Ammonium Uptake by Rice Roots (I. Fluxes and Subcellular Distribution of 13NH4+)

The time course of 13NH4+ uptake and the distribution of 13NH4+ among plant parts and subcellular compartments was determined for 3-week-old rice (Oryza sativa L. cv M202) plants grown hydroponically in modified Johnson's nutrient solution containing 2,100, or 1000 [mu]M NH4+ (referred to hereafter as G2, G100, or G1000 plants, respectively). At steady state, the influx of 13NH4+ was determined to be 1.31, 5.78, and 10.11 [mu]mol g-1 fresh weight h-1, respectively, for G2, G100, and G1000 plants; efflux was 11, 20, and 29%, respectively, of influx. The NH4+ flux to the vacuole was calculated to be between 1 and 1.4 [mu]mol g-1 fresh weight h-1. By means of 13NH4+ efflux analysis, three kinetically distinct phases (superficial, cell wall, and cytoplasm) were identified, with t1/2 for 13NH4+ exchange of approximately 3 s and 1 and 8 min, respectively. Cytoplasmic [NH4+] was estimated to be 3.72, 20.55, and 38.08 mM for G2, G100, and G1000 plants, respectively. These concentrations were higher than vacuolar [NH4+], yet 72 to 92% of total root NH4+ was located in the vacuole. Distributions of newly absorbed 13NH4+ between plant parts and among the compartments were also examined. During a 30-min period G100 plants metabolized 19% of the influxed 13NH4+. The remainder (81%) was partitioned among the vacuole (20%), cytoplasm (41%), and efflux (20%). Of the metabolized 13N, roughly one-half was translocated to the shoots.     

Ammonium Uptake by Rice Roots (III. Electrophysiology)

The transmembrane electrical potential differences ([delta][psi]) were measured in epidermal and cortical cells of intact roots of 3-week-old rice (Oryza sativa L. cv M202) seedlings grown in 2 or 100 [mu]M NH4+ (G2 or G100 plants, respectively). In modified Johnson's nutrient solution containing no nitrogen, [delta][psi] was in the range of -120 to -140 mV. Introducing NH4+ to the bathing medium caused a rapid depolarization. At the steady state, average [delta][psi] of G2 and G100 plants were -116 and -89 mV, respectively. This depolarization exhibited a biphasic response to external NH4+ concentration similar to that reported for 13NH4+ influx isotherms (M.Y. Wang, M.Y. Siddiqi, T.J. Ruth, A.D.M. Glass [1993] Plant Physiol 103: 1259-1267). Plots of membrane depolarization versus 13NH4+ influx were also biphasic, indicating distinct coupling processes for the two transport systems, with a breakpoint between two concentration ranges around 1 mM NH4+. The extent of depolarization was also influenced by nitrogen status, which was larger for G2 plants than for G100 plants. Depolarization of [delta][psi] due to NH4+ uptake was eliminated by a protonophore (carboxylcyanide-m-chlorophenylhydrazone), inhibitors of ATP synthesis (sodium cyanide plus salicylhydroxamic acid), or an ATPase inhibitor (diethylstilbestrol). The results of these observations are discussed in the context of the mechanisms of NH4+ uptake by high- and low-affinity transport systems operating across the plasma membranes of root cells.     

Varietal Differences in the Kinetics of Iron Uptake by Excised Rice Roots

Iron uptake by excised rice roots can be described in kineticterms of active absorption and the formation of ion-carriercomplexes. Conventional interpretation indicates two carriers,one of which is responsible for iron absorption at low concentrations,with the second primarily functioning at high iron levels. Themaximum absorption of both carriers vanes greatly between varieties,the variety Pebifun having much greater absorptive capacitythan either Siam-29 or Paldal. Varietal differences in uptakecapacity primarily depend on total carrier concentration, 0.93,0.40,and 0.31 µ moles per g roots of Pebifun, Siam-29, andPaldal respectively. Although the kinetic treatment suggeststwo carriers, the chromatographic evidence indicates the presenceof the iron primarily in one major initial complex, with suggestionsof accumulation rather than turnover. An alternative to thecarrier hypothesis is therefore put forward which regards theaccumulation of iron as binding initially on to one or severalclosely related substances, followed by incorporation into secondaryproducts when a critical level of the initial product is exceeded.Manganese was found to competitively inhibit iron uptake byrice roots. Inhibition was more severe in Siam-29 than in Paldaland can be explained on the basis of carrier concentration andcompetition for the same carrier site. Copper was found notto be competitive for the iron absorption sites. The effectof copper was not significant with the low levels of iron butonly with the 5 x 10^–4 M Fe level. The effect varied betweenvarieties, in Pebifun both 5 x 10^–5 and 5 x 10^–6M Cu inhibited iron uptake compared with the control. In thecase of Siam-29 both copper levels accelerated iron uptake,5 x 10^–5 M Cu giving the greatest uptake. With 5 x 10^–5M Cu Pebifun took up much less iron than Siam-29. The mecharosmof copper inhibition/stimulation is highly speculative.     

