不同氮源对悬浮培养玫瑰茄细胞的生长及硝态氮同化指征的影响

本文通过改变培养基中的氮源组成,来研究氮源的变化对悬浮培养玫瑰茄细胞的生长及硝态氮同化指征（活体内硝酸还原酶活力）的影响。实验表明,仅在含ＮＯ－３的培养基中能检测到硝酸还原酶活力,而仅含ＮＨ＋４的配方不能检测到该酶活力,表明硝酸还原酶是底物诱导酶。还探讨了培养基中不同的氮源组成对细胞生物量及ｐＨ的影响。     

Light and dark assimilation of nitrate in plants

Abstract. Heterotrophic assimilation of nitrate in roots and leaves in darkness is closely linked with the oxidative pentose phosphate pathway. The supply of glucose-6-phosphate to roots and chloroplasts in leaves in darkness is essential for assimilation of nitrite into amino acids. When green leaves are exposed to light, the key enzyme, glucoses-phosphate dehydrogenase, is inhibited by reduction with thioredoxin. Hence the dark nitrate assimilatory pathway is inhibited under photoautotrophic conditions and replaced by regulatory reactions functioning in light. On account of direct photo-synthetic reduction of nitrite in chloroplasts and availability of excess NADH for nitrate reduclase, the rate of nitrate assimilation is extremely rapid in light. Under dark anaerobic conditions also nitrate is equally rapidly reduced to nitrite on account of abolition of competition for NADH between nitrate reductase and mitochondrial oxidation.     

Regulation of nitrate reductase cellular levels in the cyanobacteria Anabaena variabilis and Synechocystis sp.

Abstract The effect of the nitrogen source on the cellular activity level of assimilatory nitrate reductase in the cyanobacteria Anabaena variabilis (ATCC29413) and Synechocystis sp. (PCC6714) has been examined. In the filamentous N₂-fixing A. variabilis , nitrate behaved as a nutritional inducer of nitrate reductase, with ammonium acting (via products of its assimilation) as an antagonist with regard to nitrate. Ammonium-promoted repression of nitrate reductase was also evident in the unicellular non-nitrogen fixer Synechocystis , but in this strain nitrate was not required as an obligatory inducer.     

Nitrate uptake and reduction in higher and lower plants

The nitrogen compounds nitrate and ammonium are the minerals that plants need in large quantities and which limit their growth in temperate zones. The nitrate assimilation pathway starts with nitrate uptake followed by nitrate reduction resulting in ammonium which is fixed into the amino acids glutamine and glutamate in most plants. This review concentrates on nitrate uptake and nitrate reduction with respect to higher and lower plants. The physiology and the progress in molecular approaches of both processes are considered. For nitrate uptake the well‐established uptake systems are discussed and special attention is drawn to nitrate sensing and the nitrate carrier. Knowledge, particularly on nitrate sensing is rare, but it seems to be the first step in a signal transduction chain triggered by nitrate. Therefore further work should consider this topic more frequently. For nitrate reductase the focus is on the post‐translational modification as a regulatory tool for nitrate assimilation, on the intersections of carbon and nitrogen metabolism and on the molecular approaches. A few remarks on how environmental conditions affect nitrate assimilation are also included. Further progress is needed to understand the transduction of positive and negative signals from the environment affecting the expression of genes coding for the nitrate assimilating pathway.     

Inactivation of nitrate reductase from wheat and rice leaves

In fresh leaves, the inactivation of nitrate reductase was rapid at high temperatures as compared to low temperatures. In leaves subjected to freeze-thaw treatment, the loss of enzyme activity was extremely rapid particularly at high temperatures. Pre-incubation with NADH not only protected the enzyme against inactivation, but also stimulated its activity. In dialysed extracts of rice leaves, NADH alone offered some protection while nitrate alone did not protect the enzyme from inactivation. Addition of both NADH and nitrate during pre-incubation enhanced the enzyme activity considerably. It is suggested that stimulation of nitrate reduction by NADH and nitrate may be of physiological significance to the plant, in the sense that in the presence of sufficient supplies of reluctant and nitrate, the process of nitrate assimilation would be accelerated.     

