A procedure for isolating vacuoles from leaves of the halophyte Suaeda maritima

Abstract. A method is described here for isolating protoplasts and vacuoles from leaves of the halophyte Suacda maritima. Integrity of the protoplasts and vacuoles was tested by staining and shown to be more than 75%, while use of biochemical markers, staining and light microscopy suggested a high degree of purity of the vacuoles. Phosphatase and NADH cytochrome- c -reductase were associated with vacuoles; phosphatase showed an eight-fold enrichment and NADH cytochrome- c -reductase a 3.5-fold enrichment relative to protoplasts. The vacuoles contained only 15% of the protein in protoplasts.     

Subcellular Localization of 2-(beta-d-Glucosyloxy)-Cinnamic Acids and the Related beta-glucosidase in Leaves of Melilotus alba Desr

The distribution of the glucosides of trans- and cis-2-hydroxy cinnamic acid and of the β-glucosidase which hydrolyzes the latter glucoside was examined in preparations of epidermal and mesophyll tissue obtained from leaves of sweet clover (Melilotus alba Desr.). The concentrations of glucosides in the two tissues were about equal when compared on the basis of fresh or dry weight. Inasmuch as the epidermal layers account for no more than 10% of the leaf volume, the mesophyll tissue contains 90% or more of the glucosides. Vacuoles isolated from mesophyll protoplasts contained all of the glucosides present initially in the protoplasts.     

Presence of the cyanogenic glucoside dhurrin in isolated vacuoles from sorghum

Large numbers of vacuoles (10⁶-10⁷) have been isolated from Sorghum bicolor protoplasts and analyzed for the cyanogenic glucoside dhurrin. Leaves from light-grown seedlings were incubated for 4 hours in 1.5% cellulysin and 0.5% macerase to yield mesophyll protoplasts which then were recovered by centrifugation, quantitated by a hemocytometer, and assayed for cyanogenic glucosides. Mature vacuoles, released from the protoplasts by osmotic shock, were purified on a discontinuous Ficoll gradient and monitored for intactness by their ability to maintain a slightly acid interior while suspended in an alkaline buffer as indicated by neutral red stain. Cyanide analysis of the protoplasts and the vacuoles obtained there from yielded equivalent values of 11 μmoles of cyanogenic glucoside dhurrin per 10⁷ protoplasts or 10⁷ vacuoles. This work supports an earlier study from this laboratory which demonstrated that the vacuole is the site of accumulation of the cyanogenic glucoside in Sorghum.     

Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells

We have shown that highly purified vacuoles of suspension-cultured tomato (Lycopersicon esculentum) cells contain RNA-oligonucleotides, using two different approaches to label and detect RNA: (a) in vivo labeling of cellular RNA with [5-³H]uridine, followed by preparation of vacuoles from protoplasts and by quantification of radioactively labeled material; and (b) in vitro labeling and analysis on sequencing gels of nucleic acids prepared from tomato vacuoles and their identification as RNA. The intravacuolar location of the RNA found in vacuolar preparations was concluded from analyzing for RNA intact organelles after repeated flotation steps as well as ribonuclease A treatment. About 3% of the RNA in protoplasts was localized within vacuoles, exceeding by severalfold the contribution made by contamination with unlysed protoplasts and subcellular organelles. Investigation of the size distribution of vacuolar RNA revealed an oligonucleotide pattern strikingly different from that which would arise from contaminating protoplasts; vacuolar RNA fragments are considerably shorter than 80 nucleotides. Characterization of these oligoribonucleotides (3′-phosphorylated termini; relatively rich in pyrimidines) as possible products of tomato vacuolar ribonuclease I action, and, in addition, enzymatic hydrolysis of vacuolar RNA by inherent enzyme activities in lysed vacuole preparations support the hypothesis that plant vacuoles are involved in cellular nucleolytic processes.     

Intracellular distribution of cardiac glycosides in leaves of Convallaria majalis

In order to investigate the intracellular distribution of the cardiac glycosides in leaves of Convallaria majalis, cell organelles were prepared by several methods. After mechanical disruption of the cells and differential centrifugation, the cardenolide content obtained was determined using the Baljet reaction. Most of the cardiac glycoside fraction was found in the soluble supernatant. However, a low but significant amount was also found in the 10 000g particles. Protoplasts and vacuoles were prepared by enzymic digestion of leaves. The cardenolide to protein ratio of vacuoles was far higher than that of protoplasts or the cytoplasmic fraction. The cardenolide content of isolated vacuoles relative to their number agreed well with the corresponding value obtained for protoplasts. This demonstrates clearly that cardiac glycosides are stored predominantly in the vacuoles of Convallaria majalis.     

