Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms

The dynamic changes of the activities of enzymes involving in starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthases (SSS), starch branching enzyme (SBE) and starch debranching enzymes (DBE) were studied, and changes of fine structure of amy- lopectin were characterized by isoamylase treatment during rice grain development, using trans anti-waxy gene rice plants. The relationships between the activities of those key enzymes were also analyzed. The amylose synthesis was significantly inhibited in transgenic Wanjing 9522, but the total starch content and final grain weight were less affected as compared with those of non-transgenic Wanjing 9522 rice cultivar. Analyses on the changes of activities of enzymes involving in starch bio- synthesis showed that different enzyme activities were expressed differently during rice endosperm development. Soluble starch synthase is relatively highly expressed in earlier stage of endosperm de- velopment, whilst maximal expression of granule-bound starch synthase (GBSS) occurred in mid-stage of endosperm development. No obvious differences in changes of the activities of AGPase and SBE between two rice cultivars investigated, except the DBEs. Distribution patterns of branches of amy- lopectin changed continually during the development of rice grains and varied between two rice culti- vars. It was suggested that amylopectin synthesis be prior to the synthesis of amylose and different enzymes have different roles in controlling syntheses of branches of amylopectin.     

Gene expression of the biosynthetic enzymes and biosynthesis of starch during Rice Grain development

Amylose and amylopectin are determinants of the physicochemical properties for starch and grain quality in rice. Their biosynthesis is catalyzed by the interplay of ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SSS), a starch branching enzyme (SBE), and a starch debranching enzyme (SDE). In this study, the genes for these enzymes were highly expressed 7 to 28 days after flowering during grain development, and their expression closely matched increases in both starch content and grain weight Among all the tested cultivars, amylose contents in the rice grains remained essentially constant throughout their development The AGPase gene was highly expressed in the high-yield cultivars of both glutinous and non-glutinous rice. The SSS gene was actively expressed when mature GBSS mRNA decreased. Genes responsible for amylopectin biosynthesis were simultaneously expressed in the late stage of grain development. We have now demonstrated that the expression patterns of starch biosynthetic genes differ between glutinous and non-glutinous rice, and between Tongil (a Japonica/ Indica hybrid) and Japonica types.     

Starch synthesis in the cereal endosperm

The pathway of starch synthesis in the cereal endosperm is unique, and requires enzyme isoforms that are not present in other cereal tissues or non-cereal plants. Recent information on the functions of individual enzyme isoforms has provided insight into how the linear chains and branch linkages in cereal starch are synthesized and distributed. Genetic analyses have led to the formulation of models for the roles of de-branching enzymes in cereal starch production, and reveal pleiotropic effects that suggest that certain enzymes may be physically associated. For the first time, tools for global analyses of starch biosynthesis are available for cereal crops, and are heralded by the draft sequence of the rice genome.     

作物淀粉生物合成与转基因修饰研究进展

淀粉是高等植物中碳水化合物的主要贮藏形式 ,也是粮食作物产品的最主要成分。淀粉虽然都由直链淀粉和枝链淀粉组成 ,但在不同作物中两者的比例和枝链淀粉结构的存在很大差异。现已明确 ,直链淀粉是在颗粒结合淀粉合成酶 (granule boundstarchsynthase,GBSS)催化下合成的 ,而枝链淀粉是四种酶共同作用的结果 ,它们分别是腺嘌呤 -葡萄糖焦磷酸化酶 (ADP glucosepyrophosphorylase ,AGP) ,可溶性淀粉合成酶 (solublestarchsynthase ,SSS) ,淀粉分枝酶 (starchbranchingenzyme ,SBE)和脱分枝酶 (starchdebranchingenzyme ,DBE)。一方面 ,在不同作物中 ,这些酶本身存在多种形式 ,如在玉米胚乳中 ,AGP有大亚基和小亚基之分 ,SBE又可分BE1,BEIIa ,BEIIb 3种 ,SSS也可分为SSI和SSIII(或SSIIa)两种 ,而DBE也有异淀粉酶 (isoamylase)和限制性糊精酶 (pullu lanase)两种。另一方面 ,控制特定酶的基因 ,在不同作物甚至在同一种作物的不同品种中也可能存在不同的复等位基因 ,如籼稻和粳稻的GBSS分别由蜡质基因Wxa 和Wxb 控制 ,两者编码的GBSS活性差异显著。此外 ,环境条件也可通过影响基因的转录使酶的含量或催化性能发生变化。迄今 ,国内外已获得多种马铃薯和水稻的转基因材料 ,对淀粉合成进行修饰 ,试图培育优质品     

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.     

