Light and Nitrate Requirements for Induction of Nitrate Reductase Activity in Hordeum vulgare

The importance of light to the induction of nitrate reductase activity in barley (Hordeum vulgare L.) was studied. Activity in etiolated leaves in darkness stayed at a low endogenous level even while large amounts of nitrate were actively accumulated. Light was required for any increase in activity, though the requirement may be satisfied to a limited extent before nitrate is available. Nitrate reductase activity was induced in the dark in green leaves which had not previously had nitrate but were supplied nitrate at the beginning of the dark period. If the nitrate then made available was sufficient, nitrate reductase activity increased until the effect of the previous light treatment was exhausted. Activity then decreased even though nitrate uptake continued. Upon returning the leaves to light, enzymatic activity increased again, as expected. Nitrate uptake was eliminated as an experimental variable by giving dark-grown plants nitrate, then detaching the leaves for induction studies. Under these conditions light saturation occurred between 3600 and 7700 lux at exemplary periods of illumination. At intensities of 3600 lux and above, activity increased sharply after a 6-hour lag period. As light intensity was decreased below 3600 lux the lag period became longer. Thus, when sufficient nitrate was available, the extent of induction of nitrate reductase activity was regulated by light.     

In vivo Nitrate Reductase Activity in Barley (Hordeum vulgare L.) during Ear Development

Experiments were conducted during the 1974–75 and 1975–76winter season with the barley (Hordeum vulgare L.) cultivarJyoti. From amongst the various plant parts, the flag leaf bladehad higher in vivo nitrate reductase (NR) activity than thelower two leaf blades, glumes, and grains. However, the potentialof a plant part to reduce NO₃^– is a function of its freshweight and the NR per unit fresh weight. On this basis, thesecond and third leaf blades could reduce more NO₃^– thanthe flag leaf blade. N fertilizer application resulted in enhancementof the activity of the leaf blades alone. N fertilizer appliedduring the reproductive phase was taken up and assimilated bythe various plant parts. The studies suggest that, even whenthe fertilizer is applied at optimum levels for obtaining maximumyields, the upper leaf blades have sub-optimal NR activity andthat there is a likelihood of either a preferential flow ofNO₃^– to the leaf blades or transnational barriers to NO₃^–movement to the ear.     

Effect of Shoot Removal and Malate on the Activity of Nitrate Reductase Assayed in Vivo in Barley Roots (Hordeum vulgare cv. Midas)

There is a diurnal variation of nitrate reductase activity (NRA) measured in vivo in barley roots (Hordeum vulgare cv. Midas). In intact plants receiving a 16-hour photoperiod, NRA increases when the light is switched on, reaches a maximum value after 7 to 8 hours, and thereafter declines. Shoot removal (detopping) at the start of the photoperiod prevents the rise in NRA; detopping after 5 hours light leads to a rapid fall in NRA. The inclusion of 10 millimolar malate in the external medium causes a rise in NRA in plants detopped at the beginning of the photoperiod and thus seems to substitute partially for the illuminated shoot. Oxalate, fumarate, and tartrate did not have this effect. Preincubation of the roots of intact plants with 10 millimolar malate for 3 hours, prior to detopping, causes an increase in the flux of amino acids into the xylem sap of detopped roots.     

硝酸盐对硝酸还原酶活性的诱导及硝酸还原酶基因的克隆

硝酸盐在植物体内的积累过多已成为影响蔬菜品质并影响人类健康的重要因素。硝酸还原酶（NR）是硝酸盐代谢中的关键酶,提高其活性有利于硝酸盐的降解。为了解植物不同组织中NR的活性,用活体测定法检测了经50mmol/L的KNO₃诱导不同时间后的油菜、豌豆和番茄幼苗根茎叶中NR活性,同时为了明确外源诱导剂浓度与植物体内NR活性的关系,检测了经不同浓度KNO₃诱导2h后的矮脚黄、抗热605、小白菜和番茄叶片中的NRA。结果表明,不同植物组织NR活性有很大差异,叶中NR活性较高,根其次,茎最低;不同植物的NR活性随诱导时间呈不同的变化趋势,相同植物不同组织的NR活性变化趋势相似;不同植物叶片NRA为最高时KNO₃浓度不同。用30mmol/L的KNO3诱导番茄苗2h后,从番茄根和叶中提取总RNA,用RT-PCR方法获得NR cDNA,全长2736bp,编码911个氨基酸。为进一步利用该基因提高植物对硝酸盐的降解能力打下基础。     

