The Regulation of Nitrate Reductase,Nitrite Reductase,and Glutamate Dehydrogenase in Excised Pea Roots by Some Exogenous Amino Acids

The effect of growth retarding amino acids (L-aspartic acid,L-leucine,L-methionine, andL-threonine) on nitrate reductase (NO₃R), nitrite reductase (NO₂R), and NADH₂ dependent glutamate dehydrogenase (GDH) in excised pea roots was followed,L-methionine andL-threonine slightly depressed NO₃R activity,L-aspartic acid enhanced NO₂R and GDH activities.L-methionine andL-threonine also slightly decreased nitrate uptake. The results obtained are discussed in connection with the growth effects of the amino acids investigated.     

The Regulation of the Synthesis of Glutamate Dehydrogenase in Excised Pea Roots by ExogenousL- aspartic andL- glutamic Acids

Exogenous L-aspartic and L-glutamic acids enhance glutamate dehydrogenase activity in isolated pea roots. The results obtained indicate that both ammo acids induce increased GDH synthesis.     

Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities.     

Effect of Phytochrome and Kinetin on Nitrite Reductase Activity in Zea mays

Red light and kinetin (10 µm) increased nitrite reductase(NIR) activity by 85 and 47% respectively in excised leavesof etiolated Zea mays. The stimulatory effect of kinetin decayedslower than that of red light. Indoleacetic acid (10 µm)had no effect on NIR activity. In the presence of abscisic acid(10 µm), the kinetin stimulated increase in NIR activitywas totally nullified, however, the red light irradiated plantsretained 20–25% increase in NIR activity over the darkcontrol. If ABA was given 2 h after kinetin treatment or redlight irradiation, it totally blocked kinetin stimulation asnoticed earlier, but red light stimulation was inhibited byonly 11%. Kinetin-treated and the red light irradiated leavesshowed 20–25% increase in nitrate accumulation, whichwas totally nullified by ABA. The experiments presented suggestan independent mode of signal transduction by kinetin and phytochromein stimulating NIR activity. (Received December 2, 1986; Accepted February 7, 1987)     

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。     

Variation in Nitrate Reductase, Nitrite, and Nitrite Reductase in Some Grasses and Cereals

Nitrite accumulation may result from unbalance between nitratereductase which produces nitrite and nitrite reductase whichremoves it. In the first experiment, using three light levelsand three nitrate levels, on Lolium, maize, and oats, both enzymesresponded to increased light, though not always significantly.The effect of nitrate was more variable. Nitrate reductase activityincreased to the intermediate or highest level of nitrate, butthere was no clear response in nitrite reductase activity orin nitrite concentration. In the second experiment, using fournitrate levels but only one, high, light intensity on Loliumand barley, the results were clearer. With increasing nitratesupply, nitrate reductase activity increased more than nitritereductase activity. This was particularly marked in Lolium,in which nitrite accumulated at the highest nitrate supply.Thus high nitrate supply unbalances the two enzymes in the directionleading to nitrite accumulation.     

Effect of l-Canavanine on Nitrate Reductase in Corn Roots

l-Canavanine inhibits the appearance of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in both root tips and mature root sections of corn (Zea mays L.). Ten-fold more canavanine was required to cause a 50% reduction in the level of nitrate reductase activity (NRA) in root tips than in mature root sections. For example with one particular batch of seeds 500 μm canavanine was effective in root tips whereas only 50 μm was required in mature root sections. In root tips arginine (1 mm) completely reversed the effect of 1 mm canavanine. In mature root sections higher concentrations of arginine (approximately 5 mm) were required for a complete reversal of the canavanine effect. Additions of canavanine to roots after a period of 3 hours with 5 mm KNO₃ resulted in a loss of NRA. NO₃⁻ protected nitrate reductase from this inactivation in both root tip and mature root sections.     

