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TCP proteins belong to the plant-specific bHLH transcription factor family, and function as key regulators of diverse developmental processes. Functional redundancy amongst family members and post-transcriptional down-regulation by miRJAW of several TCP genes complicate their functional characterization. Here, we explore the role of TCP3 by analyzing transgenic plants expressing miRJAW-resistant mTCP3 and dominant-negative TCP3SRDX. Seedlings and seeds of mTCP3 plants were found to hyper-accumulate flavonols, anthocyanins and proanthocyanidins, whereas levels of proanthocyanidins were slightly reduced in TCP3SRDX plants. R2R3-MYB proteins control not only early flavonoid biosynthetic steps but also activate late flavonoid biosynthetic genes by forming ternary R2R3-MYB/bHLH/WD40 (MBW) complexes. TCP3 interacted in yeast with R2R3-MYB proteins, which was further confirmed in planta using BiFC experiments. Yeast three-hybrid assays revealed that TCP3 significantly strengthened the transcriptional activation capacity of R2R3-MYBs bound by the bHLH protein TT8. Transcriptome analysis of mTCP3 and TCP3SRDX plants supported a role for TCP3 in enhancing flavonoid biosynthesis. Moreover, several auxin-related developmental abnormalities were observed in mTCP3 plants. Transcriptome data coupled with studies of an auxin response reporter and auxin efflux carriers showed that TCP3 negatively modulates the auxin response, probably by compromising auxin transport capacity. Genetic experiments revealed that the chalcone synthase mutant tt4-11 lacking flavonoid biosynthesis abrogated the auxin-related defects caused by mTCP3. Together, these data suggest that TCP3 interactions with R2R3-MYBs lead to enhanced flavonoid production, which further negatively modulates the auxin response.
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The plant-specific TCP family of transcription factors is named after the first three identified members: TEOSINTE BRANCHED1 in maize (Zea mays), CYCLOIDEA (CYC) in Antirrhinum majus, and PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR in rice (Oryza sativa). Repeated gene duplications during land plant evolution have enlarged this family of bHLH-containing DNA-binding proteins (Martín-Trillo and Cubas, 2010). The Arabidopsis thaliana genome encodes 24 TCP transcription factors. Based on the structure of their conserved TCP domains, 13 TCP proteins have been identified as members of class I (TCP-P) and have been suggested to function as positive regulators of cell proliferation (Kosugi and Ohashi, 2002). The other 11 TCP proteins belong to class II (TCP-C). CYC controls floral symmetry formation in Antirrhinum (Luo et al., 1996), and its Arabidopsis ortholog TCP1 participates in brassinosteroid biosynthesis and modulates longitudinal elongation of leaves and stems (Guo et al., 2010; Koyama et al., 2010b). The products of two class II/TCP-C genes, TCP12 (BRANCHED2) and TCP18 (BRANCHED1), operate as integrators of branching signals within axillary buds (Aguilar-Martinez et al., 2007), and TCP12 has also been shown to repress the floral transition of axillary meristems (Niwa et al., 2013). Generation of miRJAW-resistant versions and dominant-negative forms of TCPs have revealed functionally redundant roles for miRJAW-targeted CINCINNATA (CIN)-TCP genes (TCP2-4, TCP10 and TCP24) in the control of leaf morphogenesis and senescence by modulating jasmonate biosynthesis (Palatnik et al., 2003; Schommer et al., 2008). Class II CIN-TCPs have also been shown to regulate the morphogenesis of shoot lateral organs as well as correct petal and stamen development (Palatnik et al., 2003; Koyama et al., 2007, 2010a; Nag et al., 2009) and defense responses (Sugio et al., 2011).
Arabidopsis plants synthesize three classes of flavonoids, including colorless to pale yellow flavonols, red to purple anthocyanins, and colorless proanthocyanidins, which become brown upon oxidative reactions. Flavonoid biosynthesis is largely regulated at the transcriptional level, and starts with the general phenylpropanoid metabolism involving phenylalanine ammonia lyase, cinnamate 4-hydroxylase and 4-coumarate:CoA ligase (4CL). Molecular dissection of flavonoid metabolism reveals that chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H) and flavanone 3′-hydroxylase are encoded by TRANSPARENT TESTA4 (TT4), TT5, TT6 and TT7, respectively. Sequential reactions catalyzed by these enzymes produce dihydroflavonols, the last common intermediates in the biosynthesis of flavonoid end-products (Figure S1). Dihydroflavonols are then oxidized to flavonols by flavonol synthase (FLS), such as quercetins and kaempferols. These early steps of the flavonoid biosynthetic pathway are controlled by the R2R3-MYB proteins MYB11, MYB12 and MYB111, which activate the early biosynthetic genes CHS, CHI, F3H and FLS1 (Mehrtens et al., 2005; Stracke et al., 2007) (Figure S1). Dihydroflavonol 4-reductase (DFR), encoded by the late biosynthetic gene TT3, reduces dihydroflavonols to leucoanthocyanidins, and downstream enzymes participate in the production of anthocyanins and proanthocyanidins (Nesi et al., 2002; Appelhagen et al., 2011) (Figure S1). Three classes of regulatory proteins, including R2R3-MYBs, bHLHs and TRANSPARENT TESTA GLABROUS1 (TTG1/WD40), form the ternary transcriptional complex MYB-bHLH-WD40 (MBW), which activates several late flavonoid biosynthetic genes (Nesi et al., 2002; Appelhagen et al., 2011). Anthocyanin biosynthesis is regulated by an MBW complex consisting of TTG1, one R2R3-MYB protein (PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1), PAP2, MYB113 or MYB114), as well as one of the bHLH proteins TT8, GLABROUS3 (GL3) or ENHANCER OF GLABRA3 (EGL3) (Gou et al., 2011). Seed-specific activation of proanthocyanidin production requires activity of an MBW complex comprising the R2R3-MYB protein TT2, TT8 and TTG1 (Nesi et al., 2002; Ramsay and Glover, 2005). Several transcription factors, such as the miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN9 (SPL9), the WIP-type zinc finger protein TT1 and the R3-MYB protein MYBL2, have been shown to interact with R2R3-MYBs or bHLHs to regulate flavonoid biosynthesis, possibly by affecting the stability of the ternary MBW complex (Dubos et al., 2008; Appelhagen et al., 2011; Gou et al., 2011).
