NADH-dependent nitrate reductase from two-row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)⁻¹ min⁻¹ at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH₂-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent K_m for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent K_m for nitrate as well as V_max whereas it did not alter the K_m for NADH.     

Developmental changes of enzymes of malate metabolism in relation to respiration, photosynthesis and nitrate assimilation in peach leaves

The developmental profile of the activities of some enzymes involved in malate metabolism, namely phosphoenolpyruvate carboxylase (PEPC; EC 4. 1. 1. 31), NAD⁺-linked (EC 1. 1. 1. 37) and NADP⁺-linked (EC 1. 1. 1. 82) malate dehydrosenase (MDH), NAD⁺linked (EC 1. 1. 1. 39) and NADP⁺-linked (EC 1. 1. 1. 40) malic enzyme (ME), has been determined in leaves of peach [ Prunus persica (L.) Batsch cv. Maycrest], a woody C₃ species. In order to study the role of these enzymes, their activities were related to developmental changes of photosynthesis, respiration, and capacity for N assimilation. Activities of PEPC, NAD(P)⁺-MDH and NADP⁺-ME were high in young expanding leaves and decreased 2- to 3-fold in mature ones, suggesting that such enzymes play some role during the early stages of leaf expansion. In leaves of peach, such a role did not seem to be linked to C₃ photosynthesis or nitrate assimilation, in that photosynthetic O₂ evolution and activities of nitrate reductase (EC 1. 6. 6. 1) and glutamine synthetase (EC 6. 3. 1. 2) increased during leaf development. In contrast, leaf respiration strongly decreased with increasing leaf age. We suggest that in expanding leaves of this woody species the enzymes associated with malate metabolism have anaplerotic functions, and that PEPC may also contribute to the recapture of respiratory CO₂.     

Regulation of nitrate reductase in detached oat leaves by light and oxygen

Regulation of nitrate reductase (NR, EC 1.6.6.1) by oxygen concentration and light was studied in segments of oat ( Avena sativa L. cv. Suregrain) leaves, using the in vivo nitrate reductase assay. The activity of NR decreased after excision in either light or darkness; the addition of cycloheximide prevented this decrease. Treatments that increased tissue permeability (anoxia, Triton X-100) also increased NR activity. There was in general less NR activity in the light than in the dark and also less under aerobic (21–100% O₂) than under anaerobic (0.3% O₂) conditions. Treatments with antioxidants improved the activity in the light, but only at high O₂ levels (21–100% O₂).
The results suggest that NR may be regulated by inhibitory proteins synthesized in either light or darkness, by permeability changes and by light-induced oxidations that occur when O₂ is present. Oxygen may control the activity by stimulating the synthesis of inhibitory proteins in the light and in the dark and by promoting oxidation of SH-groups in the light.     

Nitrate metabolism in soybean root nodules

The nitrate metabolism in nodules induced by Bradyrhizobium japonicum strain PJ17 on roots of soybean [ Glycine max (L.) Merr. cv. Hodgson] has been characterized by the nitrate reductase (NR; EC 1.6.6.1 and EC 1.6.6.3) activity of both partners of the symbiosis. NR activities of bacteroids and nodular cytosol were comparable and significantly higher than those of the roots. Nitrate reduction led to nitrite accumulation in root nodules, which was maximum after pod filling. The nodule had the capacity to metabolize nitrite via nitrite reductase (NiR; EC 1.6.6.4), at least in the cytosolic fraction. This activity was partly inhibited by the low content of free O₂ in the nodule. Indeed, nitrite accumulation decreased in the presence of an increased external pressure of O₂.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

Differential response of four tree species to ozone-induced acceleration of foliar senescence

