Nitrate restricts the expression of non-symbiotic leghemoglobin through inhibition of nodule inception protein in nodules of peanut ( Arachis hypogaea )

An exquisite symbiotic relationship between legumes and rhizobia leads to the development of nitrogen-fixing special organelles known as nodules in nitrate-deficient environments, whereas a high level of nitrate in soil negatively regulates the pleiotropic phases of root nodule symbiosis (RNS), including rhizobial infection, nodule organogenesis and leghemoglobin synthesis. Here, we identified a special group of nodule-specific non-symbiotic leghemoglobin genes ( AhLghs ) in the crack entry legume peanut; however, their functional role and transcriptional regulation remain enigmatic. A comparative transcriptomic analysis revealed that the downregulation of nodule inception ( AhNIN ) and non-symbiotic leghemoglobin ( AhLghs ) genes played a pivotal role in the nitrate-mediated inhibition of nodulation in peanut. The knockdown of AhLghs and overexpression of AhLgh1 resulted in lower and higher leghemoglobin content, respectively, corroborating their roles as positive regulators of nitrogen fixation in peanut. On the other hand, knockdown of AhNINs not only inhibited root nodulation but also decreased leghemoglobin content in peanut. Further, the DNA-affinity purification sequencing (DAP-Seq) analysis identified various nodulation genes, including AhLghs , as targets of AhNINs. After validating DNA-protein interaction by EMSA, the transactivation assay revealed that AhNINs can positively regulate AhLgh1 after binding to the NIN RESPONSIVE CIS ELEMENT (NRCE) of its promoter. Our work bridges a critical gap in understanding how nitrate influences non-symbiotic leghemoglobin expression by targeting rhizobia-induced NINs in peanut, and offers a potential model suggesting that the nitrate-NIN-Lgh module might represent a key evolutionary event in fine-tuning root nodulation.