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First published online April 24, 2009; 10.1104/pp.109.139071 Plant Physiology 150:924-941 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
The Wound-, Pathogen-, and Ultraviolet B-Responsive MYB134 Gene Encodes an R2R3 MYB Transcription Factor That Regulates Proanthocyanidin Synthesis in Poplar1,[W],[OA]Centre for Forest Biology and Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 3N5 (R.D.M., L.T.T., C.P.C.); and Department of Cell and Systems Biology (M.B.P., M.M.C.) and Centre for the Analysis of Genome Evolution and Function (M.B.P., M.M.C.), University of Toronto, Toronto, Ontario, Canada M5S 3B2
In poplar (Populus spp.), the major defense phenolics produced in leaves are the flavonoid-derived proanthocyanidins (PAs) and the salicin-based phenolic glycosides. Transcriptional activation of PA biosynthetic genes leading to PA accumulation in leaves occurs following herbivore damage and mechanical wounding as well as infection by the fungal biotroph Melampsora medusae. In this study, we have identified a poplar R2R3 MYB transcription factor gene, MYB134, that exhibits close sequence similarity to the Arabidopsis (Arabidopsis thaliana) PA regulator TRANSPARENT TESTA2 and that is coinduced with PA biosynthetic genes following mechanical wounding, M. medusae infection, and exposure to elevated ultraviolet B light. Overexpression of MYB134 in poplar resulted in transcriptional activation of the full PA biosynthetic pathway and a significant plant-wide increase in PA levels, and electrophoretic mobility shift assays showed that recombinant MYB134 protein is able to bind to promoter regions of PA pathway genes. MYB134-overexpressing plants exhibited a concomitant reduction in phenolic glycoside concentrations and other minor alterations to levels of small phenylpropanoid metabolites. Our data provide insight into the regulatory mechanisms controlling stress-induced PA metabolism in poplar, and the identification of a regulator of stress-responsive PA biosynthesis constitutes a valuable tool for manipulating PA metabolism in poplar and investigating the biological functions of PAs in resistance to biotic and abiotic stresses.
Plant secondary metabolites play important ecological roles and in many plants constitute a critical component of defenses against biotic and abiotic stress. Many secondary metabolic pathways are responsive to environmental conditions and can be rapidly activated by stresses such as pathogen infection, elevated light, and herbivory. The phenylpropanoid pathway in particular leads to the synthesis of a large and diverse class of plant secondary metabolites, many of which are stress induced (Dixon and Paiva, 1995  ). Synthesis of phenylpropanoids and other secondary metabolites following stress is typically mediated by the transcriptional activation of suites of biosynthetic genes coordinately regulated by transcription factor proteins (Weisshaar and Jenkins, 1998; Davies and Schwinn, 2003). The possibility of identifying transcription factors that control entire pathways is motivating many studies in plant stress biology, since such regulators would be valuable for the metabolic engineering of plants for both plant and human health (Dixon, 2005; Sharma and Dixon, 2005; Yu and McGonigle, 2005).
Populus species (cottonwoods, poplars, and aspens, hereafter referred to collectively as poplar) are often ecological foundation species and include the most widely distributed trees in the Northern Hemisphere. The phenolic metabolites produced by poplar are thought to be important determinants of community structure and ecosystem dynamics (Lindroth and Hwang, 1996
PAs are polymers of flavan-3-ols or flavan-3,4-diols condensed with flavan-3-ol starter units and usually deposited in the vacuole (Stafford, 1990 ; Xie and Dixon, 2005; Fig. 1). With the exception of late biosynthetic steps, including the transport and condensation of PA monomers, the enzymatic steps in PA biosynthesis have been well characterized, as most are also involved in the production of other well-studied flavonoid classes such as anthocyanins (Xie and Dixon, 2005; Fig. 1). The Arabidopsis (Arabidopsis thaliana) transparent testa (tt) and tannin-deficient seed mutants have been invaluable in identifying genes involved in PA biosynthesis and its regulation (Lepiniec et al., 2006). Recent major advances in PA biochemistry include the identification of the PA-specific enzymes anthocyanidin reductase (ANR; encoded by the BANYULS [BAN] gene in Arabidopsis) and leucoanthocyanidin reductase (LAR), which are involved in the production of the flavan-3-ol PA starter units with 2,3-cis- or 2,3-trans stereochemistry of the heterocyclic C ring, respectively (Tanner et al., 2003; Xie et al., 2003; Fig. 1). Additional genes implicated in PA biosynthesis in Arabidopsis include TT12, encoding a multidrug and toxic compound extrusion (MATE) family transporter (Debeaujon et al., 2001), TT19, encoding a glutathione S-transferase (Kitamura et al., 2004), AUTO-INHIBITED H+-ATPase ISOFORM10 (AHA10), encoding a proton pump involved in vacuolar biogenesis (Baxter et al., 2005), and TT10, a predicted laccase-like polyphenol oxidase functioning in PA oxidation (Pourcel et al., 2005). Recently, UGT72L1, a glycosyltransferase specifically active toward the PA precursor (–)-epicatechin, was identified in hairy roots of Medicago truncatula in which the Arabidopsis PA regulator TT2 was expressed (Pang et al., 2008). This study illustrates the utility of using PA regulatory factors to discover new genes involved in the PA biosynthetic pathway.
