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Light modulates the transcriptomic accumulation of anthocyanin biosynthetic pathway genes in red and white grapes
J Plant Biotechnol 2022;49:292-299
Published online December 31, 2022
© 2022 The Korean Society for Plant Biotechnology.

Puspa Raj Poudel · Kazuya Koyama · Nami Goto-Yamamoto

National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan
Tribhuvan University, Institute of Agriculture and Animal Science, Paklihawa Campus, Siddharthanagar-1, Rupandehi, Lumbini, Nepal
Received October 27, 2022; Revised December 1, 2022; Accepted December 3, 2022.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Anthocyanin, an important component in the grape berry skin, strongly affects grape quality. The transcription factors VvMYBA1 and VvMYBA2 (VvMYBA1/2) control anthocyanin biosynthesis. In addition, cultivation and environmental factors, such as light, influence anthocyanin accumulation. The present study aimed to clarify the effect of shading (reduced light condition) on the transcriptomic regulation of anthocyanin biosynthesis using a red-wine grape cultivar, Vitis vinifera ‘Pinot Noir’, and its white mutant, ‘Pinot Blanc’, caused by the deletion of the red allele of VvMYBA1/2. The grape berry skins were analyzed for anthocyanin content and global gene transcription accumulation. The microarray data were later validated by quantitative real-time PCR. A decisive influence of VvMYBA1/2 on the expression of an anthocyanin-specific gene, UDP glucose: flavonoid 3-O-glucosyltransferase, was observed as expected. In contrast, upstream genes of the pathway, which are shared by other flavonoids, were also expressed in ‘Pinot Blanc’, and the mRNA levels of some of these genes decreased in both cultivars on shading. Thus, the involvement of light-sensitive transcription factor(s) other than VvMYBA1/2 was suggested for the expression control of the upstream genes of the anthocyanin biosynthetic pathway. Furthermore, it was suggested that the effects of these factors are different among isogenes.
Keywords : flavonoids, gene expression, shading, VvMYBAs
Introduction

The quality of grape and grape products such as juice and wine largely depends on the amount and composition of flavonoid phenolics, i.e., anthocyanins, proanthocyanidins and flavonols, in the grape berries. Proanthocyanidins, or condensed tannins, contribute to astringency, whereas anthocyanins are responsible for their color. The accumulation of anthocyanins in grape berry skins starts from veraison and increases gradually until just before full maturity. On the other hand, the proanthocyanidins start to accumulate soon after the berry formation and decline during ripening (Downey et al. 2003a). Another flavonoid group, flavonols, is synthesized around flowering and mainly during ripening in the skin (Downey et al. 2003b). These flavonoid compounds are synthesized through the multi-step phenylpropanoid pathway (Fig. 1). The expression of structural genes in the phenylpropanoid pathway is controlled by transcription factors. For example, the expression of the genes specific for anthocyanin biosynthesis such as UDP-glucose: flavonoid 3-O-glucosyltransferase (VvUFGT) is regulated by the MYB transcription factors, i.e. VvMYBA1 and VvMYBA2 (VvMYBA1/2) (Azuma et al. 2008). In addition, it was also reported that a series of structural genes of the anthocyanin pathway are systematically expressed in red color grapes (Ageorges et al. 2006; Kobayashi et al. 2001; Poudel et al. 2021). Likewise, genes involved in proanthocyanidins biosynthesis are regulated by the transcription factors such as VvMYB5a, VvMYB5b, VvMYBPA1, VvMYBPA2 and VvMYBPAR (Bogs et al. 2007; Deluc et al. 2006, 2008; Koyama et al. 2014; Terrier et al. 2009), while VvMYBF1 is known for flavonols (Czemmel et al. 2009).

Fig. 1. Simplified scheme of the flavonoid biosynthetic pathway of grapes. CHS, chalcone synthase; F3H, flavanone-3-hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase; UFGT, UDP-glucose flavonoid:3-O-glucosyltransferase

To study the function of VvMYBA1/2, a red-wine grape cultivar, ‘Pinot Noir’ and its white mutant ‘Pinot Blanc’ are appropriate objects. It was reported that a red allele of ‘Pinot Noir’ is missing from ‘Pinot Blanc’ (Vezzulli et al. 2012; Yakushiji et al. 2006). A white allele remains in ‘Pinot Blanc’, and it consists of VvMYBA1, which is not transcribed because of a retrotransposon in its promoter (Kobayashi et al. 2004), and VvMYBA2, which is not functional because of a mutation (Walker et al. 2007). Thus, except for the VvMYBA1/2 region, these two varieties should have the almost same genetic background.

