A review of target gene specificity of flavonoid R2R3-MYB transcription factors and a discussion of factors contributing to the target gene selectivity

Abstract

Flavonoid biosynthetic genes are often coordinately regulated in a temporal manner during flower or fruit development, resulting in specific accumulation profiles of flavonoid compounds. R2R3-MYB-type transcription factors (TFs) “recruit” a set of biosynthetic genes to produce flavonoids, and, therefore, R2R3-MYBs are responsible for the coordinated expression of structural genes. Although a wealth of information regarding the identified and functionally characterized R2R3-MYBs that are involved in flavonoid accumulation is available to date, this is the first review on the global regulation of MYB factors in the flavonoid pathway. The data presented in this review demonstrate that anthocyanin, flavone/flavonol/3-deoxyflavonoid (FFD), proanthocyanidin (PA), and isoflavonoid are independently regulated by different subgroups of R2R3-MYBs. Furthermore, FFD-specific R2R3-MYBs have a preference for early biosynthetic genes (EBGs) as their target genes; anthocyanin-specific R2R3-MYBs from dicot species essentially regulate late biosynthetic genes (LBGs); the remaining R2R3-MYBs have a wider range of target gene specificity. To elucidate the nature of the differential target gene specificity between R2R3-MYBs, we analyzed the DNA binding domain (also termed the MYB-domain) of R2R3-MYBs and the distribution of the recognition cis-elements. We identified four conserved amino acid residues located in or just before helix-3 of dicot anthocyanin R2R3-MYBs that might account for the different recognition DNA sequence and subsequently the different target gene specificity to the remaining R2R3-MYB TFs.

Keywords

MYB / cis-element / DNA-binding domain / flavonoid / transcription factor / target gene specificity

Introduction

Flavonoids comprise one of the most abundant and important groups of secondary metabolites, which are widely distributed in plants (Ferrer et al., 2008). The flavonoid biosynthetic pathway is illustrated in Fig. 1. Chalcone synthase (CHS) is positioned at the entry point in the pathway and introduces the metabolic flux from the general phenylpropanoid pathway. After the step catalyzed by CHS, the flavonoid pathway branches off into seven branch-pathways, leading to anthocyanins, proanthocyanidins (or condensed tannins), 3-deoxyflavonoids (such as phlobaphene), aurones, flavones, flavonols, and isoflavonoids (Winkel-Shirley 2001). Chalcone and flavandiol (or leucoanthocyanidin) are another two common flavonoids, and flavonoid-related stilbenes, formed by CHS-like stilbene synthase (STS) (Schröder et al., 1988), are often mentioned in reviews of flavonoids. There has long been great interest in the flavonoid biosynthetic pathway because flavonoids provide important colors, and thereby beauty, to the plant kingdom, and many human health benefits have been ascribed to these molecules (Mol et al., 1998; Koes et al., 2005). Flavonoids are also closely related to the defense mechanism of plants against biotic and abiotic stress (Winkel-Shirley 2002). The flavonoid biosynthetic pathway has been extensively studied over the past 30 years and is arguably the best understood pathway of secondary metabolites in higher plants (Quattrocchio et al., 2006).

A common denominator in the regulation of structural flavonoid pathway genes is the R2R3-MYB transcription factor (TF), which is characterized by a structurally conserved DNA binding domain (also termed the MYB-domain) consisting of two imperfect repeats R2 and R3. It is known that MYB proteins regulating the anthocyanin and PA pathways require bHLH (basic Helix Loop Helix) and WD-repeat protein partners to synergistically enhance the expression of structural genes (Hichri et al., 2011a). They form a MYB-bHLH-WD40 (MBW) complex in the transcriptional regulation. In the complex, MYB proteins are the key components to activate discrete subsets of anthocyanin biosynthesis genes and therefore determine target genes selectivity; bHLH proteins may have overlapping regulatory targets; the WD40 regulators are expressed more or less ubiquitously and may not directly be involved in transcriptional activation. Recent studies indicate bHLH can also determine target genes selectivity in some regulation (Hichri et al., 2011b; Kong et al., 2012). For flavone/flavonol/3-deoxyflavonoid (FFD) regulation, however, the two partner factors are not required.

The R2R3-MYB regulatory genes constitute a large family of more than 100 members in higher plant species, including Arabidopsis (Stracke et al., 2001), maize (Du et al., 2012a), soybean (Du et al., 2012b), and Camellia sinensis (Zhao et al., 2013). The study of plant R2R3-MYBs began with the identification of maize (Zea mays) C1 (Cone et al., 1986; Paz-Ares et al., 1987) and PL (Cone et al., 1993) genes that regulate anthocyanin synthesis in different tissues. Grotewold et al., (1991) determined the sequence of the P gene, which controls phlobaphene pigmentation in maize floral organs. Although P and C1 regulate distinct flavonoid branches, these genes show a high homology (70%) in the MYB-domain. In contrast to the wealth of knowledge available for the R2R3-MYB regulation of the anthocyanin branch, a long period elapsed before much was known regarding the regulation of other flavonoid branches. The R2R3-MYB regulation of flavones, flavonols, and PAs has only been elucidated in a number of plant species in recent decade, see Table 1. These studies indicate that anthocyanins, FFDs, and PAs are independently regulated and that this differential regulation is managed via R2R3-MYB TFs. R2R3-MYB factors were previously categorised into dozens of subgroup on the basis of the conserved MYB-domain and amino acid sequence motifs present to the C-terminal side of MYB-domain (Stracke et al., 2001; Jia et al., 2004; Dubos et al., 2010). R2R3-MYB factors within a subgroup are usually conserved in regulating a similar flavonoid branch, but this does not mean they regulate the same set of flavonoid genes. How about the conservation of target gene specificity in each R2R3-MYB subgroup? Can MYB domain explain target gene specificity? To address these questions, we present a deep review on the target gene specificity of the flavonoid R2R3-MYBs identified and characterized to date. We analyzed the DNA binding domain of R2R3-MYBs and the cis-element distribution in promoters of flavonoid structural genes. This review provides information for the understanding of how R2R3-MYBs regulate flavonoid accumulation by controlling a set of structural genes and how R2R3-MYB regulation determines the accumulation pattern of flavonoid metabolites.

R2R3-MYB regulation of flavonoid branches

Flavones, flavonols, PAs, and anthocyanins are widely distributed among higher plant species. Isoflavonoids are only found in legumes and a small number of non-legume plants, and aurones are found in a few species of a range of plant genera, particularly in Scrophulariaceae, Plumbaginaceae, and Compositae (Falcone Ferreyra et al., 2012). A few species, including sorghum, maize, and Gloxinia, produce 3-deoxyflavonoids, and a number of unrelated plant species, including peanut, grape, and pine, produce stilbene (Falcone Ferreyra et al., 2012). The flavonoid metabolites that accumulate in a limited number of plant species might have evolved multiple times or might have been lost from specific plant lineages over the course of evolution (Winkel-Shirley 2001).

