Isolation of Mesophyll Cells from Sedum telephium Leaves

Abstract

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 C02 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. Crassulacean acid metabolism plants generally are characterized by the accumulation of titratable acidity at night, followed by a loss of acidity during the day in their leaves, while the leaf stomata open principally at night, and starch accumulation and degradation occur principally in the day and night, respectively (2,5,16 rooted then transplanted in a medium of soil, sand, and vermiculite (1 :1:1 v/v/v) in a house with a light intensity between 4000 and 6000 ft-c. Day temperatures ranged from 21 to 25 C, and night temperatures ranged from 17 to 20 C. The plants were watered two times per week in the morning.CO2 Incorporation with Isolated Cells. In addition to substrates, cells, and cofactors. as specified in each experimental setup, the total mixture of 250 ,ul contained: 50 mm Trizma base adjusted with MES to pH 8.0, 2 mm EDTA, 1 mM MnCl2, 2 mm NaNO.,, 5 mm MgCl2. 5 mM K2HPO4, and 350 mM sorbitol. The vial was placed in a 30 C temperature-controlled water bath. Light intensity was 2500 ft-c at the reaction mixture surface. Two minutes were allowed for temperature adjustment, and then "4C-labeled NaH CO. was pipetted into the mixture at a final concentration of 5 mm to start the reaction. Each vial was shaken a few times during the course of the experiments to prevent cell precipitation. Samples of 50 p.1 were taken at specific times and put in a vial containing 50 ,ul of 20% (w/v) trichloroacetic acid. Scintillation liquid (11 ml) was added. and unincorporated "CO2 removed by N. flushing for I min before the samples were counted for appropriate times. Liquid scintillation solutions were prepared by adding 4 g of BBOT (2,5-tert-butyl-benzoxyzalythiophene) to 1 liter of 700 ml of toluene plus 300 ml of ethanol solution and kept at 10 C in the dark (8. 17). RESULTSA number of CAM plant leaves were surveyed to see if intact photosynthetic cells could be released by mechanical means using a mortar and pestle. The plants examined included: 97

An Evaluation of Selected Physical Characteristics and Metabolism of Enzymatically Separated Mesophyll Cells and Minor Veins of Tobacco

“…At present the full potential of isolated cells has not been realized because the procedures used have not yielded cells capable of metabolic activity comparable to the in vivo situation. Recent work by Gnanam and Kulandaivelu (1969), Edwards andBlack (1971), andRouhani, Vines, andBlack (1973) exemplifies the need for improved techniques. They used mechanical grinding procedures to obtain mesophyll cells from species employing different CO 2 fixation pathways C a , C 4 , and Crassulacean species).…”

Equilibration of Label in Malate during Dark Fixation of CO2 in Kalanchoë fedtschenkoi

“…For the in vitro experiments, isolated cell aggregates of K. fedtschenkoi were used since preparations of isolated cells (9) contain up to 40% of damaged cells, which in the presence of RuDP and CO2 will give rise to 3-PGA as shown by this author. The composition of dark 14CO2 fixation products of cell aggregates was not altered significantly by addition of various substrates; only the fixation rate was stimulated when PEP was supplied to the cells.…”

Physiological Studies with Isolated Leaf Cells

“…One of the methods generally adopted in recent years for obtaining large quantities of leaf cells is that of hydrolyzing enzymes specific to cell wall materials (11,18). The leaf tissue is macerated with these enzymes in a hypertonic medium.…”