Nitrate reductase in agrostemma githago comparison of the inductive effects of nitrate and cytokinin

The effect of nitrate and cytokinin on the induction of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in embryos of Agrostemma githago was compared. Increased enzyme levels in response to treatment with the cytokinin benzyladenine were not correlated with a general stimulation of protein synthesis or a general reduction of protein breakdown. Actinomycin D did not inhibit the formation of nitrate reductase in response to nitrate or the cytokinin. Cycloheximide and puromycin inhibited the induction by the hormone to the same extent as, or even more than, the incorporation of [¹⁴C]leucine into protein. Induction of nitrate reductase by nitrate was much less susceptible to inhibition by cycloheximide and puromycin than induction of the enzyme by benzyladenine. When induction of nitrate reductase was carried out in the presence of ²H₂O, isopycnic equilibrium centrifugation in CsCl showed that incorporation of ²H into the enzyme had occured. The increase in the buoyant density of nitrate reductase was the same whether the enzyme was induced by nitrate or by benzyladenine, indicating that at least part of the nitrate reductase molecule was newly synthesized in both instances.     

Induction of nitrate reductase by cytokinin and ethylene in Agrostemma githago L. embryos

In dark-grown, isolated embryos of Agrostemma githago, a transient period of nitrate-reductase (NR) (NADH: nitrate oxidoreductase, EC 1.6.6.1) activity occurred from 6 to 36 h after the start of imbibition. During this period, NR activity was enhanced by nitrate, 6-benzylamino-purine and ethylene. Ethylene and 6-benzylamino-purine acted synergistically, whereas ethylene had no effect on nitrate induction. Aminoethoxyvinyl-glycine, an inhibitor of ethylene biosynthesis, inhibited the cytokinin-induced increase of NR activity, but had no effect on the nitrate-induced increase. The inhibition by aminoethoxyvinylglycine was overcome completely by ethylene. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid had the same effect on NR activity as ethylene. Our data indicate that NR induction by cytokinins only occurs in the presence of ethylene, and that nitrate enhances NR activity through a mechanism which is distinct from the induction by hormones.Abbreviations ACC 1-aminocycloproparte-1-carboxylic acid - AVG aminoethoxyvinylglycine - BAP 6-benzylaminopurine - c.p. cotyledonary pair - NR nitrate reductase This article was finalized by the second author two weeks before his death. It was translated and adapted by Dr. G.J. de Klerk, Research School of Biological Sciences, Australian National University, Canberra. Reprint requests should be sent to Dr. de Klerk at his present address: Bulb Research Centre, Vennestraat 22, 2160 AB Lisse, The NetherlandsDeceased 4 September 1985     

Cytokinin-induzierte de novo-Synthese der Nitratreductase in Embryonen von Agrostemma githago

Summary Nitrate reductase can be induced in isolated embryos of Agrostemma githago by treatment with cytokinin. By labeling proteins with H₂ ¹⁸O and subsequent isopycnic density gradient centrifugation it was proved that the benzyladenine induction of nitrate reductase is caused by a de-novo synthesis of the enzyme.     

Changes in protein synthesis during the development of Agrostemma githago seeds

The transition from the growth to the maturation phase in developing seeds of Agrostemma githago L. was found to coincide with major changes in the rate of protein synthesis, the kinds of proteins synthesized, and the composition of the non-proteinbound amino acid pool. Coincident changes were observed in viability and the ability to withstand desiccation. Desiccated mature Agrostemma seeds are dormant and need at least three months of after-ripening. In imbibed dormant and after-ripened seeds no synthesis of storage proteins was observed with the exception of one particular set of storage proteins. Dormant and after-ripened seeds synthesized the same kinds of proteins during early imbibition, indicating an almost identical metabolic state which differs considerably from that of developing seeds.     

