TaWRKY33 Positively Regulates TaERF1‐A, Thereby Activating TaP5CS 2 − Mediated Proline Biosynthesis, Which Enhances Drought Tolerance in Wheat ( Triticum aestivum L.)

Drought stress severely limits global wheat productivity. Herein, the function of TaERF1-A, encoded by a previously undescribed wheat ERF gene, in enhancing drought stress tolerance was analyzed. TaERF1-A exhibited nuclear localisation and transcriptional activation activity in planta, although no transactivation was detected in yeast. Notably, TaERF1-A was upregulated under drought and salt stress. TaERF1-A overexpression (OE) in Arabidopsis enhanced drought stress resistance, resulting in increased transcription levels of stress- and antioxidant-enzyme-related genes. BSMV-TaERF1-A-silenced wheat plants exhibited exacerbated wilting under drought, accompanied by elevated malondialdehyde levels, reduced proline accumulation, and decreased antioxidant enzyme activity levels. In contrast, TaERF1-A OE wheat lines exhibited enhanced drought tolerance, with opposite physiological trends. Notably, RNAi induced a weakened response in wheat compared to wild-type wheat. Yeast one-hybrid, electrophoretic mobility shift, dual-luciferase, and qRT-PCR assays demonstrated that TaWRKY33 directly binds the TaERF1-A promoter, thereby activating its transcription. TaWRKY33 overexpression also enhanced drought tolerance in wheat. TaERF1-A subsequently regulated TaP5CS2 expression, which stimulated proline biosynthesis, thereby contributing to improved drought resistance. This study elucidates the mechanism by which the TaWRKY33-TaERF1-A-TaP5CS2 module positively regulates drought tolerance and highlights the candidate genes that can be explored for developing elite drought-resistant wheat varieties.