Nitrate Assimilation and Growth of Steptoe Barley and one of its Nitrate Reductase Deficient Mutants

Barley (Hordeum vulgare L. cv. Steptoe) and a nitrate reductasedeficient mutant (narla) were grown in a nutrient film systemwith three concentrations of nitrate. Comparisons were madewith respect to growth, yield, activities of enzymes of nitrateassimilation and accumulation of nitrate and total nitrogen.In nutrient film, grain yeild of the wild-type was greater thanthat of narla. for any treatment. Nitrate reductase activitiesof narla, measured in vivo, were higher than might be expectedin an NR-deficient mutant both in leaves and especially in roots.In all treatments, narla accumulated more nitrate than did thewild-type. No significant genotypic differences were observedin nitrite reductase or glutamine synthetase activities. Whenthe two genotypes were grown in soil (i.e. when availabilityof nitrate to the roots was less than in nutrient film) differencesin growth were insignificant. Hordeum vulgare L., mutant, nitrate status, assimilation and accumulation, growth, yield     

Reduction of Cytokinin Binding-protein in a Nitrate Reductase Deficient Mutant Cell Line of Datura inoxia

Suspension culture cells initiated from haploid Datura inoxia seedlings were transferred on a paper and were treated with UV. The nitrate reductase (NR) deficient mutants were isolated by selection for chlorate resistance. The NR activity could not be recovered, even though the mutants were transferred into the medium without selective pressure for three years. Isoelectrofocusing gel showed that the gene of NR was not destroyed by the treatment of UV. The mutant cells were defective in the cytokinin binding protein. The cytokinin binding-protein was isolated from wheat seedlings with the aid of 6BA immobilized on the epoxy-sepharose colunm. An addition of binding-protein, together with 6BA, to the medium for synthesis of RNA in vitro brough about an activation of RNA-polymerase. In wild type cells the NR activity was accelerated by the addition of cytokinin to the culture medium. In contrast, cytokinin was of no effect on the synthesis of NR in mutant cells. It is, therefore, suggested that the effect of cytokinin on the RNA synthesis and NR formation was regulated by the content of cytokinin binding-protein in Datura inoxia mutant cells.     

Extraction and Affinity Purification of NADH:Nitrate Reductase from Barley (Hordeum distichum L.) Roots

Conditions suited for the extraction and purification of NADH:nitratereductase (NR) from barley (Hordeum distichum L.) roots wereexamined. The addition of 10 mM EDTA to the extraction mediumproduced an 8-fold increase in the NR activity in the crudeextract, whereas the presence of cysteine in the medium causedan appreciable decrease in this activity. EDTA and FAD stimulatedNR activity in the crude extract; cysteine inhibited it. Theeffect of EDTA seemed to be due to the inhibition of the contaminatingNADH-oxidizing system. The NADH:NR was purified 300-fold by ammonium sulfate fractionationand blue dextran-Sepharose affinity chromatography. The specificactivity was 1,420 nmol nitrite formed min^–1 mg protein^–1at 30?C; the highest specific activity among the NR preparationsobtained thus far from root tissues of higher plants. EDTA,as well as cysteine behaved as an inhibitor for the purifiedNR. (Received January 27, 1982; Accepted June 21, 1982)     

Characterization of Nitrate Reductase Deficient Mutants of Chlorella sorokiniana

After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO₃⁻ as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH₄⁺-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein.     

Comparative Induction of Nitrate Reductase by Nitrate and Nitrite in Barley Leaves

The comparative induction of nitrate reductase (NR) by ambient NO₃⁻ and NO₂⁻ as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley (Hordeum valgare L.) seedlings was determined. The dynamic interaction of NO₃⁻ influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO₃⁻. As the ambient concentration of NO₃⁻ increased, the relative influences imposed by influx and reduction on NO₃⁻ accumulation changed with influx becoming a more predominant regulant. Significant levels of NO₃⁻ accumulated in NO₂⁻-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO₂⁻, about 60% more NO₃⁻ accumulated in the leaves than in the absence of the inhibitors. In NO₃⁻-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 mm. No NR induction occurred in leaves supplied with NO₂⁻ until the ambient NO₂⁻ concentration was 0.5 mm. In fact, NR induction from NO₂⁻ solutions was not seen until NO₃⁻ was detected in the leaves. The amount of NO₃⁻ accumulating in NO₂⁻-fed leaves induced similar levels of NR as did equivalent amounts of NO₃⁻ accumulating from NO₃⁻-fed leaves. In all cases the internal concentration of NO₃⁻, but not NO₂⁻, was highly correlated with the amount of NR induced. The evidence indicated that NO₃⁻ was a more likely inducer of NR than was NO₂⁻.     

