Regulation of the inducible soybean nitrate reductase isoform in mutants lacking constitutive isoform(s).

In soybean, three nitrate reductase isoforms have been identified based on metabolic regulation, substrate specificity, and kinetic parameters. Two isoforms have been termed constitutive, as their activities are present without the addition exogenous nitrate to soybean seedlings. The third activity is termed inducible, as its activity is present only when soybean plants have been supplied with nitrate. The purpose of this study was to examine the regulation of the inducible nitrate reductase isoform in soybean mutants lacking one or both of the constitutive isoforms. Based on evidence obtained through measurements of enzyme activity, Western blotting, and RNA determinations, the absence of one or both of the constitutive nitrate reductase isoforms has no effect on the metabolite regulation of the inducible nitrate reductase isoform.

Regulation of the inducible nitrate reductase isoform from soybeans.

The activity of the pH 7.5 NADH-linked nitrate reductase isoform from soybeans is termed inducible. Activity can be observed only in seedlings which have been supplied nitrate. Steady-state levels of mRNA for this isoform also show an absolute requirement for nitrate. Nitrate reductase specific mRNA can be observed within 2 h after nitrate treatment. Levels peaked 48 h after nitrate treatment, while the addition of glutamine to nitrate diminished amounts of nitrate reductase specific mRNA. Using nuclear runoff transcription assays, we have shown that one level of control of nitrate reductase synthesis is transcriptional.

Iron assimilation in plants: reduction of a ferriphytosiderophore by NADH:nitrate reductase from squash.

NADH:nitrate reductase (EC 1.6.6.1) from squash (Cucurbita maxima Duch., cv. Buttercup) can catalyze the reduction of a ferriphytosiderophore from barley (Hordeum vulgare L. cv. Europa). Maximal activity occurs at pH 6, with an apparentK m andV max of 76 µM and 21 nmol·min(-1)·(mg protein)(-1), respectively. The ferriphytosiderophore strongly inhibits nitrate reduction catalyzed by nitrate reductase at the optimal pH for nitrate reduction, i.e. 7.5. On the contrary, nitrate is a poor inhibitor of ferriphytosiderophore reduction catalyzed by nitrate reductase at the optimal pH for this reaction, pH 6.0. Thus, squash has the potential to assimilate the iron from a ferriphytosiderophore synthesized by another plant.

Transcriptional control of the inducible nitrate reductase isoform from soybeans.

The activity of the pH 7.5 NADH-linked nitrate reductase isoform from soybean seedlings is termed inducible. Activity is present only in the leaves of seedlings which have been supplied nitrate. A cDNA clone that encoded part of the mRNA for squash nitrate reductase hybridized specifically with mRNA for this inducible nitrate reductase isoform. Nitrate induction resulted in an increase in the steady-state levels of mRNA for this isoform after 24 hours, while the addition of glutamine to the nitrate diminished steady-steady state levels of this mRNA.

Response of various cucurbits to infection by plasmid-harboring strains of agrobacterium.

Tumor formation in cucurbit cultivars resulting from infection by various strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes is environmentally affected. In all instances, tumors could be induced on excised cotyledons while inoculating attached cotyledons or stems resulted in no tumor formation. In addition, buttercup squash (Cucurbita maxima Duch. buttercup) was most susceptible to tumor formation, while butterbush squash (Cucurbita maxima Duch. butterbush) failed to form tumors when inoculated with any of the strains of Agrobacterium. Other tested cucurbit cultivars showed intermediate susceptibility to tumor induction by the various Agrobacterium strains.

A kinetic analysis of Drosophila melanogaster dopa decarboxylase.

