Pierre Crespi

Guanosine triphosphate-binding proteins on the plasmalemma of spinach leaf cells

The molecular mechanism of light perception through phytochrome is not well understood. This red-light photosensor has been implicated in various physiological processes, including the photoinduction of flowering. A few recent studies have shown that phytochrome initiates signal transduction chains via guanosine triphosphate (GTP)-binding proteins (G-proteins). We show here by different approaches that G-proteins exist in spinach (Spinacia oleracea L. cv. Nobel). Binding of GTP on the plasmalemma has been partially characterized and its possible regulation by red light examined by in-vitro assays. These experiments indicate a clear regulation of GTP binding by red light and also by Mastoparan. At least three G-proteins or protein subunits were found to be associated with the plasmalemma of leaf cells. The use of an antibody raised against an animal Gβ subunit confirmed the presence of heterotrimeric G-proteins. Separation of a crude membrane extract by free-flow electrophoresis also showed that some G-proteins could exist on the tonoplast.

Sterols and Plasmalemma Modifications in Spinach Apex during Transition to Flowering

Summary Plasmalemma thickness was determined in the shoot apex of the long day plant Spinacia oleracea during transition to flowering. This membrane was shown to become thicker a few hours following the extension of the light period. In plants submitted to 24 h of continuous light, the plasmalemma was 10 to 15 thicker than in cells from vegetative plants maintained in short days. After the extension of the light period the numerical density of particles exposed on the extracellular and protoplasmic fracture faces of the plasmalemma remains unchanged. The total sterol content of the apex was analysed in parallel and showed a net increase during transition to flowering. This change was observed at approximately the time of the plasmalemma thickening during the critical photoperiod. The results strengthen the suggestion that changes in plasmalemma might play an important role in the establishment of the floral state of the whole plant.

Changes in spinach plasmalemma after gibberellic acid treatment

The effect on plasmalemma of exogeneously applied gibberellic acid (GA3) was investigated in spinach (Spinacia oleracea). Highly purified plasmalemma was obtained by free-flow electrophoresis and identified on the basis of both activity of the enzyme markers 1,3-β-glucan synthase and membrane morphology. Total lipids were extracted and the sterols separated by thin-layer chromatography (TLC). Thickening of the plasmalemma and modifications in its sterol content were observed in leaves in response to gibberellic acid treatment. The changes in sterol amount were quantified by reverse phase high performance liquid chromatography (RPHPLC). For these determinations pure plasmalemma was recovered by partionning in an aqueous two-phase system. The results were discussed with those previously reported for the plasma membrane of spinach induced to flowering by a transfer to continuous light.

Detection and characterization of GTP-binding proteins on tonoplast of Spinacia oleracea

Abstract Despite the growing interest in GTP-binding proteins and their role in higher plants, no compartimental analysis has been performed on tonoplast, until now. After successive extraction with a cushion of saccharose and a glycerol gradient, the tonoplast of Spinacia oleracea was checked by electron microscopy and the purity of the preparations verified by enzyme marker analyses. Guanosine triphosphate (GTP) binding assays were carried out on the extract with guanosine 5′[γ-thio] triphosphate, [35S] (GTPγ35S) as the non-hydrolysable substrate. A specific binding was observed and a KD of 0.3 mM was estimated by displacement curves. The characteristic enhancement by Mas 7 was also observed. Two dimensional gel electrophoresis of the GTP-binding test permitted localization of proteins between 20 and 55 kDa which bound specifically to GTPγ35S. Immunodetection was performed in the same experimental conditions with an antibody raised against the conserved consensus sequence of the GTP-binding site of the animal Gα subunit of the heterotrimeric G-Protein. It confirmed the presence of proteins of 41–44 kDa which correspond to those detected with the GTP-binding test.

In vitro modification of spinach plasmalemma thickness

Floral induction in the long day plant spinach (Spinacia oleracea) has been shown to be accompanied by a thickening of plasmalemma. This change was observed at early evocation, in both shoot apices and leaves, as well as after inducing GA3 treatment. To get further information on this thickening, plasma membranes from spinach leaves were isolated, in the present study, using aqueous two phase partitioning and the effect of variousin vitro treatments on their thickness was investigated. The average plasmalemma thickness was unaffected by Na+ and K+ ions. It was increased upon the effect of either Ca2+ or gibberellic acid. A thickening of plasmalemma was also observed when plasma membranes from vegetative plants were incubated with a cytosolic preparation from photoinduced plants. The results were discussed in relation with the plasmalemma modifications previously reported in spinach.

Plasma membrane sterols and flowering induction

Highly purified plasma membrane was obtained by phase partitioning and their relative content of sterol was investigated during flowering induction of spinach (Spinacia oleracea L. cv. Nobel). For this purpose, vegetative spinach was submitted to an inductive photoperiod or to a treatment with gibberellic acid (GA3). Quantitative and qualitative modifications of plasmalemma sterol content were observed during the first hours of an inductive photoperiod and after 24-h of continuous light. The main alteration of membrane sterols was in their saturation level. This change, obtained during critical photoperiod, was inhibited by 2-isopropyl-4-dimethylamino- 5-methylphenyl-1-piperidine carboxylate methyl chloride (AMO 1618). Modifications of plasmalemma sterols were also obtained after a 72-h gibberellic acid treatment of the plants maintained under short day. Some differences exist with those observed during inductive prolongation of light. These different results are discussed in relation with the previously reported thickening of plasmalemma induced by a transfer of spinach to continuous light and by a GA3 treatment.

Effect of ozone and sulphur dioxide on membrane enzyme activities of Pinus sylvestris needles

Abstract One- and 2-year-old Pinus sylvestris saplings were exposed to chronic doses of ozone (O3) and sulphur dioxide (SO2) in short-term (3 months) and long-term (18 months) experiments. Microsomal and plasma membrane fractions were purified by phase partitioning from current-year needles. The following membrane enzyme activities were determined in the microsomal and/or purified plasma membrane fractions: K+, Mg2+-ATPase (EC 3.6.1.3), NADH ferricyanide oxidoreductase (EC 1.6.99.3), NADH-duroquinone reductase (EC 1.6.5.1), NADH oxidase type I (EC 1.6.99.2), NADH oxidase type II or peroxidase-like enzyme (EC 1.11.1.7) and pyrophosphatase (EC 3.6.1.1). NADH oxidase type I was slightly stimulated in the microsomal fraction after a short-term exposure to O3 whereas NADH-dependent duroquinone reductase was not affected by this pollutant. However, in the long term experiment, NADH oxidase type II measured in the plasma membrane fraction was more than 2-fold stimulated in the SO2 treated pines and more than 4-fold when O3 was added to SO2. However, pyrophosphatase was decreased by 50% in trees treated with SO2+O3 and remained unchanged in the SO2 treatment, indicating that this enzyme is probably sensitive to oxidation. K+, Mg2+-ATPase showed a trend towards an enhancement of activity when exposed to chronic concentrations of air pollutants, this enhancement was more important in the long-term experiment after the combined effect of SO2 and O3. However, the K+-stimulated component was inhibited by the combination of both pollutants. Finally, NADH ferricyanide reductase was significantly enhanced after O3 and SO2+O3 exposures appearing as the most sensitive oxidoreductase to these air pollutants. The stimulation of ATPase and membrane oxidoreductases could facilitate the adaptation and defense of trees by maintaining an adequate redox potential in the plasma membrane region and perhaps stimulating the reduction of extracellular electron acceptors generated by the exposure to air pollutants.