miR535 negatively regulates cold tolerance in rice

The miR156/miR529/miR535 superfamily, showing extremely high sequence identity, has been well documented to modulate growth and development. However, their roles in abiotic stress responses are rarely reported. Here, in this study, we reported the negative regulatory function of OsmiR535 in cold stress responses. The induced expression of OsmiR535 by cold stress was identified through semi-quantitative RT-PCR and quantitative real-time PCR analyses. By comparing the phenotype of the wild type and OsmiR535 overexpression lines, we showed that OsmiR535 overexpression repressed the early seedling growth under cold stress. Our studies further revealed that OsmiR535 overexpression aggravated the cold-induced cell death, affected the ROS accumulation and scavenging, and influenced the osmotic regulation under cold stress. In addition, OsmiR535 overexpression altered the expression of the OsCBF1, OsCBF2, and OsCBF3 genes, the core components of the CBF-mediated cold signaling, and the cold stress–responsive marker genes downstream of the CBF signaling pathway. Expectedly, the transcriptional levels of three SPL genes OsSPL14/11/4, which were predicted to be OsmiR535 targets, were downregulated in the OsmiR535 overexpression lines. Taken together, results in this study suggest that OsmiR535 negatively regulates rice cold stress responses possibly by targeting the SPL target genes and mediating the CBF-mediated cold signaling pathway.