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Plant Physiology 66:379-382 (1980)
© 1980 American Society of Plant Biologists

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Articles

The Rapid Isolation of Vacuoles from Leaves of Crassulacean Acid Metabolism Plants 1

Randi Kringstad2, William H. Kenyon3 and Clanton C. Black, Jr.

Department of Biochemistry, University of Georgia, Athens, Georgia 30602, Department of Botany, University of Georgia, Athens, Georgia 30602

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 millimolar 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 three-step Ficoll-400 gradient consisting of 5, 10, and 15% (w/v) Ficoll-400. A majority of the vacuoles accumulate at the 5 to 10% Ficoll interface, whereas a smaller proportion sediments at the 10 to 15% Ficoll-400 interface. The total time required for vacuole isolation is 2 to 2.5 hours, 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 hours when left in Ficoll; however, 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 has been used to isolate leaf cell vacuoles from the following Crassulacean acid metabolism plants: pineapple, Kalanchoë fedtschenkoi, and Echeveria elegans. Spinach leaves also were used successfully.

2 Permanent address: Institute of Pharmacy, University of Oslo, Oslo 3, Norway. Recipient of travel grants from Norges Almenvitenskapelige Forskningsraad, Norsk Farmaceutisk Selskap and the University of Oslo.

3 Recipient of a Graduate Assistantship in Botany.

1 This work was supported by National Science Foundation Grant PCM 770 8548 and by the Mobil Foundation.




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W. Roos, R. Schulze, and J. Steighardt
Dynamic Compartmentation of Vacuolar Amino Acids in Penicillium cyclopium. CYTOSOLIC ADENYLATES ACT AS A CONTROL SIGNAL FOR EFFLUX INTO THE CYTOSOL
J. Biol. Chem., June 20, 1997; 272(25): 15849 - 15855.
[Abstract] [Full Text] [PDF]


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