Expression of granule‐bound starch synthase in developing rice grain

The expression of granule‐bound starch synthase (GBSS) in rice (Oryza sativa) genotype Tainung 67 (200 g kg^?1 grain amylose content) and its two NaN₃‐induced mutants SA419 and SA418 were examined. G/T polymorphism analysis indicated that SA418 (300 g kg^?1 grain amylose content) carried Wx^a allele. The insertion of 23 base pairs sequence was found only in SA419 (80 g kg^?1 grain amylose content), suggesting that it was a waxy mutant. Microsatellite polymorphisms (CT)_n were also detected on the Wx gene encoding GBSS in the tested genotypes. The activities of several key enzymes involving starch biosynthesis in developing grains of field‐grown rice plants were also compared during grain filling period. Significant genotypic differences were only found in the expression of GBSS. The content of amylose in SA418 grain was higher than Tainung 67 and SA419 grains throughout the entire grain filling period, possibly due to its superiority to synthesize amylose through GBSS. The lowest amylose content of SA 419 grain was attributable to its extremely low activity of GBSS in comparison with the two other genotypes. The mutation effects on the expression of GBSS were confirmed by two‐dimensional electrophoresis and mass spectrometry. Copyright © 2007 Society of Chemical Industry     

Endosperm Structure and Physicochemical Properties of Starches from Normal,Waxy, and Super-Sweet Maize

Maize is a main botanical source used for extraction of starch in the world market. New maize cultivars with different amylose contents and special starch metabolism characteristics have been generated. Three types of maize cultivars, namely, normal maize, waxy maize (wxwx homozygous mutant), and super-sweet maize (sh2sh2 homozygous mutant), were investigated to determine differences in endosperm structures, morphologies, and physicochemical properties of starches. Maize kernels exhibited significantly different contents of total starch, soluble sugar, and amylose. Normal maize kernels contained the largest proportion of floury endosperm, followed by waxy maize and then super-sweet maize. Normal maize starch and waxy maize starch were larger in size than super-sweet maize starch. Normal maize starch and waxy maize starch were spherical and polygonal in floury and vitreous endosperms, respectively. Super-sweet maize starch was spherical both in floury and vitreous endosperms. Waxy maize starch showed the strongest birefringence patterns, the highest crystallinity and the largest proportion of ordered structure in external region of granules, and the largest proportion of double helix components, followed by normal maize starch and then super-sweet maize starch. Waxy maize starch showed the highest peak viscosity, trough viscosity, breakdown viscosity, gelatinization temperatures (i.e., gelatinization conclusion temperature, gelatinization onset temperature, gelatinization peak temperature, and gelatinization enthalpy). By contrast, super-sweet maize starch showed the lowest corresponding values for these parameters.     

The Interaction of Endosperm Genotype and Genetic Background. Part I. Differences in Chromatographic Profiles of Starches from Nonmutant and Mutant Endosperms

The effect of maize endosperm genotype and genetic background on variation of endosperm starch properties has been examined by gel filtration. Nonmutant, and single, double and triple mutant combinations of the endosperm genes amylose-extender (ae), dull (du), sugary (su), and waxy (wx) in four maize inbred lines were compared. The major effects of endosperm genes on starch properties did not vary as a result of genetic background. Starches from wx endosperms contained no amylose. The mutants ae, du and su resulted in starches with increased amylose content. Genetic background did affect starch properties in predictable ways. For example, the production of amylose in mutant endosperms was higher in the dent inbred background, followed by the sweet corn inbreds. However, the production of low molecular weight amylopectin and intermediate polysaccharide fractions was greatest in a sweet corn inbred background, We conclude that the material included in this study will be valuable in future investigations designed to delineate the interaction of genetic background and endosperm genotype in starch biosynthesis and starch properties.     

