Journal Article

Production and Scavenging of Nitric Oxide by Barley Root Mitochondria

Kapuganti J. Gupta and Werner M. Kaiser

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 51, issue 4, pages 576-584
Published in print April 2010 | ISSN: 0032-0781
Published online April 2010 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcq022
Production and Scavenging of Nitric Oxide by Barley Root Mitochondria

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We examined whether root mitochondria and mitochondrial membranes produce nitric oxide (NO) exclusively by reduction of nitrite or also via a nitric oxide synthase (NOS), and to what extent direct NO measurements could become undetectable due to NO oxidation. Chemiluminescence detection of NO in the gas phase was used to monitor NO emission from suspensions (i.e. direct chemiluminescence). For comparison, diaminofluorescein (DAF) and diaminorhodamine (DAR) were used as NO indicators. NO oxidation to nitrite and nitrate was quantified after reduction of nitrite + nitrate to NO by vandium (III) with subsequent chemiluminescence detection (i.e. indirect chemiluminescence). Nitrite and NADH consumption were also measured. Anaerobic nitrite-dependent NO emission was exclusively associated with the membrane fraction, without participation of matrix components. Rates of nitrite and NADH consumption matched, whereas the rate of NO emission was lower. In air, mitochondria apparently produced no nitrite-dependent NO, and no NOS activity was detected by direct or indirect chemiluminescence. In contrast, with DAF-2 or DAR-4M, an l-arginine-dependent fluorescence increase took place. However, the response of this apparent low NOS activity to inhibitors, substrates and cofactors was untypical when compared with commercial inducible NOS (iNOS), and the existence of NOS in root mitochondria is therefore doubtful. In a solution of commercial iNOS, about two-thirds of the NO (measured as NADPH consumption) were oxidized to nitrite + nitrate. Addition of mitochondria to iNOS decreased the apparent NO emission, but without a concomitant increase in nitrite + nitrate formation. Thus, mitochondria appeared to accelerate oxidation of NO to volatile intermediates.

Keywords: Chemiluminescence; Fluorescence; Mitochondria; Nitric oxide; Nitric oxide synthase; Nitrite

Journal Article.  5067 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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