The MYB182 protein downregulates proanthocyanidin and anthocyanin biosynthesis in poplar by repressing both structural and regulatory flavonoid genes Authors:

Trees in the genus Populus contain phenolic secondary metabolites including the proanthocyanidins (PAs) which help to adapt these widespread trees to diverse environments. The transcriptional activation of proanthocyanidin (PA) biosynthesis in response to herbivory and UV 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 expression of key flavonoid genes. Similarly, a reduced accumulation of transcripts of a MYB PA activator and a bHLH co-factor 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 bHLH-binding motif of MYB182 was essential for repression. Microarray analysis of transgenic plants demonstrated that downregulated 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. www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved. Yoshida et al., p. 5 Introduction Flavonoids are widely distributed secondary plant metabolites that include the anthocyanins, flavonols, flavones, and proanthocyanidins (Koes et al., 2005). Collectively, these compounds play many roles in the interaction of plants with their environment, such as protection against ultraviolet (UV) and visible light stress, resistance to herbivores and pathogens, attraction of pollinators and seed dispersers, and modulation of physiological and developmental signals (Mol et al., 1998). The proanthocyanidins (PAs), also known as condensed tannins, are polymers of flavan-3-ols, typically consisting of 2-50 subunits. PAs are present in many plants but are especially abundant in trees, where they are found in vegetative tissues such as leaves, bark, and roots (Barbehenn and Constabel 2011). They are also present in seeds and many types of fruit, where they are thought to discourage premature consumption by frugivores or prevent spoilage by fungi (Cipollini and Stiles, 1993). In humans, a diet high in PAs has been linked to reduced risk of chronic cardiovascular diseases (Rasmussen et al., 2005; Scalbert et al., 2005). PAs are thought to prevent atherosclerosis by inhibiting the oxidation of low-density lipoproteins (LDLs), based on their electron-donating properties and ability to scavenge reactive oxygen species (Scalbert et al., 2005). Berry fruits, whole grains, wine and beer are considered good sources of PAs (Prior and Gu, 2005). In the diets of ruminants such as sheep and cattle, PAs can be important modulators of excess rumen bacterial activity, preventing bloat and improving protein use efficiency (Min et al., 2003). This effect makes these compounds important components of silage and forage crops, and targets for manipulation via agricultural biotechnology. In nature, the PAs are associated with diverse ecological functions. They typically possess broad antimicrobial properties, and by inhibiting bacterial activity in soils they affect nutrient cycling. As a result, the PA content of litter from keystone trees species such as poplar correlates strongly with community structure and ecosystem dynamics (Schweitzer et al., 2008). Many studies have attempted to show that PAs are important in defense against insect herbivores. The results of such experiments are mixed, however, as PAs do not consistently show strong effects against major tree pests (Barbehenn and Constabel, 2011; Boeckler et al., 2014). By contrast, they can adversely affect performance and reproductive success in mammals due to their ability to bind dietary protein in mammalian digestive systems (Barbehenn and Constabel, 2011). In planta, PAs may also function to inhibit pathogenic microorganisms (Constabel et al., 2014). Tannins are also often found in the roots of trees, and though their roles here are poorly defined. The flavonoids, including PAs, are derived from phenylpropanoids and malonyl-CoA, which serve as substrates for chalcone synthase (CHS), the first enzyme in the flavonoid biosynthetic pathway. Following isomerization and hydroxylation by flavanone 3-hydroxylase (F3H), www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved. Yoshida et al., p. 6 intermediates are reduced by dihydroflavonol 4-reductase (DFR) (Marles et al., 2003). Anthocyanin synthase (ANS) then catalyses the last common step in the biosynthesis of anthocyanins and PAs. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are specific to the PA branch of the pathway and produce flavan-3-ols, typically epicatechin and catechin, respectively (Abrahams et al., 2003; Xie et al., 2003). These are polymerized via an unknown mechanism and stored in the vacuole. Genetic studies in Arabidopsis revealed that MATE efflux family proteins TT12, glutathione S-transferase TT19 and Autoinhibited H1-ATPase isoform 10 are required for transport of PAs to the vacuole. The biosynthesis of anthocyanins and PAs is regulated by three types of transcription factors: R2R3-MYB factors, basic helix–loop–helix (bHLH) proteins, and conserved WD40 repeat (WDR) proteins (Ramsay and Glover, 2005; Koes et al., 2005). The MYB, bHLH and WDR proteins physically interact to form the MBW complex that activates transcription, and which has best been described using genetic tools in Arabidopsis. The MBW complex interacts directly with promoter DNA via multiple cis-elements. Known target sequences include the MRE (Myb-response element) and AC-elements for R2R3-MYB proteins, and the E-box or bHLH-binding motif which is bound by the bHLH proteins (Feller et al., 2011; Lai et al., 2013, Xu et al., 2014). In contrast, WDR proteins have not been shown to bind DNA, but are thought to function by interacting with MYB and bHLH proteins (Baudry et al., 2004). It is the specific combination of interacting R2R3-MYB, bHLH and WDR factors within the complex that determines which target genes are expressed in a given cell (Baudry et al., 2004; Broun, 2005; Koes et al., 2005; Ramsay and Glover 2005). The bHLH and WDR cofactors are adaptable and can be involved in multiple processes. For example, the bHLH factor TT8 is required for PA synthesis in Arabidopsis when it interacts with the PA-specific R2R3 MYB TT2, but is also involved in the synthesis of anthocyanins and seed coat mucilage when combined with other co-factors (Nesi et al., 2000). A second Arabidopsis bHLH protein, GL3, belongs to a distinct bHLH subgroup and has several non-overlapping functions in determining epidermal cell fate (Zhang et al., 2003. TT8 and GL3 are partially redundant, however, and can compensate for each other in knock-out mutants (Broun, 2005). In petunia, both subgroups of bHLH cofactors are represented by AN1 and JAF13, respectively (Koes et al., 2005). Both TT8 and GL3 have been shown to directly interact with the WDR protein TTG1 (Zhang et al., 2003). TTG1 has roles in the regulation of trichome and root hair formation in addition to its function in the synthesis of Arabidopsis seed coat PAs (Ramsay and Glover, 2005). By contrast, the MYBs are functionally divergent and appear to be the major determinants of whether a gene or pathway is expressed in a given cell type (Koes et al., 2005). In Arabidopsis, the R2R3-MYB factor WER functions exclusively in root hair patterning (Lee and Schiefelbein, www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved. Yoshida et al., p. 7 1999), while TT2 is specific to PA biosynthesis and PAP1 regulates only anthocyanin biosynthesis (Nesi et al., 2001; Teng et al., 2005). Nevertheless, these MYBs can all interact with the same bHLH protein such as TT8 to then activate distinct target promoters (Nesi et al., 2000; Gonzalez et al., 2008). In addition, a given MYB can interact with several different bHLHs, providing multiple functional combinations (Zimmermann et al., 2004). Homologs of Arabidopsis TT2 and PAP1 are found in many species of plants, where they have conserved roles as PA and anthocyanin regulators, respectively. TT2-type PA regulators have been characterized in fruit, including DkMYB2 from persimmon, VvMYBPA2 from grapevine, and FaMYB9 in strawberry (Akagi et al., 2010; Terrier et al., 2009; Schaart et al., 2013). MYBs from the TT2 group are also active in regulating PAs in vegetative tissues, such as LjTT2 from Japanese lotus, TaMYB14 from clover, and MYB134 from poplar (Yoshida et al., 2008; Hancock et al., 2012; Mellway et al., 2009). These MYB transcription factors act as part of the MBW complex that promotes flavonoid gene expression. For MYB134, we demonstrated direct binding to AC-like elements that are found in phenylpropanoid gene promoters (Mellway et al., 2009). A second type of PA MYB regulator