This review is written for plant biologists, biochemists and biotechnologists. It presents recent results and views on the metabolism of transitory and reserve starch. We discuss several topics related to starch biosynthesis and focus on the elongation of α-glucan chains as mediated by the various starch synthases and by the plastidial phosphorylase. We briefly discuss the two main glucosyl donors, i.e. ADPglucose or glucose 1-phosphate, that are used by the two types of glucosyl transferases. In the next section, we present a novel theoretical approach to analyse reiterating reactions mediated by a single enzyme. This type of reactions occurs frequently in carbohydrate metabolism of pro- and eukaryotic cells. We then give a schematic presentation of the structure of starch synthases from Arabidopsis thaliana and discuss the action of the different isoforms. The two types of phosphorylases that are common in plants are described and their in vivo actions are presented. We describe functional properties of heterogeneous protein complexes and explain why they are needed in starch metabolism. Finally, we discuss phenotypical analyses of starch-related mutants and explain some of the difficulties inherent to the attempt to deduce biochemical paths from phenotypical features.

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Phototrophic eukaryotes were established by the engulfment of oxygenic phototrophic prokaryotes (cyanobacteria) by a heterotrophic host. This process, called primary endosymbiosis, gave rise to the taxon Archaeplastida, which comprises green plants, rhodophytes and glaucophytes. Further rounds of endosymbiotic events produced a variety of phototrophic organisms, which could accumulate α-1,4-/α-1,6-glucans (including starch) or β-1,3-/β-1,6-glucans. In this article, we review the recent progress in the study of the intracellular localization and molecular forms of storage glucan, especially starch, among the known phototrophs and related organisms.

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The starch granules in rice endosperm are of the compound-type, which comprises up to several dozen granules per amyloplast. In contrast, the granules in the endosperm of maize and wheat are of the simple-type, which comprises only a single granule. The molecular mechanisms that control granule-type have long been an open question. Laser scanning confocal microscopy (LSCM) is advantageous over conventional microscopy when combined with the expression of fluorescent proteins (FPs) in the tissue under investigation. We have analyzed numerous transgenic rice expressing FPs fused to amyloplast-targeted proteins in endosperm. The fluorescent signals of these fusion proteins provided high-quality images of the internal structures of amyloplasts. Interestingly, changing expression levels of plastid-division proteins or isoamylase3 alter both amyloplast division and granule synthesis. On the basis of these results, a model for the synthesis of compound-type granules in rice has been proposed. In this model, the septum-like structure (SLS) is synthesized beneath the outer limit envelope prior to de novo granule synthesis, where it functions like a partition-wall in the stroma, casting the growing granules into their characteristic polyhedral, sharp-edged shape. LSCM analyses using FPs fused to starch synthase I (SSI-GFP) and the transit peptide of granule-bound starch synthase I (tp^GBSSICherry) revealed that SSI-GFP and tp^GBSSI Cherry are respectively targeted to the granule surface in the stroma and the SLS sandwiched between the inner envelope membranes. This review examines the technical issues of LSCM and transgenic analysis, summarizes models for compound-type granule synthesis, and presents future questions.

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Starch forms transparent grains, referred to as starch grains (SGs), in amyloplasts. Despite the simple glucose polymer composition of starch, SGs exhibit different morphologies depending on plant species, especially in the endosperm of the family Poaceae. In previous comprehensive work, SG morphologies of Poaceae endosperm were classified into four types: compound grains, bimodal simple grains, uniform simple grains and a mixed configuration comprised of compound and simple grains. Despite these observations, the phylogenetic relationships among the SG types remained unclear. In this report, we analyzed the previous observations with respect to current knowledge of the molecular phylogeny of Poaceae to determine how SG morphological types are clustered and scattered in the phylogenetic tree. We also obtained clear images of SGs of 26 species belonging to the four genera Hordeum, Elymus, Triticum and Bromus. Previously published work indicated that the SG morphologies of these genera were clustered in the phylogenetic tree and specific for the bimodal simple grain type. However, our work identified novel morphological types of SGs within Bromus in addition to the known bimodal simple grain type. A discussion of the molecular phylogenetic analysis and the remarkable intrageneric variation found in this study will contribute to a greater understanding of SG morphological diversity.

