Trees in the genus Populus (poplar) contain phenolic secondary metabolites including the proanthocyanidins (PAs), which help to adapt these widespread trees to diverse environments. The transcriptional activation of PA biosynthesis in response to herbivory and ultraviolet light stress has been documented in poplar leaves, and a regulator of this process, the R2R3-MYB transcription factor MYB134, has been identified. MYB134-overexpressing transgenic plants show a strong high-PA phenotype. Analysis of these transgenic plants suggested the involvement of additional MYB transcription factors, including repressor-like MYB factors. Here, MYB182, a subgroup 4 MYB factor, was found to act as a negative regulator of the flavonoid pathway. Overexpression of MYB182 in hairy root culture and whole poplar plants led to reduced PA and anthocyanin levels as well as a reduction in the expression of key flavonoid genes. Similarly, a reduced accumulation of transcripts of a MYB PA activator and a basic helix-loop-helix cofactor was observed in MYB182-overexpressing hairy roots. Transient promoter activation assays in poplar cell culture demonstrated that MYB182 can disrupt transcriptional activation by MYB134 and that the basic helix-loop-helix-binding motif of MYB182 was essential for repression. Microarray analysis of transgenic plants demonstrated that down-regulated targets of MYB182 also include shikimate pathway genes. This work shows that MYB182 plays an important role in the fine-tuning of MYB134-mediated flavonoid metabolism.

The accumulation of proanthocyanidins is regulated by a complex of transcription factors composed of R2R3 MYB, basic helix-loop-helix, and WD40 proteins that activate the promoters of biosynthetic genes. In poplar (genus Populus), MYB134 is known to regulate proanthocyanidin biosynthesis by activating key flavonoid genes. Here, we characterize a second MYB regulator of proanthocyanidins, MYB115. Transgenic poplar overexpressing MYB115 showed a highproanthocyanidin phenotype and reduced salicinoid accumulation, similar to the effects of MYB134 overexpression. Transcriptomic analysis of MYB115-and MYB134-overexpressing poplar plants identified a set of common up-regulated genes encoding proanthocyanidin biosynthetic enzymes and several novel uncharacterized MYB transcriptional repressors. Transient expression experiments demonstrated the capacity of both MYB134 and MYB115 to activate flavonoid promoters, but only in the presence of a basic helix-loop-helix cofactor. Yeast two-hybrid experiments confirmed the direct interaction of these transcription factors. The unexpected identification of dihydromyricetin in leaf extracts of both MYB115-and MYB134-overexpressing poplar led to the discovery of enhanced flavonoid B-ring hydroxylation and an increased proportion of prodelphinidins in proanthocyanidin of the transgenics. The dramatic hydroxylation phenotype of MYB115 overexpressors is likely due to the up-regulation of both flavonoid 39,59-hydroxylases and cytochrome b 5 . Overall, this work provides new insight into the complexity of the gene regulatory network for proanthocyanidin synthesis in poplar.Proanthocyanidins (PAs), also known as condensed tannins, are widespread polyphenols with diverse ecological functions. They are polymers of flavan-3-ols and, thus, end products of the phenylpropanoid and flavonoid pathways (Dixon et al., 2005). The PAs are the most broadly distributed secondary metabolites and are especially prominent in forest trees and woody plants ( Barbehenn and Constabel, 2011). PA accumulation in trees can be substantial; for example, in some species of poplar (genus Populus), PAs can constitute 25% of leaf dry weight. However, the accumulation of PAs also is highly plastic and varies with genotype and growth conditions (Hwang and Lindroth, 1997;Osier and Lindroth, 2006). In trees, PAs are common constituents of vegetative organs, including roots, leaves, bark, and flowers. Seasonal leaf drop in autumn and turnover of roots thus lead to substantial tannin input into forest soils, where it has been shown to slow litter decomposition and nutrient cycling (Schweitzer et al., 2008). In herbaceous plants, PAs are more restricted in distribution, but they can be found in leaves of legumes, 1 This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Max Planck Society, the Academy of Finland, and the Canadian Genomics R&D Initiative.2 These authors contributed equally to the article. * Address correspondence to cpc@uvic.ca. The author responsible for distribution ...

The phenylpropanoid pathway leads to the production of many important plant secondary metabolites including lignin, chlorogenic acids, flavonoids, and phenolic glycosides. Early studies have demonstrated that flavonoid biosynthesis is transcriptionally regulated, often by a MYB, bHLH, and WDR transcription factor complex. In poplar, several R2R3 MYB transcription factors are known to be involved in flavonoid biosynthesis. Previous work determined that poplar MYB134 and MYB115 are major activators of the proanthocyanidin pathway, and also induce the expression of repressor-like MYB transcription factors. Here we characterize two new repressor MYBs, poplar MYB165 and MYB194, paralogs which comprise a subgroup of R2R3-MYBs distinct from previously reported poplar repressors. Both MYB165 and MYB194 repressed the activation of flavonoid promoters by MYB134 in transient activation assays, and both interacted with a co-expressed bHLH transcription factor, bHLH131, in yeast two-hybrid assays. Overexpression of MYB165 and MYB194 in hybrid poplar resulted in greatly reduced accumulation of several phenylpropanoids including anthocyanins, proanthocyanidins, phenolic glycosides, and hydroxycinnamic acid esters. Transcriptome analysis of MYB165- and MYB194-overexpressing poplars confirmed repression of many phenylpropanoid enzyme genes. In addition, other MYB genes as well as several shikimate pathway enzyme genes were downregulated by MYB165-overexpression. By contrast, leaf aromatic amino acid concentrations were greater in MYB165-overexpressing poplars. Our findings indicate that MYB165 is a major repressor of the flavonoid and phenylpropanoid pathway in poplar, and may also affect the shikimate pathway. The coordinated action of repressor and activator MYBs could be important for the fine tuning of proanthocyanidin biosynthesis during development or following stress.

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Leguminous plants have many paralogous genes encoding enzymes involved in the flavonoid biosynthetic pathway. Duplicate genes are predicted to contribute to the production of various flavonoid compounds and to have resulted in a diversity of legume species. We identified gene duplication in the transcription factors regulating flavonoid biosynthesis in the model legume Lotus japonicus. Three copies of a homolog of Arabidopsis thaliana TRANSPARENT TESTA2 (TT2), which is a MYB transcription factor that regulates proanthocyanidin biosynthesis, were present in the L. japonicus genome. The organ specificity and stress responsiveness differed among the three LjTT2s, and correlations between proanthocyanidin accumulation and the expression levels of LjTT2s were observed during seedling development. Moreover, three LjTT2s functionally complemented TT2 in transient expression experiments in A. thaliana leaf cells. The different reporter activity caused by LjTT2a was consistent with the affinity of physical interactions with TT8 and TTG1 in yeast two-hybrid experiments as well as the branching pattern of the phylogenetic tree. These results suggest that LjTT2 factors have diverse functions in the tissues in which they are expressed; in particular, LjTT2a is predicted to have evolved flexibility in interaction with other transcription regulators to resist environmental stresses.