Protoplasma (2012) 249 (Suppl 2):S109–S118 DOI 10.1007/s00709-012-0380-z

REVIEW ARTICLE

R2R3 MYB transcription factors: key regulators of the flavonoid biosynthetic pathway in grapevine Stefan Czemmel & Simon C. Heppel & Jochen Bogs

Received: 6 December 2011 / Accepted: 19 January 2012 / Published online: 4 February 2012 # Springer-Verlag 2012

Abstract Flavonoids compose one of the most abundant and important subgroups of secondary metabolites with more than 6,000 compounds detected so far in higher plants. They are found in various compositions and concentrations in nearly all plant tissues. Besides the attraction of pollinators and dispersers to fruits and flowers, flavonoids also protect against a plethora of stresses including pathogen attack, wounding and UV irradiation. Flavonoid content and composition of fruits such as grapes, bilberries, strawberries and apples as well as food extracts such as green tea, wine and chocolate have been associated with fruit quality including taste, colour and health-promoting effects. To unravel the beneficial potentials of flavonoids on fruit quality, research has been focused recently on the molecular basis of flavonoid biosynthesis and regulation in economically important fruit-producing plants such as grapevine (Vitis vinifera L.). Transcription factors and genes encoding biosynthetic enzymes have been characterized, studies that set a benchmark for future research on the regulatory networks controlling flavonoid biosynthesis and diversity. This review summarizes Handling Editor: Kang Chong S. Czemmel (*) : S. C. Heppel Centre for Organismal Studies Heidelberg (COS Heidelberg), Im Neuenheimer Feld 360, 69120 Heidelberg, Germany e-mail: [email protected] J. Bogs Dienstleistungszentrum Ländlicher Raum (DLR) Rheinpfalz, Viticulture and Enology group, Breitenweg 71, 67435 Neustadt/W, Germany J. Bogs Fachhochschule Bingen, Berlinstr. 109, 55411 Bingen am Rhein, Germany

recent advances in the knowledge of regulatory cascades involved in flavonoid biosynthesis in grapevine. Transcriptional regulation of flavonoid biosynthesis during berry development is highlighted, with a particular focus on MYB transcription factors as molecular clocks, key regulators and powerful biotechnological tools to identify novel pathway enzymes to optimize flavonoid content and composition in grapes. Keywords Flavonoid . MYB . Transcription factor . Gene regulation . Grapevine

Biological functions of flavonoids The various facets of the phenylpropanoid pathway, which is unique to plants, enable them to synthesize a tremendous variety of aromatic metabolites including coumarins, phenolic volatiles or hydrolyzable tannins and most prominently, lignins and flavonoids (Vogt 2010). Amongst them, flavonoids have received significant attention within the past years of research. As major pigments of flowers and fruit, flavonoids provided a natural reporter tool for groundbreaking scientists: Gregor Johann Mendel used flower pigmentation to study genetic inheritance in pea, and Barbara McClintock discovered the mechanism of gene silencing through altered flavonoid pigmentation patterns caused by genetic transposition in maize (reviewed in Grotewold 2006). Besides being major attractors for pollinators and dispersers, flavonoids rapidly accumulate in response to a manifold of stresses in planta: high and low temperatures (Mori et al. 2007; Yamane et al. 2006), water stress (Castellarin et al. 2007) and excessive UV light (Price et al. 1995; Downey et al. 2004; Czemmel et al. 2009). In grapevine, the influence of light and temperature on flavonoid profiles is also strongly

