Ultrastructural features associated with secretion in the salt glands of Frankenia grandifolia (Frankeniaceae) and Avicennia germinans (Avicenniaceae)

Using stereological procedures, a detailed analysis was made from thin section electron micrographs of secreting and nonsecreting salt glands of Frankenia grandifolia (Cham. and Schlecht) and Avicennia germinans (L.) Stem. In F. grandifolia secretory cells, vacuolar volume significantly decreased, while the volume of endoplasmic reticulum increased in secreting glands. Numerous minivacuoles were predominantly located along the periphery of secreting secretory cells, some in apparent fusion with the plasma membrane. No difference was found in mitochondrial volume in the secretory cells between secreting and nonsecreting glands. In A. germinans, there was a significant decrease in vacuolar volume in secreting secretory cells. The volume of the endoplasmic reticulum and mitochondria also increased in these cells. However, no evidence of mini-vacuolar fusion with the plasma membrane was observed. These results indicate that the physical process of secretion may differ between F. grandifolia and A. germinans; in both, however, the ultrastructural observations support the contention that specific structural parameters are correlated with the process of secretion.     

Epidermal Peels of Avicennia germinans (L.) Stearn: A Useful System to Study the Function of Salt Glands

Adaxial peels were made from fully expanded leaves of Avicenniagerminans (L). Stearn. The peels consisted of the epidermis,epidermal salt glands, and underlying hypodermal cells. Electronmicroscopic examinations showed that the structural integrity(except for the innermost hypodermal cells) of all cell typeswas not altered in making the peels. When the peels were floatedon salt solutions, the glands were shown to be functionallycompetent in secretion. Secretion also occurred when the peelswere floated on distilled water, presumably from salts storedin the hypodermis. Secretion occurred in the dark and sinceall cell types in the peels lacked chloroplasts, glandular functioncould not be directly coupled energetically to photosynthesis.However, secretion was shown to be temperature dependent andinhibited by azide and dinitrophenol, which indicates that theenergy cost underlying secretion is mitochondrial and most likelycoming from the mitochondria-enriched gland cells. Inhibitorsknown to affect membrane H⁺ ATPase activity and membrane transportalso inhibited secretion, indicating that membrane transportis probably the primary mechanism underlying secretion. Lanthanum,a membrane calcium antagonist, also inhibited secretion. Avicennia germinans (Avicenniaccac), salt glands, metabolic inhibitors, ultrastructure, secretion     

Identification of tonoplast and plasma membrane in membrane fractions from garden cress (Lepidium sativum L.) with and without filipin treatment

Membranes from roots of Lepidium sativum L. were investigated in situ and after fractionation by applying morphological and biochemical methods. After freeze-fracture combined with filipin labelling the tonoplast and the plasma membrane could be easily characterized by the frequency of intramembranous particles and the arrangement of filipin-induced lesions. On tonoplast vesicles, the filipin-induced lesions were arranged in clusters of different size whereas they were evenly distributed on plasma membrane vesicles. Enrichment of tonoplast and plasma membrane in different fractions was documented by filipin labelling, phosphotungstic acid staining and by the profiles of marker enzyme activities and ATP-dependent H⁺-transport. Additionally, the presence of rightside-out and inside-out vesicles of both tonoplast and plasma membrane could be demonstrated. It was found that filipin labelling used in combination with freeze-fracturing is suitable for quantitative determinations of the percentages of tonoplast and plasma membrane in membrane fractions, which have been found to be more than 40% for the tonoplast and about 40% for plasma membrane in the respective enriched fractions.Abbreviations EF extraplasmatic fracture face - FIL filipin induced lesion - IMP intramembranous particle - PF plasmatic fracture face - PTA phosphotungstic acid-chromic acid stain - UDPG uridine 5-diphosphate glucose A preliminary report was presented at the joint Annual Meeting of the Belgian and German Societies for Cell Biology, Bonn, March 1985Dedicated to Professor Augustin Betz on the occasion of his 66th birthday     

Proteomic analysis of plasma membrane and tonoplast from the leaves of mangrove plant Avicennia officinalis

