Genome-wide identification of sORFs in indica rice and their comparative transcriptomic analysis under stress

Small open reading frames (sORFs) represent an underexplored source of functional peptides in plant genomes. This study aimed to identify drought-responsive sORFs in Oryza sativa subsp. indica and to evaluate their potential roles in drought stress adaptation, addressing the question of whether sORFs contribute to physiological and molecular drought tolerance mechanisms in rice. Stress-responsive sORFs were identified through in silico prediction combined with transcriptomic analysis across multiple abiotic stress conditions. Functional annotation was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and signal peptide prediction was used to infer potential secretory functions. Three indica rice varieties (MR 219, MR 220, and MR 297) were subjected to drought stress for physiological assessment. Oxidative stress markers and antioxidant gene expression were analyzed, and candidate sORF expression was validated using semi-quantitative RT-PCR. Differentially expressed sORFs (DE sORFs) were significantly enriched in GO terms related to structural molecule activity and binding, while KEGG analysis highlighted MAPK signaling and antioxidant defense pathways. Several sORFs were predicted to encode secretory peptides. Among the tested varieties, MR 297 showed enhanced drought tolerance with reduced leaf rolling and stable chlorophyll content, whereas MR 219 exhibited higher oxidative damage, indicated by elevated malondialdehyde and proline levels. Antioxidant genes were strongly upregulated under drought stress, particularly in MR 219. Expression of selected sORFs ( OsisORF_0050 , OsisORF_3394 , and OsisORF_3007 ) was confirmed under drought conditions. This study reveals sORFs as previously overlooked contributors to drought stress responses in indica rice and provides a foundation for their functional characterization and potential application in crop improvement.