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modulated by viticultural practices such as leaf removal and cluster thinning (Matus et al. 2008; Mori et al. 2007; Guidoni et al. 2008). Different fertilization regimens, which are used to modulate the mineral status of the soil, have been also shown to have a strong impact on flavonoid, especially anthocyanin and flavonol concentrations in plants (Stewart et al. 2001; Delgado et al. 2004; Lea et al. 2007). The third major class of flavonoids, the proanthocyanidins (PAs), have been shown to be important defence molecules that rapidly accumulate in response to pathogen or herbivore attack (Dai et al. 1995; Ali et al. 2009). Besides being important defence molecules, transgenic approaches using forage crops showed that moderate levels of PAs can be used to protect ruminants against the occurrence of pasture bloat and to promote increased dietary protein nitrogen utilization (reviewed in Dixon and Pasinetti 2010). Considering their function for plant development, flavonoid compounds, especially flavonol aglycones, have been implicated in negative regulation of the transport of the phytohormone auxin (Buer and Muday 2004; Peer and Murphy 2007). This well-established interplay between auxin transport and flavonols may underlie the influence of flavonols on certain developmental processes such as reproduction (Mo et al. 1992; Ylstra et al. 1992; Thompson et al. 2010) presumably by increasing intracellular concentration of auxin, which in turn promotes polar tube growth. Due to this versatility of functions, a large surge in research has been conducted on the mechanisms regulating flavonoid production in economically important fruit-producing plants. Studies currently focus on plants which are part of our daily nutrition and accumulate high amounts of potent antioxidant flavonoid compounds: apple (Malus x domestica, Boyer and Liu 2004; Espley et al. 2007), strawberry, Vaccinium species (Vvedenskaya et al. 2003; Häkkinen and Törrönen 2000; Jaakola et al. 2002) and grapevine (Vitis vinifera L., Downey et al. 2004; Mattivi et al. 2006). Developing grape berries have received considerable industrial scrutiny because of the manifold of marketabilities of the ripe fruit including table grapes, fruit juices, wine and raisins which allow the industry to serve and react on region-specific consumer behaviours and preferences. In particular, red colouration of fruit skin became an important determinant of food quality and is arguably nowhere more important than in the differences between white and red grapes (Allan et al. 2008). The anthocyanins, which are lacking in white grapes but are responsible for the colouration of red grapes (Walker et al. 2007), constitute one of the three major classes of flavonoids encompassing also the subclasses PAs and flavonols (Fig. 1). Flavonoids accumulate preferentially in the skin and seeds of grapes (Downey et al. 2004) not only determining the colour of the wine but also influencing the final flavour and astringency of red and white wine when extracted from the ripe berries which makes them potent food

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quality determinants (Ristic et al. 2010; Sánchez-Moreno et al. 2003). Furthermore, flavonoids extracted from grape berries and seeds exhibit strong nutraceutical potential with a broad spectrum of pharmacological and therapeutic effects (reviewed in Nassiri-Asl and Hosseinzadeh 2009). Although it is generally assumed that the benefit of a diet rich in fruits and vegetables is attributed to the additive and synergistic effects of many phytochemicals and nutraceuticals present in whole foods (Liu 2004), in vitro studies using single flavonoids on human cell cultures indicate that these polyphenols alone appear to have potent antioxidative and hydrogendonating (radical-scavenging) potential (Bagchi et al. 2000, 2003; Grotewold 2006). With respect to these properties and their already established marketability, natural origin, widespread occurrence and diversity in many fruits, flavonoids could be appropriate therapeutic agents to assist in the treatment of various diseases when studied in more detail using epidemiologic investigations and human clinical trials. Eventually, extracts from plants with optimized flavonoid content and composition could be used within human intervention studies to ask specific questions concerning the biological activity of distinct flavonoid compounds when provided not as food supplements but within a common chemical and physical food matrix (Traka and Mithen 2011).

