Starchless mutants of Chlamydomonas reinhardtii lack the small subunit of a heterotetrameric ADP-glucose pyrophosphorylase.

ADP-glucose synthesis through ADP-glucose pyrophosphorylase defines the major rate-controlling step of storage polysaccharide synthesis in both bacteria and plants. We have isolated mutant strains defective in the STA6 locus of the monocellular green alga Chlamydomonas reinhardtii that fail to accumulate starch and lack ADP-glucose pyrophosphorylase activity. We show that this locus encodes a 514-amino-acid polypeptide corresponding to a mature 50-kDa protein with homology to vascular plant ADP-glucose pyrophosphorylase small-subunit sequences. This gene segregates independently from the previously characterized STA1 locus that encodes the large 53-kDa subunit of the same heterotetramer enzyme. Because STA1 locus mutants have retained an AGPase but exhibit lower sensitivity to 3-phosphoglyceric acid activation, we suggest that the small and large subunits of the enzyme define, respectively, the catalytic and regulatory subunits of AGPase in unicellular green algae. We provide preliminary evidence that both the small-subunit mRNA abundance and enzyme activity, and therefore also starch metabolism, may be controlled by the circadian clock.

The debranching enzyme complex missing in glycogen accumulating mutants of Chlamydomonas reinhardtii displays an isoamylase-type specificity.

To investigate the functions of debranching enzymes in starch biosynthesis, we have partially purified and characterized these activities from wild type and mutant sta7 Chlamydomonas reinhardtii. Mutants of the STA7 locus substitute synthesis of insoluble granular starch by that of small amounts of glycogen-like material. The mutants were previously shown to lack an 88 kDa debranching enzyme. Two distinct debranching activities were detected in wild-type strains. The 88 kDa debranching enzyme subunit missing in glycogen-producing mutants (CIS1) is shown to be part of a multimeric enzyme complex. A monomeric 95 kDa debranching enzyme (CLD1) cleaved alpha-1,6 linkages separated by as few as three glucose residues while the multimeric complex was unable to do so. Both enzymes were able to debranch amylopectin while the alpha-1,6 linkages of glycogen were completely debranched by the multimeric complex only. Therefore CLD1 and the multimeric debranching enzyme display respectively the limit-dextrinase (pullulanase) and isoamylase-type specificities. Various mutations in the STA7 locus caused the loss of both CIS1 and of the multimeric isoamylase complex. In contrast to rice and maize mutants that accumulate phytoglycogen owing to mutation of an isoamylase-type DBE, isoamylase depletion in Chlamydomonas did not result in any qualitative or quantitative difference in pullulanase activity.

Genetic and biochemical evidence for the involvement of alpha-1,4 glucanotransferases in amylopectin synthesis

We describe a novel mutation in the Chlamydomonas reinhardtii STA11 gene, which results in significantly reduced granular starch deposition and major modifications in amylopectin structure and granule shape. This defect simultaneously leads to the accumulation of linear malto-oligosaccharides. The sta11-1 mutation causes the absence of an alpha-1,4 glucanotransferase known as disproportionating enzyme (D-enzyme). D-enzyme activity was found to be correlated with the amount of wild-type allele doses in gene dosage experiments. All other enzymes involved in starch biosynthesis, including ADP-glucose pyrophosphorylase, debranching enzymes, soluble and granule-bound starch synthases, branching enzymes, phosphorylases, alpha-glucosidases (maltases), and amylases, were unaffected by the mutation. These data indicate that the D-enzyme is required for normal starch granule biogenesis in the monocellular alga C. reinhardtii.

Preamylopectin Processing: A Mandatory Step for Starch Biosynthesis in Plants.

