Reversible inactivation of maize leaf nitrate reductase

Preincubation of maize leaves crude extracts with NADH resulted in a progressive accumulation of nitrite which mimicked a rapid and lineal activation of nitrate reductase. Nevertheless, in partially purified preparations it was found that preincubation at pH 8.8 with NADH promoted a gradual inactivation of nitrate reductase. At pH 7.5, the enzyme was not inactivated by the presence of NADH alone, but, with the simultaneous presence of a low concentration of cyanide, a fast inactivation took place. The NADH-cyanide-inactivated nitrate reductase remained inactive after removing the excess of NADH and cyanide by filtration through Sephadex G-25. However, it could be readily reactivated by incubation with ferricyanide or by a short exposure to light in the presence of FAD. Prolonged irradiation caused a progressive inactivation of the photoreactivated enzyme.     

A model of isoprene emission based on energetic requirements for isoprene synthesis and leaf photosynthetic properties for Liquidambar and Quercus

We present a physiological model of isoprene (2-methyl-1,3-butadiene) emission which considers the cost for isoprene synthesis, and the production of reductive equivalents in reactions of photosynthetic electron transport for Liquidambar styraciflua L. and for North American and European deciduous temperate Quercus species. In the model, we differentiate between leaf morphology (leaf dry mass per area, M_A, g m ^{? 2}) altering the content of enzymes of isoprene synthesis pathway per unit leaf area, and biochemical potentials of average leaf cells determining their capacity for isoprene emission. Isoprene emission rate per unit leaf area ( μ mol m ^{? 2} s ^{? 1}) is calculated as the product of M_A, the fraction of total electron flow used for isoprene synthesis ( ? , mol mol ^{? 1}), the rate of photosynthetic electron transport (J) per unit leaf dry mass (J_m, μ mol g ^{? 1} s ^{? 1}), and the reciprocal of the electron cost of isoprene synthesis [mol isoprene (mol electrons ^{? 1})]. The initial estimate of electron cost of isoprene synthesis is calculated according to the 1-deoxy- D -xylulose-5-phosphate pathway recently discovered in the chloroplasts, and is further modified to account for extra electron requirements because of photorespiration. The rate of photosynthetic electron transport is calculated by a process-based leaf photosynthesis model. A satisfactory fit to the light-dependence of isoprene emission is obtained using the light response curve of J, and a single value of ? , that is dependent on the isoprene synthase activity in the leaves. Temperature dependence of isoprene emission is obtained by combining the temperature response curves of photosynthetic electron transport, the shape of which is related to long-term temperature during leaf growth and development, and the specific activity of isoprene synthase, which is considered as essentially constant for all plants. The results of simulations demonstrate that the variety of temperature responses of isoprene emission observed within and among the species in previous studies may be explained by different optimum temperatures of J and/or limited maximum fraction of electrons used for isoprene synthesis. The model provides good fits to diurnal courses of field measurements of isoprene emission, and is also able to describe the changes in isoprene emission under stress conditions, for example, the decline in isoprene emission in water-stressed leaves.     

Strong correlation between isoprene emission and gross photosynthetic capacity during leaf phenology of the tropical tree species Hymenaea courbaril with fundamental changes in volatile organic compounds emission composition during early leaf development

