Structural and physicochemical properties of starch gels prepared from partially modified starches using Thermus aquaticus 4-α-glucanotransferase

The modified starch gels prepared from partial enzyme treatments (1, 3, and 6 U/g starch; 2-h incubation) of the corn and rice starch pastes using Thermus aquaticus 4-α-glucanotransferase (TAαGT) were investigated for their molecular characteristics, microstructures, and physicochemical properties. Unlike the native and partially modified normal starches, the native and partially modified waxy starches could not form gels strong enough for textural analysis after 24 h for gel setting. Features of the partially modified normal starches were the specific apparent amylose contents and maximum iodine absorption wavelength (λ_max, ∼567 nm), as well as the tri-modal molecular weight profiles and flatter side-chain distributions. Also, the partially modified normal starch gels possessed fractured surfaces with discontinuous crystalline fibrous assembly that differed from the native starch gels’ porous continuous network, which resulted in more brittle, rigid, and resilient gels compared with the native gels.     

Quantification of total iodine in intact granular starches of different botanical origin exposed to iodine vapor at various water activities

Iodine has been used as an effective tool for studying both the structure and composition of dispersed starch and starch granules. In addition to being employed to assess relative amylose contents for starch samples, it has been used to look at the molecular mobility of the glucose polymers within intact starch granules based on exposure to iodine vapor equilibrated at different water activities. Starches of different botanical origin including corn, high amylose corn, waxy corn, potato, waxy potato, tapioca, wheat, rice, waxy rice, chick pea and mung bean were equilibrated to 0.33, 0.75, 0.97 water activities, exposed to iodine vapor and then absorbance spectra and LAB color were determined. In addition, a new iodine quantification method sensitive to <0.1% iodine (w/w) was employed to measure bound iodine within intact granular starch. Amylose content, particle size distribution of granules, and the density of the starch were also determined to explore whether high levels of long linear glucose chains and the surface area-to-volume ratio were important factors relating to the granular iodine binding. Results showed, in all cases, starches complexed more iodine as water content increased and waxy starches bound less iodine than their normal starch counterparts. However, much more bound iodine could be measured chemically with waxy starches than was expected based on colorimetric determination. Surface area appeared to be a factor as smaller rice and waxy rice starch granules complexed more iodine, while the larger potato and waxy potato granules complexed less than would be expected based on measured amylose contents. Corn, high amylose corn, and wheat, known to have starch granules with extensive surface pores, bound higher levels of iodine suggesting pores and channels may be an important factor giving iodine vapor greater access to bind within the granules. Exposing iodine vapor to moisture-equilibrated native starches is an effective tool to explore starch granule architecture.     

Differential regulation of waxy gene expression in rice endosperm

Summary In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression.     

Alkaliviscogram and Other Properties of Starch of Tropical Rice

In the alkaliviscogram of starch of 26 nonwaxy rices grown in the tropics, gelatinization normality correlated positively with final gelatinization temperature (BEPT) of starch (r=0.969^**) and negatively with alkali spreading value of milled rice (r= ?0.931^**). Peak viscosity was not linearly related to amylose content. Among samples of rice starch having a high amylose (>28%) content, peak viscosity was correlated with the gel consistency of starch (r=?0.690^**) and of milled rice (r=?0.644^**) (n = 18). These high-amylose starches showed the widest variation in peak viscosity. Amylose content, and gel consistency were inherited from the same parent in all nine varieties and lines studied, whereas peak viscosity, gelatinization normality and the final BEPT were inherited from either parent. The starch of five waxy rices showed higher peak viscosities even at a concentration of 1.8% as compared with a 2.0% nonwaxy rice starch.     

