Leaf NADH Research Articles

Role of glutamate availability on cadmium induced changes of nitrogen and glutamate in tomato plants (Solanum lycopersicon)

Maximal glutamine synthetase (GS, EC 6.3.1.2) activity and free aminoacid accumulation occur together in the leaves and roots. GS and specific GS activities although declining in the stressed leaves. In the other hand, the decrease of GS activity in the roots is contrarily accompanied by an increase in specific GS activity in roots in treated plants. Maximal GS activity is correlated with the peak degradation of the free aminoacid and protein in the two organs. Western blots analysis from Cd treated plants shows a decrease in chloroplastic GS (GS2) and an increase in cytosolic GS (GS1) proteins. In the roots, free ami- noacids accumulated as NADH—dependent glutamate synthase (NADH-GOGAT, EC 2.6.1.5.3) and specific activities increase. Maximal leaf NADH—GOGAT activity occurs simultaneously with maximal leaf aminating glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) activity.

Conditional modulation of NAD levels and metabolite profiles in Nicotiana sylvestris by mitochondrial electron transport and carbon/nitrogen supply

Environmental controls on leaf NAD status remain poorly understood. Here, we analyzed the effects of two key environmental variables, CO(2) and nitrogen, on leaf metabolite profiles, NAD status and the abundance of key transcripts involved in de novo NAD synthesis in wild-type (WT) Nicotiana sylvestris and the CMSII mutant that lacks respiratory complex I. High CO(2) and increased N supply both significantly enhanced NAD(+) and NADH pools in WT leaves. In nitrogen-sufficient conditions, CMSII leaves were enriched in NAD(+) and NADH compared to the WT, but the differences in NADH were smaller at high CO(2) than in air because high CO(2) increased WT NADH/NAD(+). The CMSII-linked increases in NAD(+) and NADH status were abolished by growth with limited nitrogen, which also depleted the nicotine and nicotinic acid pools in the CMSII leaves. Few statistically significant genotype and N-dependent differences were detected in NAD synthesis transcripts, with effects only on aspartate oxidase and NAD synthetase mRNAs. Non-targeted metabolite profiling as well as quantitative amine analysis showed that NAD(+) and NADH contents correlated tightly with leaf amino acid contents across all samples. The results reveal considerable genotype- and condition-dependent plasticity in leaf NAD(+) and NADH contents that is not linked to modified expression of NAD synthesis genes at the transcript level and show that NAD(+) and NADH contents are tightly integrated with nitrogen metabolism. A regulatory two-way feedback circuit between nitrogen and NAD in the regulation of N assimilation is proposed that potentially links the nutritional status to NAD-dependent signaling pathways.

14-3-3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform-specific manner and reduce dephosphorylation of Ser-543 by endogenous protein phosphatases

Three lines of evidence indicate that the 14-3-3 proteins that inactivate the phosphorylated form of spinach leaf NADH:nitrate reductase (NR) bind to the enzyme at the regulatory phosphorylation site (Ser-543). First, a phosphorylated synthetic peptide based on the regulatory site can prevent and also reverse the inactivation of phospho-NR caused by 14-3-3 proteins. Second, sequence-specific and phosphorylation-dependent binding of the aforementioned synthetic peptide to the 14-3-3 proteins was demonstrated in vitro. Third, 14-3-3 proteins were required for the ATP-dependent phosphorylation of NR (as assessed by activity measurements) in the presence of NR-kinase and leaf protein phosphatases. Lastly, we demonstrate specificity of recombinant Arabidopsis 14-3-3 isoforms in the interaction with phospho-NR: ω > χ > ν ⋙ gf, ψ.

The inhibitor protein of phosphorylated nitrate reductase from spinach ( Spinacia oleracea) leaves is a 14-3-3 protein

The inhibitor protein (IP) that inactivates spinach leaf NADH:nitrate reductase (NR) has been identified for the first time as a member of the eukaryotic 14-3-3 protein family based on three lines of evidence. First, the sequence of an eight amino acid tryptic peptide, obtained from immunopurified IP, matched that of a highly conserved region of the 14-3-3 proteins. Second, an authentic member of the 14-3-3 family, recombinant Arabidopsis GF14ω, caused inactivation of phospho-NR in a magnesium-dependent manner identical to IP. Third, an anti-GF14 monoclonal antibody cross-reacted with IP and anti-IP monoclonal antibodies cross-reacted with GF14ω.