Endogenous flavonols are considered to be natural regulators of cellular auxin efflux and polar auxin transport (Murphy et al., 2000; Peer et al., 2004; Lazar and Goodman, 2006; Santelia et al., 2008; Petrasek and Friml, 2009). Transcriptome analysis of young seedlings expressing the inducible dominant-negative version of TCP3 (TCP3SRDX) revealed an altered auxin response during Arabidopsis leaf differentiation, such as up-regulation of PIN-FORMED1 (PIN1), PIN5 and PIN6 as well as down-regulation of PIN3, PIN4 and PIN7 (Koyama et al., 2010a). In this work, transgenic plants expressing miRJAW-resistant mTCP3 and dominant-negative TCP3SRDX were characterized throughout the plant life cycle, and newly observed phenotypes were further analyzed. Our results indicate that TCP3 stimulates flavonoid biosynthesis and negatively modulates the auxin response, thereby causing a plethora of auxin-related developmental defects.
Phenotypic analysis of transgenic plants expressing mTCP3 and TCP3SRDX
Arabidopsis thaliana plants over-expressing TCP3 (p35S::TCP3) as well as knockout mutants of TCP3 show no visible phenotypic alterations as a consequence of miRJAW-guided cleavage of TCP mRNAs and the functional redundancy of TCP transcription factors (Table S1) (Koyama et al., 2007). To reveal the developmental role of TCP3, the miRJAW-resistant version mTCP3 and the dominant-negative repressor TCP3SRDX were engineered by synonymous substitutions in the miRJAW target site and fusion of the EAR motif repression domain to the TCP3 C-terminus, respectively (Hiratsu et al. 2003; Palatnik et al., 2003). In agreement with previous reports (Koyama et al., 2007), 24.2% of the p35S::mTCP3 plants formed fused cotyledons, leading to early death within 4 days after germination (Table S1), and 95.7% of the p35S::TCP3SRDX plants developed wavy leaves and irregular cotyledon vasculature (Table S2). Additionally, the majority of the surviving transgenic plants (Tables S1 and S2) showed phenotypes that were not observed previously, supporting further functions for TCP3 during plant development. Wild-type and p35S::TCP3SRDX transgenic plants formed a spiral phyllotaxy, with an angle of 137.5° between the leaf initials (Figure 1a,c). In contrast, 65.0% of the p35S::mTCP3 plants showed decussate phyllotaxy, whereby each pair of leaves formed a 90° angle with the previous pair (Figure 1b). In contrast to the regular vascular patterns observed in wild-type cotyledons (Figure 1d), p35S::mTCP3 and p35S::TCP3SRDX transgenic cotyledons developed irregular vascular xylem strands (Figure 1e,f). Vascular breaks were also observed for the first true leaf expressing p35S::mTCP3 (Figure S2e). p35S::mTCP3 plants showed reduced primary root growth and formed fewer lateral roots (Figure 1h). By contrast, p35S::TCP3SRDX plants (Figure 1i) developed more lateral roots than wild-type plants (Figure 1g). Furthermore, transgenic plants expressing p35S::mTCP3 displayed a bushy stature with reduced apical dominance, and developed a mean of 29 shoots of similar length at maturity (Figure 1m), in contrast to seven and nine shoots for wild-type and p35S::TCP3SRDX-expressing plants, respectively (Figure 1m; 25 plants analyzed). Compared to the brown wild-type seeds (Figure 1j), plants harboring p35S::mTCP3 developed smaller and darker brown seeds (Figure 1k). In contrast, transgenic plants expressing p35S::TCP3SRDX formed pale brown seeds (Figure 1l) that were similar in size to wild-type seeds (Figure 1j). Additionally, p35S::mTCP3 plants produced smaller dark-green leaves without an obvious distinction between the leaf petiole and the leaf lamina (Figure S2b), smaller petals (Figure S2j), shorter inflorescences with reduced internode lengths, and shorter siliques with a defect in floral organ abscission (Figure S2 m,q), as well as thinner green stems and thinner roots (Figure S2r,u). In addition to developing bigger leaves and petals with wavy margins as well as crinkled siliques (Koyama et al., 2007) (Figure S2c,l,p), p35S::TCP3SRDX-expressing plants also formed irregular bulges on the adaxial side of leaves (Figure S2c).