Ozone (O₃)-induced accelerated senescence of leaves was measured in four tree species: black cherry ( Prunus serotina ), hybrid poplar ( Populus maximowizii x trichocarpa , clone 245), northern red oak ( Quercus rubra ) and sugar maple ( Acer saccharum ). Seedlings or ramets of the four species were subjected to chronic O₃ exposures and designated leaves harvested periodically from emergence to senescence. Gas exchange was analysed, and concentrations of total soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase were measured as indicators of leaf senescence. Total antioxidant potential and ascorbate peroxidase and glutathione reductase activities also were determined. Black cherry and hybrid poplar exhibited O₃-induced accelerated leaf senescence, whereas sugar maple and northern red oak did not. When the O₃ effects were related to cumulative uptake of the gas, black cherry was the most sensitive of the four species. Although hybrid poplar exhibited similar symptoms of O₃-induced accelerated senescence after the same exposure period as did black cherry, this species took up much greater quantities of O₃ to achieve the same response. The O₃-induced increase in glutathione reductase activity in hybrid poplar was consistent with the capacity of this species to take up high concentrations of the gas. Relative tolerance of northern red oak and sugar maple could be explained only in part by lower cumulative O₃ uptake and lower rate of uptake. Sugar maple had the highest antioxidant potential of all four species, which may have contributed to O₃ tolerance of this species. Ascorbate peroxidase activity, when expressed on a fresh weight basis, could not account for differential sensitivity among the four species.     

Nitrate metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrate uptake and reductase by ammonia

Nitrogen regulation of nitrate uptake and nitrate reductase (EC 1.7.99.4) was studied in the cyanobacterium Anabaena cycadeae Reinke and its glutamine auxotroph. Development of the nitrate uptake system preceded, and was independent of, the development of the nitrate reductase system. The levels of both systems were several-fold higher in the glutamine auxotroph lacking glutamine synthetase (EC 6.3.1.2) than in the wild type strain having normal glutamine synthetase activity. The nitrate uptake system was found to be NH4-repressible and the nitrate reductase system NO₃⁻-inducible. NH₄⁺ was the initial repressor signal for the uptake process which was involved in the control of the NO₃⁻inducible reductase system.     

Adjustments of net photosynthesis in Solanum tuberosum in response to reciprocal changes in ambient and elevated growth CO2 partial pressures

Single leaf photosynthetic rates and various leaf components of potato ( Solanum tuberosum L.) were studied 1–3 days after reciprocally transferring plants between the ambient and elevated growth CO₂ treatments. Plants were raised from individual tuber sections in controlled environment chambers at either ambient (36 Pa) or elevated (72 Pa) CO₂. One half of the plants in each growth CO₂ treatment were transferred to the opposite CO₂ treatment 34 days after sowing (DAS). Net photosynthesis (P_n) rates and various leaf components were then measured 34, 35 and 37 DAS at both 36 and 72 Pa CO₂. Three-day means of single leaf P_n rates, leaf starch, glucose, initial and total Rubisco activity, Rubisco protein, chlorophyll ( a + b ), chlorophyll ( a/b ), α -amino N, and nitrate levels differed significantly in the continuous ambient and elevated CO₂ treatments. Acclimation of single leaf P_n rates was partially to completely reversed 3 days after elevated CO₂-grown plants were shifted to ambient CO₂, whereas there was little evidence of photosynthetic acclimation 3 days after ambient CO₂-grown plants were shifted to elevated CO₂. In a four-way comparison of the 36, 72, 36 to 72 (shifted up) and 72 to 36 (shifted down) Pa CO₂ treatments 37 DAS, leaf starch, soluble carbohydrates, Rubisco protein and nitrate were the only photosynthetic factors that differed significantly. Simple and multiple regression analyses suggested that negative changes of P_n in response to growth CO₂ treatment were most closely correlated with increased leaf starch levels.     

Isolation of Capsicum annuum leaf nitrate reductase and characterization of the effect of adenine nucleotides and NADH on its activity

Abstract. In the preliminary purification of Capsicum leaf nitrate reductase (EC 1.6.6.1), treatment of the crude extract on Sephadex G-25 was necessary to prevent a gelling of the extract and sedimentation of the enzyme. Its K_m values for NADH and nitrate were estimated to be 9.3 and 105mmol m⁻³ ADP and ATP gave hyperbolic competitive inhibition, with respect to NADH, while the inhibition by AMP was linear competitive. K_i values calculated were: ADP and ATP approximately lmol m⁻³ and AMP 2.3 mol m⁻³. Inhibition by ADP was not altered by reduced glutathione.
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity.     