Transcriptional regulation of flavonoid and PA biosynthetic genes involves combinatorial interactions between several classes of transcription factor proteins (Mol et al., 1998
The R2R3 MYBs constitute large gene families in plants, with 126 members in Arabidopsis (Stracke et al., 2001
Constitutive expression of TT2 in Arabidopsis does not induce plant-wide PA accumulation, indicating that PA synthesis is controlled by the tissue- and cell type-specific expression patterns of additional regulatory genes (Nesi et al., 2001
We previously showed that the stress induction of PAs in poplar leaves follows the transcriptional activation of PA biosynthetic genes (Peters and Constabel, 2002
Identification of Putative PA Regulatory R2R3 MYB Genes in Poplar
Based on our knowledge that PA synthesis is activated by wounding and of the central role of R2R3 MYB factors in regulating flavonoid metabolism, we began a search for a MYB regulator of wound-induced PA metabolism. We first identified an EST sequence predicted to encode an R2R3 MYB domain transcription factor in an EST library made from systemically wounded poplar leaves (GenBank accession no. CN192773; Christopher et al., 2004
A phylogenetic tree was constructed using the predicted amino acid sequences of the R2R3 MYB domains of putative flavonoid regulatory MYBs from poplar as well as a number of R2R3 MYB proteins from other species (Fig. 2A
). The four candidate MYBs (MYB097, MYB086, MYB134, and MYB183) are most closely related to members of the N08 group rather than to other flavonoid regulatory MYB subgroups such as N09, G20, or G5 (Fig. 2A). These findings are congruent with a recently published phylogenetic analysis of the entire P. trichocarpa R2R3 MYB family (Wilkins et al., 2009
MYB134 and MYB086 encode proteins containing a sequence motif similar to the Vx2IRTKA[IL]RC[SN] motif located C terminal to the R2R3 MYB domain in Arabidopsis TT2 (Nesi et al., 2001 ; Fig. 2B). A similar conserved sequence motif is found in other N08 subgroup proteins, including the recently identified lotus TT2 proteins (Yoshida et al., 2008), the grapevine MYBPA2 (Terrier et al., 2009), and several uncharacterized predicted MYB proteins including cotton (Gossypium hirsutum) MYB36 (GenBank accession no. AAK19617), Fagus crenata MYB251 (BAG75107), and Malus x domestica MYB11 (AAZ20431) and MYB9 (ABB84757). The [DE]Lx2[RK]x3Lx6Lx3R motif involved in the interaction of MYB proteins with bHLH partners (Grotewold et al., 2000; Zimmermann et al., 2004) is present in all four putative poplar PA regulatory MYBs, which indicates that, like TT2, these MYBs likely require the presence of bHLH cofactors to function (Fig. 2B).
Mechanical wounding of poplar leaves has been shown to induce the up-regulation of flavonoid biosynthetic genes leading to PA accumulation (Peters and Constabel, 2002
As expected, mechanical wounding of leaf margins resulted in an up-regulation of phenylpropanoid and flavonoid genes (Fig. 3A). Gene expression was monitored at 1, 3, 6, 12, 24, and 48 h after wounding. PAL1, the wound-inducible flavonoid-associated PAL gene characterized by Kao et al. (2002) , was rapidly up-regulated (Fig. 3A). General flavonoid biosynthetic genes, including CHS, F3H, ANS1, and the DFR1 gene previously implicated in herbivore-induced PA accumulation (Peters and Constabel, 2002), were up-regulated within 24 h of wounding, as were the PA-specific ANR1 and LAR1 genes. FLS4, a flavonol synthase gene, was not responsive to wounding. A putative poplar homolog of the Arabidopsis MATE transporter gene TT12, previously found to be coinduced with PA biosynthetic genes following infection of poplar leaves with the fungal biotroph Melampsora medusae (Miranda et al., 2007), was also up-regulated following wounding (Fig. 3A). The putative PA regulatory genes MYB097, MYB134, and MYB183 all exhibited wound-induced transcript accumulation that correlated with the activation of flavonoid structural genes (Fig. 3A).
