Characterization of VvmiR166s-Targets Modules and their Interaction Mechanism in Modulation of GA-Induced Grape Seedless Berry Process

GA (Gibberellin) is one key hormone of inducing grape seedless berries. VvmiR166s were revealed to be the important regulator of responding to GA in grapes, but their roles remain unclear yet. Here, the precise sequences of VvmiR166s and their potential targets VvREV, VvHB15 and VvHOX32, were determined in grape cv. ‘Rosario Bianco’. The cleavage interactions and variations of VvmiR166s:(VvHB15, VvHOX32, VvREV) were validated by RLM-RACE and PPM-RACE. Promoters cis-elements analysis supported that VvMIR166s and targets containing similar GA-related cis-elements, might play roles in responding to GA.Subsequently, spatio-temporal expression modes analysis of VvMIR166s and their targets in grape berries suggest that, almost all VvMIR166s possessed the lowest expression level at 46DAF (stone hardening stage) in seeds and peels under GA treatment except for VvMIR166h. Interestingly, their target gene VvHB15 exhibited the opposite expression, indicating that GA promoted the negative regulatory roles of VvmiR166s on VvHB15. Subsequently, over-expressions of VvMIR166b/h in Tobaccos, decreased the expression of its target VvHB15 and obviously strengthened some key genes in lignin synthesis pathway, while over-expression of VvHB15 showed the reverse regulatory modes, confirming that VvmiR166b/h might be amongst main factors of regulating lignin synthesis by mediating the expression of VvHB15. The results was deduced a key signal pathway of “GA-VvmiR166b/h-VvHB15- lignin synthesis genes”. Our findings revealed a novel insight into the roles of VvmiR166s response to GA in modulating the lignin synthesis of grape seed-stone development, which is essential for the molecular breeding of seedless grape berry.