It has been generally assumed that the [alpha]-(1->4)-linked and [alpha]-(1->6)-branched glucans of starch are generated by the coordinated action of elongation (starch synthases) and branching enzymes. We have identified a novel Chlamydomonas locus (STA7) that when defective leads to a wipeout of starch and its replacement by a small amount of glycogen-like material. Our efforts to understand the enzymological basis of this phenotype have led us to determine the selective disappearance of an 88-kD starch hydrolytic activity. We further demonstrate that this enzyme is a debranching enzyme. Cleavage of the [alpha]-(1->6) linkage in a branched precursor of amylopectin (preamylopectin) has provided us with the ground rules for understanding starch biosynthesis in plants. Therefore, we propose that amylopectin clusters are synthesized by a discontinuous mechanism involving a highly specific glucan trimming mechanism.

Control of starch composition and structure through substrate supply in the monocellular alga Chlamydomonas reinhardtii.

In Chlamydomonas, as in higher plants, synthesis of ADP glucose catalyzed by ADP-glucose pyrophosphorylase is rate-limiting for the building of starch in the chloroplast. We have isolated disruptions of the STA1 ADP-glucose pyrophosphorylase structural gene that rendered the enzyme less responsive to the allosteric activator 3-phosphoglycerate. The structure and composition of the residual starch synthesized by all mutants of the STA1 locus is dramatically altered. The residual polysaccharide is shown to be devoid of amylose despite the presence of granule-bound starch synthase, the amylose biosynthetic enzyme. In addition, the fine structure of the mutant amylopectin revealed the presence of an altered chain-length distribution. This distribution mimicks that which is observed during growth and photosynthesis and differs markedly from that observed during storage. We therefore propose that low nucleotide sugar concentrations are either directly or indirectly responsible for the major differences observed in the composition or structure of starch during storage and photosynthesis.

Storage, Photosynthesis, and Growth: The Conditional Nature of Mutations Affecting Starch Synthesis and Structure in Chlamydomonas.

Growth-arrested Chlamydomonas cells accumulate a storage polysaccharide that bears strong structural and functional resemblance to higher plant storage starch. It is synthesized by similar enzymes and responds in an identical fashion to the presence of mutations affecting these activities. We found that log-phase photosynthetically active algae accumulate granular [alpha](1->4)-linked, [alpha](1->6)-branched glucans whose shape, cellular location, and structure differ markedly from those of storage starch. That synthesis of these two types of polysaccharides is controlled by both a common and a specific set of genes was evidenced by the identification of a new Chlamydomonas (STA4) locus specifically involved in the biosynthesis of storage starch. Mutants defective in STA4 accumulated a new type of high-amylose storage starch displaying an altered amylopectin chain size distribution. It is expected that the dual nature and functions of starch synthesis in unicellular green algae will yield new insights into the biological reasons for the emergence of starch in the eukaryotic plant cell.

Toward an understanding of the biogenesis of the starch granule. Determination of granule-bound and soluble starch synthase functions in amylopectin synthesis.

Plant starch synthesis can be distinguished from those of bacterial, fungal, and animal glycogen by the presence of multiple elongation (starch synthases) and branching enzymes. This complexity has precluded genetic assignment of functions to the various soluble starch synthases in the building of amylopectin. In Chlamydomonas, we have recently shown that defects in the major soluble starch synthase lead to a specific decrease in the amount of a subset of amylopectin chains whose length ranges between 8 and 40 glucose residues (Fontaine, T., D'Hulst, C., Maddelein, M.-L., Routier, F., Marianne-Pepin, T., Decq, A., Wieruszeski, J. M., Delrue, B., Van Den Koornhuyse, N., Bossu, J.-P., Fournet, B., and Ball, S. G. (1993) J. Biol. Chem. 268, 16223-16230). We now demonstrate that granule-bound starch synthase, the enzyme that was thought to be solely responsible for amylose synthesis, is involved in amylopectin synthesis. Disruption of the Chlamydomonas granule-bound starch synthase structural gene establishes that synthesis of long chains by this enzyme can become an absolute requirement for amylopectin synthesis in particular mutant backgrounds. In the sole presence of soluble starch synthase I, Chlamydomonas directs the synthesis of a major water-soluble polysaccharide fraction and minute amounts of a new type of highly branched granular material, whose structure is intermediate between those of glycogen and amylopectin. These results lead us to propose that the nature of the elongation enzyme conditions the synthesis of distinct size classes of glucans in all starch fractions.