The kinetic mechanism of dopa decarboxylase (3,4-dihydroxy-L-phenylalanine carboxy-lyase, EC 4.1.1.28) was investigated in Drosophila melanogaster. Based on initial velocity and product inhibition studies, an ordered reaction is proposed for dopa decarboxylase. This kinetic mechanism is interpreted in the context of measured enzyme activities and the catecholamine pools in Drosophila. The 1(2)amd gene is immediately adjacent to the gene coding for dopa decarboxylase (Ddc) and determines hypersensitivity to alpha-methyldopa in Drosophila. Dopa decarboxylase does not decarboxylate alpha-methyldopa and hence does not generate a toxic product capable of inhibiting 1(2)amd gene function. We propose that the 1(2)amd gene is involved with an unknown catecholamine pathway involving dopa but not dopamine.

Siderophore reduction catalyzed by higher plant NADH:nitrate reductase.

Squash cotyledon NADH:nitrate reductase catalyzes the reduction of the siderophore ferrioxamine B. The enzyme also reduced ferric ion in a buffer system containing the chelators oxalate and maleate. Ferrioxamine B reduction was maximal at pH 4; ferric ion reduction was maximal at pH 8. The present study indicates that iron assimilation by higher plants may occur with microbial siderophores serving as ferric ion sources and nitrate reductase functioning as the siderophore reductase.

Immunological approach to structural comparisons of assimilatory nitrate reductases.

Homogeneous squash cotyledon reduced nicotinamide-adenine dinucleotide (NADH):nitrate reductase (NR) was isolated using blue-Sepharose and polyacrylamide gel electrophoresis. Gel slices containing NR were pulverized and injected into a previously unimmunized rabbit. This process was repeated weekly and antiserum to NR was obtained after four weeks. Analysis of the antiserum by Ouchterlony double diffusion using a blue-Sepharose preparation of NR resulted in a single precipitin band while immunoelectrophoresis revealed two minor contaminants. The antiserum was found to inhibit the NR reaction and the partial reactions to different degrees. When the NADH:NR and the reduced methyl viologen:NR activities were inhibited 90% by specifically diluted antiserum, the reduction of cytochrome c was inhibited 50%, and the reduction of ferricyanide was inhibited only 30%. Antiserum was also used to compare the cross reactivities of NR from squash cotyledons, spinach, corn, and soybean leaves, Chlorella vulgaris, and Neurospora crassa. These tests revealed a high degree of similarity between NADH:NR from the squash and spinach, while NADH:NR from corn and soybean and the NAD(P)H:NR from soybean were less closely related to the squash NADH:NR. The green algal (C. vulgaris) NADH:NR and the fungal (N. crassa) NADPH:NR were very low in cross reactivity and are apparently quite different from squash NADH:NR in antigenicity. Antiserum to N. crassa NADPH:NR failed to give a positive Ouchterlony result with higher plant or C. vulgaris NADH:NR, but this antiserum did inhibit the activity of squash NR. Thus, it can be concluded from these immunological comparisons that all seven forms of assimilatory NR studied here have antigenic determinants in common and are probably derived from a common ancestor. Although these assimilatory NR have similar catalytic characteristics, they appear to have diverged to a great degree in their structural features.

Purification and Kinetics of Higher Plant NADH:Nitrate Reductase.

Squash cotyledon (Cucurbita pepo L.) NADH:nitrate reductase (NR) was purified 150-fold with 50% recovery by a single step procedure based on the affinity of the NR for blue-Sepharose. Blue-Sepharose, which is prepared by direct coupling of Cibacron blue to Sepharose, appears to bind squash NR at the NADH site. The NR can be purified in 2 to 3 hours to a specific activity of 2 mumol of NADH oxidized/minute * milligram of protein. Corn (Zea mays L.) leaf NR was also purified to a specific activity of 6.9 mumol of NADH oxidized/minute * milligram of protein using a blue-Sepharose affinity step. The blue-Sepharose method offers the advantages of a rapid purification of plant NR to a high specific activity with reasonable recovery of total activity.The kinetic mechanism of higher plant NR was investigated using these highly purified squash and corn NR preparations. Based on initial velocity and product inhibition studies utilizing both enzymes, a two-site ping-pong mechanism is proposed for NR. This kinetic mechanism incorporates the concept of the reduced NR transferring electrons from the NADH site to a physically separated nitrate site.