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International Journal of Applied Science and Engineering
2006.4, 3: 221-233
Int. J. Appl. Sci. Eng., 2006. 4, 3 221
Populus, the New Model System for Investigating Phenyl-
propanoid Complexity
Chung-Jui Tsai *, Walid El Kayal and Scott A. Harding
Biotechnology Research Center, School of Forest Resources and Environmental Science,
Michigan Technological University,
Houghton, MI 49931, U.S.A.
Abstract: Plant secondary metabolism affects ecosystem diversity and the yield and quality of
feedstocks for biomass and biofuel, through an elaborate network of pathways that share com-
mon precursors. Until recently, functional dissection of these networks has depended largely on
molecular information stored in the genome of Arabidopsis, an annual herb. Now that the Popu-
lus genome sequence is available, the potential for understanding and exploiting secondary me-
tabolism in tree species comes closer to realization. In the present overview, genomic informa-
tion pointing to greatly expanded gene complexity and function of the phenylpropanoid pathway
in Populus is summarized. Phenylpropanoid-derived flavonoid and salicylate phenolics occur in
numerous functionally distinct forms, and can account for 50% of leaf biomass in Populus and
other fast-growing tree taxa. Their potential effects on tree growth, and their documented im-
pacts on ecosystem diversity and productivity justify molecular dissection of secondary metabo-
lism in Populus. Biosynthesis of salicylate phenolics remains poorly understood. By contrast, in
silico promoter analysis of flavonoid genes, and in situ flavonoid localization in Populus re-
ported here, augment published gene expression data, and illustrate that intra and intercellular
regulatory components dramatically affect secondary carbon partitioning in this woody peren-
nial.
Keywords: lignin; phenolic glycosides; condensed tannins; Populus genome.
* Corresponding author; e-mail: chtsai@mtu.edu.tw Accepted for Publication: November 29, 2006
© 2006 Chaoyang University of Technology, ISSN 1727-2394
Introduction
Phenylpropanoid metabolism supplies a
wide array of general as well as spe-
cies-specific phenolic compounds that are
central to the success of land plants and
plant-based industrial applications [1]. Al-
though traditionally classified as “secondary
compounds”, phenylpropanoid products are
now recognized for their significant roles
during plant growth, development, reproduc-
tion, adaptation, and defense. Major classes of
phenylpropanoid products include lignins as
cell wall structural components; lignans as
defense compounds or antioxidants; flavon-
oids as pigments, signaling molecules, and
protectants against biotic and abiotic stresses;
and hydroxycinnamate derivatives, both free
and conjugated, for structural and protective
functions. The phenylpropanoid pathway thus
offers opportunities for metabolic engineering
of a range of agronomically important pheno-
lics, affecting traits from disease resistance to
fiber and wood quality, and providing the ba-
Chung-Jui Tsai, Walid El Kayal,and Scott A. Harding
222 Int. J. Appl. Sci. Eng., 2006. 4, 3
sis for novel flavor/fragrance compounds, nu-
triceuticals and pharmaceuticals. Phenylpro-
panoid metabolism is also of prime interest in
the emerging area of ecological genomics, as
it underpins plant interactions with the envi-
ronment. For long-lived species like trees, al-
location of the phenylpropanoid pools during
development and in response to the perennial
environmental fluctuations represents a major
fitness trait, one that may not be adequately
modeled on the basis of the herbaceous an-
nual paradigm exemplified in Arabidopsis or
maize. Thanks to the release of the Populus
genome sequence [2], the ecological rele-
vance of phenylpropanoid metabolism in
perennial woody species can finally be tack-
led at the genomics level. This overview will
cover the three major phenylpropanoid pools
of Populus (Figure 1), lignin, salicy-
late-derived phenolic glycosides (PGs), and
flavonoid-derived condensed tannins (CTs),
with a special emphasis on CTs. The readers
are referred to other excellent recent reviews
[3-5] for in-depth coverage.
Lignin
Lignin is the major phenolic sink in the stem,
accounting for 18-25% of dry woody biomass
[6]. As a structural component of the cell wall,
lignins limit forage digestibility by ruminants
and interfere with cellulosic-based biomass
conversion for bioenergy and pulp. Positive
attributes of lignins include their min-
eral-/protein-binding activities, which slow
decomposition and release of C into the at-
mosphere by plant detritus [1]. Lignins can
replace petroleum-based sources for use as
biobased resin in the fabrication of printed
wiring board for the electronics industry [7].
Lignins can also be used as filler in biode-
gradable plastics or package materials [8].
The branch pathways leading to the biosyn-
thesis of monolignols (Figure 1) have been
extensively characterized in trees, due to the
commercial significance of lignin modifica-
tion. The entire suite of putative lignin bio-
synthetic pathway genes identified from the
Populus genome is listed in Table 1. All be-
long to multi-gene families, and only a hand-
ful of genes have been functionally character-
ized. However, expression and kinetic data
suggest that in many cases individual gene
family members have functionally distinct
roles and are differentially involved in lignin
and non-lignin phenolic metabolism, as ex-
emplified for PAL [9] and 4CL [10, 11]. Al-
though only a handful of genes have been
targeted for genetic manipulation of lignin to
date, both qualitative and quantitative modi-
fications have been reported in transgenic
Populus. Lignin structural modification has
been reported in almost all cases, but a sub-
stantial increase in syringyl-to-guaiacyl lignin
(S/G) ratio, a characteristic positively corre-
lated with pulping efficiency [12], has only
been achieved by over-expression of F5H [13].
Reduction of lignin content was reported fol-
lowing down-regulation of 4CL [13, 14],
CCoAOMT [15] and CCR [3]. In addition,
transgenic poplars with reduced CAD did not
exhibit significant change in lignin content or
S/G ratio, but there was an increase in the in-
corporation of both coniferyl and sinapyl al-
dehydes into the lignin [16, 17]. Kraft pulping
of CAD-deficient transgenic poplar grown at
two European field sites for 4 years showed
improved pulp yields and reduced cellulose
degradation compared to the control [18].
Commercial-scale application of transgenic
poplars with improved lignin characteristics is
expected to reduce environmental burdens
associated with pulping.
Phenolic glycosides
Salicylate-derived PGs do not accumulate in
Arabidopsis or other important herbaceous
model systems, but are highly characteristic
of the Salicaceae family of fast-growing
woody species, including Salix (willows) and
Populus [19]. The wide use of willow and
poplar barks in herbal remedies can be attrib-
uted to the abundance of PGs in these species.
Populus, the New Model System for Investigating Phenylpropanoid Complexity
Int. J. Appl. Sci. Eng., 2006. 4, 3 223
Salicin, the first PG identified from plants, is
the pain-relief ingredient in willow extracts
[reviewed in 20]. In poplars and willows, PGs
serve primarily protective functions, having
been associated with insect defense [21],
UV-B protection [22] and drought response
[23]. The putative PG precursor salicylic acid
(SA) is widespread in higher plants and plays
a central role in defense signal transduction.
However, biosynthesis of PGs and SA re-
mains poorly understood. It has long been
thought that SA is biosynthesized from cin-
namate via benzoate [24], and requires PAL.
