Search

Main Conclusion: Linear glucans with degree of polymerization of up to 23 were detected in rice endosperm at the very early developmental stage of endosperm and considered to play an important role in the de novo synthesis of branched glucans. Little is known concerning the contribution of malto-oligosaccharides (MOS) and longer linear glucans to the starch biosynthesis in cereal endosperm. In the present study, the changes in the amount of major metabolic intermediates including MOS and linear glucans with a degree of polymerization (DP) of ≤ 9 and ≥ 10, respectively, in rice endosperm were measured during the development. Significant amounts of linear glucans of at least DP23 were present in the endosperm at 3 and 5 days after pollination (DAP), whereas most MOS of DP up to 8 were detected in the endosperm throughout the development up to 20 DAP. It was also found that a significant amount of simple sugars such as sucrose, glucose, and fructose, and organic acids such as malic acid, citric acid, and succinic acid were present in the developing endosperm. Although the levels of metabolites are not directly related to the extent of the metabolic flux, the present results suggest that MOS and linear glucans as well as these sugars and organic acids are involved in starch biosynthesis of rice endosperm. It is thought that linear glucans might play a role in starch biosynthesis in rice endosperm, presumably as the precursor for the subsequent synthesis of branched glucans involved in the initiation process that is possibly active in the endosperm at the very early developmental stage.

Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein-protein interactions in maize and wheat amyloplasts. This study investigated whether protein-protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200-400kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs-BEs, and, among BE isozymes, BEIIa-Pho1, and pullulanase-type DBE-BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

To investigate the functional properties of 10 α-glucan branching enzymes (BEs) from various sources, we determined the chain-length distribution of BE enzymatic products and their phosphorylase-limit dextrins (Φ-LD). All BEs could be classified into either of the three rice BE isozymes: OsBEI, OsBEIIa, or OsBEIIb. Escherichia coli BE (EcoBE) had the same enzymatic properties as OsBEI, while Synechococcus elongatus BE (ScoBE) and Chlorella kessleri BE (ChlBE) had BEIIb-type properties. Human BE (HosBE), yeast BE (SacBE), and two Porphyridium purpureum BEs (PopBE1 and PopBE2) exhibited the OsBEIIa-type properties. Analysis of chain-length profile of Φ-LD of the BE reaction products revealed that EcoBE, ScoBE, PopBE1, and PopBE2 preferred A-chains as acceptors, while OsBEIIb used B-chains more frequently than A-chains. Both EcoBE and ScoBE specifically formed the branch linkages at the third glucose residue from the reducing end of the acceptor chain. The present results provide evidence for the first time that great variation exists as to the preference of BEs for their acceptor chain, either A-chain or B-chain. In addition, EcoBE and ScoBE recognize the location of branching points in their acceptor chain during their branching reaction. Nevertheless, no correlation exists between the primary structure of BE proteins and their enzymatic characteristics., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The present study was conducted to characterize the functions of the major starch synthase (SS) isozymes SSI, SSIIa, and SSIIIa in rice endosperm and their functional interaction with starch branching enzyme (BE), by using their purified recombinant proteins. All the SS isozymes had similarly significant activities toward branched glucans such as amylopecin and glycogen whereas they scarcely showed activities toward maltohexaose. In vitro studies indicate that SSI mainly attacked A and B chains with degree of polymerization (DP) of 6 and 7 in their external segments and elongated them to DP8. It is likely that SSIIa and SSIIIa produced wider ranges of intermediate chains and long chains, respectively. This study also revealed that without addition of exogenous primer, the glucan synthesis of SSI in the presence of ≧0.3 M citrate was accelerated by the addition of any of the rice BE isozymes- BEI, BEIIa, or BEIIb, whereas no such interaction occurred between SSIIa or SSIIIa with any of the BEs. The SSI-BE unprimed glucan synthesis absolutely required citrate. The interaction between SSI and BE was established by stimulation of SSI activity with BE and by activation of the BE activity by SSI., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

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.

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.

