Application of Synephrine to Grape Increases Anthocyanin via Production of Hydrogen Peroxide, Not Phytohormones.

Global warming has caused such problems as the poor coloration of grape skin and the decreased production of high-quality berries. We investigated the effect of synephrine (Syn) on anthocyanin accumulation. Anthocyanin accumulation in cultured grape cells treated with Syn at concentrations of 1 mM or higher showed no significant difference, indicating that the accumulation was concentration-independent. On the other hand, anthocyanin accumulation was dependent on the compound used for treatment. The sugar/acid ratio of the juice from berries treated with Syn did not differ from the control. The expression of anthocyanin-biosynthesis-related genes, but not phytohormones, was increased by the treatment with Syn at 24 h or later. The Syn treatment of cultured cells increased SOD3 expression and hydrogen peroxide (H2O2) production from 3 to 24 h after treatment. Subsequently, the expression of CAT and APX6 encoding H2O2-scavenging enzymes was also increased. Treatment of cultured cells with Syn and H2O2 increased the expression of the H2O2-responsive gene Chit4 and the anthocyanin-biosynthesis-related genes mybA1 and UFGT 4 days after the treatment and increased anthocyanin accumulation 7 days after the treatment. On the other hand, the treatment of berries with Syn and H2O2 increased anthocyanin accumulation after 9 days. These results suggest that Syn increases anthocyanin accumulation through H2O2 production without changing phytohormone biosynthesis. Syn is expected to improve grape skin coloration and contribute to high-quality berry production.

Grape cytochrome P450 CYP90D1 regulates brassinosteroid biosynthesis and increases vegetative growth.

Vine vigor or vegetative growth is an important factor related to berry quality and vinicultural training management, but brassinosteroid (BR)-induced molecular mechanisms underlying vine growth remain unclear. In this study, the hypothesis that the Vitis vinifera CYP90D1 gene VvCYP90D1, one of the genes for BR biosynthesis, plays a critical role in shoot elongation was tested. RNA sequencing analysis of shoots collected from the vigorous cultivar Koshu (KO) and the reference cultivar Pinot Noir (PN) 7 days after bud break showed higher expression levels of various genes in the BR biosynthesis pathway in KO than in PN. The VvCYP90D1 expression level in KO was highest in meristems, followed by internodes and leaves. Cluster analysis of amino acid sequences including those in other plant species showed that the isolated gene belonged to the CYP90D1 group. The vegetative growth and the endogenous BR (brassinolide; BL) content were significantly higher in VvCYP90D1-overexpressing Arabidopsis than in wild type. VvCYP90D1-overexpressing Arabidopsis treated with brassinazole (Brz), a BR biosynthesis inhibitor, showed recovery of vegetative growth. These results indicate that VvCYP90D1 in grapevine has a vegetative growth promoting effect via BR biosynthesis. Our findings on the mechanism of BR-induced grape shoot growth will contribute to the development of new shoot control techniques for grapevine.

Diversity of endophytic bacterial microbiota in grapevine shoot xylems varies depending on wine grape-growing region, cultivar, and shoot growth stage.

Next-generation sequencing technology may clarify microbiota that are as yet poorly understood in the soil, the rhizosphere, and the phyllosphere of vineyards. To provide new information on the interaction between grapevine and microorganisms, we focused on the endophytic microbiota in grapevine. We performed endophytic microbiome analysis of the shoot xylems of four cultivars, Vitis vinifera cvs. Chardonnay, Pinot Noir, Cabernet Sauvignon, and Vitis sp. cv. Koshu, grown in eleven vineyards in Japan. The number of endophytic fungal species was small in the grapevine shoot xylems and could not be analyzed further, whereas a total of 7,019,600 amplicon sequences (46,642-285,003 per shoot xylem) and 1305 bacterial operational taxonomic units were obtained by analysis of the V3-V4 region of the bacterial 16S rRNA gene. Gammaproteobacteria was predominant in the shoot xylems at the shoot elongation stage irrespective of the cultivar, whereas Alphaproteobacteria and Oxyphotobacteria were predominant at véraison. Actinobacteria, Bacteroidia, Bacilli, and Clostridia were also detected in the shoot xylems. The endophytic bacterial microbiota in Koshu and Pinot Noir shoot xylems were similar irrespective of the grapevine-growing region. In contrast, the endophytic bacterial microbiota in Chardonnay and Cabernet Sauvignon showed diversity and complexity among grapevine-growing regions. Alpha diversity analysis revealed that Koshu shoot xylems had a higher diversity of endophytic bacterial microbiota than Pinot Noir, Chardonnay, and Cabernet Sauvignon shoot xylems, and that grapevine shoot xylems at the shoot elongation stage had a higher diversity of endophytic bacterial microbiota than those at véraison. Principal coordinate analysis (PCoA) demonstrated that the profiles of the endophytic bacterial microbiota in grapevine shoot xylems at véraison were relatively uniform compared with those at the shoot elongation stage. Multidimensional scaling analysis showed that the plots of all cultivars were generally apart from each other at the shoot elongation stage and then became close to each other at véraison. The plots of all grapevine-growing regions cultivating Koshu were close to each other, whereas those of grapevine-growing regions cultivating Chardonnay and Cabernet Sauvignon were apart from each other. The findings of this study suggest that the endophytic bacterial microbiota in grapevine shoot xylems varied depending on the cultivar and the grapevine-growing region even for the same cultivars, and that the microbiota fluctuated depending on the shoot growth stage.

