Introduction

Anthocyanins are flavonoid compounds found in plants. Their diverse colors, ranging from red to blue, can be observed in horticultural crops, such as ornamentals, fruits, and vegetables. Anthocyanins play substantial roles in the functions of horticultural plants, and their presence determines fruit quality and flower colors as well as providing nutritional and pharmaceutical properties in vegetables, features which are of interest to flower enthusiasts and plant breeders.

Generally, anthocyanins (cyanidin, pelargonidin, and delphinidin derivatives) are synthesized via three main branches of the biosynthetic pathway (Fig. 1), and transcriptional regulation of the structural genes [such as chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS)] involved in the three main branches leads to the production of the different flavonoid pigments in many plant species.

Fig. 1
figure 1

Three main branches of anthocyanin biosynthetic pathway in plants. CHS chalcone synthase enzyme, CHI chalcone isomerase, F3H flavanone 3-hydroxylase, F3′H flavanone 3′-hydroxylase, F3′5′H flavanone 3′,5′-hydroxylase, DFR Dihydroflavonol 4-reductase, ANS anthocyanidin synthase, UFGT UDP-glucose: flavonoid 3-O-glucosyltransferase

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Over the past decade, the roles of transcription factors (TFs) in the regulation of transcript levels of structural genes have been identified in many plant species (Allan et al. 2008). Based on their DNA binding domains, TFs are generally classified into three main families, namely, R2R3-MYB domains, basic helix-loop-helix (bHLH) domains, and conserved WD40 repeats (WDR) (Ramsay and Glover 2005). R2R3-MYBs have been shown to be involved in phenylpropanoid metabolism (Hichri et al. 2011) and activate the structural genes involved in the anthocyanin biosynthetic pathway in many plants, including model and horticultural crops (Albert et al. 2014; Xie et al. 2014; Zhang et al. 2017). Metabolic genetic engineering via anthocyanin regulatory MYBs can change cell metabolism by altering its pathway enzyme(s) or regulatory protein(s) using recombinant DNA technology; thus, metabolic genetic engineering of MYBs has generated diverse colors and color patterns in several ornamental plants and produced anthocyanin-rich fruits and vegetables with high nutritional and pharmaceutical properties (Laitinen et al. 2008; Hichri et al. 2011). However, MYBs that suppress the structural genes in some plant species have also been identified. In horticultural plants, differential roles of MYBs regarding transcriptional control of the structural genes have not been thoroughly reviewed. In addition, amino acid sequences of MYBs that exhibit different functional roles have not been phylogenetically analyzed.

In the present review, we describe which R2R3-MYBs promote or suppress the expression of the structural genes involved in the anthocyanin biosynthesis pathway of ornamental plants, fruits, and vegetables and analyze the phylogenetic relationships of the MYBs. In addition, we highlight studies that show the enhancement of anthocyanin contents in horticultural crops by metabolic genetic engineering of anthocyanin regulatory MYBs.

Functional roles of R2R3-MYBs in anthocyanin production

Ornamental plants

Diverse flower colors produced via different branches of the anthocyanin pathway are found in ornamental plants, in which TFs such as MYB, bHLH, and WDR individually or coordinately promote transcript levels of structural genes that determine flower colors. Although several R2R3-MYBs that are involved in color pigmentation of many ornamental plants have been identified, some MYBs that suppress anthocyanins have also been reported in some ornamental plants. Nevertheless, those MYBs that have been reported so far have not been thoroughly summarized to date. In the present review, MYBs that promote or suppress structural genes involved in the anthocyanin production pathway of ornamental plants are well summarized (Table 1). In addition, the phylogenetic relationships of the MYBs associated with the anthocyanin production or suppression in ornamental plants are also presented (Fig. 2).

Table 1 Identified R2R3-MYBs that promote or suppress anthocyanin structural genes in ornamental plants
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Fig. 2
figure 2

Phylogenetic analysis of protein sequences of R2R3-MYB transcription factors associated with regulation or suppression of anthocyanin in ornamental plants. The evolutionary history was inferred using the Neighbor-joining method. The bootstrap consensus tree inferred from 1500 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% of the bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1500 replicates) are shown next to the branches (Felsenstein 1985). The evolutionary distances were computed using the p-distance method and are in units of the number of amino acid differences per site. The analysis included 37 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 434 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). All R2R3-MYB sequences were retrieved from the GenBank database and accession numbers are as follows: Petunias PhAN2 (ABO21074), PhAN4 (ADQ00392), PhDPL (ADW94950), PhPHZ (ADW94951), PhMYB27 (AHX24372); Rose RhMYB10 (ABX79949); Gentian triflora GtMYB3 (BAF96933); Mimulus guttatus MgPELAN (AHJ80987), MgNEGAN (AHJ80988), MgLAR1 (ALP48587); Morning glory IpMYB1 (BAE94388); Asiatic lily LbMYB6 (BAJ05399), LbMYB12 (BAJ05398), LbMYB12-Lat (BAO04194); Oriental lily LhMYB12 (BAJ22983), LrMYB15 (BAU2993); Antirrhinum majus AmROSEA1 (ABB83826), AmROSEA2 (ABB83827), AmVENOSA (ABB83828), AmMYB305 (P81391), AmMYB340 (P81396); Torenia fournieri TfMYB1 (BAN17385); Anthurium andraeanum AaMYB2 (AML84515); Chrysanthemum CmMYB6 (AKP06190), CmMYB1 (AEO27497); Gerbera GmMYB10 (CAD87010), GmMYB11 (ACG69457); Phalaenopsis equestription PeMYB2 (AIS35919); Phalaenopsis equestris PeMYB11 (AIS35928), PeMYB12 (AIS35929); Oncidium grower ramsey OgMYB12 (ABS58501); Dendrobium spp. DwMYB2 (AAO49411); Lotus Nelumbo nucifera NnMYB5 (ALU11263); Epimedium sagittatum EsMYBA1 (AGT39060), EsAN2 (ALO24363); Poplar MYB182 (AJI76863); Arabidopsis thaliana AtTT2 (NP_198405.1), which is involved in regulation of proanthocyanidin biosynthesis, is included as an outgroup

