## 3 Results
### Regulatory Mechanism of Hormones on Anthocyanin Synthesis  
Hormone is one of the most important factors affecting anthocyanin synthesis [37; 38]. Studies have shown that plant endogenous hormones can affect regulatory genes to control anthocyanin biosynthesis. Meanwhile, the accumulation of anthocyanin can also be controlled by activating or inhibiting the expression of genes related to anthocyanin synthesis through the application of exogenous hormones [39]. Thus far, eight kinds of plant hormones have been found: auxin (IAA), gibberellin (GA), cytokinin (CTK), abscisic acid (ABA), ethylene (ETH), brassinosterol (BR), jasmonic acid (JA), and salicylic acid (SA). According to its effect, plant hormones can be divided into growth regulators and growth inhibitors [40; 41; 42]. A large number of studies have shown that ABA has an obvious effect on anthocyanin accumulation in non-climacteric fruits (such as grape, cherry, strawberry, and litchi), while it had no significant effect on climacteric fruits (such as apple) [43; 44; 45]. Exogenous ABA treatment can induce the expression of structural genes and regulatory genes for anthocyanin in grape skins and cell suspension, and promote the accumulation of anthocyanin [46]. Ethylene plays an important role in the biosynthesis of anthocyanin, 2-chloroethyl phosphonic acid treatment can upregulate anthocyanin synthesis structure genes (_CHS_, _F3H_, _LDOX_, and _UFGT_), and increase the content of anthocyanin [47]. Jasmonic acid (JA) can also induce anthocyanin biosynthesis. In _Arabidopsis thaliana_, JA has synergistic regulatory effects on sucrose-induced _DFR_ gene expression and anthocyanin synthesis [48]. JAZ protein can interact with bHLHs (_GL3_, _EGL3_, _TT8_) and R2R3-MYBs (_PAP1_, _PAP2_) transcription factors in the MBW complex to inhibit anthocyanin synthesis [49]. Auxin is also an important hormone that affects anthocyanin synthesis. After NAA treatment, the relative expression levels of anthocyanin synthesis-related structural genes (_CHS_, _CHI_, _F3H_, _F3 '5' H_, _DFR_, _LDOX_, _UFGT_) in grape skins were lower than that in the control group, indicating that exogenous NAA treatment inhibited anthocyanin synthesis [50]. There are few studies on the effect of GA on fruit coloring. DELLA protein (encoded by _GAI_ gene) is a negative regulator of GA signal. _gai_ mutants do not affect GA synthesis but affect GA signal transduction. The expression level of the _DFR_ gene in seedlings and leaves of the _gai_ mutant was much higher than that of the wild-type, indicating that GA regulates anthocyanin biosynthesis through the DELLA protein [48]. Similar to protein-coding genes, the promoter region of plant miRNA genes contains a variety of important cis-acting elements, which are key to the regulation of gene expression. Through promoter action element analysis, we found that the promoter sequence of vvmiR156b/c/d contained a large number of hormone-related elements including ABA (ABRE), GA (GARE-motif, P-box, TATC-box), MeJA (CGTCA-motif, TGACG-motif), and auxin (TGA-element) responsive cis-elements. Of these hormone cis-acting elements, ABA and MeJA had the most cis-acting elements, followed by GA, while NAA had the least cis-acting elements. Based on this, ABA, GA, MeJA, and NAA were used to treat grape leaves and fruits, respectively, to verify the short-term and long-term effects of VvmiR156b/c/d and _VvSPL9_ response hormones. Interestingly, GA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early color-change period, but significantly upregulated the expression in the middle color-change and ripening stages. In addition, GA upregulated the expression levels of anthocyanin structural genes in the middle color-change stage and ripening stage, so GA mainly promoted grape berry coloring in the middle- and late-ripening stages. After ABA and MeJA treatment, grape berries can quickly color.