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

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 K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). 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.

Light Regulation of Nitrate uptake, Assimilation and Metabolism

“…Nitrite is mutagenic and accumulation of NO 2 -may be detrimental to the plant in the long run. Furthermore, NR reduces not only NO 3 -to NO 2 - (Ruoff and Lillo 1990), but under certain conditions reduces also O 2 to form O 2 -(superoxide anion) and NO 2 -to form NO (nitric oxide) (Yamasaki 2000, Rockel et al 2002. Nitric oxide may have both detrimental and beneficial effects in plants, and the subject is far from fully understood.…”

Nitrogen regulates CRY1 phosphorylation and circadian clock input pathways

“…NR produces NO from nitrite by NAD(P)H-dependent manner. [21][22][23] Therefore, our previous study cannot rule out the side-effect of NO generated by the N assimilation. NO is involved in photoperiod and autonomous flowering pathway (NO represses the amplitudes of output pathway components CO and GI).…”

Nitric oxide production and its functional link with OIPK in tobacco defense response elicited by chitooligosaccharide

“…Our later work showed that NO mediated by NR did not participate in defense response induced by COS, indicating that NO synthesized by NR may not act as a second messenger but have other functions. It had reported that NR might be responsible for the basal level of NO production in the leaves and roots of several plant species (Rockel et al 2002;Vanin et al 2004). Our previous work found a novel Ser/Thr protein kinase gene OIPK.…”