Effect of sulfur and nitrogen nutrition on derepression of ferredoxin-sulfite reductase in leek seedlings

The ferredoxin-sulfite reductase (Fd-SiR; hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1) activities of shoot and root of leek (Allium tuberosum) were increased by sulfate limitation in the early stage of growth. Western blot analysis demonstrated an increased amount of SiRs in root under sulfate limitation, suggesting that SiRs were derepressed. The derepression was observed in shoot when 1.5 mM nitrate was supplied to the plants under sulfate limitation, and clearly in root when 15 mM nitrate was supplied under sulfate limitation. When nitrate was absent from the nutrient solution, the SiR activity in both tissues was very low. Combined with the results of the sulfate- or nitrate-limitation experiments, it is suggested that the degree of the derepression of SiR in both tissue under sulfate limitation is affected by the concentration of nitrate, and further that the mechanism of regulation of the SiR activity is different in each tissue. The decreases in the ratios of the total SiR activities (shoot/root) in the latter stage of seedling growth indicate that root play a very important role in sulfate assimilation.     

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。     

Isolation,sequence and expression in Escherichia coli of the nitrite reductase gene from the filamentous,thermophilic cyanobacterium Phormidium laminosum

The nitrite reductase (NiR) gene (nirA) has been isolated and sequenced from the filamentous, thermophilic non-N₂-fixing cyanobacterium Phormidium laminosum. Putative promoter-like and Shine-Dalgarno sequences appear at the 5 end of the 1533 bp long nir-coding region. The deduced amino acid sequence of NiR from P. laminosum corresponds to a 56 kDa polypeptide, a size identical to the molecular mass previously determined for the pure enzyme, and shows a high identity with amino acid sequences from ferredoxin-dependent NiR. This cyanobacterial NiR gene has been efficiently expressed in Escherichia coli DH5 from the E. coli lac promoter and probably from the P. laminosum NiR promoter.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - NiR nitrite reductase - NR nitrate reductase - NT nitrate transport - SiR sulfite reductase     

Cysteine does not repress adenosine-5′-phosphosulfate reductase through its conversion to either sulfate or glutathione

Cysteine (Cys) represses the activity of several key regulatory enzymes in the plant sulfate assimilatory pathway. However, it is not clear whether this effect arises from Cys itself or through its conversion to either sulfate or glutathione (GSH). Therefore, we examined this phenomenon by analyzing the activity of adenosine-5′-phosphosulfate (APS) reductase. Both APS reductase (AR) activity and mRNA levels were decreased by treatingArabidopsis thaliana roots with 1 mM Cys. The intracellular sulfate concentration was not affected, whereas enzymatic activity and, to some extent, the mRNA level, declined. Cys treatment in sulfur-starved plants also diminished both parameters. However, this response to Cys was more efficient than when plants were treated with an equal amount of sulfate. When Cys was removed from both Cys-and sulfate-fed plants, AR activity was recovered; the same removal of sulfate was not so effective. Moreover, buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, did not influence the repression of AR by Cys. Finally the AR enzyme was inhibited by cysteinein vitro. These results indicate that Cys represses AR by inhibiting mRNA expression and by directly repressing enzymatic activity, rather than through its conversion to either sulfate or GSH.     

Nitrate assimilation in rice seedlings as affected by mineral nutrition

Deficiencies of each macronutrient (N, P, K, Ca, Mg and Fe)in the culture solution depressed the specific activities ofnitrate reductase (NR) and nitrite reductase (NiR) from riceseedlings. Nitrate and potassium deficiencies especially loweredNR induction, whereas phosphorus deficiency caused the leastdecrease in enzyme induction. On the other hand the activityof NiR was decreased most by deficiencies of nitrate and phosphorus.Potassium deficiency was not as effective in suppressing theinduction of NiR. Sulfur deficiency slightly promoted the inductionof both NR and NiR. Generally, micronutrient deficiencies didnot affect either enzyme. NR induction was slightly decreasedby B, Zn, Cu and Mo deficiencies, and increased by Mn deficiency;whereas NiR activity was slightly increased by B and Cu deficiencies,and was not affected by other micronutrients. Nitrate contentwas decreased by deficiencies of N, P, K, Ca, and micronutrients,and unaffected by Mg, Fe and S deficiencies. Glutamic acid dehydrogenase(GDH) activity was increased by N, Fe and P deficiencies, anddecreased by Mo and Zn deficiencies, and unaffected by othernutrient treatments. (Received August 25, 1976; )     

