Ammonium and nitrate uptake by soybean during recovery from nitrogen deprivation

Soybean [Glycine max (L.) Merrill] plants that had been subjected to 15 d of nitrogen deprivation were resupplied for 10 d with 1.0 mol m-3 nitrogen provided as NO3-, NH4+, or NH4(+) + NO3- in flowing hydroponic culture. Plants in a fourth hydroponic system received 1.0 mol m-3 NO3- during both stress and resupply periods. Concentrations of soluble carbohydrates and organic acids in roots increased 210 and 370%, respectively, during stress. For the first day of resupply, however, specific uptake rates of nitrogen, determined by ion chromatography as depletion from solution, were lower for stressed than for non-stressed plants by 43% for NO3- resupply, by 32% for NH4(+) + NO3- resupply, and 86% for NH4+ resupply. When specific uptake of nitrogen for stressed plants recovered to rates for non-stressed plants at 6 to 8 d after nitrogen resupply, carbohydrates and organic acids in their roots had declined to concentrations lower than those of non-stressed plants. Recovery of nitrogen uptake capacity of roots thus does not appear to be regulated simply by the content of soluble carbon compounds within roots. Solution concentrations of NH4+ and NO3- were monitored at 62.5 min intervals during the first 3 d of resupply. Intermittent 'hourly' intervals of net influx and net efflux occurred. Rates of uptake during influx intervals were greater for the NH4(+)-resupplied than for the NO3(-)-resupplied plants. For NH4(+)-resupplied plants, however, the hourly intervals of efflux were more numerous than for NO3(-)-resupplied plants. It thus is possible that, instead of repressing NH4+ influx, increased accumulation of amino acids and NH4+ in NH4(+)-resupplied plants inhibited net uptake by stimulation of efflux on NH4+ absorbed in excess of availability of carbon skeletons for assimilation. Entry of NH4+ into root cytoplasm appeared to be less restricted than translocation of amino acids from the cytoplasm into the xylem.     

Na+/H+ exchange mechanism in the basolateral membrane of the rat enterocyte

Basolateral membrane vesicles from rat jejunal enterocytes, especially purified of brush-border contamination, were used for Na+ uptake. The basolateral membrane vesicles are osmotically active and under our experimental conditions Na+ binding is much lower than transport. An outwardly directed proton gradient stimulates Na+ uptake at both 5 microM and 5 mM concentrations. The proton gradient effect can be inhibited completely by 2 mM amiloride and partially by either FCCP or NH4Cl (NH3 diffusion). Membrane potential effects can be excluded by having valinomycin plus K+ on both sides of the vesicles. These results suggest that there is an Na+/H+ exchanger in the basolateral membrane of rat enterocytes.     

Identification of a Transport Mechanism for NH4+ in the Symbiosome Membrane of Pea Root Nodules

Symbiosome membrane vesicles, facing bacteroid-side-out, were purified from pea (Pisum sativum L.) root nodules and used to study NH4+ transport across the membrane by recording vesicle uptake of the NH4+ analog [14C]methylamine (MA). Membrane potentials ([delta][psi]) were imposed on the vesicles using K+ concentration gradients and valinomycin, and the size of the imposed [delta][psi] was determined by measuring vesicle uptake of [14C]tetraphenylphosphonium. Vesicle uptake of MA was driven by a negative [delta][psi] and was stimulated by a low extravesicular pH. Protonophore-induced collapse of the pH gradient indicated that uptake of MA was not related to the presence of a pH gradient. The MA-uptake mechanism appeared to have a large capacity for transport, and saturation was not observed at MA concentrations in the range of 25 [mu]M to 150 mM. MA uptake could be inhibited by NH4+, which indicates that NH4+ and MA compete for the same uptake mechanism. The observed fluxes suggest that voltage-driven channels are operating in the symbiosome membrane and that these are capable of transporting NH4+ at high rates from the bacteroid side of the membrane to the plant cytosol. The pH of the symbiosome space is likely to be involved in regulation of the flux.     

Ferulic acid uptake by soybean root in nutrient culture

Ferulic acid uptake by soybean root in nutrient culture was investigated by the depletion method at different concentrations, temperatures and pH. Results showed that soybean roots absorbed this compound at greater rates in the concentrations between 0.05-mM and 1.0-mM and it was concentration dependent. Ferulic acid uptake was unaffected at pH 4.5 or 6.0 but reduced at pH 7.0. At pH 6.0, uptake rates decreased significantly with increasing temperature of nutrient solution.     

High-affinity K+ uptake in pepper plants

High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.     

