INVITED REVIEW
Genetic and Environmental Impacts on the Biosynthesis of Anthocyanins in Grapes
2018 Volume 87 Issue 1 Pages 1-17
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2018 Volume 87 Issue 1 Pages 1-17
Because of the commercial importance of grapes (Vitis spp.), it is important to understand how grape coloration is affected by genetic and environmental factors, as this knowledge may contribute to more stable production of high-quality grapes. The color of berry skins is determined mainly by the quantity and composition of anthocyanins. This review summarizes the results of recent studies of the genetic and environmental regulation of anthocyanin biosynthesis in grape berry skin: (i) The myeloblastosis (MYB) haplotype composition at the color locus is the major genetic factor that determines the anthocyanin content. (ii) The MYB haplotype composition at the color locus and the anthocyanin O-methyltransferase locus are major genetic determinants of the ratios of tri- to di-hydroxylated anthocyanins and of methylated to non-methylated anthocyanins. (iii) The accumulation of anthocyanins depends on both low temperature and light, and the two factors have a synergistic effect on the expression of genes within the anthocyanin biosynthesis pathway. (iv) Comprehensive transcriptome analysis using a grape oligo-DNA microarray let my research group identify many candidate genes involved in low-temperature-induced abscisic acid signaling and light signaling networks related to anthocyanin accumulation in grape berry skin. These findings will allow prediction of the skin color of grapes from seedlings at a very young stage by examining the MYB haplotype composition. Furthermore, these results will contribute to a fuller understanding of how grape coloration is affected by environmental factors, thereby helping grape growers to develop cultivation techniques that contribute to the production of highly pigmented grapes.
The European species Vitis vinifera L. is the dominant grape used to produce table grapes, wine, and raisins around the world. In contrast, unique breeding programs for table grape in Japan have been conducted to cross-hybridize Vitis × labruscana Bailey with V. vinifera to produce new accessions that combine high eating quality with high resistance to diseases and berry cracking. As a result, many interspecific hybrid grapes (V. × labruscana × V. vinifera), such as ‘Kyoho’ and ‘Pione’, have been developed, and have become popular in Japan and other parts of Asia.
Skin color is an important quality that is used as the basis for selection during breeding programs because consumers generally prefer well pigmented grapes, thus the high marketability of these fruits is important for farmers. The color of berry skin is determined by the quantity and composition of anthocyanins. Color-skinned accessions accumulate anthocyanins in their skin, whereas white-skinned accessions do not synthesize these pigments (Boss et al., 1996a). As a result of hybridization and human selection, skin color in grapes has become greatly diversified, with colors ranging from black to red, pink, and “white” (yellow-green). In color-skinned grapes, the accumulation of anthocyanins in the skin begins after the onset of ripening (veraison) and is affected by the environmental conditions in the vineyard at that time (Kliewer and Torres, 1972). Temperature and light are important environmental factors that affect anthocyanin biosynthesis. Low ambient temperature during the maturation stages of grape berries increases anthocyanin accumulation, but high temperature decreases it (Kataoka et al., 1984; Mori et al., 2005; Tomana et al., 1979a, b). Furthermore, exposure of grape bunches to light significantly increases anthocyanin accumulation, whereas shading reduces it (Cortell and Kennedy, 2006; Downey et al., 2004; Fujita et al., 2006; Jeong et al., 2004; Kataoka et al., 2003; Matus et al., 2009).
Recently, decreased grape quality, such as poor coloration, has become a common problem, caused mainly by high temperatures during the maturation stages in regions with a warm climate (Teixeira et al., 2013; Winkler et al., 1962). Because of the commercial importance of grape, it is important to understand how grape coloration is affected by genetic and environmental factors, as this knowledge will contribute to more stable production of high-quality grapes despite global atmospheric warming. This review summarizes recent studies of the genetic and environmental impacts on the regulation of anthocyanin biosynthesis in grape berry skin.
The anthocyanin biosynthesis pathway in many plants is controlled by regulatory genes that control three major classes of transcription factors (TFs), namely the myeloblastosis (MYB), basic helix-loop-helix (bHLH), and WD40 classes (Koes et al., 2005). Some genes for MYB TFs that regulate anthocyanin biosynthesis, such as VvMYBA1, VvMYBA2, VlMYBA1-2, VlMYBA1-3, and VlMYBA2, have been identified in V. vinifera and interspecific hybrid grapes (Azuma et al., 2008, 2011; Kobayashi et al., 2002, 2004, 2005; Walker et al., 2007). Boss et al. (1996a, b) showed that expression of the gene for UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) is critical for anthocyanin biosynthesis in grapes, and Kobayashi et al. (2002) found that the abovementioned MYB genes regulate the expression of UFGT (Fig. 1).
Simplified illustration of the main pathways involved in flavonoid biosynthesis and its regulation in grape berries by the products of characterized MYB genes (shown in blue). The five major anthocyanins are indicated in colored boxes; the box color indicates the skin color they produce. CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3'-hydroxylase; F3'5'H, flavonoid 3'5'-hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanin dioxygenase; UFGT, UDP-glucose: flavonoid 3-O-glucosyltransferase; AOMT, anthocyanin O-methyltransferase; GST, glutathione S-transferase; antho-MATE, anthocyanin multidrug and toxic extrusion; FLS, flavonol synthase.
The grape color locus appears to be a cluster of MYB genes that span a 200-kb region of chromosome 2 (Azuma et al., 2009; Fournier-Level et al., 2009; Matus et al., 2008). This cluster includes many MYB genes, among which the VvMYBA1 locus (with the alleles VvMYBA1a, VvMYBA1b, and VvMYBA1c) and the VvMYBA2 locus (with the alleles VvMYBA2r and VvMYBA2w) are functionally important for berry pigmentation in V. vinifera (Kobayashi et al., 2004; Walker et al., 2007; Yakushiji et al., 2006). The functional allele VvMYBA1c is most likely the original sequence at the VvMYBA1 locus (Yakushiji et al., 2006). On the other hand, a Gret1 retrotransposon insertion in the promoter region of VvMYBA1c appears to lead to transcriptional inactivation, resulting in the non-functional allele VvMYBA1a (Kobayashi et al., 2004, 2005). VvMYBA1b has a single long terminal repeat (solo LTR), which may have occurred as a result of intra-recombination between the 5'LTR and 3'LTR of Gret1 in the 5'-flanking region near the coding region of VvMYBA1, and is a functional allele (Kobayashi et al., 2004, 2005). Walker et al. (2007) reported that a single-nucleotide polymorphism (SNP) mutation and a frame-shift mutation in the coding sequence of the functional VvMYBA2r allele inactivated gene transcription, and named the resulting non-functional allele VvMYBA2w.
