Microarray Analysis for the Screening of Genes Inducible by Light or Low Temperature in Post-veraison Grape Berries

Abstract

Flavonoid biosynthesis in grape (Vitis spp.) berry skin is affected by environmental factors such as light and temperature. However, the components of the light signaling and low-temperature-induced abscisic acid (ABA) signaling networks related to flavonoid accumulation in grape berry skin have not been fully elucidated, and how environmental conditions affect these components remains unclear. To clarify the details of the possible light- and ABA-related signal transduction networks, we developed a grape oligo-DNA microarray (38,549 independent probes) using the publicly available genomic sequence of grape, and performed comprehensive transcriptome analysis using detached ‘Pione’ grape (Vitis labruscana × V. vinifera) berries cultured under different light and temperature conditions. Using the microarray data, we explored the light-inducible and low-temperature-inducible genes in post-veraison grape berries. We identified 40 light-inducible genes, 55 low-temperature-inducible genes, and 34 genes induced by light plus low temperature. Among these, we selected elongated hypocotyl 5 (HY5), open stomata 1 (OST1), and enhanced response to ABA 1 (ERA1) as candidate light-inducible, low-temperature-inducible, and light- plus low-temperature-inducible genes, respectively. We investigated their detailed expression characteristics in grape accessions by means of quantitative real-time polymerase chain reaction analyses, and hypothesized that HY5, OST1, and ERA1 might be involved in flavonoid biosynthesis via light signaling and low-temperature signaling networks. We also established an extensive catalog of gene expression patterns to support future investigations of other candidate genes that respond to light and temperature in grape berry skin.

Introduction

Anthocyanins and flavonols, which are two classes of flavonoids, are secondary plant metabolites that accumulate in various plant organs. The skin color of grape (black, red, or white) is mainly determined by the content and composition of anthocyanins, and flavonols contribute to wine color through co-pigmentation with anthocyanins (Baranac et al., 1997). Coloration of grape berry skin is an important determinant of consumer preference and marketability. However, in regions with a warm climate, decreased grape quality, such as poor coloration, is a common problem mainly caused by high temperatures during the maturation stages (Teixeira et al., 2013; Winkler et al., 1962). Because of the commercial importance of grape, it is important to learn how grape coloration is affected by environmental factors, as this knowledge may contribute to more stable production of high-quality grapes despite global atmospheric warming.

Many genes that encode the enzymes of the flavonoid biosynthesis pathway in grape have been isolated (Sparvoli et al., 1994). Some MYB-related transcription factors such as VvMYBA1, VvMYBA2, VlMYBA1-2, VlMYBA1-3, and VlMYBA2 have been identified and shown to regulate anthocyanin biosynthesis (Azuma et al., 2008; Kobayashi et al., 2004; Walker et al., 2007). The grape color locus has been suggested to be a cluster of MYB genes spanning a 200-kb region of chromosome 2 (Azuma et al., 2009; Fournier-Level et al., 2009). Since the adjacent MYB genes in the color locus are inherited together, these can be regarded as an MYB haplotype (Azuma et al., 2008, 2011). Haplotype A is non-functional, and white-skinned accessions are homozygous for it. E1 and E2 are functional haplotypes, and we found that the genotypes of tetraploid black grapes, such as ‘Pione’ and ‘Kyoho’, are A/A/E1/E2 (Azuma et al., 2011). On the other hand, tetraploid red grapes, such as ‘Aki Queen’ and ‘Queen Nina’, were shown to contain only a single functional haplotype (A/A/A/E1). Thus, anthocyanin content is mainly determined by the type and number of MYB haplotypes. It has been suggested that the activity of flavonol synthase (FLS) is required for flavonol biosynthesis because the expression of FLS increases during ripening, coincident with the accumulation of flavonols in grape berry skin (Downey et al., 2003). Czemmel et al. (2009) found that the MYB-related transcription factor MYBF1 is a transcriptional regulator of FLS. In addition, several reports have suggested that other MYB-family proteins, such as MYB5a, MYB5b, MYBPA1, and MYBPA2, appear to regulate several genes in the common steps of the flavonoid biosynthesis pathway (Bogs et al., 2007; Deluc et al., 2006, 2008; Terrier et al., 2009).

Previous reports indicated that light and temperature are important environmental factors that affect flavonoid biosynthesis in many plants (Bowler and Chua, 1994; Christie and Jenkins, 1996; Christie et al., 1994; Dela et al., 2003; Ubi et al., 2006). In many grape accessions, such as black grapes ‘Pione’ and ‘Kyoho’, and red grape ‘Aki Queen’, the accumulation of anthocyanins in berry skin begins after the onset of ripening (veraison) and is affected by light and temperature conditions (Azuma et al., 2012; Kliewer and Torres, 1972; Matus et al., 2009; Tomana et al., 1979a, b; Yamane et al., 2006). Several studies found that the expression of the flavonoid biosynthetic genes and MYB-related genes in grape berry skin was induced by light, low temperature, or both, but was suppressed by shading and high temperature (Azuma et al., 2012; Jeong et al., 2004; Matus et al., 2009; Mori et al., 2005; Yamane et al., 2006). Recently, we found that the accumulation of anthocyanins depended on both light and low temperature (Azuma et al., 2012). However, the dependence of the effect of low temperature on light-dependent anthocyanin accumulation, and vice versa, remains poorly understood.

