Action of protein synthesis inhibitors in blocking electrogenic h efflux from corn roots

The block in the electrogenic H⁺ efflux produced by protein synthesis inhibitors in corn root tissue can be released or by-passed by addition of fusicoccin or nigericin. The inhibition also lowers cell potential, and the release repolarizes. Associated with the inhibition of H⁺ efflux is inhibition of K⁺ influx and the growth of the root tip; fusicoccin partially relieves these inhibitions, but nigericin does not. The inhibition of H⁺ efflux which arises from blocking the proton channel of the ATPase by oligomycin or N,N′-dicyclohexylcarbodiimide can also be partially relieved by fusicoccin, but not by nigericin; the inhibition produced by diethylstilbestrol is not relieved by fusicoccin.     

Hydrolysis of polyphosphates by corn roots

Summary The hydrolysis of seven linear oligomers (P₂, P₃, P₅, P₁₅, P₂₅, P₃₅ and P₆₅) and one cyclic polyphosphate, trimetaphosphate (TMP), by corn (Zea mays) roots was investigated. In these experiments, corn-root homogenate or intact roots were incubated in a polyphosphate solution containing 1 mM polyphosphate or 50 mg P/L, respectively, and the amount of orthophosphate produced was determined. Results showed that the optimal pH value for hydrolysis of P₃, P₅, and TMP by corn-root homogenate was 5.0, whereas for the hydrolysis of P₂, P₁₅, P₂₅, P₃₅ and P₆₅, it was 6.0. The rate of polyphosphate hydrolysis by cornroot homogenate was temperature dependent up to the point of enzyme inactivation (>50°C). Nonsterile intact roots showed higher rates of hydrolysis than sterile roots, especially with P₂. The hydrolysis of all oligomers by sterile and nonsterile intact roots was very slow during the first 18h at 30°C, but increased rapidly after 18h with the oligomers P≦25. The oligomers P₃₅ and P₆₅ were quite resistant to hydrolysis by sterile and nonsterile roots after 48h incubation at 30°C. An experiment with sterile intact roots in pyrophosphate solution suggested that pyrophosphatase was induced in corn roots in the presence of its substrate. The order of hydrolysis rates of the oligomers by intact sterile corn roots was: P₂>P₃>P₅> TMP>P₁₅>P₂₅>P₃₅>P₆₅.     

Partitioning of previously-accumulated nitrate to translocation,reduction, and efflux in corn roots

The effect of nitrate uptake, or its absence, on the utilization of nitrate previously accumulated by dark-grown, decpitated maize (Zea mays L., cv. DeKalb XL-45) seedlings was examined. Five-d-old plants that had been pretreated with 50 mM ¹⁴NO ₃ ^? for 20 h were exposed for 8 h to nutrient solutions containing either no nitrate or 50 mM ¹⁵NO ₃ ^? , 98.7 atom % ¹⁵N. The ambient solution, xylem exudate, and plant tissue were analyzed to determine the quantities of previously-accumulated (endogenous) ¹⁴NO ₃ ^? that were translocated to the xylem, lost to the solution, or reduced within the tissue during the 8-h period. Energy was continuously available to the roots from the attached endosperm. In the absence of incoming nitrate, appreciable reduction and translocation of the endogenous ¹⁴NO ₃ ^? occurred, but efflux of ¹⁴NO ₃ ^? to the external solution was minimal. In contrast, during ¹⁵NO ₃ ^? uptake, there was considerable efflux of ¹⁴NO ₃ ^? as well as translocation of ¹⁴NO ₃ ^? to the xylem, but little ¹⁴NO ₃ ^? was reduced. Thus there appeared to be an inverse relationship between ¹⁴NO ₃ ^? efflux and reduction. The data are tentatively interpreted on the basis of a model which envisages (a) two storage locations within roots, one of which primarily supplies nitrate for translocation and the other of which primarily supplies nitrate for outward passage through plasmalemma, and (b) the majority of nitrate reduction as occurring during or immediately following influx across the plasmalemma, with endogenous ¹⁴NO ₃ ^? initially moving outward being recycled inward and thereby being reduced.     

