The photomorphogenic factors UV-B RECEPTOR 1, ELONGATED HYPOCOTYL 5, and HY5 HOMOLOGUE are part of the UV-B signalling pathway in grapevine and mediate flavonol accumulation in response to the environment.

Grapevine (Vitis vinifera L.) is a species well known for its adaptation to radiation. However, photomorphogenic factors related to UV-B responses have not been molecularly characterized. We cloned and studied the role of UV-B RECEPTOR (UVR1), ELONGATED HYPOCOTYL 5 (HY5), and HY5 HOMOLOGUE (HYH) from V. vinifera We performed gene functional characterizations, generated co-expression networks, and tested them in different environmental conditions. These genes complemented the Arabidopsis uvr8 and hy5 mutants in morphological and secondary metabolic responses to radiation. We combined microarray and RNA sequencing (RNA-seq) data with promoter inspections to identify HY5 and HYH putative target genes and their DNA binding preferences. Despite sharing a large set of common co-expressed genes, we found different hierarchies for HY5 and HYH depending on the organ and stress condition, reflecting both co-operative and partially redundant roles. New candidate UV-B gene markers were supported by the presence of HY5-binding sites. These included a set of flavonol-related genes that were up-regulated in a HY5 transient expression assay. We irradiated in vitro plantlets and fruits from old potted vines with high and low UV-B exposures and followed the accumulation of flavonols and changes in gene expression in comparison with non-irradiated conditions. UVR1, HY5, and HYH expression varied with organ, developmental stage, and type of radiation. Surprisingly, UVR1 expression was modulated by shading and temperature in berries, but not by UV-B radiation. We propose that the UV-B response machinery favours berry flavonol accumulation through the activation of HY5 and HYH at different developmental stages at both high and low UV-B exposures.

Diversity of flavour characteristics of table grapes and their contributing volatile compounds analysed by the solvent-assisted flavour evaporation method.

To identify the compounds that contribute to the diverse flavours of table grapes, the flavours and volatile compounds of 38 grape cultivars harvested over 3 years are evaluated through sensory analysis and solvent-assisted flavour evaporation (SAFE). The cultivars are characterized and grouped into seven clusters by hierarchical cluster analysis (HCA) using sensory evaluation data with a flavour wheel specific to table grapes. These clusters were similar to conventional flavour classifications, except that the foxy and neutral cultivars form multiple clusters, highlighting the flavour diversity of table grapes. The SAFE method provides a comprehensive profile of the volatile compounds, including slightly volatile compounds whose profiles are lacking in hybrid grapes and Vitis rotundifolia. The sensory evaluation is supported by the volatile compound profiles, and relationships between the datasets are clarified by multivariate analysis. Specific accumulations and combinations of compounds (α-pinene, ß-pinene, phenylethyl alcohol, furaneol, mesifurane, methyl N-formylanthranilate, and mixed ethyl ester and monoterpenoid) were also identified that contribute to the diversity of flavours (fresh green, floral, fruity, fatty green, sweet, fermented/sour) in table grapes, including linalool and linalool analogues (muscat flavour) along with ethyl ester and hydroxyethyl esters (foxy flavour). The accumulation of these compounds was positively related to a higher flavour intensity. Their specific accumulation and combination supported the flavour diversity of table grapes. This study identified novel flavour-associated compound profiles in table grapes through in-depth volatile compound analysis and non-conventional multivariate analysis.

Targeted deletion of grape retrotransposon associated with fruit skin color via CRISPR/Cas9 in Vitis labrascana 'Shine Muscat'.

Transposition of transposable elements affect expression levels, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus. For example, in grape, presence of the Gret1 retrotransposon in the promoter region of the VvMYBA1a allele at the VvMYBA1 locus suppress the expression of the VvMYBA1 transcription factor gene for the anthocyanin biosynthesis and this transposon insertion is responsible for the green berry skin color of Vitis labrascana, 'Shine Muscat', a major grape cultivar in Japan. To prove that transposons in grape genome can be removed by genome editing, we focused on Gret1 in the VvMYBA1a allele as a target of CRISPR/Cas9 mediated transposon removal. PCR amplification and sequencing detected Gret1 eliminated cells in 19 of 45 transgenic plants. Although we have not yet confirmed any effects on grape berry skin color, we were successful in demonstrating that cleaving the long terminal repeat (LTR) present at both ends of Gret1 can efficiently eliminate the transposon.

Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions.