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Several MYBs [anthocyanin 2 (PhAN2), anthocyanin 4 (PhAN4), purple haze (PhPHZ), and deep purple (PhDPL)] determine the flower color of petunias (Quattrocchio et al. 1993; Spelt et al. 2000; Albert et al. 2009, 2011), with PhAN2 being responsible for the red coloration in the corolla and PhAN4 inducing the same in the anthers and corolla tubes by interacting with bHLH TF anthocyanin 1 (PhAN1). However, their activation in specific tissues depends on the light conditions because they induce weak pigmentation in the flower bud surface, but strong pigmentation in the leaves, under high light conditions (Albert et al. 2009, 2011). PhDPL and PhPHZ also activate the transcript levels of the same structural genes; however, the former determines the color pattern venation in the corolla tubes, whereas the latter is responsible for blushing of the flower bud (Albert et al. 2011) and induces pigmentation under light conditions where PhAN1 is highly expressed. Albert et al. (2011) suggested that both MYBs have the amino acid signature motif ([DE]Lx2[RK]x3Lx6Lx3R), which has been identified as being important for interaction with another co-regulator, bHLH, in Arabidopsis (Zimmermann et al. 2004). Thus, in the petunia, the production of anthocyanin by anthocyanin MYBs would occur via interaction with the co-transcription factor bHLH. Although the abovementioned MYBs are grouped in a clade and participate in the induction of anthocyanin biosynthesis (Fig. 2), PhMYB27, which is not related to the above MYBs, appeared to be an anthocyanin suppressor because it was highly expressed under non-anthocyanin-inductive shade conditions and was suppressed during anthocyanin-inductive light conditions (Albert et al. 2011, 2014). In addition, its homologous expression in the petunia also resulted in the reduction of anthocyanin in the flowers (Fig. 3) (Albert et al. 2014). Moreover, PhMYB27 has strong sequence similarity to that of CmMYB1 from chrysanthemums and PtMYB182 from poplars, which also appeared to be MYB suppressors because they have been shown to downregulate structural genes involved in anthocyanin pathway (Zhu et al. 2013; Yoshida et al. 2015). In addition, the suppressor CmMYB1 is not grouped in the same clade as the anthocyanin regulator CmMYB6 (Liu et al. 2015). Similarly, GmMYB10 from the gerbera, which is a homolog of CmMYB6 and produces pelargonidin in vegetative and flower tissues by interacting with GmMYC1 (Laitinen et al. 2008), is not grouped in the same clade as GmMYB11, which is not involved in anthocyanin production (Elomaa et al. 2003). In fact, GmMYB11 has been found to be a homolog of the suppressor PhMYB27. This indicates that the MYBs associated with anthocyanin production or suppression in flowers can be identified by phylogenetic analysis.

Fig. 3
figure 3

PhMYB27 is a suppressor of anthocyanin pigmentation. This image was taken from a publication by Albert et al. (2014) and is copyrighted by the American Society of Plant Biologists

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Morita et al. (2006) suggested that MYB1 (a homolog of PhAN2) activated anthocyanin structural genes in the flower limbs and tubes of the morning glory by interacting with the co-TFs WDR1 and bHLH2. Similarly, in the gentian, GtMYB3 (a homolog of PhAN2) interacted with GtbHLH1 (a homolog of PhAN1) and produced anthocyanin via upregulation of the structural genes (Nakatsuka et al. 2008). A similar regulatory role of anthocyanin-related MYBs has been confirmed in tobacco. It is likely that these MYBs, which are homologs of PhAN2, regulate the structural genes by interacting with co-TFs in a similar way to PhAN2.

