Interaction between electron transport at the plasma membrane and nitrate uptake by maize (Zea mays L.) roots

Summary In the present study nitrate uptake by maize (Zea mays L.) roots was investigated in the presence or absence of ferricyanide (hexacyanoferrate III) or dicumarol. Nitrate uptake caused an alkalization of the medium. Nitrate uptake of intact maize seedlings was inhibited by ferricyanide while the effect of dicumarol was not very pronounced. Nitrite was not detected in the incubation medium, neither with dicumarol-treated nor with control plants after application of 100 M nitrate to the incubation solution. In a second set of experiments interactions between nitrate and ferricyanide were investigated in vivo and in vitro. Nitrate (1 or 3 mM) did neither influence ferricyanide reductase activity of intact maize roots nor NADH-ferricyanide oxidoreductase activity of isolated plasma membranes. Nitrate reductase activity of plasma-membrane-enriched fractions was slightly stimulated by 25 M dicumarol but was not altered by 100 M dicumarol, while NADH-ferricyanide oxidoreductase activity was inhibited in the presence of dicumarol. These data suggest that plasma-membrane-bound standard-ferricyanide reductase and nitrate reductase activities of maize roots may be different. A possible regulation of nitrate uptake by plasmalemma redox activity, as proposed by other groups, is discussed.Abbreviations ADH alcohol dehydrogenase - HCF III hexacyanoferrate III (ferricyanide) - ME NADP-dependent malic enzyme - NR nitrate reductase - PM plasma membrane - PM NR nitrate reductase copurifying with plasma membranes     

Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize

To enhance our understanding of the genetic basis of nitrogen use efficiency in maize (Zea mays), we have developed a quantitative genetic approach by associating metabolic functions and agronomic traits to DNA markers. In this study, leaves of vegetative recombinant inbred lines of maize, already assessed for their agronomic performance, were analyzed for physiological traits such as nitrate content, nitrate reductase (NR), and glutamine synthetase (GS) activities. A significant genotypic variation was found for these traits and a positive correlation was observed between nitrate content, GS activity and yield, and its components. NR activity, on the other hand, was negatively correlated. These results suggest that increased productivity in maize genotypes was due to their ability to accumulate nitrate in their leaves during vegetative growth and to efficiently remobilize this stored nitrogen during grain filling. Quantitative trait loci (QTL) for various agronomic and physiological traits were searched for and located on the genetic map of maize. Coincidences of QTL for yield and its components with genes encoding cytosolic GS and the corresponding enzyme activity were detected. In particular, it appears that the GS locus on chromosome 5 is a good candidate gene that can, at least partially, explain variations in yield or kernel weight. Because at this locus coincidences of QTLs for grain yield, GS, NR activity, and nitrate content were also observed, we hypothesize that leaf nitrate accumulation and the reactions catalyzed by NR and GS are coregulated and represent key elements controlling nitrogen use efficiency in maize.     

Nitrogen assimilation in Lolium perenne colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum

To investigate nitrogen assimilation in Lolium perenne L. colonized by the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum (Thax. sensu Gerd.), nitrate uptake, key enzyme activities, and ¹⁵N incorporation into free amino acids were measured. After a 4-h labelling period with [¹⁵N]nitrate, ¹⁵N content was higher in roots and shoots of AM-plants than in those of control plants. Glutamine synthetase (GS) and nitrate reductase (NR) activities were increased in shoots of AM-plants, but not in roots. More label was incorporated into amino acids in shoots of AM plants. Glutamine, glutamate, alanine and γ-aminobutyric acid were the major sinks for ¹⁵N in roots and shoots of control and AM plants. Interactions between mycorrhizal colonization, phosphate and nitrate nutrition and NR activity were investigated in plants which received different amounts of phosphate or nitrate. In shoots of control plants, NR activity was not stimulated by high levels of phosphate nutrition but was stimulated by high levels of nitrate. At 4 m M nitrate in the nutrient solution, NR activity was similar in control and AM plants. We concluded that mycorrhizal effects on nitrate assimilation are not mediated via improved phosphate nutrition, but could be due to improved nitrogen uptake and translocation.     