In order to analyze MYB gene expression under a flavonoid-activating stress different from mechanical wounding, we moved plants from the greenhouse into full natural sunlight, where they were exposed to elevated levels of both visible and UV-B (290–320 nm) radiation (together termed "high light" [HL]). Intense visible and UV-B radiation are known to stimulate multiple branches of phenylpropanoid and flavonoid metabolism (Jordan, 1996 None of the candidate PA regulatory MYB genes was coactivated with the rapidly induced flavonol biosynthetic genes. However, MYB134 and MYB183 both exhibited coactivation with the late flavonoid and PA biosynthetic genes after HL exposure (Fig. 3A). MYB086 and MYB134 are similar at the nucleotide sequence level, such that expression may not be reliably distinguishable using northern-blot analysis. Therefore, we analyzed their expression and stress responsiveness using real-time PCR with primers specific to MYB134 or MYB086. Expression of MYB097 and MYB183 was also monitored. We determined relative transcript abundance in untreated control leaf tissue as well as leaves 24 h after wounding and 48 h after movement into HL conditions, with triplicate biological and technical replication (Fig. 3C). MYB086 exhibited a very low, constitutive level of expression, while MYB134 was found to be wound and HL induced (Fig. 3C; MYB086 expression was too low relative to the other genes to be visible with the scale of this graph, but it was visible with gel electrophoresis). The wound- and HL-induced expression of MYB097 and MYB183 was also confirmed (Fig. 3C). To pursue the distinct HL-induced patterns of early and late flavonoid structural gene expression in more detail, we examined flavonoid gene expression in poplar leaves exposed to increased UV-B irradiance in a growth chamber. Northern-blot analysis of gene expression revealed an activation of flavonol biosynthetic genes by 3 h, followed by up-regulation of PA-specific biosynthetic genes by 48 h (Fig. 4A , left and center). PA and total flavonol glycoside concentrations were determined after 7 d of stress treatment and were again found to have increased significantly (Fig. 4B). None of the poplar MYB genes tested exhibited a coinduction with FLS4 and the early flavonoid biosynthetic genes. Of the wound- and light-induced MYB genes, only MYB134 was coinduced with the late flavonoid biosynthetic genes under elevated UV-B light.
Since we observed the coactivation of PA biosynthetic genes and putative MYB regulators following both wounding and exposure to elevated light levels, we sought to determine whether this coactivation would occur following an additional PA-activating stress. We had previously found that infection of poplar leaves by the fungal biotroph M. medusae results in a significant up-regulation of PA biosynthetic genes and a corresponding increase in foliar PAs (Miranda et al., 2007 ). Profiling of flavonoid and putative PA regulatory MYB gene expression in leaf tissue 6 d after inoculation revealed that MYB134 was again most strongly coinduced with PA structural genes (Fig. 4A, right). As with mechanical wounding, FLS4 was not strongly up-regulated by M. medusae infection, confirming that expression of MYB134 is not correlated with the activation of flavonol metabolism (Fig. 4A). Overall, our data show that several members of the poplar N08 MYB subgroup exhibit some stress-induced coactivation with PA biosynthetic genes. However, MYB134 expression was the most correlated with PA activation under all conditions analyzed. Of the inducible MYB genes, the predicted protein encoded by MYB134 also exhibited the highest homology to TT2 within the R2R3 MYB domain and shared a C-terminal sequence motif. Based on these observations, we hypothesized that MYB134 may be a regulator of stress-induced PA metabolism and sought to further characterize its functions using transgenic plants.
In order to investigate the role of MYB134 in the regulation of stress-induced PA metabolism, we overexpressed this gene in poplar under the control of a double cauliflower mosaic virus 35S promoter. GUS-overexpressing lines were also produced as controls. Two genotypes, Populus tremula x P. tremuloides clone 353-38 and P. tremula x Populus alba clone 717-1-B4, were chosen for Agrobacterium tumefaciens-mediated transformation because of differences they exhibit in wound-induced PA metabolism. Under our growth conditions, the stress-induced transcriptional activation of the PA biosynthetic pathway is stronger in the 353-38 clone than in the 717-1-B4 clone (R.D. Mellway and C.P. Constabel, unpublished data). Both are characterized by relatively low leaf PA levels when grown under standard greenhouse conditions. Multiple independently transformed lines were generated for both genotypes, and confirmed transgenics were moved to a greenhouse for analysis. For plants of both genotypes, grown and maintained under our standard greenhouse conditions for up to 6 months (see "Materials and Methods"), MYB134 overexpression did not lead to any discernible gross phenotypic abnormalities (Supplemental Fig. S1). However, analysis of PA levels in leaves revealed that MYB134 overexpression resulted in a dramatic increase in PA concentrations (Fig. 5A
