Insights into wheat-starch biosynthesis from two-dimensional macromolecular structure.

Starch structure is often characterized by the chain-length distribution (CLD) of the linear molecules formed by breaking each branch-point. More information can be obtained by expanding into a second dimension: in the present case, the total undebranched-molecule size. This enables answers to questions unobtainable by considering only one variable. The questions considered here are: (i) are the events independent which control total size and CLD, and (ii) do ultra-long amylopectin (AP) chains exist (these chains cannot be distinguished from amylose chains using simple size separation). This was applied here to characterize the structures of one normal (RS01) wheat and two high-amylose (AM) mutant wheats (an SBEIIa knockout and an SBEIIa and SBEIIb knockout). Absolute ethanol was used to precipitate collected fractions, then size-exclusion chromatography for total molecular size and for the size of branches. The SBEIIa and SBEIIb mutations significantly increased AM and IC contents and chain length. The 2D plots indicated the presence of small but significant amounts of long-chain amylopectin, and the asymmetry of these plots shows that the corresponding mechanisms share some causal effects. These results could be used to develop plants producing improved starches, because different ranges of the chain-length distribution contribute independently to functional properties.

Soluble and insoluble α-glucan synthesis in yeast by enzyme suites derived exclusively from maize endosperm.

Molecular mechanisms that distinguish the synthesis of semi-crystalline α-glucan polymers found in plant starch granules from the synthesis of water-soluble polymers by nonplant species are not well understood. To address this, starch biosynthetic enzymes from maize (Zea mays L.) endosperm were isolated in a reconstituted environment using yeast (Saccharomyces cerevisiae) as a test bed. Ninety strains were constructed containing unique combinations of 11 synthetic transcription units specifying maize starch synthase (SS), starch phosphorylase (PHO), starch branching enzyme (SBE), or isoamylase-type starch debranching enzyme (ISA). Soluble and insoluble branched α-glucans accumulated in varying proportions depending on the enzyme suite, with ISA function stimulating distribution into the insoluble form. Among the SS isoforms, SSIIa, SSIII, and SSIV individually supported the accumulation of glucan polymer. Neither SSI nor SSV alone produced polymers; however, synergistic effects demonstrated that both isoforms can stimulate α-glucan accumulation. PHO did not support α-glucan production by itself, but it had either positive or negative effects on polymer content depending on which SS or a combination thereof was present. The complete suite of maize enzymes generated insoluble particles resembling native starch granules in size, shape, and crystallinity. Ultrastructural analysis revealed a hierarchical assembly starting with subparticles of approximately 50 nm diameter that coalesce into discrete structures of approximately 200 nm diameter. These are assembled into semi-crystalline α-glucan superstructures up to 4 µm in length filling most of the yeast cytosol. ISA was not essential for the formation of such particles, but their abundance was increased dramatically by ISA presence.

Tuning heterologous glucan biosynthesis in yeast to understand and exploit plant starch diversity.

BACKGROUND: Starch, a vital plant-derived polysaccharide comprised of branched glucans, is essential in nutrition and many industrial applications. Starch is often modified post-extraction to alter its structure and enhance its functionality. Targeted metabolic engineering of crops to produce valuable and versatile starches requires knowledge of the relationships between starch biosynthesis, structure, and properties, but systematic studies to obtain this knowledge are difficult to conduct in plants. Here we used Saccharomyces cerevisiae as a testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. RESULTS: We explored yeast promoters and terminators to tune the expression levels of the starch-biosynthesis machinery from Arabidopsis thaliana. We systematically modulated the expression of each starch synthase (SS) together with a branching enzyme (BE) in yeast. Protein quantification by parallel reaction monitoring (targeted proteomics) revealed unexpected effects of glucan biosynthesis on protein abundances but showed that the anticipated broad range of SS/BE enzyme ratios was maintained during the biosynthetic process. The different SS/BE ratios clearly influenced glucan structure and solubility: The higher the SS/BE ratio, the longer the glucan chains and the more glucans were partitioned into the insoluble fraction. This effect was irrespective of the SS isoform, demonstrating that the elongation/branching ratio controls glucan properties separate from enzyme specificity. CONCLUSIONS: Our results provide a quantitative framework for the in silico design of improved starch biosynthetic processes in plants. Our study also exemplifies a workflow for the rational tuning of a complex pathway in yeast, starting from the selection and evaluation of expression modules to multi-gene assembly and targeted protein monitoring during the biosynthetic process.