In addition to the genetic control, anthocyanins accumulation in grape berry skins depends on various viticultural factors such as canopy management and irrigation, as well as environmental factors such as temperature and light (Brillante et al. 2018; Goto-Yamamoto et al. 2010; Koyama and Goto-Yamamoto 2008; Mori et al. 2007; Poudel et al. 2009; Yang et al. 2020). Among these factors, light is one of the important abiotic factors that regulate the synthesis of flavonoid compounds in grape berries. It has been demonstrated that a shading condition reduced the amount of anthocyanin and mRNA level of anthocyanin-pathway genes (Jeong et al. 2004). However, its control mechanism is not fully understood. Thus, in order to know if only VvMYBA1/2 are responsible for the control of anthocyanin biosynthesis under different light regimes, and if some other factors are involved, how they influence the expression of each isogene, we carried out a bunch-shading experiment using ‘Pinot Noir’ and ‘Pinot Blanc’ grapes and compared the effects on mRNA levels of related genes. Here, we used very robust and high throughput microarray technology to identify the regulation light induced MYB transcription in grape berries. Further, the microarray data were validated by quantitative reverse-transcription real time polymerase chain reaction (qPCR).

Materials and Methods

Plant materials, experimental settings and berry sampling

To determine the influence of light on transcriptomic changes in the skin of grape berries during ripening, the ‘Pinot Noir’ and ‘Pinot Blanc’ grapevines cultivated at the National Research Institute of Brewing, Higashi-Hiroshima, Japan were used. Branches facing the same direction from north to south orientation rows were taken for both treatments. A single bunch was taken as a replicate and nine bunches from three grapevines of each cultivar with similar size and berry numbers at veraison were selected and covered with three layers of Victoria lawn. This shading treatment reduced the light intensity during the daytime to 18%-20% (Jeong et al. 2004). The non-shaded bunches of the same grapevines were taken as the control. To confirm the temperature variation between shaded and non-shaded bunches, the daytime temperature was measured at 16:00, and it was revealed that shading had a negligible effect on temperature during the daytime. Three bunches each were sampled at veraison, two weeks after veraison (WAV) and 4 WAV. For each replicate (n = 3), 30 berries were collected randomly. The skins peeled from the 30 berries were immediately frozen in liquid nitrogen and kept at -80°C until use. The frozen skins were crushed for homogenization and used for RNA extraction and anthocyanin quantification. The results of 4 WAV of non-shaded ‘Pinot Noir’ and ‘Pinot Blanc’ were used in another publication with a different objective (Poudel et al. 2021), hence those data are not presented in this paper and used for discussion purpose only.

Anthocyanin extraction and quantification using high performance liquid chromatography (HPLC)

Anthocyanin was extracted from 0.2 g of berry skin in 2.5 mL of 2% formic acid in 70% methanol (v/v) solution for 20 min with sonication. A total of 400 µL aliquot was used for HPLC analysis after centrifugation at 15,000 rpm for 10 min and filtration with a 0.45 µM micro-membrane filter (Toyo Roshi Kaisha Ltd., Japan). The anthocyanin quantification method used in this study was similar to those described by Ali and Strommer (2003). A Hewlett Packard Series 1100 HPLC system and a Zorbax SB-C18 (5 µm, 2.1 × 150 mm) column were used to separate and quantify the anthocyanin based on peak area. The total anthocyanin concentration was expressed as a milligram of malvidin-3-glucoside (Extrasynthese, France) equivalents per gram of fresh berry skin weight.

RNA extraction

Total RNA from berry skins for qRT-PCR was isolated according to the protocol reported by Reid et al. (2006) and purified using an RNeasy Plant Mini Kit (Qiagen, USA) following the manufacturer’s protocol. The total RNA isolated from 1 g of berry skin was quality assessed and quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA) as well as an RNA Nano chip and RNA 6000 Nano Assay on an Agilent 2100 Bioanalyzer (Agilent Technologies, USA).

