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.     

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.     

Nitrate reduction and compartmentation in tomato leaves. I. Nitrate accumulation in leaf sections in aqueous and gaseous medium

Nitrate pools in tomato ( Lycopersicon esculentum Mill. cv. Azes) leaf sections were estimated. Nitrite accumulation in aqueous medium was found to be an inadequate estimate of nitrate pools in tomato leaves. The main reason for the cessation of nitrite accumulation was not depletion of nitrate in the metabolic pool but rather a rapid decay of nitrate reductase (NR) activity as measured by nitrite accumulation in vivo and in vitro. Nitrate diffuses out of the tissue into the medium at a rate higher than the accumulation of nitrite in the tissue. Nitrate leakage from the tissue accelerates the loss of NR activity. Nitrite accumulation in leaf sections kept in an anaerobic gaseous atmosphere ceased earlier than in aqueous medium, at a time when NR activity was still relatively high. Measuring nitrite accumulation in gaseous atmosphere is preferable since NR is more stable and movements of nitrate between pools more restricted.     

Nitrate Reductase mRNA Regulation in Nicotiana plumbaginifolia Nitrate Reductase-Deficient Mutants

Light and substrate regulation of nitrate reductase (NR) expression were compared in wild type and mutant lines of Nicotiana plumbaginifolia. Mutants affected in the NR structural gene (nia) or in the biosynthesis of the NR molybdenum cofactor (cnx) were examined. nia mutants expressing a defective apoenzyme, as well as cnx mutants, overexpressed NR mRNA, whereas nia mutants devoid of detectable NR protein had reduced or undetectable NR mRNA levels. Diurnal fluctuations of NR mRNA were specifically abolished in nia and cnx mutants, suggesting that the integrity of NR catalytic activity is required for the expression of diurnal oscillations. Unlike some fungal mutants, the nia and cnx mutants examined retained nitrate inducibility of NR expression. The possibility of autogenous control of NR expression in higher plants is discussed.     

Mechanism of Nitrate Tolerance in Tn5 Mutants of Cicer-Rhizobium Strains

The activities of nitrate reductase (NR) and nitrite reductase (NiR) and production of indole-3-acetic acid (IAA) by symblotic nitrate tolerant Tn5 mutant AC-10 of Cicer-Rhizobium strain F-75 and mutants BC-35 and BC-46 of strain G36-84 developed earlier, have been studied under ex planta condition. The rhizobiaI mutants and their parental strains were grown with nitrate (0.0, 0.5, 1, 2 or 4 mM), aerobically and microaerobically. The overall activities of NR were 70–91% lower in aerobically grown and 78–87% lower in microaerobically grown mutant cells compared to their parental strains. Similarly, the overall activities of NiR were 36–55% and 27–37% lower in aerobically and microaerobically grown mutant cells, respectively, compared to their parental strains. On the contrary, the overall production of IAA in the culture medium by aerobically grown mutant cells was significantly higher compared to their parental strains. Based on these results, it has been suggested that impaired NR activity and a favourable NiR/NR ratio preventing nitrite accumulation in the rhizobial mutants, may be responsible for imparting nitrate tolerance to chickpea - Rhizobium symbiotic system.     

A new inducible expression system in a transformed green alga, Chlorella vulgaris

Genetic transformation is useful for basic research and applied biotechnology. However, genetic transformation of microalgae is usually quite difficult due to the technical limitations of existing methods. We cloned the promoter and terminator of the nitrate reductase gene from the microalga Phaeodactylum tricornutum and used them for optimization of a transformation system of the microalga Chlorella vulgaris. This species has been used for food production and is a promising candidate as a bioreactor for large-scale production of value-added proteins. A construct was made containing the CAT (chloramphenicol acetyltransferase) reporter gene driven by the nitrate reductase promoter. This construct was transferred into the C. vulgaris genome by electroporation. Expression of CAT in transgenic Chlorella conferred resistance to the antibiotic chloramphenicol and enabled growth in selective media. Overall efficiency for the transformation was estimated to be approximately 0.03%, which is relatively high compared with other available Chlorella transformation systems. Expression of CAT was induced in the presence of nitrate and inhibited in the presence of ammonium as a sole nitrogen source. This study presented an inducible recombinant gene expression system, also providing more gene regulation elements with potential for biotechnological applications.     

利用椭圆小球藻硝酸还原酶缺失突变体为生物反应器表达兔防御素NP-1蛋白

利用转基因小球藻为生物反应器生产兔防御素NP-1蛋白具有重要的应用价值。本研究利用椭圆小球藻(Chlorella ellipsoidea)硝酸还原酶(nitrate reductase)缺失突变体为受体, 构建了包含NPTII基因和硝酸还原酶基因两个筛选标记的兔防御素蛋白表达载体, 采用电激法将目的基因转入椭圆小球藻硝酸还原酶缺失突变体nrm-4, 获得了正确表达防御素蛋白的转基因藻, 从而表明通过硝酸还原酶作为筛选标记基因并结合硝酸还原酶缺失突变体可作为较好的小球藻生物反应器生产模式。     

Synthesis and Degradation of Nitrate Reductase during the Cell Cycle of Chlorella Sorokiniana

Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [³⁵S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.     

