Sorption of copper and zinc to the plasma membrane of wheat root.

Sorption of Cu(2+) and Zn(2+) to the plasma membrane (PM) of wheat root (Triticum aestivum L cv. Scout 66) vesicles was measured at different pH values and in the presence of organic acids and other metals. The results were analyzed using a Gouy-Chapman-Stem model for competitive sorption (binding and electrostatic attraction) to a negative binding site. The binding constants for the two investigated cations as evaluated from the sorption experiments were 5 M(-1) for Zn(2+) and 400 M(-1) for Cu(2+). Thus, the sorption affinity of Cu(2+) to the PM is considerably larger than that of Ca(2+), Mg(2+) or Zn(2+). The greater binding affinity of Cu(2+) was confirmed by experiments in which competition with La(3+) for sorption sites was followed. The amount of sorbed Cu(2+) decreased with increasing K(+), Ca(2+), or La(3+) concentrations, suggesting that all these cations competed with Cu(2+) for sorption at the PM binding sites, albeit with considerable differences among these cations in effectiveness as competitors with Cu(2+). The sorption of Cu(2+) and Zn(2+) to the PM decreased in the presence of citric acid or malic acid. Citric acid (as well as pH) affected the sorption of Cu(2+) or Zn(2+) to PM more strongly then did malic acid.

Characteristics of the quenching of 9-aminoacridine fluorescence by liposomes made from plant lipids.

Several laboratories have determined the surface charge density of membranes utilizing methods based on vesicle-induced quenching of the fluorescence of 9-aminoacridine and its relief by other cations. However, the computational methods by which surface charge density were calculated have not been verified in a model system. In this study, the quenching of 9-aminoacridine fluorescence by liposomes made from varying amounts of digalactosyldiacylglyceride and phosphatidic acid and relief of quenching by salts was examined. Quenching of 9-aminoacridine fluorescence increased with increasing amounts of phosphatidic acid added, independent of the composition of the added liposomes. In certain instances, the computational methods did not yield the surface charge density of the liposomes expected from their composition. However, when the effects of background ionic strength on surface potential were considered, there was a positive correlation between expected and calculated values. Therefore, the data support the contention that changes in the fluorescence of 9-aminoacridine can be used to calculate surface charge density of membranes.

Binding and electrostatic attraction of lanthanum (La3+) and aluminum (Al3+) to wheat root plasma membranes.

A general model for the sorption of trivalent cations to wheat-root (Triticum aestivum L cv. Scout 66) plasma membranes (PM) has been developed and includes the first published coefficients for La3+ and Al3+ binding to a biological membrane. Both ions are rhizotoxic, and the latter ion is the principal contributor to the toxicity of acidic soils around the world. The model takes into account both the electrostatic attraction and the binding of cations to the negatively charged PM surface. Ion binding is modeled as the reaction P- + IZ <==> 'PIZ-1 in which P- represents a negatively charged PM ligand, located in an estimated area of 540 A2, and IZ represents an ion of charge Z. Binding constants for the reaction were assigned for K+ (1 M-1) and Ca2+ (30 M-1) and evaluated experimentally for La3+ (2200 M-1) and H+ (21,500 M-1). Al sorption is complicated by Al3+ hydrolysis that yields hydroxoaluminum species that are also sorbed. Binding constants of 30 and 1 M-1 were assigned for AlOH2+ and Al(OH)+2, respectively, then a constant for Al3+ (20,000 m-1) was evaluated experimentally using the previously obtained values for K+, Ca2+ and H+ binding. Electrostatic attraction was modeled according to Gouy-Chapman theory. Evaluation of parameters was based upon the sorption of ions to PM vesicles suspended in solutions containing variable concentrations of H+, Ca2+ and La3+ or Al3+. Use of small volumes, and improved assay techniques, allowed the measurement of concentration depletions caused by sorption to vesicles. Some independent confirmation of our model is provided by substantial agreement between our computations and two published reports of La3+ effects upon zeta potentials of plant protoplasts. The single published report concerning the electrostatic effects of Al on cell membranes is in essential agreement with the model.

Sorption of Aluminum to Plasma Membrane Vesicles Isolated from Roots of Scout 66 and Atlas 66 Cultivars of Wheat.

