Abstract
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.
Highlights
In plants, yeasts, algae, and fungi, nitrate reductase (NR) is a key enzyme of the nitrogen (N) reduction and assimilation pathway
In the presence of Tg, the ATP/ADP ratio is significantly decreased only in 4 wpi nodules, indicating that the inhibition of Nitrate reductase (NR) partially affects the energy state in mature nodules, but not in 10 hpi and 4 dpi roots. This decrease in the ATP/ADP ratio is not observed when 4 wpi nodules are incubated in the presence of both Tg and NO2−, which means that the supply of NO2− makes it possible to maintain the nodule energy state. Considered together, these results indicate that 1) both the NR and the mitochondrial electron transport chain (ETC) are involved in the production of nitric oxide (NO), probably via the reduction of nitrate to nitrite by NR and the subsequent reduction of nitrite to NO at the mitochondrial ETC level, and 2) NR activity is linked to energy regeneration processes in mature nodules, but not during the first steps of the symbiosis
Medicago truncatula NRs Are Involved in NO Production
Summary
Yeasts, algae, and fungi, nitrate reductase (NR) is a key enzyme of the nitrogen (N) reduction and assimilation pathway. It catalyzes the reduction of nitrate (NO3−) to nitrite (NO2−), which is itself reduced to ammonia (NH4+) by nitrite reductase (NiR), before being assimilated into the amino acids and the nitrogen compounds of the cell (Campbell, 1999). Sixty-five million years ago, the legume family developed a beneficial mutual relationship with soil bacteria, the Rhizobia, which directly reduce atmospheric nitrogen (N2) to ammonia (NH4+) through the activity of the nitrogenase under nitrogen deficiency situations (Wang et al, 2018). Many studies have reported high NR expression and activity in symbiotic nodules (see Streeter, 1985a; Streeter, 1985b; Arrese-Igor et al, 1990; Silveira et al, 2001; Kato et al, 2003; Sanchez et al, 2010; Horchani et al, 2011, and references therein) and the question arose—and still arises—of what the NR can be used in the N2-fixing symbiosis, where nitrogen reduction is ensured by nitrogenase
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