Evidence for an Inactivating System of Nitrate Reductase in Hordeum vulgare L. during Darkness That Requires Protein Synthesis

The disappearance of nitrate reductase activity in leaves of Hordeum vulgare L. during darkness was inhibited by cycloheximide, actinomycin D, and low temperature. Thus, protein synthesis was probably required for the disappearance of nitrate reductase in the dark. Since chloramphenicol did not affect the rate of loss of activity, the degradation or inactivation apparently required protein synthesis by the cytoplasmic ribosomal system. Consistent with this observation, nitrate reductase is also reportedly located in the cytoplasm. Thus, the amount of nitrate reductase activity present in leaves of barley may be controlled by a balance between activating and inactivating systems.     

Variation in Nitrate Reductase Activity in Lolium

Nitrate reductase activity was studied in the leaves and rootsof Lolium perenne. Growth temperatures of 8, 15, or 20 °Cdid not affect activity, but the same temperatures during assayhad differential effects on the nitroso couple used to measureenzyme activity. Activity increased with increasing light intensity,reaching a high plateau value at around 40 W m^–2. Nitratecontent of leaves, also measured in this experiment, did notvary significantly with different light intensities. Increasingnitrate in the nutrient solution up to 0.5 mM N also increasedactivity. Adding ammonium chloride at similar levels to thenitrate caused no marked repression of activity. Removal ofnitrate from the nutrient solution decreased enzyme activitywithin 24 h. Marked diurnal fluctuations occurred in activity,apparently in response to light intensity, since the nitratelevel in the plant varied little. The enzyme activity of rootswas much less than that of leaves. In the parents and progeny from a half diallel cross, the parentalgenotypes differed significantly in activity, but the numberof families involved was too small for the regression of progenyon parents (b = 1.74) and the correlation coefficient (r = 0.44NS) to achieve significance. In this experiment a significantpositive regression was obtained between nitrate reductase activityand dry matter yield.     

硝酸盐对球形棕囊藻生长和硝酸还原酶活性的影响

以我国南海海域分离的赤潮原因种——球形棕囊藻(Phaeocystis globosa)为材料, 研究了不同硝酸盐浓度下藻细胞生长及硝酸还原酶活性的变化。当培养基中不含硝酸盐时, 藻细胞内硝酸还原酶的活性保持在非常低的水平, 藻细胞的生长受到限制, 不能形成正常的生长曲线: 当培养基中硝酸盐浓度为3.62 mmol.L-1时, 藻细胞的硝酸还原酶活性和比生长速率达到最大。在含有硝酸盐的培养基中, 接种培养后第9天藻细胞硝酸还原酶活性达到最大值, 并且在4种不同硝酸盐浓度下, 藻细胞硝酸还原酶活性的差异性达到极显著水平(P<0.01)。在接种培养第16天藻细胞密度达到最大值, 并且4种不同硝酸盐浓度培养的藻细胞密度之间的差异性也达到极显著水平(P<0.01)。实验结果表明, 在培养基中添加不同浓度的硝酸盐, 对球形棕囊藻细胞硝酸还原酶的活性和藻细胞的生长有极显著的影响, 含有较高硝酸盐的富营养化海域有利于球形棕囊藻细胞的持续生长。     

硝酸盐对球形棕囊藻生长和硝酸还原酶活性的影响

以我国南海海域分离的赤潮原因种——球形棕囊藻(Phaeocystisglobosa)为材料,研究了不同硝酸盐浓度下藻细胞生长及硝酸还原酶活性的变化。当培养基中不含硝酸盐时,藻细胞内硝酸还原酶的活性保持在非常低的水平,藻细胞的生长受到限制,不能形成正常的生长曲线:当培养基中硝酸盐浓度为3.62μmol.L-1时,藻细胞的硝酸还原酶活性和比生长速率达到最大。在含有硝酸盐的培养基中,接种培养后第9天藻细胞硝酸还原酶活性达到最大值,并且在4种不同硝酸盐浓度下,藻细胞硝酸还原酶活性的差异性达到极显著水平(P<0.01)。在接种培养第16天藻细胞密度达到最大值,并且4种不同硝酸盐浓度培养的藻细胞密度之间的差异性也达到极显著水平(P<0.01)。实验结果表明,在培养基中添加不同浓度的硝酸盐,对球形棕囊藻细胞硝酸还原酶的活性和藻细胞的生长有极显著的影响,含有较高硝酸盐的富营养化海域有利于球形棕囊藻细胞的持续生长。     