The level of the plant hormone abscisic acid (ABA) increases at veraison and enhances anthocyanin biosynthesis in grape berry skin (Coombe and Hale, 1973). It has also been found that low temperature accelerates anthocyanin biosynthesis and that the endogenous ABA content in grape berry skin is positively correlated with the anthocyanin content in it (Koshita et al., 2007; Yamane et al., 2006). On the other hand, our previous research suggested that light had a weaker effect than temperature on ABA content, although dark treatment greatly decreased the anthocyanin content (Azuma et al., 2012). Thus, many reports have described the impact of environmental factors and ABA on flavonoid accumulation in grape berry skin. However, the components of the light signaling and low-temperature-induced ABA signaling networks related to flavonoid accumulation in grape berry skin have not been elucidated, and how environmental conditions affect these components remains poorly understood.

The aim of the present study was to identify light- and low-temperature-inducible genes in post-veraison grape berries. As a first step, we developed a grape oligo-DNA microarray, and performed comprehensive transcriptome analysis using detached grape berries cultured under different light and temperature conditions. This allowed us to identify many light- and low-temperature-inducible genes, as well as genes induced by both factors. Next, we investigated the expression levels of these candidate genes during several developmental stages in berry skin and in several organs, and determined some of the transcriptional characteristics of each gene.

Materials and Methods

Light and temperature treatments using detached grape berries

In vitro environmental experiments using detached grape berries were performed as described previously (Azuma et al., 2012). Briefly, grape berries just beginning to show color were collected from mature vines of black table grape ‘Pione’ (Vitis labruscana × V. vinifera) and red table grape ‘Aki Queen’ (V. labruscana × V. vinifera) growing in a vineyard at the Grape and Persimmon Research Station, National Agriculture and Food Research Organization (NARO) Institute of Fruit Tree Science, Hiroshima, Japan. The surface-sterilized berries were placed on 0.7% agar medium, and 15 berries per treatment were incubated in a multi-incubator (TG-180CCFL-5LD; Nippon Medical and Chemical Instruments, Osaka, Japan) for 10 days under one of four treatments: 15°C/Light (15/L), 15°C/Dark (15/D), 35°C/Light (35/L), and 35°C/Dark (35/D). The light was a mixture of white light (cold-cathode fluorescent lamps) and UV light (FL10BLB; Toshiba, Tokyo, Japan) with continuous irradiation at 80 μmol·m⁻²·s⁻¹. After incubation, peeled berry skin was immediately frozen in liquid nitrogen and kept at −80°C until analysis. The ‘Pione’ data were obtained from our previous study (Azuma et al., 2012), but the grapes from ‘Aki Queen’ were newly analyzed in the present study.

Analysis of anthocyanin and flavonol contents

The total anthocyanin content in grape skin was analyzed by the method of Shiraishi et al. (2007), and was expressed as mg of cyanidin-3-monoglucoside (Extrasynthèse, Genay, France) equivalent per g of fresh berry skin. Total flavonol contents in grape skin were analyzed by the method of Mazza et al. (1999), with the slight modifications described by Azuma et al. (2012). Statistical tests were performed using version 9.0.0 of the JMP statistical software (SAS Institute Inc., Cary, NC, USA).

Construction of a grape oligo-DNA microarray

A grape oligo-DNA microarray was constructed using the eArray system (Agilent; Santa Clara, CA, USA; https://earray.chem.agilent.com/earray/) according to the standard system protocols. Probes (60 oligonucleotides per probe) were constructed using the sequence data in public databases: 30,434 assembled mRNAs (8× coverage) in the Genoscope Grape Genome Browser (French National Sequencing Center; http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/entry_ggb.html; Jaillon et al., 2007) and 23,152 unique gene sequences in NCBI UniGene Vitis vinifera Build #8 (http://www.ncbi.nlm.nih.gov/unigene). In total, 38,549 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. The remaining probes (17.1%) could not be functionally annotated or had no homologs in other species. After we had developed the microarray, the grape genome sequence data (12× coverage) were released by Genoscope (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/). The 30,434 assembled mRNAs (8× data) were compared with the 26,346 assembled mRNAs from the 12× sequence data using BLASTN (Altshul et al., 1990). In total, 25,880 of the 30,434 mRNAs from the 8× data (i.e., 85%) were similar to those in the 12× data (BLASTN e-value cut-off 1.1 × 10⁻⁴). The summarized full correspondence data are available online (http://cse.naro.affrc.go.jp/hfujii/grape_seq_data_8x&12x/grape_seq_data_8x&12x.htm).

Microarray experiments and analyses

Total RNA was extracted from the grape skin of ‘Pione’ as described by Reid et al. (2006) using three biological samples from each of the four experimental conditions. The RNA concentration was measured spectrophotometrically (NanoDrop 1000; Thermo Fisher Scientific, Waltham, MA, USA), and the sample integrity was assessed using a Bioanalyzer (Agilent). Total RNA (250 ng) was labeled with Cy3 according to the instructions for the Quick Amp Labeling Kit (Agilent). Labeled cRNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The labeled samples were hybridized to the oligoarray slides using hybridization buffer from the Gene Expression Hybridization Kit (Agilent) at 65°C for 17 h in a hybridization chamber. After hybridization, the arrays were washed with Gene Expression Wash Buffer (Agilent) according to the manufacturer’s instructions. After drying, the hybridized slides were scanned with a G2565A microarray scanner and the images were extracted using version 9.5 of the Feature Extraction software (Agilent).