In order to understand the salt tolerance and secretion in mangrove plant species, gel electrophoresis coupled with LC‐MS‐based proteomics was used to identify key transport proteins in the plasma membrane (PM) and tonoplast fractions of Avicennia officinalis leaves. PM and tonoplast proteins were purified using two‐aqueous‐phase partitioning and density gradient centrifugation, respectively. Forty of the 254 PM proteins and 31 of the 165 tonoplast proteins identified were predicted to have transmembrane domains. About 95% of the identified proteins could be classified based on their functions. The major classes of proteins were predicted to be involved in transport, metabolic processes, defense/stress response, and signal transduction, while a few of the proteins were predicted to be involved in other functions such as membrane trafficking. The main classes of transporter proteins identified included H⁺‐ATPases, ATP‐binding cassette transporters, and aquaporins, all of which could play a role in salt secretion. These data will serve as the baseline membrane proteomic dataset for Avicennia species. Further, this information can contribute to future studies on understanding the mechanism of salt tolerance in halophytes in addition to salt secretion in mangroves. All MS data have been deposited in the ProteomeXchange with identifier PXD000837 ( http://proteomecentral.proteomexchange.org/dataset/PXD000837 ).     

枣品种耐盐性与叶片生物膜结合态多胺水平和H~＋-ATP酶活性的关系

120mmol·L^-1NaCl胁迫30d,耐盐性强的‘金丝小枣’叶片细胞质膜、液泡膜共价结合态腐胺（Put）、亚精胺（Spd）、精胺（Spm）含量及多胺（PAs）总水平与对照无显著性差异,但耐盐性弱的‘冬枣’叶片质膜共价结合态Put、Spd、Spm含量和PAs总水平及液泡膜Spd含量均显著降低;‘金丝小枣’叶片类囊体膜共价结合态Put含量、PAs总水平较对照显著降低,‘冬枣’则是Put、Spd、Spm含量及PAs总水平均显著降低。盐胁迫下,‘金丝小枣’叶片细胞质膜、液泡膜、类囊体膜非共价结合态Put、Spd、Spm含量及PAs总水平下降,但其中仅类囊体膜Spd含量显著低于对照,而‘冬枣’的3种膜上非共价结合态的这些多胺及其总水平均显著低于对照。与对照相比,盐胁迫下耐盐性不同的2个枣品种,叶片细胞质膜、液泡膜和类囊体膜H＋-ATP酶活性均降低,但降低幅度因枣品种和生物膜种类不同而异,且H＋-ATP酶活性与相应膜结合态多胺水平存在极紧密的正相关关系。结果表明,膜结合态多胺参与枣品种耐盐性的表达,调节盐胁迫下枣叶细胞中溶质的跨膜运输。     

Density and size distributions of the intramembranous particles in the cytoplasmic membrane of human peripheral blood lymphocytes. II. Ultrastructural differences between T and B cells

Separated T and B lymphocytes from human peripheral blood were studied using the freeze-fracture technique. Quantitative analysis performed on density and size of intramembranous particles (IMPs) present on both fracture faces of the plasma membrane has revealed remarkable differences between cells belonging to the two main lymphocyte populations. In particular: (a) both fracture faces of the cytoplasmic membrane of B lymphocytes exhibit larger particles than T lymphocytes; (b) the mean densities, on both protoplasmic (PF) and external (EF) fracture faces, in B lymphocytes are lower than in T lymphocytes; (c) in B cells the partition ratio of particles between PF and EF is reversed with respect to T cells; (d) on both fracture faces of B lymphocytes, the IMP densities present a normal distribution while on T cells, density values show bimodal distributions indicating the existence of two cell subsets differing in particle density.     

Membrane alterations in winter rye and Brassica napus cells during lethal freezing and plasmolysis

Abstract Cells fixed during freezing or plasmolysis were used to study membrane alterations in hardened and non-hardened Brassica napus suspension-cultured cells and rye leaf mesophyll cells. The plasmalemma in non-hardened rye mesophyll cells formed multilamellar vesicles during lethal freezing at high subzero temperatures (–5°C). These vesicles became highly condensed at lower subzero temperatures (–10°C). Conversely, cold-hardened rye mesophyll cells did not undergo membrane alterations at these temperatures. The results from plasmolysis of B. napus and rye mesophyll cells hardened by ABA at 25 °C and low temperature (2°C), respectively, verify the cell response to lethal freezing. Again there was a continuum of responses with 1 kmol m^?3 balanced salt causing multilamellar protrusions. Appression of the plasmalemma against the tonoplast to form multilamellar vesicles and the invagination of these vesicles into the tonoplast were also observed in rye cells undergoing lethal plasmolysis. Increasing the plasmolysing solution to 3 kmol m^?3 occasionally caused the formation of multilamellar vesicles on the cell surface of hardened rye mesophyll cells.     