Effect of Nitrate, Ammonium and Some Amino Acids on Growth and Nitrate Reductase Activity in Suspension Cultures of Atropa belladonna

Growth and nitrate reductase activity (NRA) of Atropa belladonnacells were studied in medium supplemented with NaNO₃, NH₄NO₃,and amino acid precursors to tropane alkaloids. Growth and NRAwere stimulated by NH₄⁺ and by proline, by proline plus ornithine,but not by glutamate, in NO₃^–-containing medium. Testedamino acids inhibited neither utilization of inorganic nitrogennor growth. (Received September 30, 1988; Accepted August 28, 1989)     

Triadimenol Increases Nitrate Levels and Nitrate Reductase Activity in Canola Leaves

Nitrate reductase activity in the first true leaves of canola(Brassica napus L.) seedlings grown in one-quarter strengthHoagland's solution from seeds pretreated with triadimenol (0.3or 30 g (a.i.) kg^–1 of seed) was higher than controlsduring the growth period of 15 to 25 d after planting. Triadimenolalso increased chlorophyll levels, the increase being more pronouncedat its lower concentration. The treatment also increased theweight and nitrate content of the leaves. When seedlings weregrown in nutrient solution containing 1 to 20 mM nitrate, theincrease in nitrate reductase activity by triadimenol was higherat lower rather than at higher nitrate concentrations. The nitratelevels and Kjeldahl nitrogen in the triadimenol-treated leaveswas higher than the controls at concentrations of added nitrateabove 2 mM. Addition of nitrate to plants grown in ammonium,increased nitrate reductase activity more in plants grown fromtriadimenol-treated seeds than controls. However, addition of10µM triadimenol for 24 h to ammonium-grown plants hadlittle effect on enzyme activity, both in the absence as wellas the presence of nitrate. This study demonstrates that triadimenolincreases nitrate reductase activity and nitrate accumulationin the leaves and at least part of the increased enzyme activityis independent of nitrate accumulation. Key words: Triazoles, nitrate content, nitrate reductase activity     

Time-course of Nitrate Uptake and Nitrate Reductase Activity in Nitrogen-depleted Dwarf Bean

The rate of nitrate uptake by N-depleted French dwarf bean (Phaseolus vulgaris L. cv. Witte Krombek) increased steadily during the first 6 h after addition of NO₃ -After this initial phase the rale remained constant for many hours. Detached root systems showed the same time-course of uptake as roots of intact plants. In vivo nitrate reductase activity (NRA) was assayed with or without exogenous NO₃^- in the incubation medium and the result ing activities were denoted potential and actual level, respectively. In roots the difference between actual and potential NRA disappeared within 15 min after addition of nitrate, and NRA increased for about 15 h. Both potential and actual NRA were initially very low. In leaves, however, potential NRA was initially very high and was not affected by ambient nitrate (0.1–5 mol m^-3) for about 10 h. Actual and potential leaf NRA became equal after the same period of time. In the course of nitrate nutrition, the two nitrate reductase activities in leaves were differentially inhibited by cycloheximide (3.6 mmol m^-3) and tungstate (1 mol m^-3). We suggest that initial potential NRA reflects the activity of pre-existing enzyme, whereas actual NRA depends on enzyme assembly during NO₃^- supply. Apparent induction of nitrate uptake and most (85%) of the actual in vivo NRA occurred in the root system during the first 6 h of nitrate utilization by dwarf bean.     

Seedling Nitrate Reductase Activity and Nitrate Partitioning in Maize (Zea mays L.) Strains Divergently Selected For Post-anthesis Leaf-Lamina Nitrate Reductase Activity

