Effects of GS3 Editing in japonica Rice ‘Nipponbare’ on Grain Morphology, Yield Components, and Response to Heat Stress at the Reproductive Stage

Rice (<i>Oryza sativa</i>), particularly the <i>japonica</i> subspecies, is a vital global food source but often suffers from short grain length and heat sensitivity, highlighting the need for genetic improvement. This study employed CRISPR/Cas9 technology to investigate the effects of <i>Grain Size3</i> (<i>GS3</i>) gene editing in the <i>japonica</i> cultivar, 'Nipponbare'. Successful <i>GS3</i> editing increased grain size across stable T₃ and T₄ generations. Importantly, different <i>GS3</i>-edited lines, even when all targeted within exon 1, resulted in varied effects on grain length and other yield components. Transcriptomic analyses revealed unique gene expression profiles for each edited line, highlighting the fact that subtle <i>GS3</i> mutations trigger diverse transcriptional cascades. While common differentially expressed genes (DEGs) were enriched in ethylene signaling and chitinase activity, line-specific KEGG analyses showed distinct pathway enrichments. Crucially, the CR-L5 line exhibited significantly enhanced heat tolerance at heading stage. Under high-temperature stress, CR-L5 maintained a higher relative seed setting rate and a 15% greater grain yield than the wild type. This enhanced thermotolerance in CR-L5 correlated with differing expressions of several wax biosynthesis and chitinase-related genes. Our study provides evidence that specific <i>gs3</i> mutations can confer enhanced reproductive-stage thermotolerance, offering a strategy for breeding climate-resilient <i>japonica</i> rice with improved grain quality and yield under stress.