). The PA levels found in these MYB134 overexpressor plants were similar to the levels found in the leaves of outdoor P. tremuloides trees and much higher than we have observed for any Populus species under our greenhouse conditions (R.D. Mellway and C.P. Constabel, unpublished data). One of the 353-38 MYB134 overexpressor lines exhibited a much lower but still significant (P = 0.003) increase in PA levels (353-38 MYB134 line 1; Fig. 5A). Southern-blot analysis using a probe complementary to the neomycin phosphotransferase II gene present on the T-DNA revealed that this line contained five or six T-DNA insertions, compared with one or two copies for the other lines (data not shown). This suggested that the lower increase in PA levels in this line may be the result of silencing of the transgene due to the high number of T-DNA insertions (Tang et al., 2007
In order to confirm that MYB134 activates genes of the PA biosynthetic pathway, we examined phenylpropanoid and flavonoid structural gene expression in leaves of control and MYB134-overexpressing plants. In both the 353-38 and 717-1-B4 clones, MYB134 overexpression was found to activate the entire phenylpropanoid pathway leading to PA production, including the flavonoid-specific general phenylpropanoid genes PAL1 and 4CL2 (Kao et al., 2002 ; Fig. 5B). Both early (CHS1, CHI1, and F3H) and late (DFR1, ANR1, ANS1, and LAR1) flavonoid biosynthetic genes were more highly expressed in the MYB134-overexpressing plants, but the level of late biosynthetic gene up-regulation was much greater (Fig. 5B). The stress-inducible MATE gene was also activated in the MYB134 overexpressors, suggesting that it is a poplar homolog of Arabidopsis TT12 and that it is important for PA biosynthesis. Consistent with our hypothesis that MYB134 functions specifically to regulate PA metabolism and does not directly regulate other flavonoid pathways, the light stress- and UV-B-induced flavonol synthase gene FLS4 was not up-regulated by MYB134 overexpression. Rather, FLS4 was expressed in leaves of both control and MYB134 overexpressor plants at similarly low levels (Fig. 5B). The expression of PAL2 and CCR (for cinnamoyl CoA-reductase), genes involved in lignin production (Kao et al., 2002; Li et al., 2005), was likewise not altered in leaves of MYB134 overexpressors (Fig. 5B). MYB097 and MYB183 were also not up-regulated in MYB134-overexpressing plants (Supplemental Fig. S2). To determine if tissues other than mature leaves also respond to MYB134 overexpression with activation of the PA biosynthetic pathway, we conducted a tissue survey of PA levels in four high-MYB134 transgene-expressing lines (353-38 MYB134 overexpressor lines 2–5). Acid-butanol PA assays revealed a significant increase in PA concentrations in all tissues analyzed (P < 0.01), with the exception of young roots (P = 0.055; Fig. 5C). In addition to the large increase in PA concentrations, HPLC analysis revealed that MYB134 overexpression also caused unexpected secondary alterations to phenolic metabolism (Fig. 6). The most pronounced effect was a reduction in PG concentrations. Total PG levels were reduced from a mean of 46.7 ± 12.2 mg g–1 fresh weight to 17.0 ± 1.9 mg g–1 fresh weight, and levels of all individual PGs (salicortin, tremuloidin, and tremulacin) reflect this change (Fig. 6B ). We also observed an increase in concentrations of total flavonol glycosides and a decrease in levels of nonflavonoid phenolic acids (Fig. 6C). These latter changes were statistically significant (P < 0.05), but they were minor in comparison with the increase in PAs. Anthocyanins were detected in neither control nor MYB134 overexpressor leaves. A number of peaks with absorption spectra and retention times corresponding to PA biosynthetic intermediates, including flavan-3-ols (catechin and epicatechin) and a flavanone/dihydroflavonol, were observed in MYB134 overexpressor leaf extracts but were undetectable in controls (Fig. 6A). The same pattern of moderately reduced nonflavonoid phenylpropanoids (PGs and phenolic acids) and a small increase in non-PA flavonoids (flavonol glycosides and PA biosynthetic intermediates) was also found in the 717-1-B4 MYB134 overexpressor lines (data not shown). Overall, the combined decreases in nonflavonoid phenylpropanoids were less than the total increase in PA and flavonoid levels; thus, MYB134 overexpression led to a net increase in total soluble phenolics in all tissues (Supplemental Table S1).
