RESUMO
The final phase in root nodule development is nodule senescence. The mechanism underlying the initiation of nodule senescence requires further elucidation. Here, we investigated the intrinsic signals governing soybean (Glycine max L. Merr.) nodule senescence, uncovering ethylene as a key signal in this intricate mechanism. Two AP2/ERF transcription factor 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. Overexpression of either GmENS1 or GmENS2 accelerated senescence in soybean nodules, whereas the knockout or knockdown of both genes delayed senescence and enhanced nitrogenase activity. Furthermore, our findings indicated that GmENS1 and GmENS2 directly bind to the promoters of GmNAC039, GmNAC018, and GmNAC030, encoding three NAC transcription factors essential for activating soybean nodule senescence. Notably, the nodule senescence process mediated by GmENS1 or GmENS2 overexpression was suppressed in the soybean nac039/018/030 triple mutant compared with the wild-type control. These data indicate GmENS1 and GmENS2 as pivotal transcription factors mediating ethylene-induced nodule senescence through the direct activation of GmNAC039/GmNAC018/GmNAC030 expression in soybean.
RESUMO
Root nodules are major sources of nitrogen for soybean (Glycine max (L.) Merr.) growth, development, production, and seed quality. Symbiotic nitrogen fixation is time-limited, as the root nodule senesces during the reproductive stage of plant development, specifically during seed development. Nodule senescence is characterized by the induction of senescence-related genes, such as papain-like cysteine proteases (CYPs), which ultimately leads to the degradation of both bacteroids and plant cells. However, how nodule senescence-related genes are activated in soybean is unknown. Here, we identified 2 paralogous NAC transcription factors, GmNAC039 and GmNAC018, as master regulators of nodule senescence. Overexpression of either gene induced soybean nodule senescence with increased cell death as detected using a TUNEL assay, whereas their knockout delayed senescence and increased nitrogenase activity. Transcriptome analysis and nCUT&Tag-qPCR assays revealed that GmNAC039 directly binds to the core motif CAC(A)A and activates the expression of 4 GmCYP genes (GmCYP35, GmCYP37, GmCYP39, and GmCYP45). Similar to GmNAC039 and GmNAC018, overexpression or knockout of GmCYP genes in nodules resulted in precocious or delayed senescence, respectively. These data provide essential insights into the regulatory mechanisms of nodule senescence, in which GmNAC039 and GmNAC018 directly activate the expression of GmCYP genes to promote nodule senescence.