MiR396 is involved in plant response to vernalization and flower development in Agrostis stolonifera

MicroRNA396 (miR396) has been demonstrated to regulate flower development by targeting growth-regulating factors (GRFs) in annual species. However, its role in perennial grasses and its potential involvement in flowering time control remain unexplored. Here we report that overexpression of miR396 in a perennial species, creeping bentgrass (Agrostis stolonifera L.), alters flower development. Most significantly, transgenic (TG) plants bypass the vernalization requirement for flowering. Gene expression analysis reveals that miR396 is induced by long-day (LD) photoperiod and vernalization. Further study identifies VRN1, VRN2, and VRN3 homologs whose expression patterns in wild-type (WT) plants are similar to those observed in wheat and barley during transition from short-day (SD) to LD, and SD to cold conditions. However, compared to WT controls, TG plants overexpressing miR396 exhibit significantly enhanced VRN1 and VRN3 expression, but repressed VRN2 expression under SD to LD conditions without vernalization, which might be associated with modified expression of methyltransferase genes. Collectively, our results unveil a potentially novel mechanism by which miR396 suppresses the vernalization requirement for flowering which might be related to the epigenetic regulation of VRN genes and provide important new insight into critical roles of a miRNA in regulating vernalization-mediated transition from vegetative to reproductive growth in monocots.