The role of different Wx and BEIIb allele combinations on fine structures and functional properties of indica rice starches.

This study examined the effects of the combinations of Waxy (Wx) and starch branching enzyme IIb (BEIIb) alleles on starch fine structure and functional properties in indica rice cultivars. The results showed that be2b mutant starches with BEIIb deficiency had higher amylose content, shorter amylose long chains, a higher proportion of amylopectin long chains and molecular order, but a lower proportion of amylopectin short chains and relative crystallinity, resulting in higher gelatinization temperature but lower enthalpy and paste viscosity. Compared with the rice lines carrying Wxb allele, Wxa allele contributed to relatively higher amylose content, longer amylopectin chains, less short-range ordered structure and lower relative crystallinity, leading to a little lower gelatinization enthalpy. This study provides new insight into structure-function relations among rice lines with different allele combinations of starch synthesis related genes, which is a useful strategy for rice quality breeding.

Starch synthases SSIIa and GBSSI control starch structure but do not determine starch granule morphology in the absence of SSIIIa and SSIVb.

KEY MESSAGE: High levels of two major starch synthases, SSIIa and GBSSI, in ss3a ss4b double mutant rice alter the starch structure but fail to recover the polygonal starch granule morphology. The endosperm starch granule is polygonal in wild-type rice but spherical in double mutant japonica rice lacking genes encoding two of the five major Starch synthase (SS) isozymes expressed in endosperm, SSIIIa and SSIVb. Japonica rice naturally has low levels of SSIIa and Granule-bound SSI (GBSSI). Therefore, introduction of active SSIIa allele and/or high-expressing GBSSI allele from indica rice into the japonica rice mutant lacking SS isozymes can help elucidate the compensatory roles of SS isozymes in starch biosynthesis. In this study, we crossed the ss3a ss4a double mutant japonica rice with the indica rice to generate three new rice lines with high and/or low SSIIa and GBSSI levels, and examined their starch structure, physicochemical properties, and levels of other starch biosynthetic enzymes. Lines with high SSIIa levels showed more SSI and SSIIa bound to starch granule, reduced levels of short amylopectin chains (7 ≤ DP ≤ 12), increased levels of amylopectin chains with DP > 13, and consequently higher gelatinization temperature. Lines with high GBSSI levels showed an increase in amylose content. The ADP-glucose content of the crude extract was high in lines with low or high SSIIa and low GBSSI levels, but was low in lines with high GBSSI. Addition of high SSIIa and GBSSI altered the starch structure and physicochemical properties but did not affect the starch granule morphology, confirming that SSIIIa and SSIVb are key enzymes affecting starch granule morphology in rice. The relationship among SS isozymes and its effect on the amount of substrate (ADP-glucose) is discussed.

Wx alleles in rice: relationship with apparent amylose content of starch and a possible role in rice domestication.

The Waxy locus of rice is a highly polymorphic region embedded with microsatellite repeats in the 5'UTR leader intron 1 region, 23-bp duplication (wx motif) in exon 2, SNPs in exons 4, 6 and 10, p-Sine-r2 element in intron 1 and TnR-1 element in inton 13. Of the 80 polymorphic sites detected on the Wx gene, 24 are located in p-Sine-r2 and TnR-1 elements, revealing a higher substitution rate of bases in these two regions. All the cultivars with chalky endosperm had the 5'-AGTTATA-3' haplotype in intron 1 and 'A' to 'G' substitution at ?497 in exon 4. The AAC of starch from grains of all the accessions showed strong correlation (r=0.967) with GBSS-I activity in the grains. Based on the polymorphic sites of the Waxy locus and the GBSS-I activities, six allelic variants were defined which included wx, Wxop, Wxb, Wxin, Wxa2 and Wxa1, respectively, corresponded to glutinous, very low, low, intermediate, highII and highI amylose classes. Phylogenetic tree developed from alignment matrix of nucleotide sequences of the Waxy locus identified wx, Wxb and Wxin alleles with japonica lineage of Oryza sativa and the Wxop, Wxa2 and Wxa1 with indica lineage.

