Thérèse Moureaux

Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.

Molybdenum Cofactor Mutants, Specifically Impaired in Xanthine Dehydrogenase Activity and Abscisic Acid Biosynthesis, Simultaneously Overexpress Nitrate Reductase'

The molybdenum cofactor is shared by nitrate reductase (NR), xanthine dehydrogenase (XDH), and abscisic acid (ABA) aldehyde oxidase in higher plants (M. Walker-Simmons, D.A. Kudrna, R.L. Warner [1989] Plant Physiol 90:728–733). In agreement with this, cnx mutants are simultaneously deficient for these three enzyme activities and have physiological characteristics of ABA-deficient plants. In this report we show that aba1 mutants, initially characterized as ABA-deficient mutants, are impaired in both ABA aldehyde oxidase and XDH activity but overexpress NR. These characteristics suggest that aba1 is in fact involved in the last step of molybdenum cofactor biosynthesis specific to XDH and ABA aldehyde oxidase; aba1 probably has the same function as hxB in Aspergillus. The significance of NR overexpression in aba1 mutants is discussed.

Cytokinin affects nitrate reductase expression through the modulation of polyadenylation of the nitrate reductase mRNA transcript

Abstract Cytokinin (CK) and low-intensity light effects in modulating nitrate reductase (NR) activity, NR protein and NR encoding mRNAs were studied in tobacco cell suspension cultures. NR activity was strikingly enhanced by CK in dark- as well as in light-grown cells whereas it was less affected by light alone. NR protein accumulation was stimulated by the hormone in the light only; then a CK light-dependent regulation of NR activity was suggested. Light enhanced the steady-state levels of hybridisable total NR mRNA and a light-inductive effect was also observed after transfer from dark to light; this effect was dependent on sucrose supply and was enhanced in CK-supplied cells. NR poly(A) mRNA were assayed in cell poly(A) RNA, purified by oligo(dT)-cellulose chromatography. In growing cells CK enhanced (i) the steady state levels of hybridisable NR poly(A) mRNA and (ii) the ratio of hybridisable NR poly(A) mRNA to total hybridisable NR mRNA. CK-induced accumulation of hybridisable NR poly(A) mRNA resulted in a correlative enhancement of the accumulation of NR protein, provided cells were grown in the light. Overall stimulatory effects of CK on the amounts of bulk poly(A) mRNA and on the mean-size of mRNA poly(A) tails were also observed. It is suggested that CK effect on gene expression involves a modulation of mRNA polyadenylation.

Diurnal and circadian fluctuation of malate levels and its close relationship to nitrate reductase activity in tobacco leaves

Abstract In the leaves of nitrate-fed tobacco plants, the malate level increased 3-fold during the day and decreased during the night. These 24-h oscillations continued to occur in plants placed in continuous light conditions. On the other hand, the malate content declined markedly and showed no rhytmic variations when plants were transferred to continuous darkness. The fluctuation pattern of malate level were similar to those of in vitro nitrate reductase (NR, EC 1.6.6.1) activity although they were not parallel. Moreover, when NR activity, and presumably nitrate-reduction, were strongly decreased either in plants grown with tungstate (an inhibitor of NR activity) or in plants starved of nitrogen, leaf malate accumulation during the day was completely suppressed. The supply of nitrate to N-starved plants induced NR activity and leaf malate accumulation unless tungstate was present in the nutrient solution. These suggest that the malate content in leaves is dependent upon the level of nitrate reduction.

Bromphenol blue: nitrate reductase activity in nicotiana plumbaginifolia: an immunochemical and genetic approach

NADH: nitrate reductase (EC 1.6.6.1) was purified from Nicotiana plumbaginifolia leaves. As recently observed with nitrate reductase from other sources, this enzyme is able to reduce nitrate using reduced bromphenol blue (rBPB) as the electron donor. In contrast to the physiological NADH-dependent activity, the rBPB-dependent activity is stable in vitro. The latter activity is non-competitively inhibited by NADH. The monoclonal antibody ZM.96(9)25, which inhibits the NADH: nitrate reductase total activity as well as the NADH: cytochrome c reductase and reduced methyl viologen (rMV): nitrate reductase partial activities, has no inhibitory effect on the rBPB: nitrate reductase activity. Conversely, the monoclonal antibody NP.17-7(6) inhibits nitrate reduction with all three electron donors: NADH, MV or BPB. Among various nitrate reductase-deficient mutants, an apoprotein gene mutant (nia. E56) shows reduced terminal activities but a highly increased rBPB:nitrate reductase activity. rBPB:nitrate reductase thus appears to be a new terminal activity of higher plant nitrate reductase and involves specific sites which are not shared by the other activities.

Antibody against octopine dehydrogenase from crown gall tumor tissue, a tool in studies of plant cell transformation

1. Introduction The enzymes octopine and nopaline dehydrogenase are responsible for the synthesis of the unusual amino acid derivatives octopine and nopaline, specific for crown-gall tumors [l-4]. The nature of the enzyme synthesized is determined by the Ti-plasmid har- boured in the

Albumins and globulins in developing maize grains

Quantitative and qualitative (amino acid composition) changes of albumins and globulins in developing grain of normal and opaque-2 (o2) maizes were examined in proteins extracted sequentially with water and 0.5 M NaCl, and isolated by salting out with trichloroacetic acid. The amount of albumins per grain reached a maximum at mid-development the declined. Globulins, virtually absent in very young grain, increased in level until maturity. The variations were more marked for o2 than normal maize. Amino acid compositions changed little with development. The selectivity and exhaustiveness of sequential extraction, and the physiological role of albumins and globulins in grain are discussed.

Biochemistry, Molecular Genetics and Regulation of Nitrate Reductase in Nicotiana Plumbaginifolia, Tobacco and Tomato

The nitrate assimilation pathway is known to be tightly controlled in plants by many endogenous and environmental factors (Beevers and Hageman 1983), the most important being the nature and availability of the nitrogen source, and light. Nitrate compartmentalization and flux between plant tissues and inside plant cells are clearly the first critical steps in that control, but are, as yet, refractory to molecular analysis. The second step, nitrate reduction into ammonium, is catalysed by two enzymes, a likely cytosolic nitrate reductase (NR), and a plastidial nitrite reductase, both induced by nitrate. Interestingly, each factor known to control the overall pathway appears to control NR, which is thus thought to play a central role in regulation of the nitrate assimilation pathway.