R2R3-MYBs that regulate the biosynthesis of flavones, flavonols, anthocyanins, and PAs have been isolated and characterized from a range of plant species, whereas those regulating 3-deoxyflavonoids and isoflavonoids have been characterized only from Gramineae plants and soybean, respectively, see Table 1. No R2R3-MYBs that regulate biosynthesis pathway of aurone and stilbene have yet been reported. Chalcones and flavan-3,4-diols are the two major detected intermediate flavonoid metabolites in plants. Their regulation systems are not likely to be independent from those of flavonoid end products. In addition to R2R3-MYBs that are specific to a single flavonoid branch, there are some contributing to multiple flavonoid braches, essentially regulating the general flavonoid pathway, see section 2.6.

Anthocyanin-specific R2R3-MYBs

There have been several important reviews on the molecular structure and biosynthetic pathway of anthocyanin (Springob et al., 2003; Grotewold 2006). After characterizing the anthocyanin structural genes, researchers began identifying the regulatory factors, and it was found that R2R3-MYB TFs play a central role in anthocyanin regulation (Allan et al., 2008). Such knowledge is the foundation for creating novel flower colors through genetic engineering (Tanaka et al., 2009; Nishihara and Nakatsuka 2011).

Under normal conditions, anthocyanins are typically produced in flowers and fruits during late developmental stage, a time when these reproductive organs undergo large physiologic changes. For flowers, it is the stage when the petals rapidly grow due to cell expansion (Martin and Gerats 1993); for small berries, it is the stage called veraison, which represents the transition from berry growth to berry ripening. The investigation of the transcription profiles of anthocyanin structural genes in various plant species suggests that the genes usually can be divided into two groups. Fox example, in gentian (Gentiana triflora) for which the major flower pigment is delphinidin-based anthocyanin, F3H, F3′5′H, DFR, ANS, UFGT, and 5AT have a transcription profile during petal development paralleling with anthocyanin biosynthesis, whereas CHS, CHI, and F3′H do not (Nakatsuka et al., 2005). This difference in transcription profile is consistent with the positions of the enzyme coding genes in flavonoid pathway (Fig. 1). CHS and CHI are upstream of F3H, F3′5′H, DFR, ANS, UFGT, and 5AT in the pathway. In eustoma, F3H is grouped with CHS and CHI instead of with F3′5′H, DFR, and ANS based on their expression profiles (Noda et al., 2004). F3H classification is different between gentian and eustoma. This is because F3H is required for flavonol in addition to anthocyanin biosynthesis in eustoma. In fact, studies in major dicot flowers, fruits and small berries reveal similar patterns of coordinated gene expression, as found in snapdragon (Schwinn et al.,, 2006), pear (Li et al.,, 2012a) and bayberry (Niu et al.,, 2010). Grape (Vitis vinifera) is an exception, as only UFGT shows an expression profile consistent with anthocyanin accumulation (Boss et al., 1996). Monocot flowers, however, exhibit a different coordinated expression pattern of anthocyanin genes from dicot species. Orchids (Oncidium) and lilies (Lilium spp.) essentially show a pigmentation-correlated expression profiles of CHS genes (Chiou and Yeh 2008; Lai et al., 2012). In this review, we divided the enzyme coding genes for anthocyanin biosynthesis (or central flavonoid pathway genes) into two groups: early biosynthetic genes (EBGs) that includes CHS and CHI and late biosynthetic genes (LBGs) that includes DFR, ANS, UFGT and AT. Genes F3H, F3′H, and F3′5′H belong to either EBGs or LBGs depending on their expression pattern. This classification method is different to the traditional method as shown in gentian and eustoma which is totally based on expression pattern.

The differential expression pattern of EBGs and LBGs in gentian flowers is due to R2R3-MYB regulation. Anthocyanin-specific GtMYB3 activates the promoters of GtF3′5′H and Gt5AT but not GtCHS in a transient expression assay, indicating that EBGs are not the target genes (Nakatsuka et al., 2008). A later study found that EBGs and F3′H are under the control of GtMYBP3, a flavone-specific R2R3-MYB TF (Nakatsuka et al., 2012). It is interesting that the primary target genes of dicot anthocyanin-specific R2R3-MYBs appear to be LBGs, at least not EBGs. Anthocyanin-specific VvMYBA can activate the VvUFGT promoter but not those of any other genes, including VvANS (Kobayashi et al., 2002; Czemmel et al., 2009). Some studies indicate that EBG expression can be closely correlated with R2R3-MYB regulator genes and R2R3-MYBs can induce EBGs in transgenic plants, but there is no evidence demonstrating that anthocyanin R2R3-MYBs of dicot species directly activate EBGs (Table 1). Overexpression evaluation method is unreliable for the determination of the primary target genes of an R2R3-MYB regulator. For example, Borevitz et al., (2000) identified AtPAP1, an anthocyanin activator in Arabidopsis seedlings, by activation tagging methods and found that entire phenylpropanoid pathway was upregulated by AtPAP1. However, plants harbouring an RNAi construct targeting AtPAP1 and similar sequences of AtPAP2, AtMYB113, and AtMYB114 exhibited the downregulation of only LBGs (Gonzalez et al., 2008). This result clarifies that the target genes of AtPAP1 are restricted to only LBGs. Therefore, we emphasize the information of target gene specificity based on direct evidences as defined in Table 1. In contrast to those of dicot species, anthocyanin-specific R2R3-MYBs of monocots seem to regulate both EBGs and LBGs, as demonstrated in Oncidium, lily, and maize (Table 1).

To date, 34 anthocyanin-specific R2R3-MYBs from species in 21 genera have been functionally characterized (Table 1), with apparent diversity in target gene specificity between monocot and dicot species with regard to anthocyanin regulation. Specifically, anthocyanin-specific R2R3-MYB TFs in dicot species have not been reported to activate EBGs such as CHS, whereas those in monocot species have a wide range of target genes. It is interesting that, based on the MYB-domain and conserved motif in the carboxy terminus, dicot R2R3-MYB TFs (except GtMYB3) belong to subgroup 6 (SG6), whereas monocot R2R3-MYB TFs (except LhMYB12) belong to SG5. The structural differences between the SG5 and SG6 proteins may account for their different target gene specificity. As the MYB-domain is known to contribute to the binding selectivity of an R2R3-MYB TF, we performed a phylogenetic tree analyses based only on the MYB-domain (Fig. 2.). Basically, utilizing only the MYB-domain or the full amino acid (AA) sequence does not make a large difference to the R2R3-MYBs group based on sequence similarity. More details will be discussed in section 3.1.

