Claude Grignon

Role of apoplast acidification by the H(+) pump : Effect on the sensitivity to pH and CO2 of iron reduction by roots of Brassica napus L.

We have studied the mechanism of the response to iron deficiency in rape (Brassica napus L.), taking into account our previous results: net H+ extrusion maintains a pH shift between the root apoplast and the solution, and the magnitude of the pH shift decreases as the buffering power in the solution increases. The ferric stress increased the ability of roots to reduce Fe[III]EDTA. Buffering the bulk solution (without change in pH) inhibited Fe[III]EDTA reduction. At constant bulk pH, the inhibition (ratio of the Fe[III]EDTA-reduction rates measured in the presence and in the absence of buffer) increased with the rate of H+ extrusion (modulated by the length of a pretreatment in 0.2 mM CaSO4). These results support the hypothesis that the apoplastic pH shift caused by H+ excretion stimulated Fe[III] reduction. The shape of the curves describing the pH-dependency of Fe[III]EDTA reduction in the presence and in the absence of a buffer fitted this hypothesis. When compared to the titration curves of Fe[III]citrate and of Fe[III]EDTA, the curves describing the dependency of the reduction rate of these chelates on pH indicated that the stimulation of Fe[III] reduction by the apoplastic pH shift due to H+ excretion could result from changes in electrostatic interactions between the chelates and the fixed chargers of the cell wall and-or plasmalemma. Blocking H+ excretion by vanadate resulted in complete inhibiton of Fe[III] reduction, even in an acidic medium in which there was neither a pH shift nor an inhibitory effect of a buffer. This indicates that the apoplastic pH shift resulting from H+ pumping is not the only mechanism which is involved in the coupling of Fe[III] reduction to H+ transport. Our results shed light on the way by which the strong buffering effect of HCO3-in some soils may be involved in iron deficiency encountered by some of the plants which grow in them.

Quantitative measurement of cationic fluxes, selectivity and membrane potential using liposomes multilabelled with fluorescent probes.

Liposomes of egg PC/PG (8:2, mol/mol) were multilabelled with PBFI, pyranine and oxonol VI, fluorescent probes for, respectively, K+, H+ and membrane potential. Monitoring fluorescence with a multichannel photoncounting spectrofluorometer during K+ filling experiments allowed to measure K+ influx, the associated H+ efflux and the membrane potential, continuously and simultaneously. The proton net efflux quantitatively mirrored the K+ net influx. The rate of the K+/H+ exchange diminished progressively as a quasi-equilibrium was reached for both K+ and H+. In the presence of valinomycin, the measured membrane potential during the K+ filling actually corresponded to the Nernst potential calculated from the observed K+ gradient. In the absence of valinomycin, it corresponded to the Nernst potential calculated from the observed H+ gradient. In the latter case, the permeability coefficient of liposomes to K+, calculated from the Goldman-Hodgkin-Katz relation, was 6.10(-13) m s-1. The selectivity sequence for alkali cations of liposomes was determined from the measured H+ efflux associated to the influx of the different cations. The selectivity sequence corresponded to the series VI of Eisenman, suggesting interaction of the cation with an anionic field of intermediate strength.

In vitro study of passive nitrate transport by native and reconstituted plasma membrane vesicles from corn root cells