Multiphasic Uptake of Ammonium by Soybean Roots

Uptake of ammonium by intact soybean (Glycine max Merr. cv. Amsoy) plants can be represented by 3 phases of a single, multiphasic mechanism in the range 1.78 × 10^-5-3.57 × 10^-3M. Each phase covers a limited concentration range and obeys Michaelis-Menten kinetics. The multiphasic pattern of NH₄⁺ uptake is remarkably consistent at all stages of soybean growth (20, 40, 60 and 80 days).     

Studies of the Uptake of Nitrate in Barley: I. Kinetics of NO(3) Influx

¹³NO₃⁻ was used to investigate patterns of NO₃⁻ influx into roots of barley plants (Hordeum vulgare L. cv Klondike) previously grown with (`induced') or without (`uninduced') a source of external NO₃⁻ ([NO₃⁻]₀). In both induced and uninduced plants, ¹³NO₃⁻ influx was biphasic in the range from 0.005 to 50 moles per cubic meter [NO₃⁻]₀. In the low concentration range (<1 mole per cubic meter for induced plants and <0.3 mole per cubic meter for uninduced plants), influx was saturable and V_max and K_m values for influx either increased or decreased according to NO₃⁻ pretreatment. By contrast, ¹³NO₃⁻ influx in the high concentration range revealed a strictly linear concentration dependence. These fluxes appeared to be mediated by a constitutive, rather than an inducible, transport system.     

Analysis of 13NH4+ Efflux in Spruce Roots (A Test Case for Phase Identification in Compartmental Analysis)

13NH4+-efflux analyses were conducted with roots of intact Picea glauca (Moench) Voss. seedlings at external NH4+ concentrations of 100 [mu]M and 1.5 mM. Three kinetically distinct phases were identified with half-lives of exchange of approximately 2 s, 30 s, and 14 min. The presumed identities of the subcellular compartments corresponding to these phases were confirmed by several techniques, including pretreatment of roots (a) at 75[deg]C or with SDS, (b) with [alpha]-keto-glutarate or L-methionine-DL-sulfoximine, (c) at elevated levels of Ca2+, and (d) at low pH or with Al3+ at low pH. Treatments a and b selectively influenced phase III without affecting phases I and II. Similarly, treatment c selectively perturbed phase II, and treatment d affected phases II and III. Based on these findings and the assumption of an in-series arrangement of root cell compartments, it was concluded that phase III corresponded to the cytoplasm, phase II corresponded to the Donnan free space, and phase I corresponded to a film of solution adhering to the root surface.     

HCO(3) Influx across the Plasmalemma of Chara corallina: Physiological and Biophysical Influence of 10 mm K

The effect of 10 mm K⁺ on the HCO₃⁻ influx in Chara corallina has been used to distinguish a Ca²⁺-dependent membrane integrity site from the HCO₃⁻ transport site which is also Ca²⁺-dependent (Lucas and Dainty, Plant Physiology 1977 60: 862-867).     

Dynamic Interactions between Root NH4+ Influx and Long-Distance N Translocation in Rice: Insights into Feedback Processes

