MiR399 plays an important role in plant growth and development. The objective of the present study was to elucidate the evolutionary characteristics of the MIR399 gene family in grapevine and investigate its role in stress response. To comprehensively investigate the functions of miR399 in grapevine, nine members of the Vvi-MIR399 family were identified based on the genome, using a miRBase database search, located on four chromosomes (Chr 2, Chr 10, Chr 15, and Chr 16). The lengths of the Vvi-miR399 precursor sequences ranged from 82 to 122 nt and they formed stable stem–loop structures, indicating that they could produce microRNAs (miRNAs). Furthermore, our results suggested that the 2 to 20 nt region of miR399 mature sequences were relatively conserved among family members. Phylogenetic analysis revealed that the Vvi-MIR399 members of dicots (Arabidopsis, tomato, and sweet orange) and monocots (rice and grapevine) could be divided into three clades, and most of the Vvi-MIR399s were closely related to sweet orange in dicots. Promoter analysis of Vvi-MIR399s showed that the majority of the predicted cis-elements were related to stress response. A total of 66.7% (6/9) of the Vvi-MIR399 promoters harbored drought, GA, and SA response elements, and 44.4% (4/9) of the Vvi-MIRR399 promoters also presented elements involved in ABA and MeJA response. The expression trend of Vvi-MIR399s was consistent in different tissues, with the lowest expression level in mature and young fruits and the highest expression level in stems and young leaves. However, nine Vvi-MIR399s and four target genes showed different expression patterns when exposed to low light, high light, heat, cold, drought, and salt stress. Interestingly, a putative target of Vvi-MIR399 targeted multiple genes; for example, seven Vvi-MIR399s simultaneously targeted VIT_213s0067g03280.1. Furthermore, overexpression of Vvi_MIR399e and Vvi_MIR399f in Arabidopsis enhanced tolerance to drought compared with wild-type (WT). In contrast, the survival rate of Vvi_MIR399d-overexpressed plants were zero after drought stress. In conclusion, Vvi-MIR399e and Vvi-MIR399f, which are related to drought tolerance in grapevine, provide candidate genes for future drought resistance breeding.

Translation is a crucial process during plant growth and morphogenesis. In grapevine (Vitis vinifera L.), many transcripts can be detected by RNA sequencing; however, their translational regulation is still largely unknown, and a great number of translation products have not yet been identified. Here, ribosome footprint sequencing was carried out to reveal the translational profile of RNAs in grapevine. A total of 8291 detected transcripts were divided into four parts, including the coding, untranslated regions (UTR), intron, and intergenic regions, and the 26 nt ribosome-protected fragments (RPFs) showed a 3 nt periodic distribution. Furthermore, the predicted proteins were identified and classified by GO analysis. More importantly, 7 heat shock-binding proteins were found to be involved in molecular chaperone DNA J families participating in abiotic stress responses. These 7 proteins have different expression patterns in grape tissues; one of them was significantly upregulated by heat stress according to bioinformatics research and was identified as DNA JA6. The subcellular localization results showed that VvDNA JA6 and VvHSP70 were both localized on the cell membrane. Therefore, we speculate that DNA JA6 may interact with HSP70. In addition, overexpression of VvDNA JA6 and VvHSP70, reduced the malondialdehyde (MDA) content, improved the antioxidant enzyme activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), increased the content of proline, an osmolyte substance, and affected the expression of the high-temperature marker genes VvHsfB1, VvHsfB2A, VvHsfC and VvHSP100. In summary, our study proved that VvDNA JA6 and the heat shock protein VvHSP70 play a positive role in the response to heat stress. This study lays a foundation for further exploring the balance between gene expression and protein translation in grapevine under heat stress.

Selecting appropriate rootstocks can enhance the adaptability and fruit quality of grafted grapevines. However, grafting studies on ‘Red Globe’, one of the major cultivated cultivars, remain limited, particularly those involving long-term and comprehensive evaluations. The present research grafted ‘Red Globe’ onto four rootstocks—‘101-14’, ‘188-08’, ‘110R’, and ‘3309C’—and systematically compared graft union healing following hardwood grafting, field performance of grafted vines, vegetative growth of mature vines, and fruit phenotypic and quality traits across multiple years. The results showed that ‘101-14’ promoted the accumulation of organic acids, which reached 1.1% in 2023, and caused an increased tendency for berry detachment from the peduncle. The RG/110R combination exhibited a higher CFI, 0.8 on average, at the basal section, and promoted shoot thickening. RG/3309C was found to have a larger shoot length exceeding 600 cm, and a significant increase in fruit weight to nearly 13 g. The grafts on ‘188-08’ showed the highest survival rate of 74% among the graft combinations, and enhanced fruit quality, as evidenced by elevated TSS (16 °Brix) and firmer pulp texture, indicating that ‘188-08’ may serve as a valuable rootstock for enhancing the local adaptability and fruit quality of ‘Red Globe’ grapevines.

Downy mildew (DM), caused by Plasmopara viticola, is one of the most serious diseases that affects grape production worldwide. Utilizing host resistance, commonly known as 'R' genes, is considered the most effective way to cure the disease. The identification of the R gene in well-known table grape cultivars is conducive to breeding varieties with better quality. In this study, a population of F1 with 246 progenies was obtained from the cross of two widely grown table grapes 'Moldova' and 'Shine Muscat', in which 'Moldova' appeared resistant to P. viticola resistance. According to the sequencing data from GBTS, a high-density genetic map containing 826 SNPs, which spanned 1,515.99 cM, was constructed. Through the analysis of MapQTL and GWAS, a resistant locus for P. viticola was mapped, located at 27.534−30.729 Mbp in chr18, and was named Rpv34. A total of 21 Resistance Gene Analogues (RGA) distributed in three clusters were identified in this locus. A SNP marker chr18_29062596 was proved to be highly associated with resistance to P. viticola. This work will improve the mechanism of resistance understanding of P. viticola in grapevine and facilitate the use of resistant germplasm in breeding.

The muscat aroma of grape is largely determined by terpenoid volatiles. However, its genetic basis in ‘Muscat Hamburg’ remains unclear. We performed comparative volatile metabolomics between ‘Muscat Hamburg’ and ‘Red Globe’ across berry development, identifying 127 differentially abundant metabolites, with upregulated ones enriched in the monoterpenoid pathway. Using an F1 segregating population (264 progeny), we quantified four key monoterpenoids (α-terpineol, linalool, geraniol, and cis-linalool oxide) over two consecutive years. Using a high-density genetic map with 7872 SNP markers generated via SLAF-seq, we detected stable major QTLs for α-terpineol, linalool, and geraniol on LG5, and a novel QTL for cis-linalool oxide on LG14. Expression analysis revealed that the transcript levels of VvDXS and VvGGPPS1 were significantly higher in ‘Muscat Hamburg’ than in ‘Red Globe’ at ripening. Heterologous overexpression in tomato enhanced terpenoid aroma accumulation without affecting fruit development. These results indicate that VvDXS and VvGGPPS1 play a crucial role in the formation of the aroma in the ‘Muscat Hamburg’ variety.