ArticlePDF Available

Abstract and Figures

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.
Content may be subject to copyright.
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-
Keywords: lignin; phenolic glycosides; condensed tannins; Populus genome.
* Corresponding author; e-mail: Accepted for Publication: November 29, 2006
© 2006 Chaoyang University of Technology, ISSN 1727-2394
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 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
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-
PAL2 estExt_fgenesh4_pg.C_LG_VIII0293 LG_VIII:1885833-1890028 CCR2 estExt_fgenesh4_kg.C_LG_III0056 LG_III:16013635-16017
PAL3 grail3.0004045401 LG_XVI:7316776-7319927 CCR3 gw1.208.126.1 scaf-
PAL4 estExt_fgenesh4_pg.C_LG_X2023 LG_X:19171449-19174898 CCR4 gw1.208.109.1 scaf-
PAL5 gw1.X.2713.1 LG_X:19181547-19184719 CCR5 estExt_fgenesh4_pg.C_2080041 scaf-
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
C4HL1 gw1.164.158.1 scaffold_164:432424-434020 SAD grail3.0004034803 LG_XVI:5830946-5834
4-Coumarate-CoA ligase
Chalcone synthase
4CL1 grail3.0100002702 LG_I:1432116-1435302 CHS1 eugene3.00140920 LG_XIV:7151030-7153
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
4CL5 fgenesh4_pg.C_LG_III001773 LG_III:17994254-17998436 CHS5 eugene3.00031461 LG_III:15672197-15673
Hydroxycinnamoyl-CoA quinate/shikimate hydroxycinnamoyltransferase CHS6 eugene3.00031462 LG_III:15678905-15680
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
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
HCT7 eugene3.18780002 scaffold_1878:6898-8809
Flavonoid 3'5'-hydroxylase
4-Coumarate 3-hydroxylase F3'5'H1 eugene3.00090961
C3H1 eugene3.36160002 scaffold_3616:2997-5408 F3'5'H2 eugene3.00011827 LG_I:19972937-199751
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
Ferulate 5-hydroxylase FNSII2 eugene3.00700209
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
F5HL1 eugene3.00090440 LG_IX:2644256-2646866 FLS2 eugene3.00020803 LG_II:6082658-608434
Caffeic acid O-methyltransferase FLS3 eugene3.01350040
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
COMT4 estExt_Genewise1_v1.C_LG_XIV1942 LG_XIV:4327267-4329146 DFR2 gw1.V.1407.1 LG_V:15923736-15925
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
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
Caffeoyl-CoA O-methyltransferase ANR2 estExt_fgenesh4_pm.C_LG_XI0107 LG_XI:3895226-38975
T1 grail3.0001059501 LG_IX:4059145-4060914
Leucoanthocyanidin reductase
T2 estExt_fgenesh4_pm.C_LG_I1023 LG_I:26412640-26415499 LAR1 grail3.0010045601 LG_VIII:7398478-7400
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)
name JGI Gene Model Locus Protein name JGI Gene Model Locus
MT3 estExt_fgenesh4_pm.C_1450034 scaffold_145:744229-746661 LAR2 eugene3.00101230 LG_X:12874015-12876
MT4 fgenesh4_pm.C_LG_X000399 LG_X:10889762-10892213 LAR3 estExt_fgenesh4_pm.C_LG_XV0077
MT5 estExt_fgenesh4_pg.C_LG_VIII1209 LG_VIII:9019120-9021383
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-
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
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 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
(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
(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
(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
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
[ 1] Croteau, R., Kutchan, T. M., and Lewis,
N.G. 2000. Natural products (secondary
metabolites). In: Buchanan B, Gruissem
W, Jones R, (Eds.) "Biochemistry and
Molecular Biology of Plants". American
Society of Plant Physiologists, Rockville,
MR: 1250-1318.
[ 2] Tuskan, G. A., DiFazio, S., Jansson, S.,
Bohlmann, J., Grigoriev, I., Hellsten, U.,
Putnam, N., Ralph, S., Rombauts, S.,
Salamov, A., Schein, J., Sterck, L., Aerts,
A., Bhalerao, R. R., Bhalerao, R. P.,
Blaudez, D., Boerjan, W., Brun, A.,
Brunner, A., Busov, V., Campbell, M.,
Carlson, J., Chalot, M., Chapman, J.,
Chen, G. L., Cooper, D., Coutinho, P. M.,
Couturier, J., Covert, S., Cronk, Q.,
Cunningham, R., Davis, J., Degroeve, S.,
Dejardin, A., dePamphilis, C., Detter, J.,
Dirks, B., Dubchak, I., Duplessis, S.,
Ehlting, J., Ellis, B., Gendler, K., Good-
stein, D., Gribskov, M., Grimwood, J.,
Groover, A., Gunter, L., Hamberger, B.,
Heinze, B., Helariutta, Y., Henrissat, B.,
Holligan, D., Holt, R., Huang, W., Is-
lam-Faridi, N., Jones, S., Jones-Rhoades,
M., Jorgensen, R., Joshi, C., Kangasjarvi,
J., Karlsson, J., Kelleher, C., Kirkpatrick,
R., Kirst, M., Kohler, A., Kalluri, U.,
Larimer, F., Leebens-Mack, J., Leple, J.
C., Locascio, P., Lou, Y., Lucas, S., Mar-
tin, F., Montanini, B., Napoli, C., Nelson,
D. R., Nelson, C., Nieminen, K., Nilsson,
O., Pereda, V., Peter, G., Philippe, R., Pi-
late, G., Poliakov, A., Razumovskaya, J.,
Richardson, P., Rinaldi, C., Ritland, K.,
Rouze, P., Ryaboy, D., Schmutz, J.,
Schrader, J., Segerman, B., Shin, H.,
Siddiqui, A., Sterky, F., Terry, A., Tsai, C.
J., Uberbacher, E., Unneberg, P., Vahala,
J., Wall, K., Wessler, S., Yang, G., Yin, T.,
Douglas, C., Marra, M., Sandberg, G.,
Van, de Peer Y., and Rokhsar, D. 2006.
