Affinity purification of sucrose binding proteins from the plant plasma membrane.

Purified plasma membranes from sugar beet leaves were solubilized by 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and loaded on a sepharose 6 B column substituted with sucrose. Elution with sucrose at pH 5.2 yielded a peak that represented 0.2% of the loaded protein. This peak did not appear when the samples were pretreated with either 0.5 mM N-ethylmaleimide (NEM) or 0.5 mM para-chloromercuribenzenesulfonic acid. It was also absent when palatinose, a sucrose analogue not recognized by the sucrose transporter, was used as the affinity ligand. The peak specifically eluted by sucrose from the sucrose-Sepharose column exhibited sucrose transport activity after reconstitution into proteoliposomes. This peak was further fractionated by ion-exchange chromatography on a Mono-Q column, and the different fractions obtained were differentially labeled by [3H]NEM in the presence of sugars recognized (sucrose, maltose) or not recognized (palatinose) by the sucrose transporter. The data allowed to identify two fractions that were enriched with two polypeptides (56 and 41 kDa) differentially labeled by NEM in the presence of sucrose.

Cutting, ageing and expression of plant membrane transporters.

The activity and the expression of sucrose, hexose and amino acid transporters were studied with fresh, cut or aged tissues and plasma membrane vesicles (PMV) of mature sugar beet (Beta vulgaris L.) leaves. Cutting and ageing both induced an increase of the transcripts coding for sucrose transporters and hexose transporters. No significant effect could be detected on the amino acid transporter transcripts with the probe used (aap1). A polyclonal serum directed against the Arabidopsis thaliana sucrose transporter (AtSUC1) reacted with a 42 kDa band of the sugar beet PMV, confirming previous biochemical identification of this band as a sucrose transporter. ELISA assays run with microsomal fractions and PMV using the AtSUC1 sucrose transporter probe indicated that ageing, and to a lesser extent cutting, increased the amount of sucrose transporter present in the plasma membrane. However, while cutting strongly stimulated proton-motive force driven uptake of sucrose in PMV, ageing only resulted in a slight stimulation. These data give evidence for transcriptional, post-transcriptional and post-translational controls of the activity of the sucrose transporter by mechanical treatments. Proton-motive force driven uptake of 3-O-methylglucose and valine in PMV was strongly stimulated in PMV from aged tissues, although previous data had shown that cutting did not affect theses processes. Therefore, the plant cells possess various levels of control mechanisms that allow them to regulate fluxes of the main assimilates across the plasma membrane when their natural environment is directly or indirectly altered.

[Transport, distribution and metabolism of auxin in Vicia faba L. roots after application of [(14)C]IAA or [ (3)H]IAA to the apical bud]. / Transport, distribution et métabolisme de l'auxine dans la racine de Vicia faba L. après application de[(14)C]AIA ou de [ (3)H]AIA sur le bourgeon.

After application of [2-(14)C]IAA or [(3)H]IAA to the apical bud of intact young broad-beans, the movement of labelled auxin into the roots was followed by liquid scintillation counting and by autoradiographic analyses. Its metabolism was studied by chromatography, and its pathways by autoradiographic analyses coupled with ringing experiments or removal of the stele.The movement of [(14)C]IAA or [(3)H]IAA was characterized by a high retention of radioactivity in tissues, particularly in very young plants. The speed, which did not exceed 9 mm·h(-1) in old roots, appeared the slower the younger the plants were. However, it seemed possible that small quantities of IAA or its derivates went into sieve tubes in which they moved downwards faster. In the apical part of the root the labelled IAA was more quickly transformed than in the other parts of this organ. 24 h after the application of the IAA, the labelled molecules gathered more densely in the cap itself than in apical meristem.At least 2/3 of the applied auxin moved within the stele, which in a crosssection represents only 1/7 of the whole area. In the older part of the root, the cambial zone located between mature phloem and mature xylem was the preferred pathway of IAA transport, although it is a zone where the hormone is immobilized, used and metabolised. In the younger part of the root, the whole stele was the preferred pathway. Therefore, the auxin is in a privileged situation to take part in the regulation of various processes, especially in the development of secondary vascular tissue, more particularly of xylem.

[Transport of [(14)C] auxin from young pods of Vicia faba L]. / Transport de l'auxine-(14)C en provenance de jeunes gousses de Vicia faba L.