Polarization Colors of Lightly Iodine‐stained Maize Starches for Amylose‐Extender and Related Genotypes in the W64A Inbred Line

Previous qualitative research showed that for some maize endosperm genotype starches the color of variably iodine‐stained starch granules observed by bright field microscopy (BFM) was different from the color of identically stained granules observed by polarized light microscopy (PLM). One objective of the present study was to determine the polarization color for a variety of high‐amylose and other starch genotypes in an identical genetic background. A secondary objective was to determine the iodine concentration dependence of polarization colors for the samples. Starches from the W64A inbred line were obtained from the following genotypes: normal, wx, ae, du, su2, ae wx, ae du, ae su2, and du su2. Starches were stained with iodine solutions ranging from 0.02 to 0.075% and viewed with BFM and PLM, using an auto‐exposure digital camera function. Most starches showed the first appreciable color at about 0.04percnt; I₂. Unlike normal and non‐ae‐containing starches, ae starch showed a pink polarization color, despite its blue color in bright field. Heterogeneity in polarization color was observed both within and among granules. Double mutant starches containing ae showed variable effects, depending on the combination. It is suggested that the pink polarization color of ae starch may be due to a lack of symmetrical orientation of iodinecomplexed amylose in these granules     

In situ Raman microscopy of starch granule structures in wild type and ae mutant maize kernels

A method developed for in situ imaging of starch granule structure in dry seeds has been applied to compare the starch granule structures found in wild type and ae mutant maize kernels. In the isogenic ae mutant the activity of the starch branching enzyme IIb is inhibited, which gives rise to a high amylose starch. The granule structures in the wild type samples have been found to be homogeneous, whereas those in the ae mutant are grossly heterogeneous within individual granules, between granules within individual cells, and between cells across the endosperm. The level of heterogeneity observed in situ appears to be more marked than that previously reported for studies on isolated ae mutant starches. Iodine/potassium iodide staining and polarised light microscopy have been used together with Raman microscopy, which has allowed high‐resolution mapping of the composition and physical state of the structures within the granules, to probe the origins of the heterogeneity of the starch structures. Although the mutation inhibits the activity of the branching enzyme within the granules, and both the composition and level of crystallinity within and between granules is variable, the major origin of the heterogeneity of the granule architecture appears to result from significant changes in the assembly and packaging of the crystalline structures within the granule. It is suggested that this arises due to the mutation of the starch branching enzyme introducing defects into the self‐assembly of the crystalline structure, resulting in an accumulation of defects and increased randomisation of the granule structure.     

Starch Granule‐Associated Proteins and Polypeptides: A Review

This paper reviews current information regarding the (non‐gluten) proteins which are naturally located in and on starch granules, and which are termed starch granule associated proteins (SGAPs). At least ten major SGAPs can be extracted from most starches and have molecular weights in the range of approximately 5 to 149 kDa. A substantial number of these proteins are located at the starch granule surface, where their presence in association with that of other minor granule components (such as lipids), appears to significantly influence the overall properties of both starch granules and starchy products. The different extraction conditions which can be exploited to selectively remove SGAPs are detailed. Although some SGAPs may serve as supplementary storage proteins, the majority of SGAPs are believed to be starch biosynthetic enzymes, a proportion of which remain trapped within the growing granule structure. The identity and quantities of the biosynthetic enzymes varies with botanical source, genetic variety and even with time. Current knowledge regarding the identities, compositions, locations, properties and functions of several of the more common SGAPs (e.g. the ∼15 kDa friabilin/puroindoline proteins, the ∼30 kDa protein, and the ∼60 kDa starch granule‐bound starch synthase (SGBSS) protein) are reviewed in detail.     

Comparative Susceptibility to Amylases of Starch Granules of Several Single Endosperm Mutants and Their Double-Mutant Combinations with Opaque-2 in Four Inbred Lines of Maize

The relative susceptibility of starch granules of single mutants and double-mutant combinations of maize and their normal counterparts to Rhizopus glucoamylase and pancreatin was compared and the relative degradation of starch granules followed by scanning electron microscopy. Starch granules of distinct endosperm mutants were resistant to the action of amylases whilst others showed pin holes on the surface and pores into the inner layers. Starch granules of the sugary, brittle, shrunken, and waxy mutants tended to be digested faster than those of their normal counterparts. The opaque-2 mutant which improves nutritional quality of endosperm proteins did not change the susceptibility of starch granules to degradation by amylases.     