Ammonium influx into roots and N translocation to the shootswere measured in 3-week-old hydroponically grown rice seedlings(Oryza sativa L., cv. IR72) under conditions of N deprivationand NH₄⁺ resupply, using ¹³NH₄⁺as a tracer. Root NH₄⁺ influxwas repressed in plants continuously supplied with NH₄⁺ (at0.1 mM), but a high proportion of absorbed N (20 to 30%) wastranslocated to the shoot in the form of N assimilates duringthe 13-min loading and desorption periods. Interruption of exogenousNH₄⁺ supply for periods of 1 to 3 d caused NH₄⁺ influx to bede-repressed. This same treatment caused N translocation tothe shoot to decline rapidly, until, by 24 h, less than 5% ofthe absorbed ¹³N was translocated to the shoot, illustratinga clear priority of root over shoot N demand under conditionsof N deprivation. Upon resupplying 1 mM NH₄⁺, root NH₄⁺ influxresponded in a distinct four-phase pattern, exhibiting periodsin which NH₄⁺ influx was first enhanced and subsequently reduced.Notably, a 25 to 40% increase in root influx, peaking at {smalltilde}2 h following re-exposure was correlated with a 4- to5-fold enhancement in shoot translocation and a repression ofroot GS activity. The transient increase of NH₄⁺ influx wasalso observed in seedlings continuously supplied with NO₃^–and subsequently transferred to NH₄⁺. Extended exposure to NH₄⁺caused root NH₄⁺ influx to decrease progressively, while shoottranslocation was restored to {small tilde}30% of incoming NH₄⁺.The nature of the feedback control of NH₄⁺ influx as well asthe question of its inducibility are discussed. (Received August 7, 1998; Accepted September 21, 1998)     

Effects of Hypoxia on 13NH4+ Fluxes in Rice Roots : Kinetics and Compartmental Analysis

Techniques of compartmental (efflux) and kinetic influx analyses with the radiotracer ¹³NH₄⁺ were used to examine the adaptation to hypoxia (15, 35, and 50% O₂ saturation) of root N uptake and metabolism in 3-week-old hydroponically grown rice (Oryza sativa L., cv IR72) seedlings. A time-dependence study of NH₄⁺ influx into rice roots after onset of hypoxia (15% O₂) revealed an initial increase in the first 1 to 2.5 h after treatment imposition, followed by a decline to less than 50% of influx in control plants by 4 d. Efflux analyses conducted 0, 1, 3, and 5 d after the treatment confirmed this adaptation pattern of NH₄⁺ uptake. Half-lives for NH₄⁺ exchange with subcellular compartments, cytoplasmic NH₄⁺ concentrations, and efflux (as percentage of influx) were unaffected by hypoxia. However, significant differences were observed in the relative amounts of N allocated to NH₄⁺ assimilation and the vacuole versus translocation to the shoot. Kinetic experiments conducted at 100, 50, 35, and 15% O₂ saturation showed no significant change in the K_m value for NH₄⁺ uptake with varying O₂ supply. However, V_max was 42% higher than controls at 50% O₂ saturation, unchanged at 35%, and 10% lower than controls at 15% O₂. The significance of these flux adaptations is discussed.     

不同营养条件下竹叶眼子菜NH4^＋-N吸收动力学的初步研究

以竹叶眼子菜(Potamogeton malaianus)无菌系种苗为试验材料,研究了不同水体营养浓度水平(低营养:TN 0.213 mg·L-1,TP 0.0093 mg·L-1;中营养:TN 0.71 mg·L-1,TP 0.031 mg·L-1;高营养:TN 7.1 mg·L-1,TP 0.31 mg·L-1)对其生长与NH+4-N的吸收动力学参数的影响.结果表明,不同浓度水体营养对竹叶眼子菜生长的影响较小,而NH+4-N的吸收动力学参数有显著差异.竹叶眼子菜在高、中和低营养培养条件下的NH+4-N最大吸收速率Vmax分别为 41.1、 29.1、 21.1 μmol·g-1·h-1,米氏常数Km分别为 0.356、 0.306、 0.122 mmol·L-1.竹叶眼子菜营养吸收动力学与其生长环境关系紧密,在低浓度生长环境中时,竹叶眼子菜可以通过降低Km值来提高对营养离子的亲和力以满足营养需求;在高浓度生长环境中,该植物通过增大吸收潜力来适应高营养.     