Inactivation of rice starch branching enzyme IIb triggers broad and unexpected changes in metabolism by transcriptional reprogramming.

Starch properties can be modified by mutating genes responsible for the synthesis of amylose and amylopectin in the endosperm. However, little is known about the effects of such targeted modifications on the overall starch biosynthesis pathway and broader metabolism. Here we investigated the effects of mutating the OsSBEIIb gene encoding starch branching enzyme IIb, which is required for amylopectin synthesis in the endosperm. As anticipated, homozygous mutant plants, in which OsSBEIIb was completely inactivated by abolishing the catalytic center and C-terminal regulatory domain, produced opaque seeds with depleted starch reserves. Amylose content in the mutant increased from 19.6 to 27.4% and resistant starch (RS) content increased from 0.2 to 17.2%. Many genes encoding isoforms of AGPase, soluble starch synthase, and other starch branching enzymes were up-regulated, either in their native tissues or in an ectopic manner, whereas genes encoding granule-bound starch synthase, debranching enzymes, pullulanase, and starch phosphorylases were largely down-regulated. There was a general increase in the accumulation of sugars, fatty acids, amino acids, and phytosterols in the mutant endosperm, suggesting that intermediates in the starch biosynthesis pathway increased flux through spillover pathways causing a profound impact on the accumulation of multiple primary and secondary metabolites. Our results provide insights into the broader implications of perturbing starch metabolism in rice endosperm and its impact on the whole plant, which will make it easier to predict the effect of metabolic engineering in cereals for nutritional improvement or the production of valuable metabolites.

STARCH SYNTHASE5, a Noncanonical Starch Synthase-Like Protein, Promotes Starch Granule Initiation in Arabidopsis.

What determines the number of starch granules in plastids is an enigmatic aspect of starch metabolism. Several structurally and functionally diverse proteins have been implicated in the granule initiation process in Arabidopsis (Arabidopsis thaliana), with each protein exerting a varying degree of influence. Here, we show that a conserved starch synthase-like protein, STARCH SYNTHASE5 (SS5), regulates the number of starch granules that form in Arabidopsis chloroplasts. Among the starch synthases, SS5 is most closely related to SS4, a major determinant of granule initiation and morphology. However, unlike SS4 and the other starch synthases, SS5 is a noncanonical isoform that lacks catalytic glycosyltransferase activity. Nevertheless, loss of SS5 reduces starch granule numbers that form per chloroplast in Arabidopsis, and ss5 mutant starch granules are larger than wild-type granules. Like SS4, SS5 has a conserved putative surface binding site for glucans and also interacts with MYOSIN-RESEMBLING CHLOROPLAST PROTEIN, a proposed structural protein influential in starch granule initiation. Phenotypic analysis of a suite of double mutants lacking both SS5 and other proteins implicated in starch granule initiation allows us to propose how SS5 may act in this process.

Development and identification of three functional markers associated with starch content in lotus (Nelumbo nucifera).

It have been significantly demonstrated that Hexokinase (HXK), Granule-bound starch synthase (GBSS) and ADP-glucose pyrophosphorylase (AGPase) are three critical enzymes in the starch biosynthetic pathway and are related to starch (amylose, amylopectin and total starch) content in lotus. It is important to develop functional markers in marker-assisted selection of lotus breeding. So far there have been few reports about lotus functional markers. In this study, based on insertion-deletions (INDELs) and single-nucleotide polymorphisms (SNPs), we developed three functional markers, FMHXK-E1, FMGBSS-I8 and FMAGPL-I1. FMHXK-E1 was developed based on polymorphisms of two haplotypes of NnHXK. 26 lotus cultivars that the 320-bp fragment presented in NnHXK had a lower content of amylose and a higher content of amylopectin. FMGBSS-I8 was developed based on polymorphisms of two haplotypes of NnGBSS. The group containing 32 lotus cultivars with the 210-bp fragment had less amylose content and more amylopectin content. FMAGPL-I1 was developed based on polymorphisms of two haplotypes of NnAGPL (ADP-glucose pyrophosphorylase large subunit gene). The group containing 40 lotus cultivars with the 362-bp fragment had less amylopectin, total starch content and more amylose content. According to the study, FMHXK-E1, FMGBSS-I8 and FMAGPL-I1 are closely related to lotus starch content. It could be provided research basis for molecular assisted selection of lotus starch content improve breeding efficiency.

