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。     

Analysis of cis-acting DNA elements mediating induction and repression of the spinach nitrite reductase gene

The expression of nitrite reductase (NiR; EC 1.7.7.1), the second enzyme in the nitrate assimilatory pathway, is regulated by nitrate as well as by end-products of nitrate assimilation, namely, glutamine (Gln) and asparagine (Asn). Nitrate induces expression of the NiR gene. Previously, using deletion analysis of the spinach (Spinacia oleracea L.) NiR gene promoter in transgenic tobacco (Nicotiana tabacum L.) and in-vivo dimethyl sulfate footprinting, we had identified the region between −230 bp and −180 bp as being critical for nitrate inducibility of this gene. In the present study, we show that the region from +1 to +67, which forms part of its untranslated leader, is important for minimal induction in the presence of nitrate. Electrophoretic mobility shift assays reveal concentration-dependent and competitive binding of a factor in tobacco nuclear extracts to this region. In the presence of Gln or Asn, the expression of spinach NiR is repressed. This repression is observed with the full-length NiR promoter (−3100 bp) as well as with the shortest promoter (−230 bp) that gives nitrate induction, which includes the +67 bp leader sequence. The repressed expression of the gene is not the result of reduced nitrate accumulation in the presence of the nitrogen metabolites. Received: 2 December 1997 / Accepted: 20 January 1998     

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots

Summary Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.     

Interaction of sulfate assimilation with nitrate assimilation as a function of nutrient status and enzymatic co-regulation inbrassica juncea roots

The interaction of sulfate assimilation with nitrate assimilation inBrassica juncea roots was analyzed by monitoring the regulation of ATP sulfurylase (AS), adenosine-5’-phosphosulfate reductase (AR), sulfite reductase (SiR), and nitrite reductase (NiR). Depending on the status of sulfur and nitrogen nutrition, AS and AR activities and mRNA levels were increased by sulfate starvation but decreased by nitrate starvation. The activation of AS and AR by sulfate starvation was inhibited by sulfate/nitrate starvation. However, the rise in steady-state mRNA levels for AS and AR by sulfate starvation was not affected by sulfate/nitrate starvation. SiR gene expression was slightly activated by both sulfate starvation and sulfate/nitrate starvation, but was decreased by nitrate starvation. Although NiR gene expression was little affected by sulfate starvation, it was diminished significantly by either nitrate or nitrate/sulfate starvation. Cysteine (Cys) also decreased AS and AR activities and mRNA levels even when plants were simultaneously starved for sulfate; in contrast, both SiR and NiR gene expressions were only slightly, if at all, affected under the same conditions. This supports our conclusion that Cys, the end-product of sulfate assimilation, is the key regulatory signal. Moreover, SiR and NiR apparently are not the linking step in the co-regulation of sulfate and nitrate assimilation in plants.     

The effect of Glc6P uptake and its subsequent oxidation within pea root plastids on nitrite reduction and glutamate synthesis

In roots, nitrate assimilation is dependent upon a supply of reductant that is initially generated by oxidative metabolism including the pentose phosphate pathway (OPPP). The uptake of nitrite into the plastids and its subsequent reduction by nitrite reductase (NiR) and glutamate synthase (GOGAT) are potentially important control points that may affect nitrate assimilation. To support the operation of the OPPP there is a need for glucose 6-phosphate (Glc6P) to be imported into the plastids by the glucose phosphate translocator (GPT). Competitive inhibitors of Glc6P uptake had little impact on the rate of Glc6P-dependent nitrite reduction. Nitrite uptake into plastids, using (13)N labelled nitrite, was shown to be by passive diffusion. Flux through the OPPP during nitrite reduction and glutamate synthesis in purified plastids was followed by monitoring the release of (14)CO(2) from [1-(14)C]-Glc6P. The results suggest that the flux through the OPPP is maximal when NiR operates at maximal capacity and could not respond further to the increased demand for reductant caused by the concurrent operation of NiR and GOGAT. Simultaneous nitrite reduction and glutamate synthesis resulted in decreased rates of both enzymatic reactions. The enzyme activity of glucose 6-phosphate dehydrogenase (G6PDH), the enzyme supporting the first step of the OPPP, was induced by external nitrate supply. The maximum catalytic activity of G6PDH was determined to be more than sufficient to support the reductant requirements of both NiR and GOGAT. These data are discussed in terms of competition between NiR and GOGAT for the provision of reductant generated by the OPPP.     