Proteins from phase-partitioned corn root plasma membrane were reconstituted into soybean lipids/egg PC (8:2, w:w) using deoxycholate and rapid gel filtration to eliminate the detergent. All (H+)ATPase molecules were inside-out reinserted and the initial activity was totally recovered in an homogeneous vesicle preparation. In addition, membrane tightness greatly increased, as shown by the size and stability of the response of the fluorescent membrane potential probe (oxonol VI) to an imposed K+ diffusion gradient. Consequently, the H(+)-pumping activity of the (H+)ATPase, monitored with the fluorescent pH probe (ACMA), increased 20-fold after reconstitution. A protein-mediated passive transport of nitrate was first demonstrated by the ability of NO3- to electrically short-circuit the (H+)ATPase in plasma membrane vesicles and not in liposomes containing only the purified enzyme. The passive transport was saturable (K(m) approximately 5 mM), thermolabile, inhibited by the arginine reagent phenylglyoxal, and selective (NO3- > I- approximately ClO3- approximately Br- > Cl- approximately NO2- > Iminodiacetate approximately SO4(2-)). Passive NO3- transport was also determined, independently of the (H+)ATPase, from the NO3(-)-dependent augmentation of the dissipation rate of imposed diffusion potentials. This second transport assay gave similar K(m) for NO3- and should be suitable to continue the functional and biochemical characterization of the NO3- transport system.

Effect of HCO - 3 concentration in the absorption solution on the energetic coupling of H+-cotransports in roots of Zea mays L.

The effect of HCO3-on ion absorption by young corn roots was studied in conditions allowing the independent control of both the pH of uptake solution and the CO2 partial pressure in air bubbled through the solution. The surface pH shift in the vicinity of the outer surface of the plasmalemma induced by active H+ excretion was estimated using the initial uptake rate of acetic acid as a pH probe (Sentenac and Grignon (1987) Plant Physiol. 84, 1367). Acetic acid and orthophosphate uptake rates and NO3-accumulation were slowed down, while 86Rb+ uptake and K+ accumulation rates were increased by HCO3-. These effects were similar to those induced by 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid/2-amino-2-(hydroxymethyl)-1,3-propanediol (Hepes-Tris). They were more pronounced when the H+ excretion was strong, were rapidly reversible and were not additive to those of Hepes-Tris. The hypothesis is advanced that the buffering system CO2/H2CO3/HCO3-accelerated the diffusion of equivalent H+ inside the cell wall towards the medium. This attenuated the surface pH shift in the vicinity the plasma membrane and affected the coupling between the proton pump and cotransport systems.

Local ionic environment of plant membranes: effects on membrane functions

Summary Electrostatic properties of the isolated plasma membrane of maize roots were studied using the fluorescent ANS probe and microelectrophoresis of membrane vesicles. Variations of the net surface charge of membrane were obtained by adding ionic surfactants or by acidifying the medium. Parallel experiments were performed on excised roots. We showed that surface electrostatic interactions can affect major membrane functions such as ATPase activity, trans-membrane PD, and permeability to NO 3 .

Variability of the response to sodium chloride of eight ecotypes of Arabidopsis thaliana

ABSTRACT The variability of the sensitivity to sodium chloride (NaCl) in relation with leaf ionic content has been explored in Arabidopsis thaliana. Seedlings of eight ecotypes (WS, COL, LER, NOK0, NOK1, NOK2, NW40, and N273) were grown on a peat substrate mixed or not with 50 mM NaCl. The effect of NaCl treatment on rosette leaf dry weight after 18 days (vegetative stage) was used to establish a scale of sensitivity to salt, where COL was the most sensitive ecotype and NOK2 the more tolerant one. The tolerance to NaCl was associated to a better growth of plants, responsible for a dilution of Na+ in leaf tissues, and to a better supply of K+ to leaves. These two characteristics were responsible for maintenance of a high K/Na ratio in leaves of to the most tolerant ecotypes (NOK1, NOK2), as compared to the other ones. Responses of leaf peroxidase activity to a five-day treatment with NaCl in light and darkness conditions suggest that oxidative status is changed only in the sensitive ecotypes.

Evidence for the contribution of surface potential to the transtonoplast potential difference measured on isolated vacuoles with microelectrodes

Summary In order to elucidate the origin of the transtonoplast potential difference (PD) measured on isolated vacuoles, the possible contribution of the surface potential to the transmembrane PD was studied. The PD and the electrophoretic mobility were measured on vacuolar preparations, isolated from Beta vulgaris roots and Acer pseudoplatanus cells. Zeta potential was calculated from the electrophoretic mobility. The changes in zeta potential, in response to pH or to a cationic surfactant, were shown to induce parallel variations in the PD. These results suggest that the surface potential could contribute to the transmembrane PD of isolated vacuoles. Various hypotheses on the origin of this transtonoplast PD are discussed.