The genome of black cottonwood,
Populus trichocarpa (Torr. & Gray). Sci-
ence, 313: 1596-1604.
[ 3] Boerjan, W., Ralph, J., and Baucher, M.
2003. Lignin biosynthesis. Annual Re-
view of Plant Biology, 54: 519-546.
[ 4] Dixon, R. A., Xie, D. Y., and Sharma, S.
B. 2005. Proanthocyanidins - a final
frontier in flavonoid research? New
Phytologist, 165: 9-28.
[ 5] Winkel-Shirley, B. 2002. Biosynthesis of
flavonoids and effects of stress. Current
Opinion in Plant Biology, 5: 218-223.
[ 6] Higuchi, T. 1997. "Biochemistry and
Molecular Biology of Wood".
Springer-Verlag, New York.
[ 7] Kosbar, L. L., Gelorme, J. D., Japp, R.
M., and Fotorny, W. T. 2000. Introducing
biobased materials into the electronics
industry: Developing a lignin-based resin
for printed wiring boards. Journal of In-
dustrial Ecology, 4: 93-105.
[ 8] Ghosh, I., Jain, R. K., and Glasser, W. G.
1999. Blends of biodegradable thermo-
plastics with lignin esters. In: Glasser,
W.G., Northey, R.A., Schultz, T.P., (Eds.)
"Lignin: Historical, Biological, and Ma-
terials Prespectives". American Chemi-
cal Society Series, 742: 331-350.
[ 9] Kao, Y. Y., Harding, S. A., and Tsai, C. J.
2002. Differential expression of two dis-
tinct phenylalanine ammonia-lyase genes
in condensed tannin-accumulating and
lignifying cells of quaking aspen. Plant
Physiology, 130: 796-807.
[10] Hu, W. J., Kawaoka, A., Tsai, C. J., Lung,
J. H., Osakabe, K., Ebinuma, H., and
Chiang, V. L. 1998. Compartmentalized
expression of two structurally and func-
tionally distinct 4-coumarate : CoA li-
Chung-Jui Tsai, Walid El Kayal,and Scott A. Harding
230 Int. J. Appl. Sci. Eng., 2006. 4, 3
gase genes in aspen (Populus tremu-
loides). Proceedings of the National
Academy of Sciences of the United States
of America, 95: 5407-5412.
[11] Harding, S. A., Leshkevich, J., Chiang, V.
L., and Tsai, C. J. 2002. Differential sub-
strate inhibition couples kinetically dis-
tinct 4-coumarate: coenzyme A ligases
with spatially distinct metabolic roles in
quaking aspen. Plant Physiology, 128:
[12] Chiang, V. L., and Funaoka, M. 1990.
The Difference between guaiacyl and
guaiacyl-syringyl lignins in their re-
sponses to Kraft delignification. Hol-
zforschung, 44: 309-313.
[13] Li, L., Zhou, Y. H., Cheng, X. F., Sun, J.
Y., Marita, J. M., Ralph, J., and Chiang,
V. L. 2003. Combinatorial modification
of multiple lignin traits in trees through
multigene cotransformation. Process Na-
tional Academic Science U S A, 100:
[14] Hu, W. J., Harding, S. A., Lung, J.,
Popko, J. L., Ralph, J., Stokke, D. D.,
Tsai, C. J., and Chiang, V. L. 1999. Re-
pression of lignin biosynthesis promotes
cellulose accumulation and growth in
transgenic trees. Nature Biotechnology,
17: 808-812.
[15] Zhong, R., Morrison, W. H., Himmels-
bach, D. S., Poole, F. L., and Ye, Z. H.
2000. Essential role of caffeoyl coen-
zyme A O-methyltransferase in lignin
biosynthesis in woody poplar plants.
Plant Physiol, 124: 563.
[16] Ralph, J., Lapierre C., Marita, J. M., Kim,
H., and Lu, F. 2001. Elucidation of new
structures in lignins of CAD- and
COMT-deficient plants by NMR. Phy-
tochemistry, 57: 993.
[17] Baucher, M., Chabbert, B., Pilate, G.,
VanDoorsselaere, J., Tollier, M. T., Pe-
titConil, M., Cornu, D., Monties, B.,
VanMontagu, M., Inze, D., Jouanin, L.,
and Boerjan, W. 1996. Red xylem and
higher lignin extractability by
down-regulating a cinnamyl alcohol de-
hydrogenase in poplar. Plant Physiol,
112: 1479-1490.
[18] Pilate, G., Guiney, E., Holt, K.,
Petit-Conil, M., and Lapierre, C. 2002.
Field and pulping performances of
transgenic trees with altered lignification.
Nature Biotechnology, 20: 607.
[19] Tsai, C. J., Harding, S. A., Tschaplinski,
T. J., Lindroth, R. L., and Yuan, Y. 2006.
Genome-wide analysis of the structural
genes regulating defense phenylpro-
panoid metabolism in Populus. New
Phytologist, 172: 47-62.
[20] Mahdi, J. G., Mahdi, A. J., Mahdi, A. J.,
and Bowen, I. D. 2006. The historical
analysis of aspirin discovery, its relation
to the willow tree and antiproliferative
and anticancer potential. Cell Prolifera-
tion, 39: 147-155.
[21] Lindroth, R. L., and Hwang, S. Y. 1996.
Diversity, redundancy and multiplicity in
chemical defense systems of aspen. In:
Romeo, J. T., Saunders, J. A., Barbosa,
P.,(Eds.) "Recent Advances in Phyto-
chemistry". Plenum Press, New York:
[22] Warren, J. M., Bassman, J. H., Fellman, J.
K., Mattinson, D. S., and Eigenbrode, S.
2003. Ultraviolet-B radiation alters phe-
nolic salicylate and flavonoid composi-
tion of Populus trichocarpa leaves. Tree
Physiology, 23: 527-535.
[23] Turtola, S., Rousi, M., Pusenius, J., Ya-
maji, K., Heiska, S., Tirkkonen, V.,
Meier, B., and Julkunen-Tiitto, R. 2005.
Clone-specific responses in leaf pheno-
lics of willows exposed to enhanced
UVB radiation and drought stress.