To further elucidate the mechanisms of differential genotypic tolerance to Al, plasma membrane (PM) vesicles were isolated from whole roots, root tips, and tipless roots of Al3+-sensitive and Al3+-tolerant cultivars (cv) of wheat (Triticum aestivum L. cv Scout 66 and cv Atlas 66, respectively). Vesicles from cv Scout root tips sorbed more Al than vesicles prepared from any other source. The intrinsic surface-charge density of vesicles isolated from cv Scout was 26% more negative than vesicles from cv Atlas (-37.2 versus -29.5 millicoulombs m-2). Growth experiments indicated that cv Scout is slightly more sensitive to La3+ than is cv Atlas, that the cultivars are equally sensitive to H+, and that cv Atlas is slightly more sensitive to SeO42-. The difference in sensitivity to Al3+ was very large; for a 50% inhibition, a 16-fold greater activity of Al3+ was required for cv Atlas. Using a newly developed Gouy-Chapman-Stern model for ion sorption to the PM together with growth-response curves, we estimate that the difference in surface-charge density can account for the slightly greater sensitivity of cv Scout to cationic toxicants and the slightly greater sensitivity of cv Atlas to anionic toxicants. According to our estimates the differences in PM surface negativity and Al sorptive capacity probably account for some of the difference in sensitivity to Al3+, but the greater part of the difference probably arises from other tolerance mechanisms expressed in cv Atlas root tips that reduce the amount of Al3+ that can reach the PM.

Oxygen-Induced Membrane Depolarizations in Legume Root Nodules (Possible Evidence for an Osmoelectrical Mechanism Controlling Nodule Gas Permeability).

Various stresses trigger rapid and reversible decreases in the O2 permeability (PO) of legume root nodules. Several possible mechanisms have been proposed, but no supporting data have previously been presented that meet the requirements for both rapidity and reversibility. Stomatal regulation of gas permeability in leaves involves electrically driven fluxes of inorganic osmoticants, so we investigated the possibility of a somewhat similar mechanism in nodules. We used microelectrodes to monitor membrane potential in intact, attached nodules of Glycine max, Medicago sativa, Lotus corniculatus, and Trifolium repens while controlling external O2 concentration and, in the case of G. max, measuring PO with a nodule oximeter. A 1- to 2-min exposure to 100 kPa O2 was found to induce rapid and reversible membrane depolarizations in nodules of each species. This depolarization (which, to our knowledge, is unique to nodules) is accompanied by reversible decreases in PO in G. max nodules. An osmoelectrical mechanism for control of nodule gas permeability, consistent with these data, is presented.

Use of a Gouy-Chapman-Stern Model for Membrane-Surface Electrical Potential to Interpret Some Features of Mineral Rhizotoxicity.

A consideration of mineral toxicity to roots only in terms of ion activities in the rooting medium can be misleading. A Gouy-Chapman-Stern model, by which relative ion activities at cell-membrane surfaces may be estimated, has been applied to problems of mineral rhizotoxicity, including the toxicity of Al3+, La3+, H+, Na+, and SeO42-, to wheat (Triticum aestivum L.) roots. The Gouy-Chapman portion of the model is expressed in the Grahame equation, which relates the charge density ([sigma]) and electrical potential (E0) at the surface of a membrane to the concentrations of ions in a contracting bulk solution. The Stern modification of the theory takes into account changes in [sigma] caused by ion binding at the membrane surface. Several theoretical problems with the model and its use are considered, including the fact that previous authors have usually related the physiological effects of an ion at a membrane surface to the computed concentration (Ci0) of the unbound ion rather than its computed activity (ai0). This practice implies the false assumption that Ci0 is proportional to ai0. It is demonstrated here that ai0, computed from external activities (ai[infinity symbol]) by a Nernst equation [ai0 = ai[infinity symbol]exp([mdash]ZiFE0/RT), where Zi is the charge on the ion, F is the Faraday constant, R is the gas constant, and T is the temperature], correlates well with ion toxicity and that Ci0 sometimes correlates poorly. These conclusions also apply to issues of mineral nutrition.

Interactive effects of Al, h, and other cations on root elongation considered in terms of cell-surface electrical potential.

The rhizotoxicities of Al(3+) and of La(3+) to wheat (Triticum aestivum L.) were similarly ameliorated by cations in the following order of effectiveness: H(+) approximately C(3+) > C(2+) > C(1+). Among tested cations of a given charge, ameliorative effectiveness was similar except that Ca(2+) was slightly more effective than other divalent cations and H(+) was much more effective than other monovalent cations. H(+) rhizotoxicity was also ameliorated by cations in the order C(3+) > C(2+) > C(1+). These results suggest a role for cell-surface electrical potential in the rhizotoxicity of Al(3+), La(3+), H(+), and other toxic cations: negatively charged cell surfaces of the root accumulate the toxic cations, and amelioration is effected by treatments that reduce the negativity of the cell-surface electrical potential by charge screening or cation binding. Membrane-surface activities of free Al(3+) or La(3+) computed according to a Gouy-Chapman-Stern model correlated well with growth inhibition, which correlated only poorly with Al(3+) or La(3+) activities in the external medium. The similar responses of Al-intoxicated and La-intoxicated roots to ameliorative treatments provide evidence that Al(3+), rather than AlOH(2+) or Al(OH)(2) (+), is the principal toxic species of mononuclear Al. Comparisons of the responses of Al-sensitive and Al-tolerant wheats to Al(3+) and to La(3+) did not support the hypothesis that varietal sensitivity to Al(3+) is based upon differences in cell-surface electrical potential.