红松,白桦的氮营养行为及其种间分异

对红松、白桦的吸收空间和吸收N素的时间 (季节 )、数量、形态的研究表明 ,在混交情况下 ,白桦表现出较典型的浅根性特征 ,吸收根主要集中在土壤表层 ;红松则具有深根性趋势 ,其吸收根在下层土壤空间的分布明显增加 .白桦吸收N素养分的季节比较集中 ,具有明显的峰期 ;而受白桦庇荫的红松则在整个生长季中一直比较平缓地吸收N素 ,峰期不甚明显 .白桦对N的消耗量较大 ;而红松对N的消耗量则相对较小 ,N利用效率比白桦高 34% .在对N素养分化学形态的偏向选择性方面 ,白桦较喜NO-3 N ,而红松则较偏好NH 4 N .     

Inhibition of plasma membrane Ca2+-atpase pump activity in cultured c6 glioma cells by halothane and xenon

We have compared the effect of two inhalational anesthetics, halothane and xenon, on Ca²⁺-ATPase (PMCA) pumping activity in plasma membrane vesicles prepared from cultured rat C6 glioma cells. Halothane, at concentrations ranging from 0.5 to 1.75 vol% (equivalent to 0.5 to 1.6 MAC), significantly inhibited Ca²⁺ uptake (transport) by plasma membrane vesicles in a dose-related fashion. Xenon, at partial pressures ranging from 0.5 to 1.5 atm (equivalent to 0.5 to 1.6 MAC), similarly inhibited PMCA pumping activity. Additive effects on suppression of PMCA pump activity were observed when C6 cell plasma membrane vesicles were exposed to increasing partial pressures of xenon in the presence of halothane (1 vol%). Halothane also inhibited PMCA pumping in cells from two other lines of neural origin, B104 (rat neuroblastoma) and PC12 (rat pheochromocytoma). Studies described in this report support the thesis that PMCA in cells of neural origin is inhibited by quite different inhalational anesthetics at clinically relevant concentrations.     

The influence of pretreatment with different cations on anaerobic nitrite production by excisedPisum sativum roots

The influence of pretreatment with some cations on anaerobic nitrite production (in an assay medium lacking nitrate) by excised primary roots of pea (Pisum sativum L., ov. Raman), detached from six-day-old seedlings germinated in distilled water, was investigated. When the excised roots were precultivated in one-salt-solutions of KNO3, then these roots produced at 9 mM and 15 mM NO3- concentrations under anaerobic conditions significantly more NO2-, than those precultivated in a nutrient solution containing besides K+ ions also Ca2+ and Mg2+ ions, and they produced nitrite for a longer time. The KNO3 dependent increase in anaerobic NO2- production was counteracted most by Ca2+ and to a lesser extent by Mg2+; Na+ was without effect. NH4+ at higher concentrations (12 and 15 mM) significantly depressed nitrite production both by roots precultivated in a solution containing besides NH4+ only K+, and by roots precultivated in a full nutrient solution containing K+, Ca2+ and Mg2+, however at lower NH4+ concentrations (0.6 and 2mMNH4+; 15mMNO3-) the decrease was more conspicuous in the KNO3 solution than in the full nutrient solution. Nitrate reductase level was not influenced by this pretreatment. When 6% and 7.5% n-propanol, which increases membrane permeability and causes mixing of storage and metabolic nitrate pools in the cells, was added to the assay medium lacking nitrate, anaerobic nitrite production increased and the differences caused by the precultivation disappeared. These results show that higher K+ concentrations in unbalanced one-salt-solutions of KNO3 can cause higher membrane permeability by accentuating Ca8+ deficiency, which results in a faster penetration of NO3- from the storage pool to the sites of its reduction and in an easier penetration of NO2- out of the roots, and that higher NH4+ concentrations can change nitrate compartmentation and diminish the metabolic NO3- pool which results in a slower nitrate reduction. Besides that, lower NH4+ concentrations in KNO3 solutions (15mMNO3-) probably partially counteract the K+ dependent increase in membrane permeability. The results obtained show that there is no simple, direct relationship between the so-called metabolic pool of nitrate (i.e. anaerobic nitrite production) and the level of nitrate reductase, but that the velocity of nitrate reduction can be influenced by nitrate compartmentation in the cell.     

