Short Communication Carbon Dioxide Fixation in Isolated Kalanchoe Chloroplastst

Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 Amoles of CO per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of C02 fixation were primarily those of the photosynthetic carbon reduction cycle. Recently, with respect to photosynthesis, the plant kingdom has been divided into three major groupings, namely, those plants which assimilate CO2 by the photosynthetic carbon reduction cycle (C-3 plants); those which utilize primarily the C-4 dicarboxylic acid pathway (C-4 plants) and those which are characterized by the term Crassulacean acid metabolism. Chloroplasts have been isolated and their carbon metabolism studied in C-3 plants and C-4 plants (5). In this brief communication, we report on the photosynthetic carbon metabolism of chloroplasts isolated from Kalanchoe diagremontiana, a Crassulacean plant. MATERIALS AND METHODS Plant Material. Kalanchoe diagremontiana was cultured from plants which were obtained in the Brandeis University greenhouse. The Kalanchoe was propagated by initiating plantlet growth on vermiculite. Both plantlets and mature plants were grown in growth chambers under 12 hr of light (1400 ft-c) at 24 C to 27 C followed by 12 hr of dark at 18 to 20 C. Light was provided by cool white fluorescent lamps supplemented with tungsten lamps. The plantlets were watered with dilute Rapidgro (23% total nitrogen, 19% P, 17% K; Rapidgro Corporation, Dansville, N.Y.), 3 to 5 days before chloroplast isolation. Mature plants were maintained on half strength Hoagland's solution with occasional applications of dilute KNO0. Preparation of Chloroplasts. Chloroplasts were prepared by a modification of the method of Jensen and Bassham (8). 'This research was supported by the United States Atomic Energy Commission Grant AT 11-1(3231). 'Trainee of the National Institutes of Health, Grant GM1586-07. Present address: Department of Biochemistry and Biophysics, University of California, Davis, Calif. 95616. Chilled leaves (3-6 g) were cut into 40 ml of isolation medium (0.33 M sorbitol; 50 mm HEPES-KOH, pH 6.8; 2 mm EDTA; 1 mM NaP2O7; 1 mm MgCl2; and 1 mm MnCl2) and ground for 2 sec with a VirTis 45 homogenizer set at 45. The homogenate was filtered through Miracloth and centrifuged for 1.5 min at 1500g. The supernatant solution was discarded and the pellet was resuspended in 5 ml of isolation medium. The suspension was centrifuged for 45 sec at 1500g. The supernatant fluid was discarded and the pellet was suspended in incubation medium which was similar to the isolation medium except for the inclusion of 50 mM HEPES-KOH at pH 7.8. An aliquot was taken for Chl determination and was estimated by the method of Arnon (1). CO2 Fixation and Product Identification: Photosynthesis was measured as 'CO2 uptake into acid-stable products using 1 50-w flood lamps to provide light. Aliquots of 0.2 to 0.4 ml of the reaction mixture were removed at appropriate intervals with an automatic pipet and were combined with 25 ,ul of concentrated formic acid to stop the reaction. Of this mixture, either 50 or 100 Al were then placed on lens paper-covered planchets and dried, and radioactivity was determined with a gas flow counter. To identify the products of fixation, 100 Al were spotted on Whatman No. 1 paper and subjected to descending two-dimensional chromatography. The first solvent was water-saturated phenol and the second was a 1: 1 mixture of n-butyl alcohol and propionic acid, each saturated with water. Radioactive areas were located with Kodak nonscreen x-ray film. Radioactivity was counted with a Nuclear Chicago end window counter. Compounds were identified by elution and chromatography with authentic standards. RESULTS AND DISCUSSION Chloroplasts isolated from mature, succulent leaves or greenhouse-grown plantlets had low rates of photosynthetic CO2 fixation. Most of the chloroplasts appeared broken when viewed by phase and electron microscopy. They contained large starch granules and tended to clump. In preliminary experiments, chloroplasts were isolated from Kalanchoe plantlets grown under different light intensities in order to control the level of starch. As shown in Table I, the differences between preparations were not in total chloroplast recovery, as monitored by plastid Chl/g fresh weight of leaf, but in the rate of CO2 fixation of the isolated chloroplasts and in the relative proportions of starch in the homogenates and chloroplast suspensions. Chloroplasts which were isolated from plants grown in full light and then transferred to shade were repeatedly the most suitable for this study. For the remainder of the experiments reported here, chloroplasts were isolated from the youngest leaves of plantlets that were grown in full light and then transferred to shade. The