Abstract

High temperature (HT) during soybean flowering can cause fertilization failure and significant yield reduction, often resulting in irreversible damage. Although SPL transcription factors are known to participate in plant growth and development, their role in regulating soybean male fertility under HT stress remains unclear. Here, we report that GmSPL4a was highly expressed in flowers and induced by HT stress during flowering in the soybean cytoplasmic male sterility (CMS)-based restorer line. Overexpression of GmSPL4a enhanced male fertility under HT stress in soybean CMS-based restorer line and its F1 hybrids with the CMS line, whereas knockout of GmSPL4a and its homolog GmSPL4b reduced male fertility in soybean. Further research showed that GmSPL4a interacts with GmbHLH and GmHSP90A1. Similarly, soybean male fertility under HT stress was improved by overexpression of GmbHLH and constrained by its knockout. Physiological measurements and RNA-seq analysis confirmed that GmSPL4a enhanced reactive oxygen species (ROS) clearance and pectin accumulation under HT stress, thereby improving HT tolerance. Correspondingly, overexpression of GmbHLH also reduced ROS accumulation in soybean anthers under HT stress. Additionally, GmNAC1 was identified as a downstream regulatory gene of GmSPL4a. Overexpression of GmNAC1 reduced male fertility in soybean CMS-based restorer line, while knockout of GmNAC1 significantly improved male fertility under HT stress. This study sheds light on the molecular network of the GmSPL4a-GmbHLH-GmNAC1 module controlling male fertility in soybean CMS-based restorer line under HT stress, offering a theoretical foundation, genetic resources, and breeding strategies for developing new heat-tolerant soybean germplasm and hybrid varieties.