Effect of potassium on uptake and incorporation of ammonium-nitrogen of rice plants

1. Rice was grown for 5 months in a sand solution culture at two different

1. K levels. The higher K supply resulted in a reduced uptake of Na +, Mg ++, and Ca++ by shoots. The uptake of NH4+-N of the shoots, however, was increased by the higher K supply.
2. In short term experiments, ill which the NH4+-N of the uptake solution was labelled by N 15, increasing K concentrations in the uptake solution did not depress the NH4 + uptake of young rice plants. Higher K concentrations in the uptake solution favoured the translocation of labelled N from the roots to the shoots. In some cases the higher K levels resulted also in an enhanced transfer rate of labelled N from the soluble to the insoluble N fraction.
3. Increasing levels of Mg++ in the uptake solution did not affect the uptake of labelled NH4-N.
4. I t is concluded that K + and NH4 + do not compete for common binding sites of the uptake mechanism in rice roots. This lacking competition suggests the speculation that NH4+-N is absorbed mainly in form of NH8 by plant cells.

     

Investigation of the Apparent Induction of Nitrate Uptake in Barley (Hordeum vulgare L.) Using NO3--Selective Microelectrodes (Modulation of Coarse Regulation of NO3- Uptake by Exogenous Application of Downstream Metabolites in the NO3- Assimilatory Pathway)

The influence of a 12-h pretreatment with either NO3-, NH4+, glutamine, or glutamate (300 [mu]M) on the apparent induction of NO3- uptake was investigated. Net fluxes of NO3- into roots of intact, 7-d-old barley (Hordeum vulgare L. cv Prato) seedlings in solution culture were estimated from ion activity gradients measured with NO3--selective microelectrodes in the unstirred layer of solution immediately external to the root surface. Control plants, pretreated with nitrogen-free nutrient solution, exhibited a sigmoidal increase in net NO3- uptake, reaching a maximum rate between 8 and 9 h after first exposure to NO3-. Plants pretreated with NH4+ or Glu exhibited a delay of several hours in the initiation of the induction process after they had been exposed to NO3-. In Gln-pretreated plants, however, responses ranged from no delay of the induction process to delays comparable to those observed following NH4+ or Glu pretreatments. Only treatment with NO3-resulted in the induction of NO3- uptake, whereas pretreatments with NH4+, Gln, or Glu tended to delay induction of NO3- uptake upon subsequent exposure to NO3-.     

In vivo and in vitro effects of copper and cadmium on the plasma membrane H+-ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots

The plasmalemma vesicles isolated from cucumber and maize roots were used to study the effect of Cu²⁺ and Cd²⁺ on the hydrolytic and proton pumping activities of ATPase. In vivo application of metal ions to the plant growth solutions resulted in stimulation of the proton transport in maize. In cucumber roots the action of metals was not the same: cadmium stimulated the H⁺ transport through plasmalemma whereas Cu²⁺ almost completely inhibited it. Copper ions decreased the hydrolytic activity of H⁺-ATPase in cucumber, without any effect on this activity in membranes isolated from maize roots. The effect of cadmium on the hydrolytic activities was opposite: ATP-hydrolysis activity in plasmalemma was not altered in cucumber, whereas in maize its stimulation was observed. The amount of accumulated metals was not the main reason of different influence of metals on H⁺-ATPase activity in tested plants. In in vitro experiments Cu²⁺ inhibited H⁺ transport in the cucumber, to a higher degree than Cd²⁺ and both metals did not change this H⁺-ATPase activity of plasmalemma isolated from corn roots. Cu²⁺ added into the incubation medium reduced the hydrolytic activity of ATPase in the plasma membrane isolated from cucumber as well as from corn roots. Cd²⁺ diminished the hydrolytic activity of ATPase in cucumber, and no effect of Cd²⁺ in the plasmalemma isolated from corn roots was found. Our results indicated different in vitro and in vivo action of both metals on H⁺-ATPase and different response of this enzyme to Cu²⁺ and Cd²⁺ in maize and cucumber.     

ABA、IAA和CaM对发育菜豆子叶质膜H~＋－ATPase活力的效应

以亲水性两相分配法从发育菜豆子叶制备的质膜制剂经冻融循环操作,部分膜微囊可转变成密闭的翻转型。取冻融４次的质膜微囊用于Ｈ＋－ＡＴＰａｓｅ试验表明,ＡＴＰａｓｅ活力为ＡＢＡ和ＣａＭ显著地激活,但受ＩＡＡ显著抑制;质子泵活力被ＡＢＡ显著促进,但为ＣａＭ显著抑制,ＩＡＡ对质子泵活力无显著效应。可以认为：ＡＢＡ促进发育菜豆子叶吸收光合同化物可能是通过促进质膜Ｈ＋－ＡＴＰａｓｅ活力,从而促进质子／蔗糖同向运输而获得;ＩＡＡ则可能对菜豆子叶的质膜Ｈ＋－ＡＴＰａｓｅ无显著效应。在激素信号传导途径中,ＣａＭ对质膜Ｈ＋－ＡＴＰａｓｅ活力可能无直接影响。     

Cyclic variations in nitrogen uptake rate of soybean plants: effects of pH and mixed nitrogen sources.

