Brassinosteroid and jasmonic acid pathways mediate the effect of high temperature on the outcrossing rate of photo-thermo-sensitive genic male-sterile rice lines

The development and widespread adoption of hybrid rice have significantly contributed to global food security. However, the relatively high cost of hybrid seeds remains a limiting factor for its broader application. Improving the outcrossing rate of photo-thermo-sensitive genic male sterile (PTGMS) rice lines is essential for increasing hybrid seed yield, which could substantially reduce seed costs. The novel PTGMS Shuang1S exhibits a high outcrossing rate and exceptional seed production yield (459.07 kg per 666.67 m2), particularly under high-temperature conditions. Among the 20 PTGMS lines evaluated, Shuang1S exhibited the highest outcrossing rate (58.20%), while most other lines remained below 30%. To elucidate the molecular basis of high outcrossing rates under high-temperature conditions and to identify novel candidate genes, we performed transcriptome sequencing of stigmas from two PTGMS lines—Shuang1S and Han2S—which differ substantially in outcrossing performance. Comparative analysis identified 578 up-regulated and 625 down-regulated differentially expressed genes (DEGs) in Shuang1S relative to Han2S. KEGG enrichment analysis indicated that these DEGs were predominantly enriched in brassinosteroid (BR) biosynthesis and plant hormone signal transduction pathways. Key DEGs included three cytochrome P450 family genes (CYP734A4, CYP724B1, and CYP90D2) involved in BR metabolism, and the JA-amino acid synthetase gene OsJAR1, which participates in jasmonic acid (JA) synthesis. Consistent with previous reports, elevated expression of CYP734A4, OsJAR1, and two flavonoid biosynthesis genes (CYP93G2 and OsF3H) is associated with significantly improved outcrossing seed-setting rates. Our findings suggest that BR and JA biosynthesis pathways play critical roles in regulating the outcrossing rate of PTGMS line under high-temperature conditions. This study provides deeper insights into the molecular and regulatory mechanisms controlling outcrossing rate and offers valuable candidate genes for breeding PTGMS line with high outcrossing rates and high seed production yields.