been conserved across large
evolutionary distances or that it is possible to override the
constraints of metabolic organization by high-level
expression of heterologous enzymes.
Conclusions
The accumulation of anthocyanin pigments in vegetative
tissues is a hallmark of plant stress, yet the role that
flavonoids play in the stress response is still poorly understood. In many cases, these compounds may provide
antioxidant activity as part of a general stress response,
which may also explain their health-promoting qualities in
animals. However, there is also evidence that flavonoids
may function in plants to screen harmful radiation, bind
phytotoxins, and help to regulate the stress response by
controlling auxin transport. Understanding the molecular
basis of flavonoid function in ameliorating stress, as well as
the regulatory and biochemical mechanisms that control
the types and amounts of flavonoids synthesized under
different conditions, continues to be a high priority for
research with an eye to engineering enhanced stress
tolerance in crop plants. These efforts are benefiting from
the integration of emerging technologies with the extensive
genetic and biochemical tools that have been developed
for this system over the years.
References and recommended reading
Rational approaches to the metabolic engineering of
flavonoid biosynthesis will certainly depend on a detailed
understanding of the enzymology of the flavonoid pathway.
A wealth of new information continues to emerge regarding
the reaction mechanisms of CHS and CHI, an area that has
been significantly bolstered by the determination of the
Papers of particular interest, published within the annual period of review,
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223
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•
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