To determine if the daily pattern of NO3- and NH4+ uptake is affected by acidity or NO3- : NH4+ ratio of the nutrient solution, non-nodulated soybean plants (Glycine max) were exposed for 21 days to replenished, complete nutrient solutions at pH 6.0, 5.5, 5.0, and 4.5 which contained either 1.0 mM NH4+, 1.0 mM NO3- [correction of NO3+], 0.67 mM NH4+ plus 0.33 mM NO3- (2:1 NH4+ : NO3-) [correction of (2:1 NH3+ : NO4-)], or 0.33 mM NH4+ plus 0.67 mM NO3- (1:2 NH4+ : NO3-). Net uptake rates of NH4+ and NO3- were measured daily by ion chromatography as depletion from the replenished solutions. When NH4+ and NO3- were supplied together, cumulative uptake of total nitrogen was not affected by pH or solution NH4+ : NO3- ratio. The cumulative proportion of nitrogen absorbed as NH4+ decreased with increasing acidity; however, the proportional uptake of NH4+ and NO3- was not constant, but varied day-to-day. This day-to-day variation in relative proportions of NH4+ and NO3- absorbed when NH4+ : NO3- ratio and pH of solution were constant indicates that the regulatory mechanism is not directly competitive. Regardless of the effect of pH on cumulative uptake of NH4+, the specific nitrogen uptake rates from mixed and from individual NH4+ and NO3- sources oscillated between maxima and minima at each pH with average periodicities similar to the expected interval of leaf emergence.     

Plasma Membrane H+-ATPase in Maize Roots Induced for NO3- Uptake

Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO3- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO3- or 1.5 mM SO4-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-100%) in vesicles isolated from NO3--induced roots. Km for Mg:ATP and pH dependency were not influenced by NO3- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO3--induced roots was not affected by 10 to 150 mM NO3- or Cl-. On the other hand, kinetics of NO3-- or Cl--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. Immunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state level of the enzyme in plasma membrane vesicles isolated from NO3--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO3-.     

Ammonium enhances resistance to salinity stress in citrus plants

In this work, we demonstrate that NH?? nutrition in citrange Carrizo plants acts as an inducer of resistance against salinity conditions. We investigated its mode of action and provide evidence that NH?? confers resistance by priming abscisic acid and polyamines, and enhances H?O? and proline basal content. Moreover, we observed reduced Cl? uptake as well as enhanced PHGPx expression after salt stress. Control and N-NH?? plants showed optimal growth. However, N-NH?? plants displayed greater dry weight and total lateral roots than control plants, but these differences were not observed for primary root length. Our results revealed that N-NH?? treatment induces a similar phenotypical response to the recent stress-induced morphogenetic response (SIMRs). The hypothesis is that N-NH?? treatment triggers mild chronic stress in citrange Carrizo plants, which might explain the SIMR observed. Moreover, we observed modulators of stress signaling, such as H?O? in N-NH?? plants, which could acts as an intermediary between stress and the development of the SIMR phenotype. This observation suggests that NH?? treatments induce a mild stress condition that primes the citrange Carrizo defense response by stress imprinting and confers protection against subsequent salt stress.     

Electrochemical potential and ion transport in vesicles of yeast plasma membrane

Vesicles from yeast plasma membrane were prepared according to Franzusoff and Cirillo [1983) J. Biol. Chem. 258, 3608), with slight modifications. When Mg-ATP was added, this preparation was able to generate a membrane potential, that was sensitive to inhibitors of the yeast H+-ATPase and uncouplers, and could be decreased by the addition of permeant anions, as measured by the fluorescence changes of the dye oxonol V. The addition of ATP could also generate a pH gradient, detectable by the fluorescence changes of the monitor aminochloromethoxyacridine. This gradient was sensitive to inhibitors of ATPase and uncouplers, and could be increased by the addition of permeant anions to the incubation mixture. When the vesicles were loaded with KCl, an increased rate of K+ efflux was produced upon the addition of ATP. Cytochrome oxidase from bovine heart could be reconstituted into the vesicles and was shown to generate a membrane potential difference, negative inside, evidenced by the fluorescence quenching of the cyanide dipropylthiacarbocyanine and the uptake of tetraphenylphosphonium. Besides, in these vesicles, K+ and Rb+, but not Na+ or NH+4 could decrease the quenching of fluorescence and the uptake of tetraphenylphosphonium produced when the electron-donor system was present. In the vesicles in which cytochrome oxidase was incorporated, upon the addition of cytochrome c and ascorbate, the uptake of 86Rb+ could be demonstrated also. This uptake was found to be saturable and inhibited by K+, and to a lesser degree by Na+. The results obtained indicate that these vesicles are reasonably sealed and capable of generating and maintaining a membrane potential. The membrane potential could be used to drive ions across the membrane of the vesicles, indicating the presence and functionality of the monovalent cation carrier. The vesicles, in general terms seem to be suitable for studying transport of ions and metabolites in yeast.     