Proanthocyanidin-specific R2R3-MYBs

PAs are oligomeric and polymeric flavan-3-ols (Fig. 1). The biosynthetic pathway leading to PAs has been reviewed previously (Dixon et al., 2005; Xie and Dixon 2005). LAR and ANR are the two key enzymes introducing the metabolic flow into the biosynthesis of catechin and epicatechin, respectively, two flavan-3-ols PA monomers. Pollination constant and non-astringent (PCNA)-type persimmons (Diospyros kaki) produce PAs in the fruit flesh within 9 weeks after blooming, whereas astringent (A)-type persimmons produce PAs throughout fruit development (Ikegami et al., 2007). This differentiated PA accumulation pattern is due to DkMYB4, a protein that regulates PA biosynthesis in the fruit flesh (Akagi et al., 2009). DkMYB4 is able to activate the DkANR promoter but not the DkLAR promoter in a dual-luciferase assay (Akagi et al., 2010). Moreover, a DkMYB4 recombinant protein bind to the promoters of DkF3′5′H and DkANS in an electrophoretic mobility shift assay (EMSA), indicating that these genes are also putative primary targets (Akagi et al., 2009). Another PA regulator, DkMYB2, is able to significantly activate the promoters of DkANR and DkLAR, though it is less expressed than DkMYB4 and its expression profile appears not to be correlated with structural gene expression under normal conditions (Akagi et al., 2010). During the fruit development of blueberry (Vaccinium corymbosum), LAR and ANR show PA-specific expression profile, while the remaining flavonoid genes that are also required for anthocyanin biosynthesis do not (Zifkin et al., 2012). VcMYBPA1 is able to activate the poplar PtANR promoter in a dual-luciferase assay, indicating its role in PA regulation, though the primary target genes of VcMYBPA1 need to be identified. VcMYBPA1 has a transcription profile that parallels PA and flavonol accumulation during the early stage and anthocyanin accumulation during the late stage, indicating this R2R3-MYB may also control flavonol and anthocyanin. However, no R2R3-MYB TFs playing a central role in multiple flavonoid branches have been identified thus far, except for maize P and C1. Grape produces PAs mainly in the berry skin during the early stages and in seeds at the veraison stage. Two major PA-specific R2R3-MYB TFs in grape have been identified, VvMYBPA1 (Bogs et al., 2007) and VvMYBPA2 (Terrier et al., 2009), and their tissue-specific expression patterns indicate they may be responsible for regulation in seeds and berry skin, respectively. VvMYBPA2 may upregulate VvMYBPA1 in vivo because plants overexpressing MYBPA2 have enhanced transcription level of MYBPA1 (Terrier et al., 2009). It is notable that neither the overexpression of VvMYBPA1 nor that of VvMYBPA2 enhances the transcription level of LAR2. Another two R2R3-MYB TFs, VvMYB5a (Deluc et al., 2006) and VvMYB5b (Deluc et al., 2008), are reported to regulate PA syntheses in grape. In contrast with other PA-specific R2R3-MYBs, VvMYB5a and VvMYB5b are suggested to participate in the regulation of multiple flavonoid products since these two genes induce strong accumulation of other flavonoids such as anthocyanins in tobacco. Here, since there are no solid evidences showing VvMYB5a and VvMYB5b control other branches and these two factors in fact are not capable of activating VvUFGT promoter, we group them with PA-specific MYBs. In strawberry (Fragaria × ananassa), FaMYB9 and FaMYB11 have recently been reported to regulate PA syntheses during the small green fruit stage (Schaart et al., 2013). The studies in fruits or berries mentioned above indicate the entire flavonoid pathway genes that are necessary for PAs syntheses are regulated by PA-specific R2R3-MYBs; especially, the study in grape has provided direct evidence to support this.

However, PA-specific R2R3-MYBs in other plant species show a different target gene range. AtTT2 controls PAs syntheses in Arabidopsis seed coat. PA accumulation begins to occur approximately 5 days after flowering (Routaboul et al., 2006). Transcripts of CHS, CHI, DFR, ANS, and ANR are detected in the seed coats, but these genes showed different expression profiles prior to the torpedo stage and, therefore, can be divided into two subsets (Devic et al., 1999). On the one hand, the transcription levels of CHS, CHI, F3H, F3′H, and FLS1 are high from the bud stage onward. On the other hand, the DFR, ANS, and BAN transcription levels peak at the globular stage, consistent with TT2 (Nesi et al., 2000). The tt2 mutation results confirm that TT2 does not regulate EBGs (Nesi et al., 2001). A recent study suggests that TaMYB14 may control PA synthesis in the young leaves of Trifolium arvense (Hancock et al., 2012). The overexpression of TaMYB14 in Trifolium repens, which does not produce PAs, results in the consistent upregulation of LBGs and PA-specific genes. Poplar (Populus tremuloides) constitutively produces a large amount of PAs in the leaves, but PA biosynthesis is also often upregulated by environmental stimuli, including herbivore attack or wounding (Peters and Constabel 2002), fungal infection (Miranda et al., 2007), and abiotic stresses of high light levels and nutrient limitation (Osier and Lindroth 2001). Poplar PtMYB134 is reported to regulate inducible PA accumulation in the leaves. The genes induced by high light stimulation can be divided into two groups according to their induced expression pattern (Mellway et al., 2009). The genes after F3H are coordinately induced with PtMYB134, indicating that PtMYB134 targets LBGs in light induction. In summary, AtTT2, TaMYB14 and PtMYB134 are likely to primarily target LBGs in addition to LAR and ANR in planta.

In conclusion, the target gene specificities of PA-specific R2R3-MYBs vary and appear to depend on plant species. Based on our phylogenetic tree analyses, PA-specific R2R3-MYBs form four clades: clade 3, clade 5, clade 6, and clade 7 (Fig. 2). DkMYB2 and PtMYB134 cluster together in clade 6 and recognize AC-elements (Mellway et al., 2009; Akagi et al., 2010). In contrast, clade 2 members seem to recognize a cis-element called MYBCORE (5′-CNGTTR-3′) that is similar to animal MYB recognition site. DkMYB4 binds to MYBCORE but not any other MYB binding cis-element (Akagi et al., 2009). VvMYBPA1 is suggested to recognize the same MYBCORE element and, thus, regulate a range of structural genes (Bogs et al., 2007). However, there seems no difference regarding target gene specificity between clade 2 and clade 6. This may be due to a wide distribution of cis-elements recognized by clade 2 and clade 6 members among flavonoid genes. Analyses of the binding ability and target gene specificity of more PA-specific R2R3-MYBs are necessary to obtain further insight.

Flavone- and flavonol-specific MYBs

The biosynthetic pathway of flavones and flavonols have been previously reviewed (Martens and Mithöfer 2005; Martens et al., 2010). Many flowers produce flavones or flavonols to protect the reproductive organs against UV damage. Gentian (Fujiwara et al., 1998; Nakatsuka et al., 2005) and snapdragon (Martin et al., 1991) produce flavones at the early flower stages when anthocyanin biosynthesis has not yet begun. The same accumulation pattern of flavonols is found in petunia (Saito et al., 2006) and Eustoma (Noda et al., 2004). FNS and FLS are specific to flavones and flavonols, respectively, and are often coordinately expressed with EBGs, as found in most flowers. GtMYBP3 and GtMYBP4 are reported to control flavone synthesis in gentian flowers (Nakatsuka et al., 2012). Both genes can activate the promoter of GtF3′H, in addition to GtCHS and GtFNS, but not GtF3′5′H.

F3′H is most likely essential for flavone formation after the step catalyzed by FNS. In lily, flowers produce a single flavonol and anthocyanin at the same time during the late stages, with the flavonol concentration being much lower than that of the anthocyanin (Lai et al., 2012). The R2R3-MYB TFs regulating flavones or flavonols in monocot flowers remain unknown; furthermore, it is not clear whether other monocot flowers have the same accumulation profiles of flavonols or flavones.

