Shuai Zhao, Fashuai Li, Kuan Cheng, Hao Qi, Mingli Song, Xuehui Qian, Yanan Tian, Xinlei Feng, HaoYue Xu, Qian Liu, Jinhua Hu, Zhanqing Wu, Wei Chen, Hassan H A Mostafa, Baozhu Li
Proline, a critical osmoprotectant and reactive oxygen species (ROS) scavenger, is vital for plant adaptation to drought and various abiotic stresses. Nevertheless, the biological functions of maize Δ¹-pyrroline-5-carboxylate synthetase (P5CS) gene family members, which catalyze the rate-limiting step in proline biosynthesis, remain largely uncharacterized. We functionally characterized ZmP5CS3 and elucidated its molecular role in drought response. ZmP5CS3 contains a conserved P5CS family domain, the protein localizes to the cytoplasm and exhibits in vitro enzymatic activity promoting proline synthesis. Drought stress markedly upregulateed ZmP5CS3 expression, leading to elevated proline accumulation. Genetic assays showed that ZmP5CS3 positively regulates drought tolerance in maize. Physiological experiments demonstrated that ZmP5CS3 increases proline accumulation, reduces ROS levels, and alleviates membrane lipid peroxidation, thereby mitigating oxidative injury under drought stress. Heterologous overexpression of ZmP5CS3 in Arabidopsis thaliana also enhanced drought tolerance, confirming the functional conservation of ZmP5CS3. Taken together, these findings reveal that ZmP5CS3 improves drought tolerance in maize seedlings by coordinating proline biosynthesis and ROS detoxification. This study provides preliminary mechanistic insights into seedling stress metabolism and offers candidate gene resources for molecular breeding of drought-resistant maize.