Effect of high temperature on fine structure of amylopectin in rice endosperm by reducing the activity of the starch branching enzyme

Rice (Oryza sativa L.) grain quality is affected by the environmental temperature it experiences. To investigate the physiological molecular mechanisms of the effect of high temperatures on rice grain, a non-waxy indica rice was grown under two temperature conditions, (29/35 degrees C) and (22/28 degrees C), during the ripening stage in two phytotrons. The activities and gene expression of key enzymes for the biosynthesis of amylose and amylopectin were examined. The activity and expression levels of soluble endosperm starch synthase I were higher at 29/35 degrees C than that at 22/28 degrees C. In contrast, the activities and expression levels of the rice branching enzyme1, the branching enzyme3 and the granule bound starch synthase of the endosperm were lower at 29/35 degrees C than those at 22/28 degrees C. These results suggest that the decreased activity of starch branching enzyme reduces the branching frequency of the branches of amylopectin, which results in the increased amount of long chains of amylopectin of endosperm in rice grain at high temperature.     

Granule-bound starch synthase I. A major enzyme involved in the biogenesis of B-crystallites in starch granules.

Starch defines a semicrystalline polymer made of two different polysaccharide fractions. The A- and B-type crystalline lattices define the distinct structures reported in cereal and tuber starches, respectively. Amylopectin, the major fraction of starch, is thought to be chiefly responsible for this semicrystalline organization while amylose is generally considered as an amorphous polymer with little or no impact on the overall crystalline organization. STA2 represents a Chlamydomonas reinhardtii gene required for both amylose biosynthesis and the presence of significant granule-bound starch synthase I (GBSSI) activity. We show that this locus encodes a 69 kDa starch synthase and report the organization of the corresponding STA2 locus. This enzyme displays a specific activity an order of magnitude higher than those reported for most vascular plants. This property enables us to report a detailed characterization of amylose synthesis both in vivo and in vitro. We show that GBSSI is capable of synthesizing a significant number of crystalline structures within starch. Quantifications of amount and type of crystals synthesized under these conditions show that GBSSI induces the formation of B-type crystals either in close association with pre-existing amorphous amylopectin or by crystallization of entirely de novo synthesized material.     

Specificity of starch synthase isoforms from potato.

In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli. Significant differences in their activities are apparent which may be important in determining their specificities in vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E. coli. These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo.     

The influence of alterations in ADP-glucose pyrophosphorylase activities on starch structure and composition in potato tubers

In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway affected various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K _m for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found, but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed. Received: 2 February 1999 / Accepted: 25 March 1999     

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.     

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。     

Function and characterization of starch synthase I using mutants in rice

Four starch synthase I (SSI)-deficient rice (Oryza sativa) mutant lines were generated using retrotransposon Tos17 insertion. The mutants exhibited different levels of SSI activities and produced significantly lower amounts of SSI protein ranging from 0% to 20% of the wild type. The mutant endosperm amylopectin showed a decrease in chains with degree of polymerization (DP) 8 to 12 and an increase in chains with DP 6 to 7 and DP 16 to 19. The degree of change in amylopectin chain-length distribution was positively correlated with the extent of decrease in SSI activity in the mutants. The structural changes in the amylopectin increased the gelatinization temperature of endosperm starch. Chain-length analysis of amylopectin in the SSI band excised from native-polyacrylamide gel electrophoresis/SS activity staining gel showed that SSI preferentially synthesized DP 7 to 11 chains by elongating DP 4 to 7 short chains of glycogen or amylopectin. These results show that SSI distinctly generates DP 8 to 12 chains from short DP 6 to 7 chains emerging from the branch point in the A or B(1) chain of amylopectin. SSI seemingly functions from the very early through the late stage of endosperm development. Yet, the complete absence of SSI, despite being a major SS isozyme in the developing endosperm, had no effect on the size and shape of seeds and starch granules and the crystallinity of endosperm starch, suggesting that other SS enzymes are probably capable of partly compensating SSI function. In summary, this study strongly suggested that amylopectin chains are synthesized by the coordinated actions of SSI, SSIIa, and SSIIIa isoforms.     