1-O-n-octyl-beta-D-glucopyranoside as a competitive inhibitor of Na+-dependent D-glucose cotransporter in the small intestine brush-border membrane

1-O-n-Octyl-beta-D-glucopyranoside is a competitive inhibitor of the Na+-dependent D-glucose uptake into rabbit, rat and human intestinal brush-border membrane vesicles. The lack of effect on the equilibrium uptake demonstrates that the detergent does not act by rupturing the vesicles; no membrane leakiness was apparent at the concentrations of octylglucopyranoside used, since D-glucose uptake is not inhibited even in the absence of the Na+ gradient (in K+ solution). There is a competitive interaction between octylglucopyranoside and D-glucose, as shown by Dixon and by Hunter and Down plots. The selectivity of the detergent effect is confirmed by its modest influence on amino acid uptake.     

Kinetics of NO3(-) net fluxes in Pinus pinaster, Rhizopogon roseolus and their ectomycorrhizal association, as affected by the presence of NO3(-) and NH4+

The impact of mineral N supply, N-free or NO3(-) with or without NH4+, on the subsequent uptake of NO3(-) by maritime pine seedlings associated with the ectomycorrhizal fungus Rhizopogon roseolus was studied using ion-selective microelectrodes. NO3(-) net fluxes into N-starved non-mycorrhizal short roots (NMSRs) were low and measurable only over the NO3(-) concentration range of 0-70 microM. The simple kinetics observed in those roots may reflect the dominant operation of a high-affinity NO3(-) transport system (HATS) which is constitutive. NO3(-) pretreatment increased the NO3(-) net fluxes and led to a complex kinetics that may reflect the operation of other HATS. A simple kinetics was observed in plants pre-incubated at high NH4+ concentration. In contrast, NO3(-) uptake kinetics presented only one saturation phase in the fungus, whether associated with the plant or not. NO3(-) uptake was greater after a pretreatment in N-free or NO3 (-) solution, but NH4+ pretreatment led to a threefold reduction in NO3 (-) uptake. These results suggest that the regulation of NO3(-) transport systems varies between the host and the fungal partner. This variation is likely to contribute to the positive effect of mycorrhizal association on N uptake in plants when the N supply is low and fluctuating.     

Fusicoccin Activates the Plasma Membrane H+-ATPase by a Mechanism Involving the C-Terminal Inhibitory Domain

Plasma membrane vesicles isolated from spinach leaves incubated with the fungal toxin fusicoccin showed a twofold increase in ATP hydrolytic activity and a threefold increase in H+ pumping compared to controls. This increase in H+-ATPase activity was largely completed within 4 min of incubation and was not due to de novo synthesis of H+-ATPase as demonstrated by immunoblotting. Incubation with fusicoccin also resulted in a decrease in the apparent Km for ATP of the H+-ATPase from 0.22 to 0.10 mM. The fusicoccin-mediated activation of H+-ATPase activity and the accompanying decrease in the Km for ATP are changes very similar to those observed upon trypsin activation of the H+-ATPase, where an autoinhibitory domain in the C-terminal region of the H+-ATPase is removed. Thus, trypsin treatment of plasma membrane vesicles from control leaves gave a twofold increase in ATP hydrolytic activity and a threefold increase in H+ pumping, as well as a decrease in the apparent Km for ATP of the H+-ATPase from 0.22 to 0.10 mM. Trypsin treatment of plasma membranes from fusicoccin-incubated leaves did not further enhance the H+-ATPase activity, however, and neither was the Km for ATP further decreased. That trypsin really removed a small segment from the fusicoccin-activated H+-ATPase was confirmed by immunoblotting, which showed the appearance of a 90-kD band in addition to the native 100-kD H+-ATPase band upon trypsin treatment. Taken together, our data suggest that in vivo activation of the H+-ATPase by fusicoccin proceeds by a mechanism involving a displacement of the C-terminal inhibitory domain.