The flavonoid biosynthetic pathway: the gateway to flavonoid chemodiversity Several studies indicate that the ability of flavonoids to scavenge free radicals, protect against damaging UV light and perform many other functions might be attributed to a specific, highly variable set of modifications on the flavonoid scaffolds that strongly affect antioxidant capacity, stability, solubility and bioavailability of the resulting derivative. To understand the in planta function of flavonoids and make use of their nutraceutical value and influence on taste and colour of fruit, it is necessary to understand flavonoid biosynthesis. Specific modifications must be linked to particular in vivo functions and their accumulation profiles during plant development explored. Pioneer work to isolate genes involved in flavonoid biosynthesis has been generated not only in Arabidopsis (Koornneef 1990) but also in maize (Zea mays), snapdragon (Antirrhinum majus) and petunia (Petunia hybrida; reviewed in Holton and Cornish 1995 and Winkel-Shirley 2001). More recently, fruit crops including apple, bilberry and grapevine gain increasing interest from researchers (Takos et al. 2006a, b; Allan et al. 2008; Jaakola et al. 2002; Boss et al. 1996a, b; Bogs et al. 2005; Hichri et al. 2011) as they possess substantial amounts of flavonoids which influence the quality of the respective fruit. In general, phenylpropanoid biosynthesis and subsequent flavonoid production are tightly linked to primary metabolism via

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Fig. 1 Simplified presentation of phenylpropanoid biosynthesis in V. vinifera highlighting the flavonoid-specific branch of the pathway. The chloroplast harbouring the precursory shikimate pathway is bordered by a green line. Note that biosynthetic steps involved in production of the aromatic amino acids phenylalanine, tryptophane and tyrosine are not indicated. The classes of phenylpropanoids are indicated in boxes including the major flavonoid subclasses: flavonols (light green), anthocyanins (blue) and proanthocyanidins (PAs, brown). Abbreviations for enzymatic steps (in red) include (in alphabetical order): 4CL 4coumaroyl-coA synthase, ANR anthocyanidin reductase, C4H cinnamate-4-hydroxylase, CHI chalcone isomerase, CHS chalcone

synthase, DFR dihydroflavonol 4-reductase, F3H flavanone-3βhydroxylase, F3′H flavonoid-3′-hydroxylase, F3′5′H flavonoid-3′,5′hydroxylase, FLS flavonol synthase, HCT hydroxycinnamoyl-coA shikimate/quinate hydroxycinnamoyl transferase, LAR leucoanthocyanidin reductase, LDOX leucoanthocyanidin dioxygenase, PAL phenylalanine ammonia lyase, STS stilbene synthase, UFGT UDP-glucose: flavonoid-3-O- glucosyltransferase, UGT UDP-glycosyltransferase. For simplification, only glycosylated forms of flavonols are depicted while flavonoids catalyzed by the enzymes DFR (leucoanthocyanidins) and LDOX (anthocyanidins) have been omitted to highlight the final products anthocyanins and PAs

the plastidial-localized shikimate acid pathway, which channels approximately 20% of the carbon fixed by photosynthesis into

aromatic amino acid production, most prominently, phenylalanine as a precursor of flavonoids, other phenolics and nitrogen-

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containing secondary metabolites (Fig. 1). Catalyzing the first committed step into the flavonoid biosynthetic pathway, chalcone synthase (CHS) plays a pivotal role to provide a common chalcone precursor for production of all intermediates and final products of the flavonoid biosynthetic pathway which are therefore biogenetically and structurally related (Fig. 1). Chalcones are minor flavonoids that are rarely detected in plants because they are either rapidly isomerized by chalcone flavanone isomerase (CHI) or spontaneously isomerise, even in the absence of CHI, to form naringenin (the basic flavanone; Holton and Cornish 1995). In most plants, including grapevine, flavanones are preferentially used as substrates for flavanone-3β-hydroxylase (F3H) which produces dihydroflavonols as an important branch point flavonoid and essential substrate for all classes of downstream compounds (Fig. 1). The biosynthesis of flavonol aglycones via flavonol synthase 1 (FLS1; Downey et al. 2003b; also named FLS4, Fujita et al. 2006) as well as the biosynthesis of PA and anthocyanin precursors via dihydroflavonol 4-reductase (DFR) employ dihydroflavonols as substrates thereby directly competing for the same substrate (Fig. 1). DFR reshuffles substrates away from flavonol biosynthesis and converts dihydroflavonols to leucoanthocyanidins which are precursors for PA- and anthocyanin biosynthesis (Martens et al. 2002). Whilst DFR is specific for the anthocyanin/PA pathway, flavonoid-3′-hydroxylase (F3′H) and flavonoid-3′,5′-hydroxylase (F3′5′H) gene products are necessary for the production of all subclasses, namely flavonols, anthocyanins and PAs. In general, hydroxylation of the B-ring of dihydroflavonols, flavanones and flavones changes the colour of the resulting anthocyanin-derived pigment and increases dramatically the chemodiversity of flavonols, PAs and anthocyanins (Holton et al. 1993; Brugliera et al. 1999; Bogs et al. 2006). By the catalytic action of DFR, leucoanthocyanidins are produced which are either converted to anthocyanidins or catechin by the competitive actions of leucoanthocyanidin dioxygenase (LDOX) or leucoanthocyanidin reductase (LAR; Fig. 1, Bogs et al. 2005). Anthocyanidins are extremely unstable and rapidly converted either to anthocyanins or epicatechin in a competitive manner by the action of UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT) and anthocyanidin reductase (ANR; Fig. 1, Bogs et al. 2005; Walker et al. 2007). The flavonoid biosynthetic pathway as shown in Fig. 1 provides several basic scaffolds of flavonoids but is unable to explain the tremendous diversity of flavonoids found in nature. However, only very limited knowledge is currently available about the underlying biosynthetic genes (Bailly et al. 1997; Ford et al. 1998; Hugueney et al. 2009; Ono et al. 2010), their developmental and environmental regulation and the physiological functions of the resulting compounds.