Two experiments were conducted to evaluate the effects of phenotypicrecurrent selection for high and low post-anthesis leaf-laminain vivo NRA on nitrate uptake, nitrate partitioning and in vitroNRA of seedling roots and leaves. In Experiment 1, intact plantsof cycle 0, 4, and 6 of the high and low NRA strains were grownon NH₄-N for 11 d, then exposed to 1.0 mol m^–3 KNO₃, andcultures sampled at 6 h and 28 h (induction and post-inductionperiods). Nitrate uptake, tissue nitrate concentration and invitro NRA were determined. The pattern of response to selectionin seedling leaf NRA was similar to that observed for in vivoNRA of field grown plants. Leaf NRA increased between 6 h and28 h. Root NRA was not affected by selection or sampling time.Treatments differed in total fresh weight but not in reductionor uptake of nitrate per unit weight, indicating a lack of correspondencebetween NRA and reduction and supporting the idea that concomitantreduction by NR is not obligatorily linked to nitrate influxin the intact plant. In Experiment 2, dark-grown plants of cycle 0, and 6 of thehigh and low NRA strains were cultured without N, detopped onday 6, transferred the following day to 0-75 mol m^–3 KNO₃and sampled at 6 h and 28 h. In contrast to Experiment 1, selectionpopulations differed in nitrate reduction and root NRA, whichby 28 h reached higher average levels than root NRA of intactplants. Translocation and reduction were inversely related amongstrains within each sampling time. The high level of translocationin detopped plants of the low NRA strain was difficult to reconcilewith its low leaf NRA level of Experiment 1. It is suggestedthat nitrate transport in detopped roots is altered relativeto the intact system in a way which permits greater NRA inductionand nitrate reduction. The results indicate that nitrate partitioningby detopped root systems should be interpreted with caution. Key words: Zea, nitrate reductase activity, nitrate uptake, nitrate reduction, nitrate partitioning, selection     

Evaluation of Nitrate Reductase Activity in Rhizobium japonicum

Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase.     

Chlorate Reducing Activity of Spinach Nitrate Reductase

The activities of 2-oxoaldehyde-metabolizing enzymes (glyoxalase I, glyoxalase II, methyl- glyoxal reductase, methylglyoxal dehydrogenase and lactaldehyde dehydrogenase) were found to be widely distributed among microorganisms. One of the enzymes, methylglyoxal reductase, which catalyzes the reductive conversion of methylglyoxal into lactaldehyde, was purified from Escherichia coli cells. The enzyme was judged to be homogeneous on polyacrylamide gel electrophoresis and was a monomer with a molecular weight of 43000. The enzyme was most active at pH 6.5 and 45°C. The enzyme utilized both NADPH and NADH for the reduction of 2- oxoaldehydes (glyoxal, methylglyoxal, phenylglyoxal and 4,5-dioxovalerate) and some aldehydes (glycolaldehyde, D,l-glyceraldehyde, propionaldehyde and acetaldehyde). The Km values of the enzyme for methylglyoxal, NADPH and NADH were 4.0 mm, 1.7 fiM and 2.8 /¿m, respectively. The product of methylglyoxal reduction was identified as lactaldehyde. The enzyme from E. coli cells was different from the yeast and goat liver enzymes in both molecular structure and substrate specificity.     

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)     

Induced Androgenesis in vitro in Mutated Populations of Barley,Hordeum vulgare

Graduated concentrations of chemomutagens ethylmethanesulphonate (EMS), N-nitroso-N-methylurea (MNU) and N-nitroso-N-ethylurea (ENU) and one concentration of sodium azide (NaN₃) were used to treat seeds of spring barley cultivars Heris, Tolar, Granát and Novum. Androgenesis in vitro was induced in mutagenised plant populations. The significant stimulation effect of mutagenic treatment on the mean number of androgenic anthers from all 36 treated variants was registered in 17 variants, on the mean number of regenerated plants in 15 variants and on the mean number of regenerated plants per androgenic anthers in seven variants only. Genotypes with a lower androgenic response were relatively more stimulated. Evaluation was made of the frequency of chlorophyll mutations within androgenic regenerants and in seed progeny of androgenic donors. Androgenesis was also induced in vital chlorophyll mutants and in the variants where the mutagen treatment resulted in less than 50% survival of the donor plants. Ratios between frequency of haploids and spontaneous dihaploids were similar in green and in albino androgenic plants. The results confirm that in barley it is possible to enhance the frequency of in vitro pollen embryogenesis by mutagenic treatment of seeds.     

Action of Thiol Proteinase on Nitrate Reductase in Leaves of Hordeum distichum L.

Nitrate reductase (NR) from the leaves of Hordeum distichumwas very susceptible to inactivation by barley leaf thiol proteinase,trypsin, and papain. A cytochrome c reductase species with amolecular weight of about 40,000 was derived from the NR complexby the proteolytic actions. The barley proteinase seemed toattack the Mo⁺-containing component of NR, just like trypsinand papain. It showed a preference for the alanine and tryptophanesters among the carbobenzoxyamino acid-nitrophenylesters tested. In vivo NR activity in the presence of leupeptin was fairlyhigher than that in its absence. Leupeptin also protected NRfrom its cleavage to small cytochrome c reductase species, suggestingthat the barley proteinase may play a role in the in vivo changein NR activity. (Received May 21, 1984; Accepted September 10, 1984)