氮素形态对番茄根系液泡膜焦磷酸酶活性的影响

采用超速梯度离心提取液泡膜微囊,研究硝态氮和铵态氮供应情况下番茄（Lycopersicon esculentum L.)根系液泡膜焦磷酸酶活性的变化。结果表明：供应铵态氮番茄根系专一性液泡膜焦磷酸酶活性比供应硝态氮的高37．7％;供应铵态氮根系液泡膜焦磷酸酶转运质子活力也明显高于供应硝态氮根系。Western blot结果表明,供应铵态氮根系液泡膜焦磷酸酶蛋白表达量也明显增加。     

Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum

Summary Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells ofChenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.     

Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum

Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells of Chenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.     

Tonoplast inorganic pyrophosphatase in Vicia faba guard cells

Lysed guard-cell protoplasts of Vicia faba L. exhibited hydrolytic activity characteristic of tonoplast inorganic pyrophosphatase (V-PPase; EC 3.6.1.1). Activity was inhibited by the specific V-PPase inhibitor aminomethylenediphosphonate, stimulated by K⁺ (K _m = 51 mM) and inhibited by Ca²⁺ (80 nM free Ca²⁺ was required for 50% inhibition at 0.27 mM free Mg²⁺). Patch-clamp measurements of electrogenic activity confirmed enzyme localisation at the tonoplast. This is the first report of V-PPase activity in guard cells; its possible involvement in stomatal opening is discussed. Received: 12 February 1998 / Accepted: 24 April 1998     

Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa.

The Chenopodiaceae Suaeda salsa L. was grown under different salt concentrations and under osmotic stress. The fresh weight was markedly stimulated by 0.1 M NaCl, 0.4 M NaCl and 0.1 M KCl and reduced by osmotic stress (PEG iso-osmotic to 0.1 M NaCl). Treatment with 0.4 M KCl severely damaged the plants. Membrane vesicle fractions containing tonoplast vesicles were isolated by sucrose gradient from leaves of the S. salsa plants and modulations of V-ATPase and V-PPase depending on the growth conditions were determined. Western blot analysis revealed that V-ATPase of S. salsa consists of at least nine subunits (apparent molecular masses 66, 55, 52, 48, 36, 35, 29, 18, and 16 kDa). This polypeptide pattern did not depend on culture conditions. V-PPase is composed of a single polypeptide (69 kDa). An additional polypeptide (54 kDa) was detected in the fractions of NaCl-, KCl- and PEG-treated plants. It turned out that the main strategy of salt-tolerance of S. salsa seems to be an up-regulation of V-ATPase activity, which is required to energize the tonoplast for ion uptake into the vacuole, while V-PPase plays only a minor role. The increase in V-ATPase activity is not obtained by structural changes of the enzyme, but by an increase in V-ATPase protein amount.     

The role of vacuolar malate-transport capacity in crassulacean acid metabolism and nitrate nutrition. Higher malate-transport capacity in ice plant after crassulacean acid metabolism-induction and in tobacco under nitrate nutrition

Anion uptake by isolated tonoplast vesicles was recorded indirectly via increased H(+)-transport by H(+)-pumping of the V-ATPase due to dissipation of the electrical component of the electrochemical proton gradient, Deltamu(H+), across the membrane. ATP hydrolysis by the V-ATPase was measured simultaneously after the Palmgren test. Normalizing for ATP-hydrolysis and effects of chloride, which was added to the assays as a stimulating effector of the V-ATPase, a parameter, J(mal)(rel), of apparent ATP-dependent malate-stimulated H(+)-transport was worked out as an indirect measure of malate transport capacity. This allowed comparison of various species and physiological conditions. J(mal)(rel) was high in the obligate crassulacean acid metabolism (CAM) species Kalancho? daigremontiana Hamet et Perrier, it increased substantially after CAM induction in ice plant (Mesembryanthemum crystallinum), and it was positively correlated with NO(3)(-) nutrition in tobacco (Nicotiana tabacum). For tobacco this was confirmed by measurements of malate transport energized via the V-PPase. In ice plant a new polypeptide of 32-kD apparent molecular mass appeared, and a 33-kD polypeptide showed higher levels after CAM induction under conditions of higher J(mal)(rel). It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO(3)(-) nutrition and that a putative malate transporter must be within the 32- to 33-kD polypeptide fraction of tonoplast proteins.     

