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Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C₃ Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism ¹

Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706, Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706

Mesembryanthemum crystallinum, a halophilic, inducible Crassulacean acid metabolism (CAM) species, was grown at NaCl concentrations of 20 and 400 millimolar in the rooting medium. Plants from the low salinity treatment showed exclusively C₃-photosynthetic net CO₂ fixation, whereas plants exposed to the high salinity level exhibited net CO₂ dark fixation involving CAM. Mesophyll protoplasts, isolated from both tissues, were gently ruptured, and the intracellular localization of enzymes was studied following differential centrifugation and Percoll density gradient centrifugation of protoplast extracts. Both centrifugation techniques resulted in the separation of intact chloroplasts, with up to 90% yield, from other organelles and the nonparticulate fraction of cells. Enzymes were identified by determination of activity and by sodium dodecyl sulfate gel electrophoresis of enzyme protein.

Experiments established the extraorganellar (cytoplasmic) location of phosphoenolpyruvate carboxylase, enolase, phosphoglyceromutase, and NADP-malic enzyme; the mitochondrial location of NAD-malic enzyme; and the chloroplastic location of pyruvate, Pi dikinase. NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphohexose isomerase, and phosphoglycerate kinase were associated with both cytoplasm and chloroplasts. NADP-dependent malate dehydrogenase activity was found in both the chloroplastic and extrachloroplastic fractions; the activity in the chloroplast showed an optimum at pH 8.0 and was dependent upon preincubation of enzyme with dithiothreitol. The extrachloroplastic activity showed an optimum at pH 6.5 and was independent of pretreatment with dithiothreitol. Protoplast extracts of M. crystallinum performing CAM exhibited higher activities (expressed per mg chlorophyll per min) of phosphoenolpyruvate carboxylase, pyruvate, Pi dikinase, NADP-malic enzyme, NAD-malic enzyme, NADP-malate dehydrogenase, enolase, phosphoglyceromutase, NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and phosphohexose isomerase than protoplast extracts from M. crystallinum not exhibiting CAM. The increase in total activity of the latter three enzymes following exposure of plants to 400 millimolar NaCl and the development of CAM was due to specific increases in the levels of activity in the cytoplasm.

¹ Supported by the Science and Education Administration, United States Department of Agriculture, under Grant 59-2531-0-1-516-0 from the Competitive Research Grants Office; and by the College of Agriculture and Life Sciences, University of Wisconsin, Madison, WI.

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