Assessing the Rhizotoxicity of the Aluminate Ion, Al(OH)(4).

Dissolved aluminum (III) in acidic soils or culture media is often rhizotoxic (inhibitory to root elongation). Alkaline solutions of Al are also sometimes rhizotoxic, and for that reason toxicity has been attributed to the aluminate ion, Al(OH)(4) (-). In the present study, seedlings of wheat (Triticum aestivum L. cv Tyler) and red clover (Trifolium pratense L. cv Kenland) were cultured in aerated aluminate solutions at pH 8.0 to 8.9. The bulk phases of these solutions were free of reactive polynuclear hydroxy-Al (including the extremely toxic species AlO(4)Al(12)[OH](24)[H(2)O](7+) (12) [Al(13)]) according to the ferron (8-hydroxy-7-iodo-5-quinolinesulfonic acid) assay. At an aluminate concentration of 25 micromolar (23 micromolar activity) and a pH of 8, root elongation was less than 40% of Al-free controls, but at pH 8.9 elongation was 100% of controls. The hypothesis is offered that aluminate is nontoxic and that the inhibition at lower pH values is attributable to Al(13) postulated to have formed in the acidic free space of the roots where the ratio /{Al(3+)/}//{H(+)/}(3) may rise above 10(10). At this value hydroxy-Al in over-saturated, alkaline solutions begins to undergo rapid conversion to polynuclear species.

Proton extrusion by wheat roots exhibiting severe aluminum toxicity symptoms.

The mechanisms of Al rhizotoxicity are not known, but disruption of membrane function has been a persistent hypothesis. The objective of this study was to establish whether cells of Al-cultured wheat roots (Triticum aestivum L. cv Tyler) exhibiting severe Al toxicity symptoms were capable of vigorous proton extrusion. The membrane electrical potential difference (E(m)) was measured in individual cells throughout the first centimeter of root tips during perfusion with Al solutions similar to or more concentrated than those of the culture medium. For both Al-cultured and control roots the resting E(m) was -100 millivolts, and 1 millimolar acetic acid induced cyanide-sensitive hyperpolarizations to -180 millivolts at a maximum rate of -30 millivolts per minute. Al, like Ca(2+), enhanced the negativity of the E(m) of cells already treated with acetic acid. Both acetic acid and fusicoccin stimulated net proton extrusion from Al-cultured and control roots, both of which also extruded protons in the absence of these stimulants. These results demonstrate that wheat roots exhibiting severe Al toxicity symptoms had an undiminished capacity to extrude protons, that the membranes were intact, and that ATP synthesis was sufficient to supply the proton-translocating ATPases.

Cation amelioration of aluminum toxicity in wheat.

Aluminum is a major constituent of most soils and limits crop productivity in many regions. Amelioration is of theoretical as well as practical interest because understanding amelioration may contribute to an understanding of the mechanisms of toxicity. In the experiments reported here 2-day-old wheat (Triticum aestivum L. cv Tyler) seedlings with 15-millimeter roots were transferred to solutions containing 0.4 millimolar CaCl(2) at pH 4.3 variously supplemented with AlCl(3) and additional amounts of a chloride salt. Root lengths, measured after 2 days in the test solutions, were a function of both Al activity and the cation activity of the added salt. Percent inhibition = 100 {Al(3+)}/({Al(3+)} + K(m) + alpha{C}(beta)) where {Al(3+)} is the activity of Al(3+) expressed in micromolar, {C} is the activity of the added cation expressed in millimolar, and K(m) (= 1.2 micromolar) is the {Al(3+)} required for 50% inhibition in the absence of added salt. For Ca(2+), Mg(2+), and Na(+) the values of alpha were 2.4, 1.6, and 0.011, respectively, and the values for beta were 1.5, 1.5, and 1.8, respectively. With regard to relative ameliorative effectiveness, Ca(2+) > Mg(2+) approximately Sr(2+) >> K(+) approximately Na(+). Other cations were tested, but La(3+), Sc(3+), Li(+), Rb(+), and Cs(+) were toxic at potentially ameliorative levels. The salt amelioration is not solely attributable to reductions in {Al(3+)} caused by increases in ionic strength. Competition between the cation and Al for external binding sites may account for most of the amelioration.

