TrWRKY41: A WRKY Transcription Factor from White Clover Improves Cold Tolerance in Transgenic Arabidopsis

Trifolium repens L. (white clover) is a widely distributed perennial legume, which is regarded as one of the most important forages for its high protein content and excellent palatability. Low temperature limits the distribution and productivity of white clover, thereby reducing its economic returns. WRKY transcription factors are key regulators in stress defense and are involved in multiple abiotic stress responses in plants. In this study, a cold inducible gene named TrWRKY41 was cloned from white clover. The TrWRKY41 protein is predominantly localized in the nucleus and functions as a hydrophilic, acidic protein. Under cold stress, the overexpression plants had significantly higher chlorophyll (CHL) and proline (Pro) contents, significantly increased activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), and malondialdehyde (MDA) content significantly decreased. Compared to wild-type Arabidopsis thaliana, TrWRKY41-overexpressing plants exhibited better cold tolerance. In addition, target genes downstream of the TrWRKY41 transcription factor were predicted utilizing BLAST alignment and AlphaFold2 (version 0.2.0) software, the expression of six genes, including AtCOR47, AtCOR6.6, and AtABI5, was significantly up-regulated under cold stress. It suggests that TrWRKY41 may enhance cold tolerance in Arabidopsis by activating the ICE-CBF-COR cascade. This study provides candidate genes for research on enhancing the cold tolerance of white clover.