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The functions of the major isozymes related to the starch biosynthesis in cereal endosperm have been resolved based on the researches using the mutant lines. For further understanding of the relationships between isozymes, the analyses of double mutant lines must be very efficient. In this study, double mutant rice line (ss1^L/isa1) between leaky starch synthase I mutant line (ss1^L) and isoamylase 1 mutant line (isa1) was generated and structures of the stored α-glucans were characterized. Cross-section of the parent isa1 and ss1^L/isa1 seeds was negative for iodine staining and more than 90% of the α-glucans was soluble in water. Chain-length distributions of the glucans were similar to each other between the two lines, and the differential plot obtained by subtraction of the chain-length distribution of isa1 from that of ss1^L/isa1 was nearly identical to the corresponding differential plot of ss1^L vs. wild type, indicating effect of SSI is simply additive on chain-length distribution of the product regardless of isoamylase1 activity. Analyses of β-amylase limit dextrins revealed slight, statistically significant difference in internal chain-length distributions of the two isa1 mutant lines in contrast to the previous result on isa1 mutant lines with or without exogenous indica-type SSIIa. This study demonstrated that the primary role of SSI in the isa1 background is outer-chain elongation with a little effect on branch-position distribution, which is the same role as the case in normal amylopectin biosynthesis in the presence of isoamylase 1. Such chain-elongation properties differ among the three major SS isozymes.

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Mutations in the starch synthesizing genes of cereal crops with altered starch properties has been utilized to widen the applications of grain, flour and starch. Here, rice spontaneous mutants that lacked starch branching enzyme 1 (BEI) activity were identified from among local upland rice cultivars. Two cultivars, Kurnai and Hiderishirazu-D, lacked BEI activity in the developing endosperm, and their reserved starch was rich in short chains of amylopectin and showed a low pasting temperature (PT) of rice flour. These features are similar to those of the induced starch branching enzyme 1 gene (Sbe1) mutant of rice. We detected polymorphisms in the Sbe1 of the Kurnai and Hiderishirazu-D cultivars and used them to develop PCR markers suitable for selecting breeding lines and cultivars with BEI deficiency. We assessed the possible utilization of BEI deficiency in waxy rice processing with the use of both the spontaneous and induced BEI-deficient lines. Rice cakes made from BEI-deficient lines maintained their softness for longer periods than those made from functional BEI lines. Our results suggest that use of BEI-deficient waxy rice could improve the quality and extend the shelf life of waxy rice products.

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We studied the translucent and opaque characters of high temperature stressed chalky grains harvested in Niigata of 2010. Appearance of chalky grains of 2009 and 2010 (ripening temperatures: 24.4 and 28.0°C, respectively) showed some resemblance, and the high temperature stress of 2010 remarkably extended the chalking of grain. Scanning electron microscopic observation showed that round-shaped starch granules with numerous small pits were loosely packed in the opaque part of chalky grain. Reduction of mechanical resistance of cooked rice of chalky grain was observed in a breaking test (apparent strain, 0-75%), suggesting that the loose packing of starch granules probably led to the softness of chalky grain. In addition, slight differences in chain-length distributions of starches were observed in the grains of 2009-to-2010, however, no significant differences in the chain-length distributions between the translucent and opaque parts of perfect and chalky grains were found in both 2009 and 2010. Furthermore, the glucose contents in opaque parts were increased by the high temperature stress of 2010, though those in perfect grains were not different regardless of the environmental changes of 2009-to-2010. Together with previous findings on the increased expression of α-amylases in endosperm, these results suggested that unusual starch degradation rather than starch synthesis is involved in occurring of chalky grains of rice under the high temperature stress during grain filling period.