Kinetics of NH 4 + uptake by the arbuscular mycorrhizal fungus Rhizophagus irregularis

The kinetics and energetics of (15)NH (4) (+) uptake by the extraradical mycelium of the arbuscular mycorrhizal fungus Rhizophagus irregularis were investigated. (15)NH (4) (+) uptake increased with increasing substrate concentration over the concentration range of 0.002 to 25?mM. Eadie-Hofstee plots showed that ammonium (NH (4) (+) ) uptake over this range was biphasic. At concentrations below 100?μM, NH (4) (+) uptake fits a Michaelis-Menten curve, typical of the activity of a saturable high-affinity transport system (HATS). At concentrations above 1?mM, NH (4) (+) influx showed a linear response typical of a nonsaturable low-affinity transport system (LATS). Both transport systems were dependent on external pH. The HATS and, to a lesser extent, the LATS were inhibited by the ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP) and the ATP-synthesis inhibitor 2,4-dinitrophenol. These data indicate that the two NH (4) (+) transport systems of R. irregularis are dependent on metabolic energy and on the electrochemical H(+) gradient. The HATS- and the LATS-mediated (15)NH (4) (+) influxes were also regulated by acetate. This first report of the existence of active high- and low-affinity NH4(+) transport systems in the extraradical mycelium of an arbuscular mycorrhizal fungus and provides novel information on the mechanisms underlying mycosymbiont uptake of nitrogen from the soil environment.     

HCO(3) Influx Across the Plasmalemma of Chara corallina: Divalent Cation Requirement

An absolute requirement for divalent cations is reported for H(14)CO(3) (-) influx in Chara corallina. Effective substitution of eluted Ca(2+) by Mg(2+) and Sr(2+) was observed, but Mn(2+) was completely ineffective in restoring H(14)CO(3) (-) transport activity. Similarly, La(3+) could not substitute for Ca(2+) in this system. Low concentrations of ethylenediaminetetraacetate (0.01 to 0.06 mm) significantly enhanced the rate at which H(14)CO(3) (-) transport capacity was lost.Examination of the response of OH(-) efflux, during Ca(2+)-free treatment, indicated that the cellular control over OH(-) efflux remained unaffected until membrane integrity became severely affected. This conclusion was supported by the response of OH(-) efflux to 10 mm K(+). Therefore, assimilation of H(14)CO(3) (-) is not rate-limited by an effect of Ca(2+) elution on the OH(-) transport system. Kinetic experiments indicated that Ca(2+) removal from the membrane resulted in noncompetitive inhibition of H(14)CO(3) (-) assimilation; the apparent Michaelis constant remained unaltered over a wide range of conditions. An hypothesis is presented which suggests that membrane integrity is necessary for HCO(3) (-) transport to occur, but Ca(2+) (Mg(2+), Sr(2+)), per se, must be bound to the transport complex before activity is established.     

Feedback Regulation of Nitrate Influx in Barley Roots by Nitrate,Nitrite, and Ammonium

The short-lived radiotracer 13N was used to study feedback regulation of nitrate influx through the inducible high-affinity transport system of barley (Hordeum vulgare L. cv Steptoe) roots. Both wild-type plants and the mutant line Az12:Az70 (genotype nar1a;nar7w), which is deficient in the NADH-specific and NAD(P)H-bispecific nitrate reductases (R.L. Warner, R.C. Huffaker [1989] Plant Physiol 91: 947-953) showed strong feedback inhibition of nitrate influx within approximately 5 d of exposure to 100 fmu]M nitrate. The result with the mutant, in which the flux of nitrogen into reduced products is greatly reduced, indicated that nitrate itself was capable of exercising feedback regulation upon its own influx. This conclusion was supported by the observation that feedback in wild-type plants occurred in both the presence and absence of L-methionine sulfoximine, an inhibitor of ammonium assimilation. Nitrite and ammonium were also found to be capable of exerting feedback inhibition upon nitrate influx, although it was not determined whether these ions themselves or subsequent metabolites were responsible for the effect. It is suggested that feed-back regulation of nitrate influx is potentially mediated through several nitrogen pools, including that of nitrate itself.     

Kinetics of Potassium and Magnesium Uptake by Intact Soybean Roots

The kinetics of uptake of K⁺ and Mg²⁺ were studied by using intact soybean [Glycine max (L.) Merr. cv. Amsoy] roots. Uptake of K⁺ in the concentration range 1.29 × 10^?5 to 1.82 × 10^?3 M can be represented by two phases of a single, multiphasic mechanism. Similarly, uptake of Mg²⁺ in the concentration range 4.10 × 10^?6 to 2.49 × 10^?4M was biphasic.     

The pathways of assimilation of 13NH4+ by the cyanobacterium, Anabaena cylindrica.