Data analysis was carried out using the GeneSpring GX 10 software (Agilent; http://biology.chem.agilent.com/) and the Subio Platform (Subio Inc., Kagoshima, Japan; http://www.subio.jp/products/platform). Data normalization was carried out according to the manufacturer’s recommended protocol (Agilent FE for 1 Color). The expression level of each gene was calculated as the mean value of three biological replicates for all treatments. 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: light-inducible genes, low-temperature-inducible genes, and light- plus low-temperature-inducible genes. Light-inducible genes were extracted from the overlap of condition 1 (15/L versus 15/D), condition 2 (15/L versus 35/D), condition 3 (35/L versus 15/D), and condition 4 (35/L versus 35/D). Low-temperature-inducible genes were extracted from the overlap of condition 2, condition 5 (15/L versus 35/L), condition 6 (15/D versus 35/L), and condition 7 (15/D versus 35/D). Light- plus low-temperature-inducible genes were extracted from the overlap of conditions 1, 2, and 5.

Using the search function provided by Subio Platform, the extracted genes were further screened by searching for the following annotation keywords: light, UV, ABA, SnRK (SNF-related kinase), PP2C (protein phosphatase 2C), flavonoid, anthocyanin, and flavonol. The statistical significance of the difference in expression levels among treatments for each extracted gene was analyzed by means of two-way ANOVA.

Quantitative real-time polymerase chain reaction (qRT-PCR) analyses

Verification of the microarray analysis

Total RNA was extracted as described above from three biological replicates for each of the four experimental conditions in ‘Pione’ and ‘Aki Queen’. cDNA was synthesized using the PrimeScript II cDNA synthesis kit (Takara, Shiga, Japan), following the protocols described in the manufacturer’s manuals. qRT-PCR was performed using a 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) and a QuantiTect SYBR Green PCR kit (Qiagen), as described in the manufacturers’ manuals. We determined the expression levels of three candidate genes [elongated hypocotyl 5 (HY5), open stomata 1 (OST1), and enhanced response to ABA 1 (ERA1)] selected based on the magnitude of the difference in expression between treatments in berry skin. Primers for amplification of these genes are shown in Table 1. Relative magnitudes of gene expression were calculated using the 2^−ΔΔCt method. The amounts of the transcripts were normalized against the levels of the Ubiquitin1 transcript (Bogs et al., 2006). qRT-PCR was carried out with three biological replicates per treatment.

Table 1

Primer sequences for qRT-PCR.

Expression levels of the candidate genes at several developmental stages in the berry skin and several organs

Berry skin was collected from black table grape ‘Kyoho’ (V. labruscana × V. vinifera) growing in a vineyard just before veraison (45 days after flowering, DAF), when the berries were beginning to color (48 DAF), when the berries had fully developed their red-purple color (52 DAF), and when the berries had fully developed their black color (77 DAF). Mature berry flesh, roots, tendrils, flowers, young leaves (expanded to ≈6 cm), and seeds (collected from the berries at 21 DAF) of ‘Kyoho’ were also collected. Total RNA extraction, cDNA synthesis, and qRT-PCR analysis were performed as described above.

Diurnal expression pattern of HY5

We used container-grown vines of ‘Pione’ in a greenhouse at our institute to examine the diurnal pattern of HY5 expression. We collected three berries per sample at 3-h intervals, from 0:00 to 21:00, on 3 August, 2011 (21 d after the onset of coloration, when the berries had fully developed their reddish-purple color). We also measured the light intensity (photosynthetic photon flux, PPF) near the grape bunches at 3-h intervals during the same period, on the same day, using a quantum radiometer photometer (LI-189; LI-COR, Lincoln, NE, USA). Total RNA extraction, cDNA synthesis, and qRT-PCR analysis were performed as described above.

Results

Total anthocyanin content and total flavonol content in the in vitro environmental experiment

Sufficient anthocyanin accumulation was observed in the grape skin of ‘Pione’ in the 15/L treatment, whereas the high-temperature (35°C) and dark treatments both greatly decreased anthocyanin accumulation (Fig. 1), as in our previous study (Azuma et al., 2012). The total anthocyanin content in the grape skin of ‘Aki Queen’ showed a similar pattern to that in ‘Pione’. The total anthocyanin contents of ‘Aki Queen’ were 0.14, 0.04, 0.04, and 0.03 mg·g⁻¹ fresh weight in the 15/L, 15/D, 35/L, and 35/D treatments, respectively. The total anthocyanin content in the 15/L treatment was significantly higher than in the other treatments in both grape accessions, although the anthocyanin content of ‘Aki Queen’ was lower than that of ‘Pione’. These results indicate that irradiation at low temperature induces sufficient anthocyanin accumulation. In the present study, we found that the total flavonol contents in the irradiation treatments were significantly higher than those in the dark treatments in ‘Aki Queen’, in line with our previous data for ‘Pione’ (Azuma et al., 2012). These results suggest that irradiation is required to induce flavonol accumulation in berry skin.

Fig. 1

Total anthocyanin contents and total flavonol contents of berry skin in ‘Pione’ and ‘Aki Queen’. Error bars represent standard errors (SE). Means labeled with different letters are significantly different among treatments in each accession (P < 0.05, Tukey-Kramer test). The data for total anthocyanin content and total flavonol content in grape skin of ‘Pione’ were previously published (Azuma et al., 2012).

Screening of the light- and low-temperature-inducible genes using microarrays

Microarray analysis was carried out using detached grape berries of ‘Pione’ that had been incubated for 10 days under the four sets of environmental conditions. The complete microarray data have been deposited in the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) series entry GSE64153.