Diverse origins of waxy foxtail millet crops in East and Southeast Asia mediated by multiple transposable element insertions

The naturally occurring waxy and low-amylose variants of foxtail millet and other cereals, like rice and barley, originated in East and Southeast Asia under human selection for sticky foods. Mutations in the GBSS1 gene for granule-bound starch synthase 1 are known to be associated with these traits. We have analyzed the gene in foxtail millet, and found that, in this species, these traits were originated by multiple independent insertions of transposable elements and by subsequent secondary insertions into these elements or deletion of parts of the elements. The structural analysis of transposable elements inserted in the GBSS1 gene revealed that the non-waxy was converted to the low-amylose phenotype once, while shifts from non-waxy to waxy occurred three times, from low amylose to waxy once and from waxy to low amylose once. The present results, and the geographical distribution of different waxy molecular types, strongly suggest that these types originated independently and were dispersed into their current distribution areas. The patterns of GBSS1 variation revealed here suggest that foxtail millet may serve as a key to solving the mystery of the origin of waxy-type cereals in Asia. The GBSS1 gene in foxtail millet provides a new example of the evolution of a gene involved in the processes of domestication and its post-domestication fate under the influence of human selection. Electronic Supplementary Material Supplementary material is available for this article at     

Damaged starch characterisation by ultracentrifugation

The relative molecular size distributions of a selection of starches (waxy maize, pea and maize) that had received differing amounts of damage from ball milling (as quantified by susceptibility to alpha-amylase) were compared using analytical ultracentrifugation. Starch samples were solubilised in 90% dimethyl sulfoxide, and relative size distributions were determined in terms of the apparent distribution of sedimentation coefficients g*(s) versus s(20,w). For comparison purposes, the sedimentation coefficients were normalised to standard conditions of density and viscosity of water at 20 degrees C, and measurements were made with a standard starch loading concentration of 8 mg/mL. The modal molecular size of the native unmilled alpha-glucans were found to be approximately 50S, 51S and 79S for the waxy maize, pea and maize amylopectin molecules, respectively, whilst the pea and maize amylose modal molecular sizes were approximately 14S and approximately 12S, respectively. As the amount of damaged starch increased, the amylopectin molecules were eventually fragmented, and several components appeared, with the smallest fractions approaching the sedimentation coefficient values of amylose. For the waxy maize starch, the 50S material (amylopectin) was gradually converted to 14S, and the degradation process included the appearance of 24S material. For the pea starch, the situation was more complicated than the waxy maize due to the presence of amylose. As the amylopectin molecules (51S) were depolymerised by damage within this starch, low-molecular-weight fragments added to the proportion of the amylose fraction (14S)--although tending towards the high-molecular-weight region of this fraction. As normal maize starch was progressively damaged, a greater number of fragments appeared to be generated compared to the other two starches. Here, the 79S amylopectin peak (native starch) was gradually converted into 61 and 46S material and eventually to 11S material with a molecular size comparable to amylose. Amylose did not appear to be degraded, implying that all the damage was focused on the amylopectin fraction in all three cases. Specific differences in the damage profiles for the pea and maize starches may reflect the effect of lipid-complexed amylose in the maize starch.     

Effects of hydrogen, oxygen, and ammonia low-pressure glow plasma on granular starches

Cassava, corn, high amylose corn, potato, rice Indica, rice Japonica, sweet potato, waxy corn, and wheat starches were exposed to low-pressure ammonia, hydrogen, and oxygen plasma. In every case, depolymerization of the starch polysaccharides was noted. The extent of the depolymerization depended on the nature of the starch as well as the type of plasma applied. Among three fractions of polysaccharides distinguished by their molecular weight average, the fraction of the highest molecular weight suffered the most efficient depolymerization. The relative depolymerization for the middle- and low-molecular fractions of polysaccharides was found to be starch and plasma specific. The chemical character of the plasma had very little influence on the starch polysaccharides. Only subtle oxidation effects were observed in oxygen plasma. Low-pressure glow plasma treatment appeared to be a convenient tool for a waste-less dextrinization of starch. Manipulation of the plasma variety and the time of exposure resulted in a wide spectrum of dextrins of various molecular weights and paste-forming properties.     

Some properties of starch branching enzyme from indica rice endosperm (Oryza sativa L.)