Since the two adjacent MYB alleles in the color locus are inherited together, these can be regarded as a MYB haplotype (Azuma et al., 2008, 2011). Haplotype (Hap) C-N is presumed to be the ancestral MYB haplotype, and consists of the functional VvMYBA1c and VvMYBA2r alleles (Fig. 2). Hap C-Rs contains the functional VvMYBA1c and the non-functional VvMYBA2w (Fournier-Level et al., 2010). Hap A contains the non-functional VvMYBA1a and VvMYBA2w. On the basis of this haplotype structure, it appears that the Gret1 insertion in the VvMYBA1 promoter region occurred after the emergence of VvMYBA2w (Fig. 2). It appears that Hap B contains VvMYBA1b and VvMYBA2w because it originated from Hap A.
A model of the evolutionary differentiation of MYB haplotypes at the color locus in Vitis species.
Other MYB haplotypes have been identified. Walker et al. (2006) and Yakushiji et al. (2006) showed that the skin color mutation responsible for changing black-skinned ‘Pinot Noir’ to white-skinned ‘Pinot Blanc’ is caused by deletion of the VvMYBA1c and VvMYBA2r alleles in Hap C-N, resulting in non-functional Hap D, which contains null alleles at the VvMYBA1 and VvMYBA2 loci (Azuma et al., 2008). The color recovery in rosy-skinned ‘Benitaka’, a bud sport of white-skinned ‘Italia’ (Hap A/Hap A), is caused by the appearance of the functional VvMYBA1BEN allele at the VvMYBA1 locus (Azuma et al., 2009). It was hypothesized that the functional VvMYBA1BEN allele resulted from homologous recombination between VvMYBA1a and VvMYBA3. We also predicted the occurrence of VvMYBA1BEN and VvMYBA2w at the color locus because VvMYBA1BEN originated from VvMYBA1a. This haplotype is named Hap G.
Accessions of V. vinifera have been eco-geographically classified into three proles: convarietas pontica, convar. occidentalis, and convar. orientalis (Negrul, 1938). Interestingly, Hap F was present only in the orientalis accessions such as ‘Sultanina’, ‘Koshu’, ‘Ryugan’, and ‘Niunai’ (Fig. 2; Table 1; Azuma et al., 2008). In addition, microsatellite analysis of the orientalis cultivars showed a clear separation in a dendrogram based on phenetic distances (Goto-Yamamoto et al., 2006). These findings suggest that Hap F differentiated in the orientalis accessions. Hap F contains a VvMYBA1SUB allele at the VvMYBA1 locus, although the allele at the VvMYBA2 locus has not been identified so far. Moreover, it has not been determined whether VvMYBA1SUB is a functional allele, because it has been detected in both white-skinned accessions (‘Niunai’ and ‘Sultanina’) and pink-skinned accessions (‘Koshu’ and ‘Ryugan’) (Table 1). Lijavetzky et al. (2006) showed that the VvMYBA1SUB sequences of these accessions are identical, indicating that the color difference could not be explained by variation within the coding sequence. These results suggest that either the existence of a polymorphism in the upstream promoter region of VvMYBA1SUB or an allele at the VvMYBA2 locus of Hap F controls anthocyanin biosynthesis in color-skinned orientalis accessions.
Relationships between the MYB haplotype composition and the color of berry skins in grape accessions.
In interspecific hybrid grape, some functional MYB alleles have been identified such as VlMYBA1-2, VlMYBA1-3, and VlMYBA2 (Azuma et al., 2008, 2011; Kobayashi et al., 2002). VlMYBA1-2 and VlMYBA1-3 lie close to each other at the color locus, and this allele combination has been named Hap E1 (Fig. 2; Azuma et al., 2008, 2011). VlMYBA2 and VlMYBA1-3 also lie close together at the color locus, and this allele combination is named Hap E2. Hap E1 and Hap E2 are found only in interspecific hybrid grapes, and no V. vinifera accession contains them (Azuma et al., 2008, 2011). Hap E1 in ‘Campbell Early’ (Hap E1/Hap E2) is likely to have been inherited from ‘Concord’ (Hap A/Hap E1), one of its parents. Although the origin of ‘Concord’ is unknown, Hap E1 might have originated from V. labrusca, because ‘Concord’ belongs to the V. labrusca group. The other parent of ‘Campbell Early’ is an F1 cross, ‘Belvidere’ × ‘Muscat Hamburg’. The haplotype composition of ‘Muscat Hamburg’ is Hap A/Hap C-Rs (Azuma et al., 2008). Therefore, Hap E2 in ‘Campbell Early’ might have originated from ‘Belvidere’ (V. labrusca). These findings indicate that Hap E1 and Hap E2 originated from V. labrusca, a North American species.
Hap A is found in many accessions of V. vinifera and interspecific hybrid grapes, but was not detected in any of the North American or East Asian Vitis species (Mitani et al., 2009). This suggests that the Hap A in interspecific hybrid grapes originated from V. vinifera. North American grapes have been classified into many species, including V. labrusca, V. aestivalis, V. cinerea, V. doaniana, V. longii, V. riparia, and V. rupestris (Winkler et al., 1974). It is unknown whether Hap E1 and Hap E2 are also found in these species. Further analysis of the genomic structure of the color locus in North American species is therefore needed. We believe that many undiscovered functional and non-functional MYB haplotypes may exist in these and other Vitis species. Further studies with a broader range of accessions, including native North American and East Asian wild grapes, would contribute to elucidating the origins and evolution of Vitis species.
2. The MYB haplotype is the major genetic determinant of anthocyanin content in grape berry skinSeveral genetic studies have revealed that white-skinned individuals are homozygous for non-functional Hap A (Hap A/Hap A), whereas color-skinned individuals contain at least one functional haplotype (Fig. 3; Azuma et al., 2007; Kobayashi et al., 2004; Lijavetzky et al., 2006; This et al., 2007). Furthermore, grape individuals with two functional haplotypes had a higher anthocyanin content than those with only a single functional haplotype (Azuma et al., 2008, 2011; Ban et al., 2014; Bayo-Canha et al., 2012; Song et al., 2014). We have investigated the relationship between the haplotype composition and anthocyanin content by crossing parents with known haplotypes (Azuma et al., 2008, 2011). We found that the total anthocyanin content in offspring with Hap C-Rs/Hap E1 (both functional) was significantly higher than that in Hap A/Hap C-Rs and Hap A/Hap E1 (Fig. 3). Accessions that have a high anthocyanin content in the berry skin, such as ‘Alphonse Lavallee’, ‘Steuben’, ‘Buffalo’, ‘Merlot’, and ‘Campbell Early’, also contained two functional haplotypes (Table 1). All accessions with two functional MYB haplotypes, including ‘Black Seedless’, ‘Almeria Nera’, ‘Dattier Noir’, and ‘Negra Tardia’, are known by names that allude to their dark skin color (Lijavetzky et al., 2006). These findings indicate that the number of functional haplotypes at the color locus affects the potential anthocyanin accumulation in grape berry skin.
A simple model of the relationship between the MYB haplotype compositions and anthocyanin biosynthesis in the offspring of interspecific hybrid crosses. Tri/Di ratio, ratio of tri- to di-hydroxylated anthocyanins; M/NM ratio, ratio of methylated to non-methylated anthocyanins.
