Physiological Studies with Isolated Leaf Cells

Abstract

MATERIALS AND METHODSPlant Materials. The plant materials used in this study were cultivated in the University botanical garden, Madurai. Fresh and fully developed green leaves were harvested and used immediately, or stored at 1 to 5 C for 1 to 2 hr before experimentation. Euglena gracilis Z, was grown autotrophically at 27 C in a modified Hutner medium (15). Chlorella vulgaris was grown in a 3-liter vessel containing 2 liters of inorganic medium (10). The cells were harvested after 6 to 9 days, washed twice with 0.01 M NaCl, and suspended in 0.05 M phosphate buffer pH 7.0.Isolation of Mesophyll Cells. Mesophyll cells were isolated and used following the procedure of Gnanam and Kulandaivelu (9). Chlorophyll content was determined by the method of Arnon (1).Absorption Spectrum. The absorption spectrum of each cell suspension was measured in a Beckman DK-2A ratio recording spectrophotometer, with opal glass placed between the slit and the sample tube for correcting the light scattering as described by Takebe et al. (19). Incorporation of Labeled Compounds by Mesophyli andAlgal Cells. "4CO2 fixation by mesophyll cells was carried out with modification of the method described by Bassham and Calvin (2). The reaction mixture (30 ml) contained 50 mm potassium phosphate buffer, pH 7.2, 35 mm NaCl, 5 mm MgCl,, 20,FCi NaH`4C0, (15 mCi/mmole), and S X 107 cells. Light was supplied by a 500 w projector-reflector lamp focused on the reaction vessel. The temperature of the reaction mixture was maintained at 20 + 1 C. The reaction mixture was stirred constantly by bubbling compressed sterile air. To obtain a steady state of CO2 fixation, cells were preilluminated for 10 min before the addition of labeled compounds.Amino acid incoporation by leaf cells was studied in a reaction mixture of 6 ml containing 50 mm tris-HCl buffer, pH 7.5, 20 mm NaCl, 10 mm MgCl2, 3 uCi of L-14C leucine (29.4 mCi/mmole), and 1.2 X 107 cells. Temperature of the reaction mixture was maintained at 25 C. Aliquots of 1 ml were pipetted at regular intervals into tubes containing 0.2 ml of 20% trichloroacetic acid and centrifuged at 3000g for 5 min. The pellet was washed twice with 5 mm 'C-leucine and suspended in 0.1 M tris-HCl buffer, pH 7.5. Cells were homogenized and the protein was precipitated by 5% trichloroacetic acid and redissolved in 0.01 N NaOH for radioactive counting.Chromatography and Radioautography. Two-dimensional paper chromatography and radioautography were carried out according to the method described by Bassham and Calvin (2). The individual compounds were identified on the paper by cochromatography with authentic compounds except for phosphoglycolate. The position of P-glycolate is marked tentatively by its relative position in the chromatograms developed using the same solvent systems by others (4

Photosynthetic Carbon Metabolism in the Palisade Parenchyma and Spongy Parenchyma of Vicia faba L.

“…Such cells incorporated about 33% of the radioactivity into the insoluble fraction in 2 min, while leaf discs only incorporated 8% into the insoluble fraction. However, cell suspensions prepared rapidly by mechanical isolation (11,16), or by only 1.5 hr of enzymatic digestion (17) did not display this difference. These data might be explained by the concept that when sucrose translocation is interrupted, a feedback mechanism may inhibit its synthesis.…”

Carbon Assimilation in Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) Grown in Still Nutrient Medium

“…For pulse-chase experiments, the cells were exposed to the labeling medium for 10 12C bicarbonate, and transferred in the light to 10 ml reaction mixture for chasing the radioactivity (16).…”