Global Change Biology, 11: 1655-1663.
[24] Leon, J., Shulaev, V., Yalpani, N.,
Lawton, M. A., and Raskin, I. 1995.
Benzoic-acid 2-hydroxylase, a soluble
oxygenase from tobacco, catalyzes sali-
cylic-acid biosynthesis. Proceedings of
the National Academy of Sciences of the
United States of America, 92:
Populus, the New Model System for Investigating Phenylpropanoid Complexity
Int. J. Appl. Sci. Eng., 2006. 4, 3 231
[25] Wildermuth, M. C., Dewdney, J., Wu, G.,
and Ausubel, F. M. 2001. Isochorismate
synthase is required to synthesize sali-
cylic acid for plant defence. Nature, 414:
[26] Ferrari, S., Plotnikova, J. M., De
Lorenzo, G., and Ausubel, F. M. 2003.
Arabidopsis local resistance to Botrytis
cinerea involves salicylic acid and
camalexin and requires EDS4 and PAD2,
but not SID2, EDS5 or PAD4. Plant
Journal, 35: 193-205.
[27] Stafford, H. A. 1991. Flavonoid evolu-
tion: An enzymic approach. Plant Physi-
ology, 96: 680-685.
[28] Winkel-Shirley, B. 2001. Flavonoid
Biosynthesis. A Colorful Model for Ge-
netics, Biochemistry, Cell Biology, and
Biotechnology. Plant Physiology, 126:
[29] Zucker, W. V. 1983. Tannins - Does
structure determine function - an eco-
logical perspective. American Naturalist,
121: 335-365.
[30] Lindroth, R. L. 1989. Biochemical de-
toxication - mechanism of differential
tiger swallowtail tolerance to phenolic
glycosides. Oecologia, 81: 219-224.
[31] Schimel, J. P., VanCleve, K., Cates, R. G.,
Clausen, T. P., and Reichardt, P. B. 1996.
Effects of balsam poplar (Populus bal-
samifera) tannins and low molecular
weight phenolics on microbial activity in
taiga floodplain soil: Implications for
changes in N cycling during succession.
Canadian Journal of Botany, 74: 84-90.
[32] Bradley, R. L., Titus, B. D., and Preston,
C. P. 2000. Changes to mineral N cycling
and microbial communities in black
spruce humus after additions of
42 4
()NH SO and condensed tannins ex-
tracted from Kalmia angustifolia and
balsam fir. Soil Biology & Biochemistry,
32: 1227-1240.
[33] Lorenz, K., and Preston, C. M. 2002.
Characterization of high-tannin fractions
from humus by carbon-13
cross-polarization and magic-angle spin-
ning nuclear magnetic resonance. Jour-
nal of Environmental Quality, 31:
[34] Stoutjesdijk, P. A., Sale, P. W., and
Larkin, P. J. 2001. Possible involvement
of condensed tannins in aluminium tol-
erance of Lotus pedunculatus. Australian
Journal of Plant Physiology, 28:
[35] Davis, M. A., Pritchard, S. G., Boyd, R.
S., and Prior, S. A. 2001. Developmental
and induced responses of nickel-based
and organic defences of the
nickel-hyperaccumulating shrub, Psy-
chotria douarrei. New Phytologist, 150:
[36] Lepiniec, L., Debeaujon, I., Routaboul, J.
M., Baudry, A., Pourcel, L., Nesi, N.,
and Caboche, M. 2006. Genetics and
biochemistry of seed flavonoids. Annual
Review of Plant Biology, 57: 405-430.
[37] Harborne, J. B. and Williams, C. A. 2000.
Advances in flavonoid research since
1992. Phytochemistry, 55: 481-504.
[38] Driebe, E. M. and Whitham, T. G. 2000.
Cottonwood hybridization affects tannin
and nitrogen content of leaf litter and al-
ters decomposition. Oecologia, 123:
[39] Whitham, T. G., Young, W. P., Martinsen,
G. D., Gehring, C. A., Schweitzer, J. A.,
Shuster, S. M., Wimp, G. M., Fischer, D.
G., Bailey, J. K., Lindroth, R. L., Wool-
bright, S., and Kuske C. R. 2003. Com-
munity and ecosystem genetics: A con-
sequence of the extended phenotype.
Ecology, 84: 559-573.
[40] Matsui, K., Tanaka, H., and
Ohme-Takagi, M. 2004. Suppression of
the biosynthesis of proanthocyanidin in
Arabidopsis by a chimeric PAP1 repres-
sor. Plant Biotechnology Journal, 2:
[41] Tanner, G. J., Francki, K. T., Abrahams,
S., Watson, J. M., Larkin, P. J., and
Chung-Jui Tsai, Walid El Kayal,and Scott A. Harding
232 Int. J. Appl. Sci. Eng., 2006. 4, 3
Ashton, A. R. 2003. Proanthocyanidin
biosynthesis in plants - Purification of
legume leucoanthocyanidin reductase
and molecular cloning of its cDNA.
Journal of Biological Chemistry, 278:
[42] Ayres, M. P., Clausen, T. P., MacLean, S.
F., Redman, A. M., and Reichardt, P. B.
1997. Diversity of structure and antiher-
bivore activity in condensed tannins.
Ecology, 78: 1696-1712.
[43] Veit, M., and Pauli, G. F. 1999. Major
Flavonoids from Arabidopsis thaliana
Leaves. Journal of Natural Products, 62:
[44] Prescott, A., Stamford, N., Wheeler, G.,
and Firmin, J. 2002. In vitro properties
of a recombinant flavonol synthase from
Arabidopsis thaliana. Phytochemistry, 60:
[45] Martensa, S. and Mithöfer, A. 2005.
Flavones and flavone synthases. Phyto-
chemistry, 66: 2399-2407.
[46] Greenaway, W., English, S., Whatley, F.
R., and Rood, S. B. 1991. Interrelation-
ships of poplars in a hybrid swarm as
studied by gas chromatography-mass
spectrometry. Canadian Journal of Bot-
any, 69: 203-208.
