Abstract
The formation of intermediary glucans, mature starch, and phytoglycogen was studied using leaves of Arabidopsis thaliana wild type and dbe mutant, which lacks plastidic isoamylase (Zeeman, S. C., Umemoto, T., Lue, W. L., Au-Yeung, P., Martin, C., Smith, A. M., and Chen, J. (1998) Plant Cell 10, 1699-1711). A new approach to the study of starch biosynthesis was developed based on "very short pulse" labeling of leaf starch through photosynthetic fixation of (14)CO(2). This allowed selective analysis of the structure of starch formed within a 30-s period. This time frame is shorter than the period required for the formation of a single crystalline amylopectin lamella and consequently permits a direct analysis of intermediary structures during granule formation. Analysis of chain length distribution showed that the most recently formed outer layer of the granules has a structure different from the mature starch. The outer layer is enriched in short chains that are 6-11 glucose residues long. Side chains with 6 glucose residues are the shortest abundant chains formed, and they are formed exclusively by transfer from donor chains of 12 glucose residues or longer. The labeling pattern shows that chain transfer resulting in branching is a rapid and efficient process, and the preferential labeling of shorter chains in the intermediary granule bound glucan is suggested to be a direct consequence of efficient branching. Although similar, the short chain intermediary structure is not identical to phytoglycogen, which is an even more highly branched molecule with very few longer chains (more than 40 glucose residues). Pulse and chase labeling profiles for the dbe mutant showed that the final structure is more highly branched than the intermediary structures, which implies that branching of phytoglycogen occurs over a longer time period than branching of starch.
Highlights
Starch is the major carbohydrate reserve in many plant storage organs, including specialized roots, stems, and endosperms in which starch may accumulate to high levels and be stored over long time periods
Plant Material—Wild type (WT)1 and a mutant line of Arabidopsis thaliana (L.) Heynh. ecotype Columbia were grown in peat soil in a controlled climate chamber with mercury halide lamps supplemented with light from incandescent lamps at a photosynthetic flux of 120 mol of photons sϪ1 mϪ2 in an 8-h photoperiod
Considerable progress has been made in the understanding of starch biosynthesis
Summary
Plant Material—Wild type (WT) and a mutant line of Arabidopsis thaliana (L.) Heynh. ecotype Columbia were grown in peat soil in a controlled climate chamber with mercury halide lamps supplemented with light from incandescent lamps at a photosynthetic flux of 120 mol of photons sϪ1 mϪ2 in an 8-h photoperiod. 3.7 MBq of 14CO2 was injected resulting in a specific activity of 0.16 GBq mmolϪ1 CO2 in the chamber During both labeling and pre-incubation, 200 mol sϪ1 mϪ2 light was provided by fluorescent light tubes. The extracted insoluble leaf material was transferred to a 1.5-ml screw-cap microtube with 300 l of 50 mM sodium acetate-HCl (pH 5.0), and the starch in the sample was gelatinized by heating it for 10 min at 90 °C. Outer chains were degraded by the addition of 2 l (4 units) of barley -amylase (Megazyme) and incubation for 1 h at 40 °C; radioactivity released to the solution was determined by counting the total radioactivity in five successive washes with 2 ml of buffer. The radioactivity released was determined in three successive washes with 2 ml of buffer, and total released activity was calculated from these data
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