Grape phytochrome-interacting factor VvPIF1 negatively regulates carotenoid biosynthesis by repressing VvPSY expression.

Phytochrome-interacting factors (PIFs) play important roles in light-mediated secondary metabolism; however, the roles of PIFs in grape fruit carotenogenesis remain unclear. Here, by identifying the PIF family genes in grapes, we focused on the role of VvPIF1 in carotenoid metabolism. During grape berry development, VvPIF1 expression was negatively correlated with carotenoid accumulation and the transcription of phytoene synthase 1/2 (VvPSY1/2), which encodes the major flux-controlling enzymes for carotenoid biosynthesis. Light significantly repressed VvPIF1 expression, but induced the expression of carotenogenic genes including VvPSY1/2. VvPIF1 functioned as a nucleus-localized protein and interacted with the light photoreceptor VvphyB. Overexpression of VvPIF1 resulted in the downregulation of the endogenous PIF1 gene, which may unexpectedly induce carotenoid accumulation and PSY expression in tobacco leaves. The transgenic grape leaves and tomato fruits with high VvPIF1 expression produced a significant decrease in carotenoid concentrations, with suppressed transcription of PSY and other carotenogenic genes. Further biochemical assays demonstrated that VvPIF1 bound directly to the promoters of VvPSY1/2 to inhibit their transcription. Collectively, we conclude that VvPIF1 negatively regulates carotenoid biosynthesis by repressing VvPSY expression in grapes. These findings shed light on the role and mode of action of PIFs in the carotenoid regulatory network of grapes.

Transcriptional Profiling of Resistant and Susceptible Cultivars of Grapevine (Vitis L.) Reveals Hypersensitive Responses to Plasmopara viticola.

Grapevine downy mildew is the most serious disease of grapevine cultivars that affects the rate of resistance/susceptibility to Plasmopara viticola. In this study, we used the susceptible cultivar "Zitian Seedless" and the resistant cultivar "Kober 5BB" as materials to determine the transcriptome differences and phenotypes of the leaves after inoculation with downy mildew. The differences in microstructures and molecular levels were compared and analyzed. Fluorescence staining and microscopic observations confirmed that hypersensitive cell death occurred around the stomata in "Kober 5BB" infected by downy mildew zoospores. Meanwhile, transcriptomic profiling indicated that there were 11,713 and 6,997 gene expression differences between the resistant and susceptible cultivars at 72 h after inoculation when compared to control (0 h), respectively. The differentially expressed genes of the two cultivars are significantly enriched in different pathways, including response to plant-pathogen interaction, mitogen-activated protein kinase (MAPK) signaling pathway, plant hormone signal transduction, phenylpropanoid, and flavonoid biosynthesis. Furthermore, the results of functional enrichment analysis showed that H2O2 metabolism, cell death, reactive oxygen response, and carbohydrate metabolism are also involved in the defense response of "Kober 5BB," wherein a total of 322 key genes have been identified. The protein interaction network showed that metacaspases (MCAs), vacuolar processing enzymes (VPEs), and Papain-like cysteine proteases (PLCPs) play an important role in the execution of hypersensitive responses (HR). In conclusion, we demonstrated that HR cell death is the key strategy in the process of grape defense against downy mildew, which may be mediated or activated by Caspase-like proteases.

Systematic analysis reveals O-methyltransferase gene family members involved in flavonoid biosynthesis in grape.

O-methyltransferases (OMTs) are an important group of enzymes involved in the methylation of various secondary metabolites, including flavonoids. However, the features and functions of OMTs have not been comprehensively studied in grape (Vitis vinifera), a rich source of methylated flavonoids. Here, 47 OMT members were identified in grape genome. They were unevenly distributed on grape chromosomes and some genes were tandem duplicated, indicating the role of duplication processes in the expansion of this gene family. Based on the phylogenetic relationship, these OMTs were clustered into CCoAOMT and COMT subclades, which were further supported by the results of conserved motif and gene structure analysis. Correlation analysis revealed that three members (VvCCoAOMT1, VvCCoAOMT4, and VvCOMT1) were potentially involved in the synthesis of most methylated flavonoids in the berry skins. Expression profiling based on RNA-seq data and qRT-PCR experiments indicated that VvCCoAOMT1 and VvCCoAOMT4 had specific and high expression in berry skins, and responded to abscisic acid and high temperature treatments; and that VvCOMT1 expression was significantly induced during berry development and UVC treatment. Cis-regulatory element analysis suggested important roles of OMTs in growth, development, and defense against stresses. We further demonstrated the transcriptional regulation of VvCCoAOMT4 by VvMYBA1, a master regulator of grape berry anthocyanin, and verified the protein localization of VvCCoAOMT4 in membrane and nucleus. These findings facilitate a better understanding of the characteristics of OMT gene family, especially of the potential members involved in the formation of O-methylated flavonoids in grape.

