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 degrees 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.