Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro

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Abstract
NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post‐translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The Km for nitrite was relatively high (100 μM) compared to nitrite concentrations in illuminated leaves (10 μM). NO production was competitively inhibited by physiological nitrate concentrations (Ki=50 μM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate‐fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01–0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5′‐AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100‐fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non‐elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration.