Charge Balance in NO(3)-Fed Soybean: Estimation of K and Carboxylate Recirculation

Soybeans (Glycine max L. Merr., cv Kingsoy) were grown on media containing NO₃⁻ or urea. The enrichments of shoots in K⁺, NO₃⁻, and total reduced N (N_r), relative to that in Ca²⁺, were compared to the ratios K⁺/Ca²⁺,NO₃⁻/Ca²⁺, and N_r/Ca²⁺ in the xylem saps, to estimate the cycling of K⁺, and N_r. The net production of carboxylates (R⁻) was estimated from the difference between the sums of the main cations and inorganic anions. The estimate for shoots was compared to the theoretical production of R⁻ associated with NO₃⁻ assimilation in these organs, and the difference was attributed to export of R⁻ to roots. The net exchange rates of H⁺ and OH⁻ between the medium and roots were monitored. The shoots were the site of more than 90% of total NO₃⁻ reduction, and N_r was cycling through the plants at a high rate. Alkalinization of the medium by NO₃⁻-fed plants was interrupted by stem girdling, and not restored by glucose addition to the medium. It was concluded that the majority of the base excreted in NO₃⁻ medium originated from R⁻ produced in the shoots, and transported to the roots together with K⁺. As expected, cycling of K⁺ and reduced N was favoured by NO₃⁻ nutrition as compared to urea nutrition.     

Evidence for Cotransport of Nitrate and Protons in Maize Roots : II. Measurement of NO(3) and H Fluxes with Ion-Selective Microelectrodes

We report here on an investigation of net nitrate and proton fluxes in root cells of maize (Zea mays L.) seedlings grown without (noninduced) and with (induced) 0.1 millimolar nitrate. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, WJ Lucas [1987] Plant Physiol 84: 1177-1184) was utilized to quantify net ionic fluxes from the measurement of electrochemical potential gradients for NO₃⁻ and H⁺ within the unstirred layer at the root surface. The nitrate-inducibility, pH dependence, and concentration dependence of net NO₃⁻ uptake correlated quite closely with the electrical response of maize roots to nitrate under the same experimental conditions (as described in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289). Additionally, it was found that potential inhibitors of the plasmalemma H⁺-ATPase (vandate, diethylstilbestrol), which were shown to abolish the electrical response to NO₃⁻ (in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289), dramatically inhibited NO₃⁻ absorption. These results strongly indicate that the NO₃⁻ electrical response is due to the operation of a NO₃⁻ transport system in the plasmalemma of maize root cells. Furthermore, the results from the H⁺-ATPase inhibitor studies indicate that the NO₃⁻ transport system is linked to the H⁺-ATPase, presumably as a NO₃⁻/H⁺ symport. This is further supported by the pH response of the NO₃⁻ transport system (inhibition at alkaline pH values) and the change in net H⁺ flux from a moderate efflux in the absence of NO₃⁻, to zero net H⁺ flux after exposing the maize root to exogenous nitrate. Although these results can be explained by other interpretations, the simplest model that fits both the electrical responses and the NO₃⁻/H⁺ flux data is a NO₃⁻/H⁺ symport with a NO₃⁻:H⁺ flux stoichiometry >1, whose operation results in the stimulation of the H⁺-ATPase due to the influx of protons through the cotransport system.     

Potassium Transport in Corn Roots : IV. Characterization of the Linear Component

A detailed examination was conducted on the linear, or first-order kinetic component for K⁺(⁸⁶Rb⁺) influx into root segments of both low- and high-salt grown corn seedlings (Zea mays [A632 × Oh 43]). In tissue from both low- and high-salt grown roots, replacement of Cl⁻ in the uptake solution by either SO₄²⁻, H₂PO₄⁻, or NO₃⁻ caused a significant (50-60%) and specific inhibition of the linear component of K⁺ influx. The anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid, was found to abolish saturable Cl⁻ influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K⁺ uptake system; this inhibition was identical to that observed when Cl⁻ was replaced by other anions in the K⁺ uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K⁺ channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K⁺ influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low- and high-salt roots to absorb tetraethylammonium.

Our present results indicate that the linear component of K⁺ influx may occur by a passive process involving transmembrane K⁺ channels. Fluxes through these K⁺ channels may be partly coupled to a saturating Cl⁻ influx mechanism.