Physiological Properties of a Mutant of Pachysolen tannophilus Deficient in NADPH-Dependent d-Xylose Reductase

A d-xylose reductase mutant of Pachysolen tannophilus was isolated on the basis of its poor growth on d-xylose but normal growth on xylitol and d-glucose. Fractionation of cell extracts indicated that the mutant was deficient in d-xylose reductase activity that used NADPH exclusively as a cofactor, but not in activity that used both NADH and NADPH. Mutant cultures grown on d-xylose as the sole carbon source exhibited some properties that would be desired in improved strains. Growth rate, growth yield, and d-xylose consumption rate of the mutant were less sensitive than those of the wild type to changes in aeration rate. d-Xylose was utilized more efficiently in that less of a by-product, xylitol, was produced. In addition, under low aeration conditions, more ethanol was produced. A disadvantage was a relatively slow rate of d-xylose utilization.     

Determination of Nitrate Reductase Activity in Barley Leaves and Roots

The inactivation of nitrate reductase in the leaves and rootsof barley (Hordeum vulgare L. cv. Mazurka) during and afterextracting was investigated. At 0 °C in the absence of casein,25 per cent of ‘total’. i.e. maximal in vitro, nitratereductase activity was lost during the 2 min extraction process,followed by a slower loss of activity while the extract wasstored in ice. Activity was maintained by adding a minimum of1 per cent casein to the extraction medium containing 0·1M phosphate (pH 7·5), 1 mM EDTA and 1 mM dithiothreitol.Nitrate reductase was stable for several hours in these extracts,but declined in a first order manner in the absence of dithiothreitol.Casein also prevented the initial loss while making root extracts,but had less effect during storage. Using casein and thiols, nitrate reductase activity in light,(as product of maximal in vitro rates and wt g^–1) in leaveswas 98 per cent of the total activity in 31-day-old plants grownwith full nutrient in water culture and 60-day-old field-grownplants receiving no fertilizer. Field-grown plants, however,exhibited only 17 per cent of the activity of culture-grownplants. Nitrate reductase in leaves of barley plants grown in waterculture had a diurnal rhythm. During the first 3 h of the lightperiod, activity increased to 1·3 x the ‘dark’value. This was followed by a temporary decrease and then byanother increase to a maximum of 1·7 x the ‘dark’value, occurring about 8 h after illumination. Activity thendecreased during the rest of the light period and in darkness. Hordeum vulgare L., barley, nitrate reductase     

Nitrate Absorption by Barley: II. Influence of Nitrate Reductase Activity

The influence of protein synthesis and nitrate reductase activity on nitrate absorption by barley (Hordeum vulgare L.) was investigated. Cycloheximide decreased nitrate absorption. Pretreatment studies showed that cycloheximide affects either energy transfer or nitrate reductase activity or both.     

Magnesium and Calcium Inhibition of Squash Leaf NADH Nitrate Reductase

This paper describes the first experimental evidence that theinhibition of nitrate reductase by Mg²⁺ or Ca²⁺ is related tothe hysteretic properties of the enzyme. The low activity formof nitrate reductase, i.e. the form of nitrate reductase showinghysteretic behaviour, was inhibited 70–90% by 5 mM Ca²⁺or Mg²⁺. However, no inhibition by Ca²⁺ or Mg²⁺ was seen afterthe enzyme was converted to its high activity form by preincubationwith substrates. Addition of thiol compounds or certain aminoacids to the assay mixture also prevented the Mg²⁺ or Ca²⁺ inhibition. (Received June 28, 1993; Accepted August 11, 1993)     

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 μmol of NADH oxidized/minute • milligram of protein. Corn (Zea mays L.) leaf NR was also purified to a specific activity of 6.9 μmol 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.

     

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 W64A×W183E) scutellum λgt11 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.     

