Genome-wide identification, characterization, and evolutionary analysis of the HSP70 gene family in rice (Oryza sativa L.)

Heat shock proteins 70 (HSP70s) are highly conserved molecular chaperones essential for maintaining protein homeostasis under environmental stress conditions. Despite rice being a staple crop for over half the world population and facing increasing climate-related stresses, comprehensive characterization of its OsHSP70 gene family has remained incomplete. We identified and characterized 32 OsHSP70 genes in the rice genome through integrated bioinformatics and experimental approaches. Phylogenetic analysis classified these genes into five distinct subfamilies (A-E), with subfamily D predominating (15 members, 46.9%). Chromosome mapping revealed non-random distribution across 9 chromosomes, with notable gene clusters on chromosomes 3 (7 genes) and 11 (10 genes), indicating tandem duplication as a major expansion mechanism. Evolutionary analysis through Ka/Ks ratios demonstrated that 83% of duplicated gene pairs evolved under purifying selection (Ka/Ks < 1), with divergence times spanning from 0.98 to 64.75 million years ago. Subcellular localization predictions identified diverse targeting patterns: 34.9% chloroplast, 32.6% cytoplasm, and 14.0% mitochondria, reflecting functional compartmentalization. Quantitative RT-PCR analysis under five abiotic stress conditions (heat, cold, drought, salt, and submergence) identified seven highly stress-responsive genes (Os01g62290, Os03g02260, Os03g11910, Os03g16860, Os03g16920, Os03g50250, Os05g35400, and Os05g38530) with maximum fold changes exceeding eightfold, particularly under heat stress. Os03g50250 (OsHSP70-13) emerged as the strongest responder across multiple stresses. Protein–protein interaction networks revealed integration with key metabolic and stress response pathways. These findings establish a comprehensive framework for understanding OsHSP70-mediated stress tolerance mechanisms in rice and identify priority candidates for developing climate-resilient varieties through targeted breeding or genome editing approaches.