Changes of the volatile organic compounds (VOC) emission capacity and composition of different developmental stages of the tropical tree species Hymenaea courbaril were investigated under field conditions at a remote Amazonian rainforest site. The basal emission capacity of isoprene changed considerably over the course of leaf development, from young to mature and to senescent leaves, ultimately spanning a wide range of observed isoprene basal emission capacities from 0.7 to 111.5 µg C g^?1 h^?1 during the course of the year. By adjusting the standard emission factors for individual days, the diel courses of instantaneous isoprene emission rates could nevertheless adequately be modelled by a current isoprene algorithm. The results demonstrate the inadequacy of using one single standard emission factor to represent the VOC emission capacity of tropical vegetation for an entire seasonal cycle. A strong linear correlation between the isoprene emission capacity and the gross photosynthetic capacity (GP_max) covering all developmental stages and seasons was observed. The present results provide evidence that leaf photosynthetic properties may confer a valuable basis to model the seasonal variation of isoprenoid emission capacity; especially in tropical regions where the environmental conditions vary less than in temperate regions. In addition to induction and variability of isoprene emission during early leaf development, considerable amounts of monoterpenes were emitted in a light‐dependent manner exclusively in the period between bud break and leaf maturity. The fundamental change in emission composition during this stage as a consequence of resource availability (supply side control) or as a plant's response to the higher defence demand of young emerging leaves (demand‐side control) is discussed. The finding of a temporary emergence of monoterpene emission may be of general interest in understanding both the ecological functions of isoprenoid production and the regulatory processes involved.     

Effects of growth under elevated UV-B on photosynthesis and isoprene emission in Quercus gambelii and Mucuna pruriens

Increasing surface levels of UV-B resulting from stratospheric ozone reduction directly affect tropospheric photochemistry. There may also be indirect tropospheric effects due to changes in emission of organic compounds from vegetation. We treated woody and herbaceous isoprene-emitting species in the field with supplemental UV-B simulating 30% ozone depletion. For Quercus gambelii, photosynthesis and isoprene emission were significantly greater in elevated UV-B treatments when expressed on a leaf area basis, but not on a leaf mass basis. Leaves of Mucuna pruriens, however, showed no significant differences in photosynthesis or isoprene emission between treatments, nor when exposed for 45 min to acute high levels of UV-B. Elevated UV-B during growth did not elicit significant isoprene emission from Acer platanoides, a non-emitting species. Other potential UV-B effects, such as changes in leaf area or species composition, which may influence regional isoprene emissions, should be examined.     

Increase in nitrate reductase activity in the presence of sucrose in bean leaf segments

The supply of sucrose to leaf segments from light-grown bean seedlings caused a substantial increase in substrate inducibility of in vivo and in vitro nitrate reductase activity but only a small increase in total protein. Cycloheximide and chloramphenicol inhibited the increase in enzyme activity by nitrate and sucrose. The in vivo decline in enzyme activity in nitrate-induced leaf segments in light and dark was protected by sucrose and nitrate. The supply of NADH also protected the decline in enzyme activity, but only in the light. In vitro stability of the extracted enzyme was, however, unaffected by sucrose. The size of the metabolic nitrate pool was also enhanced by sucrose. The experiments demonstrate that sucrose has a stimulatory effect on activity or in vivo stability ' of nitrate reductase in bean leaf segments, which is perhaps mediated through increased NADH level and/or mobilization of nitrate to the metabolic pool.     

Nitrate regulation of nitrate uptake and nitrate reductase expression in barley grown at different nitrate:ammonium ratios at constant relative nitrogen addition rate

Barley (Hordeum vulgare L. cv. Golf) was cultured using the relative addition rate technique, where nitrogen is added in a fixed relation to the nitrogen already bound in biomass. The relative rate of total nitrogen addition was 0.09 day^?1 (growth limiting by 35%), while the nitrate addition was varied by means of different nitrate: ammonium ratios. In 3- to 4-week-old plants, these ratios of nitrate to ammonium supported nitrate fluxes ranging from 0 to 22 μmol g^?1 root dry weight h^?1, whereas the total N flux was 21.8 ± 0.25 μmol g^?1 root dry weight h^?1 for all treatments. The external nitrate concentrations varied between 0.18 and 1.5 μM. The relative growth rate, root to total biomass dry weight ratios, as well as Kjeldahl nitrogen in roots and shoots were unaffected by the nitrate:ammonium ratio. Tissue nitrate concentration in roots were comparable in all treatments. Shoot nitrate concentration increased with increasing nitrate supply, indicating increased translocation of nitrate to the shoot. The apparent V_max for net nitrate uptake increased with increased nitrate fluxes. Uptake activity was recorded also after growth at zero nitrate addition. This activity may have been induced by the small, but detectable, nitrate concentration in the medium under these conditions. In contrast, nitrate reductase (NR) activity in roots was unaffected by different nitrate fluxes, whereas NR activity in the shoot increased with increased nitrate supply. NR-mRNA was detected in roots from all cultures and showed no significant response to the nitrate flux, corroborating the data for NR activity. The data show that an extremely low amount of nitrate is required to elicit expression of NR and uptake activity. However, the uptake system and root NR respond differentially to increased nitrate flux at constant total N nutrition. It appears that root NR expression under these conditions is additionally controlled by factors related to the total N flux or the internal N status of the root and/or plant. The method used in this study may facilitate separation of nitrate-specific responses from the nutritional effect of nitrate.     