     

Calcium uptake by excised maize roots and interactions with alkali cations

Ca²⁺ uptake was studied in short-term experiments using 5-day-old excised maize roots. This tissue readily absorbs Ca²⁺, and inhibition by dinitrophenol and low temperature shows that the process is metabolically mediated. The uptake of Ca²⁺, like that of other cations, is influenced by the counter ion, the pH and concentration of the ambient solution, and the presence of other cations. The rate of uptake from various salts decreases in the following order: NO₃⁻ > Cl⁻ = Br⁻ > SO₄²⁻. K⁺ and H⁺ greatly interfere with Ca²⁺ absorption, while Li⁺ and Na⁺ have only slight effects.     

Isolation and transport properties of protoplasts from cortical cells of corn roots

A procedure was developed for the enzymic isolation of large quantities of protoplasts from the cortex of Zea mays L. WF9 × MO 17 roots. Cortex was separated from the primary root, sectioned, and the cell walls digested for 3.5 hours in 2% (w/v) Cellulysin, 0.1% Pectolyase Y-23, 1 millimolar CaCl₂, 0.05% bovine serum albumin, 0.5 millimolar dithiothreitol in 0.6 molar mannitol (pH 5.6). Cortical cell protoplasts were collected by centrifugation and purified by flotation in a Ficoll step gradient. The yield of protoplasts was approximately 650 × 10³/gram fresh tissue. To obtain maximum yield it was essential to include an effective pectinase (Pectolyase Y-23) and protectants (bovine serum albumin and dithiothreitol) in the digestion medium.

Cortical cell protoplasts exhibited energy-dependent uptake of K⁺ (⁸⁶Rb), H₂³²PO₄⁻, and ³⁶Cl⁻ as well as net H⁺ extrusion. Ion fluxes were sustained for at least 3 hours. Influx of K⁺ was highest between pH 7.5 and 8.0, whereas the influx of H₂PO₄⁻ was greatest between pH 4.0 and 5.0. K⁺ and H₂PO₄⁻ influx and net H⁺ efflux were inhibited by respiratory poisons such as cyanide (0.1 millimolar) and oligomycin (5 micrograms per milliliter), and by inhibitors of plasma membrane ATPase such as diethylstilbestrol (50 micromolar). Calculated flux for Cl⁻ was low, but not greatly different from that observed for other plant cells. K⁺ flux was somewhat high, probably because the K⁺ concentration in the cortical cells was below steady-state. The results indicate that isolated cortical cell protoplasts retain transport properties which are similar to those of root tissue.

     

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): II. Regulation of NO(3) Efflux by NH(4)

The influence of NH₄⁺, in the external medium, on fluxes of NO₃⁻ and K⁺ were investigated using barley (Hordeum vulgare cv Betzes) plants. NH₄⁺ was without effect on NO₃⁻ (³⁶ClO₃⁻) influx whereas inhibition of net uptake appeared to be a function of previous NO₃⁻ provision. Plants grown at 10 micromolar NO₃⁻ were sensitive to external NH₄⁺ when uptake was measured in 100 micromolar NO₃⁻. By contrast, NO₃⁻ uptake (from 100 micromolar NO₃⁻) by plants previously grown at this concentration was not reduced by NH₄⁺ treatment. Plants pretreated for 2 days with 5 millimolar NO₃⁻ showed net efflux of NO₃⁻ when roots were transferred to 100 micromolar NO₃⁻. This efflux was stimulated in the presence of NH₄⁺. NH₄⁺ also stimulated NO₃⁻ efflux from plants pretreated with relatively low nitrate concentrations. It is proposed that short term effects on net uptake of NO₃⁻ occur via effects upon efflux. By contrast to the situation for NO₃⁻, net K⁺ uptake and influx of ³⁶Rb⁺-labeled K⁺ was inhibited by NH₄⁺ regardless of the nutrient history of the plants. Inhibition of net K⁺ uptake reached its maximum value within 2 minutes of NH₄⁺ addition. It is concluded that the latter ion exerts a direct effect upon K⁺ influx.     