We also analyzed berries from most of the offspring from the interspecific hybrid cross ‘Muscat of Alexandria’ (Hap A/Hap A) × ‘Campbell Early’ (Hap E1/Hap E2). Offspring with Hap A/Hap E1 tended to be red-skinned, whereas those with Hap A/Hap E2 were black-skinned (Fig. 4; Azuma et al., 2011). As was suggested by the phenotypes, the anthocyanin contents of the berries from Hap A/Hap E2 offspring were significantly higher than those from Hap A/Hap E1 offspring. Among the diploid grape accessions, black-skinned accessions, such as ‘Houman’ and ‘North Black’, contained Hap A/Hap E2, whereas red-skinned accessions, such as 626-84 ([‘Katta Kurgan’ × ‘Takasago’] × [‘Takasago’ × ‘Campbell Early’]) and ‘North Red’, contained Hap A/Hap E1 (Table 1). These results suggest that the ability of Hap E2 to induce anthocyanin biosynthesis is stronger than that of Hap E1. In V. vinifera grapes, Fournier-Level et al. (2010) also reported that black-skinned accessions tended to have Hap C-N, whereas red-skinned accessions tended to have Hap C-Rs. We confirmed that red-skinned accessions, such as ‘Rizamat’, ‘Kaiji’, and ‘Sekirei’, had Hap A/Hap C-Rs, whereas black-skinned accessions, such as ‘Pinot Noir’ and ‘Cabernet Sauvignon’, had Hap A/Hap C-N (Table 1). These findings indicate that the combination of functional haplotypes at the color locus affects the quantity of anthocyanins.
A simple model of the relationship between the MYB haplotype composition and the anthocyanin content in offspring of the ‘Muscat of Alexandria’ × ‘Campbell Early’ cross.
The difference in potential anthocyanin accumulation between Hap C-N and Hap C-Rs grapes can be explained by the number of functional MYB alleles in each haplotype. Hap C-N has two functional alleles (VvMYBA1c and VvMYBA2r; Fig. 2), whereas Hap C-Rs has only one (VvMYBA1c). Although our data show no firm evidence that explains the difference in color between grapes with Hap E1 and Hap E2, we have proposed three hypotheses. First, different expression levels of VlMYBA1-2 and VlMYBA2 may lead to the color difference between Hap E1 and Hap E2 berries. Several reports have indicated that the expression level of MYB genes correlates with the anthocyanin content in berry skin (Azuma et al., 2009; Jeong et al., 2004; Matus et al., 2009; Yamane et al., 2006). Second, different MYB genes may cause differential regulation of genes in the anthocyanin biosynthesis pathway. Kobayashi et al. (2002) suggested that the sequence of the coding region differed between VlMYBA1-2 and VlMYBA2. The resulting amino acid substitution in the coding region may affect the binding or promoting ability of these TFs with respect to the promoters of the anthocyanin biosynthesis pathway genes. Third, Hap E1, Hap E2, or both may have some unidentified color-related genes, which might affect the color conferred by each haplotype. Fournier-Level et al. (2009) reported that the continuous variation in anthocyanin content in V. vinifera was explained mainly by a single gene cluster of three VvMYBA genes (VvMYBA1, VvMYBA2, and VvMYBA3) at the color locus, although it is not yet clear whether VvMYBA3 is functional. Unfortunately, it is not easy to investigate whether Hap E1 and Hap E2 contain novel color-related genes or DNA polymorphisms because the genomic sequence of the color locus in North American species, especially V. labrusca, is not yet known. More detailed analysis of the structures of Hap E1 and Hap E2 would be needed to clarify why the berry color differs between these haplotypes.
3. Development of a method to determine the MYB haplotype compositions of tetraploid grapesThe MYB haplotype composition at the color locus of diploid grapes can be detected by means of a PCR-based method (Azuma et al., 2008). However, this method cannot determine the haplotype composition of tetraploid grapes because it can detect only the presence or absence of a particular haplotype, not how many copies are present in the tetraploid genome. Recently, we developed a method to determine the haplotype compositions in tetraploid grapes by means of quantitative real-time PCR (qRT-PCR), and investigated the relationship between the haplotype composition at the color locus and skin color in tetraploid grapes using this method (Azuma et al., 2011).
VvMYBA1a, VlMYBA1-2, and VlMYBA2 are unique to Hap A, Hap E1, and Hap E2, respectively (Fig. 2). On the other hand, VlMYBA1-3 is present in both Hap E1 and Hap E2. Using this knowledge, my group investigated the presence or absence of these alleles and their relative DNA amounts in tetraploid grapes, and used this information to predict the MYB haplotype composition at the color locus. For example, in white-skinned ‘Hakuho’, only the non-functional VvMYBA1a was detected. The relative amount of VvMYBA1a DNA in ‘Hakuho’ was 4.00, and the haplotype composition of ‘Hakuho’ was determined to be Hap A/Hap A/Hap A/Hap A (Table 1). In black-skinned ‘Black Beat’, VvMYBA1a was not detected, whereas VlMYBA1-3, VlMYBA1-2, and VlMYBA2 were detected. Thus, the relative amount of VlMYBA1-3 DNA in ‘Black Beat’ was assumed to be 4.00 because VlMYBA1-2 and VlMYBA2 were always accompanied by a copy of VlMYBA1-3 (Fig. 2). In black-skinned ‘Kyoho’, VvMYBA1a, VlMYBA1-3, VlMYBA1-2, and VlMYBA2 were all detected. The relative amount of VvMYBA1a DNA in ‘Kyoho’ was 2.17, compared with 4.00 in ‘Hakuho’, and the relative amount of VlMYBA1-3 DNA in ‘Kyoho’ was 2.07, compared with 4.00 in ‘Black Beat’. These results suggest that the haplotype composition of ‘Kyoho’ is Hap A/Hap A/Hap E1/Hap E2 (Table 1); on this basis, the relative amounts of VlMYBA1-2 and VlMYBA2 DNA were both 1.00. The relative amounts of VlMYBA1-2 and VlMYBA2 DNA in ‘Black Beat’ were 2.38 and 1.90, respectively, compared with 1.00 each in ‘Kyoho’. From these results, the haplotype composition of ‘Black Beat’ is Hap E1/Hap E1/Hap E2/Hap E2 (Table 1). In ‘Fujiminori’ and ‘Pione’, the relative amounts of DNA of the four alleles were similar to those in ‘Kyoho’, suggesting that their haplotype composition was Hap A/Hap A/Hap E1/Hap E2. In red-skinned ‘Aki Queen’, ‘Benizuiho’, ‘Ruby Roman’, and ‘Ryuho’, the relative amounts of VvMYBA1a, VlMYBA1-3, and VlMYBA1-2 DNA were 2.72 to 3.34, 0.72 to 1.31, and 0.79 to 0.99, respectively. VlMYBA2 was not detected in any of these accessions. From these results, the haplotype composition of these accessions was determined to be Hap A/Hap A/Hap A/Hap E1 (Table 1).
All of the red-skinned tetraploid accessions had Hap A/Hap A/Hap A/Hap E1, and many of the black-skinned accessions had Hap A/Hap A/Hap E1/Hap E2 (Table 1). Interestingly, high-anthocyanin accessions, such as ‘Black Beat’ and ‘Akitsu 30’, had three or four functional haplotypes. These findings indicate that the number and kind of functional haplotypes at the color locus are the major genetic factors that determine the anthocyanin content of tetraploid grapes, as is the case for diploid grapes.
4. Other genetic factors that affect the anthocyanin contentAlthough grape individuals with many functional haplotypes tend to have increased anthocyanin accumulation in the berry skin, the total anthocyanin content in individual berries displays continuous variation even within the same haplotype composition. This suggests that in addition to the MYB haplotype, other genetic and environmental factors are involved in determining the final anthocyanin content in the skin. Several reports suggest that VvMYB5a, VvMYB5b, VvMYBPA1, and VvMYBPA2 regulate several genes in the common steps of the flavonoid pathway (Bogs et al., 2007; Deluc et al., 2006, 2008; Terrier et al., 2009). Matus et al. (2008) performed genome modeling and identified that nine anthocyanin-related MYB gene models were distributed on chromosomes 2 and 14. Ban et al. (2014) detected quantitative trait loci (QTLs) for anthocyanin content in linkage groups (LGs) 2 (MYB haplotype), 8, and 14 in interspecific hybrid grape, and suggested that the QTLs in LGs 8 and 14 could be novel loci that affect the anthocyanin content in berry skin. Costantini et al. (2015) also detected many minor QTLs for the total anthocyanin content in LGs 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 17, 18, and 19 in a V. vinifera cross (‘Syrah’ × ‘Pinot Noir’). Nevertheless, the relatively low contributions of these QTLs indicate that the number and kind of functional MYB haplotypes at the color locus are the major genetic factors that determine the variation of anthocyanin content in grape berry skin.
Genetic factors that regulate anthocyanin compositionsIn addition to the anthocyanin content, the anthocyanin composition is an important factor that affects the color variation of grape berry skin. The five major anthocyanins in grape differ from each other in the number and positions of the hydroxyl and methoxyl groups on the B-ring. Cyanidin and peonidin are di-hydroxylated precursors of red anthocyanins (Fig. 1; Deng and Qu, 1996). Delphinidin, petunidin, and malvidin are tri-hydroxylated precursors of blue and purple anthocyanins. Methylation stabilizes the phenolic B-ring, and causes a red shift in the anthocyanin absorption spectrum (Jackman and Smith, 1996; Sarni et al., 1995).
Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) competitively control the synthesis of di- and tri-hydroxylated anthocyanins, respectively, whereas anthocyanin O-methyltransferase (AOMT) methylates anthocyanins of both groups (Fig. 1). Some reports have suggested a relationship between the anthocyanin composition and the expression levels of the genes in the anthocyanin biosynthesis pathway. For example, the ratio of F3'5'H expression to F3'H expression is similar to the ratio of tri-hydroxylated anthocyanins to di-hydroxylated anthocyanins in grape skin (Castellarin and Gaspero, 2007; Castellarin et al., 2006; Jeong et al., 2006). The berries of grape accessions with higher AOMT expression levels accumulate higher amounts of methylated than non-methylated anthocyanins (Castellarin and Gaspero, 2007). Thus, anthocyanin composition is affected by the expression of genes that encode F3'H, F3'5'H, and AOMT.
1. QTLs that affect the anthocyanin compositionTo elucidate the genetic mechanism that determines the ratios of tri- to di-hydroxylated anthocyanins and of methylated to non-methylated anthocyanins in grape skin, analyses to detect major QTLs for anthocyanin composition were performed (Azuma et al., 2015a). One major QTL for the ratio of tri- to di-hydroxylated anthocyanins was found in LG 2 in an interspecific hybrid grape. Interestingly, the position of the QTL peak coincided with the MYB haplotype at the color locus. This QTL explained 26.6% to 62.8% of the phenotypic variance in the ratio of tri- to di-hydroxylated anthocyanins, with a maximum LOD score of 4.2 to 23.8 in both the parental and consensus linkage maps. Four minor QTLs in LGs 6, 7, 13, and 18 were also detected. They explained 3.3% to 8.9% of the phenotypic variance, with a maximum LOD score of 2.5 to 7.6. One of them was located in LG 6, and the SSR marker closest to the QTL peak was VMC5G1.1. The NCBI Map Viewer public database (http://www.ncbi.nlm.nih.gov/mapview/) places the F3'5'H cluster described by Falginella et al. (2010) within 695 kb of VMC5G1.1 in the reference genome (IGGP build 2). Costantini et al. (2015) also proposed that the QTL in LG 6 controls the hydroxylation level of anthocyanins in V. vinifera. These findings suggest that polymorphisms in the F3'5'H allele affect the ratios of tri- to di-hydroxylated anthocyanins in grape skin.
Two major QTLs for the ratio of methylated to non-methylated anthocyanins have been detected in V. vinifera and an interspecific hybrid grape: one near the color locus in LG 2 and one close to the AOMT locus in LG 1 (Azuma et al., 2015a; Fournier-Level et al., 2011). The QTL in LG 1 explained 32.5% to 57.1% of the phenotypic variance in the ratio of methylated to non-methylated anthocyanins, with a maximum LOD score of 5.1 to 22.3, and was located within 1 Mb of the AOMT locus, which affects the anthocyanin methylation level. The second QTL was found in LG 2, and the QTL peak was always close to or coincided with the MYB haplotype at the color locus. This QTL explained 13.1% to 15.8% of the phenotypic variance, with a maximum LOD score of 4.6 to 9.3. These findings suggest that the MYB haplotype at the color locus in LG 2 and the AOMT locus in LG 1 contribute to the genetic determination of the anthocyanin composition in grape skin.
2. The MYB haplotype affects the anthocyanin composition through trans-regulation of the F3'5'H/F3'H expression ratio and AOMT expressionWe next investigated the relationship between the MYB haplotype composition and the anthocyanin composition in interspecific grape populations and accessions (Azuma et al., 2015a). We found that Hap A/Hap E1 and Hap C-Rs/Hap E1 offspring had a higher ratio of tri- to di-hydroxylated anthocyanins and a higher ratio of methylated to non-methylated anthocyanins than Hap A/Hap C-Rs offspring (Fig. 3). We also found that Hap A/Hap C-N and Hap C-N/Hap C-N accessions had higher ratios than Hap A/Hap C-Rs, Hap A/Hap E1, and Hap A/Hap B accessions. These results indicate that the MYB haplotype composition affects both ratios. We also compared the F3'5'H/F3'H expression ratios in grape skin of offspring with four MYB haplotype compositions in two populations. The expression ratio was higher in Hap A/Hap E1 offspring than in Hap A/Hap C-Rs offspring. F3'5'H expression was undetectable in Hap A/Hap A offspring. The relative expression of AOMT tended to be higher in Hap A/Hap E1 and Hap C-Rs/Hap E1 offspring than in Hap A/Hap C-Rs offspring, and expression was undetectable in Hap A/Hap A offspring. These findings suggest that the MYB haplotype composition affects the ratios of tri- to di-hydroxylated anthocyanins and of methylated to non-methylated anthocyanins through trans-regulation of the F3'5'H/F3'H expression ratio and AOMT expression (Fig. 5).
