In recent years, more and more reports have shown that the miR156-SPL module can participate in the regulation of anthocyanin synthesis in plants. However, little is known about how this module responds to hormonal signals manipulating this process in grapes. In this study, exogenous GA, ABA, MeJA, and NAA were used to treat the ‘Wink’ grape berries before color conversion, anthocyanin and other related quality physiological indexes (such as sugar, aroma) were determined, and spatio-temporal expression patterns of related genes were analyzed. The results showed that the expression levels of VvmiR156b/c/d showed a gradually rising trend with the ripening and color formation of grape berries, and the highest expression levels were detected at day 28 after treatment, while the expression level of VvSPL9 exhibited an opposite trend as a whole, which further verifies that VvmiR156b/c/d can negatively regulate VvSPL9. Besides, VvmiR156b/c/d was positively correlated with anthocyanin content and related genes levels, while the expression pattern of VvSPL9 showed a negative correlation. Analysis of promoter cis-elements and GUS staining showed that VvmiR156b/c/d contained a large number of hormone response cis-elements (ABA, GA, SA, MeJA, and NAA) and were involved in hormone regulation. Exogenous ABA and MeJA treatments significantly upregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early stage of color conversion and made grape berries quickly colored. Interestingly, GA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early color-change period, but significantly upregulated in the middle color-change and ripening stages, therefore GA mainly modulated grape berry coloring in the middle- and late-ripening stages. Furthermore, NAA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes and delayed the peak expression of genes. Meanwhile, to further recognize the potential functions of VvmiR156b/c/d, the mature tomato transient trangenetic system was utilized in this work. Results showed that transient overexpression of VvmiR156b/c/d in tomato promoted fruit coloring and overexpression of VvSPL9 inhibited fruit coloration. Finally, a regulatory network of the VvmiR156b/c/d-VvSPL9 module responsive to hormones modulating anthocyanin synthesis was developed. In conclusion, VvmiR156b/c/d-mediated VvSPL9 participated in the formation of grape color in response to multi-hormone signals.

MicroRNA156 (miR156) is an important conserved miRNA family in plants. Recently, we revealed VvmiR156a could involve in the modulation of gibberellin (GA)‐mediated flower and berry development process of grapevine (Vitis vinifera L.). However, how to manipulate this process is unclear. For this, we used the GA‐induced grapevine parthenocarpy system to investigate the regulatory roles of VvmiR156a during this process. Here, we cloned the mature and precursor sequences of VvmiR156a in Wink grape and identified its potential target gene VvAGL80, which belongs to the MADS‐box gene family. Moreover, using RNA ligase‐mediated 5′ rapid amplification of cDNA ends (RLM‐RACE) and poly(A)polymerase‐mediated 3′ rapid amplification of cDNA (PPM‐RACE) technologies, it confirmed that VvAGL80 was the true target gene of VvmiR159a. Analysis of promoter cis‐elements and β‐glucuronidase (GUS) staining showed that both VvmiR156a and VvAGL80 contained GA‐responsive elements and could respond to GA treatments. Quantitative real‐time–polymerase chain reaction (qRT‐PCR) analysis exhibited the VvmiR156a and VvAGL80 showed opposite expression trends during grapevine flower and berry development, indicating that VvmiR156a negatively regulated the expression of VvAGL80 during this process. After GA treatment, the expression of miR156 in flowers was downregulated significantly, while that of VvAGL80 was upregulated, thereby accelerating grapevine flowering. Furthermore, GA treatment enhanced the negative regulation of VvmiR156a on VvAGL80 in seed, especially at the seed‐coat hardening stage, which was the key period of seed growth and development. Our findings enriched the knowledge of the regulatory mechanism of the miRNA‐mediated grapevine parthenocarpy process.

Exogenous gibberellin (GA) was widely used to improve berry quality through inducing parthenocarpic seedless berries in grapes. We revealed that auxin response factors (ARFs), the key transcription factors in response to auxin, might respond to GA involving modulation of grape parthenocarpy. However, the underlying molecular mechanism in this process remains yet unclear. Here, a total of 19 VvARF members were identified in the ovaries during GA-induced grapes’ parthenocarpy. Interestingly, almost all members were GA-responsive factors, of which 9 could be classified in plant hormone signal transduction (KO04075) and involved in the tryptophan metabolic pathway (K14486). Moreover, VvARFs were predicted to have 310 interacted proteins involved in 19 KEGG pathways. Of them, 32 interacted proteins participated in the KO04075 pathway, including auxin (IAA), salicylic acid (SA), abscisic acid (ABA), cytokinin (CTK), and ethylene signaling pathways by responding to GA-mediated multi-hormone crosstalk. Further analysis demonstrated that VvARF4-2 might be the major factor in the modulation of GA-induced parthenocarpy via the crosstalk of IAA, CTK, SA, and ethylene signaling, followed by VvARF6-1 and VvARF9 involved in SA and ABA signaling pathways, respectively. Finally, we developed a VvARFs-mediated regulatory network by responding to GA-mediated multi-hormone crosstalk during grape parthenocarpy. Collectively, our findings provided novel insights into the regulatory network of VvARFs in GA-guided multi-hormone signaling to modulate grape parthenocarpy, which has great implications for the molecular breeding of high quality seedless grape berries.