According to phylogenetic analysis, sequences of the anthocyanin regulatory MYBs (MgPELAN, MgNEGAN, AmVENOSA, AmROSEA1, and AmROSEA2) existing in Mimulus guttatus and Antirrhinum majus are related by divergent evolution from a common ancestor; however, the sequences within the same species are more homologous. In Mimulus guttatus, MgPELAN is responsible for pigmentation in the petal lobe, whereas MgNEGAN induces anthocyanin spots in the nectar guide (Lowry et al. 2012; Yuan et al. 2014, 2016). In addition, the functional role of MgPELAN can be confirmed by its low transcript level in yellow flowers (Yuan et al. 2014). In A. majus, AmROSEA1 induced strong red coloration in both epidermal layers of the corolla by activating the transcript levels of F3H, F3′H, DFR, FLS, and UFGT; AmROSEA2 induced weak pigmentation in the inner epidermal layer of the corolla by increasing the expression level of F3′H; and AmVENOSA influenced the venation color patterns in the petals by enhancing the transcript levels of CHI, F3H, F3′H, FLS, ANS, UFGT, and AT (Schwinn et al. 2006; Shang et al. 2011). Interestingly, AmMYB305 and AmMYB340 are not phylogenetically related to AmVENOSA, AmROSEA1, and AmROSEA2; however, they are related to MgLAR1 from Mimulus guttatus and participate in anthocyanin regulation, in which MgLAR1 regulates the flavonoid synthase that determines corolla patterning (Lowry et al. 2012; Yuan et al. 2014, 2016), whereas AmMYB305 and AmMYB340 promote anthocyanin production (Moyano et al. 1996) by activating a subset of anthocyanin structural genes. Although the MYBs participated in anthocyanin production, the reasons why they are not grouped in the same clade as other MYBs from the same species is still unclear. TfMYB1 from Torenia fournieri controls the red pigmentation in the torenia flower by elevating the transcript levels of early (TfCHS and TfF3H) and late (TfDFR, TfANS, and TfUFGT) biosynthetic genes (Nishijima et al. 2013), and is a member of the clade containing the MYBs MgPELAN, MgNEGAN, AmVENOSA, AmROSEA1, and AmROSEA2.

As for the abovementioned ornamental plants, color pattern variations (such as red, pink, and purple) in Anthurium andraeanum (Hort.) were determined by AaMYB2 and were associated with its differential expression (Li et al. 2016), in which AaMYB2 mainly upregulated the transcript levels of AaF3H and AaANS. The same functional role of AaMYB2 has been shown in tobacco by the induction of anthocyanin via transcriptional activation of the same structural genes. In fact, most of the sequences of anthocyanin regulatory MYBs are variations of the AaMYB2 sequence, which is synonymous with EsMYBA1 and EsAN1 from Epimedium, which influence pigmentation in the leaves, flowers, and flower buds (Huang et al. 2013, 2016). In addition, NnMYB5 from the lotus (Nelumbo Adans; Nelumbonaceae), which has strong sequence similarity with EsAN2, induced attractive flower color (Sun et al. 2016). An identical functional role of this MYB was observed in Arabidopsis, resulting in anthocyanin accumulation in the flower stalks.

It has been well demonstrated that anthocyanin production in the lily is controlled via activation of the transcript levels of structural genes by several MYBs whose sequences are phylogenetically related to each other (Nakatsuka et al. 2009; Yamagishi et al. 2010, 2012, 2014; Yamagishi 2011, 2016; Suzuki et al. 2016); however, their functional roles are slightly different. For example, in the Asiatic lily, LhMYB6 controlled the formation of red spots in the tepals and leaves, whereas LhMYB12 did so in the tepals, filaments, and styles (Nakatsuka et al. 2009; Yamagishi et al. 2010; Suzuki et al. 2016). LhMYB12-Lat (an allele of LhMYB12 from the Asiatic lily) and LhMYB12 (from the Oriental lily) that are grouped in the same clade as LhMYB12 from the Asiatic lily exhibited the same pigmentation in the tepals as that of LhMYB12 in the Asiatic lily by enhancing the transcript levels of the structural genes (Yamagishi 2011; Yamagishi et al. 2012, 2014). These MYBs interacted with LhbHLH2, which was confirmed by co-expression of LhbHLH2 and MYB6 or MYB12 in the lily bulb scales. However, LrMYB15 from Lilium regale, which is a homolog of LhMYB6, was thought to be responsible for anthocyanin production (Yamagishi 2016) because anthocyanin accumulation was observed only in the organs where LrMYB15 was expressed, whereas LhbHLH2 was expressed in all the organs. However, this appeared to be a light-induced MYB because under shade conditions the MYB could not be detected, and anthocyanin production was lower than that under light conditions. A similar function of LrMYB15 has been confirmed in tobacco. RhMYB10 from the rose, which is a divergent of the above MYBs from the lily, also regulated anthocyanin production; however, its functional roles have not yet been clearly revealed (Lin-Wang et al. 2010).

Like the abovementioned MYBs in lilies, sequences of the anthocyanin regulatory MYBs from orchids are also phylogenetically related to each other. In Phalaenopsis spp., MYBs (PeMYB2, PeMYB11, and PeMYB12) pigmented the flower by activating the structural genes PeF3H5, PeDFR1, and PeANS3 (Hsu et al. 2015), and their inactivation resulted in the loss of color pigmentation. Moreover, they determined the induction of distinct color patterns in floral organs such as the sepals, petals, and lip; however, the formation of red spots on the lip was determined by PeMYB11 and full pigmentation in the central lobe of the lip was influenced by PeMYB12. In most species, the co-expression of MYB and bHLH, rather than an expression of a single factor, strongly induces anthocyanin production; however, in this species, co-expression of PeMYBs and PebHLHs did not affect anthocyanin production, which was probably controlled by the MYBs. OgMYB1 from Oncidium spp., which is a homolog of PeMYB11, also enhanced red pigments in lip tissues via activation of the structural genes OgCHI and OgDFR (Chiou and Yeh 2008). DwMYB2 from Dendrobium spp. enhanced pigmentation in flowers via regulation of DFR and ANS genes (Li et al. 2017) and was phylogenetically related to the anthocyanin regulatory MYB MgLAR1, but not related to the above MYBs. Recently, PmMYBa1 from Prunus mume that was previously identified as a R2R3-MYB domain increased anthocyanin in petals by regulation of late biosynthesis genes (LBGs) (Zhang et al. 2017).