Effects of NADH and thiol compounds on wheat leaf nitrate reductase

The initial activity of wheat leaf nitrate reductase was depressed on inclusion of the following thiol compounds; dithiothreitol, dithioerythreitol or mercaptoethanol, but not cysteine and glutathione. This thiol effect simply resulted from an interference with the chemical determination of nitrite. Preincubation of the enzyme with NAD⁺ and these thiols enhanced the inhibition of nitrate reductase activity. This effect was mediated by NADH production by the thiol reduction of NAD⁺. The inactivation by NAD⁺ in the presence of thiol compounds which was enhanced by cyanide ions could be reversed by ferricyanide, as has been observed previously for NADH-mediated inactivation of nitrate reductase.     

Isoprene emission from a subarctic peatland under enhanced UV-B radiation

Isoprene is a reactive hydrocarbon with an important role in atmospheric chemistry, and emissions from vegetation contribute to atmospheric carbon fluxes. The magnitude of isoprene emissions from arctic peatlands is not known, and it may be altered by increasing UV-B radiation. Isoprene emission was measured with the dynamic chamber method from a subarctic peatland under long-term enhancement of UV-B radiation targeted to correspond to a 20% loss in the stratospheric ozone layer. The site type of the peatland was a flark fen dominated by the moss Warnstorfia exannulata and sedges Eriophorum russeolum and Carex limosa. The relationship between species densities and the emission was also assessed. Isoprene emissions were significantly increased by enhanced UV-B radiation during the second (2004) and the fourth (2006) growing seasons under the UV-B exposure. Emissions were related to the density of E. russeolum. The dominant moss, W. exannulata, proved to emit small amounts of isoprene in a laboratory trial. Subarctic fens, even without Sphagnum moss, are a significant source of isoprene to the atmosphere, especially under periods of warm weather. Warming of the Arctic together with enhanced UV-B radiation may substantially increase the emissions.     

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.     

植物氮代谢硝酸还原酶水平调控机制的研究进展

氮代谢是植株体内最基本的物质代谢之一,硝酸还原酶是植物氮代谢的关键酶。主要对植物氮代谢在硝酸还原酶水平上调控的研究新进展,尤其是其合成／降解及活性调控机制进行了较为系统的综述。硝酸还原酶合成的调控主要发生在转录水平和翻译水平上,硝酸还原酶降解的调控主要发生在翻译后水平上,同时NO3^-及光在硝酸还原酶转录水平调控上的作用重大,硝酸还原酶编码基因转录的mRNA的稳定性强弱影响植物的氮代谢,而影响mRNA稳定性的因素很多,机理复杂;磷酸化／去磷酸化在硝酸还原酶活性调控中占举足轻重的地位,研究也比较深入。钝化蛋白也能够影响硝酸还原酶活性,许多小分子物质对硝酸还原酶活性有影响。     

Ammonium can stimulate nitrate and nitrite reductase in the absence of nitrate in Clematis vitalba

Nitrogen assimilation was studied in the deciduous, perennial climber Clematis vitalba. When solely supplied with NO₃^– in a hydroponic system, growth and N-assimilation characteristics were similar to those reported for a range of other species. When solely supplied with NH₄⁺, however, nitrate reductase (NR) activity dramatically increased in shoot tissue, and particularly leaf tissue, to up to three times the maximum level achieved in NO₃^– supplied plants. NO₃^– was not detected in plant material that had been solely supplied with NH₄⁺, there was no NO₃^– contamination of the hydroponic system, and the NH₄⁺-induced activity did not occur in tobacco or barley grown under similar conditions. Western Blot analysis revealed that the induction of NR activity, either by NO₃^– or NH₄⁺, was matched by NR and nitrite reductase protein synthesis, but this was not the case for the ammonium assimilation enzyme glutamine synthetase. Exposure of leaf disks to N revealed that NO₃^– assimilation was induced in leaves directly by NO₃^– and NH₄⁺ but not glutamine. Our results suggest that the NH₄⁺-induced potential for NO₃^– assimilation occurs when externally sourced NH₄⁺ is assimilated in the absence of any NO₃^– assimilation. These data show that the potential for nitrate assimilation in C. vitalba is induced by a nitrogenous compound in the absence of its substrate and suggest that NO₃^– assimilation in C. vitalba may have a significant role beyond the supply of reduced N for growth.     

Induction of nitrate uptake and nitrate reductase activity in trembling aspen and lodgepole pine

¹³NO₃^– influx into the roots and in vivo nitrate reductase activity (NRA) in the roots and leaves have been measured in trembling aspen (Populus tremuloides Michx.) and lodgepole pine (Pinus contorta Dougl.) seedlings after exposure to either 0·1 or 1·5 mol m^–3 NO₃^– for varying periods up to 20 d. Both NO₃^– influx and NRA were inducible in these species and, in trembling aspen, peak induction of nitrate influx and NRA were achieved within 12 h, compared to 2–4 d for influx and 4–12 d for NRA in lodgepole pine. In trembling aspen, ≈ 30% of the total ¹³N absorbed during a 10 min influx period followed by 2 min of desorption was translocated to the shoot. In lodgepole pine, by contrast, translocation of ¹³N to the shoot was undetectable during the same time period. Root NRA as well as NO₃^– influx from 0·1 mol m^–3 NO₃^– were substantially higher in trembling aspen than in lodgepole pine at all stages of NO₃^– exposure, i.e. during the uninduced, the peak induction, and steady-state stages. In order to examine whether the lower rates of NO₃^– influx and NRA were related to proportionately fewer young (unsuberized) roots in lodgepole pine, we determined these parameters in young and old (suberized) roots of this species separately. Induction of influx and NRA were initially greater in young roots but at steady-state there were only minor differences between the young and the old roots. However, even the elevated initial rates in the young roots of lodgepole pine were substantially lower than those of aspen. In pine, influx at 1·5 mol m^–3 NO₃^– was ~ 6-fold higher than at 0·1 mol m^–3 NO₃^– and appeared to be mostly via a constitutive system. By contrast, in aspen, steady-state influxes at 0·1 and 1·5 mol m^–3 were not significantly different, being similar to the rate attained by pine at only the higher [NO₃^–]. In aspen, leaf NRA was ~ 2-fold higher than that of roots. In lodgepole pine NRA of the needles was below the detection limit. These results show that trembling aspen seedlings are better adapted for NO₃^– acquisition and utilization than lodgepole pine seedlings.     

Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species

BACKGROUND AND AIMS: Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. METHODS: Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. RESULTS: Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron-deficient conditions, compared with the same species grown under iron-sufficient conditions or with V. arboreum grown under either iron condition. CONCLUSIONS: V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum.     