The miRJAW-resistant version of TCP3SRDX was also generated, and expression of the p35S::mTCP3SRDX construct in wild-type plants produced the same phenotypes as observed in p35S::TCP3SRDX plants (Table S2). To exclude exogenous effects caused by ubiquitous expression driven by the CaMV 35S promoter, both miRJAW-resistant and -sensitive versions of TCP3 and TCP3SRDX were also expressed in the context of the TCP3 locus (pTCP3::TCP3, pTCP3::mTCP3, pTCP3::TCP3SRDX and pTCP3::mTCP3SRDX). Phenotypic investigation of these transgenic populations revealed similar activities of the CaMV 35S promoter and TCP3 regulatory sequences (Tables S1 and S2), as supported by the observation that the TCP3 promoter drives a ubiquitous GUS expression (Figure S3). Further analysis was therefore focused on p35S::mTCP3 and p35S::TCP3SRDX plants.
Analysis of young mTCP3 seedlings confirmed the fused cotelydon phenotype described by Koyama et al. (2007), and further observation of surviving transgenic plants revealed diverse phenotypes that were not reported previously, such as altered leaf phyllotaxy, defects in vascular patterning, reduced apical dominance, bushy architecture, and impaired root growth and development, as well as reduced organ size, implicating a possible role for TCP3 in auxin biosynthesis or signaling.
Flavonoid accumulation in mTCP3 and TCP3SRDX transgenic plants
In p35S::mTCP3 seedlings, hyper-accumulation of purple pigments was observed at the junction between the elongated hypocotyl and cotyledons (Figures 1h and 2b), whereas the corresponding junctions of wild-type and p35S::TCP3SRDX plants were green (Figures 1g,i and 2a,c). Anthocyanin contents were not significantly affected in p35S::TCP3SRDX seedlings but were ninefold enhanced in p35S::mTCP3 seedlings compared to wild-type (Figure 2d). The flavonol accumulation in the cotyledonary node, the hypocotyl–root transition zone and the root tip of wild-type and transgenic seedlings was analyzed using the flavonol-specific dye diphenylboric acid-2-aminoethyl ester (DPBA). Significantly increased flavonol levels in these three regions were observed in p35S::mTCP3 plants (Figure 2d,f,i,l). By contrast, the flavonol level of p35S::TCP3SRDX plants (Figure 2d,g,j,m) was similar to that of wild-type seedlings (Figure 2d,e,h,k). Proanthocyanidins belong to the third group of flavonoids and are located specifically in the innermost cell layer of the seed coat, the endothelium (Devic et al., 1999). Uncolored proanthocyanidins and their precursors in the endothelium of immature wild-type seeds were stained dark red by vanillin staining (Figure 2n). Expression of mTCP3 caused not only increased accumulation of proanthocyanidins in the endothelium, but also ectopic deposition in the outer layers of the seed coat (Figure 2o), which accounts for the dark brown color of mature transgenic seeds (Figure 1k). Conversely, TCP3SRDX seeds exhibited reduced levels of these products in the endothelium (Figure 2p), consistent with the attenuated brown coloration observed in mature seeds (Figure 1l). Collectively, hyper-accumulation of anthocyanins, proanthocyanidins and flavonols in mTCP3 plants, as well as reduced levels of proanthocyanidins in TCP3SRDX plants, indicate that TCP3 probably participates in regulation of the flavonoid biosynthetic pathway.
Auxin response of mTCP3 and TCP3SRDX transgenic plants
The observed auxin-related developmental defects of mTCP3 plants prompted us to compare the expression pattern of the auxin response reporter pDR5::GUS in wild-type, mTCP3 and TCP3SRDX plants. The pDR5::GUS reporter was mainly expressed at the apices and vascular tissues of wild-type leaves (Figure 3a,e) (Mattsson et al., 1999). Relative to wild-type plants (Figure 3a,d), expression of mTCP3 induced a theeefold reduction in the pDR5::GUS reporter activity (Figure 3b,d). Transgenic plants expressing TCP3SRDX showed no significant changes in reporter activity (Figure 3c,d). To investigate whether addition of exogenous auxin is able to rescue the reduced auxin response observed in mTCP3 plants, wild-type, mTCP3 and TCP3SRDX plantlets expressing pDR5::GUS were exposed for 15 h prior to GUS staining to 1-naphthaleneacetic acid (NAA), which is more long-lived than indole-3-acetic acid (IAA). Incubation of mTCP3 seedlings in the presence of NAA did not result in increased activity of the pDR5::GUS reporter (Figure 3d,g,h), in contrast with the significantly elevated expression of this reporter in wild-type (Figure 3d,e,f) and TCP3SRDX plants (Figure 3d,i,j). These results suggest that expression of mTCP3 reduces auxin transport capacity and/or sensitivity.
Membrane-localized PIN proteins are known to mediate polar auxin transport, and visualization of these proteins fused to GFP allows detailed characterization of their distribution and abundance (Teale et al., 2006). To further define the effect of expressing mTCP3 on auxin transport, functional pPIN1::PIN1-GFP and pPIN2::PIN2-GFP constructs were introduced into mTCP3 plants. In comparison to wild-type roots (Figure S4a), PIN1 showed an overall decreased abundance but an unaltered localization pattern in mTCP3 roots (Figure S4b). By contrast, the PIN2 localization and abundance in mTCP3 roots (Figure S4d) was similar to that observed in wild-type roots (Figure S4c). These observations are consistent with a previous report showing that PIN1 localization and abundance were flavonol-sensitive but PIN2 localization and abundance were not (Peer et al., 2004). The reduced abundance of PIN1 therefore probably accounts for reduced auxin transport capacity observed in mTCP3 plants.