In summary, two or more MYBs have been shown to control anthocyanin production in several ornamental plants via transcriptional regulation of the structural genes, however, only one anthocyanin regulatory MYB has been shown to date in some important ornamental plants. Based on phylogenetic results, sequences of most MYBs are related by divergent evolution from a common ancestor. Generally, most MYBs within the same species are more homologous than MYBs of different species; however, some MYBs within the same species that act as suppressors are not related to anthocyanin regulatory MYBs but are grouped in a clade with MYB suppressors from other species.

Fruits

In fruit crops, fruit color that is determined by anthocyanins not only indicates maturity and quality but also increases consumer preference, because the marketability of fruit highly depends on the red color. The quality of wine that is made from fruits such as grapes and apples is determined by the anthocyanin concentration, which affects the bitterness, astringency, and color. Klatsky (2002) suggested that cardiovascular disease and cancer might be ameliorated by moderate consumption of red wine. In addition, anthocyanins provide nutrients and health benefits for humans by increasing the antioxidant properties of cells and tissues (Bagchi et al. 2003; Pervaiz 2003). Thus, regulation of the color development of fruits is of particular interest. Recently, MYBs that are involved in the promotion or suppression of anthocyanin production in fruit crops have been identified. Here, we describe which MYBs are associated with anthocyanin production or suppression in many fruit crops (Table 2) as well as showing their phylogenetic relationships (Fig. 4).

Table 2 Identified R2R3-MYBs that promote or suppress anthocyanin structural genes in fruit plants
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Fig. 4
figure 4

Phylogenetic analysis of protein sequences of R2R3-MYB transcription factors associated with regulation or suppression of anthocyanin in fruit plants. The evolutionary history was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1500 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% of the bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1500 replicates) are shown next to the branches (Felsenstein 1985). The evolutionary distances were computed using the p-distance method and are in units of the number of amino acid differences per site. The analysis included 36 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 430 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). All R2R3-MYB sequences were retrieved from the GenBank database and accession numbers are as follows: Grapevine VvMYBA1 (BAD18977), VvMYBA2 (BAD18978), VvMYBA5a (AAS68190), VvMYBA5b (AAX51291), VvMYBC2-L1 (AFX64995), VvMYBC2-L3 (AIP98385); Pear Pyrus communis PcMYB10 (ABX71487); Mangosteen Garcinia mangostana GmMYB10 (ACM62751); Apple Malus domestica MdMYB (BAF80582), MdMYB1 (ABK58136), MdMYB6 (AAZ20429), MdMYB9 (ABB84757), MdMYB10 (ACQ45201), MdMYB110a (AFC88038), MdMYB11 (AAZ20431); Cherry Prunus avium PavMYBA (AHL45015), PavMYB1 (XP_021829544), PavMYB10 (ALH21137), PavMYB10.1 (ALM31949); Chinese bayberry Morella rubra MrMYB1 (ADG21957); Peach Prunus persica PpMYB9 (ALO81019), PpMYB10.1 (XP_007216530); Prunus cerasifera PpMYB10.2 (AKV89248), PpMYB10.3 (AKV89249); Prunus persica PpMYB10.4 (AHL68405), PpMYB17 (ALO81020), PpMYB18 (ALO81021), PpMYB19 (ALO81022), PpMYB20 (ALO81023); Citrus sinensis CsRuby (AFB73913); Kiwifruit AcMYB110a (AHY00342); Strawberry Fragaria ananassa FaMYB10 (ABX79947), FaMYB1 (AAK84064); Litchi Fragaria chiloensis FcMYB1 (ADK56163); Litchi chinensis LcMYB1 (APP94121); Arabidopsis thaliana AtTT2 (NP_198405.1), which is involved in regulation of proanthocyanidin biosynthesis, is included as an outgroup

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Diverse fruit colors determined by different levels of anthocyanin accumulation, such as white, red, and dark red–purple, are observed in different Chinese bayberry (Myrica rubra) cultivars and are strongly associated with the transcript level of MrMYB1, which suggests that it is the transcription level that determines the anthocyanin content in ripe fruit (Niu et al. 2009). According to phylogenetic analysis, the sequences of MYBs from kiwifruit (AcMYB110a), Chinese berry (MrMYB1), and grape (VvMYBA1 and VvMYBA2) are phylogenetically related and the MYB strongly influences the red pigmentation in the respective fruits as described for MrMYB1 (Fraser et al. 2013; This et al. 2007). Similarly, other genetically related MYBs (CsRuby and LcMYB1) have been shown to enhance anthocyanin in blood oranges and litchi fruits (Butelli et al. 2012; Lai et al. 2014). In fact, these MYBs are variations of GmMYB10, which induces red pigmentation in mangosteen (Palapol et al. 2009). Based on the phylogenetic results, VvMYBA1 and VvMYBA2 are homologous to each other, as are VvMYB5a and VvMYB5b; however, the two pairs are not grouped in the same clade and their roles in anthocyanin regulation are also slightly different. VvMYBA1 and VvMYBA2 regulate anthocyanin in grape berries by activating the expression of the last LBG, UFGT (This et al. 2007), whereas VvMYB5a and VvMYB5b are involved in tannin production during the early stage of berry development, but potentially regulate anthocyanin in ripe berry tissues (Deluc et al. 2006). However, VvMYBC2-L1 and VvMYBC2-L3, which are not related to the above MYBs from grapes, suppressed proanthocyanidin accumulation in grapevines by downregulation of structural genes (Huang et al. 2014; Cavallini et al. 2015) and are grouped in the same clade as MYBs from the peach (PpMYB17, PpMYB18, PpMYB19, and PpMYB20) and the strawberry (FaMYB1 and FcMYB1) that have also been reported as anthocyanin suppressor MYBs in their respective species (Zhou et al. 2016). Silencing FaMYB1 in the fruits significantly increased anthocyanin content and, conversely, its overexpression lowered the anthocyanin content by reducing transcript levels of ANS (Kadomura-Ishikawa et al. 2015). Similarly, high expression of FcMYB1 imparted a white color to the fruit (Salvatierra et al. 2013) and its downregulation increased anthocyanin by upregulation of ANS and strong suppression of the transcript levels of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). The phylogenetic analysis results (Fig. 2) support the evolutionary relationships among the anthocyanin MYB suppressors (PpMYB17, PpMYB18, PpMYB19, PpMYB20, FaMYB1, FcMYB1, VvMYBC2-L1, and VvMYBC2-L3).

