Gérard Vansuyt

The changing sensitivity to auxin of the plasma-membrane H(+)-ATPase: Relationship between plant development and ATPase content of membranes.

The auxin sensitivity of the plasma-membrane H+-ATPase from tobacco leaves (Nicotiana tabacum L. cv. Xanthi) depends on the physiological state of the plant (Santoni et al., 1990, Plant Sci. 68, 33–38). Results based on the study of auxin sensitivity according to culture conditions which accelerate or delay tobacco development demonstrate that the highest auxin sensitivity is always associated with the end of the period of induction to flowering. Auxin stimulation of H+-translocation activity corresponds to an increase of the apparent ATPase affinity for ATP. The plasma-membrane H+-ATPase content, measured with an enzyme-linked immunosorbent assay using a specific anti-H+-ATPase antibody, varies according to plant development, and was found to increase by 100% during floral induction. The specific molecular ATPase activity also changes according to plant development; more particularly, the decrease in molecular ATPase activity upto and during the floral-induction period parallels the increase of sensitivity to indole-3-acetic acid.

Differential auxin sensitivity of proton translocation by plasma membrane H+-ATPase from tobacco leaves

Abstract Auxin effects on the H + -ATPase promoted proton translocation in purified tobacco leaves plasma membrane vesicles were investigated according to the hormone concentration. Addition of IAA at low auxin concentrations was shown to induce a stimulation of the H + translocation. However, an opposite effect was also observed at higher concentrations. In addition, auxin concentrations inducing both effects appeared to depend dramatically on the plant age: at the end of the induction to flowering period, the sensitivity is nearly 1000-fold higher than that of vegetative or flowering plants. Thus proton translocation may be either stimulated or inhibited by auxins, according to the hormone concentration and/or the physiological state of the plant.

Soil-dependent variability of leaf iron accumulation in transgenic tobacco overexpressing ferritin

Ferritin overexpression in transgenic plants has been recently reported to increase leaf and seed iron content. We investigated the influence of various soil conditions on this increase in leaf iron content. One control transgenic tobacco and two transgenic tobaccos overexpressing ferritin in the plastids or in the cytoplasm, respectively, were grown on five different soils, two of them being amended with sewage sludge. Although a significant increase in leaf iron concentration was measured in transgenics overexpressing ferritin grown on three out of five soils, this increase was not a general rule. On some soils, leaf iron concentration of control plants was as high as in transgenics grown on other soils. In addition, an increased phosphorus concentration in the two sewage sludge amended soils correlated with a high leaf iron concentration in control plants, similar to the one measured in ferritin transformed plants. Indeed, growing plants in vitro with various increasing phosphate concentrations revealed a direct P involvement in iron loading of control plants, at a similar level as overexpressing ferritin plants. Also, with one of the soil tested, not only iron but also manganese, zinc and cadmium, and to a much lesser extent copper, nickel and lead were found more abundantly in ferritin transformed plants than in control plants. These data indicate that the iron fortification of leaves, based on ferritin overexpression, could be limited in its biotechnological application because of its high soil dependence.

rol A modulates the sensitivity to auxin of the proton translocation catalyzed by the plasma membrane H+-ATPase in transformed tobacco

In order to investigate the effect of the rol A gene product on the plasma membrane response to auxin, a clone of rol A‐transformed tobacco was prepared. Auxin sensitivity was measured by the auxin concentration which induced the highest stimulation of H+‐ATPase‐mediated proton translocation on isolated plasma membrane vesicles. Both transformed and control plants exhibited identical auxin‐sensitivity changes during vegetative and induction to flowering periods. However the sensitivity of flowering‐transformed plants Was 100‐times higher than that of control plants. Consistent observations were also made when using rol A+B+C‐transformed plants. These results suggest that the rol A gene product could either participate in or affect the reception/transduction pathway of auxin signal at the plasma membrane.