As a result of this analysis, 40 genes were identified as light-inducible genes (Table 2). We found seven genes related to flavonoid biosynthesis (annotation keywords anthocyanin, flavonoid, and flavonol). The expression of FLS was especially drastically up-regulated by light. Interestingly, genes in the light-signaling pathway, such as HY5 and constitutive photomorphogenic 1 (COP1), were found in this group. We also found that the 15/L treatment induced the expression of UV resistance locus 8 (UVR8: EC990084-C, GSVIVT00019292001), whereas dark treatment suppressed this expression even though this gene was not detected as a light-inducible gene in the present study based on our criteria (data not shown). On the other hand, genes related to ABA biosynthesis and signaling were absent. These results suggest that the effect of light on the expression levels of ABA-related genes is either weak or not significant.

Table 2

Genes that were identified as light-inducible genes in ‘Pione’.

An additional 55 genes were identified as low-temperature-inducible genes (Table 3). A total of 20 ABA-related genes (annotation keywords ABA, SnRK, and PP2C) were up-regulated by the low-temperature treatment. Interestingly, ABA-responsive genes, such as OST1, protein phosphatase 2C (PP2C), and responsive to desiccation 22 (RD22), were found in this group. These results suggest a strong effect of temperature on the expression levels of ABA-related genes. We also found that 35 genes related to flavonoid biosynthesis, including chalcone synthase (CHS), transparent testa 6 (TT6), and dihydroflavonol 4-reductase (DFR), were drastically up-regulated by the low-temperature treatment.

Table 3

Genes that were identified as low-temperature-inducible genes in ‘Pione’.

A total of 34 genes were identified as light- plus low-temperature-inducible genes (Table 4). Four ABA-related genes, such as ERA1, and 14 light (including UV)-responsive genes were up-regulated; 16 genes related to flavonoid biosynthesis, such as transparent testa 7 (TT7) and CHS, were induced by this treatment.

Table 4

Genes that were identified as light- plus low-temperature-inducible genes in ‘Pione’.

Our particular focus in the present study is on candidate genes involved in the light-signaling and low-temperature-induced ABA signaling networks related to flavonoid accumulation. We selected HY5, OST1, and ERA1 as the candidate genes for light, temperature, and light plus temperature responses, respectively, and investigated their expression in more detail.

qRT-PCR analyses

Verification of the microarray analysis

qRT-PCR analysis was carried out to validate the expression patterns of the three candidate genes (HY5, OST1, and ERA1) in the grape skin of both ‘Pione’ and ‘Aki Queen’. The results are summarized in Figure 2. Expression levels of HY5 in the 15/L and 35/L treatments were significantly higher than those in the 15/D and 35/D treatments in both accessions. This result demonstrates that the expression level of HY5 is up-regulated by light. The expression of OST1 was up-regulated by the low-temperature treatment; this effect was independent of irradiation in ‘Aki Queen’. The 15/L treatment up-regulated the expression of ERA1, whereas the dark and high-temperature treatments both significantly and severely down-regulated the expression of this gene in both accessions. These results suggest that both low temperature and light are needed to induce ERA1 expression. These results demonstrate that the results of our microarray analysis are reliable and that the expression patterns of these three genes are similar between black and red grape accessions.

Fig. 2

Relative expression levels of HY5 (light-inducible), OST1 (low-temperature-inducible), and ERA1 (light- plus low-temperature-inducible) in grape berry skin, as measured by qRT-PCR. All data are presented as means of three biological replicates. Error bars represent standard errors (SE). Means labeled with different letters are significantly different among treatments in each accession (P < 0.05, Tukey-Kramer test). All expression levels are relative to the expression of Ubiquitin1.

Expression levels of the candidate genes at several developmental stages in berry skin and several organs

The expression levels of HY5, OST1, and ERA1 at several developmental stages in grape skin and in different organs of ‘Kyoho’ (a black table grape like ‘Pione’) are shown in Figure 3. The expression level of HY5 in grape skin was high throughout the maturation stages, and was higher than in the other organs. The expression level of OST1 in grape skin tended to increase towards harvest time, and was much higher than that in other organs. These results suggest that HY5 and OST1 are highly expressed in grape skin during maturation. On the other hand, the expression level of ERA1 tended to decrease towards harvest, and was also high in the tendril, flower, and leaf tissues, although it was almost undetectable in the root and seed tissues.

Fig. 3

Relative expression levels of HY5 (light-inducible), OST1 (low-temperature-inducible), and ERA1 (light- plus low-temperature-inducible) in berry skin of ‘Kyoho’ at several stages of development, and from several organs of ‘Kyoho’ vines, as measured by qRT-PCR. Capital letters indicate the maturation stages of the grape berries (B just before veraison, S beginning to color, R full development of reddish-purple color, M full development of black color). All data are presented as the means of three biological replicates. Error bars represent standard errors (SE). All expression levels are relative to the expression of Ubiquitin1.

Diurnal expression of HY5

Our diurnal expression analysis showed that the expression of HY5 was up-regulated from morning (starting when PPF increased above 0) until evening, and was severely down-regulated during the night (Fig. 4). We investigated the light intensity change near the grape bunches on 3 August, and confirmed that the PPF was high from 09:00 to 15:00 and near-zero during the night. These results suggest that the expression level of HY5 might follow a circadian rhythm in grape skin, and that light exposure from morning until evening is important in the induction of HY5 expression.

