Amylose Biosynthesis Research Articles

Dosage effects of Waxy gene on the structures and properties of corn starch

The objective of this study was to understand dosage effects of the Waxy gene on the structures of amylose and amylopectin and on the properties of corn starch. Reciprocal crossing of isogenic normal and waxy corn lines was conducted to develop hybrids with different dosages (0, 1, 2, 3) of Waxy gene in the endosperm. The amylose content of starch and proportions of branch chains of DP 17–30 and extra-long branch chains (DP>100) of amylopectin were positively correlated with the Waxy-gene dosage. Proportions of short (DP<17) and long branch-chains (DP 30–80), however, were negatively correlated with the Waxy-gene dosage. The gelatinization conclusion-temperature and temperature-range of the starch were negatively correlated with the Waxy-gene dosage, indicating that amylose facilitated dissociation of the surrounding crystalline regions. These results helped us understand the function of granule-bound starch synthase I in the biosynthesis of amylose and amylopectin and impacts of Waxy-gene dosages on the properties of corn starch.

Improved understanding of rice amylose biosynthesis from advanced starch structural characterization.

BackgroundIt has been shown from the chain length distributions (CLDs) that amylose chains can be divided into at least two groups: long and short amylose chains. These molecular structures influence some functional properties of starch, such as digestibility and mouth-feel. GBSSI is the key enzyme for the elongation of amylose chains; however, the effect of other starch biosynthesis enzymes in amylose synthesis is still not fully understood. Two advanced starch characterization techniques, size exclusion chromatography (SEC) and fluorophore-assissted carbohydrate electrophoresis (FACE), together with a newly developed starch biosynthesis model, are used to improve understanding of amylose biosynthesis.ResultsSEC and FACE were used to determine the CLD of amylose and amylopectin in various native and mutant rice starches. The types of starch branching enzymes (SBEs) involved in the synthesis of the distinct features seen for shorter degrees of polymerization, DP, < 2000, and longer (DP > 2000) amylose chains are identified by combining these data with a mathematical model of amylopectin biosynthesis. The model enables each feature in the amylopectin CLD to be parameterized in terms of relative SBE activities, which are used to explain differences in the genotypes.ConclusionsThe results suggest that while GBSSI is the predominant enzyme controlling the synthesis of longer amylose chains, some branching enzymes (such as BEI and BEIIb) also play important roles in the synthesis of shorter amylose chains.

Effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice.

High temperature during grain filling affects yield, starch amylose content and activity of starch biosynthesis enzymes in basmati rice. To investigate the physiological mechanisms underpinning the effects of high temperature on rice grain, basmati rice was grown under two temperature conditions - 32 and 22 °C - during grain filling. High temperature decreased the grain filling period from 32 to 26 days, reducing yield by 6%, and caused a reduction in total starch (3.1%) and amylose content (22%). Measurable activities of key enzymes involved in sucrose to starch conversion, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and soluble starch synthase in endosperms developed at 32 °C were lower than those at 22 °C compared with similar ripening stage on an endosperm basis. In particular, granule-bound starch synthase (GBSS) activity was significantly lower than corresponding activity in endosperms developing at 22 °C during all developmental stages analyzed. Results suggest changes in amylose/amylopectin ratio observed in plants grown at 32 °C was attributable to a reduction in activity of GBSS, the sole enzyme responsible for amylose biosynthesis.

Dynamic development of starch granules and the regulation of starch biosynthesis in Brachypodium distachyon: comparison with common wheat and Aegilops peregrina

BackgroundThorough understanding of seed starch biosynthesis and accumulation mechanisms is of great importance for agriculture and crop improvement strategies. We conducted the first comprehensive study of the dynamic development of starch granules and the regulation of starch biosynthesis in Brachypodium distachyon and compared the findings with those reported for common wheat (Chinese Spring, CS) and Aegilops peregrina.ResultsOnly B-granules were identified in Brachypodium Bd21, and the shape variation and development of starch granules were similar in the B-granules of CS and Bd21. Phylogenetic analysis showed that most of the Bd21 starch synthesis-related genes were more similar to those in wheat than in rice. Early expression of key genes in Bd21 starch biosynthesis mediate starch synthesis in the pericarp; intermediate-stage expression increases the number and size of starch granules. In contrast, these enzymes in CS and Ae. peregrina were mostly expressed at intermediate stages, driving production of new B-granules and increasing the granule size, respectively. Immunogold labeling showed that granule-bound starch synthase (GBSSI; related to amylose synthesis) was mainly present in starch granules: at lower levels in the B-granules of Bd21 than in CS. Furthermore, GBSSI was phosphorylated at threonine 183 and tyrosine 185 in the starch synthase catalytic domain in CS and Ae. peregrina, but neither site was phosphorylated in Bd21, suggesting GBSSI phosphorylation could improve amylose biosynthesis.ConclusionsBd21 contains only B-granules, and the expression of key genes in the three studied genera is consistent with the dynamic development of starch granules. GBSSI is present in greater amounts in the B-granules of CS than in Bd21; two phosphorylation sites (Thr183 and Tyr185) were found in Triticum and Aegilops; these sites were not phosphorylated in Bd21. GBSSI phosphorylation may reflect its importance in amylose synthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0198-2) contains supplementary material, which is available to authorized users.