After the injection of [(14)C]indole acetic acid (IAA) into very young pods of broad-bean (Vicia faba L.) the movement of the (14)C in the peduncle and stem was followed by autoradiography. In samples with only one young pod the basipetal transport was always clearly dominant. Most of the radioactivity was found in the bundles, particularly in the outer region of the bundle and also in the inner region (protoxylem parenchyma). The progression of the tracer was relatively complex. The rate of movement of the radioactive «front¼ could be as much as 2 cm·h(-1) but most of the (14)C moved towards the base at rates clearly less than that of the «front¼. Chromatograms with several solvent systems showed that IAA was the main or the only mobile radioactive substance. During transport, a part of IAA was converted into indole-3-aldehyde (IAld) and indole-3-acetyl-aspartic acid (IAAsp). IAAsp and possibly also IAld, which were found mainly near the donor pod, seemed immobile. This work is part of a study on the interchange of phytohormones between fruit and plant.

Characterization of leucine-leucine transport in leaf tissues.

Uptake of the dipeptide [(3)H]Leu-Leu into leaf discs from mature broad bean (Vicia faba L.) was characterized. Uptake was maximal at pH 6.0 and appeared to be mediated by three systems with apparent K(m) values of 20 µM, 350 µM and 43 mM, respectively. Leu-Leu uptake was sensitive to N-ethylmaleimide, p-chloromercuribenzenesulphonic acid, diethylpyrocarbonate, and carbonyl-cyanide-m-chlorophenylhydrazone. Nitrate did not compete with peptide uptake, although the peptide transporter and the nitrate transporter have been reported to be homologous. The ability of leaf tissues to take up peptides strongly decreased with leaf age, and the phloem export of peptides as measured by exudation experiments was very low. It is concluded that the leaf tissues contain a peptide transporter that may take up some peptides with a high affinity, but that this transporter is not involved in the long-distance transport of nitrogen under the form of di- or tri-peptides.

Hgt1p, a high affinity glutathione transporter from the yeast Saccharomyces cerevisiae.

A high affinity glutathione transporter has been identified, cloned, and characterized from the yeast Saccharomyces cerevisiae. This transporter, Hgt1p, represents the first high affinity glutathione transporter to be described from any system so far. The strategy for the identification involved investigating candidate glutathione transporters from the yeast genome sequence project followed by genetic and physiological investigations. This approach revealed HGT1 (open reading frame YJL212c) as encoding a high affinity glutathione transporter. Yeast strains deleted in HGT1 did not show any detectable plasma membrane glutathione transport, and hgt1Delta disruptants were non-viable in a glutathione biosynthetic mutant (gsh1Delta) background. The glutathione repressible transport activity observed in wild type cells was also absent in the hgt1Delta strains. The transporter was cloned and kinetic studies indicated that Hgt1p had a high affinity for glutathione (K(m) = 54 micrometer)) and was not sensitive to competition by amino acids, dipeptides, or other tripeptides. Significant inhibition was observed, however, with oxidized glutathione and glutathione conjugates. The transporter reveals a novel class of transporters that has homologues in other yeasts and plants but with no apparent homologues in either Escherichia coli or in higher eukaryotes other than plants.

Glutathione depletion leads to delayed growth stasis in Saccharomyces cerevisiae: evidence of a partially overlapping role for thioredoxin.

Disruption of the first enzyme of glutathione biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a glutathione auxotrophy phenotype on plates. However, growth experiments in liquid medium revealed that the cessation of growth resulting from glutathione depletion in these yeasts is very delayed in S. cerevisiae compared to S. pombe. Glutathione metabolism was investigated to understand this delayed growth stasis in S. cerevisiae. The assimilation of reduced and oxidized glutathione, the intracellular storage pools of glutathione and the turnover of this compound were investigated and found to be similar in both yeasts. A possible overlapping role of intracellular thioredoxin in causing delayed stasis was studied. Yeast thioredoxin was overexpressed in S. cerevisiae and was found to partially relieve the dependence of S. cerevisiae glutathione auxotrophs on extracellular glutathione in glucose-grown cultures, as well as in glycerol-grown cultures where conditions of increased glutathione requirements exists in the cell. By partially, but not completely, compensating for glutathione deficiency in this yeast, thioredoxin thus appeared to be the major factor that was causing the delayed growth stasis following glutathione depletion in this yeast.