Response of net photosynthesis in bean (Phaseolus vulgaris) leaves to the elevation of the partial pressures of oxygen and carbon dioxide

Bean ( Phaseolus vulgaris L. cv. Golden Saxa) plants were grown under low artificial light or under natural daylight. The rate of net photosynthesis (P_N) was measured at: CO₂ partial pressure, p(CO₂), of 0.03, 0.09 or 0.15 kPa; O₂ partial pressure, p(O₂), of 2, 21 or 31 kPa and at light intensities of 350 or 1000 μmol m⁻² s⁻¹ (photosynthetically active radiation). In plants which had been grown under natural light, stimulation of P_N at 21 kPa p(O₂) was found only at elevated p(CO₂) and high light. It is proposed that this phenomenon is dependent on a high capacity of the photosynthetic apparatus to regenerate ribulose 1.5-bisphosphate.     

Effects of supplemental UV-B radiation on growth and leaf photosynthetic reactions of soybean (Glycine max)

Experiments were conducted under greenhouse conditions to investigate the effects of enhanced UV-B radiation (280 to 320 nm) on height, fresh and dry weights, leaf chlorophyll and carotenoids, CO₂ uptake rates, and Hill activity in soybean ( Glycine max L. cv. Bragg). Plants were exposed for 6 h continuously from midmorning to midafternoon each day to UV-B radiation which was provided by Westinghouse FS-40 sun lamps filtered with 0.127-mm cellulose acetate film (UV-B enhanced) or 0.127-mm Mylar S film (UV-B Mylar control). Three different UV-B enhanced radiation levels were tested: 1.09 (treatment T₁), 1.36 (treatment T₂), and 1.83 (treatment T₃) UV-B sun equivalent units (UV-B_sec) where 1 UV-B_sec= 15.98 mW·m⁻² of solar UV-B obtained by applying EXP -[(α-265)/21]², a weighting function that simulates the DNA absorption spectrum, to the UV-B lamp systems. These UV-B levels correspond to a calculated decrease in stratospheric ozone content of 6%, 21%, and 36% for treatment T₁, T₂, and T₃, respectively.
Daily exposure of soybean plants to UV-B radiation significantly decreased height, fresh and dry weights, leaf chlorophyll and carotenoid contents, and CO₂ uptake rates. Leaf pigment extracted in 80% acetone from UV-B-treated soybean plants showed considerable increase in absorption in the wavelength region of 330 to 400 nm with increased UV-B radiation levels. Chloroplast preparations from leaves of T₂ and T₃ plants showed significant reductions in Hill reaction measurements.     

Kinetics of leaf oxygen uptake represent in planta activities of respiratory electron transport and terminal oxidases

We present, for the first time, the oxygen response kinetics of mitochondrial respiration measured in intact leaves (sunflower and aspen). Low O₂ concentrations in N₂ (9–1500 ppm) were preset in a flow-through gas exchange measurement system, and the decrease in O₂ concentration and the increase in CO₂ concentration as result of leaf respiration were measured by a zirconium cell O₂ analyser and infrared-absorption CO₂ analyser, respectively. The low O₂ concentrations little influenced the rate of CO₂ evolution during the 60-s exposure. The initial slope of the O₂ uptake curve on the dissolved O₂ concentration basis was relatively constant in leaves of a single species, 1.5 mm s⁻¹ in sunflower and 1.8 mm s⁻¹ in aspen. The apparent K _0.5(O₂) values ranged from 0.33 to 0.67 μ M in sunflower and from 0.33 to 1.1 μ M in aspen, mainly because of the variation of the maximum rate, V _max (leaf temperature 22°C). The initial slope of the O₂ response of respiration characterizes the catalytic efficiency of terminal oxidases, an important parameter of the respiratory machinery in leaves. The plateau of the response characterizes the activity of the mitochondrial electron transport chain and is subject to regulations in accordance with the necessity for ATP production. The relatively low oxygen conductivity of terminal oxidases means that in leaves, less than 10% of the photosynthetic oxygen can be reassimilated by mitochondria.     