Deficiencies in both starch synthase IIIa and branching enzyme IIb lead to a significant increase in amylose in SSIIa-inactive japonica rice seeds.

Starch synthase (SS) IIIa has the second highest activity of the total soluble SS activity in developing rice endosperm. Branching enzyme (BE) IIb is the major BE isozyme, and is strongly expressed in developing rice endosperm. A mutant (ss3a/be2b) was generated from wild-type japonica rice which lacks SSIIa activity. The seed weight of ss3a/be2b was 74-94% of that of the wild type, whereas the be2b seed weight was 59-73% of that of the wild type. There were significantly fewer amylopectin short chains [degree of polymerization (DP) ≤13] in ss3a/be2b compared with the wild type. In contrast, the amount of long chains (DP ≥25) connecting clusters of amylopectin in ss3a/be2b was higher than in the wild type and lower than in be2b. The apparent amylose content of ss3a/be2b was 45%, which was >1.5 times greater than that of either ss3a or be2b. Both SSIIIa and BEIIb deficiencies led to higher activity of ADP-glucose pyrophosphorylase (AGPase) and granule-bound starch synthase I (GBSSI), which partly explains the high amylose content in the ss3a/be2b endosperm. The percentage apparent amylose content of ss3a and ss3a/be2b at 10 days after flowering (DAF) was higher than that of the wild type and be2b. At 20 DAF, amylopectin biosynthesis in be2b and ss3a/be2b was not observed, whereas amylose biosynthesis in these lines was accelerated at 30 DAF. These data suggest that the high amylose content in the ss3a/be2b mutant results from higher amylose biosynthesis at two stages, up to 20 DAF and from 30 DAF to maturity.

Genetic controls on starch amylose content in wheat and rice grains.

Starch accumulates in plants as granules in chloroplasts of source organs such as leaves (transitory starch) or in amyloplasts of sink organs such as seeds, tubers and roots (storage starch). Starch is composed of two types of glucose polymers: the essentially linear polymer amylose and highly branched amylopectin. The amylose content of wheat and rice seeds is an important quality trait, affecting the nutritional and sensory quality of two of the world's most important crops. In this review, we focus on the relationship between amylose biosynthesis and the structure, physical behaviour and functionality of wheat and rice grains. We briefly describe the structure and composition of starch and then in more detail describe what is known about the mechanism of amylose synthesis and how the amount of amylose in starch might be controlled. This more specifically includes analysis of GBSS alleles, the relationship between waxy allelic forms and amylose, and related quantitative trait loci. Finally, different methods for increasing or lowering amylose content are evaluated.

Tracking sulfur and phosphorus within single starch granules using synchrotron X-ray microfluorescence mapping

BackgroundNative starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2–0.5%) and proteins (0.1–0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis. MethodsSynchrotron micro-X-ray fluorescence (μXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme. ResultsWild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3–5μm in diameter. ConclusionsImaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before. General significanceμXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds.

Diversification of the Waxy Gene is Closely Related to Variations in Rice Eating and Cooking Quality

