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Grape is an economically important crop but recalcitrant to Agrobacterium-mediated genetic transformation and in vitro regeneration. Here, we have developed a protocol for transient transformation of grapes by investigating the effects of explant pre-culture and duration of vacuum infiltration on transformation efficiency. Using sliced grape berries of "Shine-Muscat" (Vitis labrusca × Vitis vinifera) between the end of fruit expansion phase and the mature stage as explants, we firstly compared the effect of pre-culture explants into a susceptible state (incubation on Murashige and Skoog (MS) agar plate in the dark at 25 ± 1 °C for 48 h) with no pre-culture and then tested different vacuum infiltration times on transformation efficiency using β-glucuronidase (GUS) reporter system. Pre-culture increased the susceptibility of explants to the agrobacteria infection and increased transient transformation efficiency as assessed by histochemical GUS activity, with intense blue coloration compared with the faint staining observed in the non-susceptible explants. Using a Circulating Water Vacuum Pump system to facilitate agrobacteria entry into berry cells, we tested vacuum durations of 5, 10, and 15 min and observed that transformation efficiency increased with vacuum duration of infiltration. These results were confirmed by relative gene expression of GUS transgene as assessed by RT-qPCR and GUS activity assay. To further confirm the usefulness of our protocol, we transiently transformed grape berries with the hydrogen peroxide sensor gene VvHPCA3, and this was confirmed by gene expression analysis as well as increased sensitivity of the explants to hydrogen peroxide treatment. Overall, this study has resulted in a simple but efficient transient transformation protocol for grape berries and would be a valuable tool for the rapid testing of gene function and the study of key regulatory networks in this important crop. [ABSTRACT FROM AUTHOR]

Grape (Vitis vinifera L.) berries are susceptible to quality deterioration after harvest. This study aimed to investigate the effect of folic acid (FA) treatment on the postharvest quality of table grape berries. Harvested 'Kyoho' (V. vinifera × V. labrusca L.) bunches were immersed in 1 mg L−1 FA solution for 15 min, then stored for sample collection and phenotype observation. Treatment with FA decreased the rates of berry abscission, water loss, and berry softening, reduced the accumulation of reactive oxygen species (ROS), and altered ROS metabolism-associated enzyme activities and gene expression levels. Transcriptome analysis revealed 507, 684, and 250 differentially expressed genes (DEGs) between FA-treated and non-treated berries at 3, 6, and 9 d after treatment, respectively. The DEGs were grouped into six modules according to a weighted correlation network analysis. A hub transcription factor, annotated as a WRKY transcription factor, was screened from the module associated with cell wall metabolism and identified as VvWRKY31. Transient transformation of grape berry slices revealed that overexpression of VvWRKY31 enhanced the relative expression level of VvRboh. The dual-luciferase assay showed that VvWRKY31 was capable of binding to VvRboh promoter. The present results provide insights into the potential molecular mechanism underlying FA-induced delay postharvest quality deterioration of grape berries. Specifically, FA inhibits expression of genes associated with ROS generation by reducing VvWRKY31 expression, thereby delaying quality deterioration induced by excessive accumulation of ROS. These results will be helpful in table grape breeding programs focused on delaying deterioration in postharvest quality. • Folic acid affects ROS metabolism in postharvest grape. • VvWRKY31 is a hub transcription factor in cell wall metabolism. • VvWRKY31 promotes VvRboh expression by binding its promoter. [ABSTRACT FROM AUTHOR]

Background: In a previous study, the early ripening of Kyoho grape following H2O2 treatment was explored at the physiological level, but the mechanism by which H2O2 promotes ripening at the molecular level is unclear. To reveal the molecular mechanism, RNA-sequencing analysis was conducted on the different developmental stages of Kyoho berry treated with H2O2. Results: In the comparison of treatment and control groups, 406 genes were up-regulated and 683 were down-regulated. Time course sequencing (TCseq) analysis showed that the expression patterns of most of the genes were similar between the treatment and control, except for some genes related to chlorophyll binding and photosynthesis. Differential expression analysis and the weighted gene co-expression network were used to screen significantly differentially expressed genes and hub genes associated with oxidative stress (heat shock protein, HSP), cell wall deacetylation (GDSL esterase/lipase, GDSL), cell wall degradation (xyloglucan endotransglucosylase/ hydrolase, XTH), and photosynthesis (chlorophyll a-b binding protein, CAB1). Gene expression was verified with RT-qPCR, and the results were largely consistent with those of RNA sequencing. Conclusions: The RNA-sequencing analysis indicated that H2O2 treatment promoted the early ripening of Kyoho berry by affecting the expression levels of HSP, GDSL, XTH, and CAB1 and- photosynthesis- pathways. [ABSTRACT FROM AUTHOR]

