Soybean ethylene response factors GmENS1 and GmENS2 promote nodule senescence

The final phase in root nodule development is nodule senescence. The mechanism underlying the initiation of nodule senescence requires further elucidation. In this study, we investigate the intrinsic signals governing soybean (Glycine max L. Merr.) nodule senescence, uncovering ethylene as a key signal in this intricate mechanism. Two AP2/ethylene response factor (ERF) transcription factor (TF) genes, GmENS1 and GmENS2 (Ethylene-responsive transcription factors required for Nodule Senescence), exhibit heightened expression levels in both aged nodules and nodules treated with ethylene. An overexpression of either GmENS1 or GmENS2 accelerates senescence in soybean nodules, whereas the knockout or knockdown of both genes delays senescence and enhances nitrogenase activity. Furthermore, our findings indicate that GmENS1 and GmENS2 directly bind to the promoters of GmNAC039, GmNAC018, and GmNAC030, encoding 3 NAC (NAM, ATAF1/2, and CUC2) TFs essential for activating soybean nodule senescence. Notably, the nodule senescence process mediated by GmENS1 or GmENS2 overexpression is suppressed in the soybean nac039/018/030 triple mutant compared with the wild-type control. These data indicate GmENS1 and GmENS2 as pivotal TFs mediating ethylene-induced nodule senescence through the direct activation of GmNAC039/GmNAC018/GmNAC030 expression in soybean.