[47] Greenaway, W., English, S., and Whatley,
F. R. 1992. Relationships of Populus X
acuminata and Populus X generosa with
their parental species examined by gas
chromatography-mass spectrometry of
bud exudates. Canadian Journal of Bot-
any, 70: 212-221.
[48] Maeda, K., Kimura, S., Demura, T., Ta-
keda, J., and Ozeki, Y. 2005. DcMYB1
acts as a transcriptional activator of the
carrot phenylalanine ammonia-lyase
gene (DcPAL1) in response to elicitor
treatment, UV-B irradiation and the dilu-
tion effect. Plant Molecular Biology, 59:
[49] Sablowski, R. W. M., Moyano, E., Cu-
lianezmacia, F. A., Schuch, W., Martin,
C., and Bevan, M. 1994. A
flower-specific Myb protein activates
transcription of phenylpropanoid bio-
synthetic genes. Embo Journal, 13:
[50] Green, P. J., Kay, S. A., and Chua, N. H.
1987. Sequence-specific interactions of a
pea nuclear factor with light-responsive
elements upstream of the Rbcs-3a gene.
EMBO Journal, 6: 2543-2549.
[51] Gilmartin, P. M., Sarokin, L., Memelink,
J., and Chua, N. H. 1990. Molecular light
switches for plant genes. Plant Cell, 2:
[52] Staiger, D., Kaulen, H., and Schell, J.
1989. A CACGTG motif of the Antir-
rhinum majus chalcone synthase pro-
moter is recognized by an evolutionarily
conserved nuclear protein. PNAS, 86:
[53] Takaiwa, F., Yamanouchi, U., Yoshihara,
T., Washida, H., Tanabe, F., Kato, A., and
Yamada, K. 1996. Characterization of
common cis-regulatory elements respon-
sible for the endosperm-specific expres-
sion of members of the rice glutelin mul-
tigene family. Plant Molecular Biology,
30: 1207-1221.
[54] Suzuki, A., Wu, C. Y., Washida, H., and
Takaiwa, F. 1998. Rice MYB protein
OSMYB5 specifically binds to the
AACA motif conserved among promot-
ers of genes for storage protein glutelin.
Plant and Cell Physiology, 39: 555-559.
[55] Debeaujon, I., Nesi, N., Perez, P., Devic,
M., Grandjean, O., Caboche, M., and
Lepiniec, L. 2003. Proanthocya-
nidin-accumulating cells in Arabidopsis
testa: Regulation of differentiation and
role in seed development. Plant Cell, 15:
[56] Inukai, Y., Sakamoto, T., Uegu-
chi-Tanaka, M., Shibata, Y., Gomi, K.,
Umemura, I., Hasegawa, Y., Ashikari, M.,
Kitano, H., and Matsuoka, M. 2005.
Crown rootless1, which is essential for
crown root formation in rice, is a target
Populus, the New Model System for Investigating Phenylpropanoid Complexity
Int. J. Appl. Sci. Eng., 2006. 4, 3 233
auxin signaling. Plant Cell, 17:
[57] Fujita, Y., Fujita, M., Satoh, R., Maru-
yama, K., Parvez, M. M., Seki, M., Hi-
ratsu, K., Ohme-Takagi, M., Shinozaki,
K., and Yamaguchi-Shinozaki, K. 2005.
AREB1 is a transcription activator of
novel ABRE-dependent ABA signaling
that enhances drought stress tolerance in
Arabidopsis. Plant Cell, 17: 3470-3488.
[58] Choi, H. I., Hong, J. H., Ha, J. O., Kang,
J. Y., and Kim, S. Y. 2000. ABFs, a fam-
ily of ABA-responsive element binding
factors. Journal of Biological Chemistry,
275: 1723-1730.
[59] Block, A., Dangl, J. L., Hahlbrock, K.,
and Schulze-Lefert, P. 1990. Functional
borders, genetic fine structure, and dis-
tance requirements of cis elements medi-
ating light responsiveness of the parsley
chalcone synthase promoter. PNAS, 87:
[60] Feucht, W., and Treutter, D. 1990. Fla-
van-3-ols in trichomes, pistils and phel-
loderm of some tree species. Annals of
Botany, 65: 225-230.
[61] Neu, R. 1956. A new reagent for differ-
entiating and determining flavones on
paper chromatograms. Naturwissen-
schaften, 43: 82.
[62] Harding, S. A., Jiang, H. Y., Jeong, M. L.,
Casado, F. L., Lin, H. W., and Tsai, C. J.
2005. Functional genomics analysis of
foliar condensed tannin and phenolic
glycoside regulation in natural cotton-
wood hybrids. Tree Physiology, 25:
... The genetic bases of metabolic pathways controlling the synthesis of many important secondary metabolites, including nicotine (Steppuhn, Gase, Krock, Halitschke, & Baldwin, 2004), various flavonoids (Saito et al., 2013), and terpenes (Redding-Johanson et al., 2011), are well characterized. However, the pathways leading to the salicinoid phenolic glycosides (SPGs) remain almost entirely undescribed (Tsai, Harding, Tschaplinski, Lindroth, & Yuan, 2006;Tsai, Kayal, & Harding, 2006;Boeckler, Gershezon, & Unsicker, 2011; but see Chedgy, Kӧllner, & Constabel, 2015). The lack of genetic data for SPGs is conspicuous given their potential agricultural, medicinal, and ecological applications. ...
... For example, salicortin, one of the SPGs in our study, has recently been investigated for potential application toward the treatment and prevention of obesity (Lee et al., 2013), insulin resistance (Harbilas et al., 2013), and inflammation (Kwon et al., 2014). Ecologically, salicortin and other SPGs serve a variety of functions (reviewed in Tsai, Kayal, et al., 2006) that include protection from UV damage (Turtola et al., 2005;Warren, Bassman, Fellman, Mattinson, & Eigenbrode, 2003), responses to drought (Turtola et al., 2005), and defense against herbivores (e.g., Lindroth & St. Clair, 2013;Holeski et al., 2016;Tahvanainen et al., 1991). In the model tree genus Populus, plant chemistry has been shown to be the key bridge between host plant genetic variation and insect community organization Bernhardson et al., 2013;Martinsen et al., 1998). ...