Studies of the relationship between isoprene emission rate and CO2 or photon-flux density using a real-time isoprene analyser

Abstract. Studies of the isoprene emission rate in response to changes in photon-flux density and CO₂ partial pressure were conducted using a recently developed on-line isoprene analyser combined with a gas exchange system and chlorophyll fluorometer. Upon darkening, the isoprene emission rate from leaves of aspen ( Populus tremuloides Michaux.) began to decline immediately, demonstrating that the internal pool of isoprene, or its precursors, is small and that the instantaneous emission rate is tightly coupled to the rate of synthesis. A post-illumination burst of isoprene was observed within 5 min after darkening and lasted for 15–20 min in four isoprene-emitting species that were examined. In leaves of eucalyptus ( Eucalyptus globulus Labill.), the magnitude of the post-illumination burst was dependent on the photon-flux density that existed before darkening, but not on ambient CO₂ partial pressure. The dependence of the post-illumination burst on photon-flux density paralleled that for the steady-state rate of isoprene emission. A step-wise increase in intercellular CO₂ partial pressure from 24.5 to 60 Pa resulted in an immediate decrease in isoprene emission rate and non-photochemical fluorescence quenching, but an increase in CO₂ assimilation rate. Given the several recent studies that link isoprene emission to chloroplastic processes, the results of this study indicate that the linkage is not dependent on the rate of CO₂ flux through the reductive pentose phosphate pathway, but rather on more complex relationships involving metabolites not appreciably influenced by CO₂ partial pressure.     

Interaction of drought and ozone exposure on isoprene emission from extensively cultivated poplar

The combined effects of ozone (O₃) and drought on isoprene emission were studied for the first time. Young hybrid poplars (clone 546, Populus deltoides cv. 55/56 x P. deltoides cv. Imperial) were exposed to O₃ (charcoal‐filtered air, CF, and non‐filtered air +40 ppb, E‐O₃) and soil water stress (well‐watered, WW, and mild drought, MD, one‐third irrigation) for 96 days. Consistent with light‐saturated photosynthesis (A_sat), intercellular CO₂ concentration (C_i) and chlorophyll content, isoprene emission depended on drought, O₃, leaf position and sampling time. Drought stimulated emission (+38.4%), and O₃ decreased it (?40.4%). Ozone increased the carbon cost per unit of isoprene emission. Ozone and drought effects were stronger in middle leaves (13th–15th from the apex) than in upper leaves (6th–8th). Only A_sat showed a significant interaction between O₃ and drought. When the responses were up‐scaled to the entire‐plant level, however, drought effects on total leaf area translated into around twice higher emission from WW plants in clean air than in E‐O₃. Our results suggest that direct effects on plant emission rates and changes in total leaf area may affect isoprene emission from intensively cultivated hybrid poplar under combined MD and O₃ exposure, with important feedbacks for air quality.     

Effects of soil applied herbicides on leaf nitrate reductase and crude protein in the leaf and seed of two cowpea cultivars

A glass-house study was conducted to determine the effects of four commonly used herbicides (pendimethalin, metobromuron, metolachlor and prometryne) applied pre-emergence at rates of 0, 0.125, 0.625 and 1.25 kg ha^–1, on leaf nitrate concentration (NO₃–C), nitrate reductase activity (NRA), leaf crude protein and seed protein in two cowpea cultivars, 60 day (60D) and Ife brown (IB).Control and treated plants of both cultivars showed separate peaks for NO₃–C and NRA, 49 days after planting (DAP) and 35 DAP for 60D and IB respectively. Herbicide treatment generally enhanced NO₃–C but tended to decrease NRA in both cultivars. Howver, metobromuron at 0.625 kg ha^–1 increased NRA throughout the growth period with an optimum increase of 52.5%, over the control, at 35 DAP. Pendimethalin increased NO₃–C NRA and leaf protein but did not influence seed protein appreciably. In contrast metobromuron increased NO₃–C, decreased NRA, but increased seed protein by 29.6% over the control at 0.125 kg ha^–1 in 60D. Metolachlor and prometryne were most inhibitory to seed protein development. In addition, metolachlor reversed the interdependence of NO₃–C and NRA.     

Extraction of nitrate reductase from leaves of ericaceae

The inability to obtain active nitrate reductase (E.C. 1.6.6.2) preparations from leaf extracts of Leucothoe and Rhododendron (Ericaceae) was due to a galloyl ester-like compound, similar in nature to tannic acid.