Electrical evidence for turgor inhibition of proton extrusion in sugar beet taproot.

Sections of sugar beet (Beta vulgaris L.) taproot were incubated in various concentrations of mannitol. At 0.4, 0.6, and 0.8 molar, the membrane electrical potential difference (E(m)) averaged about -130 millivolts; at 0.2 molar, about -90 millivolts; and at 0 molar, between -60 and -80 millivolts. Additions of 10 millivolts acetate to the incubation solutions (all at pH 5) enhanced the membrane polarity to about -200 millivolts. We conclude from these and previous findings that high turgor inhibits proton extrusion in the sugar beet, but that proton extrusion can be activated in fully turgid tissue by acidification of the cytoplasm. A possible function of this turgor effect may be the control of turgor itself.

A Quantitative Simulation Model for H-Amino Acid Cotransport To Interpret the Effects of Amino Acids on Membrane Potential and Extracellular pH.

The H(+) cotransport of neutral and acidic amino acids induces transient depolarizations of oat coleoptile (Avena sativa L., var Victory) plasma membranes. The depolarizations, which are completed within 1 or 2 minutes, are followed by repolarizations that are nearly completed within another 2 or 3 minutes. Cysteine induced a two-phased alkalinization of the tissue free space during the electrical changes. The first phase was a rapid, linear increase in pH that coincided with the depolarization; the second phase was a slower, also linear, increase in pH that coincided with the repolarization. Reacidification did not occur until cysteine was withdrawn. Five other acidic, basic, and neutral amino acids also induced persistent alkalinization of the free space.The notable features of these measurements are that free-space pH was measured more directly than previously, that pH changes corresponded in time to the electrical potential changes, and that reacidification of the free space did not occur. The latter observation indicates that net H(+) efflux did not occur during repolarization and that the repolarizing current was carried by some other ion. We propose that repolarization could have depended upon depolarization-induced changes in passive K(+) fluxes combined with an enhanced H(+) extrusion that increased until it equaled, but did not exceed, the enhanced influx of H(+).In support of the feasibility of our hypothesis, we present a quantitative simulation model for cotransport. The simulation model also provides an interpretation of the unique electrical effects of histidine and the basic amino acids. In addition, the model focuses attention upon the difficulties of interpreting H(+)-anion cotransport.

Energy Coupling in H-Amino Acid Cotransport : ATP DEPENDENCE OF THE SPONTANEOUS ELECTRICAL REPOLARIZATION OF THE CELL MEMBRANES IN OAT COLEOPTILES.

Experiments were undertaken in order to test the mechanism of energy coupling for amino acid uptake proposed in the cotransport hypothesis. According to the hypothesis an electrochemical potential difference in H(+) is established by active H(+) extrusion. That potential difference then drives the cotransport of H(+) and amino acids into the cells. Application of amino acids to oat (Avena sativa var. Victory) coleoptiles induced transient depolarizations of the cell membrane electrical potentials considered to reflect the joint uptake of H(+) and amino acids followed by an enhanced H(+) extrusion. In the presence of KCN, cysteine induced strong depolarizations, but the rate of repolarization depended linearly upon the cyanide-adjusted ATP level of the tissue. At an ATP level 44% of normal, the membrane potential was 74% of normal, but the repolarization after cysteine-induced depolarization was practically nil. Sudden transitions from room temperature to temperatures below 15 degrees C induced sharp depolarizations of the membrane which then repolarized within 3 min; the ATP content of the tissues was unaffected. Cysteine and alanine induced strong depolarizations at temperatures between 5 and 25 degrees C, and the Q(10) for the rate of depolarization was 1.5 for cysteine and 1.6 for alanine. The Q(10) for the rate of repolarization was 3.0 for cysteine and 2.0 for alanine. These experiments support the prevailing view that the depolarizations are caused by the passive joint influx of H(+) and amino acids and that the repolarizations depend upon the ATP-dependent extrusion of H(+).

Interamino Acid Inhibition of Transport in Higher Plants : EVIDENCE FOR TWO TRANSPORT CHANNELS WITH ASCERTAINABLE AFFINITIES FOR AMINO ACIDS.