Identification of differentially expressed genes in mTCP3 and TCP3SRDX plants
Microarray experiments were performed using ATH1 GeneChips, and transcriptome comparison between wild-type, mTCP3 and TCP3SRDX seedlings revealed a large number of de-regulated genes that were grouped into 11 functional categories (308 and 75 genes for mTCP3 plants and 126 and 208 genes for TCP3SRDX plants were up- and down-regulated, respectively; Figure S5a–d). Expression of mTCP3 affected transcription of genes in the categories ‘flavonoid biosynthesis’ (3% up-regulated; Figure S5a) and ‘auxin signaling’ (9% down-regulated; Figure S5b). In contrast, among the up-regulated genes in TCP3SRDX plants, no flavonoid biosynthesis-related genes were identified, but 2% belong to the category ‘auxin signaling’ (Figure S5c). Among the down-regulated genes in TCP3SRDX plants, no auxin signaling-related genes were detected, but 2% of the genes fall into the category ‘flavonoid biosynthesis’ (Figure S5d). To explore potential TCP3 target genes that contribute to observed alterations of flavonoid accumulation and auxin response, further analysis focused on candidate genes that were associated with flavonoid metabolism and auxin signaling. Nineteen genes showing a more than twofold expression change (P <0.05) between wild-type and mTCP3 seedlings were identified (Table 1). Ten genes involved in the flavonoid biosynthetic pathway were up-regulated 3.27- to 24.35-fold in mTCP3 plants (Table 1). These include the regulatory R2R3-MYB gene PAP1 and structural genes encoding key flavonoid biosynthetic enzymes, such as 4CL, CHS/TT4, F3H/TT6, DFR/TT3 and LDOX/TT18. The late flavonoid genes DFR/TT3 and LDOX/TT18 participate in anthocyanin biosynthesis and were found to be highly up-regulated (Table 1), consistent with the strong anthocyanin accumulation observed in mTCP3 seedlings. In TCP3SRDX seedlings, these genes were either unaffected or were affected in the opposite direction, showing a 1.24- to 4.90-fold down-regulation (Table 1). A 2.07- to 8.62-fold down-regulation was observed for auxin signaling-related genes in mTCP3 plants. These genes encode proteins that participate in polar auxin transport (AUX1, ATP-binding cassette transporter 1/PGP1 and ATP-binding cassette transporter 19/PGP19) or encode the indole-3-acetic acid amido synthase (GH3.17), the auxin receptor (TIR1) or the auxin-inducible transcription factor (AUX/IAA29) (Table 1). Genes that negatively affect the auxin response, such as CYP83B1/SUR2, which encodes a negative regulator of auxin production (Bak et al., 2001), and the auxin-responsive gene SAUR (At2 g46690), which possibly encodes a negative regulator of auxin synthesis and transport (Kant et al., 2009), were up-regulated 3.27- and 2.05-fold, respectively (Table 1). Enhanced expression was observed for auxin-related genes in TCP3SRDX plants, such as AUX/IAA29 (3.24-fold) and PAR1 (3.14-fold), that encodes a transcriptional repressor of the indole-3-acetic acid-responsive genes and SAUR68. The transcriptome data thus imply that TCP3 regulates flavonoid biosynthesis positively and modulates the auxin response negatively.
Table 1. De-regulated genes involved in the flavonoid biosynthetic pathway and auxin signaling in mTCP3 and TCP3SRDX plants
De-regulated genes of both pathways with at least a twofold change (P <0.05) in expression levels are shown.
+ and – indicate increases or decreases, respectively.
Anthocyanin 5-aromatic acyltransferase 1
MYB transcription factor 75
4-Coumarate:CoA ligase 1
Auxin-responsive SAUR protein
Transcriptional repressor of SAUR genes
ATP-binding cassette transporter 19
Indole-3-acetic acid amido synthetase
ATP-binding cassette transporter 1
Auxin-inducible transcription factor
Auxin influx transporter
The consensus sequence GT/CGGNCCC has been determined to be a DNA binding site for class II TCP proteins, with no more than two mismatches allowed for successful recognition (Kosugi and Ohashi, 2002; Schommer et al., 2008). The distribution of this consensus site was examined in the regions extending 1000 bp upstream of the respective genes as well as in their introns. None of the 19 de-regulated genes was found to possess such a binding motif with fewer than three mismatches. Therefore, TCP3 proteins may regulate transcription of these genes by participation in a transcription factor complex in which TCP3 itself does not bind to a class II consensus recognition site.
Identification of proteins interacting with TCP3
Expression of the early flavonoid biosynthetic genes is controlled by the three closely related R2R3-MYBs: MYB11, MYB12 and MYB111 (Stracke et al., 2007). For transcriptional activation of the late flavonoid biosynthetic genes, the R2R3-MYB–bHLH–WD40 (MBW) complex is required (Dubos et al., 2010). Some transcription factors, such as miR156-targeted SPL9, the WIP-type zinc finger protein TT1 and the R3-MYB protein MYBL2, have been shown to participate in the regulation of flavonoid biosynthesis by associating with R2R3-MYBs or bHLHs to alter the stability of this ternary complex (Dubos et al., 2008; Appelhagen et al., 2011; Gou et al., 2011). Based on these reports, we considered that TCP3 may also interact with these regulatory proteins to participate in control of the flavonoid biosynthetic pathway. Yeast two-hybrid assays were performed using TCP3 as bait fused to the GAL4 DNA-binding domain (BD), and key regulators of early flavonoid biosynthetic genes (MYB12 and MYB111) and late flavonoid biosynthetic genes (TT2, PAP1, PAP2, MYB113, MYB114, TT8, TTG1, TT1 and MYBL2) as well as the trichome initiation regulator GLABROUS1 (GL1) as prey fused to the GAL4 activation domain (AD) (Figure 4a). Except for GL1, strong interactions of TCP3 with all other analyzed R2R3-MYBs (MYB12, MYB111, TT2, PAP1, PAP2, MYB113 and MYB114) were observed. However, TCP3 did not interact directly with the bHLH protein TT8, the WD40 domain protein TTG1 or GL1 (Figure 4a). TCP3 was also found to form a heterodimer with the smaller R3-MYB protein MYBL2 but not with TT1 (Figure 4a).