Proso millet (Panicum miliaceum L.): An Evaluation of the Microstructural Changes in the Endosperm during the Malting Process by Using Scanning‐Electron and Confocal Laser Microscopy

Scanning electron microscopy (SEM) and confocal scanning laser microscopy (CLSM) were used to investigate the micro‐structural changes in the endosperm during the malting process. SEM was a suitable tool to characterise the microstructural constitution of starch granules in proso millet and the changes occurring during malting. An early visible degradation (after 24 h) of starch granules located in the floury endosperm, which is close to the embryo, could be observed. Due to this degradation, using confocal scanning laser microscopy, a less dense packaging of this part of the endosperm was observed. Degradation of starch granules in the vitreous endosperm was obvious at a distinct later stage of malting (78 h) and a preferred attack of small granules (>2.5 μm) was detected. Changes in protein structure could not be detected by either SEM or CLSM.     

Physicochemical and Biochemical Characterization of Starch Granules Isolated of Pigmented Maize Hybrids

In this study, the morphological and physicochemical of pigmented maizes as well as the initial characterization of the corresponding starch granule enzymes are described. Starch granules were isolated from blue, black, and white maize. They were analyzed using scanning electron microscopy, particle size distribution, pasting characteristics, sorption isotherms, differential scanning calorimetry, and two‐dimensional gel electrophoresis. The morphology of the starch granules of pigmented maizes was different from the granules of white maize; the pattern was related to the endosperm type of these varieties. The average starch granule size was higher for black than for white and blue maizes. The average gelatinization temperature was similar in the three starches, but the pigmented maizes had higher gelatinization enthalpy; black maize starch showed the lowest enthalpy of retrogradation. These results indicated that the starches from the three maizes analyzed had different organization level. Black maize starch showed the highest peak viscosity followed by white and blue maize starches. In the gel electrophoresis three starch granules presented one main spot at pI of 5 and MW of 60 kDa that corresponds to the granule‐bound starch synthase. Blue and white starches presented some spots near 97 kDa at pI of 5.3–5.7 (white maize) and 5.1–5.5 (blue maize), spots that were not observed for black maize starch. The morphological and physicochemical characteristics of maize starch are related to the enzymes involved in its biosynthesis.     

Chain‐length Distribution Profiles of Amylopectin Isolated from Endosperm Starch of Waxy and Low‐amylose Bread Wheat (Triticum aestivum L.) Lines with Common Genetic Background

Large A‐type endosperm starch granules were isolated from near‐isogenic waxy and non‐waxy lines and low‐amylose mutant lines of bread wheat with a common genetic background. The amylose contents of A‐type starch ranged from 2.6% to 23.6%. Amylopectin was isolated by concanavalin A (Con A) precipitation from the isolated starch. The λ_max (range: 532‐538 nm) and blue values at 680 nm (range: 0.026‐0.037) of the iodine‐amylopectin complex were not significantly different among the isolated amylopectins, indicating that amylopectins from non‐waxy and low‐amylose lines did not contain such long chains as amylose or extra‐long chains of amylopectin affecting iodine complex properties. Chain‐length distribution profiles measured by both high‐performance size‐exclusion chromatography (HPSEC) and high‐performance anion‐exchange chromatography (HPAEC) showed that the amylopectin structures of these lines were indistinguishable from each other. Extra‐long chains were not detected in the amylopectins by HPSEC measurement. The side‐chains measured by HPAEC were classified into four groups according to their degree of polymerization (DP), and the proportion of each group were in the following ranges: DP 6‐12, 26.5‐27.5%; DP 13‐24, 43.6‐44.1%; DP 25‐36, 13.6‐14.2%, and DP 37‐60, 11.0‐11.7%. The alleles on the Wx‐D1 locus, i.e., Wx‐D1a, Wx‐D1d, Wx‐D1f, and Wx‐D1g, responsible for granule‐bound starch synthase (GBSS I) biosynthesis had no influence on the properties of iodine‐amylopectin complex and the chain‐length distribution profiles of amylopectin.     