Proposed model of the regulation of anthocyanin composition in grape berry skin. The MYB haplotype composition affects the ratios of tri- to di-hydroxylated anthocyanins (Tri/Di ratio) and of methylated to non-methylated anthocyanins (M/NM ratio) through trans-regulation of the F3'5'H/F3'H expression ratio and AOMT expression. The AOMT genotype affects the M/NM ratio through cis-regulation of AOMT expression.
Hap A/Hap B accessions tended to have low ratios of tri- to di-hydroxylated anthocyanins and of methylated to non-methylated anthocyanins (Azuma et al., 2015a). Both red-skinned ‘Ruby Okuyama’ (Hap A/Hap B) and rosy-skinned ‘Benitaka’ (Hap A/Hap G) are bud sports of white-skinned ‘Italia’ (Hap A/Hap A) (Azuma et al., 2009; Kobayashi et al., 2004). ‘Ruby Okuyama’ had predominantly di-hydroxylated non-methylated anthocyanins, whereas ‘Benitaka’ had predominantly methylated anthocyanins with a moderate amount of tri-hydroxylated anthocyanins, and the F3'5'H/F3'H expression ratio and AOMT expression were much higher in ‘Benitaka’ than in ‘Ruby Okuyama’ (Azuma et al., 2009). These findings support the hypothesis that the MYB haplotype composition affects the F3'5'H/F3'H expression ratio and AOMT expression. We further hypothesize that polymorphisms in the coding regions of MYB genes may lead to differential regulation of the F3'5'H/F3'H expression ratio and AOMT expression. For example, the coding sequences differ between VvMYBA1, VvMYBA2, VlMYBA1-3, VlMYBA1-2, and VlMYBA2 (Azuma et al., 2011; Kobayashi et al., 2002; Walker et al., 2007), and this may affect TF binding or activity. Further studies are needed to clarify whether the differences in anthocyanin composition are influenced by different characteristics of these MYB TFs.
3. The AOMT genotype affects the ratio of methylated to non-methylated anthocyanins by regulating AOMT expressionThe SSR marker VMC9F2 (which is close to the AOMT locus) had three allele sizes (a, 217 bp; b, 295 bp; c, 310 bp) in two populations (Azuma et al., 2015a). The a/b offspring contained little to no methylated anthocyanins regardless of their MYB haplotype composition. On the other hand, the c/c offspring had significantly higher ratios of methylated to non-methylated anthocyanins than other haplotype compositions in the populations. These results suggest that the AOMT genotype might affect the ratio of methylated to non-methylated anthocyanins. Analysis of AOMT expression in four VMC9F2 genotypes showed that c/c offspring tended to have higher expression levels than offspring with other genotypes, and AOMT expression was almost undetectable in a/b offspring. These results suggest that alleles a and b are linked with a non-functional AOMT allele. In contrast, allele c appears to be linked with the functional AOMT allele. These findings suggest that the AOMT genotype might affect the ratios of methylated to non-methylated anthocyanins by regulating AOMT expression (Fig. 5). Multiple AOMT2 polymorphisms (including two SNPs in the coding region associated with variation in the ratio of methylated to non-methylated anthocyanins) have been identified (Fournier-Level et al., 2011). In addition to the alleles a, b, and c, we have detected various allele sizes at the VMC9F2 locus from grape accessions (unpublished data). Therefore, we believe that many alleles exist at the AOMT locus.
The genetic basis for the ratio of methylated to non-methylated anthocyanins in grape skin can be explained by an epistatic interaction between the MYB haplotype composition at the color locus (X) and the AOMT locus (Y). X–Y– individuals would be expected to accumulate methylated anthocyanins, whereas X–yy individuals would accumulate almost exclusively non-methylated anthocyanins, and xxY– and xxyy individuals would not accumulate anthocyanins. In a recessive epistasis model, the expected segregation ratio for X–Y–:X–yy:(xxY–, xxyy) is 9:3:4; the chi-squared test showed that the observed ratio was not significantly different from the expected ratio in these two populations (Azuma et al., 2015a). This suggests that the MYB haplotype affects the ratio of methylated to non-methylated anthocyanins when the AOMT locus has at least one functional allele.
These findings indicate that the MYB haplotype at the color locus and the allele at the AOMT locus are major genetic determinants of the ratios of tri- to di-hydroxylated anthocyanins and of methylated to non-methylated anthocyanins in grape skin. This research provides new knowledge about the genetic control of anthocyanin biosynthesis and contributes to a better understanding of the genetic mechanisms that control grape skin color.
Anthocyanin accumulation in grape berry skin begins after veraison, and is affected by environmental conditions such as temperature and light. Low ambient temperatures during the maturation stages of grape berries increased anthocyanin accumulation in grape skin, whereas high ambient temperatures decreased it (Mori et al., 2005; Yamane et al., 2006). Exposure of grape bunches to light increased the anthocyanin accumulation and the expression of their biosynthesis genes, whereas shading reduced both (Cortell and Kennedy, 2006; Downey et al., 2004; Fujita et al., 2006; Jeong et al., 2004; Matus et al., 2009). Although many studies have been performed to determine the impact of environmental factors on anthocyanin accumulation in grape berry skin, the interrelationships between temperature and light in terms of their effects on anthocyanin biosynthesis have not been fully elucidated at the molecular level. Furthermore, it has not yet been elucidated how genes in the anthocyanin biosynthesis pathway and MYB genes respond to various combinations of temperature and light. Therefore, we investigated the effects of temperature and light conditions on anthocyanin accumulation and on the expression of genes related to anthocyanin biosynthesis in an in vitro environmental experiment using detached ‘Pione’ grape berries (Azuma et al., 2012b). After 10 days’ incubation, low temperature (15°C) plus light (white light + UV light) promoted anthocyanin accumulation in the grape skin, whereas high-temperature (35°C) or dark treatment severely suppressed it (Table 2). This difference indicates that the accumulation of anthocyanins requires both low temperature and light.
