High-temperature (HT) stress at flowering stage causes significant damage to soybean, including pollen abortion and fertilization failure, but few genes involved in male fertility regulation under HT stress in soybean have been characterized. Here, we demonstrated that miR156b–GmSPL2b module involved in male fertility regulation of soybean cytoplasmic male sterility (CMS)-based restorer line under HT stress. Overexpression of miR156b decreased male fertility in soybean CMS-based restorer line and its hybrid F₁ with CMS line under HT stress. RNA-seq analysis found that miR156b mediated male fertility regulation in soybean under HT stress by regulating the expression of pollen development and HT response related genes. Metabolomic analysis of miR156bOE revealed reduction in flavonoid content under HT stress. Integrated transcriptomic and metabolomic analysis showed that the overexpression of miR156b caused flavonoid metabolism disorder in soybean flower bud under HT stress. Knockout of GmSPL2b also decreased the thermotolerance of soybean CMS-based restorer line during flowering. Moreover, GmSPL2b turned out to be directly bounded to the promoter of GmHSFA6b. Further verification indicated that GmHSFA6b overexpression enhanced HT tolerance in Arabidopsis during flowering. Substance content and gene expression analysis revealed that miR156b–GmSPL2b may mediate reactive oxygen species clearance by regulating flavonoid metabolism, thus participating in the regulation of male fertility in soybean under HT stress. This study not only provided important progress for understanding the molecular mechanism of miR156b–GmSPL2b regulating the male fertility of soybean CMS-based restorer line under HT stress, but also provided genetic resources and theoretical basis for creating HT-tolerant strong restorer lines.

中文翻译：

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miR156b-GmSPL2b模块介导高温胁迫下大豆细胞质雄性不育恢复系的雄性育性调节

开花期的高温（HT）胁迫会对大豆造成严重损害，包括花粉败育和受精失败，但在大豆高温胁迫下参与雄性育性调节的基因很少被表征。在这里，我们证明了 miR156b–GmSPL2b 模块参与 HT 胁迫下基于大豆细胞质雄性不育 (CMS) 的恢复系的雄性育性调节。在HT胁迫下，miR156b的过表达降低了基于CMS的大豆恢复系及其与CMS系的杂种F _{1的雄性育性。}RNA-seq分析发现，miR156b通​​过调节花粉发育和HT反应相关基因的表达来介导HT胁迫下大豆的雄性育性调节。miR156bOE的代谢组学分析揭示了 HT 胁迫下类黄酮含量的减少。综合转录组学和代谢组学分析表明，HT胁迫下miR156b的过表达导致大豆花芽中黄酮代谢紊乱。GmSPL2b的敲除还降低了基于大豆 CMS 的恢复系在开花期间的耐热性。此外，GmSPL2b 被证明直接结合至GmHSFA6b的启动子。进一步验证表明，GmHSFA6b过表达增强了拟南芥开花期间的HT耐受性。物质含量和基因表达分析表明，miR156b–GmSPL2b可能通过调节类黄酮代谢介导活性氧清除，从而参与HT胁迫下大豆雄性育性的调节。该研究不仅为理解HT胁迫下miR156b-GmSPL2b调节大豆CMS恢复系雄性育性的分子机制提供了重要进展，而且为创建耐HT强恢复系提供了遗传资源和理论基础。