Isolation of protoplasts and vacuoles from storage tissue of red beet

A fast and efficient method is described for the isolation of protoplasts and vacuoles from storage tissue of Beta vulgaris L. The viability of the isolated protoplasts is indicated by the development within a few hours of plasma strands with active cyclosis as well as by transport activity.     

Ultrastructure of fusion products from soybean cell culture and sweet clover leaf protoplasts

Summary Protoplasts from cultured cells of soybean (Glycine max L.) and from sweet clover (Melilotus officinalis L.) mesophyll cells were fused with polyethylene glycol and subsequently cultured for six days. The resulting fusion products as well as unfused protoplasts of each parental species regenerated cell walls and divided. The fusion products were characterized by the presence of soybean leucoplasts and sweet clover chloroplasts. The chloroplasts appeared to be degenerating but other cytoplasmic organelles were typical of actively growing plant cells. The fate of individual nuclei could not be determined.Supported by National Research Council of Canada, Grant A6304     

Intracellular Localization of Two Enzymes Involved in Coumarin Biosynthesis in Melilotus alba

The localization of phenylalanine ammonia-lyase [EC 4.3.1.5] within sweet clover (Melilotus alba) leaves was investigated. Apical buds and axillary leaves contained 15 to 30 times more enzyme activity than did mature leaves. Mesophyll protoplasts were prepared by digesting young leaves with Cellulysin and Macerase and were gently ruptured yielding intact chloroplasts. These chloroplast preparations exhibited neither phenylalanine ammonia-lyase nor o-coumaric acid O-glucosyltransferase activities. The general enzymic properties of sweet clover leaf phenylalanine ammonia-lyase were similar to those described for this enzyme isolated from other plant species. The conversion of l-phenylalanine to trans-cinnamic acid, which occurred at an optimum pH of about 8.7, was strongly inhibited by the metabolites trans-cinnamic and o-coumaric acids. In contrast, o-coumaric acid glucoside, coumarin, p-coumaric acid, and melilotic acid had no significant effect on the reaction rate.     

Localization and Substrate Specificity of Glycosidases in Vacuoles of Nicotiana rustica

Vacuoles isolated from Nicotiana rustica var brasilia have been shown to contain significant levels of glycosidase activity when assayed using p-nitrophenyl-glycosides as substrates. The substrate specificity for the glycosidases in the vacuolar fraction closely paralleled that found in the protoplasts, and the leaf tissue from which the vacuoles were isolated. The substrate specificity of the vacuolar enzyme(s) was different from glycosidic activity found in the commercial digestive enzyme preparations used to isolate the protoplasts from leaf tissue. It was demonstrated that 70 to 90% of the glycosidases that were found in the protoplasts appeared to be localized within the vacuole, when the p-nitrophenyl substrates α- and β-;d-galactose, β-d-glucose, and α-d-mannose were used. Neither the vacuolar nor the protoplast enzymes were active towards the naturally occurring phenolic glycoside, rutin. α-Mannosidase appears to be a valuable marker enzyme for vacuoles isolated from mesophyll leaf cells of tobacco.     

Hydrolysis of Intracellular Proteins in Vacuoles Isolated from Acer pseudoplatanus L. Cells

Acer pseudoplatanus cell suspension cultures were used to examine the ability of vacuoles isolated from protoplasts to hydrolyze their endogenous proteins. Total cell proteins were labeled by addition of [³H]leucine to the culture medium. After preparation of the protoplasts, vacuoles were isolated and were shown to be essentially free from other cellular components. Up to 30% of the [³H]leucine-labeled newly synthesized proteins were recovered in the vacuoles. When incubated for 6 hours at 20°C, the vacuoles degraded half of these proteins. The protein breakdown was temperature and pH dependent. Analysis by electrophoresis, in denaturing polyacrylamide gels, revealed that most of the vacuolar proteins were degraded. However, some vacuolar proteins were unaffected during a 6-hour incubation period. The results indicate that vacuoles are able to acquire and degrade intracellular proteins.     

Hydrolytic enzymes in the central vacuole of plant cells

The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and of pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast. (a) Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. (b) Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation. (c) Vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated.     

Electrophoretic analysis of protoplast, vacuole, and tonoplast vesicle proteins in crassulacean Acid metabolism plants

Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant.     

Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue

Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner G.J. and Siegelman, H.W. (1975) Science 190, (1298–1299). The ATPase activity of fresh vacuole suspensions was found to be 2–3 times that of protoplasts from the same tissue. 70–80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6 μg/10⁶ vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N′-dicyclohexylcarbodiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits.Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tulipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K⁺, Na⁺, Mg²⁺, Cl^?, and Ca²⁺ respectively, which are about the same as those in protoplasts.     

Translocation of photosynthates into vacuoles in spinach leaf protoplasts

A method was developed for the isolation of vacuoles from the mesophyll protoplasts of spinach leaf, employing the discontinuous Ficoll density gradient centrifugation technique. Isolated vacuole preparations were judged to be free from other organellar fractions based on the assays of marker enzyme activities of individual organelles.

Using this isolation method, a time-dependent translocation of ¹⁴C-labeled photosynthates into vacuoles was determined. In contrast to a significant transport of ¹⁴C organic acids such as malate and citrate within 10 to 15 minutes ¹⁴C neutral sugars and amino acids were barely transported into vacuoles during 40 minutes incubation, in spite of the fact that a relatively large amount of these compounds are found in the vacuoles. It was also found that a majority of [¹⁴C]sucrose remains in the cytosol, apparently not actively moving into the vacuoles. Overall results appear to suggest that vacuoles are not actively engaged in photosynthetic carbon metabolism in spinach leaf protoplasts.

     

Evidence that a part of cellular uridine of a tomato (Lycopersicon esculentum) cell suspension culture is located in the vacuoles

Cells, protoplasts and isolated vacuoles of a tomato (Lycopersicon esculentum) cell suspension culture were analyzed by high pressure liquid chromatography (HPLC) for the presence of uridine. It was found that the uridine content in 10⁸ cells or protoplasts varied between 70 and 150 nmol for different growth stages. The vacuolar location of a part of cellular uridine was evidenced by its co-migration (i) with α-mannosidase, a soluble vacuolar marker, in the gradient used for the purification of vacuoles and (ii) with α-mannosidase and vacuoles (counted microscopically) during repeated centrifugation of isolated vacuoles. Quantitatively, vacuoles sequestered about 13–35% of the amount of uridine present in protoplasts of different culture age. The possible origin of uridine in the vacuoles is discussed.     

Content and vacuole/extravacuole distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts

Neutral sugar, free amino acid, and anthocyanin levels and vacuole/extravacuole distribution were determined for Hippeastrum and Tulipa petal and Tulipa leaf protoplasts. Glucose and fructose, the predominant neutral monosaccharides observed, were primarily vacuolar in location. Glutamine, the predominant free amino acid found, was primarily extravacuolar. γ-Methyleneglutamate was identified as a major constituent of Tulipa protoplasts. Qualitative characterization of Hippeastrum petal and vacuole organic acids indicated the presence of oxalic, malic, citric, and isocitric acids. Data are presented which indicate that vacuoles obtained by gentle osmotic shock of protoplasts in dibasic phosphate have good purity and retain their contents.     

The effect of high temperatures on leaf cells of Valerianella: relative heat stability of the tonoplast membrane of mesophyll vacuoles

The effect of short-term heat stress on the tonoplast membrane of lamb's lettuce (Valerianella locusta (L.) Betcke) mesophyll vacuoles has been investigated. The maintainance of a proton concentration difference (δpH) across the tonoplast membrane served as a criterion for the integrity of the vacuoles. After heat treatment, δpH was measured at room temperature using the fluorescent amine, 9-aminoacridine. It was found with this method that thermal damage to isolated vacuoles mainly occurred in the temperature range above 50°C. Compared with this results, the photosynthetic functions of isolated lettuce protoplasts proved to be markedly more thermolabile, e.g. photosynthetic CO₂ fixation and light-induced chlorophyll fluorescence were drastically reduced at temperatures between 40° and 50°C. Heating of whole leaves and protoplasts and subsequent isolation of vacuoles showed that tonoplast-membrane integrity is not affected by heat stress in situ up to 45°C. Measurement of 9-aminoacridine fluorescence in protoplasts, which allowed conclusions to be drawn regarding the integrity of the tonoplast membrane in its natural cytoplasmic environment, revealed that heat treatment up to 55°C did not significantly affect vacuolar compartmentation. The data provide evidence that the tonoplast membrane is relatively heat stable compared with photosynthetic membranes.     