was defined by the discovery of VvMYBPA1 from grapevine (Bogs et al., 2009). Orthologs of this MYB were subsequently described in other species (Akagi et al., 2009). These PA MYBs appear to act in parallel as well as in tandem with the TT2-type PA MYBs; in grapevine, both PA MYBs regulate flavonoid promoters directly, but VvMYPA2 overexpression also induces transcripts of VvMYBPA1 (Terrier et al., 2009). Most species examined have both types of PA MYBs; Arabidopsis is a notable exception and has only the TT2-type. A further subgroup of MYB factors are the PAP1-like anthocyanin regulators. In Arabidopsis, PAP1 regulates stress-induced anthocyanins (Teng et al., 2005), and PAP1 orthologs are responsible for coloration in a variety of fruit (Lin-Wang et al., 2010). The floral color regulators AN2 from petunia and ROSEA1/2 from snapdragon are also part of this MYB clade (Koes et al., 2005; Ramsay and Glover, 2005). In poplar, Wilkins et al. (2009) identified a group of PAP1-like MYBs that are highly expressed in pigmented tissues, suggesting conserved functions in anthocyanin synthesis, but none have been characterized to date. Control of gene expression by the MBW complex can be further modulated by the involvement of repressor MYB proteins. The first such negative MYB regulators to be characterized include Antirrhinum MYB308 and MYB330, which negatively affect phenolic acid and lignin biosynthesis (Tamagnone et al., 1998), and Arabidopsis MYB4 and MYB32, which regulate sinapate esters and lignin biosynthesis (Jin et al., 2000; Preston et al., 2004). These MYB repressors defined subgroup 4 within the MYB gene family, which to date includes only negative regulators. Recently, Arabidopsis MYB7, the downstream target of MYB4, was identified as a repressor of flavonol biosynthesis and demonstrated to directly target DFR and UGT genes www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved. Yoshida et al., p. 8 (Fornalé et al., 2014). Chrysanthemum MYB1, the repressor of lignin biosynthesis, also downregulates the flavonoid pathway (Zhu et al., 2013). Repressor MYBs of the anthocyanin pathway have also been identified. These include strawberry FaMYB1 and FcMYB1, and petunia PhMYB27 (Aharoni et al., 2001; Salvatierra et al., 2013; Albert et al, 2014). Overexpression of FaMYB1 causes the suppression of anthocyanin synthesis in tobacco, and suppression of FcMYB1 by transient RNAi in strawberry fruit increased the accumulation of anthocyanin. Likewise, RNAi-suppression of PhMYB27 increases the accumulation of anthocyanin in Petunia flowers and vegetative tissues (Albert et al., 2014). In phylogenies, these flavonoid R2R3 repressor MYBs all cluster together within MYB subgroup 4 (Stracke et al., 2001), and all share the C2-motif [pfLNLD/ELxiG/S] with a core consensus sequence of LxLxL or DLNxxP (Dubos et al., 2010; Aharoni et al., 2001). These consensus sequences are the signature patterns of the EAR (ERF-associated amphiphilic repression) motif, the predominant form of transcriptional repressor motif identified in plants (Kagale and Rozwadowski, 2011). Elegant work with PhMYB27 has begun to illuminate the mechanism of action of the subgroup 4 R2R3 repressor MYBs. In yeast two-hybrid assays, PhMYB27 interacts directly with bHLHs of both the GL3 and TT8 subgroups, though with different affinities (Albert et al., 2014). Furthermore, deletion of the DLNxxP-type EAR motif reduced PhMYB27 repressor activity; the function of this motif was not defined here but may involve chromatin remodeling (Kagale and Rozwadowski, 2011). Recently, the first suppressor of the PA pathway, VvMYBC2-L1, was identified in grapevine as a new locus co-located with eQTLs for PA-related genes (Huang et al., 2014). This subgroup 4 repressor MYB downregulates PAs and flavonoid biosynthetic genes when overexpressed in hairy roots. Little is known about its mechanism of action, but it contains a partial LxLxL-type EAR motif. In addition to subgroup 4 R2R3 MYB repressors, the Arabidopsis single repeat R3 MYB repressors CPC and ETC1 were shown to be involved in the downregulation of anthocyanin biosynthesis (Zhu et al., 2009, Nemie-Feyissa et al., 2014). In petunia, the R3 MYB factor MYBx acts as negative regulator of anthocyanin accumulation in parallel to PhMYB27 (Albert et al., 