Supplementary file 1_Overexpression and silencing of the cotton GhABA2 gene reveal its role in salt stress tolerance.docx

The GhABA2 gene encodes a short-chain dehydrogenase/reductase involved in abscisic acid (ABA) biosynthesis and plays a crucial role in plant salt stress responses. To explore its function in cotton salt tolerance, we performed bioinformatic analysis, heterologous overexpression in Arabidopsis, and virus-induced gene silencing (VIGS) in cotton. Bioinformatic prediction revealed that the promoter region of the GhABA2 contains multiple cis-acting elements associated with ABA, light, and stress responses. Subcellular localization analysis indicated that GhABA2 was predominantly localized in the cytoplasm. Overexpression of GhABA2 in Arabidopsis significantly enhanced salt tolerance, as manifested by increased germination rates and root elongation. This was accompanied by a reinforced antioxidant system, with elevated activities of catalase (CAT), peroxidase (POD), glutathione reductase (GR), and ascorbate peroxidase (APX), alongside reduced accumulation of malondialdehyde (MDA) and H2O2. Furthermore, the transgenic lines exhibited higher relative water content (RWC), proline accumulation, and increased endogenous abscisic acid (ABA) levels under salt stress. Consistently, the expression of stress-responsive genes and ABA biosynthesis genes (AtNCED3, AtAAO3) was upregulated. Conversely, virus-induced gene silencing (VIGS) of GhABA2 in cotton compromised salt tolerance, characterized by diminished antioxidant enzyme activities (superoxide dismutase SOD, POD, GR, APX), reduced ABA content, and lower RWC and proline levels, but higher MDA and H2O2 accumulation. Correspondingly, the transcript levels of ROS-related genes and cotton ABA biosynthesis genes (GhNCED3a, GhNCED3c, GhAAO3) were downregulated. These findings demonstrate that GhABA2 positively regulates salt tolerance by modulating ABA biosynthesis and the antioxidant defense system. These findings elucidate the role of GhABA2 in modulating cotton salt stress responses and highlight its potential as a genetic target for breeding salt-tolerant cotton varieties.