Nitrogen Metabolism of Lemna minor. II. Enzymes of Nitrate Assimilation and Some Aspects of Their Regulation

In L. minor grown in sterile culture, the primary enzymes of nitrate assimilation, nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) change in response to nitrogen source. NR and NiR levels are low when grown on amino acids (hydrolyzed casein) or ammonia; both enzymes are rapidly induced on addition of nitrate, while addition of nitrite induces NiR only. Ammonia represses the nitrate induced synthesis of both NR and NiR.NADH dependent GDH activity is low when grown on amino acids and high when grown on nitrate or ammonia, but the activities of NADPH dependent GDH and Alanine dehydro-genase (AIDH) are much less affected by nitrogen source. NADH-GDH and AIDH are induced by ammonia, and it is suggested that these enzymes are involved in primary nitrogen assimilation.     

Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants

Nitrite reductase (NiR) is the second enzyme in the nitrate assimilatory pathway reducing nitrite to ammonium. The expression of the NiR gene is induced upon the addition of nitrate. In an earlier study, a 130 bp upstream region of the spinach NiR gene promoter, located between –330 to –200, was shown to be necessary for nitrate induction of -glucuronidase (GUS) expression in tissue-specific manner in transgenic tobacco plant [28]. To further delineate the cis-acting elements involved in nitrate regulation of NiR gene expression, transgenic tobacco plants were generated with 5 deletions in the–330 to –200 region of the spinach NiR gene promoter fused to the GUS gene. Plants with the NiR promoter deleted to –230 showed a considerable increase in GUS activity in the presence of nitrate, indicating that the 30 bp region between –230 to –200 is crucial for nitrate-regulated expression of NiR. In vivo DMS footprinting of the –300 to –130 region of the NiR promoter in leaf tissues from two independent transgenic lines revealed several nitrate-inducible footprints. Footprinting within the –230 to –181 region revealed factor binding to two adjacent GATA elements separated by 24 bp. This arrangement of GATA elements is analogous to cis-regulatory sequences found in the promoters of nitrate-inducible genes of Neurospora crassa, regulated by the NIT2 Zn-finger protein. The –240 to –110 fragment of the NiR promoter, which contains two NIT2 consensus core elements, bound in vitro to a fusion protein comprising the zinc finger domain of the N. crassa NIT2 protein. The data presented here show that nitrate-inducible expression of the NiR gene is mediated by nitrate-specific binding of trans-acting factors to sequences preserved between fungi and higher plants.     

Expression of a fully functional cd1 nitrite reductase from Pseudomonas aeruginosa in Pseudomonas stutzeri

Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.     

Molecular cloning of complementary DNA encoding maize nitrite reductase: molecular analysis and nitrate induction

Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize.     

Expression of the Arabidopsis thaliana invertase gene family

Two new loci have been found to be clustered with five other genes for the nitrate assimilation pathway in the Chlamydomonas reinhardtii genome. One gene, located close to the 3′-end of the high-affinity nitrate transporter (HANT) gene Nrt2;2, corresponds to the nitrite reductase (NiR) structural gene Nii1. This is supported by a number of experimental findings: (i) NiR-deficient mutants have lost Nii1 gene expression; (ii) Nii1 mRNA accumulation is co-regulated with the expression of other structural genes of the nitrate assimilation pathway; (iii) nitrite (nitrate) utilization ability is recovered in the NiR mutants by functional complementation with a wild-type Nii1 gene; (iv) the elucidated NII1 amino acid sequence is highly similar to that of the cyanobacterial and higher-plant enzyme, and contains the predicted domains for plastidic ferredoxin-NiRs. Thus, the mutant phenotype and the mRNA sequence and expression of the Nii1 gene have been unequivocally related. Accumulation of mRNA for the second locus identified, Lde1 (light-dependent expression), was not regulated by nitrogen, but like nitrate-assimilation clustered genes, its expression was down-regulated in the dark. Received: 27 November 1997 / Accepted: 19 January 1998     