To determine PA localization in MYB134-overexpressing and control poplar plants, leaf, petiole, and stem sections were stained with dimethylaminocinnamaldehyde (DMACA), which reacts specifically with PAs and flavan-3-ols to form a blue chromophore (Feucht and Treutter, 1990 ). As expected, DMACA stained the MYB134 overexpressor tissues more intensely than control tissues (Fig. 7
). In leaves of control plants, DMACA staining revealed PA accumulation primarily in the abaxial epidermis, with very light staining in the adaxial epidermis (Fig. 7A). In MYB134 overexpressor leaves, PAs were also present in both the adaxial and abaxial epidermal layers but at much greater concentrations. Most notably, DMACA staining was also very abundant in the upper layer of palisade mesophyll cells and sporadically within the spongy mesophyll (Fig. 7B). In petioles, staining was observed only in the epidermal cells in controls (Fig. 7C), while in MYB134 overexpressors, strong staining was observed in the epidermis with sporadic staining in the cortex and vascular tissues (Fig. 7D). In stem sections (fourth internode), PAs were localized only to the epidermis of control plants (Fig. 7E), while staining was observed in the epidermis, cortex, xylem, and pith in the MYB134 overexpressors (Fig. 7F). The pattern of PA distribution within the MYB134-overexpressing plants thus includes multiple cell types. Although our control plants showed relatively faint staining, based on previous work (Kao et al., 2002) it appears that in MYB134 overexpressors many of the same cell types that are competent to produce PAs are now stimulated to produce much higher levels.
MYB134 Binds to Promoter Regions of PA Biosynthetic Genes
Consistent with the hypothesis that PA biosynthetic pathway genes function as downstream targets of MYB134, electrophoretic mobility shift assays (EMSAs) revealed that recombinant MYB134 protein bound to 180-bp DNA regions residing upstream of the transcriptional start of the three putative target genes (Fig. 8
). These target genes were chosen to represent general phenylpropanoid/early PA metabolism (PAL1), late flavonoid metabolism (DFR1), and the PA-specific branch of flavonoid metabolism (ANR2). Candidate MYB134-binding sites in the regulatory regions of these genes were identified by visual examination of the upstream genomic sequence and comparison with characterized phenylpropanoid promoters as well as with a search of the PLACE plant cis-element database (http://www.dna.affrc.go.jp/PLACE/signalscan.html) using SIGNAL SCAN (Prestridge, 1991
EMSA experiments showed that the recombinant MYB134 protein specifically bound the 180-bp upstream regulatory sequences (Fig. 8B). Two shifted bands were observed for the PAL1 and ANR2 180-bp probes, while only one was seen with the DFR1 probe (Fig. 8B). It is possible that the MYB134 protein binds both of the overlapping AC elements in the PAL1 promoter and both the AC element-like sequence and the c-myb-binding site in the ANR2 promoter. A sequence containing a canonical AC element was an effective competitor and eliminated MYB134 binding (Fig. 8B), and recombinant MYB134 also bound to this element in a specific manner (Fig. 8C). Thus, MYB134 appears to bind to the gene regulatory regions of putative target genes in an AC motif-dependent fashion.
Identification of MYB134, a Stress-Induced Regulator of PA Metabolism in Poplar PAs are important molecules for plant adaptation to the environment and for human health, and there is considerable interest in the control of their biosynthesis. Here, we present evidence that MYB134 plays a role in the regulation of PA synthesis in poplar leaves, the first example, to our knowledge, of such a regulator implicated in stress induction of PAs in vegetative tissues. We believe MYB134 to be a PA regulator for the following reasons. First, of all the predicted R2R3 MYB genes in the P. trichocarpa genome, MYB134 exhibits the highest sequence similarity to TT2, the Arabidopsis MYB required for PA synthesis in the seed coat. Second, transcript profiling following stresses that induce the PA biosynthetic pathway demonstrated that, unlike other TT2-like poplar MYB genes, MYB134 was consistently coinduced with the PA biosynthetic pathway genes, including the PA-specific genes LAR1 and ANR1. Third, overexpression of MYB134 in transgenic poplar behind a strong constitutive promoter led to a large plant-wide accumulation of PAs. This was the result of the activation of the full PA biosynthetic pathway, including general phenylpropanoid genes, early and late flavonoid genes, and PA-specific genes. Fourth, our EMSA experiments show that MYB134 binds to representative early and late PA pathway gene regulatory regions containing putative MYB-binding sites, indicating that these genes may be directly activated by MYB134.
