Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein–protein interactions

amylose extender (ae−) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae− maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein–protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae− mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272–Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16–20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-32P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn2+-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule

Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.ANR Génoplante GPLA0611GEuropean Union-FEDER, Région Nord Pas de Calais ARCir PlantTEQ5National Science Foundation DBI-0209789Comisión Interministerial de Ciencia y Tecnología BIO2009-07040Junta de Andalucía P09-CVI-470

Association mapping of starch physicochemical properties with starch synthesis-related gene markers in nonwaxy rice (Oryza sativa L.)

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CHARACTERIZATION OF \u3ci\u3eDE NOVO\u3c/i\u3e FATTY ACID BIOSYNthesis IN SOYBEAN SOMATIC EMBRYO PLASTIDS

A method for the isolation of intact physiologically active plastids from rapidly developing soybean (Glycine max L.) somatic embryos has been developed for the in vitro study of lipid metabolism. Using de novo fatty acid biosynthesis from 14C-acetate as a marker for physiological functionality, the greatest rates of fatty acid biosynthesis were recovered in 3000 x g fractions that were isolated in the presence of 0.5 M sorbitol, with essentially no activity occurring in the 3000 x g supernatant. Plastids purified on 10% Percoll were approximately 70 and 97 % free from mitochondrial and ER contamination, respectively, as judged by marker enzymes analysis. Isolated plastids have an absolute requirement for exogenously supplied ATP, coenzyme A and bicarbonate for in vitro fatty acid biosynthesis. The greatest rates of fatty acid biosynthesis were observed in the presence of up to 7 mM, 0.35 mM and 60 mM of each of these cofactors, respectively. Although not required for activity, fatty acid biosynthesis was improved by about 100 % by the addition of both MgCl2 and glycerol-3-phosphate. The addition or omission of NADH and NADPH had little or no effect. Fatty acid biosynthesis was optimal at pH 8 in 50 mM Tricine buffer. Under optimum conditions, maximum rates of fatty acid biosynthesis ranged from 400 to 800 nmoles acetate/hr-mg chlorophyll. In comparison to acetate, there was a strong preference for pyruvate as a precursor for fatty acid biosynthesis which was followed by glucose-6-phosphate and glucose, while malate was relatively ineffective as a precursor. Radioactivity from 14C-acetate was recovered almost exclusively in palmitic and oleic acid. Similarly, radioactivity from 14C-acetate or glycerol-3-phosphate was recovered mainly in phosphatidylcholine, phosphatidic acid and neutral lipids, with smaller amounts of phosphatidylglycerol and the plant sulfolipid. Although plastids from soybean somatic embryos are pigmented green with chlorophyll, light has essentially no effect on plastid lipid metabolism. Our observations indicate that soybean embryo plastids more closely physiologically resemble heterotrophic plastids than photosynthetic plastids

Proteome and phosphoproteome analysis of starch granule-associated proteins from normal maize and mutants affected in starch biosynthesis

In addition to the exclusively granule-bound starch synthase GBSSI, starch granules also bind significant proportions of other starch biosynthetic enzymes, particularly starch synthases (SS) SSI and SSIIa, and starch branching enzyme (BE) BEIIb. Whether this association is a functional aspect of starch biosynthesis, or results from non-specific entrapment during amylopectin crystallization, is not known. This study utilized genetic, immunological, and proteomic approaches to investigate comprehensively the proteome and phosphoproteome of Zea mays endosperm starch granules. SSIII, BEI, BEIIa, and starch phosphorylase were identified as internal granule-associated proteins in maize endosperm, along with the previously identified proteins GBSS, SSI, SSIIa, and BEIIb. Genetic analyses revealed three instances in which granule association of one protein is affected by the absence of another biosynthetic enzyme. First, eliminating SSIIa caused reduced granule association of SSI and BEIIb, without affecting GBSS abundance. Second, eliminating SSIII caused the appearance of two distinct electrophoretic mobility forms of BEIIb, whereas only a single migration form of BEIIb was observed in wild type or any other mutant granules examined. Third, eliminating BEIIb caused significant increases in the abundance of BEI, BEIIa, SSIII, and starch phosphorylase in the granule, without affecting SSI or SSIIa. Analysis of the granule phosphoproteome with a phosphorylation-specific dye indicated that GBSS, BEIIb, and starch phosphorylase are all phosphorylated as they occur in the granule. These results suggest the possibility that starch metabolic enzymes located in granules are regulated by post-translational modification and/or protein–protein interactions

Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method

Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80–90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published Km values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective Km values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass–action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways

Deficiency of maize starch-branching enzyme i results in altered starch fine structure, decreased digestibility and reduced coleoptile growth during germination