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.     

Maize starch-branching enzyme isoforms and amylopectin structure. In the absence of starch-branching enzyme IIb, the further absence of starch-branching enzyme Ia leads to increased branching

Previous studies indicated that the deficiency of starch-branching enzyme (SBE) Ia in the single mutant sbe1a::Mu (sbe1a) has no impact on endosperm starch structure, whereas the deficiency of SBEIIb in the ae mutant is well known to reduce the branching of starch. We hypothesized that in maize (Zea mays) endosperm, the function of SBEIIb is predominant to that of SBEIa, and SBEIa would have an observable effect only on amylopectin structure in the absence of SBEIIb. To test this hypothesis, the mutant sbe1a was introgressed into lines containing either wx (lacking the granule-bound starch synthase GBSSI) or ae wx (lacking both SBEIIb and GBSSI) in the W64A background. Both western blotting and zymogram analysis confirmed the SBEIa deficiency in sbe1a wx and sbe1a ae wx, and the SBEIIb deficiency in ae wx and sbe1a ae wx. Using zymogram analysis, no pleiotropic effects of sbe1a genes on SBEIIa, starch synthase, or starch-debranching enzyme isoforms were observed. High-performance size exclusion chromatography analysis shows that the chain-length profiles of amylopectin as well as beta-limit dextrin were indistinguishable between wx and sbe1a wx, whereas significant differences for both were observed between ae wx and sbe1a ae wx, suggesting an effect of SBEIa on amylopectin biosynthesis that is observable only in the absence of SBEIIb. The amylopectin branch density and the average number of branches per cluster were both higher in endosperm starch from sbe1a ae wx than from ae wx. These results indicate possible functional interactions between SBE isoforms that may involve enzymatic inhibition. Both the cluster repeat distance and the distance between branch points on the short intracluster chains were similar for all genotypes however, suggesting a similar pattern of individual SBE isoforms in cluster initiation and the determination of branch point location.     

Relative turnover numbers of maize endosperm and potato tuber ADP-glucose pyrophosphorylases in the absence and presence of 3-phosphoglyceric acid

Adenosine diphosphate glucose pyrophosphorylase (AGPase; EC 2.7.7.27) synthesizes the starch precursor, ADP-glucose. It is a rate-limiting enzyme in starch biosynthesis and its activation by 3-phosphoglyceric acid (3PGA) and/or inhibition by inorganic phosphate (Pi) are believed to be physiologically important. Leaf, tuber and cereal embryo AGPases are highly sensitive to these effectors, whereas endosperm AGPases are much less responsive. Two hypotheses can explain the 3PGA activation differences. Compared to leaf AGPases, endosperm AGPases (i) lack the marked ability to be activated by 3PGA or (ii) they are less dependent on 3PGA for activity. The absence of purified preparations has heretofore negated answering this question. To resolve this issue, heterotetrameric maize ( Zea mays L.) endosperm and potato ( Solanum tuberosum L.) tuber AGPases expressed in Escherichia coli were isolated and the relative amounts of enzyme protein were measured by reaction to antibodies against a motif resident in both small subunits. Resulting reaction rates of both AGPases are comparable in the presence but not in the absence of 3PGA when expressed on an active-protein basis. We also placed the potato tuber UpReg1 mutation into the maize AGPase. This mutation greatly enhances 3PGA sensitivity of the potato AGPase but it has little effect on the maize AGPase. Thirdly, lysines known to bind 3PGA in potato tuber AGPase, but missing from the maize endosperm AGPase, were introduced into the maize enzyme. These had minimal effect on maize endosperm activity. In conclusion, the maize endosperm AGPase is not nearly as dependent on 3PGA for activity as is the potato tuber AGPase.     