To further investigate TCP3 interactions with key regulatory proteins of the flavonoid pathway in planta, bimolecular fluorescence complementation (BiFC) assays were performed. The N-terminus of YFP (YN) was cloned upstream of TCP3, and the C-terminus of YFP (YC) was N-terminally fused to TT8, TTG1 and each of the six tested R2R3-MYBs (TT2, PAP1, PAP2, MYB113, MYB114 and GL1). Reconstitution of nuclear YFP fluorescence was detected for co-expression of TCP3 with TT2, PAP1, PAP2, MYB113 and MYB114, but not with GL1, TT8 and TTG1 (Figure 4c–g and Figure S6), demonstrating that TCP3 binds to flavonoid R2R3-MYBs in the nucleus in planta and may thus affect transcriptional processes. As negative controls, co-expression of non-fused YN with non-fused YC or one of the fusion proteins with non-fused YN or non-fused YC failed to reconstitute a fluorescent YFP chromophore (Figure 4b).
Analysis of ternary complex formation among TCP3, R2R3-MYBs and TT8
TTG1 provides a scaffold that allows TT8 binding to R2R3-MYBs and thus transcriptional activation of target genes (Ramsay and Glover, 2005; Baudry et al., 2006; Feller et al., 2011). To examine whether TCP3 is able to strengthen TT8 interactions with R2R3-MYBs, yeast three-hybrid assays were performed to compare interaction strengths of the tested proteins by monitoring reporter expression using β-galactosidase assays. In addition to containing a site for integrating TT8 as a bait, the yeast three-hybrid vector pBRIDGE also possesses a second site for conditional expression of TCP3. Transcription of TCP3 was repressed in the presence of 1 mM methionine (TCP3–), whereas the absence of methionine activated TCP3 expression (TCP3+). For yeast three-hybrid assays, TT2, PAP1 and PAP2 were individually fused to the activation domain (AD-TT2, AD-PAP1 and AD-PAP2), and the pBRIDGE vector with only the TT8 integration was used as a control (BD-TT8). Consistent with previous reports (Zimmermann et al., 2004), protein interactions between each of the three R2R3-MYBs and TT8 were observed for each of the three control experiments lacking TCP3 expression, with interaction strengths varying from 54.45 to 69.23 Miller units (Figure 4h). However, co-expression of TCP3 with TT8 and each of the three tested R2R3-MYB genes led to significantly enhanced reporter activity (93.62–107.51 Miller units; Figure 4h), suggesting that TCP3 influences the stability of the MBW complex and thereby enhances its transcriptional activation activity.
Phenotypic analysis of tt4-11 mutants expressing mTCP3 and TCP3SRDX
tt4-11 null mutant plants fail to produce any class of flavonoids, as CHS, which is encoded by TT4, catalyzes the first step of the flavonoid pathway (Shirley et al., 1995; Buer et al., 2006). To test whether the observed morphological abnormalities in mTCP3 and TCP3SRDX plants were caused by altered flavonoid biosynthesis, mTCP3/tt4-11 and TCP3SRDX/tt4-11 plants were generated.
Both young and adult tt4-11 mutant plants (Figure 5a and Figure S7d,j) looked very similar to wild-type plants (Figure 2a and Figure S7a,g), except that adult tt4-11 plants exhibited slightly reduced plant height (Figure S7g versus Figure S7j), as also observed for the tt4-1 null mutant (Buer and Djordjevic, 2009). As expected, no flavonoid accumulation was observed for seedlings and seeds of tt4-11 mutants (Figure 5a,d), with the tt4-11 seeds being yellow in color (Figure 5d) and similar in size to wild-type seeds (Figure 1j versus Figure 5d). The tt4-11 cotyledons developed a vascular pattern (Figure 5g) similar to that seen in wild-type cotyledons (Figure 1d). mTCP3/tt4-11 plants resembled the tt4-11 mutant (Figure 5b,e,h and Figures S7e,k and S8e–h), and auxin-related defects, such as altered phyllotaxy, abnormal leaf vasculature and a bushy appearance caused by reduced apical dominance, were not observed. These results imply that TCP3 regulates the auxin response by positively modulating flavonoid biosynthesis. Expression of TCP3SRDX in the tt4-11 mutant (TCP3SRDX/tt4-11) showed additive effects, such as wavy leaf margins (Figure 5c and Figure S7f) and a lack of flavonoid accumulation (Figure 5c,f), suggesting that TCP3SRDX controls the development of crinkled organs independently of the flavonoid biosynthetic pathway.