Expression patterns of MYB-related genes and of flavonoid biosynthesis-related genes in grape berry skins (Azuma et al., 2012b).
qRT-PCR analysis has shown that the expression of VlMYBA1-3 was strongly affected by both temperature and light conditions (Table 2). On the other hand, the expression of VlMYBA1-2 was affected primarily by light, and that of VlMYBA2 by temperature. Thus, the expression of these three MYB genes responds differently to temperature and light. Although the expression pattern of VlMYBA1-3 was most similar to the pattern of anthocyanin content, the expression level of VlMYBA1-3 was much lower than that of the other MYB genes (Azuma et al., 2012b). On the other hand, the expression patterns of VlMYBA1-2 and VlMYBA2 were quite different from the pattern of anthocyanin content, suggesting that the final anthocyanin content in grape skin is not determined solely by the expression levels of these MYB genes. Other MYB genes (MYBPA1, MYB5a, MYB5b, MYBF1, and MYB4), whose products regulate genes for the biosynthesis of flavonoids (including anthocyanins, flavonols, and proanthocyanidins), also show different expression patterns in response to temperature and light (Table 2). The biosynthesis of anthocyanins, flavonols, and proanthocyanidins share common steps in the flavonoid biosynthesis pathway (Boss et al., 1996a; Holton and Cornish, 1995; Shirley et al., 1992; Winkel-Shirley, 2001). MYBPA1, MYB5a, MYB5b, and MYBF1 encode transcriptional activators of genes in that pathway. The expression of MYBPA1 was highest in the low temperature plus light treatment and was significantly reduced by both high temperature and dark treatment. MYBPA1 is reported to activate the promoters of genes in the general flavonoid pathway but not the promoter of UFGT (Bogs et al., 2007). Therefore, MYBPA1 might affect genes in the early steps of the flavonoid biosynthesis pathway, and thereby affect the anthocyanin contents.
On the other hand, the expression of MYB5a was highest in the high temperature plus dark treatment, and was not correlated with anthocyanin level. MYB5a controls a number of different branches of the flavonoid pathway, but is expressed only before veraison (Deluc et al., 2006, 2008). The expression of MYB5a was relatively low (Azuma et al., 2012b), and its responsiveness to temperature and light might have decreased at this stage. The expression of MYB5b was highest in the low temperature plus light treatment and lowest in the high temperature plus dark treatment. MYB5b activates several genes in the flavonoid biosynthesis pathway in ripening grape berries (Deluc et al., 2008). Therefore, variation in the expression of MYB5b would also affect flavonoid biosynthesis during ripening. Spayd et al. (2002) reported that temperature had little effect on the flavonol content of grape berry skin. However, light increased the flavonol content and upregulated the expression of MYBF1, which encodes a transcriptional regulator of flavonol synthase (FLS) 4 (Azuma et al., 2012b). FLS4 was also expressed only in the light treatments. Czemmel et al. (2009) reported that expression of both MYBF1 and FLS4 was highly induced in grapevine cell culture after light irradiation; they also suggested that transcription of MYBF1 in the skin of ripening berries was highly correlated with the accumulation of flavonols and the expression of FLS4. The results of our data (Azuma et al., 2012b) support those of Czemmel et al. (2009), who found that the effect of light on the expression of flavonol biosynthesis–related genes was much greater than the effect of temperature. The expression of MYB4, which is a repressor of UFGT, was significantly upregulated in the high-temperature treatments but was unaffected by light level. This suggests that upregulation of repressor genes, such as MYB4, may contribute to the inhibition of anthocyanin biosynthesis under high temperatures.
2. Anthocyanin biosynthesis pathway genes show different expression patterns in response to temperature and lightThe expression levels of many genes in the anthocyanin biosynthesis pathway were upregulated independently by the low temperature and light treatment (Table 2). The expression levels of chalcone synthase (CHS) 3, chalcone isomerase (CHI) 1, CHI2, flavanone 3-hydroxylase (F3H) 1, and anthocyanin multidrug and toxic extrusion (antho-MATE) were significantly higher under low temperature (15°C) than under high temperature (35°C), but no clear effect of light level was observed. Under low temperature, the expression levels of F3'H and leucoanthocyanin dioxygenase (LDOX) were not affected by light, but under high temperature, dark treatment significantly decreased them. The expression levels of CHS2, F3H2, and F3'5'H were highest in the low temperature plus light treatment and were significantly downregulated by the high temperature or dark treatments, indicating that both low temperature and light are required to induce the expression of these genes. Both high temperature and darkness downregulated expression of dihydroflavonol 4-reductase (DFR), UFGT, and AOMT, but the effect of high temperature was particularly dramatic. Although the expression level of glutathione-S-transferase (GST) was highest in the low temperature plus light treatment, the effects of temperature and light were unclear.
Thus, a number of different expression patterns were observed among the genes of the flavonoid biosynthesis pathway. Winkel-Shirley (1999) reported that multi-enzyme complexes are involved in flavonoid biosynthesis, which implies that an increase in the expression of a specific gene involved in the flavonoid pathway would probably not result in increased anthocyanin accumulation. These findings suggest that low temperature and light have synergistic effects on the expression of genes in the anthocyanin biosynthesis pathway and thus on the accumulation of anthocyanins.
3. Temperature and light conditions affect the anthocyanin composition in grape berry skinThe variation in anthocyanin composition is affected by the expression of genes for the two flavonoid hydroxylases (F3'H and F3'5'H) and for AOMT (Fig. 1). Downregulation of F3'5'H expression in the low temperature plus dark treatment and in the high temperature plus light treatment was correlated with a decrease in the percentage of malvidin derivatives, which are delphinidin-based anthocyanins (Azuma et al., 2012b). In addition, the downregulation of AOMT expression in the high temperature plus light treatment was correlated with a decrease in the percentage of peonidin derivatives (di-hydroxylated methylated anthocyanins). These results suggest that the anthocyanin compositions in grape berry skin are affected by temperature and light conditions through changes in the expression of genes in the flavonoid biosynthesis pathway. Interestingly, the percentages of peonidin-3-(p-coumarylglucoside)-5-glucoside (Pn3pG5G) in the low temperature plus dark treatment and in the high temperature plus light treatment were higher than those in the low temperature plus light treatment. The methoxylation, glycosylation, and acylation of anthocyanins lead to an increase in their stability (Jackman and Smith, 1996). Therefore, the relatively high level of Pn3pG5G under conditions when total anthocyanin levels are low might be caused by its stability relative to that of other anthocyanins.
4. ABA content and expression pattern of NCED1The level of the plant hormone abscisic acid (ABA) increases at the start of veraison, and this increase enhances anthocyanin biosynthesis in the grape berry (Coombe and Hale, 1973). It has also been reported that low temperature accelerates anthocyanin biosynthesis and that the ABA content in the skin is positively correlated with the degree of grape coloration (Koshita et al., 2007; Yamane et al., 2006). ABA levels were higher under low temperature than under high temperature (Table 2; Azuma et al., 2012b). On the other hand, the effect of light on the ABA content was lower than the effect of temperature, although anthocyanin accumulation was severely suppressed in the dark treatments. The ABA content in the high temperature plus light treatment was significantly lower than that in the low temperature plus light treatment, but the expression level of 9-cis-epoxycarotenoid dioxygenase (NCED) 1, which encodes a key enzyme in ABA biosynthesis, was not significantly lower than in the low temperature plus light treatment (Table 2).
