The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in ‘Chardonnay’ suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape.

Cultivated grapevine (Vitis) is a highly valued horticultural crop, and cold stress affects its growth and productivity. Wild Amur grape (Vitis amurensis) PAT1 (Phytochrome A signal transduction 1, VaPAT1) is induced by low temperature, and ectopic expression of VaPAT1 enhances cold tolerance in Arabidopsis (Arabidopsis thaliana). However, little is known about the molecular mechanism of VaPAT1 during the cold stress response in grapevine. Here, we confirmed the overexpression of VaPAT1 in transformed grape calli enhanced cold tolerance. Yeast two-hybrid and bimolecular fluorescence complementation assays highlighted an interaction between VaPAT1 with INDETERMINATE-DOMAIN 3 (VaIDD3). A role of VaIDD3 in cold tolerance was also indicated. Transcriptome analysis revealed VaPAT1 and VaIDD3 overexpression and cold treatment coordinately modulate the expression of stress-related genes including lipoxygenase 3 (LOX3), a gene encoding a key jasmonate biosynthesis enzyme. Co-expression network analysis indicated LOX3 might be a downstream target of VaPAT1. Both electrophoretic mobility shift and dual luciferase reporter assays showed the VaPAT1-IDD3 complex binds to the IDD-box (AGACAAA) in the VaLOX3 promoter to activate its expression. Overexpression of both VaPAT1 and VaIDD3 increased the transcription of VaLOX3 and JA levels in transgenic grape calli. Conversely, VaPAT1-SRDX (dominant repression) and CRISPR/Cas9-mediated mutagenesis of PAT1-ED causing the loss of the C-terminus in grape calli dramatically prohibited the accumulation of VaLOX3 and JA levels during cold treatment. Together, these findings point to a pivotal role of VaPAT1 in the cold stress response in grape by regulating JA biosynthesis.

Sugar accumulation is a critical event during grape berry ripening that determines the grape market values. Berry cells are highly dependent on sugar transporters to mediate cross-membrane transport. However, the role of sugar transporters in improving sugar accumulation in berries is not well established in grapes. Herein we report that a Sugars Will Eventually be Exported Transporter (SWEET), that is, VvSWEET10, was strongly expressed at the onset of ripening (véraison) and can improve grape sugar content. VvSWEET10 encodes a plasma membrane-localized transporter, and the heterologous expression of VvSWEET10 indicates that VvSWEET10 is a hexose-affinity transporter and has a broad spectrum of sugar transport functions. VvSWEET10 overexpression in grapevine calli and tomatoes increased the glucose, fructose, and total sugar levels significantly. The RNA sequencing results of grapevine transgenic calli showed that many sugar transporter genes and invertase genes were upregulated and suggest that VvSWEET10 may mediate sugar accumulation. These findings elucidated the role of VvSWEET10 in sugar accumulation and will be beneficial for the improvement of grape berry quality in the future.

Clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system is an efficient targeted genome editing method. Although CRISPR/Cas9-mediated mutagenesis has been applied successfully in grape, few studies have examined the technique’s efficiency. To optimize CRISPR/Cas9 editing efficiency in Vitis vinifera, we surveyed three key parameters: GC content of single guide RNA (sgRNA), variety of transformant cells used, and SpCas9 expression levels in transgenic cell mass. Four sgRNAs with differing GC content were designed to target exon sites of the V. vinifera phytoene desaturase gene. Suspension cells of ‘Chardonnay’ and ‘41B’ varieties were used as the transgenic cell mass. Both T7EI and PCR/RE assays showed that CRISPR/Cas9 editing efficiency increases proportionally with sgRNA GC content with 65% GC content yielding highest editing efficiency in both varieties. Additionally, gene editing was more efficient in ‘41B’ than in ‘Chardonnay.’ CRISPR/Cas9 systems with different editing efficiency showed different SpCas9 expression level, but compared with GC content of sgRNA, SpCas9 expression level has less influence on editing efficiency. Taken together, these results help optimize of CRISPR/Cas9 performance in grape.

Nuclear factor Y (NF-Y) transcription factor is composed of three distinct subunits: NF-YA, NF-YB and NF-YC. Many members of NF-Y family have been reported to be key regulators in plant development, phytohormone signaling and drought tolerance. However, the function of the NF-Y family is less known in grape (Vitis vinifera L.). A total of 34 grape NF-Y genes that distributed unevenly on grape (V. vinifera) chromosomes were identified in this study. Phylogenetic analysis was performed to predict functional similarities between Arabidopsis thaliana and grape NF-Y genes. Comparison of the structures of grape NF-Y genes (VvNF-Ys) revealed their functional conservation and alteration. Furthermore, we investigated the expression profiles of VvNF-Ys in response to various stresses, phytohormone treatments, and in leaves and grape berries with various sugar contents at different developmental stages. The relationship between VvNF-Y transcript levels and sugar content was examined to select candidates for exogenous sugar treatments. Quantitative real-time PCR (qPCR) indicated that many VvNF-Ys responded to different sugar stimuli with variations in transcript abundance. qPCR and publicly available microarray data suggest that VvNF-Ys exhibit distinct expression patterns in different grape organs and developmental stages, and a number of VvNF-Ys may participate in responses to multiple abiotic and biotic stresses, phytohormone treatments and sugar accumulation or metabolism. In this study, we characterized 34 VvNF-Ys based on their distributions on chromosomes, gene structures, phylogenetic relationship with Arabidopsis NF-Y genes, and their expression patterns. The potential roles of VvNF-Ys in sugar accumulation or metabolism were also investigated. Altogether, the data provide significant insights on VvNF-Ys, and lay foundations for further functional studies of NF-Y genes in grape.