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MYB factors: key regulators of flavonoid biosynthesis and biotechnological tools to study and manipulate flavonoid chemodiversity in grapes Despite the intermittent knowledge considering biosynthesis of flavonoids, their accumulation patterns during berry development are well studied (Fig. 2). Grape flavonoids localize preferentially in both the skin and seeds of ripening berries but only to a negligible amount in the mesocarp (Adams 2006; Conde et al. 2007; Braidot et al. 2008) although some red-fleshed varieties exist which accumulate anthocyanins in the mesocarp (Castellarin et al. 2011). While anthocyanins and flavonols are not detectable in seeds, PAs are largely present as free flavan-3-ol monomers (especially catechin and to a minor extent also epicatechin) and PA polymers, which are extracted to the final product wine (Downey et al. 2003a, b; Bogs et al. 2005). PA biosynthesis and accumulation strongly differ between Arabidopsis and grapes: while in Arabidopsis, PAs have been solely detected as monomeric or polymeric epicatechin forms in the seed coat (only ANR, not LAR is encoded in the Arabidopsis genome;

Fig. 2 Schematic representation of the accumulation of flavonoids in grape skin during berry development. Flavonoid accumulation during berry development is colour-coded with flavonols in light green, proanthocyanidins in brown and anthocyanins in blue (refer also to Fig. 1). Note that flavonol and PA accumulation and underlying gene expression profiles have been measured earliest 10 weeks before the onset of ripening which is indicated by an arrow, leaving open the possibility that both compounds accumulate even earlier. As only the flavonoid class PA is present in seeds, only comparative flavonoid accumulation patterns in skins are shown