Wheeler et al. (2009) reported that the ABA content in grape berries was not significantly correlated with the expression level of NCED1. ABA can be degraded through the irreversible pathway starting with 8' hydroxylation, catalyzed by ABA 8'-hydroxylase (CYP707As) (Nambara and Marion-Poll, 2005). It has been reported that the endogenous ABA concentration is modulated by a dynamic balance between the biosynthesis and catabolism, which are regulated by NCEDs and CYP707As transcripts, respectively (Sun et al., 2010; Zhou et al., 2004). Thus, the final ABA content in grape berries might be determined by more complex regulation. Several groups have reported that the application of exogenous ABA to grape clusters enhances anthocyanin accumulation by activating genes in the anthocyanin biosynthesis pathway (Ban et al., 2003; Jeong et al., 2004; Terrier et al., 2005). In studies by my research group, however, the expression patterns of VlMYBA1-2, VlMYBA1-3, and VlMYBA2 did not resemble the pattern of endogenous ABA levels (Azuma et al., 2012b). This suggests that the final anthocyanin content and the expression levels of related genes under natural conditions are determined by complex interactions among internal and external factors such as temperature, light, water status, sugar content, and endogenous ABA content (Castellarin et al., 2007; Gambetta et al., 2010; Hiratsuka et al., 2001; Jeong et al., 2004; Kataoka et al., 1984, 2003; Kliewer and Torres, 1972; Matus et al., 2009; Mori et al., 2007; Yamane et al., 2006). It also suggests that artificial ABA treatment can enhance the expression of MYB genes and of genes in the anthocyanin biosynthesis pathway.
In conclusion, the accumulation of anthocyanins depends on both low temperature and light, and many genes related to anthocyanin biosynthesis are upregulated independently by both. These findings suggest that low temperature and light have a synergistic effect on the expression of genes within the anthocyanin biosynthesis pathway.
Exploring the novel low-temperature- and light-inducible genes related to anthocyanin accumulation in grape berry skinAs described above, the accumulation of anthocyanins depended on both low temperature and light (Azuma et al., 2012b). The expression of the flavonoid biosynthesis genes and MYB genes in grape berry skin was induced by low temperature, light, or both, but was suppressed by high temperature and darkness. Many reports have described the impact of environmental factors and ABA on flavonoid accumulation in grape berry skin. However, the components of the low-temperature-induced ABA signaling and light signaling networks related to anthocyanin accumulation in grape berry skin have not been elucidated, and how environmental conditions affect these components remains poorly understood. To identify low-temperature- and light-inducible genes in post-veraison grape berries, we developed a grape oligo-DNA microarray, and performed comprehensive transcriptome analysis using detached grape berries cultured under different temperature and light conditions (Azuma et al., 2015b).
1. Construction of a grape oligo-DNA microarrayWe constructed a grape oligo-DNA microarray using the eArray system (Agilent Technologies, Santa Clara, CA; https://earray.chem.agilent.com/earray/). Probes (60 oligonucleotides each) were constructed using the sequence data from public databases: 30434 assembled mRNAs (8× coverage) in the Genoscope Grape Genome Browser (French National Sequencing Center, Évry, France; http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/entry_ggb.html; Jaillon et al., 2007) and 23152 unique gene sequences in NCBI UniGene Vitis vinifera Build 8 (http://www.ncbi.nlm.nih.gov/unigene). In total, 38549 independent probes were used in designing the custom grape oligo-DNA microarray in the 4×44K format of the Agilent system. Most of the probes (82.9%) were functionally annotated by using information available for other plants, predominantly Arabidopsis thaliana.
2. Microarray experiments and analysesMicroarray analysis was carried out using detached grape berries of ‘Pione’ (Hap A/Hap A/Hap E1/Hap E2) that had been incubated for 10 days under four sets of conditions: 15°C/light (15/L), 15°C/dark (15/D), 35°C/light (35/L), and 35°C/dark (35/D). Genes with up- or down-regulation >3.0 times the value in another treatment were considered to have been significantly differentially expressed between the treatments. Using Venn diagram analysis based on seven conditions (pairs of treatments), differentially expressed genes were extracted into three groups: low-temperature-inducible genes, light-inducible genes, and low-temperature-plus light-inducible genes (Fig. 6). Low-temperature-inducible genes were extracted from the overlap of condition 1 (15/L versus 35/D), condition 2 (15/L vs. 35/L), condition 3 (15/D vs. 35/L), and condition 4 (15/D vs. 35/D) (Fig. 6). Light-inducible genes were extracted from the overlap of condition 1, condition 5 (15/L vs. 15/D), condition 6 (35/L vs. 15/D), and condition 7 (35/L vs. 35/D). Low-temperature-plus light-inducible genes were extracted from the overlap of conditions 1, 2, and 5. Using the search function provided by the Subio Platform (Subio Inc., Kagoshima, Japan; http://www.subio.jp/products/platform), the extracted genes were further screened by searching for the following annotation keywords: ABA, SnRK (SNF-related kinase), PP2C (protein phosphatase 2C), light, UV, flavonoid, anthocyanin, and flavonol.
Genes that were identified as low-temperature-inducible, light-inducible, and low-temperature plus light-inducible in ‘Pione’ (Azuma et al., 2015b). Numbers at the center of each diagram represent the total number of genes induced by (left to right) low temperature, light, and both. Treatments: 15/L, 15°C/light; 15/D, 15°C/dark; 35/L, 35°C/light; 35/D, 35°C/dark.
The 55 low-temperature-inducible genes included many genes from the flavonoid biosynthesis pathway, such as CHS and antho-MATE (Fig. 6). Interestingly, many ABA-related genes, such as open stomata 1 (OST1), PP2C, and responsive to desiccation 22 (RD22), were present in this group, but none were identified in the group of light-inducible genes. Temperature appears to have a greater effect than light on the ABA content in grape berry skin (Azuma et al., 2012b). These findings suggest that ABA-related genes in grape berries are affected mainly by temperature. OST1 is an ABA-activated protein kinase (a homolog of SnRK2.2/SnRK2.3) that positively regulates ABA signal transduction in Arabidopsis (Mustilli et al., 2002; Yoshida et al., 2002). The ABA level in grape berries increases at the start of veraison, and this change promotes anthocyanin biosynthesis (Coombe and Hale, 1973). In addition, low temperature increases ABA biosynthesis in grape berry skin, and anthocyanin accumulation is positively correlated with the ABA content (Koshita et al., 2007). OST1 was highly expressed in berry skin, and its expression level increased after veraison (Azuma et al., 2015b). In addition, it was up-regulated by low temperature. However, its expression pattern was not correlated with anthocyanin content in the skin of ‘Pione’. Previous results by my research group suggest that ABA levels in detached grape berries are higher at low temperature than at high temperature (Azuma et al., 2012b). On the other hand, the effect of light on the ABA content was negligible compared with the effect of temperature, although anthocyanin accumulation was severely suppressed in the dark treatments. These findings indicate that OST1 might be involved in anthocyanin biosynthesis via low-temperature-induced ABA signaling in berry skin.
