The role mechanism of miRNA regulating anther dehiscence in soybean under high temperature stress

• Identified distinct miRNA expression profiles in thermotolerant vs. sensitive soybean anthers under high temperature (HT), with the sensitive line exhibiting more differentially expressed miRNAs (DEMs). • Functional validation revealed miR226-5p, miR5037c , and miR159e-5p overexpression causes premature anther non-dehiscence under HT in Arabidopsis . • Established miR159e-5p as a central regulator of soybean anther HT response by suppressing heat shock factors (HSFA1s) and proteins (HSPs). High-temperature (HT) stress impairs soybean yield by disrupting anther function. miRNAs are critical regulators of plant stress responses, but their roles in soybean anther thermotolerance remain largely unexplored. We performed small RNA sequencing of anthers from HT-tolerant (JD21) and HT-sensitive (HD14) soybean lines under HT and control conditions. Comparative analysis revealed that HT- stress JD21 anthers (TJA) exhibited 16 up-regulated and 19 down-regulated differentially expressed miRNAs (DEMs) compared to controls (CJA), while HT-stress HD14 anthers (THA) showed 17 up-regulated and 24 down-regulated DEMs relative to its control (CHA). The HD14 exhibited more DEMs, potentially explaining JD21′s superior HT resistance. Integrated analysis of miRNA and mRNA expression identified 23 DEMs with targeted regulatory relationships to 43 differentially expressed genes (DEGs) ( p<0.05 ). Bioinformatics analysis indicated that these target genes were primarily enriched in metabolic and cellular processes, response to stimuli, calvin cycle carbon fixation, and carbon metabolism. From this group, eight candidate miRNAs, including novel-m0226-5p ( miR226-5p ), miR5037c , and miR159e-5p , were selected for further investigation. Functional validation in Arabidopsis thaliana demonstrated that overexpression of miR226-5p, miR5037c , or miR159e-5p resulted in premature anthers non-dehiscence under HT stress, respectively. Furthermore, miR159e-5p overexpression lines specifically showed significantly reduced expression of heat shock transcription factors (HSFA1s) and heat shock proteins (HSPs), supporting a role for this miRNA in regulating thermotolerance. This study establishes as a central regulator of the soybean anther HT response via modulation of the HSFA1s-HSPs network, providing molecular insights into thermotolerance and potential targets for breeding HT-resistant soybean varieties.