Organelle purification and selective permeabilisation of the plasma membrane: two different approaches to study vacuoles of the filamentous fungus Ashbya gossypii

Two different approaches to prepare and characterise vacuoles from the filamentous fungus Ashbya gossypii are described, i.e. the isolation of vacuoles from hyphal cells and the controlled permeabilisation of the plasma membrane. By mechanical lysis of protoplasts and separation of the organelles on a stepped density gradient, we obtained a vacuolar fraction virtually free of contamination by other organelles, unlysed protoplasts and cell debris. The integrity of the isolated organelles was characterised by vital-staining, the presence of α-mannosidase, and retained accumulation of basic amino acids. In a second approach, the cell membrane of the fungus was selectively permeabilised by use of the saponin digitonin leaving the vacuoles in their physiological surrounding, i.e. protected by the rigid cell wall. The permeabilisation was monitored by the latency of predominantly cytosolic amino acids and the ATP status of the cells. Functional intactness of the vacuoles within the permeabilised hyphae was demonstrated by maintenance of the pH gradient across the vacuolar membrane as detected by accumulation of the fluorescent dye, Acridine orange. These two methods are well-suited tools for the in situ assay of intracellular compartmentation of metabolites, for vacuolar transmembrane fluxes in Ashbya gossypii, as well as for the direct access to vacuolar membranes and enzymes of this fungus.     

Characterization of Intravacuolar Pigmented Structures in Anthocyanin-Containing Cells of Sweet Potato Suspension Cultures

Intravacuolar pigmented structures occurred in anthocyanin-producingcultured cells of sweet potato (Ipomoea batatas) were characterized.Formation of the pigmented structures in sweet potato cellswas induced by transfer of callus cultured in 2,4-D containingagar medium into 2,4-D free liquid medium under continuous illumination.These structures were found in the vacuoles. The pigmented structureswere isolated from the protoplasts by precipitation in 60% (w/w)sucrose after centrifugation. Electron microscopic observationsof the anthocyanin-containing cultured cells showed these structureshad neither membrane boundary nor internal structures, and werefound as strongly osmiophilic globules in vacuoles. Numeroussmall osmiophilic globules were observed in central vacuolesat the early stage of anthocyanin accumulation, but not foundin cytoplasm. Similar pigmented structures in vacuoles werealso formed by treatment with neutral red. These observationsindicate that these pigmented structure is the high densityand insoluble globules highly concentrated with anthocyanin,which was synthesized in cytoplasm and transported to the centralvacuoles. ⁴Present address: Department of Cell Biology, National Institutefor Basic Biology Myodaijicho, Okazaki, 444 Japan     

Transport and subcellular localization of polyamines in carrot protoplasts and vacuoles

Putrescine and spermidine uptake in carrot (Daucus carota L., cv “Tip top”) protoplasts and isolated vacuoles was studied. Protoplasts and vacuoles accumulated polyamines very quickly, with maximum absorption within 1 to 2 minutes. The insertion of a washing layer containing 100 millimolar unlabeled putrescine or spermidine did not change this pattern, but strongly reduced the uptake of putrescine and spermidine in protoplasts and in vacuoles. The dependence of spermidine uptake on the external concentration was linear up to the highest concentrations tested in protoplasts, while that in vacuoles showed saturation kinetics below 1 millimolar (K_m = 61.8 micromolar) and a linear component from 1 to 50 millimolar. Spermidine uptake in protoplasts increased linearly between pH 5.5 and 7.0, while there was a distinct optimum at pH 7.0 for vacuoles. Preincubation of protoplasts with 1 millimolar Ca²⁺ affected only surface binding but not transport into the cells. Nonpermeant polycations such as La³⁺ and polylysine inhibited spermidine uptake into protoplasts. Compartmentation studies showed that putrescine and spermidine were partly vacuolar in location and that exogenously applied spermidine could be recovered inside the cells. The characteristics of the protoplast and vacuolar uptake system induce us to put forward the hypothesis of a passive influx of polyamines through the plasmalemma and of the presence of a carrier-mediated transport system localized in the tonoplast.