Proton transport in isolated vacuoles from corn coleoptiles

Vacuoles were isolated from corn coleoptile protoplasts and ATP-dependent proton transport was measured by quinacrine fluorescence quenching or by the uptake of [¹⁴C]methylamine. Intact vacuoles were judged to be free of a surrounding plasma membrane based on fluorescent staining with fluoroscein-diacetate. Essentially all of the detectable ATP-stimulated methylamine uptake and α-mannosidase activities present in intact protoplasts were recovered in isolated vacuoles. In contrast, the activities of marker enzymes for plasma membranes, Golgi, endoplasmic reticulum, and mitochondria were reduced to 5 to 17% in vacuolar preparations. The characteristics of proton pumping by isolated vacuoles were compared to those of light microsomal membranes possibly derived from the tonoplast. ATP-dependent proton pumping by both isolated vacuoles and light microsomal vesicles was stimulated by Cl⁻, and inhibited by NO₃⁻, carbonyl cyanide-m-chlorophenylhydrazone, N,N′-dicyclohexylcarbodiimide, N-ethylmaleimide, 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid, diethylstilbestrol, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but not by vanadate. Both activities also showed substrate specificity for Mg-ATP. Finally, proton transport activities of vacuolar and microsomal fractions exhibited similar profiles after flotation in linear dextran gradients. We conclude that the microsomal proton pump previously characterized in corn coleoptiles (Mettler et al. 1982 Plant Physiol 70: 1738-1742) is derived from the tonoplast.     

Effects of ouabain and low temperature on the sodium efflux pump in excised corn roots

Ouabain (0.05 millimolar) and low temperature (4 C) both caused the tissue Na⁺ content of excised 5-day-old corn roots to increase, indicating that there is an inhibition of the Na⁺ efflux pump. Na⁺ efflux was measured utilizing three different methods. Each method gave similar results in terms of rate and ouabain sensitivity. With one of these methods, the compartmental efflux method, it was demonstrated that rates for Na⁺ efflux increase as the external Na⁺ concentration is increased; e.g. the efflux rates are 0.529, 1.78, and 3.64 microequivalents per gram fresh weight per hour for external NaCl concentrations of 1, 10, and 30 millimolar, respectively. The data indicate that the Na⁺ efflux pump is located in the plasmalemma of root cells.     

Multiphasic uptake of phosphate by corn roots

Abstract The concentration dependence of phosphate uptake was studied using root sections of corn (Zea mays L. cv. Ganga 5). Detailed and wide-range (57 concentrations in the range 1 μmol m^?3-75 mol m^?3), precise (average SEM < 2.5%, n= 6) and reproducible (similar patterns in three independent experiments and for 5, 10, 15, 20, 25 and 30°C) data revealed six (or seven) concentration-dependent phases separated by ‘jumps’ or sharp breaks. These transitions were independent of temperature and occurred over relatively narrow concentration ranges (0.0001–0.0004, 0.08–0.31, 1.0–3.5, (7.5–10), 18–20 and 57–59 mol m^?3). The intermediate phases obeyed Michaelis-Menten kinetics, whereas sigmoidal kinetics were observed at lower concentrations. Uptake within each of the two highest phases increased more rapidly with increasing external phosphate concentration than predicted from Michaelis-Menten kinetics but also saturated more rapidly. The latter finding is not consistent with free diffusion across the plasmalemma at high external phosphate concentrations. Kinetic models yielding continuous isotherms, e.g. the sum of one or two Michaelis-Menten terms and a diffusion term, cannot account for the data.     

