Electrophoretic Analysis of Protoplast, Vacuole, and Tonoplast Vesicle Proteins in Crassulacean Acid Metabolism Plants

Abstract

ABSTRACrProtoplasts 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 onedimensional 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. lutoids and sugar uptake into vacuoles from beet root and pea mesophyll also have been shown (8,15). The association of ATPase and permeases with the tonoplast suggests that this membrane may have a more complex composition than previously thought. The polypeptide composition ofthe tonoplast has been examined in other plant species, e.g. beet root (16) and Hippeastrum petals (27), but not in CAM plants.The vacuoles of CAM plants are unique because they are directly involved in photosynthetic carbon assimilation (9-1 1). CAM leaf vacuoles participate in the nocturnal accumulation and diurnal release of large amounts of malic acid (10) and in pineapple hexoses and malate fluctuate reciprocally each day (9). During the night malic acid accumulates against a steep concentration gradient implying that malic acid is actively transported into vacuoles. Evidence is available for a malate permease on the tonoplast ofK. daigremontiana (4). The relationship between this permease, ATPase activity known to be present on tonoplast vesicles (2, 23), and malate transport, however, remains to be determined.Characterization of the protein composition of the vacuole and tonoplast is an initial step in understanding the processes by which the vacuole functions as an active metabolic compartment within the plant cell. This study was undertaken to characterize the proteins and polypeptides found in tonoplast vesicles and vacuoles from two CAM plants.The vacuole in higher plant cells is a multifunctional organelle which often may occupy more than 90% of the mature cell volume. It is involved in osmotic regulation of cell volume, storage of ions and metabolites, compartmentation of specific enzymes including hydrolases, sequestration ofsecondary m...

Characterization of Tonoplast Polypeptides Isolated from Corn Seedling Roots

“…By using two-dimensional gel electrophoresis, they have provided a detailed description of the polypeptide composition of the vacuolar membranes of barley mesophyll cells. One-dimensional gel electrophoretographs have also been presented for the polypeptides from Hordeum, Ananas, and Sedum (14,15). However, these gels have limited resolution.…”

Immunological Characterization of Two Dominant Tonoplast Polypeptides

“…On the other hand, the polypeptide composition of the tonoplast membrane has been resolved by one-and twodimensional gel electrophoresis (4,7,8). However, it is difficult to relate membrane protein structure to biochemical function.…”