Iron Mediated Nitrate Reductase Activity in Different Parts of Young Maize Seedlings

Incubation of 5-d-old maize seedlings in the half-strength Hoagland's nutrient solution containing 10 mM KNO₃ with FeCl₃ or FeSO₄ (0.5 or 2.0 mM) caused a significant increase in nitrate reductase (NR) activity and slightly increased total protein content in root, shoot and scutellum. In case of root, NADPH:NR activity was inhibited contrary to the NADH:NR activity. In spite of NR activity, nitrate uptake was inhibited from 13 to 37 % by the iron. The results presented demonstrate an isoform specific, organ specific, and to some extent salt specific responses of NR to iron.     

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.     

DCMU inhibits in vivo nitrate reduction in illuminated barley (C(3)) leaves but not in maize (C(4)): a new mechanism for the role of light?

The leaves of C(4) plants possess a superior metabolic efficiency not only in terms of photosynthetic carbon assimilation, but also in terms of inorganic nitrogen assimilation, when compared to C(3)plants. In vivo nitrate assimilation efficiency of leaves is dependent on light, but the obligatory presence of light has been debated and its role remains confounded. This problem has not been addressed from the standpoint of the C(3) vs. C(4) nature of the species investigated, which may actually hold the key to resolve the controversy. Here, we present the first report providing evidence for differential photo-regulation of leaf nitrate reduction in barley ( Hordeum vulgare L.) vs. maize ( Zea mays L.) plants, which may help explain the superior nitrogen-use efficiency (and hence superior productivity) of maize plants. The novel finding that carbohydrate-depleted maize leaves were able to reduce nitrate when photosynthesis was inhibited by 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) in the presence of light, raises a very important question about the possibilities of a new photo-regulatory mechanism for supporting nitrate reduction in maize leaves operating independently of photosynthetic carbon dioxide fixation. On the other hand, leaves of barley could not carry out any in vivo nitrate assimilation, whatsoever, under these conditions. We find another fundamental difference between the two species in terms of differential regulation of nitrate reductase (NR; EC 1.6.6.1). In barley leaves, NR activity and activation state remained unaffected due to DCMU, but in sharp contrast, both were appreciably upregulated in maize. Collectively, the results indicate that enzyme capacity is not limiting for nitrate reduction in leaves, as the NR activity was higher in barley than in maize. The maize leaves may have had a selective advantage due to C(4) morphology/metabolism in terms of maintaining a better reductant/carbon skeleton supply for nitrate reduction.     

The Relation Between the Denatured Nitrate Reductase and the Nitrate Reductase-Inhibiting Protein