单半乳糖甘油二脂缺失对烟草光合特性的影响

以单半乳糖甘油二脂(MGDG)相对含量比野生烟草显著降低的突变体(M18)及野生型烟草为材料,通过对转基因烟草叶绿体类囊体膜的低温荧光、放氧活性以及叶片的叶绿素荧光分析,研究MGDG部分缺失对烟草叶片光合特性的影响。结果表明,在低温下(77K)MGDG部分缺失并不影响烟草叶绿素荧光发射峰(F683和F730)的位置,但使光系统Ⅱ(PSⅡ)及光系统Ⅰ(PSⅠ)的荧光发射峰的强度减弱,F683/F730比值降低,直接影响激发能在PSⅡ和PSⅠ之间的均衡分配,使叶绿素a和叶绿素b之间的能量传递受阻,降低光能转化效率;MGDG部分缺失使PSⅡ放氧活性下降了72.9%;转基因烟草叶绿素荧光参数中最大光化学效率(Fv/Fm)、暗适应最大荧光(Fm)、实际光化学效率(Yield)、光化学猝灭系数(qP)比野生型烟草分别降低了7%、49%、32%和18%,并以Fm降幅最大。研究证明,MGDG在维持植物叶绿体类囊体膜的功能,特别是PSⅡ的功能方面起着重要的作用。     

Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase

Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M₂ seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH₂-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c₁NR), constitutive NADH:NR (c₂NR), and nitrate-inducible NR (iNR).     

Nitrate Reductase in Barley Roots under Sterile, Low Oxygen Conditions

Levels of nitrate reductase activity (EC 1.9.6.1.) as high as 11 μmoles nitrite produced/hour gram fresh weight were found in barley (Hordeum vulgare cv. Compana) roots grown under low oxygen conditions. Roots of plants given identical treatment under sterile conditions did not develop the high levels of nitrate reductase activity. The results suggest that the buildup of particulate, reduced viologen-utilizing nitrate reductase reported in barley roots may be caused by bacterial contamination. The nitrate reductase activity in roots grown under low oxygen conditions was not specific for reduced nicotinamide adenine dinucleotide like the assimilatory nitrate reductase (EC 1.6.6.1.) normally found in aerated plant roots.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

Inverse Correlation of Thiol Proteinase with Nitrate Reductase Activities in Barley Leaves

Experiments conducted to determine the effects of leupeptin,a specific inhibitor of thiol proteinase, on extractable nitratereductase (NR) activity in leaves of Hordeum distichum duringdarkness revealed that leupeptin (0.01 mg.ml^–1) appliedto detached leaves significantly reduced the loss of NR activity.At the same time it also reduced the formation of small cytochromec reductase species, which is a degradation product of NR complex,Upon nitrate induction, extractable NR activity increased butthe content of thiol proteinase decreased. This inverse correlationwas also observed upon transfer of nitrate-grown barley seedlingsto nitrate-free nutrient solution. Furthermore, cycloheximide(0.1 mg.ml^–1) treatment of barley seedlings reduced thecontent of thiol proteinase and retarded the loss of NR activityunder noninducing conditions. These results suggest that invivo changes in NR content in leaves of Hordeum distichum arethe result of proteolysis by an endogenous thiol proteinase. (Received May 16, 1985; Accepted July 22, 1985)     

Purification of Squash NADH:Nitrate Reductase by Zinc Chelate Affinity Chromatography

NADH:nitrate reductase (EC 1.6.6.1) was isolated and purified from the green cotyledons of 5-day-old squash seedlings (Cucurbita maxima L.). The 10-hour purification procedure consisted of two steps: direct application of crude enzyme to blue Sepharose and specific elution with NADH followed by direct application of this effluent to a Zn²⁺ column with elution by decreasing the pH of the phosphate buffer from 7.0 to 6.2. The high specific activity (100 micromoles per minute per milligram protein) and high recovery (15-25%) of electrophoretically homogeneous nitrate reductase show that the enzyme was not damaged by exposure to the bound zinc. With this procedure, homogeneous nitrate reductase can be obtained in yields of 0.5 milligram per kilogram cotyledons.     

Inactivation of Sunflower NADH:Nitrate Reductase by White Light-Activated Rose Bengal

Chemical modification of purified nitrate reductase (NR) from sunflower leaves by white light-irradiated rose bengal was studied. NADH:NR activity was inhibited by light-activated rose bengal in both a concentration- and time-dependent manner. MV:NR activity was less sensitive to inhibition than NADH:NR activity, especially when the enzyme was preincubated with NADH. Preincubation of the enzyme with FAD protected inhibition of NADH:NR activity but not the MV:NR activity. These results suggest that sunflower NR contains sensitive histidine residue which interacts with reduced FAD during catalytic electron transfer. Most importantly, NADH-reduced NR was more sensitive to the irradiated dye, indicating that conformation of the oxidized and reduced enzyme forms were different.