RESUMEN
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 Vmax but with minor effects on substrate Kd and Km 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.
Asunto(s)
Endospermo/metabolismo , Proteínas de Plantas/metabolismo , Almidón Sintasa/metabolismo , Zea mays/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Endospermo/enzimología , Glucanos/metabolismo , Inmunoprecipitación , Fosforilación , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/fisiología , Plastidios/metabolismo , Almidón/metabolismo , Almidón Sintasa/aislamiento & purificación , Almidón Sintasa/fisiología , Zea mays/enzimologíaRESUMEN
The mechanisms underlying starch granule initiation remain unknown. We have recently reported that mutation of soluble starch synthase IV (SSIV) in Arabidopsis thaliana results in restriction of the number of starch granules to a single, large, particle per plastid, thereby defining an important component of the starch priming machinery. In this work, we provide further evidence for the function of SSIV in the priming process of starch granule formation and show that SSIV is necessary and sufficient to establish the correct number of starch granules observed in wild-type chloroplasts. The role of SSIV in granule seeding can be replaced, in part, by the phylogenetically related SSIII. Indeed, the simultaneous elimination of both proteins prevents Arabidopsis from synthesizing starch, thus demonstrating that other starch synthases cannot support starch synthesis despite remaining enzymatically active. Herein, we describe the substrate specificity and kinetic properties of SSIV and its subchloroplastic localization in specific regions associated with the edges of starch granules. The data presented in this work point to a complex mechanism for starch granule formation and to the different abilities of SSIV and SSIII to support this process in Arabidopsis leaves.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Plastidios/metabolismo , Almidón Sintasa/fisiología , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Microscopía Confocal , Microscopía Electrónica de Transmisión , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/ultraestructura , Plastidios/genética , Plastidios/ultraestructura , Almidón Sintasa/genéticaRESUMEN
Starch synthases (SS) are responsible for elongating the alpha-1,4 glucan chains of starch. A doubled haploid population was generated by crossing a line of wheat, which lacks functional ssIIa genes on each genome (abd), and an Australian wheat cultivar, Sunco, with wild type ssIIa alleles on each genome (ABD). Evidence has been presented previously indicating that the SGP-1 (starch granule protein-1) proteins present in the starch granule in wheat are products of the ssIIa genes. Analysis of 100 progeny lines demonstrated co-segregation of the ssIIa alleles from the three genomes with the SGP-1 proteins, providing further evidence that the SGP-1 proteins are the products of the ssIIa genes. From the progeny lines, 40 doubled haploid lines representing the eight possible genotypes for SSIIa (ABD, aBD, AbD, ABd, abD, aBd, Abd, abd) were characterized for their grain weight, protein content, total starch content and starch properties. For some properties (chain length distribution, pasting properties, swelling power, and gelatinization properties), a progressive change was observed across the four classes of genotypes (wild type, single nulls, double nulls and triple nulls). However, for other grain properties (seed weight and protein content) and starch properties (total starch content, granule morphology and crystallinity, granule size distribution, amylose content, amylose-lipid dissociation properties), a statistically significant change only occurred for the triple nulls, indicating that all three genes had to be missing or inactive for a change to occur. These results illustrate the importance of SSIIa in controlling grain and starch properties and the importance of amylopectin fine structure in controlling starch granule properties in wheat.
Asunto(s)
Dosificación de Gen , Proteínas de Plantas/metabolismo , Semillas/genética , Almidón Sintasa/genética , Almidón/biosíntesis , Triticum/genética , Biomarcadores , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Reacción en Cadena de la Polimerasa , Semillas/enzimología , Semillas/metabolismo , Almidón/química , Almidón Sintasa/fisiología , Triticum/química , Triticum/enzimologíaRESUMEN
All plants and green algae synthesize starch through the action of the same five classes of elongation enzymes: the starch synthases. Arabidopsis mutants defective for the synthesis of the soluble starch synthase IV (SSIV) type of elongation enzyme have now been characterized. The mutant plants displayed a severe growth defect but nonetheless accumulated near to normal levels of polysaccharide storage. Detailed structural analysis has failed to yield any change in starch granule structure. However, the number of granules per plastid has dramatically decreased leading to a large increase in their size. These results, which distinguish the SSIV mutants from all other mutants reported to date, suggest a specific function of this enzyme class in the control of granule numbers. We speculate therefore that SSIV could be selectively involved in the priming of starch granule formation.