Geraniol as a Potential Stimulant for Improving Anthocyanin Accumulation in Grape Berry Skin through ABA Membrane Transport.

Climate change, particularly warmer temperature, has resulted in reduced anthocyanin accumulation in grape berry skin. Because anthocyanin is a crucial determinant of red wine quality, viticulturists need to devise a solution for mitigating the poor coloration of red/black grape berry skin under elevated temperature conditions. In this study, we investigated the effects of geraniol on anthocyanin accumulation in grape berry skins of field-grown grapevines and elucidated the molecular mechanisms of the geraniol-triggered anthocyanin accumulation. Geraniol-treated bunches showed enhanced anthocyanin accumulation in berry skins at harvest (50 days after treatment). Geraniol treatment upregulated the transcription of MybA1 and UFGT, which encode the key factors in anthocyanin biosynthesis, in berry skins. Geraniol treatment also improved anthocyanin accumulation in grape cultured cells. We isolated grape ATP-binding cassette transporter G family protein VvABCG40, encoding abscisic acid (ABA) membrane transporter, from geraniol-treated grape cultured cells. VvABCG40 transcription was upregulated in berry skins 40 days after treatment. Geraniol treatment also upregulated the transcription of VvPP2C24, which encodes ABA-responsible type 2C protein phosphatases, in berry skins, but not the transcription of VvNCED1, which encodes a key enzyme in ABA biosynthesis. Taken together, geraniol-triggered anthocyanin accumulation in berry skins is promoted by ABA membrane transport and not by ABA biosynthesis, and geraniol treatment of field-grown grape bunches may contribute to alleviating the poor coloration of berry skin as a novel technique in viticulture.

Ethylene Induced by Sound Stimulation Enhances Anthocyanin Accumulation in Grape Berry Skin through Direct Upregulation of UDP-Glucose: Flavonoid 3-O-Glucosyltransferase.

Global warming has resulted in the loss of anthocyanin accumulation in berry skin. Sound stimulation can be used as a potential method for enhancing fruit color development since many plants recognize sound vibration as an external stimulus and alter their physiological status in response to it. Sound stimulation (sine wave sound at 1000 Hz) enhanced anthocyanin accumulation in grape cultured cells and berry skins in field-grown grapevines at the early stage of ripening. The transcription of UFGT and ACO2, which encode the key enzymes in anthocyanin and ethylene biosynthesis, respectively, was upregulated in grape cultured cells exposed to sound stimulation. In contrast, the transcription of MybA1 and NCED1, which encode a transcription factor for UFGT and a key enzyme in abscisic acid biosynthesis, respectively, was not affected by the sound stimulation. A treatment with an ethylene biosynthesis inhibitor, aminoethoxyvinyl glycine hydrochloride, revered the enhancement of anthocyanin accumulation by sound stimulation. As the promoter assay using a GUS reporter gene demonstrated that UFGT promoter was directly activated by the ethylene-releasing compound ethephon, which enhanced anthocyanin accumulation in grape cultured cells, we conclude that sound stimulation enhanced anthocyanin accumulation through the direct upregulation of UFGT by ethylene biosynthesis. Our findings suggest that sound stimulation contributes to alleviating poor coloration in berry skin as a novel and innovative practical technique in viticulture.

Genomic Characterization of the Japanese Indigenous Wine Grape Vitis sp. cv. Koshu.

