The aim of this study was to identify grapevine miR164s (VvmiR164a/b/c/d) and their target genes, and to elucidate the regulatory roles of VvmiR164s and their target genes during exogenous GA-induced grape parthenocarpic process.

Using ‘Wink’ grapes (Vitis vinifera L. Wink) as the test material, miR-RACE, PCR, RLM-RACE and PPM-RACE, qRT-PCR and bioinformatics were used to analyze the spatio-temporal expression of VvmiR164s-VvNAC100 module in response to exogenous GA and its potential functions during grape parthenocarpic process.

Gibberellin application on ‘Wink’ grapes before flowering induced parthenocarpy, leading to seedless berries. The precise sequence of VvmiR164a/b/c/d was cloned and identified, and four of its target genes were predicted, including VvNAC100-1, VvNAC100-2, VvNAC098, and VvNAC021. Combining the degree of match with the comprehensive analysis of the previous data, this work focused on the analysis of the target gene VvNAC100-2, which was named as VvNAC100. The VvNAC100 cleavage site was located at position 9 and position 11 of the 5′ end of miRNA, with a cleavage frequency of 17/20 and 11/20, respectively, and was localized on Chr14, encoding 363 amino acids and containing a NAM. The VvNAC100 protein is highly conserved in amino acid sequence and functionally similar to other species, among which it is more closely related to species such as pepper and tobacco. The promoters of VvMIR164a/b/c/d and its target gene VvNAC100 both contain multiple hormone-acting elements, suggesting that it might be involved in the regulation of grape growth and development by responding to mult-hormones. The RT-qPCR results showed that the expression level of VvmiR164b tended to decrease as the grape ovary developed, while its target gene VvNAC100 showed an increasing expression trend in the early stage of ovary development with a certain negative correlation. On the other hand, VvmiR164a/c/d showed a similar expression pattern with VvNAC100 with a certain positive correlation. However, after GA treatment, the expression of VvmiR164a/c/d was highly significantly increased during parthenocarpy in the grape ovary, which also significantly suppressed the expression of VvNAC100 during this period, promoting a negative correlation between VvmiR164a/c/d-VvNAC100 their expression levels. VvmiR164b showed a decreasing trend after GA treatment, and it’s the expression levels showed some positive correlation with those of VvNAC100, indicating that GA treatment promoted the negative regulation of VvmiR164a/c/d on VvNAC100, but repressed the negative regulation of VvmiR164b on VvNAC100.

VvNAC100 was the true target gene of VvmiR164 family. During grape parthenocarpy, GA induced the negative regulation of VvmiR164a/c/d on the target gene VvNAC100 and suppressed that of VvmiR164b on the target gene VvNAC100. VvmiR164a/c/d were the main effectors of the VvmiR164 family involved in modulation of GA-induced grape parthenocarpy.