An additional, PAL-independent pathway
utilizing plastidic isochorismate synthase
(ICS) appears to operate in certain, but not all,
SA-mediated defense responses of Arabidop-
sis [25, 26]. There are two Arabidopsis ICS
genes, a plastid-localized AtICS1 and a cyto-
solic AtICS2, but neither appears to be ex-
pressed in healthy leaves, and only AtICS1 is
pathogen-inducible [25]. Interestingly, the
poplar genome contains a single, likely plas-
tid-targeted ICS gene that is
Figure 1. Biosynthetic pathways of major phenylpropanoid end products, lignins, phenolic glycosides and
condensed tannins in Populus. Enzyme abbreviations are listed in Table 1. Enzymatic steps that
are not yet identified are shown as dashed arrows
Chung-Jui Tsai, Walid El Kayal,and Scott A. Harding
224 Int. J. Appl. Sci. Eng., 2006. 4, 3
Table 1. List of phenylpropanoid pathway genes in Populus
Protein
name JGI Gene Model Locus Protein name JGI Gene Model Locus
Phenylalanine ammonia-lyase Cinnamoyl-CoA reductase
PAL1 estExt_Genewise1_v1.C_280661 scaffold_28:2031644-2035061 CCR1 fgenesh4_pg.C_scaffold_208000034 scaf-
fold_208:275317-27778
PAL2 estExt_fgenesh4_pg.C_LG_VIII0293 LG_VIII:1885833-1890028 CCR2 estExt_fgenesh4_kg.C_LG_III0056 LG_III:16013635-16017
348
PAL3 grail3.0004045401 LG_XVI:7316776-7319927 CCR3 gw1.208.126.1 scaf-
fold_208:329845-33231
PAL4 estExt_fgenesh4_pg.C_LG_X2023 LG_X:19171449-19174898 CCR4 gw1.208.109.1 scaf-
fold_208:292935-29538
1
PAL5 gw1.X.2713.1 LG_X:19181547-19184719 CCR5 estExt_fgenesh4_pg.C_2080041 scaf-
fold_208:343600-34630
Cinnamate 4-hydroxylase CCR6 estExt_fgenesh4_pg.C_LG_I0389 LG_I:3184141-3186481
C4H1 estExt_fgenesh4_pg.C_LG_XIII0519 LG_XIII:12820991-12825303
Cinnamyl alcohol dehydrogenase
C4H2 grail3.0094002901 LG_XIX:10989662-10992697 CAD estExt_Genewise1_v1.C_LG_IX2359 LG_IX:4268866-427113
7
C4HL1 gw1.164.158.1 scaffold_164:432424-434020 SAD grail3.0004034803 LG_XVI:5830946-5834
884
4-Coumarate-CoA ligase
Chalcone synthase
4CL1 grail3.0100002702 LG_I:1432116-1435302 CHS1 eugene3.00140920 LG_XIV:7151030-7153
182
4CL2 grail3.0099003002 LG_XIX:4083532-4087345 CHS2 estExt_fgenesh4_pg.C_LG_I0449 LG_I:3683042-3684777
4CL3 estExt_fgenesh4_pg.C_1210004 scaffold_121:49867-56929 CHS3 estExt_fgenesh4_pg.C_LG_I0450 LG_I:3690127-3692694
4CL4 gw1.XVIII.2818.1 LG_XVIII:9666118-9671112 CHS4 eugene3.00031460 LG_III:15665340-15667
449
4CL5 fgenesh4_pg.C_LG_III001773 LG_III:17994254-17998436 CHS5 eugene3.00031461 LG_III:15672197-15673
829
Hydroxycinnamoyl-CoA quinate/shikimate hydroxycinnamoyltransferase CHS6 eugene3.00031462 LG_III:15678905-15680
833
HCT1 fgenesh4_pg.C_LG_III001559 LG_III:16193170-16196683
Chalcone isomerase
HCT2 estExt_fgenesh4_pm.C_LG_XVIII0344 LG_XVIII:10668161-10672154 CHI1 estExt_Genewise1_v1.C_LG_X2396 LG_X:18468933-18471
655
HCT3 estExt_fgenesh4_pg.C_LG_XVIII0910 LG_XVIII:10642781-10645134
Flavanone 3-hydroxylase
HCT4 eugene3.00180947 LG_XVIII:10631699-10633511 F3H gw1.57.31.1 scaf-
fold 57:716253-717841
HCT5 fgenesh4_pg.C_scaffold_133000007 scaffold_133:86412-88323
Flavonoid 3'-hydroxylase
HCT6 eugene3.02080010 scaffold_208:113566-117563 F3'H estExt_fgenesh4_pg.C_LG_XIII0337 LG_XIII:6197800-6200
