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

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.

Plant phenotyping relevance

マメ科根粒のNO状態を測定するEPRと蛍光プローブを比較・検証し、アーティファクトや検出の信頼性まで評価しており、植物状態の取得方法が研究の中心である。

Code and data availability

The paper reports EPR spectroscopy and DAF-2 DA fluorescence measurements of nitric oxide in legume nodules. No public phenotype/trait dataset, image collection, analysis code, or trained model is mentioned. Supplementary EPR spectra figures are referenced only as 'available at JXB online' with no explicit URL or depos

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