Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies.

For the first time, the (31)P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 x 10(6) vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 +/- 0.06 and a mean trans-tonoplast DeltapH of 1.56 +/- 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H(+) exchanges at the tonoplast. The (31)P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 +/- 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.

Regulation of Vacuolar pH of Plant Cells: II. A P NMR Study of the Modifications of Vacuolar pH in Isolated Vacuoles Induced by Proton Pumping and Cation/H Exchanges.

The vacuolar pH and the trans-tonoplast DeltapH modifications induced by the activity of the two proton pumps H(+)-ATPase and H(+)-PPase and by the proton exchanges catalyzed by the Na(+)/H(+) and Ca(2+)/H(+) antiports at the tonoplast of isolated intact vacuoles prepared from Catharanthus roseus cells enriched in inorganic phosphate (Y Mathieu et al 1988 Plant Physiol [in press]) were measured using the (31)P NMR technique. The H(+)-ATPase induced an intravacuolar acidification as large as 0.8 pH unit, building a trans-tonoplast DeltapH up to 2.2 pH units. The hydrolysis of the phosphorylated substrate and the vacuolar acidification were monitored simultaneously to estimate kinetically the apparent stoichiometry between the vectorial proton pumping and the hydrolytic activity of the H(+)-ATPase. A ratio of H(+) translocated/ATP hydrolyzed of 1.97 +/- 0.06 (mean +/- standard error) was calculated. Pyrophosphate-treated vacuoles were also acidified to a significant extent. The H(+)-PPase at 2 millimolar PPi displayed hydrolytic and vectorial activities comparable to those of the H(+)-ATPase, building a steady state DeltapH of 2.1 pH units. Vacuoles incubated in the presence of 10 millimolar Na(+) were alkalinized by 0.4 to 0.8 pH unit. It has been shown by using (23)Na NMR that sodium uptake was coupled to the H(+) efflux and occurred against rather large concentration gradients. For the first time, the activity of the Ca(2+)/H(+) antiport has been measured on isolated intact vacuoles. Ca(2+) uptake was strongly inhibited by NH(4)Cl or gramicidin. Vacuoles incubated with 1 millimolar Ca(2+) were alkalinized by about 0.6 pH unit and this H(+) efflux was associated to a Ca(2+) uptake as demonstrated by measuring the external Ca(2+) concentration with a calcium specific electrode. Steady state accumulation ratios of Ca(2+) as high as 100 were reached for steady state external concentrations about 200 micromolar. The rate of Ca(2+) uptake appeared markedly amplified in intact vacuoles when compared to tonoplast vesicles but the antiport displayed a much lower affinity for calcium. The different behavior of intact vacuoles compared to vesicles appears mainly to be due to differences in the surface to volume ratio and in the rates of dissipation of the pH gradient. Despite its low affinity, the Ca(2+)/H(+) antiport has a high potential capacity to regulate cytoplasmic concentration of calcium.

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12-starved cells.

Vitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12-deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.

[Nucleic acids during tumorous transformation in plants]. / Les acides nucléiques au cours de la transformation tumorale chez les végétaux

Crown-gall, one of the "plant cancer" is induced in the presence of a soil bacterium Agrobacterium tumefaciens which elaborates a Tumor inducing principle (T.I.P.), the nature of which is unknown. Several informations suggest that some DNA sequences of bacterial origin are included in tumorous cells of tissue cultures. A ribonuclease inhibits induction. An RNA extracted from Agrobacterium induces some hyperplasia transplantable by graft.

Isolation of the tumor-inducing RNA from oncogenic and nononcogenic Agrobacterium tumefaciens.

Two RNA fractions have been isolated and purified from both oncogenic and nononcogenic strains of Agrobacterium tumefaciens. Both RNAs are capable of inducing the formation of transplantable tumors when introduced at wound sites in stems of Datura stramonium plants. One of these RNA fractions was found to be bound to an RNA-directed DNA polymerase, while the other was associated with the bacterial DNA. Physical evidence suggests that both are single stranded and small in size; linear sucrose gradients show that their size corresponds to a value of 5-6 S. A concentration of 4-5 mug of the RNAs dissolved in 0.01 ml of water is effective in initiating the formation of transplantable tumors in Datura plants.

Transformation of Agrobacterium tumefaciens into a non-oncogenic species by an Escherichia coli RNA.

Transforming RNA excreted by showdomycin-resistant Escherichia coli induces a persistent, heritable, and spectacular change in Agrobacterium tumefaciens B(6), a bacterium that carries the oncogenic principle for tumor induction in plants. Transformants possessing new physiological and biochemical properties have completely or partially lost the capacity for tumor induction. They synthetize new ribosomes whose components are profoundly modified. On the basis of biological and biochemical characteristics, one is inclined to consider the completely transformed Agrobacterium tumefaciens as a "new species".

Caracterisations immunochimiques de deux auxine oxydases extraites de tumeurs vegetables.

Two auxin oxidases with distinct antigenic specificities have been identified in extracts of plant tumor induced in Datura stramonium by Agrobacterium tumefaciens. Auxin oxidase and peroxidase activities are demonstrated for both enzymes. The adaptation of an immunochemical method allows the quantitative evaluation of both enzymes in different plant tissue extracts. The amount of these enzymes which is low in the organ of the healthy plant increases during tumorisation, healing and in vitro culture of healthy tissues.