禾谷类作物胚乳的淀粉合成

禾谷类作物胚乳拥有独特的淀粉合成途径,需要多种特异性同工酶的参与,这些酶在禾谷类其他组织或非禾谷类作物中是不存在的。近来明确了单个淀粉同工酶的功能,这有助于我们更深入地了解禾谷类淀粉直链、支链的合成与分布。在对禾谷类淀粉合成进行遗传分析的基础上,提出了脱分支酶作用模型。以水稻全基因组序列草图为背景,本文首次全面分析了禾谷类作物的淀粉合成。     

Starch biosynthesis in cereal endosperm

Stored starch generally consists of two d-glucose homopolymers, the linear polymer amylose and a highly branched glucan amylopectin that connects linear chains. Amylopectin structurally contributes to the crystalline organization of the starch granule in cereals. In the endosperm, amylopectin biosynthesis requires the proper execution of a coordinated series of enzymatic reactions involving ADP glucose pyrophosphorylase (AGPase), soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), whereas amylose is synthesized by AGPase and granule-bound starch synthase (GBSS). It is highly possible that plastidial starch phosphorylase (Pho1) plays an important role in the formation of primers for starch biosynthesis in the endosperm. Recent advances in our understanding of the functions of individual enzyme isoforms have provided new insights into how linear polymer chains and branch linkages are synthesized in cereals. In particular, genetic analyses of a suite of mutants have formed the basis of a new model outlining the role of various enzyme isoforms in cereal starch production. In our current review, we summarize the recent research findings related to starch biosynthesis in cereal endosperm, with a particular focus on rice.     

Amylolytic Activities in Cereal Seeds under Aerobic and Anaerobic Conditions

An adequate carbohydrate supply contributes to the survival of seeds under conditions of limited oxygen availability. The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, with starch representing the main storage compound. Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes and only through the concerted action of [alpha]-amylase (EC 3.2.1.1), [beta]-amylase (EC 3.2.1.2), debranching enzyme (EC 3.2.1.41), and [alpha]-glucosidase (EC 3.2.1.20) can starch be hydrolyzed completely. We present here data concerning the complete set of starch-degrading enzymes in three cereals, rice (Oryza sativa L.), which is tolerant to anaerobiosis, and wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), which are unable to germinate under anoxia. Among the cereal seeds tested under anoxia, only rice is able to degrade nonboiled, soluble starch, reflecting the ability to degrade the starch granules in vivo. This is explained by the presence of the complete set of enzymes needed to degrade starch completely either as the result of de novo synthesis ([alpha]-amylase, [beta]-amylase) or activation of preexisting, inactive forms of the enzyme (debranching enzyme, [alpha]-glucosidase). These enzymes are either absent or inactive in wheat and barley seeds kept under anaerobic conditions.     

植物支链淀粉合成的关键酶——淀粉分支酶

支链淀粉是植物淀粉的主要成分,而淀粉分支酶是其合成的关键酶。淀粉分支酶可分为两同形体家族,本文从酶学特性、染色体定位、基因及基因表达方面阐明了它们之间的联系和区别,并证实不同同形体在植物支链淀粉合成和结构决定上所起作用不同。开展对该酶的深入研究不论是在基础理论研究领域还是在现实应用方面都具重要意义。     

A Parameterized Model of Amylopectin Synthesis Provides Key Insights into the Synthesis of Granular Starch

A core set of genes involved in starch synthesis has been defined by genetic studies, but the complexity of starch biosynthesis has frustrated attempts to elucidate the precise functional roles of the enzymes encoded. The chain-length distribution (CLD) of amylopectin in cereal endosperm is modeled here on the basis that the CLD is produced by concerted actions of three enzyme types: starch synthases, branching and debranching enzymes, including their respective isoforms. The model, together with fitting to experiment, provides four key insights. (1) To generate crystalline starch, defined restrictions on particular ratios of enzymatic activities apply. (2) An independent confirmation of the conclusion, previously reached solely from genetic studies, of the absolute requirement for debranching enzyme in crystalline amylopectin synthesis. (3) The model provides a mechanistic basis for understanding how successive arrays of crystalline lamellae are formed, based on the identification of two independent types of long amylopectin chains, one type remaining in the amorphous lamella, while the other propagates into, and is integral to the formation of, an adjacent crystalline lamella. (4) The model provides a means by which a small number of key parameters defining the core enzymatic activities can be derived from the amylopectin CLD, providing the basis for focusing studies on the enzymatic requirements for generating starches of a particular structure. The modeling approach provides both a new tool to accelerate efforts to understand granular starch biosynthesis and a basis for focusing efforts to manipulate starch structure and functionality using a series of testable predictions based on a robust mechanistic framework.     