Variant Cell Lines of Haplopappus gracilis with Disturbed Activities of Nitrate Reductase and Nitrite Reductase

Selected variant cell lines of Haplopappus gracilis (Nutt) Gray that showed disturbed growth after transfer from an alanine medium to NO₃⁻ medium were characterized. The in vivo NO₃⁻ reductase activity (NRA) was lower in these lines than in the wild type. In vitro NRA assays suggest that decreased in vivo NRA was not caused by a lower amount of active enzyme. Cells of the variant lines revealed up to 75% lower extractable activity of NO₂⁻ reductase as compared with the wild type. This coincided with higher accumulation of NO₂⁻ by the variant than by the wild type cells after transfer from alanine medium to NO₃⁻ medium. NO₂⁻ accumulation was transient or continuous, depending on cell line, metabolic state of the cells, and light conditions.     

Effect of Chlorate Treatment on Nitrate Reductase and Nitrite Reductase Gene Expression in Arabidopsis thaliana

The herbicide chlorate has been used extensively to isolate mutants that are defective in nitrate reduction. Chlorate is a substrate for the enzyme nitrate reductase (NR), which reduces chlorate to the toxic chlorite. Because NR is a substrate (NO₃⁻)-inducible enzyme, we investigated the possibility that chlorate may also act as an inducer. Irrigation of ammonia-grown Arabidopsis plants with chlorate leads to an increase in NR mRNA in the leaves. No such increase was observed for nitrite reductase mRNA following chlorate treatment; thus, the effect seems to be specific to NR. The increase in NR mRNA did not depend on the presence of wild-type levels of NR activity or molybdenum-cofactor, as a molybdenum-cofactor mutant with low levels of NR activity displayed the same increase in NR mRNA following chlorate treatment. Even though NR mRNA levels were found to increase after chlorate treatment, no increase in NR protein was detected and the level of NR activity dropped. The lack of increase in NR protein was not due to inactivation of the cells' translational machinery, as pulse labeling experiments demonstrated that total protein synthesis was unaffected by the chlorate treatment during the time course of the experiment. Chlorate-treated plants still retain the capacity to make functional NR because NR activity could be restored by irrigating the chlorate-treated plants with nitrate. The low levels of NR protein and activity may be due to inactivation of NR by chlorite, leading to rapid degradation of the enzyme. Thus, chlorate treatment stimulates NR gene expression in Arabidopsis that is manifested only at the mRNA level and not at the protein or activity level.     

Chlorophyll Formation in Excised Roots of Cucumber and Pea

The effects of red and blue light on the formation of chlorophyll in excised roots of cucumber and pea was investigated and compared with previously reported experiments on wheat roots. All three kinds of roots are similar in that in continuous red light no or very little chlorophyll is formed. and in blue light a considerable amount of chlorophyll is produced. Roots of cucumber and pea differ from those of wheat, in that red light is ineffective (or nearly so) even when combined with blue.     

硝态氮对黄瓜子叶谷氨酰胺合成酶和谷氨酸脱氢酶活性的影响

在硝态氮存在或缺乏的条件下,测定了黄瓜(Cucumis sativus L．)种子萌发和子叶发育过程中子叶可溶性蛋白质含量以及谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(NAD(H)-GDH)活性的变化。在子叶发育初期,无论外源氮存在与否,每对子叶可溶性蛋白质含量和GS、NADH—GDH、NAD^ -GDH活性随发育上升。在外源氮存在下,第4d后,可溶性蛋白质含量虽有所下降,但基本保持恒定;第6d后,GS和NADH—GDH活性逐渐降低,NAD^ -GDH却相反增高。但在无外源氮条件下,于第4d后,可溶性蛋白质水平以及GS、NADH—GDH和NAD^ -GDH活性都逐渐降低。在子叶发育的整个过程中,外源氮对GS和NAD^ -GDH活性有促进作用,尤其是在子叶发育的后期对NAD^ -GDH活性的促进更为明显。     

Variation in Nitrate Accumulation, Nitrate Reductase Activity and Nitrite Reductase Activity along Primary and Nodal Roots of Corn (Zea mays L.) Seedlings