New insights into amylose and amylopectin biosynthesis in rice endosperm.

How various isoforms of rice-starch biosynthesis enzymes interact during amylose and amylopectin synthesis is explored. The chain-length distributions of amylopectin and amylose from 95 varieties with different environmental and genetic backgrounds were obtained using size- exclusion chromatography, and fitted with biosynthesis-derived models based on isoforms of starch synthase (SSI-SSIV), starch branching enzyme (SBE, including SBEI and SBEII) and granule-bound starch synthase (GBSS) that are involved in amylose and amylopectin synthesis. It is usually thought that these are synthesized by separate enzymes. However, the amount of longer amylopectin chains correlated with that of shorter amylose chains, indicating that GBSS, SBE and SS affect both amylose and amylopectin synthesis. Further, the activity of GBSS in amylose correlated with that of SS in amylopectin. This new understanding of which enzymes are suggested by the statistics to be involved in both amylose and amylopectin synthesis could help rice breeders develop cereals with targeted properties.

Molecular characterisation of two novel starch granule proteins 1 in wild and cultivated diploid A genome wheat species.

Starch synthase IIa, also known as starch granule protein 1 (SGP-1), plays a key role in amylopectin biosynthesis. The absence of SGP-1 in cereal grains is correlated to dramatic changes in the grains' starch content, structure, and composition. An extensive investigation of starch granule proteins in this study revealed a polymorphism in the electrophoretic mobility of SGP-1 between two species of wheat, Triticum urartu and T. monococcum; this protein was, however, conserved among all other Triticum species that share the A genome inherited from their progenitor T. urartu. Two different electrophoretic profiles were identified: SGP-A1 proteins of T. urartu accessions had a SDS-PAGE mobility similar to those of tetraploid and hexaploid wheat species; conversely, SGP-A1 proteins of T. monococcum ssp. monococcum and ssp. boeoticum accessions showed a different electrophoretic mobility. The entire coding region of the two genes was isolated and sequenced in an attempt to explain the polymorphism identified. Several single nucleotide polymorphisms (SNPs) responsible for amino acid changes were identified, but no indel polymorphism was observed to explain the difference in electrophoretic mobility. Amylose content did not differ significantly among T. urartu, T. monococcum ssp. boeoticum and T. monococcum ssp. monococcum, except in one accession of the ssp. boeoticum. Conversely, several interspecific differences were observed in viscosity properties (investigated as viscosity profiles using a rapid visco analyzer-RVA profiles) of these cereal grains. T. monococcum ssp. boeoticum accessions had the lowest RVA profiles, T. urartu accessions had an intermediate RVA profile, whereas T. monococcum ssp. monococcum showed the highest RVA profile. These differences could be associated with the numerous amino acid and structural changes evident among the SGP-1 proteins.

Distinct Functions of STARCH SYNTHASE 4 Domains in Starch Granule Formation.

The formation of normal starch granules in Arabidopsis (Arabidopsis thaliana) leaf chloroplasts requires STARCH SYNTHASE 4 (SS4). In plants lacking SS4, chloroplasts typically produce only one round granule rather than multiple lenticular granules. The mechanisms by which SS4 determines granule number and morphology are not understood. The N-terminal region of SS4 is unique among SS isoforms and contains several long coiled-coil motifs, typically implicated in protein-protein interactions. The C-terminal region contains the catalytic glucosyltransferase domains, which are widely conserved in plant SS and bacterial glycogen synthase (GS) isoforms. We investigated the specific roles of the N- and C-terminal regions of SS4 by expressing truncated versions of SS4 and a fusion between the N-terminal region of SS4 and GS in the Arabidopsis ss4 mutant. Expression of the N-terminal region of SS4 alone did not alter the ss4 mutant phenotype. Expression of the C-terminal region of SS4 alone increased granule initiation but did not rescue their aberrant round morphology. Expression of a self-priming GS from Agrobacterium tumefaciens also increased the number of round granules. Remarkably, fusion of the N-terminal region of SS4 to A. tumefaciens GS restored the development of wild-type-like lenticular starch granules. Interestingly, the N-terminal region of SS4 alone or when fused to GS conferred a patchy subchloroplastic localization similar to that of the full-length SS4 protein. Considered together, these data suggest that, while the glucosyltransferase activity of SS4 is important for granule initiation, the N-terminal part of SS4 serves to establish the correct granule morphology by properly localizing this activity.

