NIN-like protein 2 regulation of nitrite reductase mediates vacuole integrity in <i>Medicago truncatula</i> nodules under high nitrate

The NIN-like protein 2 (NLP2) transcription factor is highly expressed in the infected cells of the N2 fixation zone of mature nodules in Medicago truncatula. In these cells, NLP2 directly activates the expression of leghemoglobin genes and is required for the full expression of nitrite reductase (NiR). Histological examination of nodules formed under different nitrate regimes revealed that the infected cells of nlp2 display abnormal vacuole morphology under high nitrate (5 mm KNO3). This phenotype is associated with starch accumulation, higher expression of starch biosynthesis genes, and increased levels of nitrite and nitric oxide. A transcriptomic comparison of nlp2 and wild-type nodules across nitrate concentrations revealed that under high nitrate, nlp2 shows lower expression of genes important for hypoxia adaptation, such as alcohol dehydrogenase and pyruvate decarboxylase (PDC), and increased expression of S-nitrosoglutathione reductase (GSNOR), which encodes a key enzyme for nitric oxide homeostasis. These changes in gene expression were associated with lower nodule ATP levels and a higher NAD+/NADH ratio, suggesting that energy metabolism is specifically compromised in nlp2 nodules under high nitrate. Transgenic expression of NiR in the nodules of nlp2-1 mutants grown under high nitrate rescued the vacuole phenotype and restored the expression of GSNOR and PDC to normal levels. Overall, our data indicate a role for NLP2 in the regulation of NiR in N2-fixing cells, suggesting a role for nitrate reduction in nodule energy homeostasis.