404
HCT7 eugene3.18780002 scaffold_1878:6898-8809
Flavonoid 3'5'-hydroxylase
4-Coumarate 3-hydroxylase F3'5'H1 eugene3.00090961
LG_IX:6110882-611272
3
C3H1 eugene3.36160002 scaffold_3616:2997-5408 F3'5'H2 eugene3.00011827 LG_I:19972937-199751
22
C3H2 eugene3.00160247 LG_XVI:1538875-1542646
Flavone synthase
C3H3 fgenesh4_pg.C_LG_VI000268 LG_VI:1979652-1982315 FNSII1 estExt_fgenesh1_pg_v1.C_LG_XIII0255
LG_XIII:1794033-1795
947
Ferulate 5-hydroxylase FNSII2 eugene3.00700209
scaf-
fold 70:1407665-14101
F5H1 estExt_fgenesh4_pm.C_570058 scaffold_57:1035361-1038589
Flavonol synthase
F5H2 eugene3.00071182 LG_VII:11484639-11486746 FLS1 grail3.0191001301 LG_XIX:123233-12606
7
F5HL1 eugene3.00090440 LG_IX:2644256-2646866 FLS2 eugene3.00020803 LG_II:6082658-608434
6
Caffeic acid O-methyltransferase FLS3 eugene3.01350040
scaf-
fold 135:427496-43062
COMT1 estExt_fgenesh4_pm.C_LG_XII0129 LG_XII:3089139-3092252 FLS4 estExt_fgenesh4_pg.C_1350039 scaf-
fold 135:441620-44432
COMT2 estExt_fgenesh4_pg.C_LG_XV0035 LG_XV:255739-258237
Dihydroflavonol 4-reductase
COMT3 fgenesh4_pg.C_LG_XIV000481 LG_XIV:4314177-4316619 DFR1 estExt_Genewise1_v1.C_LG_II0799 LG_II:2174492-217652
0
COMT4 estExt_Genewise1_v1.C_LG_XIV1942 LG_XIV:4327267-4329146 DFR2 gw1.V.1407.1 LG_V:15923736-15925
503
COMT5 eugene3.00021675 LG_II:14132201-14134094
Anthocyanidin synthase
COMT6 fgenesh4_pm.C_LG_II000840 LG_II:14167706-14169393 ANS1 grail3.0018022801 LG_III:11400392-11401
847
COMT7 eugene3.00012911 LG_I:33700503-33702138 ANS2 eugene3.00010988 LG_I:8507517-8509264
COMT8 gw1.XVI.3248.1 LG_XVI:9185925-9187398
Anthocyanidin reductase
COMT9 fgenesh4_pm.C_LG_XI000417 LG_XI:14176611-14178062 ANR1 estExt_fgenesh4_pm.C_LG_IV0055 LG_IV:1671017-167330
2
Caffeoyl-CoA O-methyltransferase ANR2 estExt_fgenesh4_pm.C_LG_XI0107 LG_XI:3895226-38975
81
CCoAOM
T1 grail3.0001059501 LG_IX:4059145-4060914
Leucoanthocyanidin reductase
CCoAOM
T2 estExt_fgenesh4_pm.C_LG_I1023 LG_I:26412640-26415499 LAR1 grail3.0010045601 LG_VIII:7398478-7400
397
Populus, the New Model System for Investigating Phenylpropanoid Complexity
Int. J. Appl. Sci. Eng., 2006. 4, 3 225
Table 1. List of phenylpropanoid pathway genes in Populus (continued)
Protein
name JGI Gene Model Locus Protein name JGI Gene Model Locus
CCoAO
MT3 estExt_fgenesh4_pm.C_1450034 scaffold_145:744229-746661 LAR2 eugene3.00101230 LG_X:12874015-12876
623
CCoAO
MT4 fgenesh4_pm.C_LG_X000399 LG_X:10889762-10892213 LAR3 estExt_fgenesh4_pm.C_LG_XV0077
LG_XV:2126571-21286
64
CCoAO
MT5 estExt_fgenesh4_pg.C_LG_VIII1209 LG_VIII:9019120-9021383
CCoAO
MT6 fgenesh4_pg.C_LG_II001689 LG_II:14407483-14409698
detected in PG-accumulating leaves and
shoots, but absent in roots [19]. In contrast to
Populus where PG stores can become very
large, e.g., up to 30% leaf dry weight in cer-
tain genotypes [27], genetic manipulation to
enhance SA-based constitutive defense out-
lays in Arabidopsis resulted in dwarfing [e.g.,
28]. It appears that Populus and Salix spp
have evolved a mechanism for the efficient
management of PG metabolism for both
growth and defense, and may serve as an at-
tractive model to understand PG and SA bio-
synthesis.