Flavonols accumulate in the peel of ripening tomato (Solanum lycopersicum) fruits. In red-fruited tomatoes, CHS, F3H, and FLS transcription levels increase during ripening, peak at the turning stage, and then decrease slightly at the red stage; the transcription level of CHI remains low and even decreases upon ripening (Bovy et al., 2002). Therefore, CHI is thought to be a rate-determining step in flavonol accumulation. Grape VvMYBF1 specifically activates the promoters of the flavonoid pathway genes involved in flavonol synthesis (Czemmel et al., 2009). Surprisingly, the promoter of the VvDFR gene that is involved in the synthesis of anthocyanins and PAs is moderately activated by VvMYBF1. DFR may participate in an unknown alternative route to produce flavonols, similar to the role of F3′H in gentian flowers.

The regulation system of flavonol accumulation has been systematically revealed in Arabidopsis. AtMYB12, AtMYB11, and AtMYB111 control flavonol biosynthesis in Arabidopsis (Mehrtens et al., 2005; Stracke et al., 2007). These three TFs show differential spatial regulation, but they bind to the same targets in a promoter assay. MYB12 mainly controls flavonol biosynthesis in roots, whereas MYB111 predominantly regulates biosynthesis in cotyledons. These three factors are required for flavonol accumulation because mutant MYB11-12-111 seedlings cannot produce flavonol compounds. The targets of the three TFs are EBGs and FLS. In general, AtMYB12 has stronger trans-activation capacity for the four target genes than AtMYB111, which, in turn, has higher activity than AtMYB11. Although F3′H is a potential target of these regulatory factors, a promoter assay study did not prove this; an unknown additional co-factor might be required to active the F3′H promoter. F3′H is a special structural gene that it is necessary for quercetin flavonols but not for kaempferol flavonols. Arabidopsis leaves and stems predominantly accumulate kaempferol flavonols, whereas inflorescences and siliques show a broad spectrum of flavonoids (Stracke et al., 2010). Therefore, plants need more regulatory factors to regulate F3′H when and where necessary the synthesis of quercetin flavonols is required. In the seed coats, the predominant flavonol is quercetin-3-O-rhamnoside (Q3R); quercetin-3-O-rhamnoside-7-O-glucoside (Q3R7G) accumulates in even higher concentrations than Q3R but disappears during seed maturation (Routaboul et al., 2006). Q3R7G accumulation begins approximately 5 days after flowering and drastically decreases in quantity at approximately 15 days after flowering, a time when the large accumulation of Q3R begins. The accumulation of Q3R7G and Q3R during the early stages appears to be mainly influenced by AtMYB12 and during the late stages by AtMYB11 and AtMYB12 (Stracke et al., 2010).

In conclusion, flavone- and flavonol-specific R2R3-MYBs are extremely conserved with regard to their target gene specificity. This is likely related to their extremely conserved MYB-domain structure since all of these regulatory proteins are clustered in clade 1 in our phylogenetic tree analyses (Fig. 2).

3-Deoxyflavonoid-specific MYBs

The 3-deoxyflavonoids include phlobaphene, 3-deoxyanthocyanidins, and C-glycosyl flavones. Phlobaphenes and 3-deoxyanthocyanidins are derived from the flavan-4-ols luteoforol or apiforol. C-glycosyl flavones consist of apimaysin and maysin and are synthesized through a different pathway than FNS, with flavanone 2-hydroxylase (F2H) catalyzing the first step (Fig. 1). Perhaps the most well-studied plant system with regard to phlobaphenes is maize; phlobaphene pigments accumulate most conspicuously in the floral organs, including the cob glumes and pericarp, though it is also found in the tassel glumes and husk (Sharma et al., 2012); C-glycosyl flavones and 3-deoxyanthocyanidins primarily accumulate in maize silks and act as defense compounds (Waiss et al., 1979).

Two tightly linked MYB homologous genes, P1 and P2, control 3-deoxyflavonoid synthesis (Zhang et al., 2000). P1 is primarily expressed in the pericarp, cob glumes, and silks, whereas the expression of P2 is restricted to the silks and anther wall. Despite the differential spatial expression patterns, these two TFs show high sequence similarity and are identical in function. In maize silks, both P1 and P2 regulate maysin synthesis because maysin cannot be detected in plants in which these genes have been deleted; maize cells transformed with either P1 or P2 synthesize phlobaphene and C-glycosyl flavones (Zhang et al., 2003). Transformation of maize callus and plants with the P1 gene provides further evidence for its function in regulating phlobaphenes and maysin (Grotewold et al., 1998; Cocciolone et al., 2005). In an initial study analyzing P1 mutant plants, CHS, CHI, and DFR were identified as the target genes of P1 (Grotewold et al., 1994), and sorghum Y1 controls the same set of genes and regulates phlobaphene in the pericarp (Boddu et al., 2006). ZmFLS1 (Falcone Ferreyra et al., 2010) and ZmF3′H (Sharma et al., 2012) are the most recently reported regulation targets of P1. ZmF3′H is known to be required for anthocyanin biosynthesis and also participates in the synthesis of 3-deoxyflavonoids; ZmF3′H adds a hydroxyl group to the 3′-position of apiforol to form luteoforol.

Recently, Morohashi et al., (2012) introduced a functional P1-rr allele into a A619 inbred line that harbours a recessive null P1-ww allele and then identified the primary target genes of P1 through a combination of chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq). These advanced techniques allow the comprehensive identification of P1-regualted genes, identifying CHS (C2), CHI1, and DFR (A1), in agreement with previous studies. New target genes were also identified: a second CHS WHP1, a second DFR A1*, and ZmF2H1, which catalyzes the key branch point in the pathway leading to 3-deoxyflavonoid. The promoters of WHP1, A1*, and CHS were activated by P1 in a luciferase reporter assay; interestingly the activation of WHP1 and A1* by the C1/R complex were not efficient. In addition to target genes in the flavonoid pathway, 1500 putative targets of P1 were identified, including phenylpropanoid genes, translation-associated genes, and TF genes, in maize pericarp and silks. P1 has many more target genes than expected and plays a role in various plant physiologic activities. This finding is in agreement with the previous finding that P1-expressing maize cells also accumulate ferulic acid (Grotewold et al., 1998) and that P1 is one of the two QTLs that exerts a major effect on chlorogenic acid accumulation (Bushman et al., 2002). In summary, 3-Deoxyflavonoid-specific MYBs regulate DFR, F3′H, F2H, as well as EBGs in flavonoid pathway.

Isoflavonoid-specific MYBs

Isoflavonoids are abundant in soybeans and other leguminous plants, but some non-legume plant species also produce isoflavonoids (Mackova et al., 2006). Isoflavonoid biosynthesis has been reviewed (Du et al. 2010). Wang (2011) recently provided a review on the structural studies of the key enzymes involved in isoflavonoid biosynthesis. In soybean (Glycine max), isoflavonoids are detected throughout the plants and show the greatest amount in embryo tissues at late stages, the mature seeds, and the leaves (Dhaubhadel et al., 2003). Changes in IFS2 transcript abundance are consistent with isoflavonoid accumulation in embryo tissues, whereas CHR, CHI, and IFS1 show steady-state constitutive transcription, indicating that these genes are activated for processes other than isoflavonoid syntheses. The GmMYB176 gene encoding an R1-MYB was identified to regulate isoflavonoid biosynthesis, and GmMYB176 is capable of binding a TAGT element in vitro and of activating the GmCHS8 promoter in Arabidopsis protoplasts (Yi et al., 2010; Dhaubhadel and Li 2013). The study of the GmMYB176 in vivo function showed that GmMYB176 is essential but insufficient to regulate isoflavonoid biosynthesis in soybean.