Wheat seedlings (Triticum aestivum var. Feng-chan 3 ) were grown on water or KNO3 medium at 24℃. Before the second leaf had grown out, the shoots of the seedlings were cut down and ground with a little quartz sand. The homogenates were filtered through a layer of nylon cloth before centrifugation at 10000g for15 min. The supematant fraction was collected (crude nitrate reductase). Isolation and purification of nitrate reductase (NR) were according to Sherrard et al with a bit modifications. Ammonium sulfate was added to the crude NR and the enzyme protein was precipitated between 20%—40% saturation. After column chromatography on Sephadex G-25, the protein was then subjected to further purification by affinity chromatography on a blue dextran-Sepharose 4B column. The fraction in the NADH (0.1 mM) eluate was the highly purified enzyme. The activity of the isolated NR was assayed in vitro according to the standard method, Nitrate reductase-inhibiting protein (NRIP) was isolated and purified according to Wallace with a little modifications. After fractional precipitation by ammonium sulfate, the protein precipitating between 20%–40% saturation was collected and dissolved in distilled water. Column chromatography on Sephadex G-100 and DEAE (DE-11) cellulose was separately used. After dialysis, condensation of the highly purified NRIP was carried out. Antiserum against NR was prepared by injecting 2 mL purified NR protein (88 nmol NO2-/30 min/0.2 mL) into a rabbit five times with an interval of 10 days. For all five injections, the enzyme was mixed with complete Freund's adjuvant. Bleeding was taken 30 days after the first injection. Antiserum against NRIP was prepared in the same way mentioned above, but purified NRIP was used instead of NR. Rocket immunoelectrophoresis was performed by the method described by Funkhouser. Agarose gels (1.5% W/V). which contained 30 mM Tris and 12.3 mM meleate (pH 8.6) and 0.2% (V/V) crude antiserum were placed on a glass plate. Wells were cut along one side of the plate and filled with 10, 20, 30, 40 μ 1of antigen. Electrophoresis was carried out at 3 mA, 10 V for 2 h at 4 ℃. The antigen-antibody reaction resulted in the formation of rocket shaped immunoprecipitates. After washing overnight in PBS the rockets were visualized by staining with coomassie blue. The procedure of immunodiffusion and immunoelectrophoresis was according to that of Clausen. Nitrate reductase is a very unstable enzyme, Our former paper showed that the crude NR lost its enzyme activity by about one half, after it had been maintained at room temperature for 30 min. In order to study the stability of NR. crude NR was prepared and kept at room temperature. After the enzyme activity had been completely lost, it was added to a fresh NR preparation with high activity. The inhibition effect of denatured enzyme was revealed according to the difference between plus or minus denatured enzymes. About 70%–80% NR activities were lost in the preparation to which 0.1 ml denatured enzyme had been added instead of 0.1 ml H2O. Therefore we think that the denatured enzyme itself behaved like an inhibiting protein of NR. Wallace demonstrated that there was an inactivating enzyme of NR in maize roots. Some characteristics of the enzyme investigated in several labs. According to Wallace's methods we got a purified NR-inactivating-protein (NRIP). Furthermore, a purified NR was obtained by an affinity-chromatography method (table 1). Single of either NR or NRIP appeared on the chromatography and their Rm were the same (fig. 2). It might conclude that the NRIP and denatured NR are the similar protein. The highly purified NR protein incubated for several hours at room tempetature also became an inhibitor (table 2). We, therefore, infer that the activated NR could be converted to NRIP at room temperature. Antiserum against NR was prepared by injecting purified NR into rabbit, and antiserum against NRIP was prepared by injecting purified NRIP. The anti-NR antibody and the anti-NRIP antibody were prepared as reagents to study the immunological relation between these two proteins. The antibody of NR gave a single precipitate band against purified NRIP and the antibody of NRIP had a similar precipitate band against purified NR (fig. 3 and 4). Rocket immunoelectrophoresis was performed. The antiserum against NR were added to agarose gel and 4 wells were filled with different amount of NRIP. The height of the rockets was increased with the amount of NRIP (fig. 5). All these results show the identity of the denatured NR and NRIP. The percent of inhibition of NRIP depended upon the concentration of NADH in the reaction mixture. Fig. 6 shows that the NRIP was a competitive inhibitor. The inhibitor and NR both competed for the same cofactor NADH. The percentage of inhibition was decreased when the concentration of NADH in the reaction system was increased. According to this result, we suggest that the NR protein has two active sites. One site binds with nitrate and the other with NADH. When the site bound with nitrate is damaged or changed, the enzyme protein can not catalyze nitrate reduction. However, the site binding with NADH is less labile and not affected by incubation at room temperature, therefore NADH can still be bound on the denatured NR protein. If the concentration of NADH in this reaction system is limited, the nitrite formation decreases. This explains how the effect of NRIP can be overcome in the reaction system at higher concentration of NADH.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

Nitrate reductase and glutamate dehydrogenase activities in Skeletonema costatum as measures of nitrogen assimilation rates