Proton efflux in human skeletal muscle during recovery from exercise

In recovery from exercise, phosphocreatine resynthesis results in the net generation of protons, while the net efflux of protons restores pH to resting values. Because proton efflux rate declines as pH increases, it appears to have an approximately linear pH-dependence. We set out to examine this in detail using recovery data from human calf muscle. Proton efflux rates were calculated from changes in pH and phosphocreatine concentration, measured by ³¹P magnetic resonance spectroscopy, after incremental dynamic exercise to exhaustion. Results were collected post hoc into five groups on the basis of end-exercise pH. Proton efflux rates declined approximately exponentially with time. These were rather similar in all groups, even when pH changes were small, so that the apparent rate constant (the ratio of efflux rate to pH change) varied widely. However, all groups showed a consistent pattern of decrease with time; the halftimes of both proton efflux rate and the apparent rate constant were longer at lower pH. At each time-point, proton efflux rates showed a significant pH-dependence [slope 17 (3) mmol · l⁻¹ · min⁻¹ · pH unit⁻¹ at the start of recovery, mean (SEM)], but also a significant intercept at resting pH [16 (3) mmol · l⁻¹ · min⁻¹ at the start of recovery]. The intercept and the slope both decreased with time, with halftimes of 0.37 (0.06) and 1.4 (0.4) min, respectively. We conclude that over a wide range of end-exercise pH, net proton efflux during recovery comprises pH-dependent and pH-independent components, both of which decline with time. Comparison with other data in the literature suggests that lactate/proton cotransport can be only a small component of this initial recovery proton efflux. Accepted: 5 May 1997     

Nitrate-induced polypeptides in membranes from corn seedling roots

The polypeptide composition of the membranes from corn (Zeamays L.) seedling roots upon nitrate induction was determinedby two-dimensional gel electrophoresis and silver-staining.The synthesis of five polypeptides (49, 48, 35, 33, and 32 kDa)in the tono-plast fraction and four polypeptides (50, 49, 38,and 33 kDa) in the plasma membrane fraction was induced by both2.5 mM Ca(NO₃)₂ and 5 mM KNO₃. Extensive washing of the membraneswith salt and NaOH demonstrated that three induced polypeptides(49, 48, and 35 kDa) in the tonoplast fraction and two inducedpolypeptides (49 and 33 kDa) in the plasma membrane fractionwere integral proteins. After incubation of seedlings in N-freemedium for 4 d, the 49 and 32 kDa polypeptides in the tonoplastfraction had disappeared. By the sixth day in N-free medium,the 35 kDa polypeptide had disappeared from the tonoplast fraction.The 50 kDa polypeptide of the plasma membrane fraction was nolonger detectable in seedlings incubated for 6 d in N-free medium.The size of the spots corresponding to the 33 kDa polypeptidesof both membrane fractions and to the 49 kDa polypeptide ofthe plasma membrane fraction was reduced following incubationof seedlings in N-free medium. The changes in nitrate-inducedpolypeptides in both membrane fractions following transfer toN-free medium correlated with a reduced capacity to take upnitrate in the treated seedlings. The results support the conclusionthat the nitrate-induced polypeptides may be involved in nitratetransport across the tonoplast and plasma membrane. Key words: Nitrate transport, induction, membrane peptides     

Boron tolerance in barley is mediated by efflux of boron from the roots

Many plants are known to reduce the toxic effects of high soil boron (B) by reducing uptake of B, but no mechanism for limiting uptake has previously been identified. The B-tolerant cultivar of barley (Hordeum vulgare L.), Sahara, was shown to be able to maintain root B concentrations up to 50% lower than in the B-sensitive cultivar, Schooner. This translated into xylem concentrations that were approximately 64% lower and leaf concentrations 73% lower in the tolerant cultivar. In both cultivars, B accumulation was rapid and reached a steady-state concentration in roots within 3 h. In Schooner, this concentration was similar to the external medium, whereas in Sahara, the root concentration was maintained at a lower concentration. For this to occur, B must be actively extruded from the root in Sahara, and this is presumed to be the basis for B tolerance in barley. The extrusion mechanism was inhibited by sodium azide but not by treatment at low temperature. Several anion channel inhibitors were also effective in limiting extrusion, but it was not clear whether they acted directly or via metabolic inhibition. The ability of Sahara to maintain lower root B concentrations was constitutive and occurred across a wide range of B concentrations. This ability was lost at high pH, and both Schooner and Sahara then had similar root B concentrations. A predictive model that is consistent with the empirical results and explains the tolerance mechanism based on the presence of a borate anion efflux transporter in Sahara is presented.     

