Localization of nitric oxide production in soybean and common bean nodules as assessed by electron paramagnetic resonance and fluorescent probes

Abstract

Nitric oxide (NO) is a gaseous free radical and signaling molecule with multiple functions in plants, including the onset of the Iegume-rhizobia symbiosis and the development and senescence of root nodules. Sorne of these functions may rest on NO acting as intermediate in the signaling cascades of most phytohormones. To gain insight into the functions of NO in nodules, it is therefore crucial to localize the sites of NO production and accumulation at the tissue, cell, and organelle levels. For this purpose, we have employed two different techniques: electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5- diaminofluorescein diacetate (DAF-2 DA). The EPR detects the highly stable nitrosylleghemoglobin (Lb2+NO) complex, while the DAF-2 DA probe detects free NO. Also, we have used Bradyrhizobium diazoefficiens mutant strains lacking the enzymes of the denitrification pathway: nitrate reductase (Nap ), ni tri te reductase (NirK), or nitric oxide reductase (Nor). EPR of intact nodules revealed that Lb2+No was absent from bean or soybean nodules regardless of nitrate supply. In contrast, NO accumulated in soybean nodules treated with nitrate that were defective in bacteria! NirK or Nor, as well as in nodules that had been exposed to ambient temperature for one hour after their harvest from the roots. These observations indicate that: (i) the bacteroids are a major source of NO in nodules treated with nitrate; (ii) the denitrification enzymes NirK and Nor are required for NO homeostasis; and (iii) 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 that were 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 of nitric oxide synthase, which could indicate a minor contribution of plant nitrate reductase and supports the existence of nitrateand arginine-independent pathways for NO production. Collectively, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants.