Tonoplasts

The smaU nucleus of the yeast ceU is surrounded by a membrane or tonoplast, which has many pores with an average diameter of about 0.085 p.m. 

It is a very large vesicle enclosed by a single membrane called the tonoplast. Vacuoles tend to be smaller in young cells, but in mature cells, they may occupy more than 50% of the cell s volume. Vacuoles occupy the center of the cell, with the cytoplasm being located peripherally around it. They resemble the lysosomes of animal cells. 

Rona, J.P., Cornel, D., Gignon, C. Heller, R. (1982). The electrical potential difference across the tonoplast of Acer pseudoplantus cells. Physiologic Vegetale, 20, 459-63. 

Blumwald, E. Poole, R.J. (1987). Salt tolerance in suspension cultures of sugar beet. Induction of Na /H" antiport activity at the tonoplasts by growth and salt. Plant Physiology, 83, 884-7. 

Wagner, G.J. (1983). Higher plant vacuoles and tonoplasts. In Isolation of Membrane and Organelles from Plant Cells, ed. J.L. Hall and A.L. Moore, pp. 83-118. London Academic Press. 

R. Pinton, Z. Varanini, G. Vizzotto, and A. Maggioni, Soil humic molecules affect transport properties of tonoplast vesicles i.solated from oat roots. Plant Soil 142 203 (1992). 

Although several allelochemicals (primarily phenolic acids and flavonoids) have been shown to inhibit mineral absorption, only the phenolic acids have been studied at the physiological and biochemical levels to attempt to determine if mineral transport across cellular membranes can be affected directly rather than indirectly. Similar and even more definitive experiments need to be conducted with other allelochemicals that are suspected of inhibiting mineral absorption. Membrane vesicles isolated from plant cells are now being used to elucidate the mechanism of mineral transport across the plasma membrane and tonoplast (67, 68). Such vesicle systems actively transport mineral ions and thus can serve as simplified systems to directly test the ability of allelochemicals to inhibit mineral absorption by plant cells. 

Vacuoles (70-78) are membrane-bound regions of the cell filled with cell sap. Vacuoles are surrounded by a tonoplast (vacuolar membranes) and are diverse with distinct functions. Most investigators believe that lysosomes and the plant vacuoles are the same. Vacuoles develop turgor pressure and maintain tissue rigidity. They are storage components for various metabolites such as reserve proteins in seeds and malic acid in crassulacean acid metabolism (CAM) plants. Vacuoles canremove toxic secondary products and are the sites of pigment deposition. 

Most organelle membranes, such as the tonoplast (6) and the Golgi apparatus (7), can be separated by density gradient ultracentrifugation of plant cell homogenates. However, other effective methods for the isolation of the plasma membrane (8,9) have been described. Moreover, another method that uses an aqueous two-phase system for the isolation of ER is also described (10). Those interested in these details for these methods should consult the original articles. 

Collect the supernatant and then ultracentrifuge at 200,000g for 20 min to precipitate the crude microsomal fraction that contains the ER, Golgi complex, tonoplast, and plasma membrane. 

The marker enzymes used in this experiment are as follows vanadate-sensitive H+-ATPase (plasma membrane), nitrate-sensitive H+-ATPase or pyrophosphatase (tonoplast), TritonX-100 stimulated-UDPase or IDPase (Golgi complex), antimycin A-insensitive NADPH cytochrome c reductase (ER), and cytochrome c oxidase (mitochondria inner membrane). NADH cytochrome c reductase activity is found to be 10 times higher than NADPH cytochrome c reductase activity. Chlorophyll content can be measured as the chloroplast marker. The chlorophyll content is calculated by the following equation. Before measurement, auto zero is performed at 750 ran. 

When nitrate-sensitive H+-ATPase (tonoplast) activity is measured, 250 mM (final concentration, 200 mM) KN03 is added to the stock solution of the substrate mixture. When vanadate-sensitive H+-ATPase (plasma membrane) activity is measured, 125 pM Na3V04 (final concentration, 100 pM) is added to the stock solution of the substrate mixture. 