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Devic et al. 1999), they are present as both catechin- and epicatechin-based PAs in the seed coat, epidermal layers of the inner integument of grape seeds and skins (Downey et al. 2003a; Bogs et al. 2005; Adams 2006; Cadot et al. 2006). In comparison to skins, total PA content is reported to be significantly higher in seeds, although the mean degree of polymerization is generally several-fold lower in the seeds at all stages of berry development (Souquet et al. 1996; Downey et al. 2003a; Bogs et al. 2005). In addition, the composition of the smaller polymers of seeds is usually different from that of the skin, comprising predominantly epicatechin in the extension subunits and several classes of flavan-3-ols (epicatechin, epicatechingallate and catechin) as terminal units (Downey et al. 2003a; Bogs et al. 2005). This suggests that PA biosynthesis differs not only from plant to plant but also from one tissue to another. In skin of ripening berries, flavonoids are not evenly distributed within the different cell layers. In the outermost cell walls of the epidermis, low levels of flavonoids but high amounts of the phenolics cutin, lignin, and suberin are present, while the inner thick-walled layers of hypodermis contain most of the skin flavonoids (Adams 2006; Braidot et al. 2008). In red grape varieties, anthocyanins and flavonols co-localize in these cell layers with PAs (Adams 2006; Doshi et al. 2006). Accumulation of PAs occurs early in grape berry development and is completed when ripening initiates. Changes in PA content during later stages of berry development have been largely attributed to the decreased extractability of PAs, which is thought to be the result of complexation of the PA polymers with other cellular components (Downey et al. 2003a; Kennedy et al. 2001). Besides their accumulation during ripening of the grape berry, flavonols have been also detected in various other grapevine organs such as stems, tendrils, pedicels, petioles and developing leaves (Hmamouchi et al. 1996; Souquet et al. 2000; Downey et al. 2003b; Fujita et al. 2006). PA biosynthesis of different polymer lengths and compositions has been reported in stems, leaves and flowers (Souquet et al. 1996; Kennedy et al. 2001; Bogs et al. 2005), while in contrast to other plants, grapevine organs such as leaves, tendrils, stems and roots do not produce significant amounts of anthocyanins during plant development (Boss et al. 1996b; Kobayashi et al. 2002). The known biosynthetic pathway of flavonoids share common enzymatic steps, whereas the activities of enzymes specific for PAs, anthocyanins or flavonols lead exclusively to the biosynthesis of the respective flavonoid by competing for common substrates (Fig. 1). Therefore, a spatiotemporal regulation of this pathway has to occur to navigate biosynthesis of different flavonoids during grape berry development to avoid contention for common substrates. This theory has been underlined by several independent studies showing not only tissue-dependent but also temporally separated flavonoid

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accumulation pattern that could be linked to the expression of flavonoid pathway genes. An increase in transcript levels of a specific LAR isoform (LARII) and ANR after anthesis determines when PA biosynthesis is initiated (Bogs et al. 2005), an accumulation which is temporally delayed to flavonol biosynthesis mediated by an increase of transcripts encoding FLS (FLS1 and maybe to a minor extent also FLS2) just before flowering (Fig. 2; Downey et al. 2003b). Later during development, the onset of UFGT expression determines véraison, the beginning of ripening which is defined by the accumulation of sugars and anthocyanin pigments in the berries of red cultivars (Fig. 2; Boss et al. 1996a, b; Robinson and Davies 2000; Kobayashi et al. 2002). In order to understand how flavonoid biosynthesis and diversity are regulated in grapevine, several transcription factors (TFs) controlling the expression of the known flavonoid biosynthetic genes have been isolated and characterized recently. This was experimentally accessible, as transcriptional control of flavonoid biosynthesis is one of the best-specified regulatory systems in plants, integrating both developmental and/or various biotic and abiotic stress signals to the promoters of flavonoid biosynthetic gene via control of TFs (reviewed in Grotewold 2006). The characterization of plant regulatory proteins has been carried out in plants that classically have been used to study flavonoid biosynthesis (e.g., in maize, snapdragon, petunia) as well as in convenient model plants like Arabidopsis (WinkelShirley 2001) and more recently in plants with potent economical use such as apple and grapevine (Takos et al. 2006a; Bogs et al. 2007; Walker et al. 2007; Czemmel et al. 2009). Grapevine is currently one of the best studied crop plant in terms of regulation of flavonoid biosynthesis by TFs in fruit which control spatiotemporal production of the appropriate compounds during plant development (Fig. 2). In all species analysed to date, the common denominators in the regulation of structural flavonoid pathway genes are members of protein families containing R2R3-MYB domains, which are common to control biosynthesis of all classes of flavonoids whereas co-factors encoding the basic helix–loop–helix (bHLH) domains (also referred to as MYC proteins) and conserved WD repeats (WDR) have been found so far exclusively in regulation of anthocyanin/PA but not flavonol biosynthesis (Weisshaar and Jenkins 1998; Stracke et al. 2001; Marles et al. 2003; Hichri et al. 2010, 2011). R2R3-MYB proteins have been identified to be the key determinants in regulatory networks controlling not only the allocation of specific gene expression patterns during flavonoid biosynthesis, but also diverse aspects of development and responses to biotic and abiotic stresses which are not related to production of secondary metabolites (Stracke et al. 2001). In vertebrates, the MYB gene family includes C-MYB, A-MYB and B-MYB, whereas in plants,