木本植物阳生和阴生叶片叶绿体O_2和NO_2~-光还原作用

在有ＰＣＲ和ＰＣＯ环活性抑制剂甘油醛和光合磷酸化解偶联剂ＮＨ４ＣＬ存在下,比较了生长于 ３种 光环境的乔木黧蒴和灌木九节幼苗阳生和阴生叶片叶绿体的Ｏ２和ＮＯ２光还原速率。全自然光下两种 植物阳生叶片的叶绿体Ｏ２的光还原速率最高,占总光合电子传递活性的６６％－６８％,ＮＯ２光还原速率 也有类似趋势占总电子传递的１１％－１５％左右。３６％和１６％自然光下阴生叶片Ｏ２和ＮＯ２光还原 速率及Ｏ２光还原电子传递的比例显著降低,但ＮＯ２光还原电子传递的比例不受影响。与ＮＯ２光还原 相关的叶片ＮｉＲ和ＮＲ活性及ＮｉＲ／ＮＲ活性比也因叶片接受光强度大小而异,随光强减弱,黧蒴的 ＮｉＲ活性降低,九节的ＮＲ活性增高,但黧蒴的ＮＲ活性和九节的ＮｉＲ活性变化未达差异显著性。     

Nitrogen assimilation in plants: current status and future prospects

Nitrogen(N) is the driving force for crop yields; however, excessive N application in agriculture not only increases production cost, but also causes severe environmental problems. Therefore, comprehensively understanding the molecular mechanisms of N use efficiency(NUE) and breeding crops with higher NUE is essential to tackle these problems. NUE of crops is determined by N uptake, transport, assimilation, and remobilization. In the process of N assimilation, nitrate reductase(NR), nitrite redu...     

Screening for Arabidopsis mutants affected in the Nii gene expression using the Gus reporter gene

A mutant screen was developed to isolate Arabidopsis thaliana mutants affected in the regulation of the nitrate assimilation pathway. A fusion between the tobacco Nii1 gene (that encodes a foliar nitrite reductase involved in nitrate assimilation) and the Gus reporter gene was introduced into A. thaliana , and shown to be properly regulated by nitrate. Moreover, β -glucuronidase (GUS) activity in the transgenic plants was essentially detected in the cotyledons and leaves, showing that the organ-specific expression of the tobacco Nii1 gene was retained in Arabidopsis . M2 plantlets derived from mutagenized seeds homozygous for the Nii-Gus fusion were screened by histochemical staining of whole plates for GUS activity after growth on nitrate or glutamine. About 250 progenies were screened, leading to the isolation of plants showing an enhanced or reduced staining compared to the control non-mutagenized plants. Several mutants were analyzed for the transmission of the phenotype to the M3 generation, as well as for levels of GUS or nitrite reductase activities or mRNA levels. A major problem encountered during the screening was the high background of false positives that reproducibly showed altered GUS histochemical staining compared to control plants and did not, however, display any changes in GUS activity levels. One interesting family of mutants was isolated that overexpressed GUS activity and Nii mRNA in the absence of nitrate. These mutants turned out to be cnx mutants impaired in the molybdenum cofactor biosynthesis that is necessary for nitrate reductase activity. These results may indicate that active nitrate reductase is necessary for a correct regulation of nitrate assimilation genes by nitrate.     