Effects of Helminthosporium carbonum Toxin on Absorption of Solutes by Corn Roots

Susceptible corn roots exposed to the host-selective toxin of Helminthosporium carbonum took up and retained more NO₃⁻, Na⁺, Cl⁻, 3-o-methylglucose, and leucine than did control roots. Stimulatory effects on uptake were more pronounced with freshly cut roots than with roots that were washed and aged. Solutes were accumulated against a concentration gradient, and toxin-treated tissues developed a steeper gradient than did control tissues. Toxin affected both the low and high affinity uptake systems for Na⁺ and Cl⁻. Toxin did not affect uptake of Na₂⁻, K⁺, Ca²⁺, phosphate ion (H₂PO₄⁻ and HPO₄⁻), SO₄⁻, and glutamic acid. No toxin-induced leakage of any solute tested was detected within 5 to 6 hr after initial exposure to toxin. The data suggest that toxin from H. carbonum does not cause the general plasma membrane derangement caused by other host-selective toxins. Instead, H. carbonum toxin may cause specific changes in characteristics of the plasmalemma, which result in increased uptake of certain solutes.     

The influence of ammonium and chloride on potassium and nitrate absorption by barley roots depends on time of exposure and cultivar

Net uptakes of K⁺ and NO₃⁻ were monitored simultaneously and continuously for two barley (Hordeum vulgare) cultivars, Prato and Olli. The cultivars had similar rates of net K⁺ and NO₃⁻ uptake in the absence of NH₄⁺ or Cl⁻. Long-term exposure (over 6 hours) to media which contained equimolar mixtures of NH₄⁺, K⁺, Cl⁻, or NO₃⁻ affected the cultivars very differently: (a) the presence of NH₄⁺ as NH₄Cl stimulated net NO₃⁻ uptake in Prato barley but inhibited net NO₃⁻ uptake in Olli barley; (b) Cl⁻ inhibited net NO₃⁻ uptake in Prato but had little effect in Olli; and (c) NH₄⁺ as (NH₄)₂SO₄ inhibited net K⁺ uptake in Prato but had little effect in Olli. Moreover, the immediate response to the addition of an ion often varied significantly from the long-term response; for example, the addition of Cl⁻ initially inhibited net K⁺ uptake in Olli barley but, after a 4 hour exposure, it was stimulatory. For both cultivars, net NH₄⁺ and Cl⁻ uptake did not change significantly with time after these ions were added to the nutrient medium. These data indicate that, even within one species, there is a high degree of genotypic variation in the control of nutrient absorption.     

Properties of the Isolated Intact Chloroplast at Cytoplasmic K Concentrations : I. Light-Induced Cation Uptake into Intact Chloroplasts is Driven by an Electrical Potential Difference

Photosynthesis, stroma-pH, and internal K⁺ and Cl⁻ concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K⁺, H⁺, Cl⁻) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).

Isolated intact chloroplasts are a Donnan system which accumulates cations (K⁺ or added Tetraphenylphosphonium⁺) and excludes anions (Cl⁻) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium⁺ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K⁺ or Na⁺ and the release of protons all become very small. Light-induced K⁺ uptake is not abolished by valinomycin suggesting that the K⁺ uptake is not primarily active. Intact chloroplasts contain higher K⁺ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO₂ fixation at high K⁺ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO₂ fixation in intact chloroplasts.

     

Mineral ion contents and cell transmembrane electropotentials of pea and oat seedling tissue

The relationships of concentration gradients to electropotential gradients resulting from passive diffusion processes, after equilibration, are described by the Nernst equation. The primary criterion for the hypothesis that any given ion is actively transported is to establish that it is not diffusing passively. A test was made of how closely the Nernst equation describes the electrochemical equilibrium in seedling tissues. Segments of roots and epicotyl internodes of pea (Pisum sativum var. Alaska) and of roots and coleoptiles of oat (Avena sativa var. Victory) seedlings were immersed and shaken in defined nutrient solutions containing eight major nutrients (K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻, NO₃⁻, H₂PO₄⁻ and SO₄²⁻) at 1-fold and 10-fold concentrations. The tissue content of each ion was assayed at 0, 8, 24, and 48 hours. A near-equilibrium condition was approached by roots for most ions; however, the segments of shoot tissue generally continued to show a net accumulation of some ions, mainly K⁺ and NO₃⁻. Only K⁺ approached a reasonable fit to the Nernst equation and this was true for the 1-fold concentration but not the 10-fold. The data suggest that for Na⁺, Mg²⁺, and Ca²⁺ the electrochemical gradient is from the external solution to the cell interior; thus passive diffusion should be in an inward direction. Consequently, some mechanism must exist in plant tissue either to exclude these cations or to extrude them (e.g., by an active efflux pump). For each of the anions the electrochemical gradient is from the tissue to the solution; thus an active influx pump for anions seems required. Root segments approach ionic equilibrium with the solution concentration in which the seedlings were grown. Segments of shoot tissue, however, are far removed from such equilibration. Thus in the intact seedling the extracellular (wall space) fluid must be very different from that of the nutrient solution bathing the segments; it would appear that the root is the site of regulation of ion uptake in the intact plant although other correlative mechanisms may be involved.     

Studies on H-Translocating ATPases in Plants of Varying Resistance to Salinity : II. K Strongly Promotes Development of Membrane Potential in Vesicles from Cotton Roots

Mg²⁺-ATP-dependent H⁺-translocation has been studied in membrane vesicles derived from the roots of Gossypium hirsutum L. var. Acala San Jose 2. Establishment of a positive membrane potential was followed by measuring SCN⁻ accumulation; establishment of ΔpH across the vesicle membranes by measuring quinacrine fluorescence quenching. High specificity for ATP was shown, and H⁺-translocation was oligomycin stable. The pH profile for H⁺-translocation showed an optimum at 5.5. The relationship between SCN⁻ accumulation and ATP concentration was approximately Michaelian; the apparent K_m was 0.7 millimolar. K-2-(N-morpholino)ethanesulfonic acid strongly promoted ATP-dependent SCN⁻ uptake (up to 180% stimulation). The effect was not given by Na-Mes. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone totally inhibited SCN⁻ accumulation, both in the presence and absence of K-2(N-morpholino)ethanesulfonic acid. Vanadate at 200 micromolar inhibited SCN⁻ uptake by about 10 to 40% in the absence of K⁺, but more strongly in its presence (about 60%). NO₃⁻ at 100 millimolar inhibited initial rate of quinacrine quenching by about 25%. The NO₃⁻ insensitive fraction was activated by K⁺; and inhibited by 200 micromolar vanadate to about 40%, provided K⁺ was present. Saline conditions during the growth of the plants had no appreciable effect on the observed characteristics of H⁺-translocation.     

Potential-dependent anion transport in tonoplast vesicles from oat roots

Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Δψ) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H⁺-pumping pyrophosphatase, which is K⁺ stimulated and anion insensitive. The relative rate of ΔΨ dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN⁻ (100) > NO₃⁻ (72) = Cl⁻ (70) > Br⁻ (62) > SO₄²⁻ (5) = H₂PO₄⁻ (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Δψ dissipation by Cl⁻ and NO₃⁻, but not by SCN⁻, was saturable. The K_m values for Cl⁻ and NO₃⁻ uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H⁺-coupled Cl⁻ transporter on the same vesicles, the potential-dependent Cl⁻ transport was insensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl⁻ transport in these vesicles. The potentials generated by the H⁺-translocating ATPase and H⁺-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl⁻ conductance on the anion-sensitive H⁺-ATPase was found.     

Ion transport in isolated protoplasts from tobacco suspension cells: I. General characteristics

An investigation was conducted into the feasibility of using enzymically isolated protoplasts from suspension-cultured cells of Nicotiana glutinosa L. to study ion transport. Transport of K⁺ (⁸⁶Rb), ³⁶Cl⁻, H₂³²PO₄⁻ and ⁴⁵Ca²⁺ from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoll step gradient. Influx of K⁺, Cl⁻, and H₂PO₄⁻ measured over a 30-minute period was reduced (up to 99%) by respiratory inhibitors such as 5 micrograms per milliliter oligomycin, 0.1 millimolar dinitrophenol, 0.1 millimolar cyanide, or N₂ gas. In contrast, Ca²⁺ influx was not tightly coupled to respiratory energy production. The influx of K⁺ was highest between pH 6.5 and 7.5 whereas the influx of H₂PO₄⁻ and Cl⁻ was greatest between pH 4.5 and 5.5. Influx of K⁺ and Cl⁻ was maximal at 35 and 45 C, respectively, and was almost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K⁺ influx by more than 200% but had no effect on the influx of either Cl⁻ or H₂PO₄⁻. Apparent H⁺ efflux, as measured by decrease in solution pH, was enhanced by K⁺, stimulated further by 0.01 millimolar fusicoccin, and inhibited by 0.1 millimolar dinitrophenol or 5 micrograms per milliliter oligomycin. The measured ionic fluxes into protoplasts were similar to those obtained with intact cultured cells. The results indicate that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast, and that it is feasible to use isolated protoplasts for studies on ion transport.     

Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg

To clarify the kinetic characteristics and ionic requirements of the tonoplast H⁺-translocating inorganic pyrophosphatase (H⁺-PPiase), PPi hydrolysis and PPi-dependent H⁺ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H⁺-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H⁺-PPiase activity was maximal in the presence of K⁺ (K₅₀ approximately 3 millimolar), with PPi-dependent H⁺ transport being more selective for K⁺ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H⁺-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg²⁺ for activation. At saturating concentrations of free Mg²⁺, H⁺-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi²⁻ but not Mg₂PPi, demonstrating that MgPPi²⁻ was the true substrate of the enzyme. The apparent K_m (MgPPi²⁻) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H⁺ transport (7 micromolar). Free Mg²⁺ was shown to be an allosteric activator of the H⁺-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H⁺ transport. Half-maximal H⁺-PPiase activity occurred at a free Mg²⁺ concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg²⁺. In contrast, cytosolic concentrations of K⁺ and MgPPi²⁻ appear to be saturating for H⁺-PPiase activity. We propose that one function of the H⁺-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H⁺-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg²⁺ would be released from the MgATP²⁻ complex, thereby activating the tonoplast H⁺-PPiase.     

Cation pretreatment effects on nitrate uptake, xylem exudate, and malate levels in wheat seedlings

Week-old wheat seedlings absorbed at least 40% NO₃⁻ from NaNO₃ when preloaded with K⁺ than when preloaded with Na⁺ or Ca²⁺. Cultures of Triticum vulgare L. cv. Arthur were grown for 5 days on 0.2 mm CaSO₄, pretreated for 48 hours with either 1 mm CaSO₄, K₂SO₄, or Na₂SO₄, and then transferred to 1 mm NaNO₃. All solutions contained 0.2 mm CaSO₄. Shoots of K⁺-preloaded plants accumulated three times more NO₃⁻ than shoots of the other two treatments. Initially, the K⁺-preloaded plants contained 10-fold more malate than either Na⁺- or Ca²⁺-preloaded seedlings. During the 48-hour treatment with NaNO₃, malate in both roots and shoots of the K⁺-preloaded seedlings decreased. Seedlings preloaded with K⁺ reduced 25% more NO₃⁻ than those preloaded with either Na⁺ or Ca²⁺. These experiments indicate that K⁺ enhanced NO₃⁻ uptake and reduction even though the absorption of K⁺ and NO₃⁻ were separated in time. Xylem exudate of K⁺-pretreated plants contained roughly equivalent concentrations of K⁺ and NO₃⁻, but exudate from Na⁺ and Ca²⁺-pretreated plants contained two to four times more NO₃⁻ than K⁺. Therefore K⁺ is not an obligatory counterion for NO₃⁻ transport in xylem.     

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl⁻ and H₂PO₄⁻ uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K⁺ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K⁺ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl⁻ FC induced intracellular acidification. Since H⁺ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K⁺/A⁻ symport or HCO₃⁻/A⁻ antiport for anion uptake. Though not suitable to reveal the actual H⁺/A⁻ stoichiometry, the results are consistent with an H⁺/A⁻ cotransport mechanism.     

Correlations between Potassium Uptake and Hydrogen Efflux in Barley Varieties : A POTENTIAL SCREENING METHOD FOR THE ISOLATION OF NUTRIENT EFFICIENT LINES

Rates of hydrogen ion secretion and potassium (⁸⁶Rb) absorption by intact roots of twenty-four barley varieties were measured in solutions containing K₂SO₄ (1 × 10⁻⁴ to 1 × 10⁻³ molar) plus 5 × 10⁻⁴ molar CaSO₄, at initial pH values in the range 5.3 to 5.5. Fluxes of H⁺ and K⁺ were strongly correlated in short-term experiments (up to 15 minutes) as well as in long-term experiments (lasting 24 hours). The observed correlations provide the basis for a preliminary screening method, designed to segregate varieties with high rates of potassium uptake by the use of an acid-base indicator (methyl red).     