Asunto(s)
Arabidopsis/enzimología , Almidón Sintasa/fisiología , Almidón/biosíntesis , Alelos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis , Mutación , Fenotipo , Plastidios/metabolismo , ARN Mensajero/metabolismoRESUMEN
Starch, the most common form of stored carbon in plants, is both the major food source for mankind and important raw material for many industries. It is composed of two types of alpha-1,4-linked glucan polymer: essentially unbranched amylose and regularly branched amylopectin, and synthesized in photosynthetic and non-photosynthetic organs. Starch is synthesized via four committed enzyme steps: ADP-Glc pyrophosphorylase, which synthesizes sugar nucleotide precursors; starch synthase, which extends the alpha-1,4-linked glucan chains using ADP-Glc; starch-branching enzymes, which introduce alpha-1,6 branch points to form amylopectin; and starch debranching enzymes, which hydrolyze alpha-1,6 branches in glucans. In this paper, recent advances in biochemical characterizations and gene engineering concerning these enzymes were reviewed, and the achievements in gene engineering involved in manipulation of starch amount and quality were also cited.
Asunto(s)
Plantas/enzimología , Almidón/biosíntesis , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/fisiología , Glucosa-1-Fosfato Adenililtransferasa/genética , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glucosa-1-Fosfato Adenililtransferasa/fisiología , Glucosidasas/genética , Glucosidasas/metabolismo , Glucosidasas/fisiología , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Sistema de la Enzima Desramificadora del Glucógeno/fisiología , Plantas/genética , Plantas/metabolismo , Almidón/metabolismo , Almidón Sintasa/genética , Almidón Sintasa/metabolismo , Almidón Sintasa/fisiologíaRESUMEN
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.
Asunto(s)
Oryza/enzimología , Proteínas de Plantas/fisiología , Almidón Sintasa/fisiología , Alelos , Amilopectina/biosíntesis , Amilopectina/química , Electroforesis en Gel de Poliacrilamida , Mutagénesis Insercional , Oryza/embriología , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Semillas/anatomía & histología , Semillas/enzimología , Semillas/genética , Almidón/biosíntesis , Almidón/ultraestructura , Almidón Sintasa/genética , Almidón Sintasa/metabolismoRESUMEN
Amylopectin, accounting for 70%-80% of storage starch, is one of the key components for quality of fruits and seeds in plant. Research on biosynthetic pathway of plant amylopectin holds great promise for modifying the structural composition of amylopectin and being used in food industry. The structure of plant amylopectin is summarized in this review and three types of amylopectin synthetase: starch branching enzyme (SBE), soluble starch synthase (SSS) and starch debranching enzyme (SDBE), which have become hotspots for research now, are expatiated in terms of genetics, enzymology and function. A model for the synthesis of amylopectin, "two-step branching and improper branch clearing model" is discussed. Problems in plant amylopectin biosynthesis and prospects for its application are also presented.
Asunto(s)
Amilopectina/biosíntesis , Plantas/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/análisis , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Enzima Ramificadora de 1,4-alfa-Glucano/fisiología , Amilopectina/química , Almidón/metabolismo , Almidón Sintasa/análisis , Almidón Sintasa/genética , Almidón Sintasa/fisiologíaRESUMEN
Plant starch synthesis can be distinguished from those of bacterial, fungal, and animal glycogen by the presence of multiple elongation (starch synthases) and branching enzymes. This complexity has precluded genetic assignment of functions to the various soluble starch synthases in the building of amylopectin. In Chlamydomonas, we have recently shown that defects in the major soluble starch synthase lead to a specific decrease in the amount of a subset of amylopectin chains whose length ranges between 8 and 40 glucose residues (Fontaine, T., D'Hulst, C., Maddelein, M.-L., Routier, F., Marianne-Pepin, T., Decq, A., Wieruszeski, J. M., Delrue, B., Van Den Koornhuyse, N., Bossu, J.-P., Fournet, B., and Ball, S. G. (1993) J. Biol. Chem. 268, 16223-16230). We now demonstrate that granule-bound starch synthase, the enzyme that was thought to be solely responsible for amylose synthesis, is involved in amylopectin synthesis. Disruption of the Chlamydomonas granule-bound starch synthase structural gene establishes that synthesis of long chains by this enzyme can become an absolute requirement for amylopectin synthesis in particular mutant backgrounds. In the sole presence of soluble starch synthase I, Chlamydomonas directs the synthesis of a major water-soluble polysaccharide fraction and minute amounts of a new type of highly branched granular material, whose structure is intermediate between those of glycogen and amylopectin. These results lead us to propose that the nature of the elongation enzyme conditions the synthesis of distinct size classes of glucans in all starch fractions.