Starch branching enzyme (α-1,4-glucan: α-1,4-glucan-6-glycosyl transferase; EC 2.4.1.18) catalyzes the formation of the α-1,6-bond in branched starch molecules such as amylopectin. Some characteristics of starch branching enzyme in rice endosperm (Oryza sativa L.) were determined because of the importance of starch structure for rice quality. Two or three peaks of starch branching enzyme activity were resolved by anion-exchange chromatography of extracts from high amylose rice. The properties of rice starch branching enzyme were similar to those found for the enzyme from other plant sources except for a much lower molecular weight. Rice branching enzyme had an apparent molecular weight of 40 000 as estimated by gel permeation chromatography. Multiple forms of starch branching enzyme could also be resolved in milled rice, suggesting that relationships between starch quality and characteristics of starch branching enzyme could be examined in the mature grain after harvest.     

Identification of SNPs in the<Emphasis Type="Italic"> waxy</Emphasis> gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice

Common non-waxy (Wx) rice cultivars contain two different alleles at the waxy locus, designated Wx ^a and Wx ^b, which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wx ^a allele was predominant in non-waxy indica cultivars, whereas the Wx ^b allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wx ^a from Wx ^b have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AGGT to AGTT) at a donor site of the first intron within the Wx gene. In the case of waxy (wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wx ^a or Wx ^b alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wx ^a allele (AGGT) but not in the Wx ^b allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wx ^a- and Wx ^b-derived alleles, but that the Wx ^b-derived allele was predominant, and its distribution was independent of indica-japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples.Communicated by J. Heslop-Harrison     

LAZY3 interacts with LAZY2 to regulate tiller angle by modulating shoot gravity perception in rice

Starch biosynthesis in gravity-sensing tissues of rice shoot determines the magnitude of rice shoot gravitropism and thus tiller angle. However, the molecular mechanism underlying starch biosynthesis in rice gravity-sensing tissues is still unclear. We characterized a novel tiller angle gene LAZY3 (LA3) in rice through map-based cloning. Biochemical, molecular and genetic studies further demonstrated the essential roles of LA3 in gravity perception of rice shoot and tiller angle control. The shoot gravitropism and lateral auxin transport were defective in la3 mutant upon gravistimulation. We showed that LA3 encodes a chloroplast-localized tryptophan-rich protein associated with starch granules via Tryptophan-rich region (TRR) domain. Moreover, LA3 could interact with the starch biosynthesis regulator LA2, determining starch granule formation in shoot gravity-sensing tissues. LA3 and LA2 negatively regulate tiller angle in the same pathway acting upstream of LA1 to mediate asymmetric distribution of auxin. Our study defined LA3 as an indispensable factor of starch biosynthesis in rice gravity-sensing tissues that greatly broadens current understanding in the molecular mechanisms underlying the starch granule formation in gravity-sensing tissues, and provides new insights into the regulatory mechanism of shoot gravitropism and rice tiller angle.     

Super-absorbent polymers from starch-polyacrylonitrile graft copolymers by acid hydrolysis before saponification

Graft copolymers (SPAN) of polyacrylonitrile (PAN) onto starch were prepared from gelatinized starch varieties with ammonium ceric nitrate as an initiator. The molecular weight of the PAN branches increased for the varieties of starches in the order high amylose maize starch < maize starch < waxy maize starch. SPAN samples were saponified with aqueous NaOH, and the aqueous solution of the resulting polymer (HSPAN) was cast into film in a forced-air oven at 35°C. The water absorbency of the HSPAN film formed from waxy maize starch was the highest (1200 g H₂O (g dry sample)⁻¹) and that from high amylose maize starch was the lowest (530 g g⁻¹). SPAN samples from maize starch were partially hydrolyzed with dilute hydrochloric acid. The resulting polyacrylonitriles with low molecular weight starch end groups (LSPAN) were also saponified. The resulting saponified product (HLSPAN) was cast into film. The absorbencies of HLSPAN films were found to be far larger (up to 6000 g g⁻¹) than those of the corresponding HSPAN films. The absorbency increased with increasing molecular weight of PAN in the initial SPAN up to a molecular weight of 1−1·5 × 10⁶. The absorbency decreased significantly when HSPAN and HLSPAN films were subjected to heat treatment at 135°C or above. The crosslinks present in HSPAN and HLSPAN films prepared at 35°C and those formed during heat treatment were considered to have different structures: the former formed between carbohydrate alkoxide ions and nitrile groups at the early stages of saponification and the latter formed between carbohydrate and copoly(acrylate-acrylamide) chains and/or between copoly(acrylate-acrylamide) chains.     