The effects of endogenous cytokinin content on benzyladenineenhanced nitrate reductase induction

Foliar application of benzyladenine (BA) has been shown to enhance nitrate-dependent induction of nitrate reductase (NR; EC 1.6.6.1) in etiolated wheat ( Triticum aestivum L.) seedlings. Whether similar enhancement occurs in light-grown plants, or whether endogenous cytokinin content affects this enhancement is unknown. Since the cytokinin content of etiolated plants probably differs from that of light-grown seedlings, the NR response of each to exogenous root- or shoot-applied BA in wheat (cv. Red Bob) was examined. Endogenous cytokinins present in untreated control tissues prior to BA application and changes that occurred after a 22 h (12 h dark followed by 10 h of light) period were determined using a combined HPLC-immunoassay method. Shoot application of BA enhanced the induction of NR in etiolated seedlings in a concentration-dependent manner but failed to enhance NR induction in light-grown plants. Root-applied BA enhanced NR induction in both etiolated and light-grown seedlings. Endogenous root cytokinin levels were similar in both etiolated and light-grown plants. In contrast, shoots of 6 day-old light-grown seedlings contained at least 20 times the amount of total cytokinins measured in shoots from etiolated plants of the same age. Total cytokinin content of the light-grown plants diminished after the 22-h period while that measured in etiolated seedlings increased. The responsiveness of seedlings to BA was correlated with endogenous cytokinin levels in that enhancement of NR induction by exogenous BA was low in tissues which contained high concentrations of cytokinin at the time of BA application. These results may prove useful in interpretation of gene responses to exogenous plant growth regulators.     

Differences in Patterns of Newly Synthesized Proteins in Imbibing Embryos of After-ripened and Aged Seeds of Agrostemma githago and Comparison of Protein Synthesis In Vivo and In Vitro

Patterns of newly synthesized proteins in imbibing after-ripenedyoung and aged Agrostemma embryos show differences in the dynamicsof cotyledons and axes. In the course of imbibition the terminationof some syntheses in the embryonal parts of aged seeds is delayed.As was shown by in vitro translation, mRNAs coding for the synthesisof some proteins are still present in the embryos when proteintranslation has already finished. Key words: Protein pattern, in vivo protein synthesis, in vitro protein synthesis, after-ripened embryos, aged embryos, Agrostemma githago     

The influence of sugars on nitrate reductase induction by exogenous nitrate or nitrite in excised pisum sativum roots

Nitrate reductase (NR) induction is enhanced by exogenously supplied sucrose in excised pea roots exposed to both exogenous nitrate and exogenous nitrite. NR synthesis is preferentially supported by sugars transported to the cells at the moment, however NR induction can take place for some time without exogenous sugar influx if roots are saturated with sugars during precultivation. Steady high NR levels are dependent on steady sugar and nitrate influxes. NR induction is low in roots precultivated for 20 h without sucrose although sugar content is still high in them. This suggests that compartmentation of sugars in the cells is of major importance during NR induction. Total nitrate content in roots exposed to nitrate is not influenced by sucrose supplied together with nitrate. Some nitrite is oxidized to nitrate in roots exposed to exogenous nitrite ; we assume that this nitrate is responsible for NR induction. Our results indicate that sugars, besides many indirect effects on NR induction, may also directly influence NR synthesis either as coinducers or as derepressors of NR synthesis. Our results further show that NR is not a product-inducible enzyme.     

曼陀罗细胞中6-BA结合蛋白与硝酸还原酶活力的关系

曼陀罗单倍体细胞,在无水层情况下,经紫外线诱变,在氯酸盐培养基上筛选得到一株缺少硝酸还原酶活力的突变细胞株。经3年培养证明突变性状稳定。对它进行生理生化分析,看到它核内细胞分裂素结合蛋白减少。结合蛋白与细胞分裂素结合后能够促进核内 RNA 聚合酶活力,加速基因转绿。在正常细胞中,细胞分裂素能够促进硝酸还原酶诱导活力,而在这些缺少核内细胞分裂素结合蛋白的突变细胞中,外源细胞分裂素对酶活力的诱导没有作用。从而猜测,在突变细胞中,由于激素不能充分与结合蛋白结合,降低了细胞中转绿的总水平,可能是硝酸还原酶活力提不高的原因。本文还讨论了激素与受体结合后在调控基因表达上的可能途径。     

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.Abbreviations EDTA ethylenediaminetetraacetic acid - FAD flavine-adenine dinucleotide - IgG immunoglobulin G - NR nitrate reductase - PM plasma membrane - TX-100 Triton X-100     

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.     

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.     

