Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

Abstract

A barley (Hordeum vulgare L.) mutant, narla (formerly AzM2), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from narla was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in narla differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nrla were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. (3, 4,7,12,23,27,28). The NR from the wild-type barley is a NADH-specific enzyme (6).The objective of this study was to characterize the NR from the NR-deficient mutant, narla, to determine whether the enzyme is the same or different from the wild-type NR. MATERIALS AND METHODSPlant Material. Barley (Hordeum vulgare L.) seedlings were grown for 6 to 7 d in a growth chamber at 16°C under continuous light (300 ,uE m 2s-'). The NR-deficient mutants, narla (formerly Az12) and nar2a (formerly Az34), were induced and selected from the cv. Steptoe, as described by Warner et aL (32). Both mutants are homozygous and genetically stable (14).NR Extraction and Assay. Seedlings were extracted with a buffer (3 ml/g fresh weight leaf tissue) containing 25 mm Tris (pH 8.4), 1.5 mm EDTA, 4.0 mm DDT, and 5 ,LM FAD. The crude homogenates were centrifuged at 27,000g for 15 min, and the supernatant was saved (crude extract). NAD(P)H NR assays were conducted as described previously (30), except that 0.2 ml of a 1:1 mixture of 0.3 mm phenazine methosulfate and 1.0 M zinc acetate was used to stop the reaction (25). To insure complete oxidation of the NAD(P)H, this mixture was vigorously mixed particularly when the NAD(P)H concentrations in the assay were greater than 0.2 mm.Assays for reduced methylviologen NR activity were performed in a medium containing 25 mm K-phosphate (pH 7.5), 10 mM potassium nitrate, 0.2 mm methylviologen, and 3.2 mm sodium dithionite. Assays were conducted by adding the enzyme to the assay medium (2 ml final volume), incubating at 30°C for 1 min, initiating the reaction with 0.1 ml of the dithionite solution (14 mg/ml of 25 mm phosphate [pH 8.2]), and incubating at 30°C for 30 min. The assay was stopped by vigorously agitating the assay mixture until the methylviologen was oxidized (about 10 s). To reduce interference from substances derived from sodium dithionite, 0.2 ml of 1.5% (v/v)

Nitrate Transport Is Independent of NADH and NAD(P)H Nitrate Reductases in Barley Seedlings

“…NR activities were assayed as previously described (8,9). Lactate dehydrogenase and pyruvate were included in the NADPH NR assay medium to competitively eliminate NADH produced by phosphatase conversion of NADPH to NADH (9,21).…”

“…Barley has two NR isozymes, one specific for NADH as the electron donor, and the other bispecific for NADH and NADPH as electron donors (8,9,16). To minimize possible pleiotopic effects of mutations in MoCo genes, we used mutants with lesions in the NR structural genes of both isozymes to develop four near isogenic lines differing in NR isozyme complement (39).…”

Contribution of shoots and roots to in vivo nitrate reduction in NADH-specific nitrate reductase-deficient mutant seedlings of barley (Hordeum vulgareL.)

“…The homogenate was centrifuged at 15,000 rpm for 20 min and the resulting supernatant was used for the NR assay. The NADH-dependent NR activity was assayed in a reaction mixture (450 f.LL) containing 50 mM HEPES-KOH (pH 7.7), 5 f.LM leupeptin, 2 mM KN0 3 , 10 11 M FAD, 1 mM EDTA, and 0.2 mM I3-NADH (Dailey et al 1982). In the case of the NADPH-dependent NR assay, a reaction mixture containing 0.1 mM I3-NADPH, 0.1 mM I3-NADP+, and 0.2 mM glucose-6-phosphate instead of I3-NADH was used, in order to allow rapid regeneration of NADPH (Savidov et al 1998).…”

“…In the shoots of Az12, the total NR activity was less than 10% of that in Steptoe, while similar levels of NR activity were observed in the roots of both genotypes. Since the NADH-NR enzyme was defective in Az 12 (Dailey et al 1982;Warner et al 1987), the presence of both NADH-and NADPHdependent NR activities in Az12 was ascribed to the NAD(P)H-NR isozyme (Sueyoshi et al 1995). …”

Controle da atividade da nitrato redutase em plantas de abacaxizeiro submetidas a baixas temperaturas em diferentes fases do ciclo diurno

“…Apesar dos valores para atividade da NR com NADPH serem muito próximos aos encontrados com o succinato e de existir conversão do NADH para o NADPH durante a reação, que pode ter sido reduzida em parte pela presença de fosfato na solução (Dailey et al, 1982), acredita-se que a NR em folhas submetidas ao frio utilizaram o NADPH como doador de elétrons, pois apenas ele foi capaz de estimular a ativação da enzima (NR ativa) comparado ao controle. É possível que a forma NAD(P)H biespecífica seja mais suscetível à ativação pelo frio que a forma NADH específica em folhas, provavelmente porque a limitação da taxa de fotossíntese pelo frio restringe o consumo do NADPH produzido, possivelmente gerando seu acúmulo (Huner et al, 1993).…”