Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules.

Faouzi Horchani,Marianne Prévot,Renaud Brouquisse,Eliane Meilhoc,Alexandre Boscari,Edouard Evangelisti,Samira Aschi-Smiti,Philippe Raymond,Claude Bruand,Alain Puppo,Eric Boncompagni

doi:10.1104/pp.110.166140

Abstract

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

Highlights

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms
In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions
Production in M. truncatula-S. meliloti nodules under normoxic and hypoxic conditions. We report that both plant and bacterial nitrate reductase (NR) and electron transfer chain (ETC) are involved in NO synthesis, and we propose the existence of a NO32-NO respiration process in nodules

Summary

Introduction

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. Hypoxic stress has pronounced effects on mitochondrial function, both from the perspective of oxygen limitation and from increased production of compounds that compete at the oxygen-binding site Among these compounds, NO has been demonstrated to be produced in hypoxic roots through a mechanism called “nitrate-NO respiration,” which involves the nonsymbiotic hemoglobin, the NR, and the electron transfer chain (ETC; Gupta et al, 2005; Igamberdiev and Hill, 2009). NO has been demonstrated to be produced in hypoxic roots through a mechanism called “nitrate-NO respiration,” which involves the nonsymbiotic hemoglobin, the NR, and the electron transfer chain (ETC; Gupta et al, 2005; Igamberdiev and Hill, 2009) Under these conditions, NO22 acts as instead of oxygen.

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