Temperature and light are important environmental factors that affect flavonoid biosynthesis in grape berry skin. However, the interrelationships between temperature and light effects on flavonoid biosynthesis have not been fully elucidated at the molecular level. Here, we investigated the effects of temperature and light conditions on the biosynthesis of flavonoids (anthocyanins and flavonols) and the expression levels of related genes in an in vitro environmental experiment using detached grape berries. Sufficient anthocyanin accumulation in the grape skin was observed under a low temperature (15 °C) plus light treatment, whereas high temperature (35 °C) or dark treatment severely suppressed anthocyanin accumulation. This indicates that the accumulation of anthocyanins is dependent on both low temperature and light. qRT-PCR analysis showed that the responses of three MYB-related genes (VlMYBA1-3, VlMYBA1-2, and VlMYBA2) to temperature and light differed greatly even though the products of all three genes had the ability to regulate anthocyanin biosynthesis pathway genes. Furthermore, the expression levels of other MYB-related genes and many flavonoid biosynthesis pathway genes were regulated independently by temperature and light. We also found that temperature and light conditions affected the anthocyanin composition in the skin through the regulation of flavonoid biosynthesis pathway genes. Our results suggest that low temperature and light have a synergistic effect on the expression of genes in the flavonoid biosynthesis pathway. These findings provide new information about the relationships between environmental factors and flavonoid accumulation in grape berry skin.

Demethylation of the 3' LTR region of retrotransposon in VvMYBA1BEN allele enhances anthocyanin biosynthesis in berry skin and flesh in 'Brazil' grape.

Black-skinned and red-fleshed grape 'Brazil' is a bud sport of rosy-skinned 'Benitaka'. 'Brazil' has a much higher anthocyanin content in the skin than that of 'Benitaka' and is characterized by the accumulation of anthocyanins in the flesh. Our genomic analysis of the VvMYBA loci, which regulate anthocyanin biosynthesis, suggested that the difference in skin and flesh color between 'Brazil' and 'Benitaka' cannot be explained by genomic alteration at the loci. Expression levels of VvMYBA1 and anthocyanin biosynthesis-related genes in skin and flesh were significantly higher in 'Brazil' than in 'Benitaka' throughout berry development. DNA methylation levels in the 3' long terminal repeat (LTR) of a retrotransposon in the upstream region of VvMYBA1BEN allele were clearly higher in the skin and flesh of 'Benitaka' than in those of 'Brazil' throughout berry development. These findings suggest that a dramatic decrease in DNA methylation level in the 3' LTR of the retrotransposon in the VvMYBA1BEN allele in 'Brazil' increases the expression levels of VvMYBA1 and anthocyanin accumulation in skin and flesh. Our findings also suggest that skin and flesh colors are inherited together and vary depending on the presence or absence of the VvMYBA1BEN allele.

De novo whole-genome assembly in an interspecific hybrid table grape, 'Shine Muscat'.

The first genome sequence of an interspecific grape hybrid (Vitis labruscana × Vitis vinifera), 'Shine Muscat', an elite table grape cultivar bred in Japan, is presented. The resultant genome assemblies included two types of sequences: a haplotype-phased sequence of the highly heterozygous genomes and an unphased sequence representing a 'pseudo-haploid' genome. The unphased sequences, assembled to the chromosome level with Hi-C reads, spanned 488.97 Mb in length, 99.1% of the estimated genome size, with 4,595 scaffold sequences and a 23.9-Mb N50 length. The phased sequences had 15,650 scaffolds spanning 1.0 Gb and a 4.2-Mb N50 length. 32,827 high-confidence genes were predicted on the unphased genomes. Clustering analysis of the 'Shine Muscat' gene sequences with three other Vitis species and Arabidopsis indicated that 11,279 orthologous gene clusters were common to Vitis spp. and Arabidopsis, 4,385 were Vitis specific, and 234 were 'Shine Muscat' specific. Whole-genome resequencing was also performed for the parental lines of 'Shine Muscat', Akitsu-21 and 'Hakunan', and parental-specific copy number variations were identified. The obtained genome resources provide new insights that could assist in cultivation and breeding strategies to produce high-quality table grapes.

Haplotype composition at the color locus is a major genetic determinant of skin color variation in Vitis × labruscana grapes.

