Isolation of Mesophyll Cells from Sedum telephium Leaves

A technique is described for mechanically isolating metabolically active individual spongy mesophyll cells from the Crassulacean acid metabolism plant, Sedum telephium. Mature leaves are selected at about 2 PM when acidity is low, and three different media are used to reduce the problem of leaf acidity and to maintain isotonic conditions. The upper and lower epidermis is peeled from chilled leaves and the leaves are suspended in a buffered "soaking medium," then gently ground with a mortar and pestle. Cells and debris are separated using a "washing medium," with cells being filtered through a 136 micron net and collected on an 80 micron net. Cells then are suspended in a "cell suspension medium" and concentrated by centrifugation. Approximately 2 hours are required for the isolation procedure, and activity in CO(2) fixation is constant for up to 4 hours after isolation. Microscopic examination shows about 65% of the isolated cells appear intact and unplasmolyzed and are similar to leaf msophyll cells. The yield of cells on a leaf chlorophyll basis is about 1%. A light micrograph of the isolated cells is given.The isolated cells upon addition of phosphoenolpyruvate, 2-phosphoglycerate, and ribulose-1, 5-diphosphate fix CO(2) as rapidly as intact leaves; however, without exogenous substrate the cells only fix CO(2) between 10 and 20% of intact leaves. The temperature and pH optima for cellular CO(2) fixation in the presence of phosphoenolpyruvate is 35 to 45 C and 7.5 to 9.0, respectively. The light and dark portions of CO(2) fixation with the isolated cells are considered in relation to a scheme for net CO(2) fixation by Crassulacean acid metabolism plants.