Abstract
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.
Highlights
Grape berry ripening is an extremely complex physiological and biochemical process, accompanied by the formation of fruit color, flavor, and aroma
We confirmed that VvSPL9 is the target gene of VvmiR156b/c/d, which is involved in the regulation of grape berry development and maturation; and miR156s could be in response to gibberellin [25,26,27]
abscisic acid (ABA) and methyl jasmonate (MeJA) treatments increased the size and weight of berries, while GA and naphthalene acetic acid (NAA) treatments inhibited the expansion of the berries (Table 1)
Summary
Grape berry ripening is an extremely complex physiological and biochemical process, accompanied by the formation of fruit color, flavor, and aroma. In litchi (Litchi chinensis Sonn.), miR156a is inversely correlated with the expression profiles of its target genes LcSPL1/2 and LcCHI, and the increased expression of miR156a decreases the transcription level of LCSPL1, which downregulates anthocyanin biosynthesis via interaction with LcMYB1 [24] All these findings demonstrate that miR156s and their targets are involved in the modulation of color formation. We confirmed that VvSPL9 is the target gene of VvmiR156b/c/d, which is involved in the regulation of grape berry development and maturation; and miR156s could be in response to gibberellin [25,26,27] These imply that VvmiR156s-mediated VvSPL might respond to the hormone to manipulate grape berry and ripening. This study provides a new research view for the hormone-mediated miRNA-target gene module involved in the regulatory mechanism of anthocyanins biosynthesis, which is conducive to the use of chemical regulation in production to promote fruit coloring
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