Physico-chemical and degradative properties of in-planta re-structured potato starch

Starch re-structured directly in potato tubers by antisense suppression of starch branching enzyme (SBE), granule bound starch synthase (GBSS) or glucan water dikinase (GWD) genes was studied with the aim at disclosing the effects on resulting physico-chemical and enzyme degradative properties. The starches were selected to provide a combined system with specific and extensive alterations in amylose and covalently esterified glucose-6-phosphate (G6P) contents. As an effect of the altered chemical composition of the starches their hydrothermal characteristics varied significantly. Despite of the extreme alterations in phosphate content, the amylose content had a major affect on swelling power, enthalpy for starch gelatinization and pasting parameters as assessed by Rapid Visco Analysis (RVA). However, a combined influence of the starch phosphate and long glucan chains as represented by high amylose or long amylopectin chain length was indicated by their positive correlation to the final viscosity and set back (RVA) demonstrating the formation of a highly hydrated and gel-forming system during re-structuring of the starch pastes. Clear inverse correlations between glucoamylase-catalyzed digestibility and amylopectin chain length and starch phosphate and lack of such correlation with amylose content indicates a combined structuring role of the phosphate groups and amylopectin chains on the starch glucan matrix.     

复合调控Wx与SBE3基因对水稻籽粒直链淀粉含量的影响

颗粒淀粉合成酶（GBSS）和淀粉分支酶3（SBE3）是淀粉合成过程中的两个关键酶,这两个酶主要由耽和SBE3两个基因分别控制,它们的表达量直接影响直链淀粉和支链淀粉的含量比例。为了探讨水稻淀粉关键酶基因耽过量与SBE3干涉复合表达对直链淀粉含量的影响,构建了Wx过量表达与SBE3干涉结合的多基因表达载体,并通过农杆菌介导的方法将其导入日本晴水稻中。经过PCR检测分析获得了65株转基因阳性植株,半定量RT—PCR检测表明转基因株系中Wx基因表达量明显增加,而SBE3基因表达量显著减少。转基因株系籽粒透明度明显降低,直链淀粉含量比野生型的平均高45％,但是千粒重变化不大,与野生型相当。遗传分析表明这些转基因株系多数可稳定遗传。     

Temperature induced changes in the starch components and biosynthetic enzymes of two rice varieties

The effects of temperature on starch and amylose accumulation, fine structure of amylopectin and activities of some enzymes related to starch synthesis in developing rice endosperms was examined. Two early indica rice varieties were used, differing in amylose concentration (AC, %), namely Jia 935 (low AC) and Jia 353 (high AC). The results showed that the effects of high temperature on AC and amylopectin fine structure were variety-dependent. High temperature caused a reduction in amylose concentration and an increase in the short chain (CL<22) proportion of amylopectin for Jia 935; while opposite was true for Jia 353. High temperature also reduced and increased the activity of granule-bound starch synthase (GBSS) in Jia 935 and in Jia 353, respectively. This suggests that a change in the ratio of amylose/starch due to temperature was attributable to a change in GBSS activity. Moreover, obvious differences between the two rice varieties were detected in the activities of sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (ADPG-Ppase), soluble starch synthase (SSS), starch branching enzyme (SBE), starch de-branching enzyme (SDBE) and starch phosphorylase (SPase) to high temperature. Accumulation rate of amylose was significantly and positively correlated with GBSS for Jia 935, but not for Jia 353. Amylose accumulation was also significantly and positively correlated with the activities of SDBE, SBE, ADPG-Ppase and SuSy for both varieties. The results suggest that the ratio of amylose to starch in rice endosperm is not only related to GBSS, but also affected by the activities of SDBE, SBE, ADPG-Ppase and SuSy.