Isolation and partial characterization of vacuoles from tobacco protoplasts

Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate-tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5′-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted.     

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.     

Effects of Ga3 and Ca2+ on barley aleurone protoplasts: a freeze-fracture study

Summary Freeze-fracture electron microscopy was used to study changes in the endomembrane system of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts. Protoplasts were used for this study because their response to calcium and the plant hormone gibberellic acid (Ga₃) can be monitored prior to rapid freezing of cells for electron microscopy. Protoplasts incubated in Ga₃ plus Ca²⁺ secrete elevated levels of a-amylase relative to cells incubated in Ga₃ or Ca²⁺ alone. The endoplasmic reticulum (ER) and Golgi apparatus of protoplasts incubated in Ga₃ plus Ca²⁺ undergo changes that are well correlated with the synthesis and secretion of a-amylase. The ER, which appears as short, single sheets of membrane in Ca²⁺-and Ga₃-treated protoplasts, exists as a series of long fenestrated stacks of membranes following incubation in Ga₃ plus Ca²⁺. The Golgi apparatus is also more highly developed in protoplasts treated with Ga₃ plus Ca²⁺. This organelle is larger and has more vesicles associated with its periphery in protoplasts that actively secrete a-amylase. Evidence that the Golgi apparatus participates in a-amylase secretion is also provided by experiments with the ionophore monensin, which causes pronounced swelling of Golgi cisternae and inhibits the secretion of a-amylase. We interpret these observations as showing that the ER and Golgi apparatus of barley aleurone participate in the intracellular transport and secretion of a-amylase. The plasmalemma (PF face) of barley aleurone protoplasts shows a high density of intramembranous particles (IMPs) which, in general, are evenly distributed. Occasionally, ordered arrays of IMPs are observed, possibly resulting fro m osmotic stress. after 48 hours the plasmalemma of some Ga₃-treated protoplasts show particle-free areas considered to be indications of senescence.abbreviations ER endoplasmic reticulum - Ga₃ gibberellic acid - IEF isoelectric focusing - IMP intramembranous particle - PF protoplasmic fracture - PL plasmalemma     

Plasmalemma Invaginations in Cultured Callus of Stevia rebaudiana: Ultrastructure and Ultracytochemical Localization of Acid Phosphatase

The plasmalemma of cells within meristematic regions was observed to possess invaginations in cultured callus of Stevia rebaudiana under differentiation. The ultrastructure and acid phosphatase (AcPase) ultracytochemistry Of these invaginations were studied. The plasmalemma invaginations occurred in the cells at various stages of vacuolation. In cells with dense protoplasm, plasmalemma appeared undulated but occasionally spherical and variable in size with conspicuous invaginations that projected into the peripheral cytoplasm. In the partially vacuolated cells, plasmalemma invagination became voluminously enlarged with increased contents and structurally complexed. In vacuolated cells, the enlarged invaginations protruded into the central vacuole but were delimitted from the tonoplast by an intermembrane zone continuous with the peripheral cytoplasm. Complex accumulations of membranes consisting of vesicular and coiled membranous Structures might develop within the plasmalemma invaginations. AcPase localization demonstrated high enzymic activity in the plasmalemma and its associated invagination. It seemed likely that these invaginations were functionally analogous to the vacuoles and therefore constituted part of the lytic compartment in these cells.     

甜菊愈伤组织中的质膜内陷:超微结构和酸性磷酸酶细胞化学定位

对在分化条件下的甜菊 (Stevia rebaudiana)愈伤组织分生区域细胞的质膜内陷进行了超微结构和酸性磷酸酶细胞化学研究。结果表明 ,在不同液泡化状态的细胞中均有质膜内陷存在。在原生质浓密的细胞中 ,质膜呈起伏的波纹状 ,某些部位发生明显内陷 ,大小不等 ,多呈圆球状。在部分液泡化细胞中 ,质膜内陷体积增大 ,内含物增多且结构复杂。在液泡化细胞中 ,质膜内陷嵌入中央液泡 ,但彼此间以一膜间隙隔开。质膜内陷中的内含物以小泡和卷绕的膜结构形式存在。酸性磷酸酶活性定位结果显示 ,质膜及其内陷含高的酶活性。推测质膜内陷在功能上与液泡相似 ,构成了这些细胞水解空间的一部分。     