• Grapevine plants were dwarfed by Chlormequat chloride (CCC). • Transcriptome data analysis showed that VviEXPA7 plays an important role. • The high expression of VviRAP2.12 transcription factor suppressed the expression of VviEXPA7 after CCC treatment, which led to the inhibition of cell elongation and plant dwarfing. Treatment with chlormequat chloride (CCC) can lead to dwarfism, which has been studied in many plants. However, the underlying molecular mechanisms have been less studied. Herein, the phenotypic changes and gene expression levels of grapevine in response to CCC were investigated. Results showed that the height of grape plants became significantly shorter after CCC treatment, especially between the first two stems above the roots. Transcriptome sequencing was then performed and analyzed, 120 differentially expressed genes (DEGs) were detected in the stems after CCC treatment compared to the control. The gene ontology (GO) enrichment results showed that processes such as cellulose synthase (UDP-forming) activity, regulation of the auxin-mediated signaling pathway, and the cellulose biosynthetic process were common. In addition, weighted gene co-expression network analysis (WGCNA) identified the strong correlation between the MEbrown module and stem development. VviEXAP7 was selected from the genes belonging to key GO pathways and WGCNA modules. The quantitative real-time PCR (qRT-PCR) results showed that VviEXPA7 was significantly downregulated because of the CCC treatment. Additionally, analysis of VviEXPA7 promoter activity showed that VviEXPA7 activated GUS gene expression, and was induced by CCC, abscisic acid (ABA), and other hormones. VviEXPA7 was used as a target gene for transcription factor prediction, and after employing the PlantTFDB and JASPAR databases, VviRAP2.12 was identified as a transcription factor that might regulate VviEXPA7. qRT-PCR revealed that VviRAP2.12 was significantly upregulated after CCC treatment. Finally, the interaction between VviEXPA7 and VviRAP2.12 was verified by the dual-luciferase assay. Thus, the current study shows that CCC decreased the expression of the cell growth-related gene VviEXPA7 by promoting the expression of VviRAP2.12, which resulted in dwarfed plants. This study generated new information that will increase our understanding of the mechanism of action of CCC and provides a scientific basis for simplified cultivation of grapes. [ABSTRACT FROM AUTHOR]

• Six genes are determined as proline biosynthesis- or proline catabolism-related genes in grapevines. • P5CS1, OAT, PDH and P5CDH can be significantly induced by osmotic and oxidative stresses. • Proline is greatly accumulated under abiotic stresses, especially osmotic and oxidative stresses. • Proline biosynthesis and proline catabolism-related genes synergistically regulate proline accumulation in response to abiotic stresses. Proline is an important osmotic regulator for plants under abiotic stresses. Proline accumulation is regulated by various enzymes, while genes encoding these enzymes have not been fully investigated in grapevines. In this study, we identified four proline biosynthesis-related genes (P5CS1, P5CS2, P5CR and OAT) and two proline catabolism-related genes (PDH and P5CDH) from grapevines, and conducted bioinformatics analysis including protein characteristic, gene structure, phylogenetic relationship and cis-acting element of promoters. Promoter analysis found that cis-acting elements related to stress and phytohormone responsiveness were present in the promoters of these genes. Tissue-specific expression analysis of these genes indicated that P5CS2, OAT and PDH expressed highly in flower, and P5CS1, P5CR and P5CDH had highest expression levels in tendril, berry and leaf respectively. Expression patterns of these genes under abiotic stresses revealed that P5CS1, OAT and PDH could be significantly induced by salt, dehydration, PEG and H 2 O 2 treatments, and P5CDH was significantly suppressed under these stress treatments. By measuring proline accumulation in grapevines, we found that proline content dramatically increased after treatments of salt, dehydration, PEG and H 2 O 2. Our results revealed the molecular responses (especially P5CS1, OAT, PDH and P5CDH) in the proline metabolism pathway to osmotic and oxidative stresses in grapevines, demonstrating that proline synthesis and proline catabolism synergistically regulate proline accumulation in response to abiotic stresses. [ABSTRACT FROM AUTHOR]