Effects of granule-bound starch synthase I-defective mutation on the morphology and structure of pyrenoidal starch in Chlamydomonas.

Lowering of the CO2 concentration in the environment induces development of a pyrenoidal starch sheath, as well as that of pyrenoid and CO2-concentrating mechanisms, in many microalgae. In the green algae Chlamydomonas and Chlorella, activity of granule-bound starch synthase (GBSS) concomitantly increases under these conditions. In this study, effects of the GBSS-defective mutation (sta2) on the development of pyrenoidal starch were investigated in Chlamydomonas. Stroma starch- and pyrenoid starch-enriched samples were obtained from log-phase cells grown with air containing 5% CO2 (high-CO2 conditions favouring stromal starch synthesis) and from those transferred to low-CO2 conditions (air level, 0.04% CO2, favouring pyrenoidal starch synthesis) for 6h, respectively. In the wild type, total starch content per culture volume did not increase during the low-CO2 conditions, in spite of the development of pyrenoidal starch, suggesting that degradation of some part of stroma starch and synthesis of pyrenoid starch simultaneously occur under these conditions. Even in the GBSS-deficient mutants, pyrenoid and pyrenoid starch enlarged after lowering of the CO2 concentration. However, the morphology of the pyrenoid starch was thinner and more fragile than the wild type, suggesting that GBSS does affect the morphology of pyrenoidal starch. Surprisingly normal GBSS activity is shown to be required to obtain the high A-type crystallinity levels that we now report for pyrenoidal starch. A model is presented explaining how GBSS-induced starch granule fusion may facilitate the formation of the pyrenoidal starch sheath.

The primitive rhodophyte Cyanidioschyzon merolae contains a semiamylopectin-type, but not an amylose-type, alpha-glucan.

The storage glucans of Cyanidioschyzon merolae [clade L-1 (cyanidian algae), order Porphyridiales, subclass Bangiophycidae], which is considered to be one of the most primitive rhodophytes, were analyzed to understand the early evolution of the glucan structure in the Rhodophyta. Chain-length distribution analysis of the glucans of cyanidian algae demonstrated that while the glucans of Cyanidium caldarium and Galdieria sulphuraria are of the glycogen type, those of C. merolae are of the semiamylopectin type, as in other lineages of the Rhodophyta. Gel permeation chromatography, however, showed that the glucans of C. merolae do not include amylose, being different from those of other Bangiophycidae species. Identification by MALDI-TOF-MS and enzyme assaying of glucan granule-bound proteins indicated that phosphorylase, but not starch synthase, is included. Thus, C. merolae has an unusual glucan and bound-protein composition for the Bangiophycidae, appearing to be a member of the Florideophycidae. The finding that the alga does not contain amylose or the related enzyme, granule-bound starch synthase, is, however, consistent with previously reported results of molecular phylogenetic analysis of starch synthases. Our results support an evolutionary scenario defined by the loss of starch and reversion to glycogen synthesis during the evolution of cyanidian algae, and suggest the possibility that a C. merolae-like primitive rhodophyte might have evolved into the Florideophycidae.

Variation in storage alpha-polyglucans of red algae: amylose and semi-amylopectin types in Porphyridium and glycogen type in Cyanidium.

Red algae are widely known to produce floridean starch but it remains unclear whether the molecular structure of this algal polyglucan is distinct from that of the starch synthesized by vascular plants and green algae. The present study shows that the unicellular species Porphyridium purpureum R-1 (order Porphyridiales, class Bangiophyceae) produces both amylopectin-type and amylose-type alpha-polyglucans. In contrast, Cyanidium caldarium (order Porphyridiales, class Bangiophyceae) synthesizes glycogen-type polyglucan, but not amylose. Detailed analysis of alpha-1,4-chain length distribution of P. purpureum polyglucan suggests that the branched polyglucan has a less ordered structure, referred to as semi-amylopectin, as compared with amylopectin of rice endosperm having a tandem-cluster structure. The P. purpureum linear amylose-type polyglucan, which has a lambda(max) of 630 nm typical of amylose-iodine complex and is resistant to Pseudomonas isoamylase digestion, accounts for less than 10% of the total polyglucans. We produced and isolated a cDNA encoding a granule-bound starch synthase (GBSS)-type protein of P. purpureum, which is probably the approximately 60-kDa protein bound tightly to the starch granules, resembling the amylose-synthesizing GBSS protein of green plants. The present investigation indicates that the class Bangiophyceae includes species producing both semi-amylopectin and amylose, and species producing glycogen alone.

Granule-bound starch synthase cDNA in Chlorella kessleri 11 h: cloning and regulation of expression by CO(2) concentration.

The cDNA for the granule-bound starch synthase (GBSS; ADP-glucose-starch glucosyltransferase, EC 2.4.1.21) of Chlorella kessleri 11 h was isolated and characterized. CkGBSS encodes a 609-amino acid polypeptide (65,627 Da) that includes an N-terminal hydrophobic signal peptide of 55 amino acids. The deduced amino acid sequence of the mature CkGBSS polypeptide shares a greater identity (65%) to that of the GBSS protein of Chlamydomonas reinhardtii, than to those of vascular plant species, but does not have the extra-long C-terminal sequence found in C. reinhardtii. When CO(2 )concentration was decreased from 3 to 0.04% (air level) in light, the levels of CkGBSS mRNA, CkGBSS protein, and GBSS activity increased. Under this condition, pyrenoid and pyrenoid starch developed, and the relative amount of amylose in starch increased. These observations suggest that low CO(2) level up-regulates GBSS biosynthesis at the transcriptional level.