Stimulation of growth and nitrate assimilation inLeucaena leucocephala seedlings in response to spermidine supply

Supply of 100 μM spermidine (Spd) in the nutrient solution containing 10 mM nitrate as the sole nitrogen source, increased growth of roots and shoots, total nitrogen content andin vivo orin vitro nitrate reductase (NR) activity of leaves of 10-d oldLeucaena leucocephala seedlings. Spd and the cytokinins benzyladenine or kinetin also increased growth, total nitrogen andin vivo NR activity of isolated cotyledons. The synergistic effects of nitrate, kinetin and Spd in increasing NR activity, indicate that the Spd acted at different level than the nitrate or cytokinin.     

Effect of Cytokinin on the Enhancement of Nitrate Reductase NR Activity in Tobacco Callus Tissues and Wheat Seedlings

The effect of cytokinin on the formation of NR activity were studied with tobacco callus tissues and wheat seedlings. Cytokinin could not induce the NR activity alone but could enhance the NR inducibility (Table 1). The enhancement of NR formation was detected in the tissues pretreated with cytokinin for over 12 hours. It showed that there was a precondition in the tissues for the induction of NR (Fig. 3). The precondition could not be improved by cytokinin when cycloheximide (inhibitor of protein synthesis) was added into the medium during cytokinin pretreatment (Table 2). Thus, it was thought that cytokinin might enhance synthesis of a protein which participated in the NR activity induction. In immunological test (Fig. 5) the existence of a nonactive apoenzyme of NR in higher plant tissues was demonstrated. It is, therefore, suggested that there are two major steps in the NR activity formation: (l) the synthesis of a nonactive NR apoenzyme, (2) the activation of this nonactive apoenzyme. The former step might be stimulated by cytokinin and the latter was mediated by nitrate.     

The influence of cytokinins in nitrate regulation of nitrate reductase activity and expression in barley

The responses of nitrate reductase (NR) activity and levels of NR-mRNA to environmental nitrate and exogenous cytokinins are characterised in roots and shoots of barley ( Hordeum vulgare L., cv. Golf), using a chemostate-like culture system for controlling nitrate nutrition. Experiments were mainly performed with split root cultures where nitrate-N was supplied at a constant relative addition rate of 0.09 day⁻¹, and distributed between the subroots in a ratio of 20%:80%. The subroot NR-mRNA level and NR activity, as well as the endogenous level of zeatin riboside (ZR), increased when the local nitrate supply to one of the subroots was increased 4-fold by reversing the nitrate addition ratio (i.e. from 20%:80% to 80%:20%). Also shoot levels of ZR, NR-mRNA and NR activity increased in response to this treatment, even though the total nitrate supply remained unaltered. External supply of ZR at 0.1 μ M caused an approximately 3-fold increase in root ZR levels within 6 h. which is comparable to the nitrate-induced increase in root ZR. External application of ZR. zeatin. isopentenyl adenine or isopentenyl adenosine at 0.1 μ M caused from insignificant to 25% increases in NR-mRNA and activity in roots and up to 100% stimulation in shoots, whereas adenine or adenosine had no effect. No synergistic effects of perturbed nitrate supply and cytokinin application were detected in either roots or shoots. The translocation of nitrate from the root to the shoot was unaffected by application of ZR or switching the nitrate distribution ratio between subroots. The data give arguments for a physiological role of cytokinins in the response of root and shoot NR to environmental nitrate availability. The nature and limitations of the physiological role of cytokinins are discussed.     

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.     

细胞分裂素促进小麦硝酸还原酶的诱导

细胞分裂素促进硝酸还原酶的诱导,脱落酸强烈抑制该酶的诱导,并抵消细胞分裂素的作用。6-苄基腺瞟呤(6-BA)的促进效应与 NO_3~-的诱导作用有关。无 NO_3~-存在时,6-BA 无直接诱导作用,适宜浓度的 NO_3~-可诱导较高的酶活性,这时6-BA 的促进作用也较强。无光照时,NO_3~-只能诱导黄化叶片产生很低的酶活性,这时6-BA 的促进作用也很弱。在1—2小时的诱导期内,环己酰亚胺抑制酶的诱导。结果表明细胞分裂素对酶诱导的促进作用不仅与 NO_3~-的存在有关,还与 NO_3~-诱导硝酸还原酶的必要条件有关,即依赖于酶的诱导过程。     

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.