植物中硝酸还原酶和亚硝酸还原酶的作用

植物通过硝酸盐同化途径以硝酸盐和氨的形式吸收氮元素。硝酸盐的同化是一个受到严格控制的过程,其中两个先后参加反应的酶——硝酸还原酶(NR)和亚硝酸还原酶(NiR)对初级氮的同化起主要调控。在高等植物中,NR和NiR基因的转录及转录后加工受到各种内在和外在因素的影响,翻译后调控是消除亚硝酸盐积累的重要机制。随着分子生物学技术的发展,可以更容易地通过突变体和转基因方式来研究NR和NiR基因的调控。     

Regulation of nitrate reductase activity in higher plants

Nitrate reductase is one of the most important enzymes in the assimilation of exogenous nitrate—the predominant form of nitrogen available to green plants growing in soil. Activity of this enzyme in plants gives a good estimate of the nitrogen status of the plant and is very often correlated with growth and yield. Although it is difficult to explain the physiological significance and the mechanism of effects of several factors on the enzyme activity, in some cases suitable postulates have been advanced. In general, the enzyme activity in a plant tissue is a balance between its relative rates of synthesis/degradation and activation/inactivation. Factors may affect the overall activity by interfering with either of these processes.     

Changes in the activities of nitrogen assimilation enzymes ofLolium perenne L. during regrowth after cutting

The activities of key enzymes involved in N assimilation were investigated after defoliation of 6-week-old ryegrass plants grown in water culture conditions. In a first experiment, nitrate reductase, glutamine synthetase and glutamate dehydrogenase activities were measured in roots, stubble and leaves on the day of cutting and at 7-day intervals over the following 5-week period of regrowth. Ammonia assimilation enzymes showed little change whereas the nitrate reductase activity sharply decreased 2 weeks after clipping. In a second experiment, the nitrate reductase activity was measured at 2- or 3-day intervals 1 week before and 3 weeks after clipping.In vivo andin vitro assays both showed an increasing activity in leaves up to 8 days after cutting while root activity decreased. The opposite changes then occurred and both organs recovered their initial nitrate reductase activity levels after 12–14 days of regrowth. These fluctuations in nitrate reductase activity were considered to be related to the capacity for C assimilation and the nitrate availability.     

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana Plumbaginifolia

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.     

Regulation of nitrate reductase by nitric oxide in Chinese cabbage pakchoi (Brassica chinensis L.)

Nitrate reductase (NR), a committed enzyme in nitrate assimilation, involves generation of nitric oxide (NO) in plants. Here we show that the NR activity was significantly enhanced by the addition of NO donors sodium nitroprusside (SNP) and NONOate (diethylamine NONOate sodium) to the culturing solution, whereas it was decreased by NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, both NO gas and SNP directly enhanced but cPTIO inhibited the NR activities of crude enzyme extracts and purified NR enzyme. The cPTIO terminated the interaction between NR-generated NO and the NR itself. Furthermore, the NR protein content was not affected by the SNP treatment. The investigation of the partial reactions catalysed by purified NR using various electron donors and acceptors indicated that the haem and molybdenum centres in NR were the two sites activated by NO. The results suggest that the activation of NR activity by NO is regulated at the post-translational level, probably via a direct interaction mechanism. Accordingly, the concentration of nitrate both in leaves and roots was decreased after 2 weeks of cultivation with SNP. The present study identifies a new mechanism of NR regulation and nitrate assimilation, which provides important new insights into the complex regulation of N-metabolism in plants.     

Molecular approaches to the analysis of nitrate assimilation

Abstract. The application of molecular approaches such as mutant analysis and recombinant DNA technology, in conjunction with immunology, are set to revolutionize our understanding of the nitrate assimilation pathway. Mutant analysis has already led to the identification of genetic loci encoding a functional nitrate reduction step and is expected to lead ultimately to the identification of genes encoding nitrate uptake and nitrite reduction. Of particular significance would be identification of genes whose products contribute to regulatory networks controlling nitrogen metabolism. Recombinant DNA techniques are particularly powerful and have already allowed the molecular cloning of the genes encoding the apoprotein of nitrate reductase and nitrite reductase. These successes allow for the first lime the possibility to study directly the role of environmental factors such as type of nitrogen source (NO₃⁻ or NH₄⁺) available to the plant, light, temperature water potential and CO₂ and O₂ tensions on nitrate assimilation gene expression and its regulation at the molecular level. This is an important advance since our current understanding of the regulation of nitrate assimilation is based largely on changes of activity of the component steps. The availability of mutants, cloned genes, and gene transfer systems will permit attempts to manipulate the nitrate assimilation pathway.     