Chilling-induced changes of vacuolar proton pumps in hypocotyls of <Emphasis Type="Italic">Vigna unguiculata</Emphasis>

Vigna unguiculata (cowpea) is a legume adapted to high temperatures and is sensitive to low temperatures. Temperature is one of the limiting factors of growth and yield for many crops but its effect on cowpea metabolism is not known. We investigated the effect of chilling on activity of vacuolar proton pumps (V-ATPase and V-PPase) and their protein content in tonoplast vesicles of cowpea hypocotyls. Seedlings grown for 7 days at 10 or 4°C were used for experiments. Chilling treatment at 10 or 4°C markedly suppressed growth of cowpea seedlings. Following chilling at 10 and 4°C, activity of both proton pumps and the relative amount of V-PPase and subunit A of V-ATPase were significantly increased. Both substrate hydrolysis and H⁺ transport activities of V-PPase remained at relatively high levels during chilling treatment. For V-ATPase, treatment at 10°C for 6 days increased the ATP hydrolysis activity. However, the H⁺ transport activity of the enzyme was increased when treated for 4 days but was markedly decreased when treated for 6 days. Our results provide evidence for different regulation for these vacuolar proton pumps, indicating that V-PPase is the more stable proton pump throughout chilling stress.     

Partial characterization of H-translocating inorganic pyrophosphatase from 3 citrus varieties differing in vacuolar pH

Vacuolar pyrophosphatase (V-PPase) from juice cells of 3 citrus varieties (differing in their vacuolar pH) were partially characterized using purified tonoplast vesicles. Total V-PPase activity was highest in vesicle samples from sweet limes with vacuolar pH of 5.0, while samples from acid limes (with lowest vacuolar pH of 2.0) had the minimal total V-PPase activity. Samples from 'Valencia' orange had intermediate V-PPase levels. When assayed at equal V-PPase activity (measured as Pi production), V-PPase was not able to generate a pH gradient (ΔpH) in vesicles from acid lime, despite its capacity to form a ΔpH in the presence of ATP. Vesicles from sweet lime and 'Valencia' orange were able to form similar ΔpHs in the presence of PPi and ATP supplied together or separately. Antibodies raised against a peptide corresponding to the catalytic site of mung bean V-PPase reacted with samples from all varieties, coinciding with their capacity to hydrolyze PPi. However, antibodies raised against the entire V-PPase polypeptide from mung bean recognized V-PPase from sweet lime and 'Valencia' orange, but did not recognize acid lime samples even at elevated protein concentrations. The structural differences highlighted by antibody recognition, substrate affinity and proton-pumping reactions of V-PPase presented here may reflect evolutionary adaptations related to its reduced function under in vivo conditions and are in agreement with our understanding of acid, sugar accumulation and vacuolar pH changes during the development and maturation of citrus fruits.     

Unusual regulatory nitrate reductase activity in cotyledons of Brassica napus seedlings: enhancement of nitrate reductase activity by ammonium supply

The effect of supplying either nitrate or ammonium on nitrate reductase activity (NRA) was investigated in Brassica napus seedlings. In roots, nitrate reductase activity (NRA) increased as a function of nitrate content in tissues and decreased when ammonium was the sole nitrogen source. Conversely, in the shoots (comprising the cotyledons and hypocotyl), NRA was shown to be independent of nitrate content. Moreover, when ammonium was supplied as the sole nitrogen source, NRA in the shoots was surprisingly higher than under nitrate supply and increased as a function of the tissue ammonium content. Under 15 mM of exogenous ammonium, the NRA was up to 2.5-fold higher than under nitrate supply after 6 d of culture. The NR mRNA accumulation under ammonium nutrition was 2-fold higher than under nitrate supply. The activation state of NR in shoots was especially high compared with roots: from nearly 80% under nitrate supply it reached 94% under ammonium. This high NR activation state under ammonium supply could be the consequence of the slight acidification observed in the shoot tissue. The effect of ammonium on NRA was only observed in cotyledons and when more than 3 mM ammonium was supplied. No such NRA increase was evident in the roots or in foliar discs. Addition of 1 mM nitrate under ammonium nutrition halved NRA and decreased the ammonium content in shoots. Thus, this unusual NRA was restricted to seedling cotyledons when nitrate was lacking in the nitrogen source.     

