Summary: Nitric oxide (NO) is perfectly suited for the role of a redox signalling molecule. A key route for NO bioactivity occurs via protein S‐nitrosation, and involves the addition of a NO moiety to a protein cysteine (Cys) thiol (–SH) to form an S‐nitrosothiol (SNO). This process is thought to underpin a myriad of cellular processes in plants that are linked to development, environmental responses and immune function. Here we collate emerging evidence showing that NO bioactivity regulates a growing number of diverse post‐translational modifications including SUMOylation, phosphorylation, persulfidation and acetylation. We provide examples of how NO orchestrates these processes to mediate plant adaptation to a variety of cellular cues.

Nitrate reductase (NR) is the first enzyme of the nitrogen reduction pathway in plants, leading to the production of ammonia. However, in the nitrogen-fixing symbiosis between legumes and rhizobia, atmospheric nitrogen (N₂) is directly reduced to ammonia by the bacterial nitrogenase, which questions the role of NR in symbiosis. Next to that, NR is the best-characterized source of nitric oxide (NO) in plants, and NO is known to be produced during the symbiosis. In the present study, we first surveyed the three NR genes (MtNR1, MtNR2, and MtNR3) present in the Medicago truncatula genome and addressed their expression, activity, and potential involvement in NO production during the symbiosis between M. truncatula and Sinorhizobium meliloti. Our results show that MtNR1 and MtNR2 gene expression and activity are correlated with NO production throughout the symbiotic process and that MtNR1 is particularly involved in NO production in mature nodules. Moreover, NRs are involved together with the mitochondrial electron transfer chain in NO production throughout the symbiotic process and energy regeneration in N₂-fixing nodules. Using an in vivo NMR spectrometric approach, we show that, in mature nodules, NRs participate also in the regulation of energy state, cytosolic pH, carbon and nitrogen metabolism under both normoxia and hypoxia. These data point to the importance of NR activity for the N₂-fixing symbiosis and provide a first explanation of its role in this process.

Summary: In legumes, phytoglobins (Phytogbs) are known to regulate nitric oxide (NO) during early phase of the nitrogen‐fixing symbiosis and to buffer oxygen in functioning nodules. However, their expression profile and respective role in NO control at each stage of the symbiosis remain little‐known. We first surveyed the Phytogb genes occurring in Medicago truncatula genome. We analyzed their expression pattern and NO production from inoculation with Sinorhizobium meliloti up to 8 wk post‐inoculation. Finally, using overexpression and silencing strategy, we addressed the role of the Phytogb1.1‐NO couple in the symbiosis. Three peaks of Phytogb expression and NO production were detected during the symbiotic process. NO upregulates Phytogbs1 expression and downregulates Lbs and Phytogbs3 ones. Phytogb1.1 silencing and overexpression experiments reveal that Phytogb1.1‐NO couple controls the progression of the symbiosis: high NO concentration promotes defense responses and nodular organogenesis, whereas low NO promotes the infection process and nodular development. Both NO excess and deficiency provoke a 30% inhibition of nodule establishment. In mature nodules, Phytogb1.1 regulates NO to limit its toxic effects while allowing the functioning of Phytogb‐NO respiration to maintain the energetic state. This work highlights the regulatory role played by Phytogb1.1–NO couple in the successive stages of symbiosis.

The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship between plant and bacteria. This is characterized by the formation of a new organ, the nodule, which facilitates the fixation of atmospheric nitrogen (N2) by nitrogenase through the creation of a hypoxic environment. Nitric oxide (NO) accumulates at each stage of the symbiotic process. NO is involved in defense responses, nodule organogenesis and development, nitrogen fixation metabolism, and senescence induction. During symbiosis, either successively or simultaneously, NO regulates gene expression, modulates enzyme activities, and acts as a metabolic intermediate in energy regeneration processes via phytoglobin-NO respiration and the bacterial denitrification pathway. Due to the transition from normoxia to hypoxia during nodule formation, and the progressive presence of the bacterial partner in the growing nodules, NO production and degradation pathways change during the symbiotic process. This review analyzes the different source and degradation pathways of NO, and highlights the role of nitrate reductases and hemoproteins of both the plant and bacterial partners in the control of NO accumulation.