Effects of Germination of Wheat,Oats, and Pearl Millet on Alpha-Amylase Activity and Starch Degradation

Hard red winter wheat, oats, and pearl millet were germinated at 15°C for periods up to 14 days. Endosperm areas in kernels fractured with a razor blade and starches isolated from the malted flours were examined with a scanning electron microscope to determine the morphology of starch granules and the nature of α-amylolytic attack on granules. Free sugars content, damaged starch content, and α-amylase activity of the flours were determined to establish relationships between physical changes in the cereal grains and formation of α-amylase during germination. Starch granules in the three cereals were degraded during germination by α-amylase accompanied by production of free sugars and increases in the damaged starch of flours milled from the germinated grains. The cementing material embedding starch granules in endosperms of wheat and oats decreased and disappeared during germination; these changes were not obvious in germinated millet endosperm. Amorphous-appearing material seemed to cover starch granules in the endosperm of wheat and oats during later stages of germination, but were not observed in the isolated starch granules. Although wheat starch granules had the highest α-amylase activity, this enzymic degradation was less than expected. Enzymic attack was evidenced as channels or pits on the surfaces of wheat and millet starch granules. Compound oat starch granules were relatively resistant to enzymic attack and no evidence of erosion of the granule surface was observed; small granula were released from the compound granules during germination. The most extensive degradation was obtained with millet starch, appearing as discrete holes leading to the interior of the granule. Concentric shells were visible in the interior of wheat and millet starch granules, but were not observed in oat starch granules.     

Biochemisch-technologische Studien über die Naßverarbeitung von Mais. 7. Mitteilung,Modellversuch zur Bestimmung hydrolytischer Reaktionen durch Maisenzyme während des technischen Maisquellprozesses

Biochemical-Technological Studies on Wet-Processing of Maize. Part 7: Model Test for the Determination of Hydrolytic Reactions by Maize Enzymes during the Technological Maize Steeping Process. In a model test enzymatic activity during the steeping process, which can be derived from the determined activity optima of maize enzymes in connection with the environmental conditions in the maize grain is confirmed. Amylolytic enzymes are qualified for degradation of maize starch only in the first steeping phase. Here, starch being closely linked to the endosperm protein is more stable than “isolated” maize starch. Proteases act throughout steeping time, but their activity decreases towards the end. It is supposed that during the steeping of maize degradation takes place mainly in the outer layers of the maize grain. It leads to a loosening of the linkage between endosperm and the germ on the one hand and the fruit hull on the other hand. These might facilitate the separation of the grain constituents. Proteolysis only plays a minor role for the dissolution of the protein matrix enclosing the starch granules.     

Characterization of Endosperm Starch from High-amylose Mutants of Rice (Oryza sativa L.)

The structure and properties of endosperm starch from high-amylose mutants of rice were examined. The starch in the mutants was characterized by a higher content of amylose and loosely branched amylopectin with longer chains compared with non-mutant starch. The starch granules in the mutants showed high temperatures of gelatinization and a type B pattern in X-ray diffractometry. These properties were similar to those of amylose-extender (ae) starch of maize. The effect of locations where rice plants were grown on the endosperm starches of one high-amylose mutant and a non-mutant was also investigated.     

STARCH DEGRADATION IN ENDOSPERMS OF DEVELOPING BARLEY KERNELS*

Shortly after anthesis, empty endosperm cells adjacent to the embryo were crushed between the developing embryo and endosperm tissues to form the crushed cell layer. Starch granules in cells adjacent to this layer were hydrolysed and the empty cells were added to the crushed layer. In this way, the crushed layer increased in thickness throughout kernel development. α-Amylase 2 was detected in the crushed cell layer region of barley endosperms during the period that starch granule hydrolysis was occurring.     

Effect of endosperm type on texture and in vitro starch digestibility of maize tortillas

Tortilla is the main staple of Mexico and it is made using diverse maize varieties, which have different endosperm types. Three maize varieties with vitreous, intermediate and floury endosperms were used. Texture and starch digestibility were evaluated in freshly prepared and stored tortillas for 24, 48 and 72 h. Tortilla made with maize of vitreous endosperm had the highest force to rupture and the lowest distance of elongation, indicating more rigid texture. Stored tortillas had lower available starch content and higher effect was shown by tortilla of vitreous endosperm, pattern that agrees with the higher increase in the resistant starch content with the storage time. Fresh tortilla of floury endosperm showed the highest hydrolysis rate during the first 15 min followed by tortillas of intermediate and vitreous endosperms. Starch hydrolysis values decreased when storage time increased, in agreement with the resistant starch content in the stored tortillas. At the longest storage time (72 h) tortilla of floury endosperm presented higher hydrolysis rate, followed by tortilla of intermediate and vitreous endosperms. The endosperm type plays an important role in the textural and starch digestibility of fresh and stored tortillas.     