Partial Purification and Characterization of a Calcium-Dependent Protein Kinase and an Inhibitor Protein Required for Inactivation of Spinach Leaf Nitrate Reductase

Evidence is accumulating that the activity of spinach (Spinacia oleracea L.) leaf NADH:nitrate reductase (NR) is modulated both in vitro and in vivo by protein phosphorylation. From the present study we report the partial purification of the two protein factors needed for NR inactivation. We identified NR-protein kinase (NR-PK) as a calcium-dependent and metabolite-regulated protein kinase and have provided additional evidence that phosphorylation of NR is necessary but not sufficient to inactivate the enzyme. The inhibitor protein required for inactivation of phospho-NR was purified 625-fold by polyethylene glycol fractionation and sequential column chromatography. Using partially purified inhibitor protein and NR-PK, we characterized NR inactivation (increased sensitivity to Mg2+ inhibition) in a reconstituted in vitro system. NR-PK activity was inhibited by a variety of metabolic phosphate esters including di-hydroxyacetone phosphate, glucose-6-phosphate, and fructose-1,6-bisphosphate. Light-to-dark transition experiments with a starchless tobacco (Nicotiana sylvestris) mutant, which accumulates phosphate esters during the photoperiod, indicated that NR inactivation in vivo might, indeed, be down-regulated by metabolites. Additionally, we postulate that cytosolic free calcium could play an important role in the regulation of NR activity in vivo.

Identification of an “essential” cysteine of nitrate reductase via mutagenesis of its recombinant cytochrome b reductase domain.

Five cysteine residues in the recombinant cytochrome b reductase domain of corn leaf NADH:nitrate reductase (EC 1.6.6.1) were modified by site-directed mutagenesis. At least two amino acid replacement mutants were generated for each of the 5 cysteines of this domain. Characteristics of the amino acid replacement mutants correlated well with the structural location of the cysteine residues in the preliminary three-dimensional model of the cytochrome b reductase domain: somewhat exposed cysteines could be replaced by hydrophilic amino acid residues, while more buried cysteines by hydrophobic residues. An exception was found for the invariant cysteine near the C terminus, which is found in all nitrate reductases and also in the closely related NADH: cytochrome b5 reductase, as well as, most other members of this flavoenzyme family. No substitution for the invariant cysteine yielded highly active enzyme, although these mutants had normal visible spectra. When the invariant cysteine was mutated to serine, the cytochrome b reductase domain was resistant to inhibition by pchloromercuribenzoate, an inhibitor of nitrate reductases. Kinetic analysis suggested that the catalytic efficiency of the mutant was markedly reduced. We concluded, the invariant cysteine plays an important role in catalysis and may be essential for high catalytic efficiency of nitrate reductases.

Magnesium and Calcium Inhibition of Squash Leaf NADH Nitrate Reductase

Magnesium and Calcium Inhibition of Squash Leaf NADH Nitrate Reductase

Regulation of maize root nitrate reductase mRNA levels

The regulation of maize (Zea mays L. cv. W64A x W182E) root nitrate reductase (NR) transcript levels has been investigated. To facilitate this process a partial nitrate reductase cDNA clone has been isolated from a maize root cDNA library. Its derived amino acid sequence was found to be more similar to that of the barley nicotinamide adenine dinucleotide (phosphate)‐reduced from [NAD(P)H] bispecific isoform of NR than to that of the maize leaf NADH:NR. The amount of NR mRNA in maize roots was found to increase very rapidly upon the addition of nitrate, reaching a maximum approximately 30 min after induction in the root tip. The level of NR mRNA was analysed in leaf and root tissue. There was very little cross‐hybridizing mRNA in leaf tissue when the root cDNA clone was used as a probe. Conversely, the leaf NADH:NR cDNA clone hybridized to an mRNA that was present at considerably higher levels in leaf tissue than in root tissue, although there was a clearly detectable level of this transcript present in the roots. Finally, the level of root NR mRNA was analyzed by using the tissue print technique to study its localization at the cellular level. The greatest amount of NR transcript was found in the region just above the root tip. At the cellular level, NR mRNA was found in all root cell types.

Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA

A cDNA clone was isolated from a maize (Zea mays L. cv W64AxW183E) scutellum lambdagt11 library using maize leaf NADH:nitrate reductase Zmnr1 cDNA clone as a hybridization probe; it was designated Zmnr1S. Zmnr1S was shown to be an NADH:nitrate reductase clone by nucleotide sequencing and comparison of its deduced amino acid sequence to Zmnr1. Zmnr1S, which is 1.8 kilobases in length and contains the code for both the cytochrome b and flavin adenine dinucleotide domains of nitrate reductase, was cloned into the EcoRI site of the Escherichia coli expression vector pET5b and expressed. The cell lysate contained NADH:cytochrome c reductase activity, which is a characteristic partial activity of NADH:nitrate reductase dependent on the cytochrome b and flavin adenine dinucleotide domains. Recombinant cytochrome c reductase was purified by immunoaffinity chromatography on monoclonal antibody Zm2(69) Sepharose. The purified cytochrome c reductase, which had a major size of 43 kilodaltons, was inhibited by polyclonal antibodies for maize leaf NADH:nitrate reductase and bound these antibodies when blotted to nitrocellulose. Ultraviolet and visible spectra of oxidized and NADH-reduced recombinant cytochrome c reductase were nearly identical with those of maize leaf NADH:nitrate reductase. These two enzyme forms also had very similar kinetic properties with respect to NADH-dependent cytochrome c and ferricyanide reduction.

High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B 5 reductase

Higher plant nitrate reductase can be divided into three functional domains representing its prosthetic groups: 1) flavin; 2) cytochrome b; and 3) Mo-pterin. The flavin domain has been synthesized by heterologous expression in Escherichia coli using a fragment of a corn leaf NADH:nitrate reductase cDNA clone, Zmnr1, which we had previously isolated and sequenced. A Xho2-BamH1 fragment was cut from Zmnr1, containing the sequence for the flavin domain, and ligated in the BamH1 site of expression vector pET3c. When this construct was expressed in E. coli, a 30 kD polypeptide was found to be newly synthesized. The flavin domain was purified to homogeneity using blue Sepharose and shown to have a molecular weight of 30 kD. The recombinant flavin domain has a ferricyanide reductase specific activity of 1000 μmol NADH oxidized/min/mg protein and a visible spectrum virtually identical to that of human NADH:cytochrome b 5 reductase.

CDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P)+:Glyceraldehyde-3-Phosphate Dehydrogenase

cDNA clones were selected from a corn (Zea mays L.) leaf lambda gt11 expression library using polyclonal antibodies for corn leaf NADH:nitrate reductase. One clone, Zmnrl, had a 2.1 kilobase insert, which hybridized to a 3.2 kilobase mRNA. The deduced amino acid sequence of Zmnrl was nearly identical to peptide sequences of corn leaf NADH:nitrate reductase. Another clone, Zm6, had an insert of 1.4 kilobase, which hybridized to a 1.4 kilobase mRNA, and its sequence coded for chloroplastic NAD(P)(+):glyceraldehyde-3-phosphate dehydrogenase based on comparisons to sequences of this enzyme from tobacco and corn. When nitrate was supplied to N-starved, etiolated corn plants, nitrate reductase, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels in leaves increased in parallel. When green leaves were treated with nitrate, only nitrate reductase mRNA levels were increased. Nitrate is a specific inducer of nitrate reductase in green leaves, but appears to have a more general effect in etiolated leaves. In the dark, nitrate induced nitrate reductase expression in both etiolated and green leaves, indicating light and functional chloroplast were not required for enzyme expression.

Differential Inhibition and Activation of Two Leaf Dihydroxyacetone Phosphate Reductases

The chloroplastic and cytosolic forms of spinach (Spinacia oleracea cv Long Standing Bloomsdale) leaf NADH:dihydroxyacetone phosphate (DHAP) reductase were separated and partially purified. The chloroplastic form was stimulated by dithiothreitol, reduced thioredoxin, dihydrolipoic acid, 6-phosphogluconate, and phosphate; the cytosolic isozyme was stimulated by fructose 2,6-bisphosphate but not by reduced thioredoxin. End product components that severely inhibited both forms of the reductase included lipids and free fatty acids, membranes, and glycerol phosphate. In addition, two groups of inhibitory peptides were obtained from the fraction precipitated by 70 to 90% saturation with (NH(4))(2)SO(4). Chromatography of this fraction on Sephadex G-50 revealed a peptide peak of about 5 kilodaltons which inhibited the chloroplastic DHAP reductase and a second peak containing peptides of about 2 kilodaltons which inhibited the cytosolic form of the enzyme. Regulation of the reduction of dihydroxyacetone phosphate from the C(3) photosynthetic carbon cycle or from glycolysis is a complex process involving activators such as thioredoxin or fructose 2,6-bisphosphate, peptide and lipid inhibitors, and intermediary metabolites. It is possible that fructose 2,6-bisphosphate increases lipid production by stimulating DHAP reductase for glycerol phosphate production as well as inhibiting fructose 1,6-bisphosphatase to stimulate glycolysis.

