Redefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes.

Laura Calvo-Begueria,Manuel Becana,Maria J Delgado,Eulogio J Bedmar,Maria C Rubio,Jesús I Martínez,Carmen Pérez-Rontomé

doi:10.1093/jxb/ery159

Abstract

Nitric oxide (NO) is a signaling molecule with multiple functions in plants. Given its critical importance and reactivity as a gaseous free radical, we have examined NO production in legume nodules using electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate. Also, in this context, we critically assess previous and current views of NO production and detection in nodules. EPR of intact nodules revealed that nitrosyl-leghemoglobin (Lb2+NO) was absent from bean or soybean nodules regardless of nitrate supply, but accumulated in soybean nodules treated with nitrate that were defective in nitrite or nitric oxide reductases or that were exposed to ambient temperature. Consequently, bacteroids are a major source of NO, denitrification enzymes are required for NO homeostasis, and Lb2+NO is not responsible for the inhibition of nitrogen fixation by nitrate. Further, we noted that Lb2+NO is artifactually generated in nodule extracts or in intact nodules not analyzed immediately after detachment. The fluorescent probe detected NO formation in bean and soybean nodule infected cells and in soybean nodule parenchyma. The NO signal was slightly decreased by inhibitors of nitrate reductase but not by those of nitric oxide synthase, which could indicate a minor contribution of plant nitrate reductase and supports the existence of nitrate- and arginine-independent pathways for NO production. Together, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants.

Highlights

Given its critical importance and reactivity as a gaseous free radical, we have examined Nitric oxide (NO) production in legume nodules using electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate
EPR of intact nodules revealed that nitrosyl-leghemoglobin (Lb2+NO) was absent from bean or soybean nodules regardless of nitrate supply, but accumulated in soybean nodules treated with nitrate that were defective in nitrite or nitric oxide reductases or that were exposed to ambient temperature
3704 | Calvo-Begueria et al In L. japonicus roots inoculated with Mesorhizobium loti, the NO concentration increases after ~4 h and decreases due to the induction of a non-symbiotic hemoglobin (LjGlb1-1) which scavenges NO and avoids triggering the defense response of the plant (Nagata et al, 2008; Fukudome et al, 2016)

Summary

Introduction

Nitric oxide (NO) is a gaseous free radical and signal mol- (Hichri et al, 2016).NO was detected after infection of ecule involved in a vast array of physiological processes of roots by rhizobia in the model legumes Lotus japonicus (Nagata plants, including legume nodule formation and development et al, 2008) and Medicago truncatula (del Giudice et al, 2011).3704 | Calvo-Begueria et al.In L. japonicus roots inoculated with Mesorhizobium loti, the NO concentration increases after ~4 h and decreases due to the induction of a non-symbiotic hemoglobin (LjGlb1-1) which scavenges NO and avoids triggering the defense response of the plant (Nagata et al, 2008; Fukudome et al, 2016). Soybean nodules were produced with the WT strain as well as with the bradyrhizobial mutants napA, nirK, and norC.To avoid artifacts, it was critical to collect intact nodules directly from the plants into the EPR tubes, while immersed in liquid nitrogen.

Results

Conclusion