General flavonoid pathway MYBs and negative regulators

Snapdragon AmMYB305 and AmMYB340 are capable of activating the promoters of AmCHI and AmF3H and bean GmPAL2 but not AmDFR and AmAS (Moyano et al., 1996). AmMYB305 and AmMYB340 are expressed only in flowers, and their temporal expression mimics exactly that of AmF3H during flower development (Jackson et al., 1991). This result indicates their roles in anthocyanin biosynthesis, but further investigation using in situ hybridization suggests that their flower tissue-specific expression patterns are correlated to sites of flavonol accumulation (Moyano et al., 1996). AmMYB305 and AmMYB340 are not highly expressed in the epidermal tissues of petals that synthesize anthocyanins. Additional evidence, particularly with regard to the activation of the FSL promoter, is needed to identify the function of AmMYB340 in flavonol biosynthesis. Because AmMYB305 and AmMYB340 do not cluster with flavonol-related clade 1 (Fig. 2), we postulate these factors participate in the regulation of general flavonoid genes for multiple purposes.

In addition to flavonoid activators, some repressor MYB regulators play an important role in balancing flavonoid biosynthesis. There are two types of MYB repressors: single R repeat MYBs and R2R3-MYBs. Arabidopsis AtMYBL2 is an R3-MYB that inhibits the flavonoid pathway by competing with R2R3-MYB for interaction with a necessary bHLH co-factor, thereby negatively regulating flavonoid synthesis (Dubos et al., 2008; Matsui et al., 2008). However, it is unlikely that this MYB repressor selects its target genes by directly interacting with the gene promoters, even though it is reported that an R3-MYB can still bind to DFR promoter (Gong et al., 1999). R2R3-MYB repressors fulfil their functions via conserved repression domains at the carboxy terminus. Arabidopsis AtMYB4 is such a repressor (Jin et al., 2000); although AtMYB4 mutant plants enhance the transcription of C4H, other genes, such as PAL and CHS, are not affected. FaMYB1 from strawberry is related to fruit ripening and contains a repression domain similar to that of AtMYB4 (Aharoni et al., 2001). The overexpression of FaMYB1 in tobacco results in a reduction of anthocyanins and flavonols in flowers and a corresponding reduction of ANS and DFR expression but not CHS and F3H. More recently, Gao et al., (2011) reported that apple MdMYB6 affects structural gene expression and inhibits Arabidopsis anthocyanin biosynthesis. These studies indicate that R2R3-MYB repressors involved in flavonoid pathway select their target genes in a manner similar to R2R3-MYB activators, though the mechanism is not clear, and there is no direct evidence showing their interaction with promoters.

Factors accounting for differential target gene specificity

To control a particular flavonoid branch, an R2R3-MYB activator targets a set of flavonoid structural genes. The selectivity of target genes relies on the two components of cis-trans regulation: a MYB-domain that directly recognizes a DNA sequence and the presence of related DNA sequences in the promoters of flavonoid genes. In addition, the interaction with co-factors is also involved in the DNA specific recognition. Here, we tentatively explain the diversity of target gene specificity of R2R3-MYB TFs by closely examining these three factors.

DNA binding site of MYBs

MYB TFs recognize the corresponding cis-elements through their MYB-domain. The MYB-domain of R2R3-MYB consists of two imperfect repeats (R2 and R3), and each is approximately 50-53 amino acids and forms three α-helix (Ogata et al., 1992). The second and third helices form a helix-turn-helix (HTH) structure that intercalates in the major groove of DNA (Rabinowicz et al., 1999; Tahirov et al., 2002). Maize C1 and P are both capable of activating the ZmDFR (A1) promoter, though only C1 targets the ZmUFGT (Bz1) promoter. However, replacement of the MYB region of C1 with that of P results in the inability to activate ZmUFGT promoter but not the ZmDFR promoter (Grotewold et al., 1994). This result clearly indicates that the MYB-domain is sufficient for specific DNA binding. In the existing literatures about R2R3-MYBs, phylogenic analyses using MYB domain is usually done to predicate their function and study their relationship. For example, Lin-Wang et al., (2010) studied the phylogenetic relationship between anthocyanin-related MYBs of rosaceous and other species. Only a few reports discussed the relation between MYB domain and DNA binding selectivity by phylogenic analyses. Agaki et al., (2010) divided PA-specific R2R3-MYBs into three groups based on MYB domain structure and discussed their subfunctionalization.

We analyzed the MYB-domains of all the available flavonoid-regulating R2R3-MYBs by constructing a phylogenetic tree (Fig. 2.). The flavonoid R2R3-MYBs form 9 clades, essentially the same result as the analyses using the full amino acid (AA) sequence of each protein. When using the full AA sequences, clade 3 is placed between clade 4 and clade 5, and clade 7 is placed between clade 5 and clade 6 (data not shown). The members within clades 1 and 9 share a similar set of flavonoid target genes. Clade 1 members are those that regulate FFD synthesis and are characterized by EBG target genes. In contrast, all clade 9 members (except for lily LhMYB12) primarily target LBGs. Clade 9 consists of all the anthocyanin-specific R2R3-MYBs from dicot species, with the exception of gentian GtMYB3, which clusters with grape PA-regulating MYBs. It is noteworthy that the anthocyanin-specific R2R3-MYBs from maize and Oncidium cluster with the PA-specific R2R3-MYBs in clade 6, suggesting that the monocot anthocyanin regulators are closer to the PA regulators than to the dicot anthocyanin regulators. All the R2R3-MYB repressors cluster together in clade 2, with the exception of apple MdMYB6 and strawberry FaMYB1. Clade 4 members are chiefly activators regulating the general flavonoid pathway. The PA-regulating R2R3-MYBs span four clades: clade 3, 5, 6, and 7. In summary, the MYB-domain feature of FFD-specific R2R3-MYBs and dicot anthocyanin-specific R2R3-MYBs show a significant correlation with the target gene selectivity.

To obtain a further understanding of the relation between MYB-domain feature and the differential target gene specificity, we generated a multiple sequence alignment of R2R3-MYB proteins (Fig. 3), including nearly all the identified flavonoid R2R3-MYBs. If a residue actually contributes to the differential target gene specificity between the different R2R3-MYB clades, it should be conserved within that clade but be different to that of other clades. We identified nine such residues that may account for the limited target range of dicot anthocyanin-specific R2R3-MYBs (Fig. 3), with residues 36, 87, 89, and 90 showing unique patterns among these MYB sequences. These amino acids are 36R, 87A, 89D, and 90V in the clade 9 R2R3-MYBs, whereas they are 36G, 87D, 89E, and 90I in the remaining R2R3-MYBs. There are four exceptions for the clade 9 MYBs, including 36L of VvMYBA2, 87G of AmVENOSA, 89A of MdMYB1, and 90I of LhMYB12, and two exceptions for the remaining MYBs, including 89A of MdMYB6 and 90V of FaMYB1. Regardless, the number of exceptions is very small when considering the large number of R2R3-MYBs analyzed, and it is rare to find such a distribution of other residues among these proteins. We must note that residues 36, 89, and 90 are positioned in helix-3 of R2 or R3 and that residue 87 is positioned just before helix-3 of R3. Experimental evidences have indicated that helix-3 plays a central role in recognizing specific base. Mutations occurring in helix-3 often abolish specific DNA binding and result in the loss of function (Saikumar et al., 1990; Frampton et al., 1991; Gabrielsen et al., 1991; Ogata et al., 1994; Sasaki et al., 2000). The difference in residues 36, 87, 89, and 90 between clade 9 and the remaining proteins may account for the different target gene specificity.