NADH Nitrate Reductase and NAD(P)H Nitrate Reductase in Genetic Variants and Regenerating Callus of Maize

Different organs of maize seedlings are known to contain different complements of NADH and NAD(P)H nitrate reductase (NR) activity. The study of the genetic programming that gives rise to such differences can be initiated by looking for genetic variants exhibiting different patterns of distribution of the above enzymes. We demonstrate in this work that scutella of very young maize seedlings contain NADH NR almost exclusively and that this activity is gradually replaced, as the seedling ages, with NAD(P)H NR. Leaves in the seedlings contain exclusively the NADH NR activity. A genetic variant is described that contains much reduced levels of NAD(P)H NR activity but not of NADH NR activity in the scutellum. This same variant exhibits a relatively low level of NAD(P)H NR but normal NADH NR activity in seedling root tips. These observations suggest that the genetic program used to specify the scutellar complement of NR activity shares some common components with the genetic program used to determine the young root tip complement of NR activities. Parts of regenerating callus at different stages of differentiation were examined to determine when the differences in NR complement begin to appear. The same pattern of NADH NR and NAD(P)H NR activities was found in unorganized as well as in organized callus, in recognizable root-like and even in green shoot-like material, both activities being present in all these tissues. An examination of the NR complement in different organs of a number of siblings originating from a cross involving transposon Mu-containing parents and having different levels of leaf NADH NR activity shows that the leaf NADH NR activity content and the scutellum NAD(P)H NR activity content are relatively independent of each other, indicating that the genetic programs specifying the NR content of these organs are not tightly coupled, if at all.

Ferric‐citrate reductase activity of nitrate reductase and it's role in iron assimilation by plants

Abstract Squash cotyledon and corn leaf NADH:nitrate reductase (NR) catalyze the reduction of Fe (III)‐citrate, as shown by trapping the reduced iron with ferrozine. The co‐identity of NR as a catalyst for both nitrate and iron reduction was shown by the co‐purification and co‐electrophoresis of the two activities with both partially pure and homogeneous enzyme forms. In addition, the Fe (III)‐citrate reductase activity had a pH optimum and substrate kinetics consistent with an enzymic reaction, and was inhibited by rabbit antiserum which had been prepared against homogeneous NADH:NR. Our previous studies with various inhibitors showed that Fe (III)‐citrate was directly reduced by NR, but at a site separate from the nitrate reducing site. A role for the Fe (III)‐citrate reductase activity of NR in the remoblization of recently transported iron is described in relation to the role of this enzyme in nitrate assimilation.

AGEING‐LINKED CHANGES IN PHOTOSYNTHETIC CAPACITY AND IN FRACTION I PROTEIN CONTENT OF THE FIRST LEAF OF PEA PISUM SATIVUM L.

SUMMARYPea seedlings (Pisum sativum L. cv. Melbourne Market) were grown under light‐limited conditions (12 h photoperiod of 1 klx; 19°C) so that both the development and senescence of the first true leaf could be studied within a period of 4 weeks from the beginning of germination. Rapid cell expansion and synthesis of protein and chlorophyll occurred between days 8 to 12. Whereas photosynthetic O2 evolution and RuBP carboxylase both showed maximum values per leaf at 16 days, declining thereafter, that of NADH:glyoxylate reductase, a peroxisomal marker, occurred between 11 and 14 days and was decreasing by 16 days. Total leaf ribose‐5‐P isomerase activity increased two‐fold to a maximum at 14 days then fell sharply before 16 days. Dark O2 uptake by leaf slices and NADH oxidase activity showed extended maxima from the earliest stages to 20 and 16 days respectively. Soluble acid phosphatase also showed an extended period of maximum activity per leaf, from 12 to 21 days, then declined slowly.As a proportion of leaf soluble protein, Fraction I protein varied from 17% (day 8) to 41% (day 10), 57%(day 14), 76%(day 16) then to about 60% from day 21 onwards. Between 14 and 16 days the amount of Fraction I protein in the leaf continued to increase, although the amount of total soluble protein had begun to decline. Aminopeptidase (leucyl β‐naphthylamidase) activities per leaf also began to decline after 14 days. Since the maximum photosynthetic capacity of the leaf is not observed until 16 days, the data indicate the onset of leaf senescence prior to the completion of chloroplast development.