Our data indicate that MYB134 functions specifically in regulating PA biosynthesis and is unlikely to be involved in activating other flavonoid branches. MYB134 transcripts were not coinduced with FLS4 and the other early flavonoid biosynthetic genes by elevated light and UV-B stress (Figs. 3 and 4). Similarly, the absence of FLS4 induction in wounded and M. medusae-infected leaves (in which MYB134 expression was induced), as well as the lack of FLS4 up-regulation in the MYB134 overexpressor plants, indicates that it is not a target of MYB134 regulation. In addition, in MYB134 overexpressors, transcript levels of the lignin-specific PAL2 (Kao et al., 2002
While MYB134 overexpression is sufficient for activation of PA biosynthetic genes, up-regulation of predicted target genes in stably transformed plants does not in itself demonstrate a direct regulation by MYB134, and it is thus possible that this protein activates some downstream genes indirectly via other factors. Data generated using stable transformation of plants with genes encoding transcription factors must be interpreted with caution, as the resulting phenotype may be confounded by off-target and indirect effects (Broun, 2004
MYB134 overexpression results in a differential degree of activation of early and late PA biosynthetic genes (Fig. 5B). This suggests that precise regulation of the full pathway in planta could involve additional MYB or other regulatory proteins (see below). The expression of the other two stress-induced poplar N08 MYB genes (MYB183 and MYB097) was unaltered in the MYB134 overexpressors (Supplemental Fig. S2), indicating that they are not required for pathway activation by MYB134. Overexpression of MYB183 and MYB097 in transgenic poplar does not result in activation of the full PA biosynthetic pathway (R.D. Mellway and C.P. Constabel, unpublished data). Since many of the flavonoid enzymes in poplar are encoded by small gene families that exhibit differential stress inducibility (Tsai et al., 2006b
In addition to this work, Arabidopsis and lotus TT2 and grapevine MYBPA1 and MYBPA2 are the only functionally characterized R2R3 MYBs shown to be involved specifically in the regulation of PA metabolism. While Arabidopsis TT2, grapevine MYBPA2, and poplar MYB134 are very similar and fall within subgroup N08, MYBPA1 does not fall within the same phylogenetic cluster (Fig. 2A). Rather, it is more closely related to poplar MYB factors such as MYB115, MYB201, and MYB153. Another well-characterized N08 member, maize C1 (and paralogues), is an anthocyanin regulator, although C1 was found to strongly activate the Arabidopsis BAN (ANR) promoter when coexpressed with the maize bHLH protein Sn in transient expression experiments (Baudry et al., 2004
Like Arabidopsis TT2, the predicted poplar MYB134, MYB097, MYB183, and MYB086 proteins contain residues in the R3 domain involved in mediating interactions with members of the bHLH transcription factor family (Fig. 2B); thus, these MYBs likely function with bHLH partners in transcription factor complexes. The accumulation of PAs in diverse organs and cell types of MYB134-overexpressing plants suggests that the additional factors required to form a functional MYB-bHLH-WDR ternary transcription complex are already present or are themselves regulated by MYB134, at least in those cell types within each tissue in which PA accumulation was observed. DMACA staining of MYB134 overexpressors revealed that PA accumulation was not uniform within tissues (Fig. 7). For example, in leaves, PAs consistently accumulated in the upper layer of palisade mesophyll cells and not in the lower layer (Fig. 7B). It is likely that cell type-specific expression patterns of additional factors, such as interacting WDR or bHLH proteins, may be integral in the regulation of the PA pathway, as has been found in Arabidopsis (Baudry et al., 2004
In silico analysis has shown that the promoter regions of the poplar flavonoid and PA biosynthetic genes contain cis elements matching the consensus sequences recognized by phenylpropanoid regulatory R2R3 MYB proteins (Tsai et al., 2006a
In activating the full suite of early and late flavonoid as well as PA biosynthetic genes, MYB134 differs from Arabidopsis TT2, which regulates a more limited set of late PA structural genes (Nesi et al., 2001
In addition to the large increase in PA levels, the up-regulation of PA biosynthesis by MYB134 overexpression resulted in other changes to the phenolic metabolite profile. Concentrations of the phenolic glycosides were consistently reduced in leaves of the MYB134 overexpressor plants, resulting in an approximately 3-fold overall decrease relative to controls. The phenolic glycosides are the most abundant soluble phenolic compounds in leaves of the control plants, so this reduction represents a significant shift in carbon flux. Although less abundant overall, levels of phenolic acids were also reduced in the MYB134 overexpressor leaves, while non-PA flavonoids were increased slightly. These differences were consistently observed in different genotypes and transformed lines.
Based on the specificity of previously characterized flavonoid regulatory R2R3 MYB factors, it seems unlikely that MYB134 controls the PG and phenolic acid pathways directly. However, it is possible that these alterations are caused by competition for common cofactors between MYB134 and MYB factors regulating other branches of phenolic metabolism. Because the biosynthetic pathway of the phenolic glycosides is as yet unknown, we do not currently have molecular probes for enzymes of this pathway that would allow us to determine if transcriptional regulation is involved. However, it seems probable that the high rate of PA synthesis is diverting metabolic resources from other phenolic pathways, so that the observed changes in non-PA phenylpropanoid levels may be the result of competition for common phenolic precursors between the up-regulated PA pathway and other branches of phenolic metabolism. Holton et al. (2003) The increase in flavonol glycoside levels but not other non-PA flavonoid end products such as flavones and anthocyanins is also consistent with the hypothesis that the high flux into PAs has indirect consequences on other pathways. The increased flux into the flavonoid pathway resulting from MYB134 overexpression and the accumulation of PA intermediates should provide higher levels of substrates for branch pathways that are already active in leaves (i.e., flavonol biosynthesis). Our data show that FLS4 was equally expressed in controls and MYB134 overexpressors (Fig. 5B) and that flavonol glycosides were present in leaves of both controls and MYB134 overexpressors (Fig. 7). With an enhanced synthesis of shared PA and flavonol intermediates such as dihydroflavonols, substrate availability for flavonol synthase should also increase. This would result in increased flavonol production without the activation of FLS4 or other flavonol-specific genes. Flavonoid branch pathways not already active in these tissues (i.e. anthocyanins) would be unaffected. However, other levels of regulation cannot be excluded. The observation that MYB134 overexpression can directly or indirectly affect multiple phenylpropanoid pathways and end products has implications for analyses of metabolic flux and control points in phenolic metabolism, and these plants may be useful tools for such studies. However, the modification of several biologically active metabolites also complicates the interpretation of the biological effects of this overexpression.
PAs have long been investigated in the context of tree defense against insect pests, but their importance is still debated in the literature. Some of the controversy is likely due to observations that the biological activities of PAs are dependent upon subtle differences in their chemical structures, which show variation among different plants (Ayres et al., 1997
The strong up-regulation of PA biosynthetic genes and accumulation of PAs following light stress and UV-B exposure was a novel result of our experiments. PAs are not usually considered to be light stress- or UV-B-protective compounds. To our knowledge, rapid induction of PA biosynthesis following UV-B stress has not been previously reported in poplar, although Lavola (1998)
Whether PAs can protect leaves of poplar, birch, and other plants against excess UV-B light directly as "sun screens" is unclear. Correlations between light levels and concentrations of PAs or other high-Mr polyphenols have been observed (Mole et al., 1988
The extensive genomics resources combined with the complexity and biological importance of phenylpropanoid metabolism in poplar make it a useful system for investigating this pathway. In this report, we describe work identifying a gene encoding an R2R3 MYB transcription factor that appears to play an important role in controlling PA biosynthesis under a variety of stress conditions. We have established that MYB factors not only regulate developmental PA production but also function to control PA synthesis in response to stress. Identifying transcriptional regulators of biosynthetic pathway genes is an important goal for metabolic engineering of secondary metabolism in plants, and the identification of a putative regulator of PA metabolism in poplar may permit new experimental approaches for evaluating the biological functions of PAs.
Plant Growth Conditions and Stress Treatments
Populus tremuloides clone A2 was collected from the vicinity of Edmonton, Alberta, Canada (Haruta et al., 2001
For wounding experiments, leaf margins totaling approximately one-fifth of the area of each leaf were crushed with pliers. For HL exposure experiments, trees were moved from the greenhouse (mean maximum photosynthetically active radiation, 400–700 nm; 377 mol m–2 s–1; biologically effective UV-B irradiance [UV-Bbe], 0.26 kJ m–2 d–1) into full natural sunlight during August in Victoria, British Columbia, Canada (mean maximum photosynthetically active radiation, 1,655 mol m–2 s–1; UV-Bbe, 3.48 kJ m–2 d–1). The wounding and light stress experiment shown was conducted in August 2005 in Victoria and replicated with equivalent results in 2006. For UV-B treatments, trees were acclimated for 1 week in a growth chamber (16-h/8-h photoperiod, 19–25°C) equipped with F40T12 UV-B lamps (Phillips Lighting) with presolarized cellulose acetate filters to block UV-C light transmission. Plants were exposed to 0.21 kJ m–2 d–1 UV-Bbe before UV-B lamps were activated and to 1.45 kJ m–2 d–1 UV-Bbe after activation. The UV-B experiment shown was conducted in October 2006. Measurements of UV-Bbe were made with an IL1700 radiometer equipped with an IL782A high-gain photomultiplier (International Light) using weighting factors from the Caldwell action spectrum normalized to 300 nm (Bjorn and Teramura, 1993
Primers for amplifying full-length sequences were designed using Vector NTI Advance version 9.0 (Invitrogen). Primer sequences are listed in Supplemental Table S2. Full-length coding sequences were amplified from P. tremuloides (clone A2) leaf cDNA and cloned into pGEM-T Easy (Promega) for sequencing. For multiple sequence alignment and phylogenetic analysis, sequences were aligned using ClustalW (Chenna et al., 2003
The coding sequence of MYB134 was PCR amplified from a P. tremuloides (clone A2) cDNA library with primers (Supplemental Table S2) containing restriction linker sites for subcloning into the vector pBI-524 between the double cauliflower mosaic virus 35S promoter with
For HPLC analysis, 0.50 g of frozen leaf tissue was ground in liquid nitrogen and extracted for 4 h in 10 mL of 80% methanol. Extracts were centrifuged to remove solid debris, and methanol was removed using a rotary evaporator, followed by cleanup with Strata-X 33-µm solid-phase extraction columns, according to the manufacturer's instructions (Phenomenex). Compounds were eluted in 2 mL of methanol:acetonitrile (1:1, v/v), and 30 µL was injected onto an HPLC system (Beckman Coulter System Gold 126 solvent module with a System Gold 168 diode array detector) with a reverse-phase Luna C18(2) column (250 x 60 mm, 5 µm; Phenomenex). Separation was performed with a linear elution gradient from 90% solvent A (0.5% methanol in 0.01 M phosphoric acid, v/v) to 40% solvent B (100% acetonitrile) over 30 min at a flow rate of 1.5 mL min–1. Although HPLC with diode array detection does not permit the precise structural identification of compounds, the subclasses of phenolic compounds present in poplar leaves are well characterized and peaks can be confidently assigned to different subclasses based on their distinctive UV/visible absorption spectra and quantified using representative standards (Mabry et al., 1970