Significant differences in NO₃ accumulation and nitrate reductaseactivity (NRA) were noted in the successive segments of developingyoung primary and nodal roots. This variation was also foundto be a function of root age. Nitrite reductase activity (NiRA)on the other hand had little variation among various segmentsof primary and nodal roots and also as a function of root age.These data suggest root NO₃ accumulation and root NRA are twoprocesses which are not directly linked. ¹ Present address: Division of Plant Physiology, Indian AgriculturalResearch Institute, New Delhi-110012, India. (Received December 3, 1983; Accepted June 18, 1983)     

Effect of Exogenous Nitrate on Anaerobic Metabolism in Excised Rice Roots: III. GLYCOLYTIC INTERMEDIATES AND ENZYMATIC ACTIVITIES

The levels of glycolytic intermediates, enzymatic activitiesand lactate concentration were estimated in excised rice rootsanaerobically grown in the presence or absence of exogenousnitrate in order to establish the conditions which permit ahigher fermentative activity in roots fed with nitrate. Thepresence of nitrate in the culture medium increased the levelof glucose, pyruvate and the activity of pyruvate kinase, anddecreased the level of lactic acid. The different amount oflactic fermentation and the consequent involvement of pH inthe regulation of glycolytic flux under anoxia are discussedwith a view to explaining the greater alcoholic fermentationcarried out by nitrate-fed roots. Key words: Anaerobiosis, nitrate, glycolytic intermediates, enzymes     

Effect of Exogenous Nitrate on Anaerobic Metabolism in Excised Rice Roots: II. FERMENTATIVE ACTIVITY AND ADENYLIC ENERGY CHARGE

Reggiani, R., Brambilla, I. and Bertani, A. 1985. Effect ofexogenous nitrate on anaerobic metabolism in excised rice roots.II Fermentative activity and adenylic energy charge.—J.exp. Bot 36: 1698–1704. The presence of nitrate in the culture medium of excised sterilerice roots stimulated CO₂ and ethanol evolution and, to a smallerextent, alanine accumulation. The increased anaerobic carbohydrateconsumption observed in roots grown on nitrate is consistentwith the constantly higher level of adenylic energy charge.An hypothesis serving to explain the evidence concerning theeffect of nitrate on anaerobically grown rice roots is proposed. Key words: Anaerobiosis, ethanol, nitrate, alanine, adenylic energy charge     

Effect of Exogenous Nitrate on Anaerobic Metabolism in Excised Rice Roots: I. NITRATE REDUCTION AND PYRIDINE NUCLEOTIDE POOLS

Reggiani, R., Brambilla, I. and Bertani, A. 1985. Effect ofexogenous nitrate on anaerobic metabolism in excised rice roots.I. Nitrate reduction and pyridine nucleotide pools.— J.exp.Bot 36:1193-1199. In apical segments of sterile rice roots, reduction of nitratein the absence of oxygen is promoted by the presence of exogenousnitrate in the growth medium in the first 3 h of oxygen deficiency.Anaerobic treatment also increased the concentration of NADH.When nitrate was made available for reduction, smaller accumulationsof NADH were observed. Oxidation of reduced nudeotides associatedwith the conversion of nitrate to nitrite is suggested as beingresponsible for this behaviour. Key words: Anaerobiosis, nitrate, pyridine nucleotides     

水稻硝酸还原酶的RNA干涉及其酶活性分析

分析水稻硝酸还原酶（NR）基因生物信息学的结果显示：水稻基因纽中有2个NR基因成员：一个为NR[NADH]（NR1）：另一个为NR[NAD（P）H]（NR2）。两者的蛋白序列相似性为70％。用RT—PCR技术从水稻cDNA中获得了NR1和NR2的cDNA片段,其大小分别为1086bp和892bp。构建RNA干涉载体（称pRNAi—NR1和pRNAi-NR2）转化水稻愈伤组织后检测转基因后代酶活性的结果表明：两种干涉植株的根叶中的NR活性均大幅度下降,并且根叶中的活性变化呈线性正相关关系。表明2个基因可能均有调控根叶中NR活性的作用。