In apples (Malus domestica), three MYBs (MdMYB1, MdMYB10, and MdMYBA) that have strong sequence homology enhance anthocyanin production in fruit skin via upregulation of structural genes (Takos et al. 2006; Ban et al. 2007; Espley et al. 2007; Telias et al. 2011; Vimolmangkang et al. 2013). These MYBs are divergent from MYB110a, which produces red coloration in the apple flesh cortex (Chagné et al. 2013). The MYBs MdMYB9 and MdMYB11, which are not phylogenetically related to the above MYBs, also promote anthocyanin accumulation by activating early biosynthesis genes (EBGs) and LBGs (MdCHS, MdCHI, MdF3H, MdANS, MdDFR, and MdANR) (An et al. 2015). PcMYB10 from pears is grouped with the anthocyanin MYBs (MdMYB1, MdMYB10, and MdMYBA) and was thought to be participating in the anthocyanin pathway (Feng et al. 2010; Pierantoni et al. 2010; Li et al. 2012) because the expression level of this MYB was significantly higher in the red-skinned pear cultivars (Aoguan and Max Red Bartlett) than in the yellow Williams pear (Feng et al. 2010; Pierantoni et al. 2010). However, in the pear cultivar Wujiuxiang, this MYB was more likely to be induced by low temperatures and enhanced transcript levels of the structural genes F3H, DFR, and ANS during fruit development (Li et al. 2012). Sequences of the MYBs from cherries (PavMYBA, PavMYB10, and PavMYB10.1), peaches (PpMYB10.1, PpMYB10.2, and PpMYB10.3), and apples (MdMYB1, MdMYB10, MdMYB110a, and MdMYBA) are related by divergent evolution from a common ancestor and, as observed in apples, the MYBs from the cherry and peach also participated in anthocyanin accumulation in their fruits (Lin-Wang et al. 2010; Sooriyapathirana et al. 2010; Ravaglia et al. 2013; Rahim et al. 2014; Shen et al. 2014; Zhou et al. 2014, 2015, 2016; Starkevič et al. 2015; Jin et al. 2016). The MYBs are more closely related within species than among species and their regulatory roles are also similar. Generally, in the cherry, PavMYBA activated the promoters of the LBGs involved in the anthocyanin pathway by interacting with bHLH (Shen et al. 2014); similarly, PavMYB10 and PavMYB10.1 also interacted with bHLH and WD40 to bind with promoters of the LBGs (Jin et al. 2016). In peaches, although PpMYB10.4 and PpMYB9 are anthocyanin regulatory MYBs, they are not closely related to other peach MYBs (PpMYB10.1, PpMYB10.2, and PpMYB10.3), and the sequences of these two MYBs are not homologous. This is related to their tissue-specificity because the MYBs (PpMYB10.1, PpMYB10.2, and PpMYB10.3) are responsible for anthocyanin accumulation in fruit, whereas PpMYB10.4 and PpMYB9 are responsible for accumulation in leaves and flowers, respectively (Zhou et al. 2014, 2016). FaMYB10 was observed to be a potential MYB for anthocyanin biosynthesis because it is primarily expressed in ripe red strawberry fruit, and when its expression was transiently silenced in fruit receptacles, anthocyanin was not produced (Fig. 5) (Medina-Puche et al. 2014).

Fig. 5
figure 5

Role of FaMYB10 in anthocyanin accumulation in strawberry. This image was taken from the study by Medina-Puche et al. (2014) and is copyrighted by Oxford University Press

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Like ornamental plants, the majority of MYBs that regulate anthocyanin in fruits are related by divergent evolution from a common ancestor and those within the same species are more homologous than those from different species (with a few exceptions). Generally, fruit anthocyanin-related MYBs from peaches, cherries, and apples are closely related, while the suppressor MYBs from different fruit species are also closely related to each other. The present review reveals the MYBs (more than 30) that enhance anthocyanin accumulation in different fruit crops via regulation of structural genes and provides information about their phylogenetic relationships.

Vegetables

Vegetables are not only one of the fundamental components of the human diet but are also important to human health because they are important sources of essential vitamins and minerals, as well as secondary metabolites such as anthocyanins that can protect against several human diseases (Hou 2003; Lila 2004). R2R3-MYBs have been considered closely related to the anthocyanin biosynthetic pathway in several plant species (Davies et al. 2012) and the regulatory roles of MYBs in anthocyanin biosynthesis have been reported for some vegetables. In the present review, we highlight the MYBs and their regulated target genes involved in anthocyanin production in vegetable crops (Table 3) and present their phylogenetic relationships (Fig. 6).