Evidence for cotransport of nitrate and protons in maize roots : I. Effects of nitrate on the membrane potential

The electrical response of nitrate-grown maize (Zea mays L.) roots to 0.1 millimolar nitrate was comprised of two sequential parts: a rapid and transient depolarization of the membrane potential, followed by a slower, net hyperpolarization to a value more negative than the original resting potential. The magnitude of the response was smaller in roots of seedlings grown in the absence of nitrate, but, within 3 hours of initial exposure to 0.1 millimolar nitrate, increased to that of nitrate-grown roots. Chloride elicited a separate electrical response with a pattern similar to that of the nitrate response. However, the results presented in this study strongly indicate that the electrical response to nitrate reflects the activity of a nitrate-inducible membrane transport system for nitrate which is distinct from that for chloride. Inhibitors of the plasmalemma H⁺-ATPase (vanadate, diethylstilbestrol) completely inhibited both parts of the electrical response to nitrate, as did alkaline external pH. The magnitude of the initial nitrate-dependent, membrane potential depolarization was independent of nitrate concentration, but the subsequent nitrate-dependent hyperpolarization showed saturable dependence with an apparent K_m of 0.05 millimolar. These results support a model for nitrate uptake in maize roots which includes a depolarizing NO₃⁻/H⁺ symport. The model proposes that the nitrate-dependent membrane potential hyperpolarization is due to the plasma membrane proton pump, which is secondarily stimulated by the operation of the NO₃⁻/H⁺ symport.     

Effects of deuterium oxide on growth, proton extrusion, potassium influx, and in vitro plasma membrane activities in maize root segments

Elongation of subapical segments of maize (Zea mays) roots was greatly inhibited by ²H₂O in the incubation medium. Short-term exposure (30 min) to ²H₂O slightly reduced O₂ uptake and significantly increased ATP levels. ²H₂O inhibited H⁺ extrusion in the presence of both low (0.05 mm) and high (5 mm) external concentrations of K⁺ (about 30 and 53%, respectively at 50% [v/v] ²H₂O). Experiments on plasma membrane vesicles showed that H⁺-pumping and ATPase activities were greatly inhibited by ²H₂O (about 35% at 50% [v/v] ²H₂O); NADH-ferricyanide reductase and 1,3-β-glucan synthase activities were inhibited to a lesser extent (less than 15%). ATPase activities present in both the tonoplast-enriched and submitochondrial particle preparations were not affected by ²H₂O. Therefore, the effect of short incubation time and low concentration of ²H₂O is not due to a general action on overall cell metabolism but involves a specific inhibition of the plasma membrane H⁺ -ATPase. K⁺ uptake was inhibited by ²H₂O only when K⁺ was present at a low (0.05 mm) external concentration where absorption is against its electrochemical potential. The transmembrane electric potential difference (E_m) was slightly hyperpolarized by ²H₂O at low K⁺, but was not affected at the higher K⁺ concentrations. These results suggest a relationship between H⁺ extrusion and K⁺ uptake at low K⁺ external concentration.     

Nitrate Reductase Activity in Shoots and Roots of Maize Seedlings as Affected by the Form of Nitrogen Nutrition and the pH of the Nutrient Solution

The effect of nitrogen form (NH₄-N, NH₄-N + NO₃⁻, NO₃⁻) on nitrate reductase activity in roots and shoots of maize (Zea mays L. cv INRA 508) seedlings was studied. Nitrate reductase activity in leaves was consistent with the well known fact that NO₃⁻ increases, and NH₄⁺ and amide-N decrease, nitrate reductase activity. Nitrate reductase activity in the roots, however, could not be explained by the root content of NO₃⁻, NH₄-N, and amide-N. In roots, nitrate reductase activity in vitro was correlated with the rate of nitrate reduction in vivo. Inasmuch as nitrate reduction results in the production of OH⁻ and stimulates the synthesis of organic anions, it was postulated that nitrate reductase activity of roots is stimulated by the released OH⁻ or by the synthesized organic anions rather than by nitrate itself. Addition of HCO₃⁻ to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO₃⁻, like OH⁻, increases pH and promotes the synthesis of organic anions, this provides circumstantial evidence that alkaline conditions and/or organic anions have a more direct impact on nitrate reductase activity than do NO₃⁻, NH₄-N, and amide-N.