Reactions of yeast cells to glycerol treatment alterations to membrane structure and glycerol uptake

Summary Ultrastructural alterations to the plasmalemma and tonoplast ofSaccharomyces cerevisiae were studied after incubation in hypertonic solutions of glycerol and sorbitol. After 20 to 30 minutes incubation in glycerol, the cells had shrunk to about 40% of their original volume. Large depressions of the plasmalemma were then always found associated with the typical plasmalemma invaginations. The vacuoles of treated cells changed to an irregular form, the tonoplast intramembranous particles were clustered, and large smooth areas appeared. After 6 to 12 hours incubation, cell and vacuole volume, as well as plasmalemma and tonoplast ultrastructure, had reverted to normal. The rate of recovery was strongly temperature dependent.Protoplasts could be similarly shrunk, but no alterations to the plasmalemma ultrastructure were then observed; however, the tonoplast revealed particle clustering as observed in whole cells. Protoplasts also reverted to normal volume and ultrastructure after prolonged incubation. Cells and protoplasts treated with sorbitol showed similar phenomena, but remained shrunken.By the use of radioactive tracers, glycerol was shown to penetrate cells, protoplasts and isolated vacuoles, but no uptake of sorbitol could be demonstrated.During the glycerol permeation period (0.5 to 6 hours), numerous vesicles were found in the cytoplasm and these were possibly engulfed by the vacuole. Associated with the engulfment, patches of tonoplast intramembranous particles were found in a semicrystalline array. Osmotic stress induced alterations to membrane ultrastructure, due to the use of cryoprotective agents, are discussed.A preliminary note of the paper was given at the Sixth European Congress on Electron Microscopy, Jerusalem, 1976.     

The Permeability of the Guard Cell Plasma Membrane and Tonoplast

Uptake experiments and efflux compartmental analysis of planthormones, osmotica and toxins using ‘isolated’ guardcells of Valerianella locusta and guard cell protoplasts (GCP)of Vicia faba were performed in order to study the permeabilityproperties of guard cell plasma membrane and tonoplast. Theplasma membrane of guard cells exhibits a higher permeabilitythan plasma membranes of mesophyll cells for most solutes investigated.The permeability coefficients (P_s calculated for the guard cellplasma membranes are also significantly higher than the P_s valuesfor the guard cell tonoplast. This applies also for protonatedABA. We suppose that the high permeability for ABAH could bepart of the target cell properties. A Collander analysis demonstratesa linear correlation between P_s, values and the ratio K_r/M_r^1,5for both plasma membrane (r = 0.87) and for the tonoplast (r=0.93). Because of deviations from the observed correlations,the permeation of some solutes (ABA, GA, IAA through the tonoplast;methylamine through the plasma membrane) seems to be facilitatedby an additional transport mechanism. The Collander analysisof the plasma membrane of GCP shows very similar results tothe analysis of the plasma membrane of ‘isolated’guard cells, indicating that isolation of protoplasts does notalter the permeability of the guard cell plasma membrane. Key words: Permeability coefficient, guard cells, plasma membrane, tonoplast     

An adaptive feature of some mangroves of Sundarbans, West Bengal

Mangrove taxa, apart from their morphological characters, have some unique leaf anatomical features which are very much related to their adaptation as the plants grow in unstable, variable and saline environments with regular tidal influence. Special stomatal structures with extended cuticles render the transpiration rate in many taxa. The presence of glandular and non-glandular hairs on the abaxial and/or adaxial leaf surfaces in some taxa are related to salt secretion of these plants. Comparatively large amounts of water storage tissues occur in the hypodermal or mesophyll tissue of the leaves, reflecting the adaptive nature of mangroves in their stressful habitat. The occurrence of terminal tracheids helps with capillary water storage within the leaf. The coriaceous nature of the leaves in some taxa is due to the presence of sclereids within the mesophyll region. It is noted thatHeritiera is unsuitable to the highly saline habitat of the Sundarbans forest region because of some anatomical peculiarities.     

Effects of Salinity Stress on the Levels of Covalently and Noncovalently Conjugated Polyamines in Plasma Membrane and Tonoplast Isolated from Barley Seedlings

When the 7-d old barley ( Hordeum vulgare L. ) seedlings were treated with different concentrations of NaC1 for 3 d, the levels of the noncovalemly conjugated polyamines (PAs) in the plasma membrane and tonoplast vesicles and the covalently conjugated PAs in the membrane proteins were promoted by NaC1 of low concentrations and suppressed by NaC1 of high concentrations. Among the noncovalenfly conjugated PAs in the vesicles, spennidine (Spd) level was the most abundant, while putrescine (Put) content was predominant among the covalenfly conjugated PAs, accounted for 40%– 70%, 35% – 60%, respectively. In addition, the TLC (thin-layer chromatography) profiles of the benzoylated PAs presented an unknown polyamine with Rf = 0.92 (X0.92), which conjugated covalently and noncovalently in root tonoplast and its content changed as well as Spd with NaC1 treatment. The total PA contents in the roots were higher than that in the leaves, and the types and contents of covalenfly and noncovalently conjugated PAs in the tonoplast were higher than those in the plasma membrane. The results showed that the above two PAs associated with the membrane might be essential in salt adaption of cells and the maintenance of membrane function.     