玉米淀粉生物合成及其遗传操纵

淀粉是许多植物重要的储藏物质。淀粉突变体以及转基因植物中淀粉变异的特点使我们对淀粉生物合成的过程有了较深入的了解,许多研究的结果揭示了玉米淀粉的生物合成涉及4类酶--ADPG焦磷酸化酶、淀粉合成酶、淀粉分支酶和去分支酶。随着编码这些酶的基因的克隆,利用转基因技术对淀粉合成过程进行遗传操纵业已成为可能,并且在提高淀粉产量以及不同特性淀粉品质的种质资源创新等方面展示出巨大的潜力。 Abstract:Starch is the most important source of calories and a vital storage component in plants.The characterization and production of starch variants from mutation and with transgenic technology has improved our understanding of the synthesis of starch granule.In starch biosynthesis in plants,four enzymes,including ADP-glucose pyrophosphorylase,starch synthase,starch branching enzyme and starch debranching enzyme,are widely accepted from an enormous amount of research aimed primarily at enzyme characterization.As many genes encoding the enzymes and their multiple isoforms in starch biosynthesis pathway have been isolated,genetic manipulation of the starch biosynthesis pathway shows to be a practical way by which starch quantity is increased and starch with novel properties can be created.     

Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue

Starch is made up of amylose (linear alpha-1,4-polyglucans) and amylopectin (alpha-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, alpha-1,6-branch formation and the subsequent alpha-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 alpha-1,6-glucosidic linkages that are occasionally formed at improper positions apart from two densely branched regions of the cluster.     

Different isoforms of starch-synthesizing enzymes controlling amylose and amylopectin content in rice (Oryza sativa L.)

Starch, composed of amylose and amylopectin, greatly influences rice cooking and textural quality, which in turn is controlled by various isoforms of several enzymes. Activity of one or more isoforms of starch‐synthesizing enzymes results in various forms of starch structure based on the amylopectin chain length and average external, internal and core chain length distribution and hence results in varying physicochemical and cooking quality. Since the synthesis of starch is highly complex, it is crucial but essential to understand its biosynthetic pathway, starch structure and effects on the physicochemical properties that control eating and cooking quality, and alongside conduct research on gene/QTL mapping for use in marker-assisted selection (MAS) with a view to improve and select cultivars with most desirable range and class of rice starch properties. This article presents the updates on current understanding of the coordination among various enzymes/isoforms towards rice starch synthesis in endosperm and their effect on rice grain physicochemical, cooking and eating qualities. The efforts in identifying regions responsible for these enzymes by mapping the gene/QTLs have provided a glimpse on their association with physicochemical and cooking properties of rice and, hence, improvement is possible by modifying the allelic pattern, resulting in down or nil regulation of a particular enzyme. The clear understanding of the tissue specific coordination between enzyme isoforms and their subsequent effect in controlling eating and cooking properties will enhance the chances to manipulate them for getting desired range of amylose content (AC) and gelatinization temperature (GT) in improved cultivars through combining desired alleles through MAS.     

Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development

cDNA clones for two isoforms of starch branching enzyme (SBEI and SBEII) have been isolated from pea embryos and sequenced. The deduced amino acid sequences of pea SBEI and SBEII are closely related to starch branching enzymes of maize, rice, potato and cassava and a number of glycogen branching enzymes from yeast, mammals and several prokaryotic species. In comparison with SBEI, the deduced amino acid sequence of SBEII lacks a flexible domain at the N-terminus of the mature protein. This domain is also present in maize SBEII and rice SBEIII and resembles one previously reported for pea granule-bound starch synthase II (GBSSII). However, in each case it is missing from the other isoform of SBE from the same species. On the basis of this structural feature (which exists in some isoforms from both monocots and dicots) and other differences in sequence, SBEs from plants may be divided into two distinct enzyme families. There is strong evidence from our own and other work that the amylopectin products of the enzymes from these two families are qualitatively different. Pea SBEI and SBEII are differentially expressed during embryo development. SBEI is relatively highly expressed in young embryos whilst maximum expression of SBEII occurs in older embryos. The differential expression of isoforms which have distinct catalytic properties means that the contribution of each SBE isoform to starch biosynthesis changes during embryo development. Qualitative measurement of amylopectin from developing and maturing embryos confirms that the nature of amylopectin changes during pea embryo development and that this correlates with the differential expression of SBE isoforms.     

A novel shrunken endosperm mutant of barley

Although mutations affecting several enzymes of the starch synthetic pathway in developing cereal endosperm have been isolated, none has a major effect on soluble starch synthase We report a new recessive shrunken endosperm mutant in barley ( Hordeum vulgare L. cv. Bomi-like), shx , which has 25% of normal starch content. We have assayed the activity of sucrose synthase (EC 2.4.1.13), ADP and UDP-glucose pyrophosphorylases (EC 2.7.7.27 and 2.7.7.9), branching enzyme (EC.2.4.1.18), and granule-bound and soluble starch synthase (EC 2.4.1.21) in shx. Sucrose synthase activity is reduced by 49% and UDP-glucose pyrrphosphorylase is 80% of the normal level. Branching enzyme and starch-bound starch synthase activities are normal, but ADP-glucose pyrophosphorylase activity is reduced by 72%. The soluble starch synthase that is primer-independent in the presence of sodium citrate shows 14% of normal activity in shx. whereas the primer-dependent form is unaffected. This lower starch synthase activity in shx cannot be explained by inhibition, substrate destruction or lack of primer. Although several starch-synthetic enzymes are affected, it is suggested that the primer independent from of soluble starch synthase may be the primary-site of the mutation in shx.     

Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans

Isoamylases are debranching enzymes that hydrolyze alpha-1,6 linkages in alpha-1,4/alpha-1,6-linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.     

Analysis of the Functional Interaction of Arabidopsis Starch Synthase and Branching Enzyme Isoforms Reveals that the Cooperative Action of SSI and BEs Results in Glucans with Polymodal Chain Length Distribution Similar to Amylopectin

Starch synthase (SS) and branching enzyme (BE) establish the two glycosidic linkages existing in starch. Both enzymes exist as several isoforms. Enzymes derived from several species were studied extensively both in vivo and in vitro over the last years, however, analyses of a functional interaction of SS and BE isoforms are missing so far. Here, we present data from in vitro studies including both interaction of leaf derived and heterologously expressed SS and BE isoforms. We found that SSI activity in native PAGE without addition of glucans was dependent on at least one of the two BE isoforms active in Arabidopsis leaves. This interaction is most likely not based on a physical association of the enzymes, as demonstrated by immunodetection and native PAGE mobility analysis of SSI, BE2, and BE3. The glucans formed by the action of SSI/BEs were analysed using leaf protein extracts from wild type and be single mutants (Atbe2 and Atbe3 mutant lines) and by different combinations of recombinant proteins. Chain length distribution (CLD) patterns of the formed glucans were irrespective of SSI and BE isoforms origin and still independent of assay conditions. Furthermore, we show that all SS isoforms (SSI-SSIV) were able to interact with BEs and form branched glucans. However, only SSI/BEs generated a polymodal distribution of glucans which was similar to CLD pattern detected in amylopectin of Arabidopsis leaf starch. We discuss the impact of the SSI/BEs interplay for the CLD pattern of amylopectin.     

