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Nitric oxide production in legume nodules examined by combining electron paramagnetic resonance and fluorescent probes

AuthorsRubio Luna, María Carmen ; Calvo-Beguería, Laura ; Martínez, Jesús I.; Pérez-Rontomé, Carmen ; Becana Ausejo, Manuel
Issue Date2019
CitationXVII National Meeting of the Spanish Society of Nitrogen Fixation (2019)
VI Portuguese-Spanish Congress on Nitrogen Fixation (2019)
AbstractNitric oxide (NO) is a gaseous free radical and signaling molecule with multiple functions in plants, including the onset of the legume-rhizobia symbiosis and the development and senescence of root nodules (Hichri et al., 2016). Some of these functions may rest on NO acting as intermediate in the signaling cascades of most phytohormones (Hill, 2012). 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 nitrosyl-leghemoglobin (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), nitrite reductase (NirK), or nitric oxide reductase (Nor) (Meakin et al., 2007). 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 bacterial 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 nitrate- and arginineindependent pathways for NO production. Collectively, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants (Calvo-Begueria et al., 2018).
DescriptionResumen del póster presentado al XVII National Meeting of the Spanish Society of Nitrogen Fixation y al VI Portuguese-Spanish Congress on Nitrogen Fixation, celebrados en Madrid (España) del 10 al 12 julio de 2019.
Appears in Collections:(EEAD) Comunicaciones congresos
(ICMA) Comunicaciones congresos
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