Microarray analysis

Microarray and q-PCR analysis methods used in this experiment were identical as those described in our previous study (Poudel et al. 2020). Briefly, for microarray analysis, complementary DNA (cDNA) was synthesized from a total of 10 µg total RNA. The cDNA was cleaned up, quantified, quality assessed and used for hybridization after labelling with Cy3-Random Nonamers (Roche NimbleGen Inc., USA). The hybridization was done with 4 µg Cy3 labelled cDNA to a NimbleGen gene expression 12 × 135 K array (Roche NimbleGen Inc., USA) according to the manufacturer’s protocol. The image data were acquired and analysed using NimbleGen MS 200 software. The data analysis was performed using GeneSpring software. To extract the differentially expressed genes, we applied a > 3.5 fold cut off. The signal intensities obtained from different replicates were averaged and the ratio between the average signal intensities of shaded to that of non-shaded was calculated. Additionally, to further extract the genes with significantly differentially expressed, a t-test was applied assuming the equal variance (< 0.05).

Quantitative reverse-transcription real-time PCR (qRT-PCR)

To determine mRNA levels of the anthocyanin pathway and related genes, qRT-PCR was carried out as described in our previous study (Poudel et al. 2020). Briefly, the qRT-PCR mixture was prepared with cDNA, upper and lower primers (Poudel et al. 2021) and SYBR Green Master Mix (Qiagen), and the final volume was adjusted to 20 µL with RNase-free water. The reaction was performed in a StepOnePlus real-time PCR system and StepOne software version 2.1 (Applied Biosystem, USA). The reaction condition was 95°C for 15 min, followed by 40 cycles at 95°C for 15 s, at each annealing temperature for 20 s and at 72°C for 20 s. The reaction was performed in at least three biological replicates and three analytical replicates for each prepared cDNA sample. The average data were normalized to the ubiquitin control gene.

Results and Discussion

Effect of shading on anthocyanin accumulation

The bunch-shading treatment was applied at veraison, the grape berries were sampled at 2 WAV and their anthocyanin compositions were analyzed using ‘Pinot Noir’. The shading treatment slightly affected the concentration of anthocyanin at 2 WAV (Table 1). However, the data on anthocyanin content were not different significantly at P < 0.05 by t test. When we compared the effect of shading at the full ripened stage, i.e., 4 WAV, the shading significantly reduced the total amount of anthocyanin, to nearly half of the control i.e., non-shaded 2.49 mg·g-1 fresh weight (Poudel et al. 2021) and shaded 1.19 mg·g-1 fresh weight. On the other hand, the composition of anthocyanin was not influenced substantially by shading. Thus, the light was found to be responsible for reducing the amount of anthocyanin rather than altering the composition in this cultivar. Similar results were reported for V. vinifera ‘Red Globe’ (Sun et al. 2020), while the shading treatment reduced the ratio of 3’4’5’-OH anthocyanin to 3’4’-OH anthocyanin of another red-wine cultivar ‘Cabernet Sauvignon’ to some extent (Koyama and Goto-Yamamoto 2008). The effects of light on the anthocyanin composition are probably different among cultivars.

Anthocyanin content (mg·g-1 fw) in the berry skin of ‘Pinot Noir’ with and without shading

Anthocyanins Non-shaded Shaded
Delphinidin 3-glucoside 0.02 ± 0.007 0.01 ± 0.001
Cyanidin 3-glucoside 0.01 ± 0.002 0.00 ± 0.000
Petunidin 3-glucoside 0.04 ± 0.010 0.02 ± 0.003
Peonidin 3-glucoside 0.15 ± 0.010 0.14 ± 0.023
Malvidin 3-glucoside 0.58 ± 0.100 0.61 ± 0.095

Total contents 0.80 ± 0.134 0.78 ± 0.116


Microarray analysis and mRNA levels of flavonoid pathway genes

The transcript level of the structural genes and transcriptional factors involved in anthocyanin biosynthesis as revealed by microarray analysis is presented in Table 2. The log2 ratio of non-shaded to that of shaded revealed that majority of the flavonoid/anthocyanin biosynthesis genes were down regulated, and this effect was much pronounced in red grape Pinot Noir compared to that of white grape Pinot Blanc (Table 2). Among the flavonoid biosynthetic pathway genes such as Chalcone synthase-3 (CHS3), flavonol synthase (VvFLS), leucoanthocyandin reductase 1 (VvLAR1), VvUFGT showed negative values in both the cultivars - Pinot Noir and Pinot Blanc under shaded condition. Likewise, the genes like phenylalanine ammonia-lyase 1 (VvPAL), chalcone isomerase 1 (CHI1), flavonoid 3’ hydroxylase (VvF3’H), flavonoid-3’, 5’-hydroxylase (VvF3’5’H), Caffeoyl-CoA O-methyltransferase, anthocyanin-O-methyltransferase (VvOMT), glutathione S-transferase (GST4), VvMYBPA2 and VvMYB5b were down regulated in red cultivars and up regulated in white grape cultivar. The genes such as CHS1, CHS2, LAR2, VvANR, MYB5a had similar pattern in both the grapes. The microarray data were later validated by qPCR. The qPCR data revealed almost similar pattern to that of microarray analysis (Fig. 2). The microarray data revealed that the shading effect was also found to be responsible to reduce the major structural genes specific to anthocyanin biosynthetic pathway (CHS3, VvFLS, VvLAR1, VvUFGT). More particularly the gene specific to anthocyanin biosynthesis such as UFGT was supressed under shading condition (Table 2). This finding was further supported by qRT-PCR data (Fig. 2). Likewise, anthocyanin biosynthetic specific genes such as VvOMT, GST4 and Caffeoyl-CoA O-methyltransferase were downregulated in red grape cultivar Pinot Noir.

Transcription level of major flavonoid biosynthetic genes as influenced by light condition

Probe ID Accession no. Gene name PN-NS/PN-S PB-NS/PB-S
CHR6_JGVV4_543_T01 GU585850 Phenylalanine ammonia-lyase 1 (VvPAL) -0.18938 0.876675
CHR13_JGVV19_80_T01 BQ796207 Phenylalanine ammonia-lyase 1 (Manihot esculenta) 0.014164 0.309994
CHR14_JGVV68_88_T01 AB015872 Chalcone synthase-1 (CHS1) 0.273898 0.822124
CHR14_JGVV68_87_T01 AB066275 Chalcone synthase-2 (CHS2) 0.177767 1.194718
CHR5_JGVV136_15_T01 AB066274 Chalcone synthase-3 (CHS3) -0.10565 -0.07754
CHR6_JGVV61_85_T01 CB971933 Chalcone synthase 1.044293 0.057527
CHR13_JGVV67_6_T01 X75963 Chalcone isomerase (CHI1) -0.04321 0.468042
CHR18_JGVV1_214_T01 AY257979 Flavonol synthase (VvFLS) -0.61008 -0.56749
CHR17_JGVV0_280_T01 DQ786632 Flavonoid 3’-hydroxylase (VvF3’H) -0.20072 0.137538
CHR6_JGVV9_81_T01 DQ786631 Flavonoid 3’,5’-hydroxylase (VvF3’5’H) -0.00846 0.034731
CHR6_JGVV9_83_T01 BM437829 Flavonoid 3’,5’-hydroxylase 0.072886 0.144696
CHR4_JGVV23_54_T01 X75965 Flavanone-3-hydroxylase (F3H1) 0.222199 -0.20184
CHR18_JGVV1_1071_T01 GU585859 Flavanone-3-hydroxylase 2 (F3H2) V. vinifera ‘Merlot’ 0.05915 0.626465
CHR18_JGVV1_928_T01 X75964 Dihydroflavonol reductase (VvDFR) 0.239058 0.073193
CHR2_JGVV25_429_T01 X75966 Leucoanthocyanidin dioxygenase (VvLDOX) 0.018039 -0.05549
CHR1_JGVV11_360_T01 AJ865335 Leucoanthocyandin reductase (VvLAR1) -0.01258 -0.3349
CHR17_JGVV0_557_T01 AB372550 Leucoanthocyandin reductase (LAR2) 0.192885 0.186066
CHRUN_JGVV361_4_T01 DQ129684 Anthocyanidin reductase (VvANR) 0.750263 0.362415
CHR16_JGVV39_148_T01 AF000372 UDP glucose:flavonoid 3-O-glucosyltransferase (VvUFGT) -0.2948 -0.55772
CHR3_JGVV63_13_T01 CF214966 Caffeoyl-CoA O-methyltransferase -0.5509 0.094469
CHR7_JGVV31_32_T01 CB347033 S-adenosylmethionine-dependent methyltransferase (Arabidopsis thaliana) -0.10558 0.031087
CHR1_JGVV10_262_T01 GU237132 Anthocyanin-O-methyltransferase (VvOMT) -0.29061 0.082774
CHR4_JGVV79_54_T01 CF518071 Glutathione S-transferase (GST4) -0.12097 0.01651
CHR12_JGVV28_290_T01 CF517304 Glutathione S-transferase (GST4) 0.025776 0.056984
CHR2_JGVV33_33_T01 AB097923 Myb-related transcription factor (VvMYBA1) 0.280151 -0.02786
CHR2_JGVV33_30_T01 CB915151 My-related transcription factor (VvMYBA1) 0.390959 0.072633
CHR2_JGVV33_31_T01 DQ886419 MYBA2 red allele 0.216586 0.185839
CHR2_JGVV33_31_T01 DQ886420 MYBA2 white allele 0.216586 0.185839
CHR15_JGVV46_313_T01 AM259485 VvMybPA1 0.071471 0.779112
CHR11_JGVV16_111_T01 EU919682 VvMybPA2 -0.0553 0.223363
CHR8_JGVV7_172_T01 AY555190 Myb transcription factor (MYB5a) 0.158022 0.43784
CHR6_JGVV4_726_T01 AY899404 MYB5b -0.20858 0.106131


