Tonoplast vesicles were isolated from Kalanchoe daigremontiana Hamet et Pierrer de la Bâthie and Mesembryanthemum crystallinum L., exhibiting constitutive and inducible crassulacean acid metabolism (CAM), respectively. Membrane-bound proteins were detergent-solubilized with 2% of Triton X-100. During CAM induction in M. crystallinum, ATPase activity increases four-fold, whereas pyrophosphatase activity decreases somewhat. With all plants, ATPase and pyrophosphatase could be separated by size-exclusion chromatography (SEC, Sephacryl S 400), and the ATPase was further purified by diethylaminoethyl-ion-exchange chromatography. Sodium-dodecyl-sulfate electrophoresis of the SEC fractions from K. daigremontiana containing maximum ATPase activity separates several protein bands, indicating subunits of 72, 56, 48, 42, 28, and 16 kDa. Purified ATPase from M. crystallinum in the C3 and CAM states shows a somewhat different protein pattern. With M. crystallinum, an increase in ATP-hydrolysis and changes in the subunit composition of the native enzyme indicate that the change from the C3 to the CAM state is accompanied by de-novo synthesis and by structural changes of the tonoplast ATPase.

Abstract. The tonoplast is usually characterized by the presence of two electrogenic proton pumps: a vacuolartype H+-ATPase and a pyrophosphatase, as well as a putative water-channel-forming protein (y-TIP). Using a post-embedding immunogold labelling technique, we have detected the presence of these transport-protein complexes not only in the tonoplast, but also in the plasma membrane and trans Golgi elements of maturing pea (Pisum sativum L.) cotyledons. These ultrastructural observations are supported by Western blotting with highly purified plasma-membrane fractions. In contrast to the vacuolar-type H+-ATPase, whose activity was not measurable, considerable pyrophosphatase activity was detected in the plasma-membrane fraction. These results are discussed in terms of a possible temporary repository for tonoplast proteins en route to the vacuole.

Tonoplast preparations were obtained from leaves of Hordeum vulgare (C3), Kalanchoe daigremontiana (obligate CAM) and Mesembryanthemum crystallinum (C, and inducible CAM). Lipid analyses showed reproducible patterns comprising free sterols, glycolipids of plastidic origin, glucose-containing lipids (steryl glucoside, acylated steryl glucoside, cerebroside) and phospholipids. Predominant components were sterols, cerebrosides, phosphatidyl choline and phosphatidyl ethanolamine. Very long chain fatty acids were found in phosphatidyl serine and hydroxy fatty acids in cerebrosides. Isolation of tonoplasts via protoplasts and vacuoles may have resulted in reduced levels of free sterols. The data show a similarity between tonoplasts and plasma membranes with respect to lipid profiles. Lipid composition was neither affected by different COzfixation mechanisms nor by salt-induced changes in Mesembryanthemum crystallinum.

The action of auxin in the promotion of growth has been suggested in the literature to depend on cell wall acidification. In a former investigation by the present authors the electrochemical balance in auxin-induced proton extrusion was shown to be maintained by potassium net uptake. The present paper reports data demonstrating that the elongation of Avena coleoptile segments is accompanied by an accumulation of malate, which is stoichiometrically correlated with potassium uptake. We concluded that this malate accumulation is required in a mechanism regulating intracellular pH.Auxin (IAA)-induced elongation of shoot cells is accompanied by a rapid release of protons from the cells (5, 21). External media of low pH (3-5) can mimic the action of auxin (3, 9, 25). These results have led to the suggestion that hydrogen ions play a role as second messengers in auxin action (9): acidification of the cell wall enhances its extensibility either by activating cell wall-loosening enzymes or by breaking some acid-labile links.Indoleacetic acid is also known to stimulate the uptake of K+ and Rb+ ions into different tissues (14-16). In a previous paper (10) we demonstrated that IAA-promoted proton efflux is electrochemically balanced by a stoichiometric influx of K+ ions. The presence of alkali, and, in particular, of K+ ions synergistically stimulates IAA-induced growth (11).A proton efflux of the observed magnitude must be associated with a mechanism regulating cytoplasmic pH. In many tissues a proton-cation exchange (or antiport), e.g., during excess cation uptake, is associated with synthesis and subsequent accumulation of malic acid by dark C02-fixation via P-enole-pyruvate carboxylases (12). A similar mechanism is reported to be involved in K+-H+-exchange regulating stomatal guard cell movement (1, 18). On the basis of these and other results, Davies (6) and Raven and Smith (19) proposed a common mechanism for stabilization of intracellular pH, which is based on synthesis and breakdown, respectively, of organic acids, especially of malate. In this respect it may appear to be pertinent that IAA-1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft. 2 The result of this paper has been communicated orally at the 1974 conference of the Deutsche Botanische Gesellschaft, Wurzburg, September 26, 1974. promoted growth is accompanied by consumption of CO2 (25) and that IAA enhances the fixation of H'4C03-by Avena coleoptile cells (4).The present paper demonstrates that IAA-induced elongation growth is paralleled by a stoichiometric potassium and malate accumulation. MATERIAL AND METHODSPlant Material. Seeds of Avena sativa (cv. "Flemings Krone") were dehusked and soaked for 3 hr in three changes of aerated 0.5 mM CaSO4. Subsequently, the seeds were placed on a steel screen floated on aerated 0.5 mm CaSO4. Germination, preparation of tissues, and experiments were carried out under dim green light at 28 + 1 C, except for a 3-hr exposure to red light at the beginning of the germination period.Exp...

Dimensions and area densities of membrane particles were studied by electron microscopy of replicas of freeze‐fractured suspensions of tonoplast vesicles of Mesembryanthemum crystallinum L. in the C‐3 state and after induction of crassulacean acid metabolism (CAM) by salinity. The results are compared with the relative contribution of tonoplast‐ATPase protein to total membrane protein obtained from integration of elution profiles in size‐exclusion chromatography. Coverage of tonoplast area by globular membrane particles was 20% and 36 % and ATPase in relation to total membrane protein was 33 % and 35 % in C‐3 and CAM M. crystallinum, respectively. Thus, by order of magnitude, it is most likely that the particles represent the ATPase. In Kalanchoë daigremontiana Hamet et Perrier de la Bâthie the ATPase also constituted 36% of total tonoplast protein. Induction of CAM in M. crystallinum was associated with an increase in specific ATPase activity of the tonoplast and an increase of the size and area coverage of tonoplast particles, whereas the relative contribution of ATPase protein to total tonoplast protein and the molecular mass of the ATPase holoenzyme, as seen in size‐exclusion chromatography, remained almost unchanged.