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Although an alternative pathway has been suggested, the prevailing view is that starch synthesis in cereal endosperm is controlled by the activity of the cytosolic isoform of ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, which code for the small (S2b) and large (L2) subunits of the heterotetrameric enzyme, respectively. In this study, we isolated several allelic missense and nonsense OsAGPL2 mutants by N-methyl-N-nitrosourea (MNU) treatment of fertilized egg cells and by TILLING (Targeting Induced Local Lesions in Genomes). Interestingly, seeds from three of the missense mutants (two containing T139I and A171V) were severely shriveled and had seed weight and starch content comparable with the shriveled seeds from OsAGPL2 null mutants. Results from kinetic analysis of the purified recombinant enzymes revealed that the catalytic and allosteric regulatory properties of these mutant enzymes were significantly impaired. The missense heterotetramer enzymes and the S2b homotetramer had lower specific (catalytic) activities and affinities for the activator 3-phosphoglycerate (3-PGA). The missense heterotetramer enzymes showed more sensitivity to inhibition by the inhibitor inorganic phosphate (Pi) than the wild-type AGPase, while the S2b homotetramer was profoundly tolerant to Pi inhibition. Thus, our results provide definitive evidence that starch biosynthesis during rice endosperm development is controlled predominantly by the catalytic activity of the cytoplasmic AGPase and its allosteric regulation by the effectors. Moreover, our results show that the L2 subunit is essential for both catalysis and allosteric regulatory properties of the heterotetramer enzyme. [ABSTRACT FROM PUBLISHER]

Unicellular, diazotrophic species of cyanobacteria, Cyanobacterium sp. NBRC 102756, Cyanothece sp. ATCC 51142 and Cyanobacterium sp. CLg1, accumulate insoluble α-glucan inside the cells as the storage polysaccharide. The purified polysaccharides showed granular morphology, with a diameter of 0.2–0.7 µm. The three α-glucan preparations all showed A-type allomorph in X-ray diffraction analysis. Distinct thermal gelatinization temperatures were observed for these polysaccharides. The α-glucans from NBRC 102756 and ATCC 51142 strains consisted solely of branched α-glucans, or semi-amylopectin, while CLg1 contained semi-amylopectin as the primary component as well as linear or scarcely branched glucan (amylose). Separation of the debranched glucan chains by gel filtration chromatography explicitly showed the presence in the semi-amylopectin molecule of long chains corresponding to B2 chains, which connect clusters in amylopectin of plants. The relative proportions of short and long glucan chains in the branched polysaccharides differed depending on the species, and the variation was intimately correlated with the physical properties of the α-glucans. The results suggested that semi-amylopectin of the three cyanobacteria exhibit essentially similar organization with a tandem cluster structure. The polysaccharides of these strains are therefore referred to as ‘cyanobacterial starch’, distinct from glycogen. [ABSTRACT FROM PUBLISHER]

The present study established the way in which plastidial α-glucan phosphorylase (Pho1) synthesizes maltodextrin (MD) which can be the primer for starch biosynthesis in rice endosperm. The synthesis of MD by Pho1 was markedly accelerated by branching enzyme (BE) isozymes, although the greatest effect was exhibited by the presence of branching isozyme I (BEI) rather than by isozyme IIa (BEIIa) or isozyme IIb (BEIIb). The enhancement of the activity of Pho1 by BE was not merely due to the supply of a non-reducing ends. At the same time, Pho1 greatly enhanced the BE activity, possibly by generating a branched carbohydrate substrate which is used by BE with a higher affinity. The addition of isoamylase to the reaction mixture did not prevent the concerted action of Pho1 and BEI. Furthermore, in the product, the branched structure was, at least to some extent, maintained. Based on these results we propose that the interaction between Pho1 and BE is not merely due to chain-elongating and chain-branching reactions, but occurs in a physically and catalytically synergistic manner by each activating the mutual capacity of the other, presumably forming a physical association of Pho1, BEI and branched MDs. This close interaction might play a crucial role in the synthesis of branched MDs and the branched MDs can act as a primer for the biosynthesis of amylopectin molecules. [ABSTRACT FROM AUTHOR]

To our knowledge the present paper shows for the first time the kinetic parameters of all the three starch branching enzyme (BE) isozymes, BEI, BEIIa and BEIIb, from rice with both amylopectin and synthetic amylose as glucan substrate. The activities of these BE isozymes with a linear glucan amylose decreased with a decrease in the molar size of amylose, and no activities of BEIIa and BEIIb were found when the degree of polymerization (DP) of amylose was lower than at least 80, whereas BEI had an activity with amylose of a DP higher than approximately 50. Detailed analyses of debranched products from BE reactions revealed the distinct chain length preferences of the individual BE isozymes. BEIIb almost exclusively transferred chains of DP7 and DP6 while BEIIa formed a wide range of short chains of DP6 to around DP15 from outer chains of amylopectin and amylose. On the other hand, BEI formed a variety of short chains and intermediate chains of a DP ≤40 by attacking not only outer chains but also inner chains of branched glucan while BEIIa or BEIIb could only scarcely or could not attack inner chains, respectively. The comprehensive in vitro studies revealed different enzymatic characteristics of the three BE isozymes and give a new insight into the distinct roles of individual BE isozymes in amylopectin biosynthesis in the endosperm. Based on these results, the functional distinction and interaction of BE isozymes during amylopectin biosynthesis in cereal endosperm is discussed. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM PUBLISHER]

It is widely accepted that green plants evolved the capacity to synthesize the highly organized branched α-polyglucan amylopectin with tandem-cluster structure, whereas animals and bacteria continued to produce random branched glycogen. Although most previous studies documented that cyanobacteria accumulate glycogen, the present study shows explicitly that some cyanobacteria such as Cyanobacterium sp. MBIC10216, Myxosarcina burmensis and Synechococcus sp. BG043511 had distinct α-polyglucans, which were designated as semi-amylopectin. The semi-amylopectin was intermediate between rice amylopectin and typical cyanobacterial glycogen in terms of chain length distribution, molecular size and length of the most abundant α-1,4-chain. It was also found that Cyanobacterium sp. MBIC10216 had no amylose-type component in its α-polyglucans. The evolutionary aspect of the structure of α-polyglucan is discussed in relation to the phylogenetic evolutionary tree of 16S rRNA sequences of cyanobacteria. [ABSTRACT FROM AUTHOR]

Starch is made up of amylose (linear α-1,4-polyglucans) and amylopectin (α-1,6-branched polyglucans). Amylopectin has a distinct fine structure called multiple cluster structure and is synthesized by multiple subunits or isoforms of four classes of enzymes: ADPglucose pyrophosphorylase, soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE). In the present paper, based on analyses of mutants and transgenic lines of rice in which each enzyme activity is affected, the contribution of the individual isoform to the fine structure of amylopectin in rice endosperm is evaluated, and a new model referred to as the “two-step branching and improper branch clearing model” is proposed to explain how amylopectin is synthesized. The model emphasizes that two sets of reactions, α-1,6-branch formation and the subsequent α-1,4-chain elongation, are catalyzed by distinct BE and SS isoforms, respectively, are fundamental to the construction of the cluster structure. The model also assesses the role of DBE, namely isoamylase or in addition pullulanase, to remove unnecessary α-1,6-glucosidic linkages that are occasionally formed at improper positions apart from two densely branched regions of the cluster. [ABSTRACT FROM PUBLISHER]