The Wx gene encoding granule-bound starch synthase is responsible for the biosynthesis of amylose, which plays an important role in determining rice eating and cooking quality. However, the functional evaluation of different Wx alleles remains unclear. In the present study, a set of near-isogenic lines (NILs) carrying four different Wx alleles—Wx a , Wx b , Wx in , and wx—was developed in the genetic background of indica rice cv. Yangfunuo 4 (YFN 4), and designated NIL-Wx a , NIL-Wx b , NIL-Wx in , and NIL-wx, respectively. These NILs show great variations in amylose content (AC), gel consistency (GC), starch viscosity, and thermodynamic property. AC showed the following trend among different NILs: NIL-Wx a > NIL-Wx in > NIL-Wx b > NIL-wx, but the opposite trend for GC. NIL-Wx a and NIL-Wx in had lower starch thermodynamic properties, such as onset temperature, peak temperature, and conclusion temperature, when compared with both NIL-Wx b and NIL-wx. The transcript and protein of Wx gene were not detectable in the NIL-wx line. The accumulation levels of Wx transcript and Wx protein were higher in NIL-Wx a than those in NIL-Wx b and NIL-Wx in . Therefore, Wx gene was closely related to starch quality parameters, such as AC, GC, starch paste viscosity, and starch thermodynamic property. Our results provide a solid foundation for rice grain quality improvement through marker-assistant selection.

Two-Dimensional Size/Branch Length Distributions of a Branched Polymer

The first two-dimensional structural distributions are reported for a hyperbranched polymer, the two dimensions being macromolecular size and individual branch length. The 2D distributions for native starch, a polymer with both short- and long-chain branched components (amylopectin and amylose), were obtained by size fractionation using size-exclusion chromatography combined with enzymatic debranching. These distributions show distinct macromolecular architectures: separate mountains corresponding to amylopectin and amylose and two foothills assigned to hybrid populations. The distributions reveal new mechanistic information on the underlying polymer synthesis. The branch-length distributions for amylopectin are independent of macromolecular size, whereas a size variation is observed for amylose. Biological imperatives in amylopectin biosynthesis force the same branching structure for all macromolecular sizes because of evolutionary pressure to provide the crystallinity indispensable for the plant survival. Amylose biosynthesis does not have such restrictive biological constraints, offering a range of branched structures throughout macromolecular sizes and revealing a previously unsuspected size dependence.

Enzymatic characterization of starch synthase III from kidney bean (Phaseolus vulgaris L.)

In plants and green algae, several starch synthase isozymes are responsible for the elongation of glucan chains in the biosynthesis of amylose and amylopectin. Multiple starch synthase isozymes, which are classified into five major classes (granule-bound starch synthases, SSI, SSII, SSIII, and SSIV) according to their primary sequences, have distinct enzymatic properties. All the starch synthase isozymes consist of a transit peptide, an N-terminal noncatalytic region (N-domain), and a C-terminal catalytic region (C-domain). To elucidate the enzymatic properties of kidney bean (Phaseolus vulgaris L.) SSIII and the function of the N-domain of kidney bean SSIII, three recombinant proteins were constructed: putative mature recombinant SSIII, recombinant kidney bean SSIII N-domain, and recombinant kidney bean SSIII C-domain. Purified recombinant kidney bean SSIII displayed high specific activities for primers as compared to the other starch synthase isozymes from kidney bean. Kinetic analysis showed that the high specific activities of recombinant kidney bean SSIII are attributable to the high k(cat) values, and that the K(m) values of recombinant kidney bean SSIII C-domain for primers were much higher than those of recombinant kidney bean recombinant SSIII. Recombinant kidney bean SSIII and recombinant kidney bean SSIII C-domain had similar chain-length specificities for the extension of glucan chains, indicating that the N-domain of kidney bean SSIII does not affect the chain-length specificity. Affinity gel electrophoresis indicated that recombinant kidney bean SSIII and recombinant kidney bean SSIII N-domain have high affinities for amylose and amylopectin. The data presented in this study provide direct evidence for the function of the N-domain of kidney bean SSIII as a carbohydrate-binding module.

Structure of starches extracted from near-isogenic wheat lines

Abstract Differential scanning calorimetry (DSC), acidic hydrolysis and different physico-chemical approaches were used to study thermodynamic and structural characteristics of starches from near-isogenic wheat lines to establish the effect of different combinations of active granule-bound starch synthase isoforms, taking part in amylose biosynthesis, on the structure and thermodynamic properties of starches. Obtained results suggest that the effect of different GBSS I combinations is realized through altered amylose localization within starch granules, reflecting in changes of melting temperature of crystalline lamellae (T m) and rates of acidic hydrolysis. It has also been demonstrated that changes in T m values for native wheat starches are determined by amylose content in amylopectin clusters.