Effect of high hydrostatic pressure on the formation of radicals in maize starches with different amylose content

Native and high pressure-treated (water suspensions, 650 MPa) waxy maize starch, containing mainly amylopectin, and Hylon VII, rich in amylose, were studied for their ability to generate free radicals upon thermal treatment at 180–230 °C. The electron paramagnetic resonance (EPR) spectroscopy was used to characterize the nature, number and stability of radicals. Various stable and short living (stabilized by N-tert-butyl-α-phenylnitrone (PBN) spin trap) radical species were formed. It was found, that at given conditions the waxy maize starch reveals higher ability to generate radicals, than Hylon VII. The presence of water and high pressure pretreatment of starches, both resulted in the reduction of the amount of thermally generated radicals. The decrease in crystallinity of waxy maize starch and of Hylon VII, occurring upon high pressure treatment, leads to the increase of the relative amount of fast rotating component in the EPR spectrum of both types of starches.     

Classification of cultivated rices into indica and japonica types by the isozyme,RFLP and two milled-rice methods

Four methods for classifying cultivated rices (Oryza sativa L.) (including IR varieties) into indica and japonica types — waxy gene product in endosperm starch, glutelin ₃ molecular weight in milled rice, RFLP polymorphism at the Wx locus and Glaszmann's isozyme method — were compared. On the basis of the two endosperm traits and the RFLP method Glaszmann's group 1 (indica) was classified as mainly indica and intermediate groups 2, 3 and 4 as exclusively indica. However, the endosperm traits classified Glaszmann's group 5 as mainly indica, while the RFLP method classified it as japonica. The RFLP waxy gene probe was closest to the isozyme method in classifying group 6 as japonicas; the waxy gene product gave mainly indica reaction even in group 6, and the glutelin ₃ method was intermediate. All IR rices were classified as being indica on the basis of Wx gene product and by Glaszmann's method, but a few were classified as japonica by the glutelin ₃ method and by the RFLP waxy gene probe.     

Improved methods for the structural analysis of the amylose-rich fraction from rice flour

Cooking and sensory properties of rice are largely determined by the amylose content and structure. For relationships between functional and structural properties, a more accurate method to determine the structure of amylose is required. Here we calibrate size exclusion chromatography (SEC) columns, using Mark-Houwink parameters for linear starch and pullulan standards, to obtain the true molecular weight distribution of linear starch. When the molecular weight distribution is reported relative to pullulan, rather than the actual molecular weight which is readily obtained from universal calibration, it is seen that the molecular weights of longer amylose chains are greatly underestimated. We validate the SEC method to enable the measurement of the hydrodynamic volume distribution of the starch by examining reproducibility and recovery. Analysis of the starch in the sample pre- and post-SEC shows that 20% of the carbohydrate is not recovered. Comparison of the weight-average degree of polymerization, X(w), of (undebranched) starch of pre- and post-SEC is made using iodine binding as well as Berry plots of data from multi-angle laser light scattering (MALLS). These both show that current SEC techniques for starch analysis lead to significant loss of high molecular weight material. Indeed, for the systems studied here, the values for X(w) after SEC are about three times lower than those before SEC. Iodine-starch complexes of pre- and post-SEC samples reveals that the SEC techniques give reliable data for the amylose fraction but not for amylopectin. We address reports in the literature suggesting that the conventional isoamylase method for debranching starch would lead to incomplete debranching and thus incorrect molecular weight distributions. However, it is shown using (1)H NMR that isoamylase can completely debranch the amylose (to within the detection limit of 0.5%), and by SEC that successive incubation with isoamylase, alpha-amylase, and beta-amylase can degrade the amylose-rich fraction completely to maltose. We develop a method to obtain a hot water soluble fraction (HWSF), rich in undamaged amylose molecules, directly from rice flour, avoiding the structural degradation of previous techniques. With appropriate sample handling, the formation of associations between starch chains is minimized. With the combination of calibrated and validated SEC methods, and an improved extraction of amylose from rice, the X(w) for both HWSF and debranched HWSF are found to be much larger than has previously been reported.     