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especially in grapevine, the MYB family is much more expanded constituting one of the most abundant groups of TFs described in plants (Matus et al. 2008). The bHLH proteins may have overlapping regulatory targets (Zimmermann et al. 2004; Koes et al. 2005) which are represented by their expression throughout grape berry development (Hichri et al. 2010), whereas the precise molecular function of WDRs in the ternary complex is still unclear with authors suggesting functions at the transcriptional (Sompornpailin et al. 2002) or post-translational level (Lloyd et al. 1992). At the early phase of grape berry development, expression of genes of the general flavonoid biosynthetic pathway and FLS1 are induced to initiate biosynthesis of flavonols (Downey et al. 2003b; Czemmel et al. 2009). Interestingly, flavonols are the only flavonoid compounds which accumulate before anthesis and even increase during early flowering stages, probably to provide UV protection to the inflorescence and pollen (Fig. 2). This is due to the expression profile of the key flavonol regulator MYBF1 which has been shown to regulate FLS1 transcription thereby ensuring flavonol production at early phases of berry development (Fig. 2; Czemmel et al. 2009). This study could demonstrate that MYBF1 is not solely responsible for flavonol biosynthesis during early grape berry development but is also lightregulated implicating a role of this MYB factor in both lightdependent and developmental-independent accumulation of flavonols in grapevine berries. After flowering, MYBF1 and, concomitantly, FLS1 transcripts and flavonols rapidly decrease whereas PA biosynthesis is induced until véraison (Downey et al. 2003a, b; Czemmel et al. 2009). PA accumulation initiates at fruit set and achieves its maximum before véraison which has been shown to be determined by the pre-véraisonal presence of MYBPA1, MYBPA2 and probably also MYB5a. These TFs control the expression of genes encoding central pathway enzymes (CHS, CHI, DFR, LDOX) and PA-specific enzymes (LAR, ANR) necessary for the accumulation of PAs from anthesis to véraison (Figs. 2 and 3; Downey et al. 2003a; Bogs et al. 2005, 2007; Terrier et al. 2009; Deluc et al. 2006, 2008). Véraison hallmarks the beginning of the second growth phase and represents a major switch in the flavonoid biosynthetic pathway from PA to anthocyanin production (Fig. 2). This is represented by TF gene expression: at véraison, transcripts of MYB5a & 5b, MYBPA2 and all PA-specific genes decrease while anthocyanin-specific gene expression (MYBA and UFGT) increases leading to the switch from PA biosynthesis to anthocyanin biosynthesis in grape berries (Kobayashi et al. 2002; Bogs et al. 2005; Walker et al. 2007; Terrier et al. 2009). Anthocyanin biosynthesis is a characteristic of ripening berries which defines véraison, the onset of ripening in red grape cultivars. Although genes encoding the TFs MYBA1 and MYBA2 as well as the last biosynthetic enzyme UFGT

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Fig. 3 Model of the genetic regulation of the flavonoid pathway by R2R3 MYB TFs leading to biosynthesis of anthocyanins, flavonols and PAs. The major flavonoids and the corresponding regulatory TFs are colour-coded with flavonols in light green, anthocyanins in blue and proanthocyanidins (PAs) in brown. Note that only previously characterized TFs are shown: anthocyanin regulators MYBA1 & A2 are summarized as MYBA and PA regulators MYBPA1 & PA2 are summarized as MYBPA. To further simplify the regulatory scheme, general regulators such as MYB5a and MYB5b have been omitted. Abbreviations for the enzymatic steps are identical to Fig. 1. To simplify the pathway, hydroxylation steps catalyzed by F3′H and F3′5′H are omitted. Subsequently, naringenin, eriodictyol and pentahydroxy-flavone are summarized as flavanones whereas dihydrokaempferol, dihydroquercetin and dihydromyricetin have been summarized as dihydroflavonols. The products of the enzymatic steps DFR (leucoanthocyanidin) and LDOX (anthocyanidin) are added (see Bogs et al. 2006, 2007)