The influence of reduced photorespiration on long-term growth and development of wheat

The long-term role of photorespiration was investigated by comparing growth, development, gas exchange characteristics and mineral nutrition of a wheat crop ( Triticum aestivum L. cv. Courtot) cultivated in a culture chamber during a life cycle, either in 4% O₂ or in normal O₂ Low O₂ pressure reduced photorespiration, but CO₂ was controlled so that net photosynthesis remained the same as in the control crop. The growth and development of the low O₂ crop was slowed down. Ear appearance was 16 days late, but the rate of tillering was the same as in the control and was maintained longer so that the final number of tillers was doubled. Pigment, ribulose bisphosphate carboxylase (EC 4.1.1.39) and soluble sugar contents were similar. The response of photosynthesis to CO₂ and O₂ was not appreciably changed by the low O₂ treatment. There was almost no seed formation, and the senescence of the leaves was delayed. It appears that in non-stress conditions most of the photorespiration can be suppressed without damage to the photosynthetic apparatus. Retardation of development and inhibition of reproduction are likely due to other effects of O₂.     

Response of nitrogen metabolism to boron toxicity in tomato plants

Boron (B) toxicity has become important in areas close to the Mediterranean Sea where intensive agriculture has been developed. The objective of this research was to study the effects of B toxicity (0.5 m m and 2.0 m m B) on nitrogen (N) assimilation of two tomato cultivars that are often used in these areas. Leaf biomass, relative leaf growth rate (RGR_L), concentration of B, nitrate (NO₃⁻), ammonium (NH₄⁺), organic N, amino acids and soluble proteins, as well as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthase (GS), glutamate synthetase (GOGAT) and glutamate dehydrogenase (GDH) activities were analysed in leaves. Boron toxicity significantly decreased leaf biomass, RGR_L, organic N, soluble proteins, and NR and NiR activities. The lowest NO₃⁻ and NH₄⁺ concentration in leaves was recorded when plants were supplied with 2.0 m m B in the root medium. Total B, amino acids, activities of GS, GOGAT and GDH increased under B toxicity. Data from the present study prove that B toxicity causes inhibition of NO₃⁻ reduction and increases NH₄⁺ assimilation in tomato plants.     

Effects of solar radiation on the endemic Mediterranean red alga Rissoella verruculosa: photosynthetic performance, pigment content and the activities of enzymes related to nutrient uptake

The effects of full-spectrum solar radiation and of solar radiation deprived of total u.v. radiation (λ<395 nm) on the endemic Mediterranean red alga Rissoella verruculosa (Bertoloni) J. Agardh were studied in situ in early summer, from sunrise to sunset. Photosynthetic performance, pigment content and the activities of enzymes related to nutrient uptake, were monitored under both radiation conditions throughout a daylight period.
The doses of solar radiation, measured on the day during which the experiments were carried out (24 June 1997), were 9228·25, 1109·70 and 13·03 kJ m⁻² for PAR (λ=400–700 nm), u.v.-A (λ=315–400 nm) and u.v.-B (λ=280–315 nm), respectively. Under these conditions, a clear daily variation in photosynthetic performance was found. However, no significant differences were detected between the two radiation conditions. At noon, strong sunlight impaired O₂ evolution by 75%, but complete recovery occurred during the afternoon, reaching similar values to those measured in the early morning. By contrast, photoinhibition of optimal quantum yield ( F _v/ F _m), and the relative electron transport rate was followed by only a slight recovery during the afternoon. The rate of photosynthesis in air (simulating emersion conditions), estimated by CO₂ exchange, showed a negative balance at noon, which was accentuated in plants exposed to the full-solar spectrum.
Significant changes in the activities of nitrate reductase and carbonic anhydrase were found throughout the day under both radiation conditions. In thalli receiving solar radiation deprived of u.v., total CA activity decreased throughout the day. However, in thalli exposed to full-spectrum solar radiation, the activity of the enzyme tended to increase in the afternoon, correlating with an increase in NR activity.     