烟草叶片液泡内氨基酸成份研究

液泡是植物细胞中特有的大型细胞器,具有重要的生理功能。本文报导了用双酶直接酶解法从烟草叶肉细胞中分离原生质体和完整液泡。在最适保存条件下,原生质体和液泡分别在３６和１２小时后,尚有一半保持活力。液泡内含有大量游离氨基酸,液泡膜ＡＴＰａｓｅ的最适ｐＨ为７．０,受Ｃｌ－激活,受ＮＯ３－抑制。     

PEG-enhanced,electric field-induced fusion of tonoplast and plasmalemma of grape protoplasts

Cultured grape cells accumulate anthocyanins in vacuoles rather than secreting them into the nutrient medium. Therefore, grape cells that contain tonoplast segments in their plasmalemma should be capable of excreting anthocyanins rather than sequestering them in their vacuoles. In initial attempts to construct such novel cells, small vacuoles were fused with the plasmalemma of cultured plant cells. Protoplasts were isolated from grape calluses that produce and accumulate anthocyanins. Small vacuoles were formed by gently rupturing vacuoles isolated from grape protoplasts. Although small vacuoles and protoplasts became aligned in an AC field, the tonoplast and plasmalemma did not readily fuse when subjected to 3 DC pulses of 1200 V cm^–1 for 50 s each. Changes in the intensity, number and/or duration of the DC pulses had no effect on the fusion process. When 1.0% polyethylene glycol was added to the electrofusion buffer, however, small vacuoles and protoplasts fused within a few minutes after the DC pulses were applied. These novel grape cells remained viable for several hours.Abbreviations BSA bovine serum albumin - 2,4-D 2,4-dichloro-phenoxyacetic acid - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-amino-ethyl ether)-N,N,N,N - MES 2-[N-Morpholino]ethanesulfonic acid - MOPS 3-[N-Morpholino]propanesulfonic acid - PVP polyvinylpyrrolidone     

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.     

Response to Phosphate Deficiency in Bean (Phaseolus vulgarisL.) Roots. Respiratory Metabolism, Sugar Localization and Changes in Ultrastructure of Bean Root Cells

Bean plants (Phaseolus vulgarisL. ‘Zlota Saxa’)were cultured on complete (+P) or phosphate-deficient (-P) nutrientmedium. A large increase in glucose concentration was foundin the meristematic zone of -P roots compared to control roots.The increased glucose concentration in the meristematic zonedid not influence total respiration rate. Glucose or uncoupler(carbonyl cyanide m-chlorophenylhydrazone) failed to increasethe respiration rate in -P root segments, but stimulated respirationin +P roots. The ultrastructure of cortical cells from the meristematicroot zone showed marked differences between +P and -P plants.Large vacuoles, invaginations of the plasmalemma and condensedforms of mitochondria were dominating features in cortical cellsof -P roots. Analysis of extracts after treating roots withdimethylsulfoxide (DMSO) indicated different localization ofsugars in the cell compartments. In roots of -P plants, mostof the reducing sugars were detected in the cytoplasm fractionwhile most sucrose was in the vacuole. Observations of the effectof 10% DMSO on cell ultrastructure indicated partial destructionof the plasmalemma but not the tonoplast. The localization ofreducing sugars in secondary vacuoles or plasmalemma invaginationsin the cells from the meristematic region of -P roots is discussed.Copyright1998 Annals of Botany Company. Bean (Phaseolus vulgarisL.), roots, Pi deficiency, respiration, meristematic zone, ultrastructure, sugar efflux, reducing sugars and sucrose localization.     

Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts,vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana

Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H⁺-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces.Abbveviations CAM Crassulacean acid metabolism - H⁺-ATPase proton-translocating ATPase     

Dynamic changes of protoplasmic volume and of fine structure during osmotic adaptation in the intertidal red alga Porphyra umbilicalis

Abstract Cells of Porphyra umbilicalis show a biphasic osmotic regulatory response. After transfer from 1 × into 3.5 × artificial seawater medium (osmotic upshock) the protoplasts shrink rapidly, then recover their original size within 3 h and continue to increase over the next 14 d. After retransfer from 3.5 × into 1 × medium (osmotic downshock) the protoplasts swell immediately and then adjust to the normal size in 1 x medium. Parallel to the shrinkage of the protoplasts after osmotic upshocks the plasmalemma at first gets a wavy surface which becomes smooth again during the following adaptation process. Immediately after osmotic upshock the vacuolar volume increases and it decreases drastically after osmotic downshock. After osmotic upshocks only small vacuoles are present at first. In adapted plants, however, the vacuolar system is mainly composed of large vacuoles. The volume of the protoplasm without the vacuoles is regulated osmotically. Parallel to the increase in the vacuolar volume after osmotic upshocks there is an increase in the number of intramembraneous particles on the PF-face of the tonoplast. This high value is reduced rapidly to the original number after osmotic downshock. The findings are discussed in relation to the function of the vacuoles as compartments of inorganic ion accumulation during osmotic adaptation.     

