The Rapid Isolation of Vacuoles from Leaves of Crassulacean Acid Metabolism Plants

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Abstract
A technique is presented for the isolation of vacuoles from Sedum telephium L. leaves. Leaf material is digested enzymically to produce protoplasts rapidly which are partially lysed by gentle osmotic shock and the inclusion of 5 mM ethyleneglycol-bis (.beta.-aminoethyl ether)N,N''-tetraacetic acid in the wash medium. Vacuoles are isolated from the partially lysed protoplasts by brief centrifugation on a 3-step Ficoll-400 gradient consisting of 5, 10 and 15% (wt/vol) Ficoll-400. A majority of the vacuoles accumulate at the 5 to 10% Ficoll interface, whereas a smaller proportion sediments at the 10-15% Ficoll-400 interface. The total time required for vacuole isolation is 2-2.5 h, beginning from leaf harvest. The yield of vacuoles is approximately 44%. The major vacuole layer is < 7% contaminated by marker enzymes from the cytoplasm and other organelles but shows no contamination by chloroplasts. Isolated vacuoles were stable for > 15 h when left in Ficoll; dispersion into media of various osmotic concentrations resulted in decreased stability. Addition of mercaptobenzothiazole, CaCl2, MgCl2, bovine serum albumin, ethylenediaminetetraacetic acid, polyethylene glycol 600 and KH2PO4 to the vacuole isolation media did not increase the stability of the isolated vacuoles. This technique with only slight modifications was used to isolate leaf cell vacuoles from the following Crassulacean acid metabolism plants: pineapple, Kalanchoe fedtschenkoi and Echeveria elegans. Spinach leaves also were used successfully.