Functional analysis of a cold-responsive rice WRKY gene, OsWRKY71

Publicly available microarray data and RNA gel blot analysis identified a rice WRKY transcription factor, OsWRKY71, that is highly upregulated in response to cold stress. Experiments with OsWRKY71 promoter:GFP transgenic rice confirmed its cold-inducible expression. Transient expression of OsWRKY71-GFP in maize mesophyll protoplasts indicated that it is localized predominantly in the nucleus and to a lesser extent in the cytosol. Transcriptional activation assays revealed that OsWRKY71 suppresses luciferase reporter activity in maize protoplasts, suggesting that it functions as a transcriptional repressor in rice. To characterize the function of OsWRKY71 in rice, we generated transgenic rice plants carrying CaMV35S promoter:OsWRKY71. Upon cold (4 °C) treatment, two selected OsWRKY71 transgenic lines, OX12 and OX21, recovered much better with respect to survival rate, photosynthetic ability, fresh weight, and dry weight than the control lines. RT-PCR analysis of known cold-responsive genes found that expression of OsTGFR and WSI76 was increased in OsWRKY71 transgenic lines in response to cold stress. Our results suggest that OsWRKY71 has a positive function in cold tolerance by regulating downstream target genes.