Data from published experiments were analyzed to determine the number and specificities of amino acid transport channels in cells of higher plants. Each experiment measured the uptake of a labeled amino acid in the presence of unlabeled amino acids, used one at a time, in the incubating medium. The observed interamino acid inhibitions can be accounted for by two transport channels, each with characteristic affinities that were computed from the observed interamino acid inhibitions. The first channel is a general transport system with the following relative affinities for the amino acids: methionine 75, alanine 75, phenylalanine 64, tyrosine 64, leucine 63, cysteine 58, serine 57, glycine 56, tryptophan 54, glutamine 51, threonine 49, valine 44, isoleucine 44, glutamic acid 44, proline 43, histidine 33, lysine 32, asparagine 22, arginine 22, aspartic acid 18. The second channel is a basic amino acid tranport system with relative affinities for arginine, lysine, and histidine of 66, 39, and 21, respectively. The affinities for the other acids in the second channel are lower. Despite considerable diversity in the species, tissues, and solute concentrations employed in the experiments, multiple regression equations (Y = alpha + beta(1)X(1) + betaX(2), in which Y is the observed transport inhibition and X(1) and X(2) are the relative transport affinities of the two channels) account for 50 to 99% of the variance in all but six experiments, five of which employed unusually high solute concentrations.

Electrical evidence for different mechanisms of uptake for basic, neutral, and acidic amino acids in oat coleoptiles.

The application of neutral or acidic amino acids to oat coleptiles induced transient depolarizations of the membrane potentials. The depolarizations are considered to reflect H(+) -amino acid co-transport, and the spontaneous repolarizations are believed to be caused by subsequent electrogenic H(+) extrusion. The basic amino acids depolarized the cell membrane strongly, but the repolarizations were weak or absent. The depolarizations induced by the basic amino acids were weakly sensitive to manipulations of the extracellular and intracellular pH. The depolarizations induced by the other amino acids, in contrast, were more strongly affected by the pH changes. Several amino acids induced distinct but diminished depolarizations in the presence of 2,4-dinitrophenol or cyanide, but the repolarizations were generally eliminated. These experiments support the co-transport theory but suggest somewhat different mechanisms for the transport of the neutral, acidic, and basic amino acids. We suggest that the neutral amino acids are co-transported with a single H(+) and that accumulation depends upon both the DeltapH and the membrane potential components of the proton motive force. The acidic amino acids appear to be accumulated by a similar mechanism except that the transport of each molecule may be associated with a cation in addition to a single proton. The permanently protonated basic amino acids appear not to be co-transported with an additional proton. Accumulation would depend only on the membrane potential component of the proton motive force.

Wounding Stimulates Cyanide-sensitive Respiration in the Highly Cyanide-resistant Leaves of Bryophyllum tubiflorum Harv.

The rate of respiration in sectioned leaves of Bryophyllum tubiflorum Harv. increases with decreasing section thickness. The rates of uninhibited respiration in 2- and 8-millimeter-thick sections are 74 and 46 microliters of O(2) per gram fresh weight of unruptured tissue per hour at 20 C, whereas the rate in the presence of cyanide is 31 microliters of O(2) in each case. The rates are unaffected by salicylhydroxamic acid, but cyanide and salicylhydroxamic acid together completely eliminate O(2) uptake. The capacity of the alternative respiratory pathway is thus initially high (estimated at 84% of the uninhibited respiratory rate in whole leaves) and remains constant but probably unexpressed subsequent to the rapid induction of wound respiration.

Restoration of Organic Acid Accumulation in Sectioned Leaves of Bryophyllum tubiflorum Harv.

When leaves of Bryophyllum tubiflorum were cut into transverse sections, and held at 20 C in the dark, the capacity to accumulate organic acid decreased with decreasing section thickness. In addition, the rate of respiration increased with decreasing section thickness and was unaffected by changes in O(2) concentration above 5% or by the presence (1%) of CO(2). It was concluded that O(2) ventilation is not a controlling factor in respiration. Malonate (0.1 m) and fluoroacetate (0.01 m) restored the capacity of sectioned leaves to accumulate acid to normal levels and depressed respiration in 1-millimeter sections. Acid accumulation in 8-millimeter sections remained essentially constant at 20, 15, and 10 C, and was equal to that in unsectioned leaves, but accumulation in 2-millimeter sections rose to normal levels as the temperature fell to 10 C. Twenty-three additional metabolic inhibitors (none specific to the tricarboxylic acid cycle) were screened, and none promoted acid accumulation in sectioned leaves at 20 C. The results suggest that sectioning stimulates a respiratory sequence which includes the tricarboxylic acid cycle. This sequence in turn competes with the synthesis or accumulation of malic acid.