Characterization of the Nitrate-Nitrite Transporter of the Major Facilitator Superfamily (the nrtP Gene Product) from the Cyanobacterium Nostoc punctiforme Strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

植物氮代谢硝酸还原酶水平调控机制的研究进展

氮代谢是植株体内最基本的物质代谢之一,硝酸还原酶是植物氮代谢的关键酶。主要对植物氮代谢在硝酸还原酶水平上调控的研究新进展,尤其是其合成／降解及活性调控机制进行了较为系统的综述。硝酸还原酶合成的调控主要发生在转录水平和翻译水平上,硝酸还原酶降解的调控主要发生在翻译后水平上,同时NO3^-及光在硝酸还原酶转录水平调控上的作用重大,硝酸还原酶编码基因转录的mRNA的稳定性强弱影响植物的氮代谢,而影响mRNA稳定性的因素很多,机理复杂;磷酸化／去磷酸化在硝酸还原酶活性调控中占举足轻重的地位,研究也比较深入。钝化蛋白也能够影响硝酸还原酶活性,许多小分子物质对硝酸还原酶活性有影响。     

Induction of nitrate assimilation in the cyanobacterium Anabaena variabilis

Filaments of Anabaena variabilis Kütz strain ATCC 29413 grown in the absence of nitrate contain nitrate reductase that is active in permeabilized filaments, but not in intact, living filaments until they have been incubated for about 40 min in the presence of nitrate. The delayed acquisition of the ability to reduce nitrate is insensitive to chloramphenicol. Thus, switching on of enzyme activity in the presence of nitrate does not involve protein synthesis and nitrate reductase activity is not regulated by the amount of enzyme present.     

Chromosomal location and function of genes affecting Pseudomonas aeruginosa nitrate assimilation

Seven known genes control Pseudomonas aeruginosa nitrate assimilation. Three of the genes, designated nas, are required for the synthesis of assimilatory nitrate reductase: nasC encodes a structural component of the enzyme; nasA and nasB encode products that participate in the biosynthesis of the molybdenum cofactor of the enzyme. A fourth gene (nis) is required for the synthesis of assimilatory nitrite reductase. The remaining three genes (ntmA, ntmB, and ntmC) control the assimilation of a number of nitrogen sources. The nas genes and two ntm genes have been located on the chromosome and are well separated from the known nar genes which encode synthesis of dissimilatory nitrate reductase. Our data support the previous conclusion that P. aeruginosa has two distinct nitrate reductase systems, one for the assimilation of nitrate and one for its dissimilation.     

Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves

The effects of red and far-red light on the enhancement of in vitro nitrate reductase activity and on nitrate accumulation in etiolated excised maize leaves were examined. Illumination for 5 min with red light followed by a 4-h dark period caused a marked increase in nitrate reductase activity, whereas a 5-min illumination with far-red light had no effect on the enzyme activity. The effect of red light was completely reversed by a subsequent illumination with the same period of far-red light. Continuous far-red light also enhanced nitrate reductase activity. Both photoreversibility by red and far-red light and the operation of high intensity reaction under continuous far-red light indicated that the induction of nitrate reductase was mediated by phytochrome. Though nitrate accumulation was slightly enhanced by red and continuous far-red light treatments by 17% and 26% respectively, this is unlikely to account for the entire increase of nitrate reductase activity. The far-red light treatments given in water, to leaves preincubated in nitrate, enhanced nitrate reductase activity considerably over the dark control. The presence of a lag phase and inhibition of increase in enzyme activity under continuous far-red light-by tungstate and inhibitors of RNA synthesis and protein synthesis-rules out the possibility of activation of nitrate reductase and suggests de novo synthesis of the enzyme affected by phytochrome.