The importance of residue 89 has been demonstrated to date. When mutation of 89D (101D in the original paper) to 89E occurred, ZmC1 became defective in DNA binding because its ability to activate UFGT and DFR promoter was reduced (Goff et al., 1991; Sainz et al. 1997). A similar DNA binding defect is caused by the same mutation in PhMYB3 (Solano et al. 1995). Most recently, Heppel et al. (2013) reported identification of key amino acids for the differential in target specificity between PA-specific R2R3-MYBs and dicot anthocyanin-specific R2R3-MYBs. Their experimental results are highly consistent with our in silico predictions. Exchange of residues 36, 87, 89, and 90 between TT2 and PAP1-4 can swap the pathway selection of TT2 and PAP1-4 (Heppel et al., 2013). We tentatively propose that clade 9 members recognize different DNA sequence to others, see section 3.2, due to their distinctive residue 36, 87, 89, and 90, and therefore are not capable of activating EBGs like most of the remaining flavonoid R2R3-MYBs. On the surface, the features of the MYB-domain cannot fully explain the observed differential target gene specificity because lily LhMYB12 contains 36R, 87A, and 89D, similar to dicot anthocyanin R2R3-MYBs, but is capable of activating CHS (Lai et al., 2012). However, we must remind that the LhCHS promoter fragment activated by LhMYB12/LhbHLH2 contains a perfect G-box but not any typical anthocyanin elements. Therefore, LhbHLH2 rather than LhMYB12 likely determine DNA binding via ACT domain OFF model, see section 3.3.

In addition to the key residues described above, several other residues have been experimentally identified to determine the differential in target specificity between AmMYB305 and vertebrate c-MYB. AmMYB305 and vertebrate c-MYB bind to two different cis-element MBSII and MBSI, respectively; PhMYB3 binds to both of these elements (Table 2). Three residues were first identified to be the key residues in the MBSI recognition by protein mutant experiment (Ogata et al., 1994); their counterparts in our multiple sequence alignment are residue 37, 91 and 92. After that, Solano et al., (1997) identified five new important residues; their counterparts in our multiple sequence alignment are residue 41, 45, 88, 95 and 96. The eight residues in total were suggested be responsible for the differential target DNA sequence between c-MYB and AmMYB305.

Cis-elements for MYB binding

The regulation of coordinated expression of flavonoid structural genes is achieved through the interaction of the MYB-domain with conserved cis-elements that are present in target genes. A limited number of such cis-regulatory motifs for flavonoid MYB binding have been well characterized and reported (Table 2). According to their sequence and cognate MYB, these motifs can basically be divided into two classes: AC-rich cis-motifs and vertebrate c-MYB sites. Several critical DNA sequences for anthocyanin regulation have been identified and they are very different to the known AC-rich motifs and vertebrate c-MYB sites (Table 2).

AC-rich cis-motifs are widely distributed and recognized by R2R3-MYBs that regulate anthocyanin, 3-deoxyflavonoid, and PA biosynthesis. The P binding site 5′-CCWACC-3′ (-64 bp to -59 bp, relative to the transcriptional start site) was first identified in the proximal ZmDFR (A1) promoter using a DNase I footprinting approach (Grotewold et al., 1994). When this region was mutated, trans-activation by P was decreased by approximately 50%. Later, in 1997, Sainz et al., found an additional P binding site, 5′-AACTACCGG-3′ (-116 bp to -124 bp), within the distal region of the ZmDFR promoter (Sainz et al., 1997). However, P only bound to a large degree to the distal site when the proximal site was mutated due to the lower affinity of the distal site. These two P binding sites in the ZmDFR promoter are also bound by C1, though the proximal site is bound with a lower affinity by C1 relative to P. When a random pool of oligonucleotides was prepared, P primarily selected sequences with an 5′-ACCWACC-3′ motif, whereas C1 selected 26 diverse sequences, with only some of the fragments having a site similar to the two binding sites in the ZmDFR prompter (Sainz et al., 1997). This result suggests that C1 has a broader DNA binding specificity than P. Indeed, ZmC1 is even capable of activating ArabidopsisAtANR (Baudry et al., 2004). The MYB-recognition element (MRE), first identified in the PcCHS promoter, is required for light induction (Feldbrügge et al., 1997), and the MRE sequence 5′-ACCTACC-3′ is very similar to P/C1-site 1. MREs are distributed among the promoters of the co-activated Arabidopsis flavonol synthesis genes AtCHS, AtCHI, AtF3H, and AtFLS1 (Hartmann et al., 2005), and MREs are recognized by AmMYB305 (Feldbrügge et al., 1997), PcMYB1 (Feldbrügge et al., 1997), ZmC1 (Hartmann et al., 2005), and AtMYB12 (Mehrtens et al., 2005). The principle of the ‘one cis-element versus many factors’ type of regulation was previously proposed to be a common feature of the large TF families found in plants. PtMYB134 and DkMYB2 that control PA biosynthesis in poplar and persimmon, respectively, was found to directly bind to AC-rich cis-motifs in an EMSA experiment (Mellway et al., 2009; Akagi et al., 2010).

The consensus sequence of 5′-CNGTTR-3′ was proven to be the recognition site of vertebrate c-MYB (Lüscher and Eisenman 1990), and some studies suggest plant R2R3-MYBs also recognize these sequences. The region from -47 to -78 bp of ZmUFGT(Bz1) is sufficient to respond to C1/R-specific regulation in transient assay under the help of CaMV35 minimal promoter (Roth et al., 1991). Inspection of this region reveals that one of the two critical cis-elements is homologous to the consensus sequence 5′-CNGTTR-3′; the other one is the bHLH biding site 5′-CANNTG-3′. DkMYB4 directly targets MYBCORE motifs that are similar to the vertebrate c-MYB site but not other representative MYB sites, including AC-rich cis-motifs (Akagi et al., 2009). MYBCORE motifs are not present in the promoters of DkLAR and DkF3′H, in agreement with the observation that the expression of DkLAR and DkF3′H is not correlated with DkMYB4 in persimmon fruits. Petunia PhMYB3 recognizes two DNA sequences: MBSI and MBSII (Solano et al., 1995). MBSI resembles the vertebrate c-MYB site; MBSII is similar to Box-P and can be recognized by AmMYB305 but not by vertebrate c-MYB.