Integrative Analyses of Metabolomes and Transcriptomes Provide Insights into Flavonoid Variation in Grape Berries.

Flavonoids in grapes contribute the quality of the berry, but the flavonoid diversity and the regulatory networks underlying the variation require a further investigation. In this study, we integrated multi-omics data to systematically explore the global metabolic and transcriptional profiles in the skins and pulps of three grape cultivars. The results revealed large-scale differences involved in the flavonoid metabolic pathway. A total of 133 flavonoids, including flavone and flavone C-glycosides, were identified. Beyond the visible differences of anthocyanins, there was large variation in other sub-branched flavonoids, most of which were positively correlated with anthocyanins in grapes. The expressions of most flavonoid biosynthetic genes and the major regulators MYBA1 were strongly consistent with the changes in flavonoids. Integrative analysis identified two novel transcription factors (MYB24 and MADS5) and two ubiquitin proteins (RHA2) as promising regulatory candidates for flavonoid biosynthesis in grapes. Further verification in various grape accessions indicated that five major genes including flavonol 3'5'-hydroxylase (F3'5'H), UDP-glucose:flavonoid 3-O-glycosyl-transferase, anthocyanin O-methyltransferase, acyltransferase (3AT), and glutathione S-transferase (GST4) controlled flavonoid variation in grape berries. These findings provide valuable information for understanding the mechanism of flavonoid biosynthesis in grape berries and the further development of grape health products.

Genome-wide identification and characterization of gibberellin metabolic and signal transduction (GA MST) pathway mediating seed and berry development (SBD) in grape (Vitis vinifera L.).

BACKGROUND: Grape is highly sensitive to gibberellin (GA), which is crucial during seed and berry development (SBD) either by itself or by interacting with other hormones, such as auxin, Abscisic acid (ABA), and Cytokinin (CK). However, no systematic analysis of GA metabolic and signal transduction (MST) pathway has been undertaken in grapevine. RESULTS: In this study, total endogenous GA3 content significantly decreased during SBD, and a total of 48 known genes in GA metabolic (GAM; 31) and signal transduction (ST; 17) pathways were identified in this process. In the GAM pathway, out of 31 genes, VvGA20ox1-1, VvGA3ox4-1, and VvGA2ox1-1 may be the major factors interacting at the green-berry stage (GBS) accompanied with higher accumulation rate. GA biosynthesis was greater than GA inactivation at GBS, confirming the importance of seeds in GA synthesis. The visible correlation between endogenous GA3 content and gene expression profiles suggested that the transcriptional regulation of GA biosynthesis pathway genes was a key mechanism of GA accumulation at the stone-hardening stage (SHS). Interestingly, we observed a negative feedback regulation between VvGA3oxs-VvGAI1-4, VvGA2oxs-VvGAI1-4, and VvGID1B-VvGAI1-4 in maintaining the balance of GA3 content in berries. Moreover, 11 miRNAs may be involved in the modulation of GA MST pathway by mediating their target genes, such as VvGA3ox, VvGID1B, and VvGAMYB. Many genes in auxin, ABA, and CK MST pathways were further identified and found to have a special pattern in the berry, and the crosstalk between GA and these hormones may modulate the complex process during SBD through the interaction gene network of the multihormone pathway. Lastly, based on the expression characterization of multihormone MST pathway genes, a proposed model of the GA-mediated multihormone regulatory network during SBD was proposed. CONCLUSIONS: Our results provided novel insights into GA-mediated regulatory networks during SBD in grape. The complexity of GA-mediated multihormone ST in SBD was also elucidated, thereby providing valuable information for future functional characterizations of specific genes in grape.