Change of granular and molecular structures of waxy maize and potato starches after treated in alcohols with or without hydrochloric acid

Starches from waxy maize and potato were treated in methanol and 2-propanol either with or without 0.36% hydrochloric acid at 65 °C for 1 h. The granule morphology, molecular structure and pasting properties of the starches were determined and the effects of treatments on the granule and molecular structures of starch were investigated. Starch treated in alcohols without acid showed loss of native order through the hilum of granules, and no obvious molecular degradation was found. However, acid–alcohol treated starch showed many cracks inside granules, and both waxy maize and potato starches showed obvious molecular degradation after treated. Furthermore, the amylose chains and long chains of amylopectin of starch were more easily degraded with acid–alcohol treatment. The pasting viscosity of acid–alcohol treated starches were also obviously less than that of their counterpart native starch and starch after alcohol treatment. The extent of degradation of molecules and the decrease of pasting viscosity on potato starch after acid–alcohol treated were more obvious than that of waxy maize starch. The result indicates that the degradation preferentially occur in the amorphous region when starch treated by acid–alcohol, and the degradation of starch molecules enhances the amorphous excretion and the occurrence of cracks inside the granules.     

Crystalline Features of Streptococcal (l→3)-α-D-Glucans of Human Saliva

Crystalline polymorphs of the backbone (l→3)-α-D-glucans of two streptococcal α-glucans were studied by X-ray diffraction measurements in comparison with that of a fungal (l→3)-α-D-glucan. The glucan produced by S. salivarius changed its polymorph from the hydrated form at 100% relative humidity to the dehydrated form under vacuum, that produced by cariogenic S. mutans took the dehydrated form only, and the fungal glucan always showed the hydrated form. The difference of polymorphic behavior was ascribed to the molecular weight of the glucan since the fungal glucan showed the highest viscosity, the saliverius glucan, middle, and the mutans glucan, the lowest.     

FLOURY ENDOSPERM16 encoding a NAD‐dependent cytosolic malate dehydrogenase plays an important role in starch synthesis and seed development in rice

Starch is the most important form of energy storage in cereal crops. Many key enzymes involved in starch biosynthesis have been identified. However, the molecular mechanisms underlying the regulation of starch biosynthesis are largely unknown. In this study, we isolated a novel floury endosperm rice (Oryza sativa) mutant flo16 with defective starch grain (SG) formation. The amylose content and amylopectin structure were both altered in the flo16 mutant. Map‐based cloning and complementation tests demonstrated that FLO16 encodes a NAD‐dependent cytosolic malate dehydrogenase (CMDH). The ATP contents were decreased in the mutant, resulting in significant reductions in the activity of starch synthesis‐related enzymes. Our results indicated that FLO16 plays a critical role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm. Overexpression of FLO16 significantly improved grain weight, suggesting a possible application of FLO16 in rice breeding. These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.     

Purification and Partial Characterization of an Endo-(1→3, 1→4)-β-glucanase from Rice,Oryza sativa L.

(1→3, 1→4)-β-Glucanase (EC 3.2.1.73), with a molecular weight of 34, 000 and an isoelectric point of 4.9, was purified to homogeneity from extracts of fresh rice bran. The enzyme specifically hydrolyzed (1→3, 1→4)-β-glucans such as barley β-glucan and lichenans, but laminarins and CM-cellulose were not substrates. Endproduct analysis using barley β-glucan as the substrate suggested that the enzyme is an endo-type (1→3, 1→4)-β-glucanase.