Nitrate reductase (NADH-NR) and glutamate dehydrogenase (NADPH-GDH)activities were measured in Skeletonema costatum (Grev.) Clevein ammonium and nitrate limited continuous cultures before andafter additions of nitrate and/or ammonium. Comparisons of enzymicactivity with nitrogen uptake and assimilation rates, externaland internal nitrate concentrations, and external ammonium concentrationswere made in order to assess the roles of NR and GDH in nitrogenassimilation and to determine their suitability as measuresof nitrogen assimilation rates. NR activity appeared to be inducedby internal rather than external nitrate concentrations. Ammoniumin the medium reduced NR activity under some environmental conditions,but not others. However, ammonium acted indirectly, perhapsby causing the accumulation of an internal pool of an intermediateof ammonium assimilation. NR activity was found to approximatenitrate assimilation rates during growth limited by the nitratesupply and undeT some conditions in the presence of high nitrateand ammonium concentrations in the medium. Under other environmentalconditions, NR activity did not agree with nitrate assimilationrates; a second nitrate reducing mechanism may operate whenthese conditions prevail. GDH activities were consistently low,representing less than 5% of the ammonium uptake and assimilationrates. Consequently, it is proposed that ODH is not the primaryammonium assimilating enzyme under most environmental conditionsand cannot be used as a measure of ammonium assimilation. ¹ Contribution number 1095 from the Department of Oceanography,University of Washington     

玉米ST和ATPS部分cDNA序列克隆及分析

硫酸盐转运蛋白(ST)和ATP硫酸化酶(ATPS)是根系吸收硫酸盐和植物体内硫酸盐同化过程的关键蛋白和酶,在硫酸盐的生物转运过程中具有重要作用.以水培玉米农大108根系为材料,并根据已报道的玉米的硫酸盐转运蛋白和ATP硫酸化酶基因保守序列分别设计PCR引物对,采用RT-PCR方法克隆到783 bp和820 bp的部分硫酸盐转运蛋白和ATP硫酸化酶cDNA片段,分别命名为ST_ND108和ATPS_ND108.序列分析和比对结果显示,ST_ND108与已报道的玉米和水稻的高亲和型硫酸盐转运蛋白基因同源性分别为99%和85%;而ATPS_ND108与已报道的玉米ATP硫酸化酶基因同源性达到97%,进化树聚类分析和预测氨基酸的BLAST结果证实ST_ND108为高亲和性硫酸盐转运蛋白基因片段,ATPS_ND108为质体ATP硫酸化酶基因片段.     

Rapid and slow modulation of nitrate reductase activity in the red macroalga <Emphasis Type="Italic">Gracilaria chilensis</Emphasis> (Gracilariales,Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.     

Recovery of uptake and assimilation of nitrate in Scenedesmus sp. previously exposed to elevated levels of Cu2+ and Zn2+

A study of the effects of elevated levels of Cu2+ and Zn2+ on NO3- uptake and nitrate reductase (NR) activity in Scenedesmus sp. was carried out. The two metals inhibited NR and NO3- uptake in a concentration-dependent manner, with the latter process being inhibited more strongly than the former. After withdrawal of metal stress, NR activity and NO3- uptake recovered in a metal ion concentration-dependent manner. Dark pretreatment of the alga enhanced the toxic effects of the metal ions on NR activity and NO3- uptake. The recovery from metal stress was slower in the dark-pretreated cells in comparison to the light-pretreated cells. No recovery of NR and NO3- uptake occurred in the presence of the photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), suggesting that photosynthesis was required for the recovery from metal stress. Cycloheximide blocked the recovery of NR activity in metal-treated alga, suggesting that new enzyme synthesis was required for the recovery from metal stress.     

Nitrate effects on nitrate reductase activity and nitrite reductase mRNA levels in maize suspension cultures

Nitrate reductase (NR) activity and nitrite reductase (NiR) mRNA levels were monitored in Black Mexican Sweet maize (Zea mays L.) suspension cultures after the addition of nitrate. Maximal induction occurred with 20 millimolar nitrate and within 2 hours. Both NR and NiR mRNA were transiently induced with levels decreasing after the 2 hours despite the continued presence of nitrate in the medium. Neither ammonia nor chlorate prevented the induction of NR. Furthermore, removal of nitrate, followed by its readdition 22 to 48 hours later, did not result in reinduction of activity or message. NR was synthesized de novo, since cycloheximide completely blocked its induction. Cycloheximide had no effect on the induction of NiR mRNA or on the transient nature of its induction. These results are similar to those reported previously for maize seedlings.     