Variability of the Effects of Salinity on Reproductive Capacity of Arabidopsis Thaliana

Abstract For this study, the diversity of some effects of salinity on rosette growth, seed production, and seed viability in Arabidopsis thaliana was explored. Plants from 5 accessions (“ecotypes”) were grown on a sand-peat mixture irrigated with nutritive medium. At the emergence of inflorescence axis, 35 mM NaCl was added to the solution. Several morphological parameters of the rosette were measured at the beginning of silique opening, irrigation was interrupted and the substrate was allowed to dry. The siliques and the mature seeds were harvested, and assayed for K+, Na+ and Cl−. Seeds were sown on pure water and germination kinetics were determined. There was a large inter-ecotype variability of the effect of salt on the rosette growth and on all factors contribution to reproduction (number of inflorescence axes, number of flower per axis, flower fertility, and seed viability). Among the five ecotypes, those with highest levels of Na+ and Cl− in the silique valves produced the less viable seeds. This comparison, (i) suggests that salt accumulation in the former organs has detrimental effect on seed maturation, and (ii) indicates that there is a natural variability for control of ion transport to fruits in A. thaliana.

GROWTH RATE AFFECTS SALT SENSITIVITY IN TWO LENTIL POPULATIONS

ABSTRACT In saline media, the building of high concentration of Na+ and Cl− in plant leaves may be delayed by rapid growth. This dilution effect may improve the salt tolerance of plant by increasing the time necessary for ion accumulation to reach toxic levels. This could permit new leaves to be produced faster than elimination of older leaves by excess salt accumulation. The present study is aimed at studying this hypothesis on lentil (Lens culinaris L.). Various mild environmental constraints have been used to restrict growth rate, and the effect on salt sensitivity was evaluated. Three week-old plants were cultivated for 4 or 8 weeks on medium supplemented with 10 or 36 mM NaCl and submitted to one of three environmental conditions aimed to limit growth: low illumination, high temperature, and competition (high planting density). Whole plant dry weights were used to estimate mean relative growth rates (RGR). Increasing NaCl concentration from 10 mM to 36 mM reduced RGR more severely under limited light, or at high temperature, or at high planting density. In the presence of 36 mM NaCl, this increased sensitivity of growth was concomitant with Na+ accumulation in leaves, leaf withering and leaf necrosis. In the case of the high temperature treatment, the increase in Na+ concentration in the leaves was clearly attributable to reduction of growth rate, since Na+ amounts imported into the leaves were independent of temperature. These results support the hypothesis that glycophyte plants growing slowly are more sensitive to salt than those growing rapidly. This behavior contrast with that of halophytes, for which slow growth rate can be of advantage to overcome salt stress.

A test for screening monoclonal antibodies to membrane proteins based on their ability to inhibit protein reconstitution into vesicles

The hypothesis that the binding of an antibody to a membrane protein is likely to prevent the reconstitution of the protein into liposomes was checked, by using the plant plasma membrane H(+)-ATPase (EC 3.6.1.35) as a model system, and two reconstitution procedures: spontaneous insertion (SI) of purified H(+)-ATPase into preformed liposomes, and a detergent-mediated reconstitution (DMR) procedure allowing the reconstitution of the whole membrane protein content. Nine monoclonal antibodies (MABs) raised against H(+)-ATPase were tested. None affected the functioning of the enzyme reconstituted in liposomes, suggesting that the probability to obtain an inhibitory MAB is low. Five MABs inhibited its SI, and seven inhibited its reconstitution in the DMR procedure. These results indicate that it is possible to screen antibodies directed against membrane protein, by making use of their ability to inhibit the reconstitution of these proteins.