施钾对甘薯氮素转移分配及氮代谢酶活性的影响

利用¹⁵N示踪技术,研究了施钾对甘薯发根结薯期、薯块膨大期地上和地下部氮素转移分配、光合特性及氮代谢酶活性的影响.结果表明: 在发根结薯期,施钾显著提高¹⁵N向地上部的转移分配,其中K₃(K₂O, 300 mg·kg^-1)处理与对照相比¹⁵N向叶片转移速率提高了76.2%,¹⁵N积累量提高了92.1%.在薯块膨大期,随施钾量增加地上部叶片¹⁵N总分配率由33.7%降低至24.4%,块根¹⁵N分配率由5.8%升高至17%,其中K₃处理块根¹⁵N积累量是对照的3倍.两个关键生长期硝酸还原酶、谷氨酸脱氢酶、谷氨酰胺合酶、谷氨酸合酶和净光合速率(P_n)均随施钾量的增加而提高.逐步回归分析表明,氮代谢酶活性和P_n是影响甘薯¹⁵N转移和分配的主要因素(R分别为0.965和0.942),通径分析表明,在发根结薯期主要通过促进硝酸还原酶和谷氨酸脱氢酶介导的氮素催化能力促进氮素向地上部分配;在薯块膨大期主要通过提高谷氨酰胺合酶/谷氨酸合酶循环介导的氮素同化能力促进氮素向地下部分配.     

Time-course of the phosphinothricin effect on gas exchange and nitrate reduction in Medicago sativa

The long-time effect of phosphinothricin (PPT) on gas exchange and nitrate metabolism in intact plants of lucerne ( Medicago sativa L. cv. Aragón) was investigated. Photosynthetic CO₂ uptake, stomatal conductance, and transpiration were measured with an Infra-Red Gas Analyzer (IRGA). Under photorespiratory conditions, CO₂ uptake continuously decreased after PPT treatment. The decrease of photosynthesis led to an increase in the internal CO₂ concentration, which in turn caused stomatal closure and a reduction of transpiration rate. Nitrate reduction from plants sprayed with PPT was assayed both in vitro and in vivo. In vivo nitrate reductase was measured with and without nitrate in the infiltration medium. Both types of nitrate reductase assays indicated that the enzyme was inhibited in plants treated with PPT; however, the enzyme appeared more affected when the in vivo assay was used than when the one in vitro was applied. The nitrate reduction was pronouncedly affected after 24 h of PPT treatment, when glutamine synthetase (GS, EC 6.3.1.2.) activity and gas exchange were inhibited by more than 60%. The data suggest that the inhibition of GS leads to inhibition of photosynthesis, which, in turn, means lack of NADPH and nitrate, the substrates for nitrate reductase. The inhibition of GS also leads to a high ammonia level, which will produce a secondary inhibition of nitrate reductase activity.     

Effect of decreased irradiance on N and C metabolism in leaves and roots of maize

The effects of decreased irradiance on fresh and dry weight, root respiration, levels of carbohydrates and N-compounds, and extractable activities of enzymes involved in C and N metabolism were evaluated in maize ( Zea mays L. cv. Plauto) seedlings during the 7 days following transfer from 450 to 200 μmol m⁻² s⁻¹ PAR. The fresh weight of roots and stems, the initiation of new leaves, root respiration rate, and the accumulation of dry matter, soluble sugars, starch, malate and amino acids in both leaves and roots were strongly reduced at low irradiance. In contrast, the level of nitrate was increased in leaves and only marginally affected in roots. Leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity started to decrease after 24–34 h, whereas ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity and chlorophyll content were unaffected or only slightly reduced. In both leaves and roots, the adjustment of N metabolism to low irradiance occurred through a relatively rapid (30% after 10 h) and large (60% after 3 days) decrease of nitrate reductase (NR; EC 1.6.6.1) activity, followed by slower and smaller changes in the activity of nitrite reductase (EC 1.7.7.1), glutamine synthetase (EC 6.3.1.2) and NAD-dependent glutamate dehydrogenase (EC 1.4.1.2). We suggest that the preferential decrease of NR activity relative to other N-assimilating enzymes may be important for preventing the accumulation of toxic N-compounds like ammonia in both leaf and root tissues.