... However, forest trees from the model genus Populus express a diverse array of SPGs and other secondary metabolites of interest (Boeckler et al., 2011;Chen, Liu, Tschaplinski, & Zhao, 2009;Constabel & Lindroth, 2010;Keefover-Ring et al., 2014). Populus species have been studied extensively in genomic (Tuskan et al.,2006), metabolomic (Morreel et al., 2006;Tsai, Kayal, et al., 2006), ecological (Boeckler et al., 2011;Caseys, Stritt, Glauser, Blanchard, & Lexer, 2015;Lindroth & St. Clair, 2013), and commercial improvement (Jansson & Douglas, 2007;Taylor, 2002;Wullschleger, Jansson, & Taylor, 2002) research. The North American black cottonwood (P. ...
Full-text available
Genomic studies have been used to identify genes underlying many important plant secondary metabolic pathways. However, genes for salicinoid phenolic glycosides (SPGs)—ecologically important compounds with significant commercial, cultural, and medicinal applications—remain largely undescribed. We used a linkage map derived from a full-sib population of hybrid cottonwoods (Populus spp.) to search for quantitative trait loci (QTL) for the SPGs salicortin and HCH-salicortin. SSR markers and primer sequences were used to anchor the map to the V3.0 P. trichocarpa genome. We discovered 21 QTL for the two traits, including a major QTL for HCH-salicortin (R2 = .52) that colocated with a QTL for salicortin on chr12. Using the V3.0 Populus genome sequence, we identified 2,983 annotated genes and 1,480 genes of unknown function within our QTL intervals. We note ten candidate genes of interest, including a BAHD-type acyltransferase that has been potentially linked to PopulusSPGs. Our results complement other recent studies in Populus with implications for gene discovery and the evolution of defensive chemistry in a model genus. To our knowledge, this is the first study to use a full-sib mapping population to identify QTL intervals and gene lists associated with SPGs.
... In many dicots, CHS is encoded by a multigene family [25][26][27]. Usually, the chalcone synthase gene forms a family of three to twelve members in most of dicots, such as apple (3 members) [28], mulberry (5 members) [29], Populus (6 members) [30], Glycine max (8~9 members) [31,32], Viola cornuta (10 members) [33], and petunia (12 members) [34]. In turnip, six CHS genes were cloned and identified, although only three were functional. ...
... After a final centrifugation at 10,000 r/min for 10 min, the 0.5 mL of methanol extract was reacted with aluminium chloride before measuring the absorbance at 415 nm with the spectrophotometer. The protocol for detecting flavanones and flavanonols was slightly modified from the method described by Chang [30]. Naringenin was used as a standard chemical to generate criterion solutions at concentrations of 50, 100, 200, 500, 1000, 3000, 4000 and 5000 μg/mL with methanol. ...
Full-text available
Background: Citrus flavonoids are considered as the important secondary metabolites because of their biological and pharmacological activities. Chalcone synthase (CHS) is a key enzyme that catalyses the first committed step in the flavonoid biosynthetic pathway. CHS genes have been isolated and characterized in many plants. Previous studies indicated that CHS is a gene superfamily. In citrus, the number of CHS members and their contribution to the production of flavonoids remains a mystery. In our previous study, the copies of CitCHS2 gene were found in different citrus species and the sequences are highly conserved, but the flavonoid content varied significantly among those species. Results: From seventy-seven CHS and CHS-like gene sequences, ten CHS members were selected as candidates according to the features of their sequences. Among these candidates, expression was detected from only three genes. A predicted CHS sequence was identified as a novel CHS gene. The structure analysis showed that the gene structure of this novel CHS is very similar to other CHS genes. All three CHS genes were highly conserved and had a basic structure that included one intron and two exons, although they had different expression patterns in different tissues and developmental stages. These genes also presented different sensitivities to methyl jasmonate (MeJA) treatment. In transgenic plants, the expression of CHS genes was significantly correlated with the production of flavonoids. The three CHS genes contributed differently to the production of flavonoids. Conclusion: Our study indicated that CitCHS is a gene superfamily including at least three functional members. The expression levels of the CHS genes are highly correlated to the biosynthesis of flavonoids. The CHS enzyme is dynamically produced from several CHS genes, and the production of total flavonoids is regulated by the overall expression of CHS family genes.
... Salicinoids are ecologically important anti-herbivory compounds in the Salicaceae family [4]. Although salicinoids are described as a defined branch of the phenylpropanoid pathway [70,72], the exact sequence of steps and molecules and their respective intermediate forms remain largely unknown. It has been proposed that salicinoids may not be synthesized within a single linear pathway but rather within a network of interconnected reactions (Fig 6; [72]). ...
... Overview of the phenylpropanoid pathway. Black arrows represent well-known branches of the phenylpropanoid pathways, their enzymes and numbers of genes in Populus [70,71]. The hypothesized metabolite network for salicinoids is represented by grey arrows, following [72]. ...
Full-text available
The mechanisms responsible for the origin, maintenance and evolution of plant secondary metabolite diversity remain largely unknown. Decades of phenotypic studies suggest hybrid-ization as a key player in generating chemical diversity in plants. Knowledge of the genetic architecture and selective constraints of phytochemical traits is key to understanding the effects of hybridization on plant chemical diversity and ecological interactions. Using the European Populus species P. alba (White poplar) and P. tremula (European aspen) and their hybrids as
... Poplars (Populus L. spp.) are economically important tree species rich in flavonoid metabolites, of which PAs and anthocyanins generally account for 30% of the leaf dry weight (Tsai et al., 2006;Tuskan et al., 2006). As with many other plant species, the flavonoid biosynthetic pathway of poplar is also conserved, but is rather more complex. ...