Skin color is one of the most important fruit traits in grape, and has become greatly diversified due to hybridization and human selection. Many studies concerning the genetic control of grape color in European species (Vitis vinifera L.), especially the role of MYB-related genes, have been reported. On the other hand, there have been few studies of the MYB-related genes in grapes belonging to V. ×labruscana L.H. Bailey, a subgroup of grapes that originated from the hybridization of V. labrusca with V. vinifera. In the present study, we found a novel functional haplotype, HapE2 (consisting of the genes VlMYBA2 and VlMYBA1-3), in diploid V. ×labruscana. Moreover, we developed a method to determine the haplotype compositions of tetraploid grapes by means of quantitative real-time PCR, and investigated the relationship between haplotype composition and skin color. The color locus in V. ×labruscana grapes usually consists of functional haplotypes (HapE1 and/or HapE2), and non-functional haplotype HapA. The number of functional haplotypes in the genome was found to be correlated with the level of anthocyanin in the skin. Anthocyanin contents of grapes that contained HapE2 were significantly higher than those containing HapE1. These results suggest that the number and kind of functional haplotypes at the color locus are the major genetic factors that determine skin color variation. These findings provide new knowledge about the unique genetic control of color in V. ×labruscana grapes, and should contribute to development of new cultivars that have the desired color and anthocyanin content.

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.

Color mutations in grape berry skin are relatively frequent events, and can be easily seen in the vineyard. Both light-red-skinned 'Ruby Okuyama' and more intense and uniform rosy-skinned 'Benitaka' (Vitis vinifera L.) are bud sports of white-skinned 'Italia'. Previously, we reported that 'Ruby Okuyama' was caused by the recovery of VvmybA1 expression, which may have occurred as a result of intra-LTR (long terminal repeat) recombination within a retrotransposon, Gret1. However, the molecular basis of the color recovery in 'Benitaka' has not been elucidated so far. Here, we found that the VvmybA1 locus of 'Benitaka' is heterozygous for the VvmybA1a allele (non-functional) and a novel VvmybA1(BEN) allele, and that VvmybA1(BEN) restored VvmybA1 transcripts. We hypothesized that VvmybA1(BEN) allele was caused by homologous recombination between VvmybA1a and VvmybA3. In addition, the content and composition of anthocyanins in berry skins differed greatly between 'Ruby Okuyama' and 'Benitaka'. The levels of expression of the genes for flavonoid 3',5'-hydroxylase (F3'5'H), O-methyltransferase (OMT), and glutathione-S-transferase (GST) were associated with differences in the anthocyanin content and composition between the two cultivars.

CRISPR/Cas9-mediated targeted mutagenesis in grape.

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape-an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape berry skin.

As a result of natural hybridization and human selection over millennia, the skin colors of grapes have become greatly diversified. The color is determined by the quantity and composition of anthocyanins. Color-skinned cultivars accumulate anthocyanins in their skins, whereas white-skinned cultivars do not. Myb-related transcription-factor genes such as VvmybA1 regulate anthocyanin biosynthesis. VvMYBA2r, VlmybA1-1, VlmybA1-2, and VlmybA2, which are homologs of VvmybA1, also regulate anthocyanin biosynthesis. In this study, we isolated a novel Myb-related sequence, VlmybA1-3, from cultivars of Vitis labruscana (Vitis vinifera x Vitis labrusca) by means of inverse PCR, and confirmed by means of transient gene expression assay that the gene regulates anthocyanin biosynthesis in grape berry skin. Seedlings of V. labruscana with two functional haplotypes at a region of berry color loci accumulated more anthocyanins than seedlings with a single functional haplotype. In addition, we investigated the haplotypes at the region in 35 cultivars (both V. vinifera and V. labruscana), and found certain typical characteristics. These findings will contribute to the selection of seedlings with high anthocyanin quantities in breeding programs for wine and table grapes, and will help elucidate the origin and evolution of Vitis species.

A skin color mutation of grapevine, from black-skinned Pinot Noir to white-skinned Pinot Blanc, is caused by deletion of the functional VvmybA1 allele.

A white-wine grape, Pinot Blanc, is thought to be a white-skinned mutant of a red-wine grape, Pinot Noir. Pinot Noir was heterozygous for VvmybA1. One allele was the non-functional VvmybA1a, and the other was the functional VvmybA1c. In Pinot Blanc, however, only VvmybA1a was observed, and the amount of VvmybA1 DNA in Pinot Blanc was half that in Pinot Noir. These findings suggest that deletion of VvmybA1c from Pinot Noir resulted in Pinot Blanc.