Table 3 Identified R2R3-MYBs that regulate anthocyanin structural genes in vegetable plants
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Fig. 6
figure 6

Phylogenetic analysis of protein sequences of R2R3-MYB transcription factors associated with regulation of anthocyanin in vegetable plants. The evolutionary history was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1500 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% of the bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1500 replicates) are shown next to the branches (Felsenstein 1985). The evolutionary distances were computed using the p-distance method and are in units of the number of amino acid differences per site. The analysis included 21 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 423 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). All R2R3-MYB sequences were retrieved from the GenBank database and accession numbers are as follows: Cabbage Brassica oleracea BoMYB1 (ADP76651), BoMYB2 (ADP76651), BoMYB3 (ADP76651); Cauliflower BoMYB2 (ADP76650); Kohlrabi BoPAP2 (ADP76650); Chinese cabbage BrMYB2 (AIP94023); Tomato Solanum lycopersicum SlANT1 (AAQ55181), SlANT2 (ACN22102); Pepper Capsicum annuum CaMYB (ABN11121), CaMYB1 (AAQ05796), CaMYB2 (CAE75745); Eggplant Solanum melongena SmMYB (AMK01804); Potato Solanum tuberosum StAN1 (AGC31676), StMYBA1 (AFD31816), StMYB113 (AND01219); Onion Allium cepa AcMYB1 (AQP25672), AcMYB4 (AQP25673), AcMYB5 (AQP25674); Radish Raphanus sativus RsMYB1 (AKM95888); Lablab purpureus LpPAP1 (AKP06425); Daucus carota DcMYB6 (ARD08872); Oenanthe javanica OjMYB1 (ASZ70608); Gerbera GmMYB12 (ACG69458); Arabidopsis thaliana AtTT2 (NP_198405.1), which is involved in regulation of proanthocyanidin biosynthesis, is included as an outgroup

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To date, several anthocyanin regulatory MYBs have been functionally characterized in various vegetable crops, mostly Brassica spp. including cabbage, cauliflower, purple kale, purple kohlrabi (B. oleracea), mustard (B. juncea), and heading Chinese cabbage (B. rapa). In cabbage, three MYBs, BoMYB1, BoMYB2, and BoMYB3, participate in anthocyanin biosynthesis; however, BoMYB2 is more likely to be related to anthocyanin production because its transcript level in red cultivars is higher than that of other MYBs (Yuan et al. 2009), together with an increase in the expression levels of most structural genes (CHS, F3H, F3′H, DFR, LDOX, and GST). In addition, upregulation of the co-TFs (bHLH) has been observed in the red cultivars. Thus, it is likely that BoMYB2 up-regulated the transcript levels of the structural genes in red cabbage by interacting with bHLH (Yuan et al. 2009). The MYB (MYB2) from cauliflower and heading Chinese cabbage, which is a homolog of BoMYB2 from cabbage, also enhanced anthocyanin production via upregulation of structural genes by interacting with bHLHs, as achieved by BoMYB2 from cabbage (Chiu et al. 2010; Chiu and Li 2012; He et al. 2016); however, BoMYB2 from cauliflower mainly induced the pigments in curds and seed endosperm (Chiu et al. 2010; Chiu and Li 2012). Moreover, BoPAP2 from purple kohlrabi (B. oleracea), which is grouped in a clade with the above three MYBs, also enhanced pigmentation via up-regulation of structural genes by interacting with bHLH (Zhang et al. 2015a). Specifically, in the leaves of the cultivar Kolibri (purple), transcript levels of BoPAP2 were 990-fold higher than those in the cultivar Winner (green), whereas in the skins of swollen stems, BoPAP2 transcript levels in Kolibri were 452-fold higher than those in Winner. RsMYB1 from radishes (Raphanus sativus), in a clade with the above MYBs, also regulated anthocyanin biosynthesis in red radish cultivars via strong induction of structural genes by interacting with bHLH (Park et al. 2011), indicating that this MYB group induce anthocyanin via upregulation of structural genes by interacting with co-TFs (bHLHs). In the case of the mustard cultivars ‘Zi Yang’ and ‘Lv Ying’ (Fig. 7), induction of purple pigments in the leaves of the cultivar Zi Yang was due to strong expression of the MYBs (BjMYB1, BjMYB2, BjMYB3, and BjMYB41), which are homologs of the anthocyanin regulatory MYBs (AtPAP1, AtPAP2, AtMYB113, and AtMYB114, respectively) that activate transcription of the structural genes F3H, F3′H, DFR, and ANS (Xie et al. 2014); however, expression of the MYBs appeared to be light-induced, because the expression levels of BjMYB1, BjMYB2, BjMYB3, and BjMYB4 were significantly higher in seedlings grown in the light than in those grown in the dark.