ULTRASTRUCTURE OF THE SHOOT APEX OF CHENOPODIUM ALBUM AND CERTAIN OTHER SEED PLANTS

The ultrastructure of cells of the vegetative shoot apices is described for Chenopodium album, Kalanchoë blossfeldiana and K. laxiflora, Bryophyllum daigremontianum, Nicotiana rustica, and N. tabacum (Maryland Mammoth), and Ginkgo biloba. A less intensive study was made of the last three listed. The structures and organelles usually associated with meristematic cells were observed: dictyosomes, plastids (in various stages of development), mitochondria, endoplasmic reticulum (ER), vacuoles, lipid droplets, and plasmalemma. In addition, spherosome-like structures were observed in all zones of the shoot apices. Also, multivesicular bodies were observed in C. album and B. daigremontianum. Ribosome density is greater in cells of the flank meristem. Proplastids, plastids with prolamellar bodies, or grana have a differential distribution in the apex, characteristic for a particular species. Confirmation could not be given to the concept that vacuoles arise as a series of local dilations in long extensions of the so called "smooth ER." The tonoplast and ER are distinguishable at the time of inception of a vacuole, although the tonoplast may arise from the ER. Rapid growth of a vacuole and/or fusion with other vacuoles may result in irregularly shaped prevacuoles. No vacuoles were observed to originate from cisternae of dictyosomes in the species studied.     

Lipid phase separations and intramembranous particle movements in the yeast tonoplast

The tonoplast of Saccharomyces cerevisiae contains regions depleted of intramembranous particles as the cells enter stationary phase. Freeze-fracture studies on intact cells from this growth stage show that a dispersed particle distribution predominates if the cell temperature is raised to 40°C but that particle-depleted areas prevail at or below the cell growth temperature of 30°C. Tonoplasts of isolated vacuoles also contain particle-depleted regions. Differential thermal analyses of lipids extracted from isolated vacuoles show an endothermic transition which encompasses the cell growth temperature. These results suggest that the tonoplast at this stage contains patches of gel-phase lipid and that these patches correspond to the intramembranous particle-depleted areas of the freeze-fractured tonoplast.     

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.     

Ultrastructural transitions associated with the development of the bladder cells of the trichomes of Atriplex

Early in development, bladder cells are characterized by the absence of a vacuole or vacuoles, the presence of autophagic vesicles, and numerous, unaggregated ribosomes. With the formation and expansion of the central vacuole, the ribosomes become aggregated and elements of rough endoplasmic reticulum become apparent. This developmental transition is probably related to the production of proteins involved in ion accumulation in the vacuole. Throughout expansion, invaginations of the tonoplast and membraneous structures are associated with the vacuole. These may be indicative of a continued lytic function for this compartment. Also, dictyosomes are continuously present and dictyosome vesicles are associated with both the plasmalemma and tonoplast, which suggest that they contribute to both membrane systems during expansion of the cell and vacuole.     

Characterization of vacuolar polypeptides of barley mesophyll cells by two-dimensional gel electrophoresis and by their affinity to lectins

Vacuoles were isolated from primary leaves of barley (Hordeum vulgare L.) by mechanical breakage of protoplasts, and their polypeptide composition analyzed by two-dimensional gel electrophoresis. Vacuoplasts which consist of the vacuole, a portion of the plasmalemma and of the cytoplasma were prepared from protoplasts by ultracentrifugation. By comparing the vacuolar polypeptide pattern with polypeptide patterns of isolated chloroplasts and of vacuoplasts, vacuolar polypeptides could clearly be distinguished from polypeptides derived from cross-contaminating cell compartments. At least 14 polypeptides of apparent molecular mass between 12 and 76 kilodaltons and an isoelectric point between 4.5 and 7.6 could be attributed to the tonoplast fraction of the vacuole, and 35 polypeptides to the soluble fraction of the vacuole. Several lectins with different specificity were employed to characterize the degree and nature of glycosylation of vacuolar polypeptides. Concanavalin A bound to a large number of polypeptides. Three out of the 14 tonoplast polypeptides exhibited detectable carbohydrate moieties and almost two-thirds of the surveyed soluble polypeptides were glycosylated.Abbreviations IEF isoelectric focussing - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Multistage disruption of protoplasts by dikegulac