Structure of starches extracted from near isogenic wheat lines

Differential scanning calorimetry (DSC), acidic hydrolysis and different physico-chemical approaches were used to study thermodynamic and structural characteristics of starches from near-isogenic wheat lines to establish the effect of different combinations of active granule-bound starch synthase isoforms, taking part in amylose biosynthesis, on the structure and thermodynamic properties of starches. Obtained results suggest that the effect of different GBSS I combinations is realized through altered amylose localization within starch granules, reflecting in changes of melting temperature of crystalline lamellae (T m ) and rates of acidic hydrolysis. It has also been demonstrated that changes in T m values for native wheat starches are determined by amylose content in amylopectin clusters.

Genetic Analysis of Long Chain Synthesis in Rice Amylopectin

The amount of long chains (LC) of amylopectin in high-amylose rice is thought to be one of the important determinants of its quality when cooked. A wide range of differences in LC content have been reported in rice varieties, which can be clearly divided into four classes based on LC and apparent amylose content: namely, amylose and LC-free, low or medium-amylose and low-LC, high-amylose and medium-LC, high-amylose and high-LC. However, genetic factors controlling LC content have not been fully understood. Here, we performed quantitative trait loci (QTL) analysis of LC content using 157 recombinant inbred lines (RILs) derived from a cross of a low-LC cultivar, Hyogokitanishiki, and a high-LC line, Hokuriku 142. By analyzing randomly selected 15 RILs, it was shown that high LC content (≥11%) was associated with high setback viscosity (≥200 RVU), and that low LC (≤ 3%) was associated with low setback viscosity (≤ 130 RVU), as measured by a Rapid Visco Analyzer. With setback viscosity as an indicator for LC content, QTL analysis was conducted using 60 DNA markers including a CAPS marker that distinguished Wx a and Wx b alleles coding for granule-bound starch synthase I (GBSSI or Wx protein), the enzyme working for amylose biosynthesis. Only one QTL with a peak log of likelihood score at the wx locus was detected, and no line showing setback viscosity corresponding to the medium-LC class appeared. The fact that wx mutants of Hokuriku 142 lacked LC in their rice starch supports the view that the functional Wx allele is indispensable for LC synthesis in addition to amylose synthesis in rice endosperm. We suggest three possible reasons why no line with medium-LC content was observed. First, the locus (loci) responsible for generation of medium-LC may be located very close to the wx locus and not able to be dissected by the population and DNA markers we used. Second, there may be special QTLs for medium-LC cultivars that do not exist in low- or high-LC cultivars. Third, medium-LC cultivars may have an as-yet unidentified Wx allele with lower capability in LC synthesis compared to the Wx allele in high-LC cultivars.

Modelling epistatic effects of embryo and endosperm QTL on seed quality traits

Coordinated expression of embryo and endosperm tissues is required for proper seed development. The coordination among these two tissues is controlled by the interaction between multiple genes expressed in the embryo and endosperm genomes. In this article, we present a statistical model for testing whether quantitative trait loci (QTL) active in different genomes, diploid embryo and triploid endosperm, epistatically affect a trait expressed on the endosperm tissue. The maximum likelihood approach, implemented with the EM algorithm, was derived to provide the maximum likelihood estimates of the locations of embryo- and endosperm-specific QTL and their main effects and epistatic effects. This model was used in a real example for rice in which two QTL, one from the embryo genome and the other from the endosperm genome, exert a significant interaction effect on gel consistency on the endosperm. Our model has successfully detected Waxy, a candidate gene in the embryo genome known to regulate one of the major steps of amylose biosynthesis in the endosperm. This model will have great implications for agricultural and evolutionary genetic research.