RNA for northern-blot and PCR analyses was isolated from leaf tissue using the cetyltrimethylammonium bromide method as described (Haruta et al., 2001
For real-time PCR analysis, 25 ng of total RNA was treated with DNase I (Invitrogen) according to the manufacturer's instructions. Five nanograms of DNase I-treated RNA was then was used for reverse transcription with SuperScript II reverse transcriptase (Invitrogen). Following validation experiments, real-time PCR analysis was performed using a Stratagene Mx4000. Triplicate reactions were run on triplicate independent experiments. Reactions (15 µL) consisted of the QuantiTect SYBRGreen mix (Qiagen) with 0.67 µM gene-specific primers and 6.25 ng of cDNA template per reaction. The amplification protocol was 95°C for 15 min followed by 40 cycles of 94°C for 30 s, 55°C for 40 s, and 72°C for 60 s. Dissociation curves were obtained and reaction products visualized using agarose gel electrophoresis to confirm that single, specific products were produced in each reaction. Cycle threshold (Ct) values were determined by Mx4000 software at a manually set fluorescence threshold of 0.019, and relative transcript abundances (2–
Fresh plant material was placed into Tissue-Tek O.C.T. Compound Embedding Medium (Sakura Finetek) and left overnight in a –20°C freezer before slicing 20-µm-thick (leaf) or 40-µm-thick (petiole and stem) sections using a Microm HM 500 cryomicrotome. PAs and flavan-3-ols were detected by staining sections for 5 min with DMACA (1% [w/v] in ethanol:6 N HCl, 1:1 [v/v]). Images were recorded using a Spot RT KE digital camera (Diagnostic Instruments) mounted on a Zeiss Universal compound microscope for leaf blade sections or a Wild M420 macroscope for stem and petiole sections.
Recombinant MYB134 protein was produced in Escherichia coli using the coding sequence cloned in-frame into the NdeI and BamHI sites of the pET15b vector (Novagen). Recombinant MYB134 protein was produced, extracted, and affinity purified as described previously for pine (Pinus spp.) MYB proteins (Patzlaff et al., 2003 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers FJ573150, FJ573151, FJ573152, and FJ588548.
The following materials are available in the online version of this article.
We thank Brent Gowen (University of Victoria) for assistance with the preparation of tissue for histochemical staining, George Newcombe (University of Idaho) for the M. medusae isolate, Richard Lindroth (University of Wisconsin-Madison) and Thomas Clausen (University of Alaska) for the purified phenolic glycosides, Silvia L'Hirondelle (Ministry of Forests and Range) for the IL1700 radiometer, and Nici T. Darychuk (University of Victoria) for critical reading of the manuscript. Received March 27, 2009; accepted April 21, 2009; published April 24, 2009.
1 This work was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery Grants to C.P.C. and M.M.C., and Undergraduate, Canada Graduate, and Postgraduate Scholarships to R.D.M.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: C. Peter Constabel (cpc{at}uvic.ca).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.139071 * Corresponding author; e-mail cpc{at}uvic.ca.
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Plant J 40: 22–34[CrossRef][Web of Science][Medline]
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max (note that some peaks present in both chromatograms are labeled in one or the other). Ph Ac, Phenolic acids; FG, flavonol glycosides. B, Phenolic glycoside (salicortin, tremuloidin, and tremulacin) concentrations in leaves of 353-38 GUS controls and MYB134 overexpressors. FW, Fresh weight. C, Total phenolic acid and flavonol glycoside concentrations in leaves of 353-38 GUS controls and MYB134 overexpressors. Bars indicate means of four independently transformed lines (GUS lines 1–4 and MYB134 overexpressor lines 2–5), with error bars indicating SE. Asterisks indicate significant differences using Student's t test (* P < 0.05).
-mosaic virus RNA4 transcriptional enhancer sequence and the nopaline synthase terminator sequence (Datla et al., 1992
Ct) were determined after normalization to a constitutively expressed Actin gene (for all real-time PCR primer sequences, see Supplemental Table S2).