MIKC-type MADS-box transcription factor OsMADS31 positively regulates salinity tolerance in rice

Rice (Oryza sativa L.) is one of the world's most vital staple crops, providing food for over 50% of the global population. As a salt-sensitive crop, rice is susceptible to damage from soil-soluble salt stress, which can severely reduce rice yield. Here, we aimed to elucidate the molecular mechanisms underlying salt tolerance in rice. Investigation of MADS-box genes involved in abiotic stress responses in rice led to the identification of OsMADS31. To investigate the role of OsMADS31 in salt stress tolerance, we generated its knockout mutant and overexpression lines in Nipponbare (Nip). Phenotypic analysis of T₂-generation OsMADS31 knockout (osmads31) mutants revealed altered panicle morphology and significant reductions in seed-setting rate, panicle length, grain number per panicle, and 1000-grain weight. Under salt stress, both during seed germination and at the three-leaf stage, osmads31 knockout mutants exhibited markedly inhibited growth, whereas OsMADS31 overexpression (OE) lines maintained normal germination and development. At the three-leaf stage, knockout mutants showed significantly lower survival rates following salt treatment and subsequent recovery. Physiological and biochemical assays demonstrated that, compared with wild-type (WT) plants, osmads31 mutants exhibited substantially decreased catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities as well as reduced proline (Pro) content. Conversely, compared with WT plants, 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining intensities as well as malondialdehyde (MDA) content were significantly higher in osmads31 mutants and significantly lower in OE lines. Transcriptome analysis of WT and osmads31 mutants under salt stress conditions, followed by Gene Ontology (GO) enrichment of the identified differentially expressed genes (DEGs), revealed the enrichment of genes encoding protein kinases, CATs, and transcription factors. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified several key pathways including carbon metabolism, amino acid biosynthesis, metabolic pathways, glycolysis/gluconeogenesis, lipid metabolism, and plant hormone signal transduction. Furthermore, weighted gene co-expression network analysis (WGCNA) of the DEGs demonstrated that OsMADS31 enhances salt tolerance by upregulating antioxidant-related genes, activating antioxidant enzymes, and reducing oxidative damage. Our results conclusively show that OsMADS31 improves salt tolerance in rice.