<p>Abstract</p> <p>Background</p> <p>Two distinct starch branching enzyme (SBE) isoforms predate the divergence of monocots and dicots and have been conserved in plants since then. This strongly suggests that both SBEI and SBEII provide unique selective advantages to plants. However, no phenotype for the SBEI mutation, <it>sbe1a</it>, had been previously observed. To explore this incongruity the objective of the present work was to characterize functional and molecular phenotypes of both <it>sbe1a </it>and wild-type (Wt) in the W64A maize inbred line.</p> <p>Results</p> <p>Endosperm starch granules from the <it>sbe1a </it>mutant were more resistant to digestion by pancreatic α-amylase, and the <it>sbe1a </it>mutant starch had an altered branching pattern for amylopectin and amylose. When kernels were germinated, the <it>sbe1a </it>mutant was associated with shorter coleoptile length and higher residual starch content, suggesting that less efficient starch utilization may have impaired growth during germination.</p> <p>Conclusions</p> <p>The present report documents for the first time a molecular phenotype due to the absence of SBEI, and suggests strongly that it is associated with altered physiological function of the starch <it>in vivo</it>. We believe that these results provide a plausible rationale for the conservation of SBEI in plants in both monocots and dicots, as greater seedling vigor would provide an important survival advantage when resources are limited.</p

Biochemical and genetic analyses of physical associations among Zea mays starch biosynthetic enzymes

Mutations affecting specific starch biosynthetic enzymes commonly have pleiotropic effects on other enzymes in the same metabolic pathway. Such genetic evidence indicates functional relationships between components of the starch biosynthetic system including starch synthases (SS), starch branching enzymes (BE), and starch debranching enzymes (DBE), however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/or DBEs associate physically with each other in multisubunit complexes. To test this hypothesis, this study sought to identify stable associations between SSI, SSIIa, SSIII, SBEI, SBEIIa, and SBEIIb from maize amyloplasts. Three separate detection methods, yeast two-hybrid, co-immunoprecipitation, and affinity purification using recombinant proteins as the solid phase ligand were used to identify specific protein-protein interactions. Numerous instances were detected of specific pairs of proteins associating either directly or indirectly in the same multi-subunit complex.;Gel permeation chromatography of proteins extracted from maize amyloplasts revealed two high molecular weight complexes of approximately 600kDa (C600) and 300kDa (C300) containing either SSIIa, SSIII, SBEIIa, and SBEIIb, or SSIIa, SBEIIa, and SBEIIb, respectively. To further characterize these interactions, genetic analyses tested the interdependence of specific starch biosynthetic enzymes on each other for assembly into the complexes. Association of SSIIa, SBEIIa, and SBEIIb into C600 was found to require the presence of SSIII, however, loss of SSIII did not affect assembly of the C300 complex. Further purification of the complexes through successive chromatography steps demonstrated SSIII, SSIIa, SBEIIa, and SBEIIb co-purified with C600 and the latter three proteins co-purified with C300. These data support the hypothesis that all four enzymes are present in C600 and that SSIII mediates assembly of the other three proteins into that quaternary structure. Additional proteins that co-purified with each complex were identified, specifically pyruvate orthophosphate dikinase (PPDK) and sucrose synthase1. Both of these proteins bound to a specific conserved, non-catalytic fragment of SSIII expressed in E. coli, and co-immunoprecipitated with SSIII. Association of PPDK and starch biosynthetic enzymes suggests a means of global regulation of carbon partitioning between protein and starch in developing seeds.;Finally, the observed, specific interactions among the SSs and SBEs were further examined. Direct protein-protein interactions were reconstituted in a minimal system. Three full-length maize enzymes (SSI, SBEIIa, and SBEIIb) and one conserved domain known to participate in complex formation (SSIIIHD) were expressed in E. coli and purified. Pull-down experiments revealed direct binding between SSIIIHD and SSI, SBEIIa, and SBEIIb. This binding occurred in the absence of any other maize factors. SSIIIHD and SSI were phosphorylated after incubation with soluble extracts of maize amyloplasts. Phosphorylation of SSIIIHD enhanced its ability to bind SSI. These data provide novel information about the specificity of interactions between starch biosynthetic enzymes, and further demonstrate that phosphorylation is likely to play a regulatory role in assembly and activity of the complexes

Development of high amylose wheat through TILLING

BACKGROUND: Wheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes. RESULTS: Using TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels compared to wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry. CONCLUSIONS: We report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents

Increasing the amylose content of durum wheat through silencing of the SBEIIa genes

<p>Abstract</p> <p>Background</p> <p>High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter.</p> <p>Results</p> <p>Amylose content was markedly increased in the durum wheat transgenic lines exhibiting <it>SBEIIa </it>gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR).</p> <p>Conclusion</p> <p>We have found that the silencing of <it>SBEIIa </it>genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.</p