Alfalfa MsGAD2 induces γ-aminobutyric acid accumulation and enhances Cd resistance in transgenic tobacco

The present study compared two alfalfa varieties with different Cd-stress resistance levels: Medicago sativa ‘Zhaodong’ (ZD, resistant) and ‘Zhongmu No.1’ (ZM, sensitive). While the varieties showed no significant difference in Cd content under Cd stress, ZD exhibited greater adaptability, indicated by lower suppression of PSII activity, photosynthetic carbon assimilation, and reduced ROS damage. Weighted gene co-expression network analysis (WGCNA) based on RNA-seq data revealed that differentially expressed genes (DEGs) associated with physiological resistance indicators were significantly enriched in KEGG pathways related to proline (PRO) and γ-aminobutyric acid (GABA) synthesis, including alanine, aspartate and glutamate metabolism (map00250), arginine biosynthesis (map00220), and arginine and proline metabolism (map00330). Notably, the accumulation of proline and GABA was higher in the resistant alfalfa variety ZD. Furthermore, a key gene in the GABA synthesis pathway, MsGAD2 , was identified as potentially playing a crucial role in the Cd-stress adaptation. To investigate its role in Cd stress resistance, alfalfa MsGAD2 was heterologously overexpressed in tobacco. MsGAD2 overexpression significantly enhanced GABA synthesis in tobacco leaves under both control and, especially, Cd stress conditions. MsGAD2 overexpression also effectively prevented the decline of chlorophyll in tobacco leaves under Cd stress. It achieved this by stabilizing the activities of PSII and PSI and optimizing energy distribution within the PSII reaction center, thereby alleviating photosynthetic inhibition. The accumulation of GABA further enhanced the activities of antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in tobacco leaves under Cd stress, thus promoting PRO accumulation and mitigating oxidative damage associated with Cd exposure. Thus, PRO and GABA accumulation likely contributed to ZD’s superior adaptability to Cd stress compared to ZM. Thus, MsGAD2 played a crucial role in enhancing Cd tolerance in transgenic tobacco. These findings not only elucidate the mechanisms by which GABA is involved in Cd-stress adaptation, but also provide valuable genetic resources for breeding Cd-resistant alfalfa. • GABA and PRO synthesis play key roles in alfalfa Cd-stress adaptation. • Alfalfa MsGAD2 is the main regulator of GABA synthesis in Cd stress adaptation. • MsGAD2 overexpression enhances Cd stress resistance in transgenic tobacco. • MsGAD2 overexpression promotes the PRO accumulation in tobacco under Cd stress.