The principal initial product of metabolism of 13N-labeled ammonium by Anabaena cylindrica grown with either NH4+ or N2 as nitrogen source is amide-labeled glutamine. The specific activity of glutamine synthetase is approximately half as great in NH4+-grown as in N2-grown filaments. After 1.5 min of exposure to 13NH4+, the ratio of 13N in glutamate to 13N in glutamine reaches a value of approximately 0.1 for N2- and 0.15 for NH4+-grown filaments, whereas after the same period of exposure to [13N]N2, that ratio has reached a value close to unity and is rising rapidly. During pulse-chase experiments, 13N is transferred from the amide group to glutamine into glutamate, and then apparently into the alpha-amino group of glutamine. Methionine sulfoximine, an inhibitor of glutamine synthetase, inhibits the formation of glutamine. In the presence of the inhibitor, direct formation of glutamate takes place, but accounts for only a few per cent of the normal rate of formation of that amino acid; and alanine is formed about as rapidly as glutamate. Azaserine reduces formation of [13N]glutamate approximately 100-fold, with relatively little effect on the formation of [13N]glutamine. Aminooxyacetate, an inhibitor of transaminase reactions blocks transfer of 13N to aspartate, citrulline, and arginine. We conclude, on the basis of these results and others in the literature, that the glutamine synthetase/glutamate synthase pathway mediates most of the initial metabolism of ammonium in A. cylindrica, and that glutamic acid dehydrogenase and alanine dehydrogenase have only a very minor role.     

铵离子对不同基因型水稻吸收硝酸根离子的影响1

NH+4可在很短时间内影响两种不同水稻亚种吸收NO-3,即可以影响NO-3的最初跨膜运输.籼型稻在NH+4存在时对NO-3吸收有促进,而粳型稻NO-3吸收则明显减少.     

Relationship of Plasmalemma Redox Activity to K+ Uptake by Dunaliella salina Cells

The plasmalemma-bond redox system localized within the plasmalemma of unicellular green alga Dunaliella salina was studied. This system oxidized exogenous NADH, increased O2 consumption to 165 % and increased the pH of the external medium, while K+ influx was inhibited. With no NADH added, ferricyanide stimulated K+ uptake about 3 folds. In the presence of exogenous NADH, ferricyanide was rapidly reduced and the external medium was acidified, generating a greater electrochemical proton gradient across the plasmalemma, thus resulting an 6-fold increase of K+ influx. Typical inhibitors of plasmalemma H+-ATPase and redox system inhibited K+ uptake to different extent. That the inhibition of K+ uptake by vanadate could be resumed partly by addition of NADH and ferricyanide indicated that plasmalemma redox system operated in association with the H+-ATPase to exert an influence on K+ transportation. A model was presented in which the implication of two possible redox chains and H+-ATPase in generating an electrochemical potential gradient for protons (△uH+) was discussed.     

Uptake and Assimilation of NO(3) and NH(4) by Nitrogen-Deficient Perennial Ryegrass Turf

Assimilation of NO₃⁻ and NH₄⁺ by perennial ryegrass (Lolium perenne L.) turf, previously deprived of N for 7 days, was examined. Nitrogen uptake rate was increased up to four- to five-fold for both forms of N by N-deprivation as compared to N-sufficient controls, with the deficiency-enhanced N absorption persisting through a 48 hour uptake period. Nitrate, but not NH₄⁺, accumulated in the roots and to a lesser degree in shoots. By 48 hours, 53% of the absorbed NO₃⁻ had been reduced, whereas 97% of the NH₄⁺ had been assimilated. During the early stages (0 to 8 hours) of NO₃⁻ uptake by N-deficient turf, reduction occurred primarily in the roots. Between 8 and 16 hours, however, the site of reduction shifted to the shoots. Nitrogen form did not affect partitioning of the absorbed N between roots (40%) and shoots (60%) but did affect growth. Compared to NO₃⁻, NH₄⁺ uptake inhibited root, but not shoot, growth. Total soluble carbohydrates decreased in both roots and shoots during the uptake period, principally the result of fructan metabolism. Ammonium uptake resulted in greater total depletion of soluble carbohydrates in the root compared to NO₃⁻ uptake. The data indicate that N assimilation by ryegrass turf utilizes stored sugars but is also dependent on current photosynthate.     

Assimilation of 13NH4+ by Azospirillum brasilense grown under nitrogen limitation and excess.

The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.