Condensed Tannins (CTs)
Flavonoids make up a large class of spe-
cies-specific phenolic compounds, and are
commonly associated with pigmentation,
stress responses, defense, reproduction and
symbiotic interactions [27, 28]. CTs, in par-
ticular, encompass the most structurally, and
functionally complex members of the flavon-
oids [29] and their protein-binding properties
account for their historical importance to the
tanning industry. They act as deterrents to mi-
crobial, insect or animal feeding [30-32]. CTs
in leaf detritus bind to organic soil constitu-
ents, slow carbon mineralization and increase
soil fertility [32, 33]. CTs also bind to poten-
tially phytotoxic forms of aluminum [34] and
other metals [35], a valuable trait to be ex-
plored for phytoremediation applica-
tions. CTs are important determinants of
seed nutritional properties [36] due to their
powerful antioxidant activity. Foods rich in
antioxidant CTs (e.g., grape, cranberry, red
wine) are of particular interest for their pro-
tective roles in human health [37].
The flavonoid biosynthetic pathways are
more complex in Populus than in Arabidopsis,
both in terms of chemical diversity and gene
regulation [19]. The pathway is well
characterized in Arabidopsis, and all
flavonoid biosynthetic enzymes, except
flavonol synthase, are encoded by single-copy
genes [28]. In contrast, a vast majority of the
flavonoid pathway enzymes are encoded by
gene families in Populus (Table 1). The only
exceptions are chalcone isomerase (CHI),
flavonoid 3’-hydroxylase (F3’H), and
flavanone 3-hydroxylase (F3H). The
expanded flavonoid gene families are
consistent with substantial accumulation of
flavonoid-derived CTs in vegetative tissues of
Populus, accounting for up to 18% leaf dry
weight in aspen (P. tremuloides) [21], and
concentrations as high as 50% have been
reported in cottonwood (P. angustifolia and
hybrids) [38, 39]. Arabidopsis, on the other
hand, produces CTs primarily in the seed coat
(~1% fresh weight), with little accumulation
(<0.004% fresh weight) in rosette leaves [40].
Populus thus offers a model system distinct
from Arabidopsis to investigate flavonoid
pathway complexity, regulation and carbon
allocation during the rapid expansion of
vegetative tissues including leaves, stems and
roots.
As shown in Figure 1, the flavonoid
biosynthetic pathway branches from
phenylpropanoid metabolism by the action of
chalcone synthase (CHS), whose family is
particularly expanded in poplar and contains
Chung-Jui Tsai, Walid El Kayal,and Scott A. Harding
226 Int. J. Appl. Sci. Eng., 2006. 4, 3
at least six genes, several of them in tandem
repeats (Table 1). In sharp contrast, enzymes
involved in conversion of chalcones to
flavanones and dihydroflavonols, as well as
B-ring hydroxylation of flavanones and
dihydroflavonols are all encoded by
single-copy genes (i.e., CHI, F3H and F3’H).
Two flavonoid 3’,5’-hydroxylase (F3’5’H)
genes are present in the Populus genome, but
exhaustive RT-PCR amplification from a wide
range of genotypes and tissues yielded no
product for F3’5’H2 (unpublished),
suggesting that F3’5’H may also be encoded
by a single functional gene (i.e., F3’5’H1).
Subsequent synthesis of the CT precursor,
2,3-cis-flavan-3-ols, requires dihydroflavonol
4-reductase (DFR), anthocyanidin synthase
(ANS) and anthocyanidin reductase (ANR),
whereas formation of the other CT precursor,
2,3-trans-flavan-3-ols is mediated by DFR
and leucoanthocyanidin reductase (LAR,
Figure 1). Interestingly, all four gene families
contain two paralogous members derived
from genome-wide duplication events [2].