Isolation of plasma membrane and tonoplast fractions from spinach leaves by preparative free-flow electrophoresis and effect of photoinduction

A membrane fraction enriched in plasma membrane and tonoplast vesicles was isolated from green leaves of Spinacia oleracea L. and subjected to subfractionation by free-flow electrophoresis. The most electronegative membrane vesicle fraction collected after the free-flow electrophoretic separation was identified as derived from tonoplast, while the least electronegative fraction was identified as derived from plasma membrane. The identification of the fractions was based on membrane morphology, and on the presence or absence of biochemical markers. The plasma membrane fraction was enriched in thick (9–11 nm) membranes which bound N-1-naphthylphthalamic acid (NPA), and reacted with phosphotungstic acid at low pH on thin sections for electron microscopy. The tonoplast fraction was enriched in vesicles with 7–9 nm thick membranes that neither bound NPA nor reacted with phosphotungstic acid at low pH. Both the plasma membrane and the tonoplast fraction were about 90% pure, with a cross-contamination of not more than 2%. Membrane vesicles originating from dictyosomes, endoplasmic reticulum, mitochondria, plastids, or peroxisomes contaminated the plasma membrane and the tonoplast fractions by a few % only. In leaves of photoinduced plants (24 h light period), the plasma membranes were thicker than in control leaves (8 h light, 16 h dark). The plasma membrane fraction obtained from photo-induced leaves by free-flow electrophoresis retained this increase in thickness, showing not only that photoinduction alters plasma membrane structure, but also that this change is stable to isolation.     

The Synapse-Like Interaction between Chloroplast, dictyosome, and Other Cell Compartments during Increased Ethylene Production in Leaves of Rye (Secale cereale L.)

Rye (Secale cereale L.) plants were treated with an ethylene releaser ethephon (2-chloroethylphosphonic acid) in concentration of 4×10⁻² M. We studied electron microscopically, if and how chloroplasts interact with well-documented sites of ethylene production/binding, i.e., with endoplasmic reticulum, dictyosomes, mitochondria, plasma membrane, and tonoplast. During the sharp increase of ethylene synthesis in mesophyll cells of rye leaves, the direct local continguity of chloroplast envelope or envelope protrusions with the above mentioned cell compartments was typical. Moreover, a large number and diversity of versatile chloroplast-dictyosome associations were conspicuous, in which both the chloroplast and each cisterna of dictyosome were capable to exo/endocytosis. The dictyosomes were directed towards the chloroplasts, plasma membrane, or tonoplast both with cis-face, trans-face, or with the rim, they could change their direction or shut up the trans-face, developing simultaneously several flexible chains of vesicular dispatches among chloroplasts and some other cell compartments. This reflects interaction of protein/ethylene producing, photosynthesising, DNA containing compartments, and regulated action of lysosomal system. Structural contacts and vesicular transport among compartments of symplastic system equalises concentrations of H⁺, Ca²⁺, etc. ions, as well as provide connection with an apoplast. We propose that ethylene functions in plant mesophyll cells are both as intra/intercellular signalling substance and as phytohormone that regulates gene expression in nuclei, chloroplasts, and mitochondria in a complicated synapse-like process and causes programmed death of leaves of the main stalks of rye for the sake of promoted growth of side shoots. This revised version was published online in August 2006 with corrections to the Cover Date.     

盐胁迫对大麦幼苗质膜,液泡膜上共价和非共价结合多胺含量的影响

用不同浓度ＮａＣｌ处理７ｄ龄大麦（Ｈｏｒｄｅｕｍ ｖｕｌｇａｒｅＬ．）幼苗３ｄ。以非共价键和共价键形式分别与质膜和液泡膜微囊及膜蛋白结合的多胺含量受低 度盐的促进而被高浓度盐所抑制。以非共价键形式与膜微囊结合的各种多胺中亚精胺（Ｓｐｄ）含量最高,占膜上多胺总量的４０％－７０％,与膜蛋白共价结合的各种多胺中腐胺（Ｐｕｔ）含量占主导地位,占膜蛋白上多胺总量的３５％－６０％。在根系液泡膜上发现一种含量丰     