Storage glucans were analyzed in the Porphyridiales which include the most primitive and phylogenetically diverged species in the Rhodophyta, to understand early evolution of the glucan structure in the Rhodophyta. The storage glucans of both Galdieria sulphuraria and Cyanidium caldarium consisted of glycogen, while those of Rhodosorus marinus, Porphyridium purpureum, P. sordidum and Rhodella violacea could be defined as semi-amylopectin. X-ray diffraction analysis of the glucans demonstrated variation in the crystalline structure: the patterns in P. purpureum and R. violacea were of A- and B-types, respectively, while alpha-glucans of R. marinus and P. sordidum displayed structures with lower crystallinity. Electron microscopic observations indicated that the alpha-glucans of P. sordidum consisted of two kinds of granules; a minor component of more dense granules with crystalline leaflets and a major component of softer ones without crystalline structure. Gel permeation chromatography showed that all the species containing the semi-amylopectin-type glucans also contained amylose, although the relative amounts of this fraction were different depending on the species. Our results are consistent with two distinct evolution scenarios defined either by the independent acquisition of semi-crystalline starch-like structures in the different plant lineages or more probably by the loss of starch and reversion to glycogen synthesis in cyanidian algae growing in hot and acid environments.

It is widely accepted that green plants evolved the capacity to synthesize the highly organized branched alpha-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 alpha-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 alpha-1,4-chain. It was also found that Cyanobacterium sp. MBIC10216 had no amylose-type component in its alpha-polyglucans. The evolutionary aspect of the structure of alpha-polyglucan is discussed in relation to the phylogenetic evolutionary tree of 16S rRNA sequences of cyanobacteria.

Functional interactions of plastidial phosphorylase (Pho1) and starch branching enzymes (BEs) from the developing rice endosperm are the focus of this study. In the presence of both Pho1 and BE, the same branched primer molecule is elongated and further branched almost simultaneously even at very low glucan concentrations present in the purified enzyme preparations. By contrast, in the absence of any BE, glucans are not, to any significant extent, elongated by Pho1. Based on our in vitro data, in the developing rice endosperm, Pho1 appears to be weakly associated with any of the BE isozymes. By using fluorophore-labeled malto-oligosaccharides, we identified maltose as the smallest possible primer for elongation by Pho1. Linear dextrins act as carbohydrate substrates for BEs. By functionally interacting with a BE, Pho1 performs two essential functions during the initiation of starch biosynthesis in the rice endosperm: First, it elongates maltodextrins up to a degree of polymerization of at least 60. Second, by closely interacting with BEs, Pho1 is able to elongate branched glucans efficiently and thereby synthesizes branched carbohydrates essential for the initiation of amylopectin biosynthesis. [ABSTRACT FROM AUTHOR]

It has been believed that isoamylase (ISA)-type α-glucan debranching enzymes (DBEs) play crucial roles not only in α-glucan degradation but also in the biosynthesis by affecting the structure of glucans, although molecular basis on distinct roles of the individual DBEs has not fully understood. In an attempt to relate the roles of DBEs to their chain-length specificities, we analyzed the chain-length distribution of DBE enzymatic reaction products by using purified DBEs from various sources including rice, cyanobacteria, and bacteria. When DBEs were incubated with phytoglycogen, their chain-length specificities were divided into three groups. First, rice endosperm ISA3 (OsISA3) and Eschericia coli GlgX (EcoGlgX) almost exclusively debranched chains having degree of polymerization (DP) of 3 and 4. Second, OsISA1, Pseudomonas amyloderamosa ISA (PsaISA), and rice pullulanase (OsPUL) could debranch a wide range of chains of DP≧3. Third, both cyanobacteria ISAs, Cyanothece ATCC 51142 ISA (CytISA) and Synechococcus elongatus PCC7942 ISA (ScoISA), showed the intermediate chain-length preference, because they removed chains of mainly DP3-4 and DP3-6, respectively, while they could also react to chains of DP5-10 and 7–13 to some extent, respectively. In contrast, all these ISAs were reactive to various chains when incubated with amylopectin. In addition to a great variation in chain-length preferences among various ISAs, their activities greatly differed depending on a variety of glucans. Most strikingly, cyannobacteria ISAs could attack branch points of pullulan to a lesser extent although no such activity was found in OsISA1, OsISA3, EcoGlgX, and PsaISA. Thus, the present study shows the high possibility that varied chain-length specificities of ISA-type DBEs among sources and isozymes are responsible for their distinct functions in glucan metabolism. [ABSTRACT FROM AUTHOR]

The relationship between the solubility, crystallinity, and length of the unit chains of plant storage α-glucan was investigated by manipulating the chain length of α-glucans accumulated in a rice mutant. Transgenic lines were produced by introducing a cDNA for starch synthase IIa (SSIIa) from an indica cultivar (SSIIaI, coding for active SSIIa) into an isoamylase1 (ISA1)-deficient mutant (isa1) that was derived from a japonica cultivar (bearing inactive SSIIa proteins). The water-soluble fraction accounted for >95% of the total α-glucan in the isa1 mutant, whereas it was only 35–70% in the transgenic SSIIaI/isa1 lines. Thus, the α-glucans from the SSIIaI/isa1 lines were fractionated into soluble and insoluble fractions prior to the following characterizations. X-ray diffraction analysis revealed a weak B-type crystallinity for the α-glucans of the insoluble fraction, while no crystallinity was confirmed for α-glucans in isa1. Concerning the degree of polymerization (DP) ≤30, the chain lengths of these α-glucans differed significantly in the order of SSIIaI/isa1 insoluble > SSIIaI/isa1 soluble > α-glucans in isa1. The amount of long chains with DP ≥33 was higher in the insoluble fraction α-glucans than in the other two α-glucans. No difference was observed in the chain length distributions of the β-amylase limit dextrins among these α-glucans. These results suggest that in the SSIIaI/isa1 transgenic lines, the unit chains of α-glucans were elongated by SSIIaI, whereas the expression of SSIIaI did not affect the branch positions. Thus, the observed insolubility and crystallinity of the insoluble fraction can be attributed to the elongated length of the outer chains due to SSIIaI. [ABSTRACT FROM PUBLISHER]

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]

Plastidial phosphorylase (Phol) accounts for ~96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Phol caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30°C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20°C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures. [ABSTRACT FROM AUTHOR]

The present study established that there are two distinct polymeric forms of isoamylase1 (ISA1) in rice endosperm: presumably a homo-pentamer of ISA1 and a hetero-hexamer composed of five ISA1 and one ISA2. The molecular sizes of the homo- and hetero-oligomers, which could be fractionated by hydrophobic chromatography, were approximately 420–480 and 510–550 kDa, respectively. The hetero-oligomer exhibited higher affinities for various branched polyglucans, especially for phytoglycogen, which had a K m value that was approximately 12 times lower relative to that with the homo-oligomer, although no marked differences were found in chain preferences for debranching of amylopectin and phytoglycogen between these forms. The hetero-oligomer was active even when incubated at 50°C for 10 min, while the homo-multimer was completely inactivated at 40°C in 10 min. When the ISA1 homo-oligomer was incubated with the ISA2 protein expressed in Escherichia coli and applied onto a nondenature polyacrylamide gel, additional debranching activity bands which were specific for the purified ISA1–ISA2 preparation were also detected, indicating that ISA1 and ISA2 combine to form a hetero-oligomer. These results suggest that the hetero-oligomer plays a predominant role in the amylopectin biosynthesis in rice endosperm although the homo-oligomer can complement the function of the hetero-oligomer at least to some extent. [ABSTRACT FROM AUTHOR]

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]

When the starch branching enzyme IIb (BEIIb) gene was introduced into a BEIIb-defective mutant, the resulting transgenic rice plants showed a wide range of BEIIb activity and the fine structure of their amylopectins showed considerable variation despite having the two other BE isoforms, BEI and BEIIa, in their endosperm at the same levels as in the wild-type. The properties of the starch granules, such as their gelatinization behaviour, morphology and X-ray diffraction pattern, also changed dramatically depending on the level of BEIIb activity, even when this was either slightly lower or higher than that of the wild-type. The over-expression of BEIIb resulted in the accumulation of excessive branched, water-soluble polysaccharides instead of amylopectin. These results imply that the manipulation of BEIIb activity is an effective strategy for the generation of novel starches for use in foodstuffs and industrial applications. [ABSTRACT FROM AUTHOR]

Up to now, no information on the structural characteristics of glucans involved in the initiation of starch biosynthesis in cereal endosperm has been available. Since amylopectin is a major constituent of starch and largely affects the starch physicochemical properties, we attempted to characterize the fine structures of branched glucans. We isolated small glucan granules with a diameter of approximately 40–80 nm from rice endosperm at the early developmental stages and analyzed the chain-length distribution of the glucans and their phosphorylase-limit dextrins. Compared with normal amylopectin, the proportion of long chains of these glucans with degree of polymerization (DP) ≥ 35 was significantly lower and these long chains did not form a peak. These structural features were commonly found in small glucan granules in three rice cultivars. Based on the present results, we propose that these immature glucans located in small granules in young endosperm are involved in the initiation process of amylopectin biosynthesis in rice endosperm. • Isolation and characterization of immature glucans in very young rice endosperm. • The glucans having structurally an immature cluster compared with amylopectin. • Possible role of immature glucans in the initial starch synthesis in rice endosperm. • New insights into the specific initial stage of starch synthesis in cereal endosperm. [ABSTRACT FROM AUTHOR]