Changes in H+-Pumps and a Tonoplast Intrinsic Protein of Vacuolar Membranes during the Development of Pear Fruit

Vacuolar H⁺-ATPase (V-ATPase) was purified from pear fruit andantibodies were raised against the subunits of 55 and 33 kDa.Antibodies against mung bean H⁺-pyro-phosphatase (V-PPase) andradish VM23, which is a tonoplast intrinsic protein (TIP) anda water channel, cross-reacted with the vacuolar membrane proteinsof pear fruit. To clarify the roles of these proteins in developmentof pear fruit, we determined their levels relative to the totalamount of protein by immunoblot analysis. The levels of subunitsof the V-ATPase increased with fruit development. By contrast,the level of V-PPase was particularly high at the cell-divisionstage and remained almost the same at other stages. The changesin the activities of V-ATPase and V-PPase corresponded to thosein their protein levels. The ratio of V-PPase activity to V-ATPaseactivity indicated that V-PPase is a major H⁺-pump of the vacuolarmembranes of young fruit and that the contribution of V-ATPaseincreases with fruit development, finally, V-ATPase becomesthe major H⁺-pump during the later stages of fruit development.The level of a protein analogous to VM23 (VM23P) was especiallyhigh during the active cell-expansion stage in young fruit,and VM23P might, therefore, play an important role in the rapidexpansion of cells as a vacuolar water channel. Our resultsshow that the levels of V-ATPase, V-PPase and VM23P change differentlyand reflect the roles of the respective proteins in the developmentof pear fruit. ³Research Fellow of the Japan Society for the Promotion of Science ⁴Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan     

Interaction of sulfur and nitrogen nutrition in tobacco (Nicotiana tabacum) plants: significance of nitrogen source and root nitrate reductase

Abstract: The significance of root nitrate reductase for sulfur assimilation was studied in tobacco (Nicotiana tabacum) plants. For this purpose, uptake, assimilation, and long-distance transport of sulfur were compared between wild-type tobacco and transformants lacking root nitrate reductase, cultivated either with nitrate or with ammonium nitrate. A recently developed empirical model of plant internal nitrogen cycling was adapted to sulfur and applied to characterise whole plant sulfur relations in wild-type tobacco and the transformant. Both transformation and nitrogen nutrition strongly affected sulfur pools and sulfur fluxes. Transformation decreased the rate of sulfate uptake in nitrate-grown plants and root sulfate and total sulfur contents in root biomass, irrespective of N nutrition. Nevertheless, glutathione levels were enhanced in the roots of transformed plants. This may be a consequence of enhanced APR activity in the leaves that also resulted in enhanced organic sulfur content in the leaves of the tranformants. The lack of nitrate reductase in the roots in the transformants caused regulatory changes in sulfur metabolism that resembled those observed under nitrogen deficiency. Nitrate nutrition reduced total sulfur content and all the major fractions analysed in the leaves, but not in the roots, compared to ammonium nitrate supply. The enhanced organic sulfur and glutathione levels in ammonium nitrate-fed plants corresponded well to elevated APR activity. But foliar sulfate contents also increased due to decreased re-allocation of sulfate into the phloem of ammonium nitrate-fed plants. Further studies will elucidate whether this decrease is achieved by downregulation of a specific sulfate transporter in vascular tissues.     

Effect of ammonium or nitrate nutrition on net photosynthesis,growth, and activity of the enzymes nitrate reductase and glutamine synthetase in blueberry,raspberry and strawberry

Blueberry, raspberry and strawberry may have evolved strategies for survival due to the different soil conditions available in their natural environment. Since this might be reflected in their response to rhizosphere pH and N form supplied, investigations were carried out in order to compare effects of nitrate and ammonium nutrition (the latter at two different pH regimes) on growth, CO2 gas exchange, and on the activity of key enzymes of the nitrogen metabolism of these plant species. Highbush blueberry (Vaccinium corymbosum L. cv. 13–16–A), raspberry (Rubus idaeus L. cv. Zeva II) and strawberry (Fragaria × ananassa Duch. cv. Senga Sengana) were grown in 10 L black polyethylene pots in quartz sand with and without 1% CaCO3 (w: v), respectively. Nutrient solutions supplied contained nitrate (6 mM) or ammonium (6 mM) as the sole nitrogen source. Compared with strawberries fed with nitrate nitrogen, supply of ammonium nitrogen caused a decrease in net photosynthesis and dry matter production when plants were grown in quartz sand without added CaCO3. In contrast, net photosynthesis and dry matter production increased in blueberries fed with ammonium nitrogen, while dry matter production of raspberries was not affected by the N form supplied. In quartz sand with CaCO3, ammonium nutrition caused less deleterious effects on strawberries, and net photosynthesis in raspberries increased as compared to plants grown in quartz sand without CaCO3 addition. Activity of nitrate reductase (NR) was low in blueberries and could only be detected in the roots of plants supplied with nitrate nitrogen. In contrast, NR activity was high in leaves, but low in roots of raspberry and strawberry plants. Ammonium nutrition caused a decrease in NR level in leaves. Activity of glutamine synthetase (GS) was high in leaves but lower in roots of blueberry, raspberry and strawberry plants. The GS level was not significantly affected by the nitrogen source supplied. The effects of nitrate or ammonium nitrogen on net photosynthesis, growth, and activity of enzymes in blueberry, raspberry and strawberry cultivars appear to reflect their different adaptability to soil pH and N form due to the conditions of their natural environment. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Interactions between nitrate and ammonium during uptake by carob seedlings and the effect of the form of earlier nitrogen nutrition

Seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were used in two sets of experiments in order to evaluate; (1) the reciprocal effects of each nitrogen form on net uptake of nitrate and ammonium, and (2) the effect of earlier nitrogen nutrition on ammonium versus nitrate uptake. In the former group of experiments we studied the kinetics of nitrate and ammonium uptake as well as the interference of each of the two forms with net uptake of ammonium and nitrate by both nitrogen depleted and nitrogen fed carob seedlings. On the whole, nitrogen depletion led to increase in both affinity and V_max of the system for both forms of nitrogen, at the same time as the effects of nitrate on uptake of ammonium and vice versa were concentration dependent. In the second group of experiments the effects of earlier nitrogen nutrition on nitrate and ammonium uptake were characterized, and in this case we observed that: (a) if only one form of N was supplied, ammonium was taken up in greater amounts than nitrate; (b) the presence of ammonium enhanced nitrate uptake; (c) ammonium uptake was inhibited by nitrate; (d) there was a significant effect of the earlier nitrogen nutrition on the response of the plants to a different nitrogen source. The latter was evident mainly as regards ammonium uptake by plants grown in ammonium nitrate. The interactions between nitrate and ammonium uptake systems are discussed on the basis of the adaptation to the nitrogen source during early growth.     

Na~ ,K~ 和ABA对盐胁迫大麦根液泡膜ATPase活性的影响

许多植物液泡膜ATPaso活性与植物抗盐性有关(Bremberger等1988,Matsumoto和Chung 1988,Gabarino和Dupont 1988)。当植物生长在高浓度NaCl环境中时,液泡膜上Na~ /K~ 交换对维持细胞质中高K~ /Na~ 起重要作用(Jeschke     

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.     

Ammonium and nitrate nutrition inPlantago lanceolata andPlantago major L. ssp.major

P. lanceolata andP. major were grown in culture solutions with nitrate or ammonium as the nitrogen source. Dry matter accumulation in the shoot was faster with nitrate than with ammonium, whilst that of the roots was not affected by the nitrogen source. As a consequence, the shoot-to-root ratio was lower with ammonium than with nitrate. InP. lanceolata, dry matter percentage of shoot and root tissue was lower with nitrate nutrition, suggesting better elongation growth than with ammonium. However, in shoot tissue ofP. major the opposite was found. The rate of root respiration declined with time, and this was almost completely due to a declining activity of the alternative path, which amounted to about 30–60% of total root respiration. Respiration via the cytochrome path was for a part of time slightly increased by ammonium, whereas the activity of the alternative path was strongly enhanced. The concentration of ethanol-soluble carbohydrates (SC) in the roots of both species was higher when nitrate was used, but no difference in the concentration of starch was found. When the plants were transferred from one nitrogen source to the other, many parameters, including the concentration of nitrate and chloride, and the shoot to root ratio, adjusted to the new situation in both species. Grassland Species Research Group, Publication no. 116.     

Nitrate transport across the tonoplast of Cucumis sativus L. root cells

Nitrate transport across the tonoplast has been studied using vacuole membranes isolated from cucumber roots grown in nitrate. The addition of NO3- ions into the tonoplast with ATP-generated transmembrane proton gradient caused the dissipation of delta pH, indicating the NO3(-)-induced proton efflux from vesicles. NO3(-)-dependent H+ efflux was almost insensitive to the transmembrane electrical potential difference, suggesting the presence of an electroneutral NO3-/H+ antiporter in the tonoplast. Apart from saturation kinetics, with respect to nitrate ions, NO3(-)-linked H+ efflux from the tonoplast of cucumber roots showed other characteristics expected of substrate-specific transporters. Experiments employing protein modifying reagents (NEM, pCMBS, PGO and SITS) indicated that a crucial role in the activity of tonoplast nitrate/proton antiporter is played by lysine residues (strong inhibition of NO3-/H+ antiport by SITS). None of the ion-channel inhibitors (NIF, ZnSO4 and TEA-Cl) used in the experiments had a direct effect on the nitrate transport into tonoplast membranes. On the other hand, every protein reagent, as well as NIF and ZnSO4, significantly affected the ATP-dependent proton transport in vesicles. Only TEA-Cl, the potassium channel blocker, had no effect on the vacuolar proton pumping activity.