VvmiR408 positively modulates seedlessness formation by cleaving VvAGL61 in grape berries

Grape seedlessness, a pivotal agronomic trait, is governed by intricate molecular mechanisms that remain partially elucidated. In this study, we identified VvmiR408 as a key regulator targeting VvAGL61 (a class D MADS-box gene) through RLM-RACE and PPM-RACE analysis, with cleavage validation at the 10th nucleotide of the miR408 complementary site, and uncover VvmiR408- VvAGL61 is a pivotal regulatory module during grape seed development, with a striking antagonism of spatial-temporal expression profiles between VvmiR408 and VvAGL61 in this process: VvmiR408 peaked during seed abortion in the seedless cultivar 'Jingkejing' (40 DAF, 3.8-fold increase), while VvAGL61 dominated during seed development in the seeded 'Wink' (40 DAF, 11-fold elevation), indicating VvmiR408 promoting seed abortion in seedless berries while VvAGL61 enhances seed development. This hypothesis was further validated by transgenic tobacco overexpressing VvMIR408 , which exhibited a significant reduction in both seed number and silique size, through the down-regulation of NtAGL61 , the tobacco ortholog of VvAGL61 . Collectively, we propose an interaction model of the miR408- AGL61 module mediating seed development and abortion in grapes and tobaccos. This model highlights key target sites for molecular breeding aimed at producing high-quality seedless grapes. • VvmiR408 directly targets VvAGL61 via cleavage at the 10th nucleotide position in grapes; • The VvmiR408- VvAGL61 module functions as a molecular switch for the seed development-abortion transition: VvmiR408 represses VvAGL61 to promote abortion in seedless grape cultivars, while VvAGL61 enhances development in seeded ones; • This mechanism is evolutionarily conserved, as VvMIR408 overexpression in tobacco repressed its AGL61 homolog and recapitulated the seedless phenotype.