Fig. 7
figure 7

Phenotype of purple cultivar “Zi Ying” expressing high BjMYB (a) and green cultivar “Lv Ying” expressing low BjMYB (b). This image was taken from a publication by Xie et al. (2014) and is copyrighted by the American Chemical Society

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The regulatory role of MYBs in anthocyanin production has also been documented in Solanum spp., including tomato, eggplant, and potato, in which the MYBs are phylogenetically related by divergent evolution from a common ancestor. Specifically, two MYBs [anthocyanin 1 (SlANT1) and anthocyanin 2 (SlANT2)] are involved in anthocyanin synthesis in tomatoes (S. lycopersicum) and are mainly responsible for the purple pigmentation in vegetative tissues and fruits; however, the production of anthocyanin due to SlANT2 can only be seen under high-light or low-temperature conditions (Mathews et al. 2003; Kiferle et al. 2015). Recently, another two MYB encoding genes, SlMYB7-like and SlMYB48-like, were identified as possible positive regulators of anthocyanin synthesis in tomatoes (Jia et al. 2015). SmMYB1 from eggplant (S. melongena) is a homolog of SlANT1 from tomato and, like SlANT1, is also responsible for the purple pigmentation in fruits via upregulation of most structural genes (Fig. 8), while overexpression of SmMYB1 enhanced the same purple pigmentation in transgenic eggplants (Zhang et al. 2014). Three MYBs (StAN1, StMYBA1, and StMYB113) that are phylogenetically related to the above MYBs are also observed to have the same function in the potato (S. tuberosum L.) (Jung et al. 2009; Liu et al. 2016); however, they interacted with the co-TFs StbHLH1 and StJAF13. In addition, several MYBs have been reported to regulate anthocyanin in other important culinary crops such as pepper (Capsicum annuum) and onion (Allium cepa). Among the MYBs (CaMYB, CaMYB1, and CaMYB2) reported in peppers, CaMYB1 and CaMYB2 participate in anthocyanin accumulation in hot peppers (purple fruit) and during fruit developmental stages (Borovsky et al. 2004; Li et al. 2011) by enhancing the expression level of CaDFR, whereas CaMYB is responsible for regulation of anthocyanin structural genes in pepper leaves (Zhang et al. 2015b). These MYBs are not homologous to each other; thus, although they are derived from the same species and involved in anthocyanin production, their existence in different groups is probably related to variation in their specific roles. In white and red onions (Allium cepa) (Fig. 9), the MYBs are associated with pigmentation, i.e., AcMYB1 is observed to be the regulatory MYB in red onion, whereas AcMYB4 and AcMYB5, which are homologous to each other but not to AcMYB1, act as suppressors. AcMYB1 has been confirmed as an activator in onion by transient overexpression and RNAi methods (Schwinn et al. 2016). Interestingly, in purple lablab pods, LpPAP1 seemed to be a dark-induced anthocyanin activator, because it is highly expressed under dark conditions and regulates most of the structural genes (LpPAL, LpF3H, LpF3′H, LpDFR, and LpANS) (Cui et al. 2016); however, the genes and the MYB were markedly suppressed when removed from dark conditions, leading to suppression of anthocyanin accumulation in the purple pods. Recently, it has been shown that DcMYB6 from Daucus carota and OjMYB1 from Oenanthe javanica, which group in the same clade as LpPAP1, enhanced the accumulation of anthocyanin in the same way as LpPAP1 (Feng et al. 2018; Xu et al. 2017) via upregulation of structural genes; however, they interacted with its co-transcription factor bHLH because they contained the conserved bHLH-interaction motif.

Fig. 8
figure 8

Phenotypes of two eggplant cultivars Zi Chang (on the left) expressing high SmMYB1 and Bai Xue (on the right) expressing low SmMYB1. This image was taken from the study by Zhang et al. (2014) and is copyrighted by the American Chemical Society

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Fig. 9
figure 9

Phenotypes of onions (white) expressing low AcMYB1 and (red) expressing high AcMYB1. This image was taken from a publication by Schwinn et al. (2016) and is copyrighted by Frontiers

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In summary, several MYBs have been shown to play critical roles in anthocyanin accumulation in vegetable crops and they are related by divergent evolution from a common ancestor. Generally, the majority of MYBs grouping in the same clade exhibited identical regulatory modes; however, some MYBs were likely to be dependent on environmental stimulus, organs, and developmental stages to induce anthocyanin.

Metabolic engineering of R2R3-MYB TFs to improve anthocyanin content

Ornamental plants

Flower color, which is determined by anthocyanins, is one of the most important commercial traits that interest flower enthusiasts and breeders. The MYBs participating in floral pigment accumulation have been well described (see above), and flower color modification in ornamental plants has been achieved by genetic engineering of the MYBs. In the present review, we describe the recent progress in color modification in important flower species by metabolic engineering of the MYBs (Table 4).

Table 4 Metabolic engineering of R2R3-MYBs to modify flower color or improve anthocyanin contents in fruit and vegetable crops
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The overexpression of MYBs from gerberas, Arabidopsis, radishes, snapdragons, petunias, and tomatoes resulted in anthocyanin production in the flowers and other tissues (Mol et al. 1998; Borevitz et al. 2000; Mathews et al. 2003; Allan et al. 2008; Laitinen et al. 2008; Ai et al. 2016; Li et al. 2016). Of these MYBs, GmMYB10 was found to mainly induce cyanidin- and pelargonidin-based anthocyanins in transgenic gerbera flowers (Elomaa et al. 2003; Laitinen et al. 2008). In addition, the single expression of C1 (MYB), or its homolog AaMYB1 from A. andraeanum, could enhance pigmentation in petals in Cymbidium orchid cultivars (Albert et al. 2010). In fact, production of pigments in the white flowers of Phalaenopsis and Doritis by co-expression of C1 (MYB) and leaf color (Lc) from maize (bHLH) was reported more than two decades ago (Griesbach and Klein 1993; Ma et al. 2008). Similarly, the expression of AmROSEA1 alone or in combination with Lc in lisianthus and petunia also resulted in higher pigmentation in the flower petals and sepals than that in the control lines, by regulating the transcript levels of CHS and ANS (Schwinn et al. 2014); however, induction of a novel color was seen in petunias, particularly in the petal throat region and anthers. Recently, overexpression of RsMYB1 from radishes in chrysanthemum, petunia, and ornamental tobacco resulted in upregulated anthocyanin structural genes (Naing et al. 2015; Kee et al. 2016; Lim et al. 2016; Ai et al. 2017), and higher pigmentation was also observed in petunias and tobacco.