Fig. 2. Abundance of the mRNA of genes related to the anthocyanin biosynthetic pathway in the berry skin of ‘Pinot Noir’ and ‘Pinot Blanc’ under shaded and non-shaded conditions. Vertical bars represent mean ± SE. PN, Pinot Noir; PB, Pinot Blanc; S, shaded; NS, Non-shaded

The suppression of anthocyanin biosynthesis genes such as F3H, FLS, DFR and UFGT in addition to transcription factors MYB10, WD40 and bHLH under dark condition have been reported in the leaves of crab apple (Lu et al. 2016). The reduced expression of DFR, LDOX and UF3GT have also been reported in model plant Arabidopsis thaliana under low light condition (Li et al 2016). Reduced expression of MYB1 under reduced light condition was reported in eggplant (Jiang et al. 2016), and oranges (Huang et al. 2019). The signal transduction by MYB transcription factors for anthocyanin regulation is quite complex to describe. The G box (CACGTC) within the promoter region of CsRuby1, a R2R3 MYB transcription factor, was found to regulate anthocyanin in fruit peel of blood orange (Huang et al. 2019). A MYB transcription factor, MYB75 was found to interact with MAP KINASE4 (MPK4) for phosphorylation activity and further playing very crucial role of MAPK pathway in light signal transduction in Arabidopsis thaliana plant (Li et al. 2016).

The microarray analysis also revealed that the genes such as LAR2, VvANR, MYBA2 (both the alleles), MYBPA1 and MYB5b were less influenced (up regulated) under the shaded condition in both the grapes. This finding was in agreement with the qRT-PCR data as well (Fig. 2). Except MYBA2, the transcription factors-MYBPA1, MYB5a and MYB5b are mainly involved in procaynidin biosynthesis (Poudel et al. 2020). It has been reported that PA related genes are less affected by shaded condition (Koyama et al. 2012). It is likely that the expression of transcription factors involved in proanthocyanidin biosynthesis are not influenced by light exclusion during the berry ripening period (two weeks after veraision).

The results of qRT-PCR are shown in Fig. 2. One-way analysis of variance or multiple comparison was not carried out, since many data are not homoscedastic. However, it is obvious that the mRNA level of VvMYBA1 of ‘Pinot Blanc’ was almost undetected, and that of ‘Pinot Noir’ was reduced to a large extent by shading. On the other hand, no clear effects of shading on VvMYBA2, VvMYBA5a and VvMYB5b were observed in both cultivars. The results of VvMYBA1 and VvMYB5b of ‘Pinot Noir’ are consistent with the study of Koyama and Goto-Yamamoto (2008) using ‘Cabernet Sauvignon’. Even though the mRNA level of VvMYBA2 was reduced by shading in that study, its extent was smaller than that of VvMYBA1. Similarly, in the study of V. × labruscana ‘Pione’ (Azuma et al. 2012), VlMybA1-3 was much reduced by a dark condition at 15°C, while the reduction of VlMYB2 was less extent. Thus, the light sensitivity is possibly different between VvMYBA1 and VvMYBA2.