Vitis sp. cv. Koshu is indigenous to Japan and used as a table and processing grape. It also constitutes an important grape cultivar in Japanese white wine making and is phylogenetically distinct from European grapes. To understand its genomic information, we compared its small and structural variations with those of the table grape cultivar "Thompson seedless" and European wine grape cultivar "Tannat" via a short-read-based resequencing approach. The Koshu genome exhibited high heterozygosity compared to these cultivars, with this characteristic being particularly prominent on chromosome 7. Furthermore, Koshu structural variation encompassed the most and largest extent of duplications and the fewest and smallest extent of deletions with regard to copy number variation and the fewest absence variations among the compared grape cultivars. Plant disease resistance related to cell death associated with hypersensitive response and environmental stress response, such as water deprivation, oxidative stress, and cell wall organization, was inferred through enrichment analysis of small and structural variations. Variant accumulation levels in Koshu indicated that phenylpropanoid, flavonoid, glutathione, and α-linolenic acid pathways were related to polyphenol and flavor components. Together, this Koshu genomic information provides a foundation for improving the quality of Koshu wine and may facilitate the use of Koshu as a genetic resource.

Crosstalk Pathway between Trehalose Metabolism and Cytokinin Degradation for the Determination of the Number of Berries per Bunch in Grapes.

In grapes, the number of flowers per inflorescence determines the compactness of grape bunches. Grape cultivars with tight bunches and thin-skinned berries easily undergo berry splitting, especially in growing areas with heavy rainfall during the grapevine growing season, such as Japan. We report herein that grape cytokinin oxidase/dehydrogenase 5 (VvCKX5) determines the number of berries per inflorescence in grapes. The number of berries per bunch was inversely proportional to the VvCKX5 expression level in juvenile inflorescences among the cultivars tested. VvCKX5 overexpression drastically decreased the number of flower buds per inflorescence in Arabidopsis plants, suggesting that VvCKX5 might be one of the negative regulators of the number of flowers per inflorescence in grapes. Similarly, the overexpression of grape sister of ramose 3 (VvSRA), which encodes trehalose 6-phosphate phosphatase that catalyzes the conversion of trehalose-6-phosphate into trehalose, upregulated AtCKX7 expression in Arabidopsis plants, leading to a decrease in the number of flower buds per Arabidopsis inflorescence. VvCKX5 gene expression was upregulated in grapevine cultured cells and juvenile grape inflorescences treated with trehalose. Finally, injecting trehalose into swelling buds nearing bud break using a microsyringe decreased the number of berries per bunch by half. VvCKX5 overexpression in Arabidopsis plants had no effect on the number of secondary inflorescences from the main inflorescence, and similarly trehalose did not affect pedicel branching on grapevine inflorescences, suggesting that VvCKX5, as well as VvSRA-mediated trehalose metabolism, regulates flower formation but not inflorescence branching. These findings may provide new information on the crosstalk between VvSRA-mediated trehalose metabolism and VvCKX-mediated cytokinin degradation for determining the number of berries per bunch. Furthermore, this study is expected to contribute to the development of innovative cultivation techniques for loosening tight bunches.

Exogenous allantoin improves anthocyanin accumulation in grape berry skin at early stage of ripening.

Anthocyanin accumulation in grape berry skin is a crucial determinant of red/black grape berry quality. Recently, viticulture has been hampered by the issue of global warming, which has led to loss of anthocyanin accumulation in berry skin. The objectives of this study were to investigate the effect of allantoin on anthocyanin accumulation in berry skins of field-grown grapevines and to elucidate the molecular mechanism of the allantoin-induced anthocyanin accumulation in the berry skins. Allantoin enhanced anthocyanin accumulation in grape cultured cells and berry skins from field-grown grapevines at the early stage of ripening. MybA1 and UFGT, which encode the key transcription factor and enzyme in anthocyanin biosynthesis, were upregulated in allantoin-treated berry skins. Allantoin seems to increase the contents of delphinidin-based anthocyanin 3-glucosides in berry skins through the upregulation of F3'5'H gene that drives the synthesis of delphinidin-based anthocyanin 3-glucosides, compared with control berry skins. Allantoin increased soluble sugar contents in berries 10 days after treatment and upregulated abscisic acid (ABA)-responsible HT6 gene that encodes a key hexose transporter in sugar accumulation during ripening, in berry skins. These results suggested that physiological changes in allantoin-treated grape berries occurred in an ABA-dependent manner. Allantoin produced bioactive ABA through the ß-glucosidase-catalyzed hydrolysis of glucose-conjugated ABA but not through the ABA biosynthesis by NCED, a key enzyme in ABA biosynthesis, in berry skins. Allantoin application in viticulture under global warming conditions is expected to contribute to mitigating loss of red/black berry skin color.