Fig. 4

Diurnal expression of HY5 in ‘Pione’ grape berry skin, as measured by qRT-PCR. Values are means ± standard errors (SE) of three biological replicates. All expression levels are relative to the expression of Ubiquitin1.

Discussion

Many studies have elucidated the impact of environmental factors on flavonoid biosynthesis in grape berries (Kliewer and Torres, 1972; Matus et al., 2009; Mori et al., 2005). However, we do not yet understand how the interactions between temperature and light affect flavonoid biosynthesis in post-veraison grapes because such studies are difficult under field conditions. On the other hand, in vitro experiments using detached berries enable us to control environmental conditions accurately. Therefore, this method has been used by many research groups to investigate the effects of environmental conditions on flavonoid biosynthesis in grape berries (Kataoka et al., 2003; Mori et al., 2007; Zheng et al., 2009). In the present study, we developed a grape oligo-DNA microarray using the publicly available genomic sequence of grape (Jaillon et al., 2007), and performed comprehensive transcriptome analysis using detached ‘Pione’ grape berries cultured under different light and temperature conditions. Using microarray data, we identified light- and temperature-inducible genes that might be involved in flavonoid biosynthesis in post-veraison grape berries.

Light-inducible genes

We identified 40 light-inducible genes by microarray analysis (Table 2). These included genes related to light signaling, such as HY5 and COP1. We also found some genes related to flavonoid biosynthesis, such as CHS and FLS, in this group. We further investigated the expression characteristics of HY5 in grape accessions by means of qRT-PCR, and found that HY5 expression was high during the maturation stages of berry skin, and was induced by light exposure (Figs. 2, 3, and 4). HY5 is a basic leucine zipper (bZIP) transcription factor, 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 is thought to regulate anthocyanin and flavonol accumulation positively 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. Previously, we reported that the expression level of VlMYBA1-2, which is responsible for anthocyanin biosynthesis in grape, was induced by light during the early stages of veraison (Azuma et al., 2012). We also found that VlMYBA1-2 had the G-box-containing promoter (our unpublished data). The expression patterns of HY5 and VlMYBA1-2 were not correlated with the levels of anthocyanins in the grape skin of both ‘Pione’ and ‘Aki Queen’ (Figs. 1 and 2; Azuma et al., 2012). Our previous study suggested that many anthocyanin biosynthesis-related genes were up-regulated independently by either low temperature or light (Azuma et al., 2012). These findings imply 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. Previously, it was found that the expression of genes related to flavonol biosynthesis, such as VvMYBF1 and FLS4, was drastically up-regulated by light (Azuma et al., 2012). We also found that the expression pattern of HY5 was correlated with the levels of VvMYBF1 and FLS4 (Fig. 2 and Azuma et al., 2012). From the present findings and our previous studies, we hypothesize that HY5 is involved in the regulation of flavonoid biosynthesis through the regulation of MYB-related genes, flavonoid biosynthetic genes, or both in grape berry skin.

COP1 was also found in the group of light-inducible genes (Table 2). It was also found that light treatment induced the expression of UVR8, whereas dark treatment suppressed this expression even though this gene was not detected as a light-inducible gene in the present study. 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. Further research is necessary to clarify the roles of these candidate genes in the light signaling networks in grape berry skin.

Low-temperature-inducible genes

We detected 55 low-temperature-inducible genes, which included many flavonoid biosynthesis pathway genes, such as CHS, DFR, and MATE (Table 3). Interestingly, many ABA-related genes, such as OST1, PP2C, and RD22, were also present in this group, whereas no ABA-related genes were identified in the group of light-inducible genes based on our criteria. Our previous study suggested that temperature has a greater effect than light on the ABA content in grape berry skin (Azuma et al., 2012). These findings suggest that ABA-related genes in grape berries might be mainly affected by temperature conditions and not by light conditions. OST1 is an ABA-activated protein kinase (a homolog of SnRK2.2/SnRK2.3), which acts as a positive regulator of ABA signal transduction in Arabidopsis (Mustilli et al., 2002; Yoshida et al., 2002). It has been suggested that the level of ABA in grape berry increases at the start of veraison and that this change promotes anthocyanin biosynthesis (Coombe and Hale, 1973). It has also been suggested that low temperature increases ABA biosynthesis in grape berry skin and that anthocyanin accumulation is positively correlated with ABA content (Koshita et al., 2007). In the present study, we found that OST1 was highly expressed in berry skin, and that the expression level increased after veraison (Fig. 3). We also found that the expression of OST1 was up-regulated by low temperature (Fig. 2). The expression pattern of OST1 was not correlated with the levels of anthocyanins and flavonols in the grape skin of both ‘Pione’ and ‘Aki Queen’ (Figs. 1 and 2). Our previous study suggested that ABA levels in detached grape berries were higher in low-temperature treatment than at high temperatures (Azuma et al., 2012). On the other hand, the effect of light on ABA content was relatively weak compared with the effect of temperature, although anthocyanin accumulation in dark treatments was severely suppressed. From these findings, we hypothesize that low temperature and light have a synergistic effect on anthocyanin content, and OST1 might be involved in flavonoid biosynthesis via low-temperature-induced ABA signaling in berry skin.