Homologous expression of PhAN2, PhDPL, and PhPHZ in petunias could increase pigmentation in flowers (Quattrocchio et al. 1998; Albert et al. 2011). Likewise, heterologous expression of the grapevine MYBs VvMYBA1 and VvMYB5b in petunias also increased color pigmentation in the petals (Cavallini et al. 2014). A similar pigmentation in petunia flowers could be obtained by overexpression of the MYB AtPAP1 (production of anthocyanin pigment 1), where it increased not only the levels of anthocyanins but also the emissions of floral volatiles (benzenoids) (Ben et al. 2008). Additionally, the role of the PAP1 was confirmed in roses, in which it significantly activated transcript levels of some structural genes and induced approximately six- to nine-fold higher anthocyanin content than that in the control (Zvi et al. 2012).

Fruit and vegetable crops

The health benefits of anthocyanins that have high antioxidant activity have been highlighted earlier in the present review; therefore, it is important to develop fruits and vegetables enriched with anthocyanins to obtain the desired quantity of health-promoting components in our daily diets. Anthocyanin production by metabolic engineering is an alternative method to obtain anthocyanin-rich fruits and vegetables. Metabolic engineering of MYBs to improve anthocyanin levels has been reported in some fruit and vegetable crops (Table 4). A common approach is to overexpress heterologous or homologous anthocyanin regulatory MYBs, when available, in the species of interest.

In grapes, the overexpression of VvMybPA1 or VvMybPA2 dramatically increased proanthocyanidin accumulation (Terrier et al. 2009), whereas, in apples, transgenic lines overexpressing MdMYB9 or MdMYB11 promoted anthocyanin accumulation (An et al. 2015). Increased anthocyanin content in the strawberry due to heterologous expression of apple MYB10 has been reported (Kortstee et al. 2011) and it has also been demonstrated that homologous expression of McMYB10 in crabapples and apples could promote anthocyanin accumulation (Tian et al. 2015).

Overexpression of BoMYB2 in the purple cauliflower enhanced anthocyanin production in the seed endosperm (Chiu et al. 2010), and overexpression of AtPAP1 in transgenic B. napus (canola) also enhanced the intense purple coloration and antioxidant capacity in the leaves up to fourfold (Akagi et al. 2010). In tomato, overexpression by activation tagging of the MYB SlANT1 increased anthocyanins in the fruit (Mathews et al. 2003). In addition, the combined expression of ROSEA1 and DELILA genes or individual expression of MYBs (SlANT1 and SlANT2) produced purple- or red-colored tomatoes by strong promotion of anthocyanin structural genes (Butelli et al. 2008; Kiferle et al. 2015). Transgenic tomato plants expressing AtPAP1 and LeAN2 significantly enhanced anthocyanin levels in the leaves, stems, roots, flowers, and fruits under normal growth conditions (Zuluaga et al. 2008; Meng et al. 2015). Similarly, compared to wild-type eggplants, transgenic eggplants overexpressing SmMYB1 under normal growth conditions exhibited higher levels of anthocyanin in the leaves, petals, stamens, fruit peels, and especially in the fruit flesh, by transcriptional activation of most of the anthocyanin structural genes (Zhang et al. 2016). Moreover, overexpression of Pr-D/BoMYB2 in cauliflowers, AcMYB1 in onions, and Lc from maize in garlic also regulated anthocyanin accumulation (Fig. 10) via activation of structural genes (Chiu and Li 2012; Schwinn et al. 2016).

Fig. 10
figure 10

Overexpression of AcMYB1 from onion in garlic enhanced anthocyanin accumulation. This image was taken from a publication by Schwinn et al. (2016) and is copyrighted by Frontiers

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Conclusions and future perspectives

There is increasing interest in the modification of flower colors and the development of anthocyanin-rich fruits and vegetables. The identification of MYBs and the characterization of their roles in controlling anthocyanin biosynthesis in crop plants are crucial prerequisites for such modifications. The present review describes MYBs that have been reported thus far as anthocyanin promoters or suppressors in horticultural crops (flowers, fruits, and vegetables) and highlights their specific roles and their targeted genes involved in anthocyanin biosynthesis. In addition, phylogenetic analysis highlighted the different relationships of MYBs that are linked to production or suppression of anthocyanins in horticultural crops. Thus, the present review facilitates a better understanding of the regulation of anthocyanin accumulation in plants by MYBs and of the phylogenetic relationships between anthocyanin promoters and suppressors. Further, our review shows that in horticultural crops, the anthocyanin pathway can be manipulated by overexpressing R2R3-MYBs, which will pave the way for the development of new varieties and other commercially important plants, as well as the production of anthocyanin-rich foods by metabolic engineering approaches.