Waxy and non-waxy barley cultivars exhibit differences in the targeting and catalytic activity of GBSS1a.

Amylose synthesis is strictly associated with activity of granule-bound starch synthase (GBSS) enzymes. Among several crops there are cultivars containing starch types with either little or no amylose known as near-waxy or waxy. This (near) amylose-free phenotype is associated with a single locus (waxy) which has been mapped to GBSS-type genes in different crops. Most waxy varieties are a result of either low or no expression of a GBSS gene. However, there are some waxy cultivars where the GBSS enzymes are expressed normally. For these types, single nucleotide polymorphisms have been hypothesized to represent amino-acid substitutions leading to loss of catalytic activity. We here confirm that the HvGBSSIa enzyme from one such waxy barley variety, CDC_Alamo, has a 90% reduction in catalytic activity. We also engineered plants with expression of transgenic C-terminal green fluorescent protein-tagged HvGBSSIa of both the non-waxy type and of the CDC_Alamo type to monitor their subcellular localization patterns in grain endosperm. HvGBSSIa from non-waxy cultivars was found to localize in discrete concentric spheres strictly within starch granules. In contrast, HvGBSSIa from waxy CDC_Alamo showed deficient starch targeting mostly into unknown subcellular bodies of 0.5-3 µm in size, indicating that the waxy phenotype of CDC_Alamo is associated with deficient targeting of HvGBSSIa into starch granules.

Identification of a novel starch synthase III from the picoalgae Ostreococcus tauri.

Hydrosoluble glycogen is the major energy storage compound in bacteria, archaea, fungi, and animal cells. In contrast, photosynthetic eukaryotes have evolved to build a highly organized semicrystalline granule of starch. Several enzymes are involved in polysaccharide synthesis, among which glycogen or starch synthase catalyze the elongation of the α-1,4-glucan chain. Ostreococcus tauri, accumulates a single starch granule and contains three starch synthase III (SSIII) isoforms, known as OsttaSSIII-A, OsttaSSIII-B and OsttaSSIII-C. After amino acids sequence analysis we found that OsttaSSIII-C lacks starch-binding domains, being 49% identical to the catalytic region of the SSIII from Arabidopsis thaliana and 32% identical to the entire Escherichia coli glycogen synthase. The recombinant, highly purified OsttaSSIII-C exhibited preference to use as a primer branched glycans (such as rabbit muscle glycogen and amylopectin), rather than amylose. Also, the enzyme displayed a high affinity toward ADP-glucose. We found a marked conservation of the amino acids located in the catalytic site, and specifically determined the role of residues R270, K275 and E352 by site-directed mutagenesis. Results show that these residues are important for OsttaSSIII-C activity, suggesting a strong similarity between the active site of the O. tauri SSIII-C isoform and other bacterial glycogen synthases.

Direct Characterization of the Maize Starch Synthase IIa Product Shows Maltodextrin Elongation Occurs at the Non-reducing End.

A comprehensive description of starch biosynthesis and granule assembly remains undefined despite the central nature of starch as an energy storage molecule in plants and as a fundamental calorie source for many animals. Multiple theories regarding the starch synthase (SS)-catalyzed assembly of (α1-4)-linked d-glucose molecules into maltodextrins generally agree that elongation occurs at the non-reducing terminus based on the degradation of radiolabeled maltodextrins, although recent reports challenge this hypothesis. Surprisingly, a direct analysis of the SS catalytic product has not been reported, to our knowledge. We expressed and characterized recombinant Zea mays SSIIa and prepared pure ADP-[13CU]glucose in a one-pot enzymatic synthesis to address the polarity of maltodextrin chain elongation. We synthesized maltoheptaose (degree of polymerization 7) using ADP-[13CU]glucose, maltohexaose (degree of polymerization 6), and SSIIa. Product analysis by ESI-MS revealed that the [13CU]glucose unit was added to the non-reducing end of the growing chain, and SSIIa demonstrated a >7,850-fold preference for addition to the non-reducing end versus the reducing end. Independent analysis of [13CU]glucose added to maltohexaose by SSIIa using solution NMR spectroscopy confirmed the polarity of maltodextrin chain elongation.

Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins.

Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.

Comparative in vitro analyses of recombinant maize starch synthases SSI, SSIIa, and SSIII reveal direct regulatory interactions and thermosensitivity.

Starch synthases SSI, SSII, and SSIII function in assembling the amylopectin component of starch, but their specific roles and means of coordination are not fully understood. Genetic analyses indicate regulatory interactions among SS classes, and physical interactions among them are known. The N terminal extension of cereal SSIII, comprising up to 1200 residues beyond the catalytic domain, is responsible at least in part for these interactions. Recombinant maize SSI, SSIIa, and full-length or truncated SSIII, were tested for functional interactions regarding enzymatic activity. Amino-terminal truncated SSIII exhibited reduced activity compared to full-length enzyme, and addition of the N terminus to the truncated protein stimulated catalytic activity. SSIII and SSI displayed a negative interaction that reduced total activity in a reconstituted system. These data demonstrate that SSIII is both a catalytic and regulatory factor. SSIII activity was reduced by approximately 50% after brief incubation at 45 °C, suggesting a role in reduced starch accumulation during growth in high temperatures. Buffer effects were tested to address a current debate regarding the SS mechanism. Glucan stimulated the SSIIa and SSIII reaction rate regardless of the buffer system, supporting the accepted mechanism in which glucosyl units are added to exogenous primer substrates.

The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species.

Underlying Mechanisms of Zymographic Diversity in Starch Synthase I and Pullulanase in Rice-Developing Endosperm.

Amylopectin is synthesized by the coordinated actions of many (iso)enzymes, including ADP-glucose pyrophosphorylase (AGPase), starch synthases (SSs), branching enzymes (BEs), and debranching enzymes (DBEs). Here, two polymorphic forms of starch synthase I (SSI) and pullulanase (PUL) in rice-developing seeds, designated as SSI-1/SSI-2 and PUL-1/PUL-2, were discovered for the first time by zymographic analysis. The SSI and PUL polymorphisms were strongly associated with the SSI microsatellite marker (p = 3.6 × 10(-37)) and PUL insertion/deletion (InDel) markers (p < 3.6 × 10(-51)). Western blotting and mass spectrometric analysis confirmed that the polymorphic bands were truly the SSI and PUL enzymes. Only one non-synonymous variation in SSI DNA sequence (the SNP A/G) causing the change of the amino acid K438 to E438 was observed, which coincided well with the polymorphic forms of SSI. Nine non-synonymous variations were found between PUL-1 and PUL-2. Two non-synonymous variations of PUL (F316L and D770E) were identified by mass spectrometric analysis, but all of the variations did not change the structure of PUL. The co-immunoprecipitation results revealed the differences in protein-protein interaction patterns, i.e., strong or weaker signals of SSI-BEI and SSI-BEIIb, between the two forms of SSI. The results will enhance our understanding of SSI and PUL properties and provide helpful information to understand their functions in starch biosynthesis in rice endosperm.

Functional demonstrations of starch binding domains present in Ostreococcus tauri starch synthases isoforms.

BACKGROUND: Starch-binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non-catalytic modules have already been described as essential for starch-binding and the catalytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII-A, SSIII-B and SSIII-C. RESULTS: In this work, using in silico and in vitro characterization techniques, we have demonstrated that Ostta SSIII-A, SSIII-B and SSIII-C contain two, three and no starch-binding domains, respectively. Additionally, our phylogenetic analysis has indicated that OsttaSSIII-B, presenting three N-terminal SBDs, is the isoform more closely related to higher plant SSIII. Furthermore, the sequence alignment and homology modeling data gathered showed that both the main 3-D structures of all the modeled domains obtained and the main amino acid residues implicated in starch binding are well conserved in O. tauri SSIII starch-binding domains. In addition, adsorption assays showed that OsttaSSIII-A D2 and SSIII-B D2 domains are the two that make the greatest contribution to amylose and amylopectin binding, while OsttaSSIII-B D1 is also important for starch binding. CONCLUSIONS: The results presented here suggest that differences between OsttaSSIII-A and SSIII-B SBDs in the number of and binding of amino acid residues may produce differential affinities for each isoform to polysaccharides. Increasing the knowledge about SBDs may lead to their employment in biomedical and industrial applications.