Fig. 3. Distribution ofvarious membrane markers (ER, tonoplast, Golgi complex, and mitochondrion) in the fractions of linear sucrose density gradient fractionation of mulberry cortical parenchyma cells in February.

Fig. 4. Localization of WAP27 and WAP20 in the crude microsome fractions and the relation with marker-enzyme activities in three organelles (ER, tonoplast, and Golgi). SDS-PAGE of fractionated proteins by isopycnic linear sucrose density gradient centrifugation of microsome fraction of mulberry cortical parenchyma cells was performed using 6-pL samples in each fraction. Immunoblot analysis was performed with anti-WAP27 and anti-WAP20 antibodies. (From ref. [1], with permission from the American Society of Plant Physiologists.)...

Yoshida S. Isolation of smooth endoplasmic reticulum and tonoplast from etiolated mung bean hypocotyls. in Methods in Enzymology, Vol. 228 (Walter H, Johansson G, ed.), Academic Press, New York, 1994, pp. 482-489. 

Abbreviations. a-M, a-mannosidase AP, acid phosphatase as-ni-ATPase, anion-stimulated, nitrate-inhibitable ATPase CCR, NAD(P)H-dependent cytochrome oreduc-tase cs-vi-ATPase, cation-stimulated, vanadate-inhibitable ATPase, CAT, catalase GS 1/11, glucan synthase 1 or 11 IDPase, inosine diphosphatase cs-PPase, cation-stimulated pyrophosphatase RNA polymerase, DNA-dependent RNA polymerase TP-25, 25 kDa tonoplast integral protein. 

Tannic acid Tannic acid can be added at 1-4% w/v in either cacodylate-buffered glutaraldehyde or glutaraldehyde-formaldehyde or between prefixation and postfixation (immerse specimens 1-2 h in tannic acid in 0.1M buffer) Tonoplast... 

Figure 6.10 The main transport processes across the plasma membrane and tonoplast of plant cells (adapted from Taiz and Zeiger, 2002). Reprodnced by permission of Sinauer Associates...

A particular ion or uncharged molecule can be transported by different transporters depending on its concentration. For example NH4+ may be absorbed by a passive low-affinity uptake system when its external concentration is large and by an active high-affinity system when its external concentration is small. Figure 6.10 summarizes the main transport processes on the plasma membrane and tonoplast of plant cells. 

All of the previous studies with cell suspension cultures of C. roseus have led to the conclusion that not all of the cells in suspension produce alkaloids, i.e., that some differentiation occurs. Neumann and co-workers at Halle 149) used fluorescence and electron microscopy to show that, like the intact plants, indole alkaloid accumulation occurs in the vacuoles of particular cells. Yet there appears to be no ultrastructural difference between these cells and those which do not produce alkaloids. It had been suggested earlier that basic alkaloids were accumulated by some kind of ion trap mechanism in acidic vacuoles (750). Indeed, a substantial pH difference was observed between those vacuoles which do accumulate alkaloids (pH 3) and those which do not (pH 5). It was concluded that the tonoplast of the alkaloid cells seemed to be highly permeable to the neutral form of the alkaloids, but only slightly permeable to the protonated forms. Cell lines which did not exhibit a difference in their vacuolar pH did not accumulate alkaloids. 

Ultrastructural examination of duckweed frond (Fig. 5) and root tissues treated with 18 (100 xM) revealed membrane damage to the tonoplast after 12 hours of exposure. The samples viewed through the transmission electron microscope showed ruptured tonoplasts, free-floating organelles and loss of cytoplasm relative to control tissues. The tonoplast may be the primary target for the phytotoxic effect of 18, which represents an unusual, if not unique, toxic mechanism among phytotoxic agents. 

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