MicroRNA156 amplifies transcription factor-associated cold stress tolerance in plant cells

MicroRNAs may increase cold stress tolerance by regulating stress-related signal transduction pathways and by modulating the expression of transcription factors. However, the molecular mechanism by which microRNAs enhance cold stress tolerance is not fully understood. Here, we report that overexpression of rice microRNA156 (OsmiR156) results in increased cell viability and growth rate under cold stress in Arabidopsis, pine, and rice. OsmiR156 increases cold stress tolerance by targeting OsSPL3. OsSPL3 positively regulates the expression of OsWRKY71, a negative regulator of the transcription factors OsMYB2 and OsMYB3R-2. OsMYB2 counteracts cold stress by activating the expression of the stress-response genes OsLEA3, OsRab16A, and OsDREB2A. OsMYB3R-2 counteracts cold stress by activating the expression of OsKNOLLE2, OsCTP1, OsCycB1.1, OsCycB2.1, and OsCDC20.1. In OsmiR156 transgenic rice cell lines, the transcript levels of OsLEA3, OsRab16A, OsDREB2A, OsKNOLLE2, OsCTP1, OsCycB1.1, OsCycB2.1, and OsCDC20.1 were increased by OsWRKY71 knockdown and inversely regulated by OsWRKY71 overexpression, indicating that OsmiR156 enhances cold stress tolerance by regulating the expression of transcription factor genes in plant cells. These results will increase our understanding of microRNA-related cold stress tolerance in different plant species, including monocotyledonous, dicotyledonous, and gymnosperm plant species, and will be valuable in plant molecular biotechnology.