DNA sequences crucial for the activation of anthocyanin-specific R2R3-MYBs are conserved within the promoter regions of anthocyanin structural genes and, therefore, have been assigned the name of an anthocyanin regulatory element (ARE). By investigating the C1/B (MYB/bHLH) activation of deleted and mutated variants of the ZmDFR (A1) promoter, Tuerck and Fromm (1994) identified that the most important region for C1/B trans-activation was between -98 to -88 bp, a region overlapping ARE^ZmDFR (-101 to -88 bp); mutation in this region reduced C1/B activation severely to below 8% of normal. It is notable that ARE^ZmDFR is located between the two P/C1 binding sites which both contribute to C1/B activation (Sainz et al., 1997). The two binding sites most likely increase the local concentration of the C1 protein on DNA and, thereby, help the ARE to recruit C1. AREs appears to be specific for anthocyanin regulation because a promoter fragment containing this motif does not confer efficient P activation of ZmUFGT (Grotewold et al., 1994). Inspection of the ZmCHS (C2), ZmANS (A2), ZmUFGT (Bz1), and ZmGST (Bz2) promoters reveals homology of the ARE motif in these promoters (Tuerck and Fromm 1994), with a consensus sequence of 5′-CGACTGGCNGGTGC-3′. Because these DNA sequences have high similarity and a similar distance to the transcription start site, they are likely conserved with regard to anthocyanin regulation in maize. Lesnick and Chandler (1998) have since proven that ARE^ZmANS (-92 to -107 bp) is also crucial for ZmANS transcription activated by C1/B, though the motif is not the motif that Tuerck and Fromm predicted by sequence comparison. Moreover, ARE^ZmANS overlaps the third binding site for C1 (5′-GGTGGTTG-3′). In gerbera, an ARE^GhDFR2 in the proximal promoter region is critical for GhMYB10 regulation (Elomaa et al., 2003). Dare et al., (2008) identified the PAP1 cis-regulatory element (PCE) as 5′-YCNCCACRWK-3′ by scanning the eight promoters that were activated by AtPAP1 in N. benthamiana leaves. Using microarray analyses, the eight genes were selected from 33 genes that showed significant alteration in transcription level due to the overexpression of the AtPAP1 gene. Deleting and mutating the PCE sequence in promoters of AtUFGT and AtGST resulted in reduced activation by PAP1. Since PCE shows a low similarity to the previously identified ARE, there is a discrepancy in recognition site between AtPAP1 and GhMYB10. GhMYB10 shows monocot type of recognition site. ARE and PCE were identified by specific mutation to the DNA sequences and therefore in our review it is still far to know how these sequence is “crucial” to regulation of anthocyanin-specific R2R3-MYBs. It is possible that other factors that are necessary for anthocyanin regulation may interact with C1 through AREs. Thus, it is too early to draw a conclusion whether anthocyanin-specific R2R3-MYBs of dicots have the same recognition site to those of monocots or not. Nonetheless, AtPAP1 and GhMYB10, the two clade 9 members with known recognition sites, do not appear to select their target genes through interacting with AC-rich elements and animal MYB sites in vivo that are widely distributed among flavonoid promoters (Table 2).

Despite the advances in identifying the DNA binding sequence or functionally crucial DNA sequence, the distribution of cis-elements alone cannot fully explain the observed target gene specificity. For example, gentian GtMYBP3 targets GtCHS, GtF3′H, and GtFNS and regulates flavone production, whereas GtMYB3 targets GtF3′5′H and GtAT and regulates anthocyanin synthesis (Nakatsuka et al., 2008; Nakatsuka et al., 2012). The GtCHS, GtF3′H, GtF3′5′H, and GtFNS promoters present P binding sites and MYBCORE binding sites, and the GtAT promoter lacks a P binding site. Such a distribution pattern of cis-elements in flavonoid genes alone cannot fully explain the target gene specificity of GtMYBP3 or GtMYB3. Indeed, the presence of cis-elements appears insufficient to confer R2R3-MYB target specificity to a structural gene in vivo.

Roles of bHLH co-factors

Kong et al., (2012) recently proposed a combinatorial gene regulatory framework that placed bHLH TFs in an important role in the coordinated regulation of genes lacking obviously conserved cis-regulatory elements. Maize R has two models to activate anthocyanin genes that depend on the homodimerization of the C-terminal ACT domain. When the ACT domain is in the dimer form (ON model), the monomeric conformation of the bHLH region is preserved. In this case, R is tethered to DNA through its interaction with C1, indicating that C1 determines DNA binding. In contrast, when the ACT domain is the monomeric form (OFF model), the bHLH region of R forms homodimers that bind the G-box motif; although C1 is still required to form a complex for activation, R determines DNA binding. It is interesting that R and C1 regulate ZmDFR via the ACT domain ON model, whereas they regulate ZmUFGT via the ACT domain OFF model. This new finding can explain some cases where genes lacking obviously conserved cis-elements are regulated coordinately. Hichri et al., (2011b) provided one another evidence that the target gene selectivity of VvMYB5b depends on the interaction with bHLH co-factors. The single residue mutated protein VvMYB5b^L lost its ability to correctly interact with VvMYC1, and thereby resulted in severely reduced trans-activation activity in tobacco stamens. However, VvMYB5b^L overexpression appeared to retain trans-activation activity in tobacco corolla and target NtCHS, NtDFR and NtANS. The tissue-dependent trans-activation activity of VvMYB5b^L indicates that bHLH or unknown co-factors affect the target gene selectivity of R2R3-MYBs.

R2R3-MYB regulation in developing flowers and fruits

The major flavonoid compounds in flowers are flavones/flavonols and anthocyanins, and in fruits and seeds they are flavones/flavonols, anthocyanins, and PAs. The flavonoids in these organs show spatiotemporal accumulation patterns, and different classes of flavonoids usually do not occur simultaneously. The spatiotemporal regulation of flavonoid branches is essential to direct the biosynthesis of different flavonoids. Metabolic channelling by R2R3-MYBs in plant flavonoid metabolism enables plants to effectively synthesize the appropriate metabolites and avoid metabolic interference. Below, we demonstrate how R2R3-MYBs regulate their target genes of necessity for flavonoid biosynthesis during development of flowers and berry skins.

Flower development

Flower development can be divided in to the early stage and the late stage as demonstrated in gentian (Nakatsuka et al., 2005) and eustoma (Noda et al., 2004). The R2R3-MYB TFs of clade 1 induce the coordinated expression of EBGs and FSN or FLS to produce flavones or flavonols during the early stage (Fig. 4A). The transcription levels of clade 1 members then drastically decrease before pigmentation, resulting in decreases in FSN or FLS but not EBGs. At the late stage, the R2R3-MYB TFs of clade 9 induce the coordinated expression of LBGs in dicot species with the help of their bHLH co-factor. However, it remains unclear which factors are responsible for the activation of EBGs because clade 9 R2R3-MYBs in dicot species never regulate these genes, and, moreover, transcripts of the clade 1 R2R3-MYBs are absent at the late stage. Some other regulatory proteins may contribute to activate EBGs at the late stage. Another likely mechanism for the expression of early genes might be light activation during photomorphogenesis. Light-independent CHS expression is mediated by regulators, such as HY5, which can bind to a minimal light-responsive region of the CHS promoter (Ang et al., 1998; Lee et al., 2007). R2R3-MYB regulation in monocot flower development remains to be further clarified. Lily synthesizes flavonol during the late stage, and LhMYB12 appears to contribute to the activation of the entire anthocyanin pathway (Lai et al., 2012).

Figure 4. An illustration of the flavonoid regulation mechanism in developing flowers (A) and berry skin of grape ‘Shiraz’ (B). The pink color indicates anthocyanin pigmentation, the orange color indicates flavone or flavonol accumulation, and the green color indicates PA accumulation. The transcript abundance changes of VvMYBA and VvUFGT (Matus et al., 2010), VvMYBF1 (Czemmel et al., 2009), VvMYBPA1(Bogs et al., 2007), VvMYBPA2 (Terrier et al., 2009), VvMYB5b (Deluc et al., 2008), VvFLS (Downey et al., 2003b), and VvLAR1, VvLAR2, VvANR and VvANS (Bogs et al., 2005) are based on real-time PCR results from the original papers; that of VvMYB5a is roughly quantified from semiquantitative RT-PCR result in the original paper (Deluc et al., 2006); thoes of VvCHS, VvCHI, VvF3H, and VvDFR were investigated through northern blots in the original paper (Boss et al., 1996). The bar indicates a 2-week period during grape berry development. 150 × 176mm (160 × 160 DPI)

Unlike flowers of big size mentioned above, grape flowers synthesizes flavonols and PAs during the late flower stage. VvMYBPA1 and VvMYB5b show a maximum of expression one week prior to anthesis, preceding maximum PA accumulation at flowering (Bogs et al., 2007; Deluc et al., 2008). The highest transcription level of VvMYBF1 also occurs at anthesis (Czemmel et al., 2009). However, it is noteworthy that entire grape buds or flowers must be used to analyze the accumulation of flavonoids and gene transcripts because these organs are small. Indeed, this observation could result in different findings from studies of large flowers that examine only flower petals.

Fruit development

Grape berry development can be divided into four phases: fruit set stage (0-2 weeks after flowering, WAF), young fruit stage (3-6 WAF), veraison stage (7-8 WAF), and post-veraison stage (8 WAF<). Grape is an important plant for the study of the biosynthesis and regulation of flavonoids because the berries are able to produce three common flavonoids: anthocyanins, flavonols, and PAs (Fig. 4B), and the temporal control of PA, anthocyanin, and flavonol synthesis needs to be coordinated during berry development. Czemmel et al., (2012) recently provided a deep review on R2R3-MYBs that regulate flavonoids in grapevine.

Vitis vinifera ‘Shiraz’ grape mainly produces quercetin-3-glycoside (Q3G) flavonols, and the deposition tissues are floral organs, tendrils, inflorescences, leaves, and berry skins but not the berry flesh and seeds (Downey et al., 2003b). In reproductive organs, the Q3G concentration is high at flowering and then decreases between flowering and berry set; the total amount of flavonols per berry increases at fruit set stage and then remains the level until post-veraison stage along with the weight and size increase of berry, indicating flavonol syntheses in berries mainly occurs at fruit set stage and post-veraison stage. In strawberry fruit, flavonols largely accumulate in the ripe fruits concomitant with anthocyanin synthesis (Schaart et al., 2013). ‘Shiraz’ grape also produces PAs and the deposition tissues are flowers, berry skin, seeds, and leaves, and seeds have impressively high concentrations of PAs (Downey et al., 2003a), but the different tissues have PAs of different subunit composition (Bogs et al., 2005). The flowers and berry skins contain mainly catechin, whereas the seeds contain epicatechin. The main accumulation period of PAs in grape berries is from fruit set to 1-2 weeks following veraison (Bogs et al., 2005). Essentially, the biosynthesis of both flavonols and PAs during early fruit developmental stage is consistent with the finding in blueberry (Zifkin et al., 2012).

VvMYBF1, VvMYBPA1 and VvMYBPA2 show decreasing levels at the fruit set stage; thereafter, VvMYBPA2 remains an undetectable level throughout berry development. Despite the drastic change of transcription levels, these three TFs are highly expressed at fruit set stage. Accordingly, transcription of their target genes CHS, CHI, F3H, DFR, ANS, FLS, LAR1, and ANR are induced, although ANS and FLS have moderate transcript abundance. At the young berry stage, the berry skin and seed can be separated, and the following narrative is about berry skins. VvMYBPA1 and VvMYBPA2 are nearly undetectable at this stage; the transcription levels of VvMYB5a and VvMYB5b both dramatically decrease and VvMYB5a transcripts then become undetectable after the veraison stage. This change of R2R3-MYBs transcription levels is consistent with the sharp decrease of all the structural genes. LAR2 is not the target gene of any the R2R3-MYBs mentioned above and therefore shows discrepant change of transcription level. At the stage just before the veraison, all the known flavonoid R2R3-MYBs are inactive except VvMYBF1. This is consistent with the fact that the transcription of all the targeted structural genes become almost undetectable (Boss et al., 1996); LAR2 shows a remarkable and rapid decrease at the same time (Bogs et al., 2005). There is a discrepancy between FLS and its regulator VvMYBF1 at this stage, which may be due to an additional function of VvMYBF1 or the repression of VvFLS by another factor as suggested by the authors (Czemmel et al., 2009). It looks like that the flavonoid biosynthesis in grape berry skins is briefly inhibited around veraison stage; it should be interesting to clarify the possible physiologic or molecular mechanism to explain this phenomenon. After veraison stage, flavonoid synthesis “switches” from PA to anthocyanin synthesis. At 10 WAF, VvMYBPA1 shows the highest transcription level, which is closely correlated with the re-activation of CHS, CHI, F3H, DFR and ANS; VvMYBA begins to drastically increase at veraison stage which should be responsible for the UFGT activation. At 12 WAF, VvMYBPA1 shows a notable decrease in transcript abundance and thereafter remains undetectable; interestingly, VvMYB5b increases its transcription simultaneously and reaches its highest transcription level at 16 WAF. It seems that VvMYBPA1 “passes” the responsibility to activate genes from CHS to ANS in the pathway onto VvMYB5b that are necessary for anthocyanin biosynthesis. At 14 WAF, the transcription profile of VvMYBF1 has a small peak, which should be responsible for the peak of that of FLS.

Conclusion

Our review demonstrates that dicot anthocyanin-specific R2R3-MYBs and FFD-specific R2R3-MYBs primarily target LBGs and EBGs, respectively, and that PA-specific R2R3-MYBs target both EBGs and LBGs or only LBGs. Our in silico analyses of conserved amino acids in MYB domain together with existing experimental data shed light into the understanding of the mechanism of target specificity of R2R3-MYBs. Three factors are involved in the DNA specific recognition of R2R3-MYBs: DNA binding domain, the presence of target cis-elements, and co-factors. We identified several unique conserved residues of dicot anthocyanin-specific R2R3-MYBs that may account for the unique recognition DNA sequence and the unique target gene specificity. Our review also demonstrates that the differential target gene specificity of R2R3-MYBs is required to “switch” the metabolic flow between the different flavonoid branches during flower and fruit development.

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