Ph. Matile

Vacuolation: Origin and development of the lysosomal apparatus in root-tip cells

SummaryThe morphology of vacuolation has been investigated in root tip cells of corn using the freeze-etching technique. The genesis of vacuoles involves the following processes: a) Formation of small, endoplasmic-reticulum (ER)-derived vesicles (provacuoles); b) fusion of provacuoles resulting in the formation of small vacuoles, and followed by fusion and expansion of vacuoles; c) incorporation of large, dictyosome-derived vesicles into vacuoles by invagination of the tonoplast; d) invagination of the tonoplast resulting in the incorporation of cytoplasmic material into vacuoles. The morphological findings are correlated with biochemical data obtained from isolated vacuoles (lysosomes). Provacuoles (ER-derived vesicles) are shown to be primary lysosomes; their hydrolases arise from the ER. Vacuoles represent secondary lysosomes (digestive vacuoles) of the higher-plant cell. The metabolic role of lytic processes proceeding in the lysosomal apparatus is discussed.

Isolation and properties of the plasmalemma in yeast

SummaryA method is described for the isolation of fragments of the plasmalemma based on differential and density gradient centrifugation using cell free extracts from anaerobically grown Saccharomyces cerevisiae. Electron microscopically investigated frozen-etched specimens of isolated plasmalemma revealed the presence of globular particles attached to the outer surface of the membrane; these particles correspond to those observed in situ.In isolated plasmalemma a high specific activity of Mg++-dependent ATPase, which is not sensitive to Oligomycin, is present. Yeast plasmalemma contains protein, lipids (including phospholipids) and an appreciable amount of polysaccharide. Hydrolysis of this polysacharide yields only mannose.The treatment of the isolated plasmalemma with detergents liberates the globular particles which can be isolated by density gradient centrifugation. Protein and polysaccharide occur in the respective fraction; therefore the globular particle represents a mannan-protein. It is concluded that the particles, which cover the plasma-membrane of plant cells, represent glycoproteins, that is, building stones to be incorporated into the fibrillar network of the cell walls.

Malic Acid in Vacuoles Isolated from Bryophyllum Leaf Cells

Summary A method for the preparation of vacuoles from mesophyll cells of Bryophyllum daigremontianum is described. It is based on the lysis of protoplasts induced upon treatment with DEAE dextran. Isolated vacuoles contain the bulk of malic acid present in the protoplasts.

Catabolism of chlorophyll: Involvement of peroxidase?

Summary Commercial horseradish peroxidase oxidizes chlorophyllides to yellow products. In the absence of hydrogenperoxide the reaction rate is low; the highest rates were observed in the presence of both hydrogenperoxide and monophenols. Manganese ions have no effect on peroxidative catabolism of chlorophyllides. It is suggested that peroxidase may be involved in the catabolism of chlorophyll in senescent leaves.

The Role of the Vacuole in Storage and Mobilization of Stachyose in Tubers of Stachys sieboldii

Summary Stachyose (gal-gal-sucrose) is the main storage carbohydrate of resting tubers of Stachys sieboldii (80% of dwt). During an 18-day sprouting period the stachyose content decreased by 70 % and was correlated with a twofold increase of the activity of α-galactosidase, the key-enzyme responsible for the degradation of galactosyl saccharides. In order to study the compartmentation of storage and mobilization of stachyose, protoplasts and vacuoles were isolated from the storage parenchyma tissue of resting Stachys tubers. Stachyose was found to be exclusively localized in the vacuoles. Galactinol (gal-inositol), the precursor of galactosyl saccharides, and sucrose were mainly present in the cytoplasm, whereas the other saccharides were evenly distributed between these two compartments. α-Galactosidase was confined entirely to the vacuoles in contrast to galactokinase, the second enzyme of the catabolism of the galactosyl saccharides, which was cytoplasmic. Free galactose present in the vacuoles is proposed to have some regulatory function as a competetive inhibitor of α-galactosidase activity during the storage and mobilization of galactosyl saccharides in these tubers.

Changes in Chloroplast Number and Chloroplast Constituents in Senescing Barley Leaves

Summary Protoplasts from primary leaves of barley were analyzed for the changes in chloroplast numbers and chloroplastic constituents in the course of senescence. Whether naturally senescing leaves or leaves induced to senesee in the dark were employed, the number of chloroplasts per protoplast decreased to a markedly smaller extent than the contents in chlorophyll, protein and ribulosebisphosphate carboxylase. This result suggests that senescent chloroplasts are capable of degrading their own constituents.

A lysosomal aminopeptidase isozyme in differentiating yeast cells and protoplasts.

SummaryCells of Saccharomyces cerevisiae that have been growing exponentially for many generations contain low activities of lysosomal enzymes. In contrast to such fully adapted cells, differentiating or resting cells contain comparatively high activities of these enzymes. Thus, the digestive enzymes seem to be involved in the process of biochemical differentiation.One of the four aminopeptidase isozymes present in extracts from yeast cells is localized in the vacuoles. This lysosomal enzyme can be separated from the other aminopeptidases by Sephadex G-150 gel filtration. Its specific activity is about 4 times higher in stationary cells than in exponentially growing cells.Upon incubating protoplasts in a buffered sorbitol medium the activities of proteases and RNase increase significantly. A corresponding increase of lysosomal aminopeptidase activity occurs in the absence of glutamic acid or casein hydrolysate. Cycloheximide and actinomycin D inhibit the increase of lysosomal hydrolase activities in differentiating protoplasts. The observed changes of enzyme activities are probably due to induced synthesis of the respective proteins.

Comparmentation of Ascorbic Acid in Vacuoles of Horseradish Root Cells. Note on Vacuolar Peroxidase

Summary In the presence of L-ascorbic acid myrosinase of horseradish root is activated by a factor of 800–1000. Activation is optimal at an ascorbate concentration of 1.8 mM. This corresponds almost exactly with the concentration of ascorbic acid in the root tissue. L-Ascorbic acid is present in isolated vacuoles. In order to quantify the vacuolar compartmentation of ascorbate, phenolic compounds and acid phosphatase activity were chosen as markers of vacuoles. The yields of vacuoles with regard to these markers were equal to the yields of L-ascorbic acid recovered in preparations of isolated vacuoles. It is concluded that in horse-radish root cells the totality of ascorbate is compartmented in the large central vacuoles. About 70% of the total peroxidase activity extractable through tissue slicing is located in vacuoles.

Vacuoles of Chelidonium Latex: Lysosomal Property and Accumulation of Alcaloids

Summary Vacuoles isolated from the latex of Chelidonium majus contain high specific activities of lysosomal enzymes. Electron micrographs of differentiating laticifers suggest a function of vacuoles in cellular lytic processes. Isolated vacuoles contain the alcaloids that cause the orange-yellow colour of the latex. Addition of sanguinarine to a suspension of isolated vacuoles results in its rapid absorption and accumulation in the vacuolar sap. This process does not require energy. It involves the exchange between sanguinarine and other alcaloids present in the vacuolar sap. These results are discussed in terms of an ion-trap mechanism of alcaloid accumulation.

Some properties of vacuoles isolated from Neurospora crassa slime variant.

A method for the isolation of vacuoles based on polybase induced lysis of protoplasts of the cell wall deficient Neurospora crassa slime variant is described. Isolated vacuoles are characterized by 12 to 50 times increased specific activities of several hydrolases as compared with the total homogenate of protoplasts. Total α-amino nitrogen, arginine, and polyphosphate are also greatly enriched in these vacuoles. Vacuoles are equipped with a permease for the transport of basic amino acids across the tonoplast.