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     

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     

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.

     

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)     

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).     

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.     

Inhibition of Nitrate Transport by Anti-Nitrate Reductase IgG Fragments and the Identification of Plasma Membrane Associated Nitrate Reductase in Roots of Barley Seedlings

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO₃⁻ uptake by more than 90% but had no effect on NO₂⁻ uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO₃⁻ uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO₃⁻ uptake. The results present the possibility that NO₃⁻ uptake and NO₃⁻ reduction in the PM of barley roots may be related.     

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.     

Somatic Hybridization of Nitrate Reductase Deficient Mutants of Nicotiana tabacum by Protoplast Fusion

Protoplasts were isolated from two mutant cell lines of Nicotiana tabacum L. cv. Gatersleben and fused with the aid of polyethylene glycol. Both mutants lacked nitrate reductase and were thus auxotrophic for reduced nitrogen. The fusion resulted in a high frequency of hybrid cells which were detected by their regained ability to grow in media containing nitrate as sole nitrogen source. Thus, the two mutants were found to complement each other in the hybrids. In control experiments, back mutation and cross-feeding were excluded as possible explanations for the occurrence of cell lines utilizing nitrate. A total of 1061 hybrid lines capable of sustained proliferation were isolated. Some of them were further characterized with respect to nitrate reductase activity, chlorate sensitivity, chromosome number, and shoot formation. The results demonstrate that protoplast fusion can be used for the genetic analysis of cell variants of higher plants and that nitrate reductase-deficient mutants provide efficient selective systems for hybrid cells.     

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

Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.     

Expression of NADH-Specific and NAD(P)H-Bispecific Nitrate Reductase Genes in Response to Nitrate in Barley

Barley (Hordeum vulgare L.) has two, differentially regulated, nitrate reductase (NR) genes, one encoding the NADH-specific NR (Nar1) and the other encoding the NAD(P)H-bispecific NR (Nar7). Regulation of the two NR genes by nitrate was investigated in wild-type Steptoe and in an NADH-specific NR structural gene mutant (Az12). Gene-specific probes were used to estimate NADH and NAD(P)H NR mRNAs. The kinetics of induction by nitrate were similar for the two NR genes; expression was generally below the limits of detection prior to induction, reached maximum levels after 1 to 2 h of induction in roots and 4 to 8 h of induction in leaves, and then declined to steady-state levels. Derepression of the NAD(P)H NR gene in leaves of the NADH-specific NR gene mutant Az12 did not appear to be associated with changes in nitrate assimilation products or nitrate flux. Nitrate deprivation resulted in rapid decreases in NADH and NAD(P)H NR mRNAs in seedling roots and leaves and equally rapid decreases in the concentration of nitrate in the xylem sap. These results indicate that factors affecting nitrate uptake and transport could have a direct influence on NR expression in barley leaves.     

In Vitro Formation of Nitrate Reductase Using Extracts of the Nitrate Reductase Mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum

In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH-nitrate reductase, FADH(2)-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10(-4) M Na(2)MoO(4) and 10(-2) M NaNO(3) to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase.