000074978 001__ 74978 000074978 005__ 20200117221656.0 000074978 0247_ $$2doi$$a10.1093/jxb/ery159 000074978 0248_ $$2sideral$$a107340 000074978 037__ $$aART-2018-107340 000074978 041__ $$aeng 000074978 100__ $$0(orcid)0000-0001-5327-4046$$aCalvo-Begueria, L. 000074978 245__ $$aRedefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes 000074978 260__ $$c2018 000074978 5060_ $$aAccess copy available to the general public$$fUnrestricted 000074978 5203_ $$aNitric oxide (NO) is a signaling molecule with multiple functions in plants. Given its critical importance and reactivity as a gaseous free radical, we have examined NO production in legume nodules using electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4, 5-diaminofluorescein diacetate. Also, in this context, we critically assess previous and current views of NO production and detection in nodules. EPR of intact nodules revealed that nitrosyl-leghemoglobin (Lb 2+ NO) was absent from bean or soybean nodules regardless of nitrate supply, but accumulated in soybean nodules treated with nitrate that were defective in nitrite or nitric oxide reductases or that were exposed to ambient temperature. Consequently, bacteroids are a major source of NO, denitrification enzymes are required for NO homeostasis, and Lb 2+ NO is not responsible for the inhibition of nitrogen fixation by nitrate. Further, we noted that Lb 2+ NO is artifactually generated in nodule extracts or in intact nodules not analyzed immediately after detachment. The fluorescent probe detected NO formation in bean and soybean nodule infected cells and in soybean nodule parenchyma. The NO signal was slightly decreased by inhibitors of nitrate reductase but not by those of nitric oxide synthase, which could indicate a minor contribution of plant nitrate reductase and supports the existence of nitrate- and arginine-independent pathways for NO production. Together, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants. 000074978 536__ $$9info:eu-repo/grantAgreement/ES/MINECO/AGL2014-53717-R$$9info:eu-repo/grantAgreement/ES/MINECO/AGL2013-45087-R$$9info:eu-repo/grantAgreement/ES/MINECO/CTQ2015-64486-R 000074978 540__ $$9info:eu-repo/semantics/openAccess$$aby$$uhttp://creativecommons.org/licenses/by/3.0/es/ 000074978 590__ $$a5.36$$b2018 000074978 591__ $$aPLANT SCIENCES$$b14 / 228 = 0.061$$c2018$$dQ1$$eT1 000074978 592__ $$a2.56$$b2018 000074978 593__ $$aPlant Science$$c2018$$dQ1 000074978 593__ $$aPhysiology$$c2018$$dQ1 000074978 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion 000074978 700__ $$aRubio, M.C. 000074978 700__ $$0(orcid)0000-0002-5406-3280$$aMartínez, J.I. 000074978 700__ $$aPérez-Rontomé, C. 000074978 700__ $$aDelgado, M.J. 000074978 700__ $$aBedmar, E.J. 000074978 700__ $$aBecana, M. 000074978 773__ $$g69, 15 (2018), 3703-3714$$pJ. Exp. Bot.$$tJournal of Experimental Botany$$x0022-0957 000074978 8564_ $$s1044839$$uhttps://zaguan.unizar.es/record/74978/files/texto_completo.pdf$$yVersión publicada 000074978 8564_ $$s13084$$uhttps://zaguan.unizar.es/record/74978/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada 000074978 909CO $$ooai:zaguan.unizar.es:74978$$particulos$$pdriver 000074978 951__ $$a2020-01-17-22:11:18 000074978 980__ $$aARTICLE