are present in both red- and white-fruited grapevine cultivars, UFGT-mediated anthocyanin biosynthesis does not occur in white-fruited vines due to mutations in the genes encoding MYBA1 and MYBA2 (Walker et al. 2007). The accumulation of anthocyanins in red varieties is related to the reinduction of transcripts of general pathway enzymes including CHS isoforms, CHI, F3H, F3′H and F3′5′H and LDOX. The ripening specific TFs MYBA1 and MYBA2 initiate specifically UFGT expression which results in a maximum pigment bulge in the latest phases of fruit maturation (Boss et al. 1996a; Goto-Yamamoto et al. 2002; Walker et al. 2007). Considering this specificity of MYBA for UFGT gene expression, it could be speculated which factors control expression of the central pathway genes important to provide substrates for UFGT-mediated glycosylation. To do that, MYBPA1 and MYB5b are likely candidates as they show peaks of expression in skin tissue around 2 and 8 weeks after véraison (Bogs et al. 2007; Deluc et al. 2008). Given the facts that anthocyanins need to be produced and PAs are still

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abundant in grape skins following véraison, MYBPA1 and, later during berry development, MYB5b might compensate for the high specificity of MYBA for UFGT expression and activate the shared genes of the flavonoid pathway (CHS, CHI, DFR and LDOX) required for PA and anthocyanin biosynthesis. Interestingly, during ripening, FLS1 expression and flavonol biosynthesis are reinduced leading to a significant accumulation of flavonols in the ripe berry, a profile which is inimitable regarding flavonoid biosynthesis in grapevine as flavonols have been shown to be the only components of the pathway whose accumulation pattern peaks before and after véraison (Fig. 2; Downey et al. 2003b). This accumulation pattern, established by increased FLS1 transcription and probably protein production, could not be explained by the expression pattern of MYBF1 (Czemmel et al. 2009) leaving open the question how FLS1, or in general the flavonol pathway, is transcriptionally or post-transcriptionally regulated during late phases of berry development. An identification of this regulatory mechanism might help us to understand the biological role of flavonols in ripe berries, presumably not only to provide UV protection but also to stabilize pigmentation by building aggregates with anthocyanins (reviewed in Boulton 2001) or prevent them from photo-bleaching (Yamasaki et al. 1996). Although the stabilization of anthocyanin-derived pigmentation by flavonols has been experimentally proven solely in aqueous solution (Baranac et al. 1997; Dimitrić Marković et al. 2005), it provides an interesting field of research because it could help us to design biotechnological tools to stabilize coloration of berries and wine. Taken together, a model can be drawn with R2R3 MYB TFs playing pinpointing roles for the determination of flavonoid accumulation profiles at defined developmental phases and in specific tissues: MYB proteins for anthocyanin– (MYBA1 & A2), PA– (MYBPA1 & PA2) or flavonol biosynthesis (MYBF1) exist which activate genes involved in biosynthesis of the respective compound. In addition, in contrast to anthocyanin regulators, PA and flavonol regulators are also able to coordinately activate genes of the central pathway to redirect substrate flow within the flavonoid pathway (Fig. 3). Beside these MYB factors, TFs exist (MYB5a & 5b) which are able to activate preferentially genes of the central pathway and are therefore not considered to be specific for genetic regulation of one distinct branch of the flavonoid pathway. Considering their accumulation pattern in ripening berries, flavonoid subclasses are present throughout berry development presumably to fulfil various biological functions (Fig. 2). However, so far, no study provided a comprehensive picture on how to unravel the differential composition of flavonoids during fruit ripening which is of great interest, as modification states significantly affect biological function by influencing stability, bioavailability, solubility and chemo-