4. Light-inducible genesThe 40 light-inducible genes included genes related to light signaling such as elongated hypocotyl 5 (HY5) and constitutive photomorphogenic 1 (COP1) (Fig. 6). Some genes related to flavonoid biosynthesis, such as CHS and FLS, were also included in this group. HY5 expression was high during the maturation stages of berry skin, and was induced by light exposure. HY5 is a basic leucine zipper (bZIP) TF, and is one of the central modulators for the coordination of light signals and regulation of the expression of many genes that have G-box-containing promoters (Lee et al., 2007). In Arabidopsis, HY5 appears to positively regulate anthocyanin accumulation by directly or indirectly binding to the promoters of genes for flavonoid biosynthesis such as CHS and DFR (Lee et al., 2007; Shin et al., 2007). Peng et al. (2013) suggested that MdHY5 in apple is involved in UV-B signaling by binding to the promoter region of MdMYBA, which is responsible for anthocyanin biosynthesis.
The expression level of VlMYBA1-2, which is responsible for anthocyanin biosynthesis in grape skin, was increased by light during the early stages of veraison (Azuma et al., 2012b). However, the expression patterns of HY5 and VlMYBA1-2 were not correlated with the anthocyanin content in the skin of ‘Pione’ (Azuma et al., 2012b). Many genes related to anthocyanin biosynthesis were up-regulated independently by either low temperature or light (Azuma et al., 2012b). These findings suggest that an increase in the expression level of a specific gene involved in the regulation of anthocyanin biosynthesis would not result in increased anthocyanin accumulation. On the other hand, Stracke et al. (2010) reported that HY5 regulates the expression of MYB12, which is a transcriptional regulator of FLS, in response to light in Arabidopsis. The expression of genes related to flavonol biosynthesis, such as MYBF1 and FLS4, was drastically up-regulated by light, and the expression pattern of HY5 was correlated with the levels of MYBF1 and FLS4 (Azuma et al., 2012b). From these findings, it appears that HY5 is involved in the regulation of flavonoid biosynthesis through the regulation of MYB genes, flavonoid biosynthesis genes, or both in grape berry skin (Azuma et al., 2015b).
COP1 was found in the group of light-inducible genes (Fig. 6). Light treatment induced the expression of UV resistance locus (UVR) 8, whereas dark treatment suppressed it. COP1 is considered to be one of the core members of the light signaling pathway, and UVR8 is a photoreceptor specific to UV-B (Osterlund et al., 2000; Rizzini et al., 2011). COP1 and HY5 act together to promote a response to light, and HY5 promotes COP1 transcription via a positive feedback loop (Favory et al., 2009; Huang et al., 2012). Favory et al. (2009) suggested that UVR8 detects UV-B, and that the resulting signal is transduced to COP1. Recently, a comprehensive analysis of co-expression and genome-wide HY5 binding sites was conducted and tested with a transient expression assay in grape, demonstrating its capacity to induce the expression of flavonoid-structural and regulatory genes that relate to flavonoid biosynthesis (Loyola et al., 2016). In addition, the grape berry UV-B response machinery favors flavonoid accumulation by activating HY5 in response to UV-B exposure. A similar UV-responsive behavior was obtained for HY5 in ‘Sauvignon Blanc’ (Liu et al., 2014). Further research is necessary to clarify the roles of these candidate genes in the light signaling networks in grape berry skin.
5. Low-temperature- plus light-inducible genesThe 34 low-temperature- plus light-inducible genes included genes related to flavonoid biosynthesis, light signaling, and ABA signaling (Fig. 6). Among them, the expression level of enhanced response to ABA 1 (ERA1), which encodes a beta subunit of farnesyl transferase that is involved in the ABA-mediated signal transduction pathway in Arabidopsis (Cutler et al., 1996), was strongly affected by both light and temperature conditions, and the expression pattern was significantly positively correlated with the anthocyanin content in the skin of ‘Pione’ (Azuma et al., 2015b). Although Koornneef et al. (1998) suggested that ERA1 is a negative regulator of ABA signal transduction proteins in Arabidopsis, there have thus far been no reports about the function of this gene in grape. More detailed functional characterization of this gene is therefore needed to clarify whether it is involved in anthocyanin biosynthesis via ABA signaling or other signaling networks in grape berry skin.
In summary, we developed a grape oligo-DNA microarray and used it to perform a comprehensive transcriptome analysis that enabled us to identify light- and low-temperature-inducible genes in post-veraison grape berries. We identified 55 low-temperature-inducible genes, 40 light-inducible genes, and 34 low-temperature-plus light-inducible genes. Based on the expression characteristics of three candidate genes, we hypothesized that HY5, OST1, and ERA1 are involved in anthocyanin biosynthesis via low-temperature-induced ABA signaling and light signaling. In addition, the extensive catalog of gene expression patterns defined in this analysis will serve as a valuable reference for future investigations, including the exploration of other candidate genes in grape berry skin that respond to temperature and light.
Genetic studies of grape skin color during the last decade have revealed that the MYB haplotype composition at the color locus is the major genetic determinant of the anthocyanin content and composition in grape berry skin. Using this knowledge, it will be possible to develop a more effective strategy for grape breeding programs such as the development of molecular markers linked to skin color. Recent findings enabled us to predict the skin color of grapes from very young seedlings by examining the MYB haplotype composition. For example, identifying seedlings that are homozygous in functional MYB haplotypes by means of PCR would let us select diploid accessions that will contain a high quantity of anthocyanins in the grape berry skin (for tetraploid accessions, qRT-PCR must instead be used for this identification). In addition, identifying seedlings that contain Hap B or Hap C-Rs would let us select accessions that have an attractive red color and that predominantly contain di-hydroxylated non-methylated anthocyanins in the skin.
Recent studies of environmental impacts on grape skin color by my research group have suggested that low temperature and light have a synergistic effect on anthocyanin accumulation and the expression of genes within the anthocyanin biosynthesis pathway. Based on these findings, we demonstrated that irradiation with light-emitting diodes (LEDs) at night, when the ambient temperature decreases, enhances the expression of genes related to anthocyanin biosynthesis and accelerates anthocyanin accumulation in grape berry skin (Azuma et al., 2012a). Furthermore, many candidate genes for low-temperature-induced ABA signaling and light signaling networks and that are related to anthocyanin accumulation in grape berry skin have been identified. Final anthocyanin content and the expression levels of related genes are determined by complex interactions among internal and external factors such as temperature, light, water status, sugar content, and endogenous ABA content. Further studies including the interactions of these factors at the molecular level will contribute to a fuller understanding of how grape coloration is affected by environmental factors, and should help grape growers to develop cultivation techniques that contribute to the production of highly pigmented grapes.
I deeply thank all the colleagues at the Division of Grape and Persimmon Research, NARO Institute of Fruit Tree and Tea Science for their valuable suggestions and discussions. Thanks are also due to the members of the Technical Support Center at our institute for preparation of plant materials.