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The present paper comparatively characterized starch and glycogen branching enzymes (BEs) from various organisms. All these BEs examined were purified from recombinant proteins expressed in Escherichia coli (E. coli) cells encoding rice BEI (OsBEI), rice BEIIa (OsBEIIa), rice BEIIb (OsBEIIb), E. coli BE (EcoBE), Synechococcus elongatus BE (ScoBE), Saccharomyces cerevisiae BE (SacBE) and human BE (HosBE). All the BEs had commonly the same properties in terms of the minimum chain-length of DP 6 for the transferred chains, that of DP 6 for the residual chain, and hence that of DP 12 for the substrate chains. Detailed analysis of chain-length distribution of the BE reaction products revealed that the chain-length preference of BE can be classified into three types. First, the OsBEI type which included EcoBE formed short to intermediate chains having degree of polymerization (DP) 6－15 with the most abundant chains of DP 6 and 10－12. Second, the OsBEIIb type into which ScoBE was classified transferred almost exclusively very short chains of DP 6 and 7. Third, the OsBEIIa type to which SacBE and HosBE belonged formed very short chains of DP 6 and 7 as OsBEIIb, but it also significantly produced short chains of DP ≦ 10. The optimal temperatures for the catalytic activities of BEs were comparatively low in the range of 20－30°C, and the activities were markedly inhibited by higher temperatures above 30°C with an exception that ScoBE maintained its high activity until 40°C.

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Analytical methods for branched structures of amylose were developed, employing enzymatic debranching followed by fluorescent labeling/high-performance size-exclusion chromatography (HPSEC). The methods allow differential measurement of size distribution of main- and side-chain of branched molecules. Main-chain distribution was obtained by HPSEC of debranched amylose that was labeled before debranching. By labeling after debranching, both main- and side-chains were detected. Side-chain distribution was obtained by subtracting detector response of the main chain from the response of main plus side chains. Main chains showed little difference in the size distribution from that of amylose. As reported previously, debranching of amylose was incomplete and the percentage of debranching was increased from 33 to 67% by prior hydrolysis with β-amylase. Chain-length distribution of side chains that were released from amylose by enzymatic debranching could be divided into long and short chains, and the latter showed similar, but not identical, distribution to that of amylopectin counterpart. Despite of the similarity, chain-length distributions of β-amylase limit dextrin of amylose were distinct from those of amylopectin prepared from the same starch preparation. Molar percentage, estimated as area% of a chromatogram by fluorescence detection, of A and B chains is considerably higher and lower, respectively, in amylose than that in amylopectin. A-chain fraction was 62% in amylose and 57% in amylopectin, and the amount of short B-chains with lengths comparable to B₁-chains in amylopectin was a half (16%) of that in amylopectin. These results imply that unlike amylopectin, short side-chains of amylose are not organized in a cluster fashion.

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Growth of Gram-positive bacteria was more strongly inhibited compared with that of Gram-negative bacteria when cultured in media containing 1,5-anhydro-D-fructose (AF). For example, the growth of Escherichia coli ML-35 was barely inhibited, with all of the AF in the medium being metabolized after being converted into 1,5-anhydro-D-glucitol (AG), whereas the growth of Staphylococcus aureus ATCC 10832 was strongly inhibited, with most of the AF being retained and only low amounts of AG being observed. Because we detected NADPH-dependent AF reductase (AFR; AG-forming) in the cell extract of both these strains, we assumed that the difference in the ability to regenerate NADPH was the main factor responsible for growth inhibition in the presence of AF. The N-terminal amino acid sequence of AFR isolated from E. coli ML-35 was consistent with that of 2,5-diketo-D-gluconate reductase, with sequences characteristic of the aldo-keto oxidoreductase family. We also found that growth of Listeria monocytogenes ATCC BAA-679 was inhibited by addition of AF and the growth of this organism was further inhibited by a combination of AF and sodium chloride. Furthermore, AF inhibited spore formation and germination in Bacillus subtilis NBRC 3009. These results demonstrate that AF is a beneficial material for the food industry.

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