Nitric oxide emission from tobacco leaves and cell suspensions: rate limiting factors and evidence for the involvement of mitochondrial electron transport

Quantitative data on nitric oxide (NO) production by plants, and knowledge of participating reactions and rate limiting factors are still rare. We quantified NO emission from tobacco (Nicotiana tabacum) wild-type leaves, from nitrate reductase (NR)- or nitrite reductase (NiR)-deficient leaves, from WT- or from NR-deficient cell suspensions and from mitochondria purified from leaves or cells, by following NO emission through chemiluminescence detection. In all systems, NO emission was exclusively due to the reduction of nitrite to NO, and the nitrite concentration was an important rate limiting factor. Using inhibitors and purified mitochondria, mitochondrial electron transport was identified as a major source for reduction of nitrite to NO, in addition to NR. NiR and xanthine dehydrogenase appeared to be not involved. At equal respiratory activity, mitochondria from suspension cells had a much higher capacity to produce NO than leaf mitochondria. NO emission in vivo by NiR-mutant leaves (which was not nitrite limited) was proportional to photosynthesis (high in light +CO(2), low in light -CO(2), or in the dark). With most systems including mitochondrial preparations, NO emission was low in air (and darkness for leaves), but high under anoxia (nitrogen). In contrast, NO emission by purified NR was not much different in air and nitrogen. The low aerobic NO emission of darkened leaves and cell suspensions was not due to low cytosolic NADH, and appeared only partly affected by oxygen-dependent NO scavenging. The relative contribution of NR and mitochondria to nitrite-dependent NO production is estimated.     

Molecular biology, genetics and regulation of nitrite reduction in higher plants

Nitrite reductase (ferredoxin:nitrite oxidoreductase, EC 1.6.6.1) carries out the six-electron reduction of nitrite to ammonium ions in the chloroplasts/plastids of higher plants. The complete or partial nucleotide sequences of a number of nitrite reductase apoprotein genes or cDNAs have been determined. Deduced amino acid sequence comparisons have identified conserved regions, one of which probably is involved in binding the sirohaem/4Fe4S centre and another in binding the electron donor, reduced ferredoxin. The nitrite reductase apoprotein is encoded by the nuclear DNA and is synthesised as a precursor carrying an N-terminal extension, the transit peptide, which acts to target the protein to, and within, the chloroplast/plastid. In those plants examined the number of nitrite reductase apoprotein genes per haploid genome ranges from one (barley, spinach) to four ( Nicotiana tabacum ). Mutants defective in the nitrite reductase apoprotein gene have been isolated in barley. During plastidogenesis in etiolated plants, synthesis of nitrite reductase is regulated by nitrate, light (phytochrome), and an uncharacterised 'plastidic factor' produced by functional chloroplasts. In leaves of green, white-light-grown plants up-regulation of nitrite reductase synthesis is achieved via nitrate and light and down-regulation by a nitrogenous end-product of nitrate assimilation, perhaps glutamine. A role for phytochrome has not been demonstrated in green, light-grown plants. Light regulation of nitrite reductase genes is related more closely to that of photosynthetic genes than to the nitrate reductase gene. In roots of green, white-light-grown plants nitrate alone is able to bring about synthesis of nitrite reductase, suggesting that the root may possess a mechanism that compensates for the light requirement seen in the leaf.     

Inhibition of tobacco nitrite reductase activity by expression of antisense RNA

A tobacco nitrite reductase (NiR) cDNA and its corresponding gene were isolated from cDNA and genomic libraries. An NiR antisense mRNA was expressed in transgenic tobacco under the control of a double 35S promoter. Transformants were obtained on a medium containing ammonium as the sole source of nitrogen. One plant growing normally on ammonium but displaying drastically reduced development and chlorotic leaves when grown on nitrate as the sole source of nitrogen was studied further. This plant accumulated nitrite fivefold over wild-type level and showed reduced amounts of ammonium (11% wild-type level), glutamine (19%), and total protein (8%). NiR mRNA and activity were below detectable levels. Under these conditions, nitrate reductase (NR) activity and mRNA were overexpressed, suggesting that N-metabolites resulting from nitrate reduction are responsible for the repression of the expression of the NR gene, independently from the presence or absence of a functional NR protein.