However, the LAR family is unique in that it
contains an additional member (LAR3) that is
phylogenetically distinct from the LAR1 and
LAR2 paralogs [19]. Arabidopsis lacks LAR
and does not accumulate
2,3-trans-flavan-3-ols [41], but both cis and
trans starter units for CTs are present in
Populus and other tree species with large
metabolic commitments to CTs [42]. Based
on gene family size and expression data [19],
the CHS and LAR families may play
important roles in modulating CT
biosynthesis, structure and functional
diversity in Populus.
Flavonols are among the most widespread
flavonoids in plants, and have been associated
with a range of physiological activities, in-
cluding UV-protection, signaling, male steril-
ity and auxin transport regulation [5]. In
Arabidopsis, they represent the only flavonoid
compounds detected in vegetative tissues [43].
Flavonols are synthesized from dihydrofla-
vonols by flavonol synthase (FLS), repre-
sented in Arabidopsis by six genes, only one
of which has been functionally characterized
[44]. Populus contains four FLS genes, three
of which are expressed in leaves [19]. Unlike
many other flavonoid biosynthetic genes,
however, FLSs are not wound-inducible in
Populus [19], suggesting a role for FLS in
flavonoid partitioning. The structurally related
flavones are also prevalent in higher plants,
but are conspicuously absent in the Brassica-
ceae, including Arabidopsis [45]. Accordingly,
flavone synthase (FNS) genes are absent in
the Arabidopsis genome. Flavones are de-
tected in bud exudes of Populus species [46,
47], consistent with the identification of 5
genes encoding FNSII of the Cytochrome
P450 family 93B in the Populus genome.
Regulation of CT biosynthesis
Coordinated expression of flavonoid bio-
synthetic pathway genes has been reported in
several species, and in the case of Populus, is
supported by in silico analysis of flavonoid
gene promoters. An AC-rich, MYB-binding
element described as L box-like (ACCWWCC)
[48] or P box-like (MACCWAMC) [49] in
many phenylpropanoid gene promoters is, as
expected, present in the promoters of most of
the Populus flavonoid genes (Table 2). The
G-box (CACGTG), found in the promoters of
ribulose 1,5-bisphosphate carboxylase/ oxy-
genase small subunit (rbcS) [50] and various
other light-induced genes [51], including CHS
[52], is also present in Populus flavonoid
gene promoters, consistent with
light-dependent regulation of flavonoid bio-
synthesis [5]. Another MYB-recognizing
AACA motif confers endosperm-specific ex-
pression of seed storage protein, glutelin, in
rice [53, 54]. The endosperm-specific activity
of glutelin promoter is reminiscent of the en-
dothelium-specific expression of the BAN
promoter and proanthocyanidin-accumulation
in Arabidopsis seed [55]. Consistent with this,
the AACA motif is found in the promoters of
all CT biosynthetic gene families, but under-
Populus, the New Model System for Investigating Phenylpropanoid Complexity
Int. J. Appl. Sci. Eng., 2006. 4, 3 227
represented in the FLS and FNS gene pro-
moters (Table 2). Flavonoids are known to
play an important signaling role during root
development and legume nodulation, proc-
esses that are intimately linked to auxin re-
sponse [5]. An auxin response element
(AuxRE) recognized by the auxin response
factor (ARF) transcription factor family in-
volved in auxin signaling [56] is found in the
promoters of most flavonoid genes, but is
poorly represented in the FLS promoters (i.e.,
present in only 1 of 4 FLS promoters). In
contrast, an ABA responsive element (ABRE)
associated with ABA-mediated dehydration or
drought tolerance [57, 58], and light-regulated
expression of parsley CHS [59] is ubiquitous
in all Populus FLS promoters (Table 2). This
is consistent with the significant up- regula-
tion of Arabidopsis FLS1 (At5g08640), but
not other flavonoid biosynthetic genes, in tran
Table 2. In silico analysis of putative regulatory elements in Populus flavonoid pathway gene promoters
Cis element CHS CHI F3H F3'H F3'5'H DFR ANS BAN LAR FLS FNSII
cis element present in most flavonoid gene promoters
L box-like
(ACCWWCC) 2/6 1/1 1/1 1/1 1/2 1/2 1/2 2/2 3/3 3/4 1/2
P box-like (MAC-
CWAMC) 5/6 1/1 1/1 1/1 1/2 1/2 0/2 2/2 3/3 2/4 0/2
G-box
(CACGTG) 6/6 1/1 0/1 1/1 2/2 1/2 2/2 1/2 0/3 2/4 0/2
cis elements underrepresented in the FLS family
AACA motif
(AACAAAC) 4/6 1/1 1/1 1/1 1/2 1/2 1/2 1/2 3/3 1/4 0/2
AuxRE
(TGTCTC) 2/6 1/1 0/1 0/1 1/2 1/2 2/2 1/2 1/3 1/4 1/2
cis elements overrepresented in the FLS family
ABRE-like
(ACGTGGC) 1/6 0/1 0/1 0/1 0/2 1/2 0/2 0/2 0/3 4/4 0/2
Each data point represents the number of gene(s) containing the specific cis element in the 2-kb promoter(s) over the total number of genes in
each family.
