Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts

Abstract

Photoassimilation of 'CO2 by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praeakum was investigated. The main watersoluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction.Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. There have been only a few reports of CO2 assimilation by isolated chloroplasts from CAM plants. Levi and Gibbs (19) reported assimilation by mechanically isolated chloroplasts from Kalanchoe daigremontiana. Nishida and Sanada (21) Plant Material. Sedum praealtum D. C., supplied by Edwards and Spalding, was propagated with stem cuttings in 15-cm pots containing a vermiculite-soil (1:1) mixture. The plants were kept in a greenhouse, but natural light was supplemented with banks of fluorescent lights in the winter months. The plants were watered when dry, approximately every 2 to 3 days. They were fertilized with one-quarter strength Hoagland solution once a week. Leaf material was not removed from the plants until 2 months after the cuttings became established. Young, but fully expanded leaves were used for chloroplast isolation. Protoplast yields diminished significantly when the established plants were older than about 8 months.Protoplast Isolation. Protoplast isolation was modeled after the procedure developed by Spalding and Edwards (29), except several changes were made in the procedure which increased the yield. Isolation was begun near the end of the solar day. The leaves were thinly sliced and placed in cold 0.3 M sorbitol. The slices were washed once, very gently vacuum-infiltrated for a few seconds, and washed once again with 0.3 M sorbitol. The slices (40g) were then transferred to 65 ml cooled digestion medium on a bed of ice. The digestion medium contained 0.3 M sorbitol, 0.9% (w/v) cellulase, 0.45% Macerozyme, and 4% BSA at pH 5.6. The next morning, the digestion mixture was manually swirled, passed through a 210-,pm nylon net, and centrifuged for 10 to 15 min at 20g in a swinging bucket rotor. The pellets were resuspended in 40 ml 30%o (w/v)

Starch degradation in intact amyloplasts isolated from cauliflower floral buds (Brassica oleracea L.)

“…Since isolated cauliflower-bud amyloplasts contain sucrose and because ct-glucosidase also hydrolyzes sucrose unspecifically we also measured fructose accumulation after hydrolysis and an equivalent amount of glucose was substracted. The observation that isolated plastids contain sucrose had already been made by analyzing chloroplasts from Sedum praealtum (Piazza et al 1982) or spinach (Stitt and Heldt 1981a).…”

Transport Processes and Corresponding Changes in Metabolite Levels in Relation to Starch Synthesis in Barley (Hordeum vulgare L.) Etioplasts

“…In addition, there is evidence for a glucose transport system with low substrate affinity (25). Furthermore, a hexose phosphate transport system in the chloroplasts of Sedum (20) and Codium (24) has been described. Information about transport processes involved in starch synthesis in plastids of heterotrophic tissue is scarce due to the fact that the isolation of these starch-containing organelles is extremely difficult (6).…”

Influence of Adenosine Phosphates and Magnesium on Photosynthesis in Chloroplasts from Peas, Sedum, and Spinach

“…As discussed above, PGA-supported 02 evolution was not inhibited by the addition of ADP to Sedum chloroplasts. To gain an understanding of the site of ADP inhibition, 14CO2 assimilation was conducted for 20 min and the labeled products were separated by anion exchange chromatography (23). For comparison, experiments were also performed with MgADP and FCCP.…”

“…Chloroplast Isolation. Sedum chloroplasts were isolated from protoplasts, as previously described (23). Spinach and pea chloroplasts were isolated by mechanical homogenization of leaves and were purified on a Percoll cushion (20).…”