H-pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots

The initial rate of quenching of quinacrine fluorescence was used to monitor Mg:ATP-dependent H⁺-pumping in membrane vesicles from corn (Zea mays L. cv WF9 × MO17) roots and obtain a preparation in which vanadate-sensitive H⁺-pumping could be observed. Separation of membranes on a linear sucrose density gradient resulted in two distinct peaks of H⁺-pumping activity: a major one, at density 1.11 grams per cubic centimeter, was sensitive to NO₃⁻ and resistant to vanadate, while a minor one, at density 1.17 grams per cubic centimeter, was substantially resistant to NO₃⁻ and sensitive to vanadate. A membrane fraction enriched in the vanadate-sensitive H⁺-pump could be obtained by washing microsomes prepared in the presence of 10% glycerol with 0.25 molar KI. The kinetics of inhibition of H⁺-pumping by vanadate in this membrane preparation indicated that most of the H⁺-pumping activity in this fraction is sensitive to inhibition by vanadate, 50% inhibition being reached at about 60 micromolar vanadate. This value is fairly close to that observed for inhibition by vanadate of the ATPase activity in similar experimental conditions (40 micromolar). The inhibitor sensitivity, divalent cation dependence, pH optimum (6.5), and K_m for ATP (0.7 millimolar) of the H⁺-pumping activity match quite closely those reported for the plasma membrane ATPase of corn roots and other plant materials.     

Nitrate absorption by corn roots : inhibition by phenylglyoxal

Nitrate transport in excised corn (Zea mays L.) roots was inhibited by phenylglyoxal, but not by 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) or fluorescein isothiocyanate (FITC). Inhibition of nitrate uptake by a 1-hour treatment with 1 millimolar phenylglyoxal was reversed after 3 hours, which was similar to the time needed for induction of nitrate uptake. If induction of nitrate uptake occurs by de novo synthesis of a nitrate carrier, then the resumption of nitrate uptake in the inhibitor-treated roots may occur because of turnover of phenylglyoxal-inactivated nitrate carrier proteins. All three chemicals inhibited chloride uptake to varying degrees, with FITC being the strongest inhibitor. While inhibition due to DIDS was reversible within 30 minutes, both FITC and phenylglyoxal showed continued inhibition of chloride uptake for up to 3 hours after removal from the uptake solution. Assuming that the anion transporter polypeptide(s) carries a positive charge density at or near the transport site, the results indicate that the nitrate carrier does not carry any lysyl residues that are accessible to DIDS or FITC, whereas the chloride carrier does. Both chloride and nitrate carriers, however, seem to possess arginyl residues that are accessible to phenylglyoxal.     

Compartmental efflux analysis and removal of extracellular cadmium from roots

Profiles of ¹⁰⁹Cd efflux from roots into three solutions were determined for young intact plants of Agrostis gigantea and maize. The solutions were (a) nutrient culture medium containing 3 micromolar Cd at room temperature, (b) ice-cold 5 millimolar CaCl₂, and (c) ice-cold 5 millimolar PbCl₂. Efflux profiles were clearly resolved into three easily discernible components having fast, medium, and slow exchange rates. These results were unexpected for the situation where some intracellular Cd was present both as extractable Cd-binding peptide and in electron-dense granules within the cytoplasm and the vacuoles. Adding a fourth compartment to the curve-fitting model produced a splitting of the fast exchanging component. Use of these efflux kinetics to estimate Cd fluxes through membranes was inappropriate. However, they were useful in determining optimal washing times for the removal of extracellular Cd. A 10 minute wash in ice-cold 5 millimolar CaCl₂ is recommended for this purpose for Agrostis and maize roots.     

Proton efflux from rat intestinal basal-lateral membrane vesicles is stimulated by ATP and Na+

1. ATP-stimulated 22Na uptake and 14C-methylamine efflux were studied in inside-out rat intestinal basal-lateral membrane vesicles (BLMV). 2. Uptake of 22Na by basal-lateral membrane vesicles was stimulated by addition of ATP and by an acidic vesicle interior. 3. Efflux of 14C-methylamine was stimulated by ATP and Na+. 4. 14C-methylamine efflux was not influenced by vanadate or amiloride by themselves but was inhibited by the presence of both agents. 5. These data are consistent with a basal-lateral proton translocation mechanism which may be responsible for alkalinization of the lateral intercellular space and implicates the Na+-pump in this mechanism.     