TCP3 interacts with R2R3-MYB proteins and promotes flavonoid biosynthesis
TCP proteins participate in the control of a large variety of developmental processes, such as floral symmetry (Luo et al., 1996), plant architecture (Kiefer et al., 2011), morphogenesis of lateral organs (Nath et al., 2003; Koyama et al., 2007; Nag et al., 2009; Sarvepalli and Nath, 2011; Danisman et al., 2012), leaf senescence (Schommer et al., 2008; Sarvepalli and Nath, 2011), seed germination (Tatematsu et al., 2008), pollen development (Takeda et al., 2006), floral transition in axillary meristems (Niwa et al., 2013), the circadian clock (Pruneda-Paz et al., 2009; Giraud et al., 2010) and defense responses (Sugio et al., 2011). Furthermore, TCPs are also involved in the biosynthesis of plant hormones, such as brassinosteroids and jasmonic acid (Schommer et al., 2008; Sugio et al., 2011).
Here, a combination of microarray data, protein interaction experiments and genetic interaction analysis together with determination of altered flavonoid levels in transgenic plants indicates a regulatory function for TCP3 in the flavonoid biosynthetic pathway. The production of flavonols, anthocyanins and proanthocyanidins shares the last common precursor dihydroflavonol, which is further oxidized by FLS to generate flavonols or reduced by DFR to ultimately synthesize anthocyanins and proanthocyanidins (Figure S1). Expression of miRJAW-resistant mTCP3 stimulated flavonoid biosynthesis, leading to hyper-accumulation of all three end-products. Proanthocyanidin hyper-production was observed not only in the endothelium of mTCP3 seeds but also occurred in the other four layers of the seed coat. Similar deposition profiles of proanthocyanidins were also found in transgenic plants over-expressing ARABIDOPSIS BSISTER (ABS)/TT16, which encodes the ARABIDOPSIS BSISTER MADS domain protein and is necessary for BANYULS expression and proanthocyanidin accumulation in the endothelium of the seed coat. with the exception of the chalazal/micropylar area (Nesi et al., 2002). Ecotopic accumulation of proanthocyanidins in mTCP3 seed coats implies that expression of mTCP3 induces the biosynthesis and accumulation of proanthocyanidins in the whole testa. In agreement with enhanced flavonoid production in mTCP3 plants, microarray data showed that expression of mTCP3 activated many enzymatic and regulatory genes involved in the early (4CL, CHS/TT4 and F3H/TT6) and late steps (DFR/TT3, LDOX/TT18, PAP1) of the flavonoid biosynthetic pathway. Consistently, transcriptome analysis detected down-regulation of these genes in TCP3SRDX plants, albeit to a lower extent compared with their up-regulation in mTCP3 plants, which may account for slightly decreased flavonoid levels in TCP3SRDX plants. Collectively, flavonoid phenotypes and transcriptome data from combinatorial analysis of mTCP3 and TCP3SRDX plants strongly indicate a regulatory role for TCP3 in flavonoid biosynthesis.
R2R3-MYB proteins activate early flavonoid biosynthetic genes and also participate in formation of the MBW complex to control expression of late flavonoid biosynthetic genes (Dubos et al., 2010). Several transcription factors have been suggested to interfere with the formation or integrity of the MBW complex by interacting with R2R3-MYBs or bHLHs (Dubos et al., 2008; Appelhagen et al., 2011; Gou et al., 2011). Yeast two-hybrid assays combined with BiFC experiments revealed nuclear interactions of TCP3 with the flavonoid R2R3-MYBs TT2, PAP1, PAP2, MYB113, MYB114, MYB12 and MYB111. The three closely related R2R3-MYBs MYB11, MYB12 and MYB111 act redundantly to activate expression of CHS/TT4, CHI/TT5, F3H/TT6 and FLS1 and thus regulate early steps of the flavonoid pathway (Stracke et al., 2007). Heterodimerization of TCP3 with MYB12 or MYB111 as detected here may contribute to the up- and down-regulation of the early biosynthetic genes CHS/TT4 and F3H/TT6 observed in mTCP3 and TCP3SRDX plants, respectively. Interactions of TCP3 with the anthocyanin-specific regulators PAP1, PAP2, MYB113 and MYB114, as well as with the proanthocyanidin-specific regulator TT2, imply a role for TCP3 in regulation of the late biosynthetic genes. The two functionally equivalent R2R3-MYBs GL1 and WEREWOLF (WER) form an MBW complex with GL3/ENHANCER OF GLABRA3 (EGL3) and TTG1 to control trichome initiation and specify the identity of root-hairless cells, respectively (Ishida et al., 2007). Our inability to detect interaction between TCP3 and GL1 is in agreement with the lack of trichome phenotypes in transgenic plants, demonstrating the specificity of TCP3 interactions with flavonoid R2R3-MYBs. No direct interactions between TCP3 with TT8 or TTG1 were detected. Furthermore, yeast three-hybrid experiments demonstrated that TCP3 strengthened TT8 interactions with TT2, PAP1 and PAP2. Given this observation, TCP3 may act in a manner similar to the WIP-type zinc finger protein TT1 to stabilize formation of the ternary MBW complex and thereby promote flavonoid production. TT1 interacts with TT2 and PAP1 but not with MYB12, TT8 or TTG1, and activates not only late steps of the flavonoid pathway in the seed coat endothelium but also early steps leading to heightened CHS expression (Appelhagen et al., 2011). In addition, over-expression of PAP genes stimulates expression of early and late structural genes of the flavonoid pathway (Borevitz et al., 2000; Tohge et al., 2005). However, expression studies using GL3:glucocorticoid receptor (GR) and TTG1:GR fusions revealed direct regulation of only the late flavonoid biosynthetic genes by the MBW complex (Gonzalez et al., 2008). Together, activation of early flavonoid biosynthetic genes by TT1 and PAPs implies indirect involvement of anthocyanin- and proanthocyanidin-specific MBW complexes in the control of early steps of the flavonoid pathway. The positive feedback regulation of TT8 expression by the MBW complex adds another layer of complexity to the regulatory mechanism of flavonoid biosynthesis (Baudry et al., 2006). Microarray data revealed a 13.08-fold up-regulation of PAP1 expression in mTCP3 plants, suggesting that TCP3 may regulate PAP1 expression by associating with R2R3-MYB proteins.
The miR156-targeted SPL9 and R3-MYB protein MYBL2 function as negative regulators of flavonoid biosynthesis, and have been shown to interact with R2R3-MYBs and bHLHs, respectively (Dubos et al., 2008; Gou et al., 2011). Molecular interactions between MYBL2 and bHLHs as well as between SPL9 and R2R3-MYBs may competitively disrupt the R2R3-MYB/bHLH protein interaction and thus counteract the regulatory activity of the MBW complex (Dubos et al., 2008; Matsui et al., 2008; Gou et al., 2011). TCP3 was also found to form a heterodimer with MYBL2 in yeast cells. Therefore, it is also possible that TCP3 promotes formation of the MBW complex by interacting with MYBL2 and thus releasing bHLHs.
TCP3 compromises the auxin response by enhancing flavonol production
Auxin regulates diverse developmental processes by altering the expression of a large number of genes, including early auxin-responsive genes of the AUX/IAA, GH3 and SAUR gene families (Petrasek and Friml, 2009). Transgenic plants expressing mTCP3 showed altered leaf phyllotaxy, abnormal vasculature patterning, reduced apical dominance, and impaired root growth and development, as well as reduced organ size. These morphological abnormalities are reminiscent of auxin-deficient or -insensitive mutants (Leyser et al., 1993; Dharmasiri et al., 2005), suggesting that TCP3 interferes with auxin signaling. Analysis of the auxin-responsive reporter pDR5::GUS revealed that expression of mTCP3 significantly reduced the auxin response. This reduction and the phenotypic defects of mTCP3 plants were not rescued by applying exogenous auxin. Further analysis of auxin efflux carriers revealed a decreased abundance of plasma membrane-localized PIN1 in mTCP3 roots. Microarray analysis of mTCP3 seedlings identified many auxin-related genes with altered expression levels, including early auxin-responsive genes (GH3.17, AUX/IAA29 and SAUR), auxin transporter protein genes (PGP19, PGP1 and AUX1) and the auxin receptor gene TIR1. Collectively, these results indicate that expression of mTCP3 weakens the auxin response, probably by impairing the auxin transport capacity. Although no significant alterations were found for TCP3SRDX seedlings in terms of the auxin response as monitored by the pDR5::GUS reporter, transcriptome analysis did reveal enhanced expression of several auxin-related genes, including AUX/IAA29, PAR1 and PGP1. Recently, inducible expression of dominant-negative TCP3SRDX led to up-regulation of PIN-FORMED1 (PIN1), PIN5 and PIN6 as well as down-regulation of SAUR genes, PIN3, PIN4 and PIN7, and AUX/IAA genes (Koyama et al., 2010a). These de-regulated auxin-related genes were not identified in this study, possibly due to the different experimental conditions. Koyama et al. (2010a) generated microarray data from 10-day-old pXVE::TCP3SRDX seedlings exposed to 5 μM estradiol for 24 h in liquid medium, whereas 7-day-old soil-grown p35S::TCP3SRDX seedlings were used in this study. Furthermore, no visible morphological abnormalities were observed for induced pXVE::TCP3SRDX seedlings, while p35S::TCP3SRDX seedlings showed phenotypic alterations, such as undulated leaves, abnormal cotyledon vasculature and improved root growth.
Naturally occurring flavonols such as quercetins and kaempferols have been established as negative regulators of polar auxin efflux (Murphy et al., 2000; Peer et al., 2004; Lazar and Goodman, 2006). Changing flavonol levels by applying exogenous flavonols to activate or inactivate the flavonoid pathway has been shown to impair auxin transport capacity and thus affects many auxin-related developmental processes (Lazar and Goodman, 2006; Santelia et al., 2008; Buer and Djordjevic, 2009). Expression of mTCP3 in wild-type plants led to enhanced flavonol levels and auxin-related developmental defects. To unravel the regulatory hierarchy of TCP3 for these two biological processes, the null mutant tt4-11 (Shirley et al., 1995), which does not produce any type of flavonoids, was utilized. The absence of flavonoid production in mTCP3/tt4-11 plants abrogated the auxin-related abnormalities observed in mTCP3 plants. Given this observation, altered auxin response in mTCP3 plants was probably caused by enhanced flavonol biosynthesis. Together, these data imply that TCP3 promotes flavonol accumulation and thus affects the auxin response.
Plant materials and growth conditions
Arabidopsis thaliana mutants and wild-type plants were grown in soil or on half-strength Murashige & Skoog medium with sucrose. Tobacco (Nicotiana benthamiana) and Arabidopsis plants were grown in the greenhouse under controlled environmental conditions.