Effects of asparagine,Irradiation and NADH on the nitrate reductase inWolffia microscopica

Nitrate concentration required for maximal extractable level of nitrate reductase (NR) inWolffia varies with the conditions prior to the nitrate treatment. Maximal enzyme activity is obtained at 2 mM nitrate concentration with asparagine grown plants and at 15 mM with nitrate grown. Both the level of enzyme activity and nitrate uptake by the tissue are increased by irradiation. The radiant energy induced increase in enzyme activity is not due to photosynthetic activity alone. An effect of radiant energy at the membrane level is sug gested. The extracted enzyme, which is labile, is protected and activated by NADH at 0 °C.     

Nitrate reductase activity and uptake of nitrate and oxygen as affected by nitrate supply to detached maize organs

Supply of 1, 2, 5, 10 or 20 mM nitrate to detached roots, scutella or shoots from 5- to 6-d-old Zea mays L. seedlings increased in vitro nitrate reductase (NR) activity in all the organs and NADPH specific NR (NADPH:NR) activity in roots and scutella but not in the shoots. Usually 2 to 5 mM nitrate supported maximum enzyme activity, the higher concentration did not increase it further. The protein content in the roots, scutella and shoots increased up to 5, 2 and 20 mM medium nitrate, respectively. Nitrate uptake also increased with increasing nitrate concentration in roots and shoots, but it increased only slightly in the scutella. In both roots and scutella, methionine sulfoximine had no effect, while cycloheximide and tungstate abolished nitrate induced NADH:NR activity completely and NADPH:NR partially. Methionine sulfoximine increased nitrate uptake by roots and scutella slightly, but other inhibitors had no effect. The depletion of dissolved oxygen from the medium was lower in the presence of nitrate than in its absence or in the presence of ammonium, especially in the scutella. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of NO3- transport and reduction on intracellular pH: an in vivo NMR study in maize roots

The effect of NO3- uptake on cellular pH was studied in maize roots by an in vivo 31P-NMR technique. In order to separate the effects on cytoplasmic pH due to NO3- uptake from those due to NO3- reduction, tungstate was used to inhibit nitrate reductase (NR). The results confirm that in maize roots tungstate inhibited NR activity. 15N-NMR in vivo experiments demonstrated the cessation of nitrogen flux from nitrate to organic compounds. Tungstate affected neither NO3- uptake nor the levels of the main phosphorylated compounds. Slight changes in cytoplasmic pH were observed during NO3- uptake and reduction (i.e. control). By contrast, in the presence of tungstate, a consistent decrease in cytoplasmic pH occurred. The vacuolar pH did not change in any of the conditions tested. These data show that NO3- uptake is an acidifying process and suggest a possible involvement of NO3- reduction in pH homeostasis. In the presence of NO3-, a transient depolarization of transmembrane electric potential difference (Em) was observed in all the conditions analysed. However, in tungstate-treated roots, a lesser depolarization accompanied by a greater ability to recover Em was found. This was related to a higher activity of the plasma membrane (PM) H+-ATPase. When NO3- was administered as potassium salt, its uptake increased and a greater depolarization of Em took place, whilst the changes in cytoplasmic pH were remarkably reduced, according to the central role played by K+ in the control of plasma membrane activities and cell pH homeostasis. A possible involvement of cytoplasmic pH in the control of PM H+-ATPase expression during nitrate exposure is suggested.     

Comparative stability of ammonium- and nitrate-induced nitrate reductase activity in maize leaves

Ammonium sulfate (5 mM) had no effect on nitrate reductase activity during a 3 hr dark incubation, but the enzyme was increased 2.5-fold during a subsequent 24 hr incubation of the maize leaves in light. The enzyme activity induced by ammonium ion declined at a slower rate under non-inducing conditions than that induced by nitrate. The decline in ammonium stimulated enzyme activity in the dark was also slower than that with nitrate. Further. cycloheximide accelerated the dark inactivation of the ammonium-enzyme while it had no effect on the nitrate-enzyme. The experiments demonstrate that increase in nitrate reductase activity by ammonium ion is different from the action of nitrate action.