-sgenic plants over-expressing an
ABRE-binding protein [57], and may suggest
a role of flavonols in ABA signaling.
Cellular Localization of flavonoids and CTs
Intercellular transport and its gene regulation
are likely to participate in CT partitioning.
Based on dimethylaminocinnamaldehyde
(DMACA) staining which is specific for CT
starter subunit flavan-3-ols [60],
stress-induced CT accumulated mostly in an
abaxial layer of the spongy mesophyll (Figure
2A). The CT content of the abaxial cells of
wound-induced cottonwood plants is higher
than observed in abaxial cells of unstressed
aspen [9]. Flavonoid epi-fluorescence in the
presence of 2-aminoethyl-diphenyl borinate
[61] revealed that flavonoid precursors to CT
were concentrated in the, upper palisade
mesophyll (Figure 2B). It appears that in ex-
panding leaves, CTs or CT precursors are
synthesized in the upper palisade cells and
then exported. CT induction in roots, appears
Chung-Jui Tsai, Walid El Kayal,and Scott A. Harding
228 Int. J. Appl. Sci. Eng., 2006. 4, 3
to deplete intracellular flavonoids, and to de-
pend more directly on flavonoid pool size
than in leaves (Figure 2C-H). What limits CT
induction in leaves where flavonoid interme-
diates are plentiful is unclear. The possible
relevance of such metabolic controls to
Populus growth is under investigation using
phytochemically distinct genotypes [62].
Concluding Remarks
Because of the economic significance of
Populus for pulp and bioenergy production,
and because of its ecological importance as a
keystone species in terrestrial ecosystems,
advances in phenylpropanoid metabolism
promise to have far-reaching impacts.
Phenylpropanoid sinks are characteristic of
the defense and overall fitness of Populus
species. Their metabolic costs to biomass
growth remain an area of uncertainty. Avail-
ability of the genome sequence, and the
ever-growing genomics resources for Populus
will accelerate research into mechanisms
governing regulation of phenylpropanoid me-
tabolism, resource competition and tradeoffs.
For example, the dynamics of CT and PG
regulation in the context of growth versus
overall plant fitness await dissection at the
molecular level. Advances in phytochemical
regulation should elevate the potential for im-
proved biomass quality and production
through genetic selection or metabolic engi-
neering in Populus.
Figure 2. A-B, Cross sections of young, systemic leaves from wounded cottonwood showing (A) CT depo-
sition (blue DMACA stain), and (B) flavonoid localization (yellow fluorescence). C-H, Cross sec-
tions of roots from cottonwood plants subjected to nitrogen replete (C, E, G) or nitrogen limiting (D,
F, H) conditions for two weeks. CT accumulation shown by DMACA staining increased during lim-
iting N (C vs. D), but flavonoid reserves decreased (E vs. F). Limiting N did not appear to affect
root lignification (G vs. H). The flavonoid and lignin fluorescence images were taken using the
same section
Populus, the New Model System for Investigating Phenylpropanoid Complexity
Int. J. Appl. Sci. Eng., 2006. 4, 3 229
Acknowledgments
Research in our laboratory is supported by
the U.S. National Science Foundation (Plant
Genome Program DBI-0421756), and the U.S.
Department of Energy (Biological and Envi-
ronmental Research Program
DE-FG02-05ER64112).
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