Full-text available
Flavonoids, which modulate plant resistance to various stresses, can be induced by high light. B-box (BBX) transcription factors (TFs) play crucial roles in the transcriptional regulation of flavonoids biosynthesis, but limited information is available on the association of BBX proteins with high light. We present a detailed overview of 45 Populus trichocarpa BBX TFs. Phylogenetic relationships, gene structure, tissue-specific expression patterns, and expression profiles were determined under 10 stress or phytohormone treatments to screen candidate BBX proteins associated with the flavonoid pathway. Sixteen candidate genes were identified, of which five were expressed predominantly in young leaves and roots, and BBX23 showed the most distinct response to high light. Overexpression of BBX23 in poplar activated expression of MYB TFs and structural genes in the flavonoid pathway, thereby promoting accumulation of proanthocyanidins and anthocyanins. CRISPR/Cas9-generated knockout of BBX23 resulted in the opposite trend. Furthermore, the phenotype induced by BBX23 overexpression was enhanced under exposure to high light. BBX23 was capable of binding directly to the promoters of proanthocyanidin- and anthocyanin-specific genes, and its interaction with HY5 enhanced activation activity. We identified novel regulators of flavonoid biosynthesis in poplar, thereby enhancing our general understanding of the transcriptional regulatory mechanisms involved. This article is protected by copyright. All rights reserved.
... Gene ontology (GO) term enrichment analysis (Supplementary Figure S3) indicated that the down-regulated genes by drought and salt treatment contained similar molecular function; interestingly, the genes annotated to be related with "cell wall" were enriched in both cases (Supplementary Figure S3A, C), suggesting that the gene for cell wall metabolism would be actively regulated by abiotic stresses. For detailed analysis of the genes involved in cell wall metabolism, we identified orthologs of genes associated with cellulose, hemicellulose and lignin biosynthesis (Dhugga 2012;Hussey et al. 2013;Pauly et al. 2013;Shi et al. 2010;Tsai et al. 2006;Vanholme et al. 2010) and extracted the transcriptomics data for these genes (Figure 4, Supplementary Figure S4). The putative poplar genes involved in cellulose and hemicellulose biosynthesis, such as CELLULOSE SYNTHASE (CESA) and IRREGULAR XYLEM (IRX) genes, were strongly repressed by the drought and salt stresses (Supplementary Figure S4), with the exception of GATL9 genes, putative pectin synthase genes, which were upregulated by the drought stress (Supplementary Figure S4). ...
Growth of biomass for lignocellulosic biofuels and biomaterials may take place on land unsuitable for foods, meaning the biomass plants are exposed to increased abiotic stresses. Thus, the understanding how this affects biomass composition and quality is important for downstream bioprocessing. Here, we analyzed the effect of drought and salt stress on cell wall biosynthesis in young shoots and xylem tissues of Populus trichocarpa using transcriptomic and biochemical methods. Following exposure to abiotic stress, stem tissues reduced vessel sizes, and young shoots increased xylem formation. Compositional analyses revealed a reduction in the total amount of cell wall polysaccharides. In contrast, the total lignin amount was unchanged, while the ratio of S/G lignin was significantly decreased in young shoots. Consistent with these observations, transcriptome analyses show that the expression of a subset of cell wall-related genes is tightly regulated by drought and salt stresses. In particular, the expression of a part of genes encoding key enzymes for S-lignin biosynthesis, caffeic acid O-methyltransferase and ferulate 5-hydroxylase, was decreased, suggesting the lower S/G ratio could be partly attributed to the down-regulation of these genes. Together, our data identifies a transcriptional abiotic stress response strategy in poplar, which results in adaptive changes to the plant cell wall.
... Of the A, B and C rings of CTs, the A and C rings (60% C) are derivatives of phenylpropanoid, while the B ring (40% C) originates from malonyl-CoA, via acetyl-CoA synthesized from pyruvate or via beta-oxidation of fatty acids (Taiz and Zeiger, 1998). The phylogenetic organization of many of the phenylpropanoid and flavonoid pathway genes expressed in Populus tissues has been reported (Tsai et al., 2006b). The regulation of genes in this pathway has also been discussed in several review papers (Dixon and Paiva, 1995;Lanot et al., 2008;Lucheta et al., 2007;Weisshaar and Jenkins, 1998 (Donaldson et al., 2006b). ...
Secondary metabolites play an important role in plant protection against biotic and abiotic stress. In Populus, phenolic glycosides (PGs) and condensed tannins (CTs) are two such groups of compounds derived from the common phenylpropanoid pathway. The basal levels and the inducibility of PGs and CTs depend on genetic as well as environmental factors, such as soil nitrogen (N) level. Carbohydrate allocation, transport and sink strength also affect PG and CT levels. A negative correlation between the levels of PGs and CTs was observed in several studies. However, the molecular mechanism underlying such relation is not known. We used a cell culture system to understand negative correlation of PGs and CTs. Under normal culture conditions, neither salicin nor higher-order PGs accumulated in cell cultures. Several factors, such as hormones, light, organelles and precursors were discussed in the context of aspen suspension cells’ inability to synthesize PGs. Salicin and its isomer, isosalicin, were detected in cell cultures fed with salicyl alcohol, salicylaldehyde and helicin. At higher levels (5 mM) of salicyl alcohol feeding, accumulation of salicins led to reduced CT production in the cells. Based on metabolic and gene expression data, the CT reduction in salicin-accumulating cells is partly a result of regulatory changes at the transcriptional level affecting carbon partitioning between growth processes, and phenylpropanoid CT biosynthesis. Based on molecular studies, the glycosyltransferases, GT1-2 and GT1-246, may function in glycosylation of simple phenolics, such as salicyl alcohol in cell cultures. The uptake of such glycosides into vacuole may be mediated to some extent by tonoplast localized multidrug-resistance associated protein transporters, PtMRP1 and PtMRP6. In Populus, sucrose is the common transported carbohydrate and its transport is possibly regulated by sucrose transporters (SUTs). SUTs are also capable of transporting simple PGs, such as salicin. Therefore, we characterized the SUT gene family in Populus and investigated, by transgenic analysis, the possible role of the most abundantly expressed member, PtSUT4, in PG-CT homeostasis using plants grown under varying nitrogen regimes. PtSUT4 transgenic plants were phenotypically similar to the wildtype plants except that the leaf area-to-stem volume ratio was higher for transgenic plants. In SUT4 transgenics, levels of non-structural carbohydrates, such as sucrose and starch, were altered in mature leaves. The levels of PGs and CTs were lower in green tissues of transgenic plants under N-replete, but were higher under N-depleted conditions, compared to the levels in wildtype plants. Based on our results, SUT4 partly regulates N-level dependent PG-CT homeostasis by differential carbohydrate allocation.