Physiological characterization of leaf and internode after bud break in Japanese indigenous Koshu grape by comparative RNA sequencing analysis.

Koshu is indigenous to Japan and considered the most important wine grape in Japan. Koshu grape berry possesses characteristics that make it unique from European V. vinifera as wine grape. However, the physiological characteristics of Koshu leaf and internode remain unknown. An understanding of those characteristics would contribute to improvements in Koshu cultivation, thereby enhancing grape berry and wine quality. To identify the genes responsible for the physiological characteristics of Koshu, we comprehensively analyzed leaf and internode differences at the transcriptome level between Koshu and Pinot Noir by RNA sequencing. A total of 248 and 131 differentially expressed genes (DEGs) were detected in leaves and internodes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of these DEGs revealed that "flavonoid biosynthesis" and "glutathione metabolism" pathways were significantly enriched in Koshu leaves. On the other hand, when internodes were compared, "flavonoid"-related GO terms were specifically detected in Koshu. KEGG pathway enrichment analysis suggested that the expression of such genes as leucoanthocyanidin reductase and flavonol synthase in the flavonoid biosynthesis pathway was higher in Koshu than Pinot Noir. Measurement of the relative expression levels of these genes by RT-qPCR validated the results obtained by RNA sequencing. The characteristics of Koshu leaf and internode, which are expected to produce flavonoids with antibacterial activity and UV protection function, would suit Japanese climate as a survival strategy.

Vanillylacetone up-regulates anthocyanin accumulation and expression of anthocyanin biosynthetic genes by inducing endogenous abscisic acid in grapevine tissues.

We investigated the effect of vanillylacetone (VA) on anthocyanin accumulation with aim of improving grape berry coloration. Spraying Vitis vinifera cv. Muscat Bailey A berries with VA at veraison increased sugar/acid ratio, an indicator of maturation and total anthocyanin accumulation. To elucidate the molecular mechanism underlying the effect of VA on anthocyanin accumulation, in vitro VA treatment of a grapevine cell culture was carried out. Endogenous abscisic acid (ABA) content was higher in the VA-treated cell cultures than in control at 3h after treatment. Consistent with this, the relative expression levels of anthocyanin-synthesis-related genes, including DFR, LDOX, MybA1 and UFGT, in VA-treated cell cultures were much higher than those in control, and high total anthocyanin accumulation was noted in the VA-treated cell cultures as well. These results suggest that VA up-regulates the expression of genes leading to anthocyanin accumulation by inducing endogenous ABA. In addition, VA increased total anthocyanin content in a dose-dependent manner. Although VA treatment in combination with exogenous ABA did not exhibit any synergistic effect, treatment with VA alone showed an equivalent effect to that with exogenous ABA alone on total anthocyanin accumulation. These findings point to the possibility of using VA for improving grape berry coloration.

High Constitutive Overexpression of Glycosyl Hydrolase Family 17 Delays Floral Transition by Enhancing FLC Expression in Transgenic Arabidopsis.

Vitis vinifera glycosyl hydrolase family 17 (VvGHF17) is a grape apoplasmic ß-1,3-glucanase, which belongs to glycosyl hydrolase family 17 in grapevines. ß-1,3-glucanase is not only involved in plant defense response but also has various physiological functions in plants. Although VvGHF17 expression is negatively related to the length of inflorescence in grapevines, the physiological functions of VvGHF17 are still uncertain. To clarify the physiological functions of VvGHF17, we conducted a phenotypic analysis of VvGHF17-overexpressing Arabidopsis plants. VvGHF17-overexpressing Arabidopsis plants showed short inflorescence, similar to grapevines. These results suggested that VvGHF17 might negatively regulate the length of inflorescence in plants. VvGHF17 expression induced a delay of floral transition in Arabidopsis plants. The expression level of FLOWERING LOCUS C (FLC), known as a floral repressor gene, in inflorescence meristem of transgenic plants were increased by approximately 10-fold as compared with wild plants. These results suggest that VvGHF17 induces a delay of floral transition by enhancing FLC expression and concomitantly decreases the length of plant inflorescence.