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  • 1
    ISSN: 1432-2048
    Keywords: ATPase ; Crassulacean acid metabolism ; Mesembryanthemum ; Polypeptide induction ; Pyrophosphatase ; Salt stress ; Tonoplast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In plants of Mesembryanthemum crystallinum the activities of the two proton pumps on the tonoplast, i.e. the ATPase and the pyrophosphatase, and the gelelectrophoretic pattern of the total tonoplast proteins were analyzed during the transition of the metabolic state from C3 photosynthesis to Crassulacean acid metabolism (CAM). In one series, CAM was induced by watering the plants with NaCl. In another series, the change of the metabolic state to CAM was a consequence of the aging of the plants. No significant differences in the specific activities of ATP hydrolysis were found in plants performing C3 photosynthesis and CAM, respectively. However, with both series the protein content of tonoplast preparations and, in parallel, the total ATP hydrolytic activity of the tonoplast ATPase were higher after the change to CAM. In contrast, the specific activity of pyrophosphate hydrolysis was maximum in the preparations of young plants and diminished after the induction of CAM in both series. Therefore the tonoplast ATPase seems to be the main enzyme responsible for the energization of malate accumulation in CAM. The tonoplast pyrophosphatase is important in the early stages of plant growth and plays a minor role in CAM. With M. crystallinum the change from C3 photosynthesis to CAM is accompanied by de-novo synthesis of tonoplast proteins. Several polypeptides with relative molecular masses (Mrs) of 55, 41, and 36 kDa were clearly more pronounced in the gel-electrophoretic pattern of the total tonoplast protein after CAM induction. These changes were independent of the CAM-inducing salt treatment or aging. Moreover, two subunits of the tonoplast ATPase with Mrs of about 27 and 31 kDa showed particularly high intensities only in the CAM state. It is assumed that the subunit composition of the tonoplast ATPase differs in the two metabolic states and that the two subunits induced modify the regulation of the ATPase in CAM. In addition, the reaction of the plants to the NaCl treatment per se was the induction at the tonoplast of a polypeptide with an Mr of 24 kDa.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-2048
    Keywords: Circadian rhythm ; Crassulacean acid metabolism ; Kalanchoe ; Model simulations ; Phase setting ; Tonoplast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Leaves of Kalanchoë daigremontiana Hamet et Perr. at a photon flux density (PFD) above 220 μmol·m−2s−1 (400–700 nm) or at leaf temperatures above 27.0 °C showed a rapid loss of rhythmicity, and a more or less pronounced damping-out of the endogenous circadian rhythm of CO2 exchange under continuous illumination. This rhythm was reinitiated after reduction of the PFD by 90–120 μmol·m−2·s−1 or reduction of leaf temperature by 3.5–11.0 °C under otherwise unchanged external conditions. The reduction in the magnitude of the external control parameter of the Crassulacean acid metabolism (CAM) rhythm (i.e. PFD or leaf temperature) set the phase of the new rhythm. The maxima of CO2 uptake occurred about 5, 28, 51, 75 h after the reduction. Simulations with a CAM model under comparable conditions showed a similar behaviour. The influence of temperature on the endogenous CAM rhythm observed in K. daigremontiana in vivo could be simulated by incorporating into the model temperature-dependent switch modes for passive efflux of malate from the vacuole to the cytoplasm. Thus, the model indicates that tonoplast function plays an important role in regulation of the endogenous CAM rhythm in K. daigremontiana.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-2048
    Keywords: Crassulacean acid metabolism ; Carboxylate transporter (reconstitution) ; Citrate ; Kalanchoe ; Malate ; Tonoplast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract When native tonoplast vesicles of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie were energized by an artificial K+ gradient establishing only an inside-positive electrical membrane potential (ΔΨ), it was shown that ΔΨ was sufficient as the sole driving force and that a proton gradient (ΔpH) is not required for malate uptake. Following [14C]malate uptake, K m-malate of the malate transporter was estimated as 2.7–3.0 mM, a value that would allow malate synthesis via phosphoenolpyruvate carboxylase and malate accumulation in vivo in view of the feed-back inhibition of cytosolic phosphoenolpyruvate carboxylase by malate. The maximum reaction velocity (V max) was found to be between 30 and 85 nmol malate·min−1·mg protein −1 , a value that would explain nocturnal malate accumulation in K. daigremontiana even if the transporter were operating below substrate saturation. Citrate (50 mM at pH 7) inhibited transport by 78%. The malate-transport protein of the tonoplast of K. daigremontiana may be a carboxylate uniporter with strong affinities for malate and citrate. From total tonoplast proteins solubilized from native tonoplast vesicles the malate transporter was functionally reconstituted into phospholipid liposomes. The malate transporter was purified and separated from the tonoplast H+-ATPase by hydroxyapatite chromatography, but not from the tonoplast H+-pyrophosphatase. The partially purified malate-transport protein was functionally reconstituted into phospholipid liposomes. In these final proteoliposomes, 0.6% of the protein of the initial tonoplast-vesicle preparation used for solubilization of membrane proteins was recovered. Using the specific rates of malate transport as a reference, i.e. rates of transport related to protein in the preparations, enrichment of the malate transporter in the final proteoliposomes obtained with the reconstitution of the hydroxyapatite eluate was 44-fold compared to the initial native tonoplast vesicles and 2000-fold compared to the liposomes reconstituted from solubilized tonoplast proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the peptides from the final proteoliposomes, which were functional in malate transport, showed only a few polypeptide bands among which the malate transporter must be found.
    Type of Medium: Electronic Resource
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