As for VvUFGT, an anthocyanin-specific biosynthetic gene, its mRNA levels were almost negligible in ‘Pinot Blanc’ and reduced by shading in ‘Pinot Noir’. This pattern explains the effect of shading on anthocyanin accumulation well. The difference between ‘Pinot Noir’ and ‘Pinot Blanc’ is explained by VvMYBA1/2 as expected. It should be noticed that VvMYBA2 of ‘Pinot Blanc’ is not functional even when it is transcribed.

In contrast, mRNA of upstream genes, i.e., VvLDOX, VvVvDFR, VvF3H1/2 and VvCHS1/2/3, which are shared by the biosynthesis of other flavonoids, i.e., flavonols and proanthocyanidins (Fig. 2), was detected from both cultivars. Thus, the expression of these genes is probably induced not only by VvMYBA1/2 but also by other transcription factors. The mRNA level of VvLDOX in ‘Pinot Blanc’ was lower than that in ‘Pinot Noir’ and reduced by shading in both cultivars, while the regularity of VvDFR was not clear. The mRNA levels of both isogenes of F3H were reduced by shading, and mRNA levels of VvF3H1 in ‘Pinot Blanc’ were consistently lower than that in ‘Pinot Noir’. This result suggests that VvMYBA1/2 influences the expression of VvF3H1 more strongly than that of VvF3H2.

Among three isogenes of CHS, mRNA levels of VvCHS2 in ‘Pinot Blanc’ were much lower than those of ‘Pinot Noir’, and those of VvCHS3 in ‘Pinot Blanc’ were almost negligible (Fig. 2). No clear influence of shading on these genes was observed. These patterns can be explained by a hypothesis that these two CHS isogenes were induced to a large extent by the function of VvMYBA1/2. Lower mRNA levels of VvCHS3 and VvCHS2 in ‘Pinot Blanc’ compared with those in ‘Pinot Noir’ were also observed using a microarray assay (Poudel et al. 2014). In contrast, a certain level of VvCHS1 mRNA was detected in ‘Pinot Blanc’, suggesting a strong influence of other transcription factors than VvMYBA1/2.

Thus, isogenes of CHS and F3H were shown to be differently regulated by VvMYBA1/2 and other transcription factors. Azuma et al. (2012) also reported some isogenes were differentially regulated during light and temperature treatments, which have a synergistic effect on the expression of genes in the pathway using ‘Pione’. In addition, the results of ‘Pinot Blanc’ suggest the involvement of light-sensitive transcription factor(s). A transcription factor for flavonols, VvMYBF1, which was reported to be strongly induced by UV light (Czemmel et al. 2009), is a reasonable candidate. LDOX and DFR, however, are not involved in flavonol biosynthesis (Fig. 1). Thus, the biosynthesis of proanthocyanidins, which are polymers of flavan-3-ols, e.g., catechin and epicatechin, is possibly related to the expression of VvLDOX and VvDFR in ‘Pinot Blanc’, even though the proanthocyanidins are mainly synthesized in the earlier stage of berry development and maturation. Otherwise, unknown factor(s) might be involved in the induction of these genes. It was reported that many genes are involved in the regulation of light-induction of anthocyanin in grape berries (Sun et al. 2020). Further research is needed to elucidate the control mechanisms of flavonoid biosynthetic genes under different light conditions. It is also interesting if these control mechanisms other than VvMYBA1/2 influence the anthocyanin biosynthesis.

Conclusion

Anthocyanin biosynthesis in the grape skin is controlled genetically and influenced by many factors such as light. Comparison of the mRNA levels of anthocyanin biosynthetic pathway genes in ‘Pinot Noir’ and ‘Pinot Blanc’ showed the decisive influence of VvMYBA1 and VvMYBA2 on the expression of an anthocyanin-specific gene, VvUFGT. On the other hand, upstream genes of the pathway, which are shared by other flavonoids, were also expressed in ‘Pinot Blanc’, a cultivar that lacks functional VvMYBA genes, and the influence of light was observed in some genes. Therefore, the involvement of light-sensitive transcription factor(s) other than VvMYBA1/2 was suggested for the expression of upper-stream genes of the anthocyanin biosynthetic pathway. Also, it was indicated that these transcriptional factors influence the expression differently among isogenes.

Acknowledgement

This research was supported by the grants from Japan Society for the Promotion of Science, Japan, to P. R. Poudel.

Conflict of interests

The authors declare that there is not any conflict of interest.

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