2014). These R3 MYB proteins do not contain the repressor motif, but act as passive repressors by binding bHLH proteins required for formation of MBW complexes (Albert et al., 2014). A distinct type of repressor from Arabidopsis, AtMYBL2, also contains only a single repeat, but is otherwise more closely related to the R2R3 MYBs than other characterized R3 MYBs (Dubos et al., 2008; Matsui et al., 2008). This protein has a unique C-terminal TLLLFR motif that is required for repressor activity, and so the mechanism appears to be distinct from the small R3 MYB repressors. In general, the flavonoid MYB repressors are associated with specific flavonoids, but may www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved. Yoshida et al., p. 9 also exert effects on related pathways. For example, FaMYB1 suppresses anthocyanin biosynthetic genes when overexpressed in tobacco, including ANS and GT (Aharoni et al., 2001), and MYBL2 targets CHS, CHI, F3H, DFR, ANS, and TT8 (Dubos et al., 2008; Matsui et al., 2008). However, when overexpressed in Lotus corniculatus, FaMYB1 also reduced PA biosynthesis (Paolocci et al., 2011), and AtMYBL2 suppresses ANR expression in the seed coat (Dubos et al., 2008). Likewise, the VvMYBC2-L1-overexpressing grapevine hairy roots have a reduction in stilbene content in addition to reduced PAs and PA precursors (Huang et al., 2014). Therefore, the determinants of target specificity and details about the mechanism of repression still remain to be elucidated, especially in perennial plants that accumulate significant concentrations of PAs. The genus Populus consists of widespread trees of the northern hemisphere that are commonly known as poplars, aspens and cottonwoods. Populus typically contain substantial amounts of phenolic metabolites, including hydroxycinnamate esters, salicinoids, and PAs, which in P. tremuloides can accumulate to 25% DW of leaves (Donaldson et al., 2006). Furthermore, in Populus PA biosynthesis and accumulation can be rapidly induced by stresses including wounding, herbivore damage, pathogen attack, nitrogen deficiency, and UV light (Osier and Lindroth, 2004, Miranda et al., 2007; Mellway et al., 2009), suggesting a complex system of regulators. Stress induction of PAs in poplar involves the upregulation of flavonoid biosynthesis genes. This response is mediated by MYB134, a TT2 type R2R3 MYB transcription factor (Mellway et al., 2009). In the poplar genome, 192 genes encode R2R3 MYB transcription factors (Wilkins et al., 2009), but very few poplar MYBs with roles in flavonoid regulation are functionally characterized. MYB134 is an activator of PA biosynthesis and stimulates the poplar ANR1 promoter in transient expression assays in Arabidopsis (Zifkin et al., 2012; Gesell et al., 2014). Transgenic poplar plants overexpressing MYB134 accumulate up to 50 times more PAs in their leaves, yet they have normal anthocyanin levels and only slightly elevated flavonol contents, suggesting MYB134 regulates PAs specifically (Mellway et al., 2009). Concurrently, microarray analysis revealed that all known early and late structural genes for PA biosynthesis were upregulated in these plants (Mellway, 2009). In addition, several MYB genes predicted to encode both positive and negative regulators were expressed at elevated levels in the MYB134 overexpressors. One such regulator is MYB115, which belongs to the MYBPA1 subgroup of R2R3 MYB activators (Terrier et al., 2009). We also identified several genes encoding R2R3 repressor MYBs of subgroup 4, and one single repeat R3 MYB. Here, we characterize the MYB182 R2R3 MYB repressor-like gene in detail. We demonstrate that, when MYB182 is overexpressed in poplar hairy roots and transgenic poplar plants, PA accumulation is reduced, as www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved. Yoshida et al., p. 10 is expression of flavonoid biosynthetic genes. Using transient expression assays, we further show that MYB182 represses gene expression induced by the activator MYBs, and that this repression requires interaction with a bHLH co-factor. In addition, MYB182 may repress other regulatory genes, as well as other enzyme-encoding genes important for phenolic metabolism in poplar. www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved. Yoshida et al., p. 11