Light- plus low-temperature-inducible genes

We detected 34 light- plus low-temperature-inducible genes, which included genes related to flavonoid biosynthesis, light, and ABA (Table 4). Among them, we focused on the expression characteristics of ERA1, which encodes a beta subunit of farnesyl transferase involved in the ABA-mediated signal transduction pathway in Arabidopsis (Cutler et al., 1996). It has also been suggested that ERA1 is a negative regulator of ABA signal transduction proteins (Koornneef et al., 1998). The results of the present qRT-PCR analyses showed that the expression level of ERA1 was strongly affected by both light and temperature conditions, and the expression pattern was correlated with the levels of anthocyanins in the grape skin of both ‘Pione’ and ‘Aki Queen’ (Figs. 1 and 2). We also found that the expression level of ERA1 in berry skin tended to decrease towards harvest time (Fig. 3). However, there have thus far been no reports about the function of this gene in grape. Therefore, more detailed functional characterization of this gene is needed to clarify whether it is involved in flavonoid 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. On the basis of this analysis, we identified 40 light-inducible genes, 55 low-temperature-inducible genes, and 34 light- plus low-temperature-inducible genes. From the expression characteristics of three candidate genes, we hypothesize that HY5, OST1, and ERA1 might be involved in flavonoid biosynthesis via light signaling and low-temperature-induced ABA signaling. In addition, the extensive catalog of gene expression patterns defined in this study will serve as a valuable reference for future investigations, including the exploration of other candidate genes in grape berry skin that respond to light and temperature. Our findings will therefore contribute to improving our understanding of how grape coloration is affected by environmental factors at the molecular level.