Regulation of sulfate assimilation by nitrogen and sulfur nutrition in poplar trees

Plants cover their need for sulfur by taking up inorganic sulfate, reducing it to sulfide, and incorporating it into the amino acid cysteine. In herbaceous plants the pathway of assimilatory sulfate reduction is highly regulated by the availability of the nutrients sulfate and nitrate. To investigate the regulation of sulfate assimilation in deciduous trees we used the poplar hybrid Populus tremula × P. alba as a model. The enzymes of the pathway are present in several isoforms, except for sulfite reductase and -glutamylcysteine synthetase; the genomic organization of the pathway is thus similar to herbaceous plants. The mRNA level of APS reductase, the key enzyme of the pathway, was induced by 3 days of sulfur deficiency and reduced by nitrogen deficiency in the roots, whereas in the leaves it was affected only by the withdrawal of nitrogen. When both nutrients were absent, the mRNA levels did not differ from those in control plants. Four weeks of sulfur deficiency did not affect growth of the poplar plants, but the content of glutathione, the most abundant low molecular thiol, was reduced compared to control plants. Sulfur limitation resulted in an increase in mRNA levels of ATP sulfurylase, APS reductase, and sulfite reductase, probably as an adaptation mechanism to increase the efficiency of the sulfate assimilation pathway. Altogether, although distinct differences were found, e.g. no effect of sulfate deficiency on APR in poplar leaves, the regulation of sulfate assimilation by nutrient availability observed in poplar was similar to the regulation described for herbaceous plants.     

Sulfur starvation and restoration affect nitrate uptake and assimilation in rapeseed

We analyzed the effect of omission of sulfur (S) from the nutrient solution and then restoration of S-source on the uptake and assimilation of nitrate in rapeseed. Incubation in nutrient solution without S for 1–6 days led to decline in uptake of nitrate, activities, and expression levels of nitrate reductase (NR) and glutamine synthetase (GS). The nitrite reductase (NiR) and glutamate synthase (GOGAT) activities were not considerably affected. There was significant enhancement in nitrate content and decline in sulfate content. Evaluation of amino acid profile under S-starvation conditions showed two- to fourfold enhancement in the contents of arginine, asparagine and O-acetyl-l-serine (OAS), whereas the contents of cysteine and methionine were reduced heavily. When the S-starved plants were subjected to restoration of S for 1, 3, 5, and 7 days, activities and expression levels of NR and GS recovered within the fifth and seventh days of restoration, respectively. Exogenous supply of metabolites (arginine, asparagine, cysteine, glutamine, OAS, and methionine) also affected the uptake and assimilation of nitrate, with a maximum for OAS. These results corroborate the tight interconnection of S-nutrition with nitrate assimilation and that OAS plays a major role in this regulation. The study must be helpful in developing a nutrient-management technology for optimization of crop productivity.     

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.     

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide and O-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. Using Lemna minor, we analyzed the effects of omission of CO(2) from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5'-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO(2) led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO(2) on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO(2), APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO(2) also recovered both enzyme activities, with OAS again influenced only APR. (35)SO(4)(2-) feeding showed that treatment in air without CO(2) severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of (35)S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of (35)S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.     

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     

Nitrate assimilation during the anaerobic germination of rice: expression of ferredoxin-dependent glutamate synthase

Ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) is the last enzyme involved in the pathway of nitrate assimilation in higher plants. This paper describes the synthesis and expression of the enzyme in anaerobic coleoptiles of rice (Oryza sativa L.) and its regulation by exogenous nitrate. The activity of Fd-GOGAT was strongly inhibited by cycloheximide between 4 and 9 d of anaerobic germination. The addition of nitrate slightly increased, in the first 5 h, the specific activity of Fd-GOGAT as well as the amount of a 160-kDa protein specifically immunoprecipitated with anti-Fd-GOGAT serum. Northern blot analysis, performed with a specific riboprobe, showed the presence of mRNA of the expected size and the inductive effect of nitrate. The role of Fd-GOGAT is discussed in relation to the anaerobic assimilation of nitrate by rice coleoptiles.Abbreviations CHX cycloheximide - Fd ferredoxin - GOGAT glutamate synthase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase The authors wish to thank Dr. J. Turner (Rothamsted Experimental Station, Harpenden, UK) for providing Fd-GOGAT antibody and Dr. H. Sakakibara (Nagoya University, Nagoya, Japan) for Fd-GOGAT clone. This research was supported by the National Research Council of Italy, special project RAISA, sub-projekt N. 2, paper N. 2174.     

Effect of applied nitrate on enzymes of ammonia assimilation in nodules ofCicer arietinum L.

Summary The relationship between N₂-fixation, nitrate reductase and various enzymes of ammonia assimilation was studied in the nodules and leaves ofC. arietinum. In the nodules of the plants growing on atmospheric nitrogen, maximum activities of glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), asparagine synthetase (AS) and aspartate aminotransferase (AAT) were recorded just prior to maximum activity of nitrogenase. In nitrate fed plants, the first major peak of GDH and AS coincided with that of nitrate reductase in the nodules. With the exception of AS, application of nitrate decreased the activities of all these enzymes in nodules but not in leaves. Activities of GS, GOGAT and AAT were affected to much greater extent than that of GDH. On comparing the plants grown without nitrate and those with nitrate, the ratios of the activities of GDH/GS and GDH/GOGAT in nitrate given plants, increased by 4 and 12 fold, respectively. The results presented in this paper suggest that in nodules of nitrate fed plants, assimilation of ammonia via GDH assumes much greater importance.     

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.     

Regulation of nitrate assimilation in the cyanobacterium Phormidium laminosum

The intracellular ratio of 2-oxoglutarate to glutamine has been analyzed under nutritional conditions leading to different activity levels of nitrate-assimilating enzymes in Phormidium laminosum (Agardh) Gom. This non-N₂-fixing cyanobacterium adapted to the available nitrogen source by modifying its nitrate reductase (NR; EC 1.7.7.2), nitrite reductase (NiR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) activities. The 2-oxoglutarate/glutamine ratio was similar in cells adapted to grow with nitrate or ammonium. However, metabolic conditions that increased this ratio [i.e., nitrogen starvation or l-methionine-d,l-sulfoximine (MSX) treatment] corresponded to high activity levels of NR, NiR, GS (except in MSX-treated cells) and glutamate synthase (GOGAT; EC 1.4.7.1). By contrast, metabolic conditions that diminished this ratio (i.e., addition of ammonium to nitrate-growing cells or addition of nitrate or ammonium to nitrogen-starved cells) resulted in low activity levels. The variation in the 2-oxoglutarate/glutamine ratio preceded the changes in enzyme activities. These results suggest that changes in the 2-oxoglutarate/glutamine ratio could be the signal that triggers the adaptation of P. laminosum cells to variations in the available nitrogen source, as occurs in enterobacteria.Abbreviations Chl chlorophyll - GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - GS glutamine synthetase (EC 6.3.1.2) - MSX l-methionine-d,l-sulfoximine - NiR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.7.7.2) - TP total protein This work has been partially supported by grants from the Spanish Ministry of Education and Science (DGICYT PB88-0300 and PB92-0464) and the University of the Basque Country (042.310-EC203/94). M.I.T. was the recipient of a fellowship from the Basque Government.     

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.     

Targeted knock-out of a gene encoding sulfite reductase in the moss Physcomitrella patens affects gametophytic and sporophytic development

A key step in sulfate assimilation into cysteine is the reduction of sulfite to sulfide by sulfite reductase (SiR). This enzyme is encoded by three genes in the moss Physcomitrella patens. To obtain a first insight into the roles of the individual isoforms, we deleted the gene encoding the SiR1 isoform in P. patens by homologous recombination and subsequently analysed the ΔSiR1 mutants. While ΔSiR1 mutants showed no obvious alteration in sulfur metabolism, their regeneration from protoplasts and their ability to produce mature spores was significantly affected, highlighting an unexpected link between moss sulfate assimilation and development, that is yet to be characterized.