Auxin effects on proton accumulation by reconstituted plasma membrane h+-atpase

Abstract Auxin effects on ionic membrane permeability, ATP hydrolysis and H + accumulation by plasma membrane H + -ATPase reconstituted into proteoliposomes were investigated because of the recently reported auxin inhibitory effect, between 10 u M and 1 mM, on proton accumulation by plasma membrane vesicles. Whilst none of the tested auxins, even at 1 mM concentration, affected ATP hydrolysis by the enzyme, all compounds, at concentrations ranging between 0.1 and 1 mM, significantly inhibited the H + -ATPase mediated proton accumulation by proteoliposomes. At concentrations similar to those inducing this latter effect, auxins significantly increased the permeability to protons of proteoliposomes. In addition 1-naphthaleneacetic acid also increased permeability to Cl − . Similar effects on ionic permeability were also observed with pure phospholipid vesicles. On the other hand none of the auxins affected the fluorescence anisotropy of 1,6-diphenyl hexa-1,3,5-triene or 1-(4-rimethylamino)phenyl-6-phenylhexa-1,3,5-triene incorporated into proteoliposomes or liposomes. Data show that the auxin induced increase of passive membrane permeability to ions may account for part of the inhibitory effect evidenced at the plasma membrane, but that another kind of phenomenon should be involved when inhibition occurs at concentrations around 10 u M.

Action de molecules a proprietes hormonales sur l'activite phenylalanine ammoniac lyase

Abstract A comparative study has been made of phenylalanine ammonia lyase (PAL) activity in plants sensitive or resistant to various herbicides (piclorame, methylchloro-phenoxyacetic acid (MCPA), atrazine). Piclorame, a herbicide with hormonal activity caused a large decrease in PAL activity of sensitive plants ( Nicotiana tabacum ), even at low concentrations (5 × 10 -9 M) whilst in resistant plants ( Triticum aestivum ) its effect is negligible; MCPA, also a herbicide with hormonal activity, similarly affects the activity of PAL, but only at higher concentrations. On the contrary, the action of atrazine, which has no hormonal activity, is lower and weaker, probably being only a secondary effect. Determinations of PAL activity during the photoperiod following piclorame application indicate that this herbicide influences principally the photodependent enzyme activity.

Differential Sensitivity to Auxin at the Plasma Membrane Level

Among growth regulators, auxins constitute the group for which the most convincing evidences were raised for an action at the membrane level. Particularly, several data argue for an action on the plasma membrane H+-ATPase (1–3) and it is now largely assumed that auxin stimulates the ATPase. In addition, this effect would involve auxin binding proteins (1–3). However one intriguing point concerns the concentrations used to demonstrate the stimulation. For instance the ATPase activity is stimulated by 1 µM IAA in membrane vesicles from zucchini hypocotyls (4) but by 100 µM IAA in membrane vesicles from pumpkin hypocotyls (5). With tobacco protoplasts, maximal hyperpolarization is observed in the presence of 5 µM NAA, and depolarization with 100 µM NAA (6). On the other hand, in studies with artificial phospholipid membranes, millimolar auxin concentrations were found to alter permeability properties (7–9). The question to know to what auxin concentrations the plasma membrane is sensitive appears so quite puzzling.

Physiological and morphological consequences of expression of the rolA gene in transgenic tobacco plants

Summary When transferred to the plant genome, the rolA gene of Agro- bacterium rhizogenes induces numerous changes in plant phenotype, including prominent leaf wrinkling and dwarfism. Cytological, physiological and molecular studies were carried out on tobacco plants expressing rolA, with the goal of explaining these developmental modifications.

Influence du piclorame sur l'activité phénylalanine-ammoniac lyase de Nicotiana tabacum induite par la réaction d'hypersensibilité au virus de la mosaïque du tabac

Abstract In Nicotiana tabacum var. Xanthi n.c. phenylalanine ammonia lyase (PAL) activity, which increases significantly during the hypersensitivity reaction to tobacco mosaic virus (TMV), is only partially affected when plants are treated with piclorame, which is known to be an inhibitor of PAL in vivo . Using piclorame together with TMV, it has been possible to distinguish between that increase in PAL activity due to the virus and that dependent on the photoperiod.