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preventive potential of the resulting derivatives. In this review, the transcriptional potential of flavonoid-specific MYB factors has been reviewed and their prodigious influence on the accumulation of distinct flavonoids during grape berry development was highlighted. Based on the above summarized knowledge, R2R3 MYB TF provides promising biotechnological tools for overexpression studies that will allow a more detailed view on the enzymes modifying basic flavonoid skeletons, data that could be conveyed to the developing berries by comparative gene expression analysis (for similar studies, see Terrier et al. 2009; CutandaPerez et al. 2009). With the grapevine model “hairy roots,” a suitable system for the determination of in vivo function is available and co-expression of modifying genes together with the respective MYB regulator will presumably lead to the accumulation of certain flavonoid compounds whereas biochemical characterization of promising target enzymes will be performed to determine substrate specificity. Extracts of hairy roots overexpressing MYB TFs, which are specifically enriched in a respective flavonoid, could be tested for their chemo-preventive effects (Lee et al. 2008) in cell- and enzyme-based in vitro marker systems relevant for inhibition of carcinogenesis (Gerhäuser et al. 2003) or their suitability as nutraceutical food supplements (reviewed in Martin et al. 2011). In order to improve viticultural applications, responsiveness of the flavonoid pathway including transcription of structural and regulatory gene expression to environmental conditions such as pathogen attack, mineral depletion, UV light stress, water availability or hormonal status of the developing berry needs to be carefully assessed in future research. Based on recent results, we conclude that flavonoid-specific R2R3 MYB TFs in combination with Next Generation sequencingbased technologies (RNA-Seq, Chip-Seq) provide ideal molecular markers and tools to analyse transcriptomic adaptations in flavonoid metabolism in response to developmental or environmental cues. Knowledge generated might provide the genetic understanding and platform to manipulate flavonoid content and composition in grape berries not only by viticultural management practices but also by molecular breeding approaches. As classical breeding of new grapevine cultivars can take up to 40–50 years and plant transformation is cost-intensive and not accepted by most European consumers, there is a growing interest in metabolic engineering strategies such as marker-assisted breeding. As the most desirable criteria for grape berry and its product wine are agricultural productivity, human nutrition, taste, colour and human health, molecular markers for traits such as sugar concentration, acidity, various aroma compounds and flavonoid content and composition would be very useful for the breeding of high-quality fruit. Therefore, genomic molecular markers are an important tool for grapevine breeding, which, during the last decades, was focussing on the resistance

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against powdery and downy mildew. The cataloguing of flavonoid chemodiversity and the underlying biosynthetic pathways will increase the ability to design molecular markers for highquality fruits, with the aim of generating plants with optimized flavonoid content and composition which are able to deal with a multitude of stresses including mineral depletion, UV irradiation and pathogen attack. This is of special interest in grapes, as a large variety of grape cultivars exist that strongly differ in flavonoid content and composition. While it is known that complete loss of anthocyanin production in white grapes arose due to a loss of function of the anthocyanin regulators MYBA1 and MYBA2 (Walker et al. 2007), only little is known about the reasons for cultivar-dependent variations in flavonoid content and composition in grapes (Mattivi et al. 2006; Sánchez-Moreno et al. 2003). Cultivar-specific allelic variation in the promoter and also the coding regions of MYBA genes are linked to colour variation between different cultivars (This et al. 2007; Fournier-Level et al. 2009). This indicates that similar links could exist also between MYBF/MYBPA genes and flavonol/PA content in different grape varieties. The promoter and coding regions of flavonoid MYB genes are therefore promising targets for the development of molecular markers for grapevine breeding with the aim of optimizing flavonoid content and composition in grapes. Acknowledgements The authors wish to apologize to those colleagues whose work could not be cited due to space constraints. We wish to thank the Bundesministerium für Bildung und Forschung (BMBF) and its GABI initiative for financial support. Conflict of interest The authors declare that they have no conflict of interest.

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