Development of mucilage cells of Araucaria angustifolia (Araucariaceae)

The roles of mucilage cells were investigated through morphological and cytological analysis during leaf development in young Araucaria angustifolia plants. Differentiation began in leaf primordia in the shoot apex, when the young cells underwent a greater increase in volume in comparison with other mesophyll cells. The mucilage polysaccharides were synthesized by dictyosomes, from where they were taken by large vesicles and released into a cavity formed by detachment of the tonoplast, which was separated from the cytoplasm. At the end of differentiation, the cell was completely filled with mucilage, a gel consisting of a denser reticular structure surrounding less dense regions. The nucleus and cytoplasm were degenerated in mature cells. The A. angustifolia mucilage cells presented some cytological resemblances to the mucilage cells of members of some dicotyledonous families; however, differences in the dictyosomes and the secretion route were observed. Translocation and water storage of solutes was suggested by the use of the hydroxy pyrenetrisulfonic acid tri-sodium salt apoplastic tracer. The tonoplast detachment, dechromatinization, nuclear condensation, and general degeneration of the membrane systems observed during maturity indicated a programmed cell death process, one not yet described for angiosperm mucilage cells.     

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.     

Milk fat globule membranes devoid of intramembranous particles

When isolated milk fat globule membranes from bovine, human, and murine (rat) milk were examined by freeze-fracturing most of the membrane faces were devoid of membrane-intercalated particles whereas a minor portion showed relatively few particles, either in clusters or in apparent random distribution. A reduced particle density was also noted in membranes of intra-alveolar milk fat globules of cows and rats, in contrast to high particle densities in the apical plasma membrane of lactating epithelial cells. The observations suggest that certain membrane constituents recognized as intramembranous particles either are displaced from the region of the apical surface of the mammary epithelial cell which is involved in milk fat globule budding or are dislocated and rearranged during the budding process.     

Isolation of mesophyll and secretory cell protoplasts of the halophyte Ceratostigma plumbaginoides (L.): a comparison of ATPase concentration and activity

Summary A method is described for isolating mesophyll protoplasts from leaves and secretory cell protoplasts from salt glands of the facultative halophyte, Ceratostigma plumbaginoides (L.). Rates of ATP hydrolysis in both cell types were determined, and levels in secretory cell protoplast preparations were fourfold higher than those in mesophyll protoplast preparations, based on total protein. The rate of ATP hydrolysis was sensitive to azide and vanadate, and stimulated by Triton-X-100. Additionally, immunoblot procedures using an antibody to the plasma membrane H⁺/ATPase was used to compare ATPase levels of the mesophyll and secretory cell protoplasts. Results indicate that secretory cells have a higher concentration of H⁺/ATPase than mesophyll cells, consistent with their putative function in salt glands.Abbreviations ATP adenosine triphosphate - BSA bovine serum albumin - DIDS diisothiocyano-2,2'-disulfonic acid stilbene - DNP dinitrophenol - DTT dithiothreitol - FITC fluorescein isothiocyanate - NAD⁺/NADH nicotinamide adenine dinucleotide - SDS sodium dodecylsulfate     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

In situ localization and tissue distribution of the replication-associated proteins of Cucumber mosaic virus in tobacco and cucumber

The replication-associated proteins encoded by Cucumber mosaic virus (CMV), the 1a and 2a proteins, were detected by immunogold labeling in two host species of this virus, tobacco (Nicotiana tabacum) and cucumber (Cucumis sativus). In both hosts, the 1a and 2a proteins colocalized predominantly to the vacuolar membranes, the tonoplast. While plus-strand CMV RNAs were found distributed throughout the cytoplasm by in situ hybridization, minus-strand CMV RNAs were barely detectable but were found associated with the tonoplast. In both cucumber and tobacco, 2a protein was detected at higher densities than 1a protein. The 1a and 2a proteins also showed quantitative differences with regard to tissue distributions in tobacco and cucumber. About three times as much 2a protein was detected in CMV-infected cucumber tissues as in CMV-infected tobacco tissues. In tobacco, high densities of these proteins were observed only in vascular bundle cells of minor veins. In contrast, in cucumber, high densities of 1a and 2a proteins were observed in mesophyll cells, followed by epidermis cells, with only low levels being observed in vascular bundle cells. Differences were also observed in the distributions of 2a protein and capsid protein in vascular bundle cells of the two host species. These observations may represent differences in the relative rates of tissue infection in different hosts or differences in the extent of virus replication in vascular tissues of different hosts.