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

Rice (<i>Oryza sativa</i> 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 <i>OsMADS31</i>. To investigate the role of <i>OsMADS31</i> in salt stress tolerance, we generated its knockout mutant and overexpression lines in Nipponbare (Nip). Phenotypic analysis of T₂-generation <i>OsMADS31</i> knockout (<i>osmads31</i>) 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, <i>osmads31</i> knockout mutants exhibited markedly inhibited growth, whereas <i>OsMADS31</i> overexpression (<i>OE</i>) 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, <i>osmads31</i> 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 <i>osmads31</i> mutants and significantly lower in <i>OE</i> lines. Transcriptome analysis of WT and <i>osmads31</i> 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 <i>OsMADS31</i> enhances salt tolerance by upregulating antioxidant-related genes, activating antioxidant enzymes, and reducing oxidative damage. Our results conclusively show that <i>OsMADS31</i> improves salt tolerance in rice.