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.

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.