Starch metabolism in leaves

Starch is the most abundant storage carbohydrate produced in plants. The initiation of transitory starch synthesis and degradation in plastids depends mainly on diurnal cycle, post-translational regulation of enzyme activity and starch phosphorylation. For the proper structure of starch granule the activities of all starch synthase isoenzymes, branching enzymes and debranching enzymes are needed. The intensity of starch biosynthesis depends mainly on the activity of AGPase (adenosine 5'-diphosphate glucose pyrophosphorylase). The key enzymes in starch degradation are beta-amylase, isoamylase 3 and disproportionating enzyme. However, it should be underlined that there are some crucial differences in starch metabolism between heterotrophic and autotrophic tissues, e.g. is the ability to build multiprotein complexes responsible for biosynthesis and degradation of starch granules in chloroplasts. The observed huge progress in understanding of starch metabolism was possible mainly due to analyses of the complete Arabidopsis and rice genomes and of numerous mutants with altered starch metabolism in leaves. The aim of this paper is to review current knowledge on transient starch metabolism in higher plants.     

淀粉合酶的酶学与分子生物学研究进展

淀粉合酶作为淀粉合成的关键酶之一,一直是淀粉研究的重要内容,这些研究多集中在对其同工型的研究,淀粉合酶的两类主要同工型分别为淀粉粒结合的淀粉合酶和可溶性淀粉合酶,这两类同工型的作用极为复杂,本文介绍了淀粉合酶同工型的酶学和分子生物学近年来的研究进展,同时也讨论了这些同工型的分类,相互关系及其在淀粉合成过程中的生理功能等内容。     

Soluble isoforms of starch synthase and starch-branching enzyme also occur within starch granules in developing pea embryos

Developing wild-type pea embryos contain two major isoforms of starch synthase and two isoforms of starch-branching enzyme. One of the starch synthases and both starch-branching enzymes occur both in the soluble fraction and tightly bound to starch granules. The other starch synthase, which is very similar to the waxy proteins of other species, is exclusively granule-bound. It is inactive when solubilized in a native form from starch granules, but activity is recovered when the SDS-denatured protein is reconstituted from polyacrylamide gels.
Evidence is presented which indicates that all of these proteins become incorporated within the structure of the granule as it grows. It is proposed that the granule-bound waxy protein is active in vivo at the granule surface, whereas the remaining proteins are active in the soluble fraction of the amyloplast. The proteins become trapped within the granule matrix as the polymers they synthesize crystallize around them, and they probably play no further part in polymer synthesis.     

淀粉合酶的酶学与分子生物学研究进展

淀粉合酶作为淀粉合成的关键酶之一,一直是淀粉研究的重要内容。这些研究多集中在对其同工型的研究,淀粉合酶的两类主要同工型分别为淀粉粒结合的淀粉合酶和可溶性淀粉合酶,这两类同工型的作用极为复杂。本文介绍了淀粉合酶同工型的酶学和分子生物学近年来的研究进展,同时也讨论了这些同工型的分类、相互关系及其在淀粉合成过程中的生理功能等内容。     

Improving wheat flour hydrolysis by an enzyme mixture from solid state fungal fermentation

In traditional cereal-based industrial processes, component separation is often incomplete resulting in a residue of mixed macromolecules including largely starch, protein, phytic acid and many others. The development of a viable cereal-based biorefinery would involve effective bioconversion of cereal components for the production of a nutrient-complete fermentation feedstock. Simultaneous starch and protein hydrolysis represents an effective approach to the production of platform chemicals from wheat. Solid state fermentations of wheat pieces and waste bread by Aspergillus oryzae and Aspergillus awamori have been combined in this study to enhance starch and protein hydrolysis. Kinetic studies confirmed that the proteolytic enzymes from A. oryzae introduced no negative effect on the stability of the amylolytic enzymes from A. awamori under the optimal conditions for starch hydrolysis. When applied to hydrolyse wheat flour, the enzyme solution from A. awamori converted nearly all of the starch into glucose and 23% of the total nitrogen (TN) into free amino nitrogen (FAN). Under the same reaction conditions the enzyme solution from A. oryzae hydrolysed 38% of the protein but only 18.5% of the starch. A mixture of the two enzyme solutions hydrolysed 34.1% of the protein, a 1.5-fold increase from that achieved by the enzyme solution from A. awamori, while maintaining a near completion of starch hydrolysis.     