硝酸盐对球形棕囊藻生长和硝酸还原酶活性的影响

以我国南海海域分离的赤潮原因种——球形棕囊藻(Phaeocystis globosa)为材料, 研究了不同硝酸盐浓度下藻细胞生长及硝酸还原酶活性的变化。当培养基中不含硝酸盐时, 藻细胞内硝酸还原酶的活性保持在非常低的水平, 藻细胞的生长受到限制, 不能形成正常的生长曲线: 当培养基中硝酸盐浓度为3.62 mmol.L-1时, 藻细胞的硝酸还原酶活性和比生长速率达到最大。在含有硝酸盐的培养基中, 接种培养后第9天藻细胞硝酸还原酶活性达到最大值, 并且在4种不同硝酸盐浓度下, 藻细胞硝酸还原酶活性的差异性达到极显著水平(P<0.01)。在接种培养第16天藻细胞密度达到最大值, 并且4种不同硝酸盐浓度培养的藻细胞密度之间的差异性也达到极显著水平(P<0.01)。实验结果表明, 在培养基中添加不同浓度的硝酸盐, 对球形棕囊藻细胞硝酸还原酶的活性和藻细胞的生长有极显著的影响, 含有较高硝酸盐的富营养化海域有利于球形棕囊藻细胞的持续生长。     

Nitrate assimilation in the forage legume Lotus japonicus L.

Nitrate assimilation in the model legume, Lotus japonicus, has been investigated using a variety of approaches. A gene encoding a nitrate-inducible nitrate reductase (NR) has been cloned and appears to be the only NR gene present in the genome. Most of the nitrate reductase activity (NRA) is found in the roots and the plant assimilates the bulk of its nitrogen in that tissue. We calculate that the observed rates of nitrate reduction are compatible with the growth requirement for reduced nitrogen. The NR mRNA, NRA and the nitrate content do not show a strong diurnal rhythm in the roots and assimilation continues during the dark period although export of assimilated N to the shoot is lower during this time. In shoots, the previous low NR activity may be further inactivated during the dark either by a phosphorylation mechanism or due to reduced nitrate flux coincident with a decreased delivery through the transpiration stream. From nitrate-sufficient conditions, the removal of nitrate from the external medium causes a rapid drop in hydraulic conductivity and a decline in nitrate and reduced-N export. Root nitrate content, NR and nitrate transporter (NRT2) mRNA decline over a period of 2 days to barely detectable levels. On resupply, a coordinated increase of NR and NRT2 mRNA, and NRA is seen within hours.     

Light-mediated Activation of Nitrate Reductase in Synchronous Chlorella

The mechanism underlying the sharp increase in activity of nitrate reductase (EC 1.6.6.1) in Chlorella vulgaris forma tertia (strain 211 8k) during the first hour of the 7 hours/5 hours light/dark cycle was investigated. Using the method of density labeling and isopycnic centrifugation, it could be demonstrated that this rapid increase in activity is based on light-mediated activation rather than de novo synthesis of the enzyme. The problematic nature of cycloheximide specificity and models of nitrate reductase activation are discussed.     

Nitrate reduction mutants of fusarium moniliforme (gibberella fujikuroi)

Twelve strains of Fusarium moniliforme were examined for their ability to sector spontaneously on toxic chlorate medium. All strains sectored frequently; 91% of over 1200 colonies examined formed chlorate-resistant, mutant sectors. Most of these mutants had lesions in the nitrate reduction pathway and were unable to utilize nitrate (nit mutants). nit mutations occurred in seven loci: a structural gene for nitrate reductase (nit1), a regulatory gene specific for the nitrate reduction pathway (nit3), and five genes controlling the production of a molybdenum-containing cofactor that is necessary for nitrate reductase activity (nit2, nit4, nit5, nit6, nit7). No mutations affecting nitrite reductase or a major nitrogen regulatory locus were found among over 1000 nit mutants. Mutations of nit1 were recovered most frequently (39-66%, depending on the strain) followed by nit3 mutations (23-42%). The frequency of isolation of each mutant type could be altered, however, by changing the source of nitrogen in the chlorate medium. We concluded that genetic control of nitrate reduction in F. moniliforme is similar to that in Aspergillus and Neurospora, but that the overall regulation of nitrogen metabolism may be different.     

Transformation of Aspergillus niger with the homologous nitrate reductase gene

A homologous transformation for Aspergillus niger was developed based on the nitrate reductase structural gene niaD. This system offered certain advantages over existing A. niger systems, such as the ease of recipient mutant isolation, absence of abortive transformants, convenient enzyme assay, ease of transformant stability testing, and complete absence of background growth. Transformation frequencies of up to 100 transformants per microgram DNA were obtained with the vector pSTA10 which carries the niaD gene of A. niger. Southern blotting analysis indicated that vector DNA had integrated into the genome of A. niger. Mitotic stability studies demonstrated that while some transformants were as stable as the wild-type (wt), others were markedly less so. No correlation was seen between plasmid integration, mitotic stability and nitrate reductase activity, which was markedly different from wt in only three of the transformants examined.