... Gene duplication has been recognized as a primary mechanism for increasing functional diversification, and the increased expression divergence in duplicated genes can substantially contribute to morphological diversification . Indeed, the phenylpropanoid pathway leads to a wide variety of compounds such as flavonoids, lignans and hydroxycinnamate derivatives produced in specific metabolic branches and participating in diverse cellular processes underlying plant growth, development, adaptation, defense and reproduction (Tsai et al., 2006). In metabolic terms, this pattern might derive from cellular strategies to adjust protein synthesis according to functional needs, keeping only one or two highly expressed genes in each family, in a particular tissue, and/or in particular environmental conditions. ...
Lignin, a major component of secondary cell walls, hinders the optimal processing of wood for industrial uses. The recent availability of the Eucalyptus grandis genome sequence allows comprehensive analysis of the genes encoding the 11 protein families specific to the lignin branch of the phenylpropanoid pathway and identification of those mainly involved in xylem developmental lignification. We performed genome-wide identification of putative members of the lignin gene families, followed by comparative phylogenetic studies focusing on bona fide clades inferred from genes functionally characterized in other species. RNA-seq and microfluid real-time quantitative PCR (RT-qPCR) expression data were used to investigate the developmental and environmental responsive expression patterns of the genes. The phylogenetic analysis revealed that 38 E. grandis genes are located in bona fide lignification clades. Four multigene families (shikimate O-hydroxycinnamoyltransferase (HCT), p-coumarate 3-hydroxylase (C3H), caffeate/5-hydroxyferulate O-methyltransferase (COMT) and phenylalanine ammonia-lyase (PAL)) are expanded by tandem gene duplication compared with other plant species. Seventeen of the 38 genes exhibited strong, preferential expression in highly lignified tissues, probably representing the E. grandis core lignification toolbox. The identification of major genes involved in lignin biosynthesis in E. grandis, the most widely planted hardwood crop world-wide, provides the foundation for the development of biotechnology approaches to develop tree varieties with enhanced processing qualities. © 2015 The Authors New Phytologist © 2015 New Phytologist Trust.
Pyrus pyrifolia, which is famous for its delicate flesh, is the most widely cultivated oriental pear. Stone cell content is an important factor affecting P. pyrifolia fruit quality. However, the mechanism underlying stone cell development remains to be fully elucidated. In this study, the content and distribution of stone cells of two cultivars of P. pyrifolia, namely, ‘Twentieth Century’ (TC) and its bud variety ‘Golden Twentieth Century’ (GTC), were compared. Results showed that the contents of stone cells and lignin in TC were significantly higher than those in GTC during the entire fruit development process. Moreover, fruits that developed 60 days after flowering had the highest stone cell content. By transcriptome sequencing, 1558 differentially expressed genes (DEGs), including 530 upregulated genes and 1028 downregulated genes, were identified. Gene Ontology (GO) enrichment analysis indicated hemicellulose metabolic process (GO: 0010410), lignin metabolic process (GO:0009808) and lignin catabolic process (GO: 0046274) were highly active in stone cell development. Kyoto Encyclopedia of Genes and Genomes metabolic pathway enrichment analysis suggested that in phenylpropanoid biosynthesis (ko00940) pathway, two enzymes (glucosyltransferase [EC:] and peroxidase [EC:]) could be important factors of lower lignin content in GTC than TC. The quantitative real-time polymerase chain reaction was used to screen out several genes and TFs, such as peroxidase 42-like gene, vinorine synthase-like gene, and Myb4-like TFs, are likely associated with stone cell development. Our study would provide a basis for the molecular mechanisms of stone cell development in P. pyrifolia cultivars and would aid in improving quality of pear fruits by breeding.
Full-text available
Plant specialized metabolites are being used worldwide as therapeutic agents against several diseases. Since the precursors for specialized metabolites come through primary metabolism, extensive investigations have been carried out to understand the detailed connection between primary and specialized metabolism at various levels. Stress regulates the expression of primary and specialized metabolism genes at the transcriptional level via transcription factors binding to specific cis-elements. The presence of varied cis-element signatures upstream to different stress-responsive genes and their transcription factor binding patterns provide a prospective molecular link among diverse metabolic pathways. The pattern of occurrence of these cis-elements (overrepresentation/common) decipher the mechanism of stress-responsive upregulation of downstream genes, simultaneously forming a molecular bridge between primary and specialized metabolisms. Though many studies have been conducted on the transcriptional regulation of stress-mediated primary or specialized metabolism genes, but not much data is available with regard to cis-element signatures and transcription factors that simultaneously modulate both pathway genes. Hence, our major focus would be to present a comprehensive analysis of the stress-mediated interconnection between primary and specialized metabolism genes via the interaction between different transcription factors and their corresponding cis-elements. In future, this study could be further utilized for the overexpression of the specific transcription factors that upregulate both primary and specialized metabolism, thereby simultaneously improving the yield and therapeutic content of plants.
Full-text available
There is increasing evidence that the structure and functioning of ecological communities and ecosystems are strongly influenced by flexible traits of individuals within species. A deep understanding of how trait flexibility alters direct and indirect species interactions is crucial for addressing key issues in basic and applied ecology. This book provides an integrated perspective on the ecological and evolutionary consequences of interactions mediated by flexible species traits across a wide range of systems. It is the first volume synthesizing the rapidly expanding research field of trait-mediated indirect effects and highlights how the conceptual framework of these effects can aid the understanding of evolutionary processes, population dynamics, community structure and stability, and ecosystem function. It not only brings out the importance of this emerging field for basic ecological questions, but also explores the implications of trait-mediated interactions for the conservation of biodiversity and the response of ecosystems to anthropogenic environmental changes.
Full-text available
Analysis of bud exudate by gas chromatography-mass spectrometry (GC–MS) of 14 clones belonging to a natural hybrid swarm involving Populus angustifolia, P. balsamifera, and P. deltoides produced results consistent with those obtained by previous analysis of leaf characteristics. Specimens that had leaves most characteristic of a pure species also produced bud exudate GC–MS profiles which were characteristic of those species. GC–MS profiles of interspecific hybrid clones were intermediate between the parental species. This demonstrates the usefulness of GC–MS analysis of bud exudate as a chemo-taxonomic method for the study of intersecific poplar hybrids and also supports the accuracy of analysis of foliar morphology for taxonomic assessment. Key words: Populus, bud exudate, gas chromatography – mass spectrometry.
Tissues from nine tree species were examined histochemically for the presence of flavan-3-ols including the catechins. It was possible to stain these phenolics selectively with p-dimethylaminocinnamaldehyde (DMACA) and to show that they were located in trichomes, pistils and shoot phelloderm. The staining intensity of the tissues was categorized into four groups. The flavan-3-ols were extracted from three tree species and the diverse components were distinguished using a combination of HPLC and chemical reaction detection (CRD). Twelve flavan-3-ols were isolated from pistils of Tilia grandifolia and 32 from leaves of Acer platanoides. The hairs of the leaves of dormant buds from Aesculus hippocastanum yielded 13 components. Epicatechin and ( + )-catechin were present in all three species.
Thermoplastic blends of several biodegradable polymers with lignin and lignin esters were prepared by solvent casting and melt processing. Among the biodegradable thermoplastics were cellulose acetate butyrate (CAB), a starch-caprolactone copolymer/blend (SCC), and poly(hydroxybutyrate) (PHB). Lignin esters included the acetate, butyrate, hexanoate, and laurate of organosolv lignin, LA, LB, LH, and LL, respectively. Blend properties were analyzed by thermal, mechanical, and optical (transmission electron microscopy, TEM) analysis. The results indicate widely different levels of interaction between the two polymer constituents. Blends of LA and LB with CAB exhibited a high level of compatibility that was lost when the acyl substituent increased in size. The addition of unmodified lignin to PHB greatly retarded crystallization and produced blends with lower melting points. The same was true for SCC blends, which were found to crystallize and melt at lower temperatures if lignin was present. However, a significantly increased modulus at room temperature resulted with the addition of lignin, and this was attributed to increased crystallinity in the presence of lignin.
We investigated the distribution of aluminium (Al) in the root tips of the Al-tolerant forage legume,Lotus pedunculatus Cav., a species that also accumulates condensed tannin (proanthocyanidin) in the roots and leaves. Clonal cuttings were grown in low ionic-strength nutrient solutions containing Al at levels that were either stimulatory or inhibitory (5–60µM ). The X-ray microanalysis of treated root apices revealed Al deposits at all Al concentrations. In freeze-fractured root samples from high Al concentrations (30 and 60 M ), deposits were found very close to the root tip. These deposits were predominantly composed of Al, phosphorus (P) and silicon (Si). At low Al concentrations (10 µM ), epoxy-embedded root samples were examined and Al deposits were also found near the meristematic areas. At lower concentrations (10 µM ), Al was found associated with P. In all osmium-fixed samples from high and low Al concentrations, Al was generally found in association with osmium-binding vacuoles. Because of the established high affinity of osmium for condensed tannin, the hypothesis is developed that condensed tannins possibly bind and detoxify Al in the root apices of L. pedunculatus.
Tannins are phenolic compounds of complex structures and widespread occurrence in higher plants. They are not known to have any physiological function. It is proposed that the enormous structural heterogeneity and natural division of tannins into 2 broad classes, the condensed and hydrolyzable, has important ecological consequences. Their structures suggest strong specificities for a variety of target molecules including proteins, digestive enzymes and polysaccharides. These specificities insure mainline defense against plant enemies, but are nevertheless subject to the coevolutionary 'arms race'. The idea that condensed tannins are reserved primarily for defense against microbes and pathogens, while the hydrolyzable tannins protect the plant against herbivores is discussed. What structure suggests about function, and how these ideas fit into the current scheme of antiherbivore chemistry, evolution of the arms race between plants and their parasites, and ecological theory is considered. -from Author
Kraft pulping of Douglas-fir (Pseudoisuga meniesii) and sweetgum (Liquidambar styraciflua) was carried out from 9()°C to a final temperature of 170°C at a heating rate of l °C/min. At various stages of delignification, pulps were analyzed by nucleus exchange and alkaline nitrobenzene oxidation reactions to reveal the difference between kraft pulping of hardwood and softwood in dissolution and condensation of lignin building units. The most significant difference between kraft pulping of hardwood and softwood was in the formation and dissolution of Condensed units. The formation of guaiacyl-syringyl types of diphenyl methane moieties occurred already at 110°C and reached the maximum at around 150°C. These Condensed units were dissolved at higher temperatures (> 160°C) and after 30 minutes at 170°C, no syringyl unit was found associated with diphenyl methane moieties in residual sweetgum lignins. At the end of pulping (∼ kappa 20). the residual sweetgum lignin consisted of 66 and 26 mol% of guaiacyl units that were associated with diphenyl methane moieties and other types of Condensed units, respectively. The formation of guaiacyl-guaiacyl types of diphenyl methane moieties during pulping of Douglas-fir occurred at 170°C and reached the maximum after 20 minutes at this temperature. After reaching its maximum, the quantity of these types of diphenyl methane moieties in residual lignins remained almost constant äs pulping proceeded. At the end of pulping (∼ kappa 30), the residual Douglas-fir lignin consisted of 54 and 37 mol% of guaiacyl units that were associated with diphenyl methane moieties and other types of Condensed units, respectively.
Trembling aspen (Populus tremuloides Michx.) is the most widely distributed tree in North America, occurring over a variety of climatic, soil and topographical conditions.1,2 Interactions between aspen and its biotic and abiotic environments play pivotal roles in the ecological dynamics of many early-suc-cessional ecosystems. These interactions, in turn, influence and are influenced by the chemical composition of aspen.