Toward an understanding of the biogenesis of the starch granule. Evidence that Chlamydomonas soluble starch synthase II controls the synthesis of intermediate size glucans of amylopectin.

Low starch mutants of Chlamydomonas reinhardtii were isolated after x-ray mutagenesis of wild-type strain 137C. The mutants accumulated 20-40% of the normal amount and displayed a 2-fold decrease of the total glycogen-primed soluble starch synthase activity. Three different mutant alleles of the st-3 gene were isolated that were characterized by similar defects and displayed a net increase in amylose content. Amylose-primed synthesis of glucan in native gels revealed a complete wipe out of one of the soluble starch synthases. Zymograms and kinetic analyses performed both in the mutant and in partially purified wild type extracts reveal at least two distinct activities that are partly analogous to higher plant soluble starch synthases I and II (SSI and II). The st-3 mutants were defective for SSII. Methylation and debranching of the purified amylopectin fraction clearly show a decrease in the number of intermediate size glucans (dp8 to 50) and an absolute and relative increase of very short glucans (dp2 to 7). These results suggest that a soluble starch synthase may be necessary for the synthesis or maintenance of intermediate size glucans that are the main component of the branched clusters of amylopectin.

Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin.

Amylose-defective mutants were selected after UV mutagenesis of Chlamydomonas reinhardtii cells. Two recessive nuclear alleles of the ST-2 gene led to the disappearance not only of amylose but also of a fraction of the amylopectin. Granule-bound starch synthase activities were markedly reduced in strains carrying either st-2-1 or st-2-2, as is the case for amylose-deficient (waxy) endosperm mutants of higher plants. The main 76-kDa protein associated with the starch granule was either missing or greatly diminished in both mutants, while st-2-1-carrying strains displayed a novel 56-kDa major protein. Methylation and nuclear magnetic resonance analysis of wild-type algal storage polysaccharide revealed a structure identical to that of higher-plant starch, while amylose-defective mutants retained a modified amylopectin fraction. We thus propose that the waxy gene product conditions not only the synthesis of amylose from endosperm storage tissue in higher-plant amyloplasts but also that of amylose and a fraction of amylopectin in all starch-accumulating plastids. The nature of the ST-2 (waxy) gene product with respect to the granule-bound starch synthase activities is discussed.

A Chlamydomonas reinhardtii low-starch mutant is defective for 3-phosphoglycerate activation and orthophosphate inhibition of ADP-glucose pyrophosphorylase.

A low-starch mutant accumulating less than 5% of wild-type amounts was isolated after X-ray mutagenesis of Chlamydomonas reinhardtii cells. The recessive st-1-1 defect segregated as a single mendelian mutation through meiosis, and led to a severe decrease in starch accumulation under all culture conditions tested, whether in the light or in darkness. Adenosine 5'-diphosphoglucose pyrophosphorylase (in the absence of 3-phosphoglycerate), starch synthase, phosphoglucomutase, phosphorylase and starch-branching enzyme were all characterized and shown to be unaffected by the mutation. However, ADP-glucose pyrophosphorylase in the mutant had its sensitivity to activation by 3-phosphoglycerate lowered dramatically and became less responsive to orthophosphate. Our results are consistent both with a mutation in a structural gene of a multisubunit enzyme or in a regulatory gene responsible for switching ADP-glucose pyrophosphorylase from a 3-phosphoglycerate-insensitive to a 3-phosphoglycerate-sensitive form. These results provide definite proof of the in-vivo requirement for 3-phosphoglycerate activation to obtain substantial starch synthesis in plants. The conclusions hold both for synthesis from CO2 in the light or from exogenous organic carbon sources in darkness. A model is presented in which the existence of a 3-phosphoglycerate gradient explains localized starch synthesis around the pyrenoid of lower plants.