植物のデンプン生合成関連酵素の酵素化学的研究

About three centuries have already passed since Antoni van Leeuwenhoek observed the gelatinization of starch granule under a microscope in 1719. Now, many people know not only the word “starch,” but also the reaction in which starch is digested by α-amylases contained in our saliva. However nobody can correctly address the question how plant starch is made. The final aims of our research were to elucidate the mechanism of plant starch biosynthesis and then to take advantage of the knowledge in our lives. For that purpose, we tried to understand the characteristics of various enzymes related to starch biosynthesis. We have isolated cDNA clones for ADPglucose pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (DBE) from kidney bean (Phaseolus vulgaris L.) plants. Northern blot analyses showed that transcripts for all AGPases and SBEs isolated in this study accumulate in both leaves and seeds, whereas the profiles of organ-specific expression for SSs and DBEs differ with each isozyme. To investigate enzymatic properties, several recombinant enzymes were purified from Escherichia coli cells. Three SS isozymes (designated rPvSSI, rPvSSIIb and rPvGBSSIa) showed distinct chain-length specificities for the extension of glucan chains. rPvGBSSIa acts on chains of approximately DP=15 of amylopectin and elongates them processively to synthesize ultimate long chains. The elongation properties of chains by PvGBSSIa isozyme must have a pivotal role in amylose biosynthesis. Two SBE isozymes (designated rPvSBE1 and rPvSBE2) were also purified from E. coli cells and their enzymatic properties were determined. Western blot analysis with antisera raised against rPvSBE1 and rPvSBE2 showed that these two SBEs were located in different amyloplast fractions of developing seeds of kidney bean: PvSBE2 was present in the soluble fraction, whereas PvSBE1 was associated with starch granule fraction. These differences in location suggest that these two SBE isozymes have different roles in amylopectin synthesis in kidney bean seeds. Moreover, we showed that a larger form of PvSBE2, LF-PvSBE2, exists which contains an extended N-terminal region. Unlike the soluble location of PvSBE2, LF-PvSBE2 is observed in both the soluble and starch-granule fractions. We also demonstrated that the extended N-terminal region in LF-PvSBE2 alters not only its subcellular location but also its kinetic properties as well. The two isoforms are encoded by the same gene, which produces two distinct transcripts generated by alternative splicing of the first two exons. Our results shed new insights on our understanding of the regulatory mechanism of amylopectin biosynthesis.

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 °C) and (22/28 °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 °C than that at 22/28 °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 °C than those at 22/28 °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.

Gene dosage effect of the wheat Wx alleles and their interaction on amylose synthesis in the endosperm

To find out gene dose effect of each of the three homoeologous Wx genes and their interaction on the production of granule-bound starch synthase (GBSS I) and amylose biosynthesis in the endosperm, Chinese Spring and its near-isogenic waxy types were crossed reciprocally and, obtained a plant population with varying doses of each Wx gene. The amount of GBSSI was increased linearly with increasing gene dose of either of Wxloci. In each of the three Wx loci, the change in amylose content was linear up to 3 doses, with a more potent capacity ofWx-B1a at any dose. Higher level of amylose production was observed in the reciprocal F1 grains than the expected effect of dose/s of each gene or additive effect of different allelic combination by artificially blend starches which have amylose produced by equivalent number ofWx alleles to that of relevant F1 cross. When Wx-B1a and Wx-A1a were combined, increase in amylase content was not in proportion to increase in gene dosage. The enhanced amylase synthesis was shown by 2-gene and 3-geneinteraction, indicating that not only type of the three Wx genes and its dose but the interaction among them have significant roles in determining the amylose content.

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.

Interaction of rice bZIP protein REB with the 5?- upstream re-gion of both rice sbe1 gene and waxy gene

Rice starch branching enzyme 1 (SBE1) and granule-bound starch synthase (GBSS) catalyzed the biosynthesis of amylopectin and amylose in rice developing endosperm respectively, and the genes encoding these two enzymes, sbe1 and waxy, are mainly expressed in the endosperm. Within the 5′-upstream region of rice sbe1 gene, we identified a 53 bp fragment C53 which could interact with the nuclear proteins extracted from rice endosperm. We also found that fragment Ha-2 in the 5′-upstream region of rice waxy gene could compete with C53 for its interaction. Further experiments demonstrated that rice bZIP protein REB could interact with two ACGT elements (G-box and Hex) in the fragment C53 as well as three ACGT elements (WG1, WG2, and WG3) in the fragment Ha-2. The WG1 element could compete well with Hex element for its interaction with REB. These results suggested that the expression of sbe1 and waxy gene in rice developing endosperm may be coordinately regulated by REB-like bZIP transcriptional factors.

Location of Starch Granule-associated Proteins Revealed by Confocal Laser Scanning Microscopy

Abstract Distinct locations of starch granule-associated proteins were revealed using a protein-specific dye with confocal laser scanning microscopy (CLSM). The dye, 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde, fluoresces only after it reacts with primary amines in proteins, thereby removing background interference from residual dye. CLSM has the capability to discern fluorescence-labelled protein distribution in an optical slice of an intact starch granule while it is still in an intact state. With these techniques, starch granule proteins were revealed to be concentrated in internal concentric spheres in potato, maize, and wheat starches. Spheres were more distinct in potato starch than in other starches. Amylose-free potato and waxy maize starches showed no protein spheres, indicating that the internal protein spheres are composed of granule-bound starch synthase (GBSS). Identification of GBSS suggests the location of biosynthesis of amylose in starch granules, as well as spatial and temporal aspects of biosynthesis.