Construction of transgenic plants
All cDNAs were isolated from a seedling-derived cDNA library. miRJAW-resistant mTCP3 was engineered as described by Palatnik et al. (2003). The dominant-negative TCP3SRDX and miRJAW-resistant mTCP3SRDX were created by fusing the SRDX domain to the C-termini of TCP3 and mTCP3. TCP3, mTCP3 and TCP3SRDX were introduced into the pBAR35S vector (Li et al., 2009) for ectopic expression. A 7000 bp genomic fragment from the TCP3 locus comprising 3557 bp upstream of the start codon and 2290 bp downstream of the stop codon was cloned into the pBAR-A vector (Li et al., 2009) to express the three TCP3 versions in their genomic context. To generate transgenic plants, constructs were introduced into Agrobacterium tumefaciens strain GV3101 (pMP90RG). Arabidopsis plants harboring pDR5::GUS were crossed with mTCP3 and TCP3SRDX plants. Cloning primers used for generation of all constructs are listed in Table S3.
Yeast two- and three-hybrid assays
For yeast-two hybrid assays, TCP3 was fused to the GAL4 DNA-binding domain (GAL4-BD) in the vector pGBKT7 (Clontec, http://www.clontech.com/). Full-length cDNAs for TT8, TTG1, TT1, MYBL2 and all the tested R2R3-MYB genes were cloned to the GAL4 activation domain (GAL4-AD) in the vector pGADT7 (Clontech). After co-transformation using Y2HGold or mating using Y187 and Y2HGold (Clontech), yeast cells were plated on selection medium (SD/-Trp-His-Leu-Ade-aureobasidin, Clontech) and incubated at 30°C for 3 days. For ternary complex analysis, TT8 and TCP3 were cloned into the MCS I and MCS II sites of the yeast three-hybrid vector pBRIDGE (Clontech), respectively. One construct with TT8 integrated into the MCS I site was used as a control. Expression of TCP3 was repressed in the presence of 1 mM methionine and activated in the absence of methionine. TT2, PAP1 and PAP2 were integrated into the pGADT7 vector. After co-transformation, yeast cells were plated on selection medium (SD/-Trp-His-Leu-Ade-aureobasidin or SD/-Trp-His-Met-Leu-Ade-aureobasidin; Clontech) and incubated at 30°C for 3 days. Quantification of the β-galactosidase activity was performed as described by Li et al. (2011).
Gateway-compatible vectors (pE-SPYNE and pE-SPYCE; Walter et al., 2004) were used to generate expression vectors using a Gateway cloning strategy. The N-terminus of YFP (YN) was cloned upstream of TCP3 in the pE-SPYNE vector, and the C-terminus of YFP (YC) was fused upstream of TT2, PAP1, PAP2, MYB113, MYB114, GL1, TT8 and TTG1 in the pE-SPYCE vector. Confocal laser scanning microscopy was performed as described by Li et al. (2009).
GUS enzymatic activity, GUS staining and vascular patterning
GUS enzymatic activity and GUS staining were performed as described by Yin et al. (2007). Vascular patterning was characterized as described by Carland et al. (1999). Representative images were obtained using a Leica M165 FC fluorescent stereomicroscope (http://www.leica-camera.de/).
Staining of flavonols and proanthocyanidins
Six-day-old Arabidopsis seedlings were stained for 20 min using saturated (0.25% w/v) diphenylboric acid-2-aminoethyl ester (DPBA, http://www.roth-gmbh.de/) with 0.005% Triton X-100. Arabidopsis immature siliques were incubated in 1% w/v vanillin (4–hydroxy-3-methoxybenzaldehyde) in 6 m HCl for 40 min at 25°C. Representative images were obtained using a Leica DM5000B fluorescent microscope.
Quantification of anthocyanins and flavonols
Anthocyanins were quantified as described by Gou et al. (2011). Flavonol levels were measured as described by Lewis et al. (2011). Focal planes of maximal DPBA fluorescence intensity for the cotyledonary node, hypocotyl/root transition zone and root tip of each seedling were imaged at identical gain and laser intensity using the fluorescein isothiocyanate filter (excitation 490 nm and emission 525 nm; Leica DM5000B fluorescence microscope). Fluorescence intensity was recorded using ImageJ (http://www.rsbweb.nih.gov/ij/), and the sum of the mean fluorescence intensity for the three analyzed flavonol-accumulating regions represents the flavonol level for each seedling. Fluorescence data were normalized and represented as fold changes over wild-type plants.
Three biological samples of total RNA were prepared from 7-day-old wild-type and transgenic seedlings expressing mTCP3 and TCP3SRDX. Probe preparation, hybridization to Arabidopsis Affymetrix ATH1 GeneChips (http://www.affymetrix.com/) and statistical data analysis were performed at the Integrated Functional Genomic Service Unit (University of Münster, Germany). Candidate genes showing a more than twofold change (down or up, P <0.05) were considered as de-regulated genes. Classification of de-regulated genes was performed based on functional annotation of each locus (Arabidopsis Information Resource, http://www.arabidopsis.org/).
We thank Bernd Weisshaar (School of Biology, University of Bielefeld) for tt4-11 mutant seeds (SALK_020583) and Klaus Palme (Botany, Institute of Biology II, University of Freiburg) for PIN1::PIN1-GFP and PIN2::PIN2-GFP seeds. This work was supported by the Deutsche Forschungsgemeinschaft.