Characterization of tonoplast polypeptides isolated from corn seedling roots

Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg²⁺ from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross-contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from corn roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast-specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions.     

Alterations in membrane potential and in proton efflux in plant roots induced byAzospirillum brasilense

Inoculation of soybean seedlings withAzospirillum brasilense Cd significantly reduced the membrane potential in every root part and was being maximal in the root elongation zone. Monitoring the proton efflux pattern of inoculated wheat roots by severalA. brasilense strains and byPseudomonas sp. for prolonged periods (up to 200h) revealed a change from the bimodal pattern of proton efflux of non inoculated roots. This change was not related to root colonization ability but to bacterial capacity to induce changes in root surface area. Continuous perfusion of the plant nutrient solution with a fresh solution (from inoculation time), eliminated the enhancing effect of inoculation on proton efflux. We propose thatA. brasilense inoculation influences membrane activity and subsequently proton efflux in roots, probably through the release of an as yet unidentified bacterial signal.     

Hydrolysis of organic phosphates by corn and soybean roots

Because of the importance of organic phosphates as sources of P for plants, this work was performed to study the hydrolysis of nine organic phosphates by sterile, intact corn (Zea mays L.) and soybean (Glycine max L.) roots. Results showed that the rates of hydrolysis ofp-nitrophenyl phosphate (PNP) in buffered solutions by roots of three varieties of corn and three varieties of soybean ranged from 13 to 22 μmol PO₄−P g⁻¹ root h⁻¹ and from 2.1 to 2.2 μmol PO₄−P 0.1 g⁻¹ root h⁻¹, respectively. The average rate of hydrolysis of PNP in nonbuffered solutions was 2- to 3-fold lower for corn roots and 6- to 10-fold lower for soybean roots as compared with those obtained with buffered solutions. The orthophosphate released from hydrolysis of organic P compounds in buffered solutions during a 48-h incubation of corn roots showed that the maximum rate of hydrolysis of PNP was 4 to 6 times greater than the commonly used substrates: α- and β-glycerophosphates, phenolphthalein diphosphate, and glucose-6-phosphate. The rates of hydrolysis of glucose-6-phosphate and glucose-1-phosphate were similar and about 6- to 12-fold lower than that of PNP. Phosphoethanolamine and phosphocholine were hydrolyzed slightly, ando-carboxyphenyl phosphate was not hydrolyzed. The rates of hydrolysis of organic P compounds in nonbuffered solutions by corn and soybean roots were 1 to 3 and 1 to 10 times lower than those in buffered solutions, respectively. The trends in rates of hydrolysis by soybean roots of buffered organic P substrates were similar to those observed with corn roots, with the exception of glucose-1-phosphate and phosphoethanolamine.     

Water efflux from the surface of field-grown grass roots. Observations by cryo-scanning electron microscopy

The objective of this study was to compare whole plant growth and physiological responses to salt stress of two Acacia nilotica subspecies (ssp. cupressiformis and ssp. tomentosa ). Salt stress was induced by adding NaCl at different concentrations to the nutrient solution: 0, 75, 100 and 200 m M . After one month under such stress, plants were still healthy and actively growing in both subspecies up to 100 m M NaCl. Water potential (Ψ) and osmotic potential (π) decreased with salinity and the lower π enabled the plants to maintain turgor. Höfler diagrams confirmed that osmotic adjustment had occurred under all treatments. Furthermore, the point of zero turgor occurred at a higher relative water content. An increase in the elastic modulus (ɛ) was observed under stress (low elasticity of the cell wall). Both osmotic adjustment and a high ɛ modified the capacity of both subspecies to maintain a positive water balance. Accumulation of ions (Na⁺, K⁺ and Cl⁻) and proline could explain such osmotic adjustment. Acacia nilotica ssp. cupressiformis showed a higher absorption of K⁺ than ssp. tomentosa up to 100 m M NaCl treatment.