Literature Cited

•  Altschul,  S. F.,  W.  Gish,  W.  Miller,  E. W.  Myers and  D. J.  Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403–410.
•  Azuma,  A.,  S.  Kobayashi,  N.  Goto-Yamamoto,  M.  Shiraishi,  N.  Mitani,  H.  Yakushiji and  Y.  Koshita. 2009. Color recovery in berries of grape (Vitis vinifera L.) ‘Benitaka’, a bud sport of ‘Italia’, is caused by a novel allele at the VvmybA1 locus. Plant Sci. 176: 470–478.
•  Azuma,  A.,  S.  Kobayashi,  N.  Mitani,  M.  Shiraishi,  M.  Yamada,  T.  Ueno,  A.  Kono,  H.  Yakushiji and  Y.  Koshita. 2008. Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape berry skin. Theor. Appl. Genet. 117: 1009–1019.
•  Azuma,  A.,  Y.  Udo,  A.  Sato,  N.  Mitani,  A.  Kono,  Y.  Ban,  H.  Yakushiji,  Y.  Koshita and  S.  Kobayashi. 2011. Haplotype composition at the color locus is a major genetic determinant of skin color variation in Vitis × labruscana grapes. Theor. Appl. Genet. 122: 1427–1438.
•  Azuma,  A.,  H.  Yakushiji,  Y.  Koshita and  S.  Kobayashi. 2012. Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions. Planta 236: 1067–1080.
•  Baranac,  J. M.,  N. A.  Petranovic and  J. M.  Dimitric-Markovic. 1997. Spectrophotometric study of anthocyan copigmentation reactions. 2. Malvidin and the nonglycosidized flavone quercetin. J. Agric. Food Chem. 45: 1694–1697.
•  Bogs,  J.,  A.  Ebadi,  D.  McDavid and  S. P.  Robinson. 2006. Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development. Plant Physiol. 140: 279–291.
•  Bogs,  J.,  F. W.  Jaffé,  A. M.  Takos,  A. R.  Walker and  S. P.  Robinson. 2007. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol. 143: 1347–1361.
•  Bowler,  C. and  N. H.  Chua. 1994. Emerging themes of plant signal transduction. Plant Cell 16: 1529–1541.
•  Christie,  P. J.,  M. R.  Alfenito and  V.  Walbot. 1994. Impact of low temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194: 541–549.
•  Christie,  J. M. and  G. I.  Jenkins. 1996. Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells. Plant Cell 8: 1555–1567.
•  Coombe,  B. G. and  C. R.  Hale. 1973. The hormone content of ripening grape berries and the effects of growth substance treatments. Plant Physiol. 51: 629–634.
•  Cutler,  S.,  M.  Ghassemian,  D.  Bonetta,  S.  Cooney and  P.  McCourt. 1996. A protein farnesyl transferase involved in abscisic acid signal transduction in Arabiopsis. Science 273: 1239–1241.
•  Czemmel,  S.,  R.  Stracke,  B.  Weisshaar,  N.  Cordon,  N. N.  Harris,  A. R.  Walker,  S. P.  Robinson and  J.  Bogs. 2009. The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries. Plant Physiol. 151: 1513–1530.
•  Dela,  G.,  E.  Or,  R.  Ovadia,  A.  Nissim-Levi,  D.  Weiss and  M.  Oren-Shamir. 2003. Changes in anthocyanin concentration and composition in ‘Jaguar’ rose flowers due to transient high-temperature conditions. Plant Sci. 164: 333–340.
•  Deluc,  L.,  F.  Barrieu,  C.  Marchive,  V.  Lauvergeat,  A.  Decendit,  T.  Richard,  J. P.  Carde,  J. M.  Merillon and  S.  Hamdi. 2006. Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway. Plant Physiol. 140: 499–511.
•  Deluc,  L.,  J.  Bogs,  A. R.  Walker,  T.  Ferrier,  A.  Decendit,  J. M.  Merillon,  S. P.  Robinson and  F.  Barrieu. 2008. The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanin biosynthesis in developing grape berries. Plant Physiol. 147: 2041–2053.
•  Downey,  M. O.,  J. S.  Harvey and  S. P.  Robinson. 2003. Synthesis of flavonols and expression of flavonol synthase genes in developing grape berries of Shiraz and Chardonnay (Vitis vinifera L.). Aust. J. Grape Wine Res. 9: 110–121.
•  Favory,  J. J.,  A.  Stec,  H.  Gruber,  L.  Rizzini,  A.  Oravecz,  M.  Funk,  A.  Albert,  C.  Cloix,  G. I.  Jenkins,  E. J.  Oakeley,  H. K.  Seidlitz,  F.  Nagy and  R.  Ulm. 2009. Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. EMBO J. 28: 591–601.
•  Fournier-Level,  A.,  L.  Le Cunff,  C.  Gomez,  A.  Doligez,  A.  Ageorges,  C.  Roux,  Y.  Bertrand,  J. M.  Souquet,  V.  Cheynier and  P.  This. 2009. Quantitative genetic bases of anthocyanin variation in grape (Vitis vinifera L. ssp. sativa) berry: a quantitative trait locus to quantitative trait nucleotide integrated study. Genetics 183: 1127–1139.
•  Huang,  W.,  P.  Perez-Garcia,  A.  Pokhilko,  A. J.  Millar,  I.  Antoshechkin,  J. L.  Riechmann and  P.  Mas. 2012. Mapping the core of the Arabidopsis circadian clock defines the network structure of the oscillator. Science 336: 75–79.
•  Jaillon,  O.,  J. M.  Aury,  B.  Noel,  A.  Policriti,  C.  Clepet,  A.  Casagrande,  N.  Choisne,  S.  Aubourg,  N.  Vitulo,  C.  Jubin,  A.  Vezzi,  F.  Legeai,  P.  Hugueney,  C.  Dasilva,  D.  Horner,  E.  Mica,  D.  Jublot,  J.  Poulain,  C.  Bruyere,  A.  Billault,  B.  Segurens,  M.  Gouyvenoux,  E.  Ugarte,  F.  Cattonaro,  V.  Anthouard,  V.  Vico,  C.  Del Fabbro,  M.  Alaux,  G.  Di Gaspero,  V.  Dumas,  N.  Felice,  S.  Paillard,  I.  Juman,  M.  Moroldo,  S.  Scalabrin,  A.  Canaguier,  I.  Le Clainche,  G.  Malacrida,  E.  Durand,  G.  Pesole,  V.  Laucou,  P.  Chatelet,  D.  Merdinoglu,  M.  Delledonne,  M.  Pezzotti,  A.  Lecharny,  C.  Scarpelli,  F.  Artiguenave,  M. E.  Pe,  G.  Valle,  M.  Morgante,  M.  Caboche,  A. F.  Adam-Blondon,  J.  Weissenbach,  F.  Quetier and  P.  Wincker. 2007. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449: 463–467.
•  Jeong,  S. T.,  N.  Goto-Yamamoto,  S.  Kobayashi and  M.  Esaka. 2004. Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins. Plant Sci. 167: 247–252.
•  Kataoka,  I.,  A.  Sugiyama and  K.  Beppu. 2003. Role of ultraviolet radiation in accumulation of anthocyanin in berries of ‘Gros Colman’ grapes (Vitis vinifera L.). J. Japan. Soc. Hort. Sci. 72: 1–6.
•  Kliewer,  W. M. and  R. E.  Torres. 1972. Effect of controlled day and night temperatures on grape coloration. Am. J. Enol. Vitic. 23: 71–77.