Starches from A to C. Chlamydomonas reinhardtii as a model microbial system to investigate the biosynthesis of the plant amylopectin crystal.

Wide-angle powder x-ray diffraction analysis was carried out on starch extracted from wild-type and mutant Chlamydomonas reinhardtii cells. Strains containing no defective starch synthases as well as mutants carrying a disrupted granule-bound starch synthase structural gene displayed the A type of diffraction pattern with a high degree of crystallinity. Mutants carrying a defect for the major soluble starch synthase (SSS), SSS II, were characterized by a switch to the B type of diffraction pattern with very low crystallinity. Mutant strains carrying SSS I as the only glucan elongation enzyme regained some of their crystallinity but switched to the C type of diffraction pattern. Differential scanning calorimetry analysis correlated tightly with the x-ray diffraction results. Together with the electron microscopy analyses, these results establish C. reinhardtii as a microbial model system displaying all aspects of cereal starch synthesis and structure. We further show that SSS II is the major enzyme involved in the synthesis of crystalline structures in starch and demonstrate that SSS I alone builds a new type of amylopectin structure.     

Modification of starch metabolism in transgenic Arabidopsis thaliana increases plant biomass and triples oilseed production

We have identified a novel means to achieve substantially increased vegetative biomass and oilseed production in the model plant Arabidopsis thaliana. Endogenous isoforms of starch branching enzyme (SBE) were substituted by either one of the endosperm‐expressed maize (Zea mays L.) branching isozymes, ZmSBEI or ZmSBEIIb. Transformants were compared with the starch‐free background and with the wild‐type plants. Each of the maize‐derived SBEs restored starch biosynthesis but both morphology and structure of starch particles were altered. Altered starch metabolism in the transformants is associated with enhanced biomass formation and more‐than‐trebled oilseed production while maintaining seed oil quality. Enhanced oilseed production is primarily due to an increased number of siliques per plant whereas oil content and seed number per silique are essentially unchanged or even modestly decreased. Introduction of cereal starch branching isozymes into oilseed plants represents a potentially useful strategy to increase biomass and oilseed production in related crops and manipulate the structure and properties of leaf starch.     

Protein phosphorylation regulates maize endosperm starch synthase IIa activity and protein−protein interactions

Starch synthesis is an elaborate process employing several isoforms of starch synthases (SSs), starch branching enzymes (SBEs) and debranching enzymes (DBEs). In cereals, some starch biosynthetic enzymes can form heteromeric complexes whose assembly is controlled by protein phosphorylation. Previous studies suggested that SSIIa forms a trimeric complex with SBEIIb, SSI, in which SBEIIb is phosphorylated. This study investigates the post-translational modification of SSIIa, and its interactions with SSI and SBEIIb in maize amyloplast stroma. SSIIa, immunopurified and shown to be free from other soluble starch synthases, was shown to be readily phosphorylated, affecting V_max but with minor effects on substrate K_d and K_m values, resulting in a 12-fold increase in activity compared with the dephosphorylated enzyme. This ATP-dependent stimulation of activity was associated with interaction with SBEIIb, suggesting that the availability of glucan branching limits SSIIa and is enhanced by physical interaction of the two enzymes. Immunoblotting of maize amyloplast extracts following non-denaturing polyacrylamide gel electrophoresis identified multiple bands of SSIIa, the electrophoretic mobilities of which were markedly altered by conditions that affected protein phosphorylation, including protein kinase inhibitors. Separation of heteromeric enzyme complexes by GPC, following alteration of protein phosphorylation states, indicated that such complexes are stable and may partition into larger and smaller complexes. The results suggest a dual role for protein phosphorylation in promoting association and dissociation of SSIIa-containing heteromeric enzyme complexes in the maize amyloplast stroma, providing new insights into the regulation of starch biosynthesis in plants.