Dikegulac (2,3:4,6 di-o-isopropylidine-2-keto-I-gulonate) is a growth regulator used to differentially kill terminal apices, and it analogously inhibits basic metabolic functions in dividing cells, but not stationary cells, in suspension culture. This report demonstrates an analogous situation in isolated tobacco protoplasts. At the lowest concentrations, dikegulac partially suppresses division of the protoplasts. Higher concentrations are required to produce visual cytoplasmic damage to the protoplasts, which probably first occurs at the level of the plasmalemma, as the vacuoles can be released intact. Later, tonoplast disruption occurs.Abbreviation FDA fluorescein diacetate     

The Ultrastructure of Ion-Secreting and Non-Secreting Salt Glands of Limonium platyphyllum

The ultrastructure of salt glands in developing leaves of Limoniumplatyphyllum is described prior to exposure to 3% NaCl solution(with non-secreting glands) and after 4.5 and 18 h exposureto the salt solution. It is shown that in most glands, the transitionto active chloride transport was accompanied by the displacementof vacuoles toward the cell periphery and by the establishmentof plasmalemma contact sites with the tonoplast which appearedsimilar to gap junctions in animal epithelial cells. No evidencefor the exocytosis of vacuoles was found. It is suggested thatgland vacuoles may have a primary role in chloride secretionand that the tonoplast may be functionally asymmetrical, sothat the free part facing the hyaloplasm bears ion pumps, whereashighly permeable ion channels are active along the zone of contactwith the plasmalemma. It follows that the active step in chloridetransport in Limonium glands is the influx of ions into thevacuoles. Within the inner cup cells of the gland, vacuolescome into contact with the plasmalemma only at sites where thecell wall is adjacent to secretory and accessory cells. Suchan asymmetry appears to ensure the directed flux of ions intothis cell wall. Wall protuberances in the gland cells are rudimentaryand presumably not involved directly in NaCl secretion. Thenucleolus is activated during secretion and the frequency offree ribosomes is significantly increased, which is suggestiveof their involvement in the synthesis of membrane transportproteins. The ultrastructure of about one-third of the glandsremained unchanged in salt-treated leaves. Key words: Salt glands, ultrastructure, ion fluxes     

Morphometric study of Lemna gibba in relation to the use of compartmental analysis and the flux-ratio equation in higher plant cells

Morphometric characteristics of Lemna gibba L. cells have been estimated. The mean relative volumes of the wall, of the cytoplasm and of the vacuoles were 0.03, 0.32 and 0.65, respectively. The distribution of the individual values has been studied: for instance 29% of the cells had a mean relative volume of the vacuoles close to 0.73, 39% to 0.68, 19% to 0.61 and 13% to 0.51. The mean value and distribution of the surface areas of the tonoplast and plasmalemma were also determined. This allows us to discuss the active or passive character of the transport of various substances at the plasmalemma and at the tonoplast, according to the usual flux-ratio approach. The feasibility of such an approach in ordinary (non-giant) living cells is discussed with regard to the degree of reliability of the measurements which can be performed with such cells.     

Cytochemical localization of peroxidase in soybean suspension culture cells and protoplasts: Intracellular vacuole differentiation and presence of peroxidase in coated vesicles and multivesicular bodies

Summary The ultrastructural localization of peroxidase in soybean (Glycine max L.) suspension culture cells and protoplasts is reported. In cells peroxidase is found primarily in the cell wall and at the tonoplast. Protoplasts and cells contain a vacuolar system which is differentiated with respect to peroxidase content since some vacuoles are found which do not contain peroxidase reaction product. The Golgi dictyosomes, coated and smooth vesicles contain peroxidase. Some of the multivesicular bodies have the reaction product as well. The results are discussed in terms of the pathways of sorting of peroxidase between the cell wall and vacuoles of cultured cells.