•  Kobayashi,  S.,  N.  Goto-Yamamoto and  H.  Hirochika. 2004. Retrotransposon-induced mutations in grape skin color. Science 304: 982.
•  Koornneef,  M.,  K. M.  Leon-Kloosterziel,  S. H.  Schwartz and  J. A. D.  Zeevaart. 1998. The genetic and molecular dissection of abscisic acid biosynthesis and signal transduction in Arabidopsis. Plant Physiol. Biochem. 36: 83–89.
•  Koshita,  Y.,  T.  Asakura,  H.  Fukuda and  Y.  Tsuchida. 2007. Nighttime temperature treatment of fruit clusters of ‘Aki Queen’ grapes during maturation and its effect on the skin color and abscisic acid content. Vitis 46: 208–209.
•  Lee,  J.,  K.  He,  V.  Stolc,  H.  Lee,  P.  Figueroa,  Y.  Gao,  W.  Tongprasit,  H.  Zhao,  I.  Lee and  X. W.  Deng. 2007. Analysis of transcription factor HY5 genomic binding sites revealed its hierarchical role in light regulation of development. Plant Cell 19: 731–749.
•  Matus,  J. T.,  R.  Loyola,  A.  Vega,  A.  Peña-Neira,  E.  Bordeu,  P.  Arce-Johnson and  J. A.  Alcalde. 2009. Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera. J. Exp. Bot. 60: 853–867.
•  Mazza,  G.,  L.  Fukumoto,  P.  Delaquis,  B.  Girard and  B.  Ewert. 1999. Anthocyanins, phenolics, and color of Cabernet Franc, Merlot, and Pinot Noir wines from British Columbia. J. Agric. Food Chem. 47: 4009–4017.
•  Mori,  K.,  N.  Goto-Yamamoto,  M.  Kitayama and  K.  Hashizume. 2007. Loss of anthocyanins in red-wine grape under high temperature. J. Exp. Bot. 58: 1935–1945.
•  Mori,  K.,  S.  Sugaya and  H.  Gemma. 2005. Decreased anthocyanin biosynthesis in grape berries grown under elevated night temperature condition. Sci. Hortic. 105: 319–330.
•  Mustilli,  A. C.,  S.  Merlot,  A.  Vavasseur,  F.  Fenzi and  J.  Giraudat. 2002. Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14: 3089–3099.
•  Osterlund,  M. T.,  N.  Wei and  X. W.  Deng. 2000. The roles of photoreceptor systems and the COP1-targeted destabilization of HY5 in light control of Arabidopsis seedling development. Plant Physiol. 124: 1520–1524.
•  Peng,  T.,  T.  Saito,  C.  Honda,  Y.  Ban,  S.  Kondo,  J. H.  Liu,  Y.  Hatsuyama and  T.  Moriguchi. 2013. Screening of UV-B-induced genes from apple peels by SSH: possible involvement of MdCOP1-mediated signaling cascade genes in anthocyanin accumulation. Physiol. Plant. 148: 432–444.
•  Reid,  K. E.,  N.  Olsson,  J.  Schlosser,  F.  Peng and  S. T.  Lund. 2006. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol. 6: 27. DOI: 10.1186/1471-2229-6-27. <http://www.biomedcentral.com/bmcplantbiol/>.
•  Rizzini,  L.,  J. J.  Favory,  C.  Cloix,  D.  Faggionato,  A.  O’Hara,  E.  Kaiserli,  R.  Baumeister,  E.  Schafer,  F.  Nagy,  G. I.  Jenkins and  R.  Ulm. 2011. Perception of UV-B by the Arabidopsis UVR8 protein. Science 332: 103–106.
•  Shin,  J.,  E.  Park and  G.  Choi. 2007. PIF3 regulates anthocyanin biosynthesis in an HY5-dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis. Plant J. 49: 981–994.
•  Shiraishi,  M.,  M.  Yamada,  N.  Mitani and  T.  Ueno. 2007. A rapid determination method for anthocyanin profiling in grape genetic resources. J. Japan. Soc. Hort. Sci. 76: 28–35.
•  Sparvoli,  F.,  C.  Martin,  A.  Scienza,  G.  Gavazzi and  C.  Tonelli. 1994. Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.). Plant Mol. Biol. 24: 743–755.
•  Stracke,  R.,  J. J.  Favory,  H.  Gruber,  L.  Bartelniewoehner,  S.  Bartels,  M.  Binkert,  M.  Funk,  B.  Weisshaar and  R.  Ulm. 2010. The Arabidopsis bZIP transcription factor HY5 regulates expression of the PFG1/MYB12 gene in response to light and ultraviolet-B radiation. Plant Cell Environ. 33: 88–103.
•  Teixeira,  A.,  J.  Eiras-Dias,  S. D.  Castellarin and  H.  Gerós. 2013. Berry phenolics of grapevine under challenging environments. Int. J. Mol. Sci. 14: 18711–18739.
•  Terrier,  N.,  L.  Torregrosa,  A.  Ageorges,  S.  Vialet,  C.  Verriès,  V.  Cheynier and  C.  Romieu. 2009. Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway. Plant Physiol. 149: 1028–1041.
•  Tomana,  T.,  N.  Utsunomiya and  I.  Kataoka. 1979a. The effect of environmental temperatures on fruit ripening on the tree. I. The effect of temperatures around whole vines and clusters on the ripening of ‘Delaware’ grapes. Studies from the Institute of Horticulture, Kyoto University 9: 1–5 (In Japanese with English summary).
•  Tomana,  T.,  N.  Utsunomiya and  I.  Kataoka. 1979b. The effect of environmental temperatures on fruit ripening on the tree. II. The effect of temperatures around whole vines and clusters on the coloration of ‘Kyoho’ grapes. J. Japan. Soc. Hort. Sci. 48: 261–266 (In Japanese with English summary).
•  Ubi,  B. E.,  C.  Honda,  H.  Bessho,  S.  Kondo,  M.  Wada,  S.  Kobayashi and  T.  Moriguchi. 2006. Expression analysis of anthocyanin biosynthetic genes in apple skin: effect of UV-B and temperature. Plant Sci. 170: 571–578.
•  Walker,  A. R.,  E.  Lee,  J.  Bogs,  D. A. J.  McDavid,  M. R.  Thomas and  S. P.  Robinson. 2007. White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J. 49: 772–785.
• Winkler, A. J., J. A. Cook, W. M. Kliewer and L. A. Lider. 1962. Development and composition of grapes. p. 141–196. In: A. J. Winkler, J. A. Cook, W. M. Kliewer and L. A. Lider (eds.). General viticulture. University of California Press. Berkeley, CA.
•  Yamane,  T.,  S. T.  Jeong,  N.  Goto-Yamamoto,  Y.  Koshita and  S.  Kobayashi. 2006. Effects of temperature on anthocyanin biosynthesis in grape berry skins. Am. J. Enol. Vitic. 57: 54–59.
•  Yoshida,  R.,  T.  Hobo,  K.  Ichimura,  T.  Mizoguchi,  F.  Takahashi,  J.  Aronso,  J. R.  Ecker and  K.  Shinozaki. 2002. ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis. Plant Cell Physiol. 43: 1473–1483.
•  Zheng,  Y.,  L.  Tian,  H.  Liu,  Q.  Pan,  J.  Zhan and  W.  Huang. 2009. Sugars induce anthocyanin accumulation and flavanone 3-hydroxylase expression in grape berries. Plant Growth Regul. 58: 251–260.