Scanning electron microscopy of Fusarium damaged kernels of spring wheat

Kernels of five wheat cultivars (Triticum aestivum) of different bread-making quality were examined. Grown under field conditions, heads of wheat were inoculated in the flowering stage with an aqueous suspension of Fusarium culmorum conidia. Wheat heads were collected from the control and inoculated plots at full maturity. Control (non-inoculated) kernels without any symptoms of disease and Fusarium damaged kernels (FDK) were examined under scanning electron microscopy (SEM). Examination of the FDK fraction confirmed localisation of Fusarium hyphae on the surface and inside the tissues of kernels. Observations of the endosperm from Fusarium infected kernels revealed presence of fungal hyphae in the endosperm and some characteristic structural changes in many of its regions, such as partial or complete lack of the protein matrix, damage to large and small starch granules caused by fungal amylolytic enzymes, disappearance of small starch granules as the colonisation progressed, complete disappearance of the starchy endosperm under severe infection. Fungal colonisation of the endosperm and structural changes in its area were highly variable traits within the FDK fraction of a given cultivar.     

Influence of α‐amylolysis on the formation of electron density inhomogeneities on the surface of starch granules

Low‐temperature nitrogen adsorption and SAXS were used to analyze the granule surface of wheat and rice starches before and after an action of Bacillus subtilis α‐amylase. Results obtained by the first method showed that α‐amylolysis caused surface changes arising of new pores or enlarging diameters of pores already existing. According to the SAXS results, the action of enzymes caused complete sharpening of a solid skeletal boundary in the case of wheat starch granules. Partial sharpening of this boundary in the case of rice starch granules could be attributed to the relatively high content of proteins in rice starch.     

Identification of hexaploid wheat cultivars based on isozyme patterns

The classification and identification of 38 wheat cultivars of Triticum aestivum L. and one cultivar of Triticum spelta L. have been carried out using polyacrylamide and starch gel electrophoresis. The analyses were carried out using different parts of dry kernels, specifically embryo plus scutellum, endosperm and 15-day-old seedling leaves. The isozymic systems studied were: peroxidases, malic dehydrogenase, alcohol dehydrogenase, acid and alkaline phosphatase in dry kernels; α-amylases in germinating kernel endosperms and phospho-glucose mutase and isomerase, esterases, leucin-aminopeptidase, glutamic oxaloacetic transaminase, malic dehydrogenase and peroxidases in 15-day-old seedling leaves. Not all the cultivars could be identified. One group had three cultivars, five groups had two cultivars, and 26 groups had only one cultivar. The classification obtained using the isozymic patterns and the similarity indexes did not show a clear relation to the ancestor-descendant relationships among related cultivars.     

Ghosts of B‐type Wheat Starch Granules in Concentrated KI/I2 Solution

When B‐type (2.0–8.0 µm) wheat starch granules containing various amounts (2.1–25%) of amylose were treated with 25% KI/10% I₂ solution, low‐amylose (below 10% amylose) B‐type wheat starch granules changed to the ghost form. It is known that A‐type (25–35 µm) wheat starch granules change to the ghost form and show a typical double structure (a black‐brown central portion and red‐brown surrounding portion), however, the B‐type wheat starch ghosts did not show the same double structure but rather a simple (red‐brown portion) sack form. The relative ghost areas in the B‐type wheat starch granules were highly correlated to the amylose content (%), which was similar to the results of A‐type starch granules. This suggests that the amylose molecule in B‐type starch contributes to the structural stability of the starch granule.     

The Possible Relationship of Starch and Phytoglycogen in Sweet Corn. II. The Role of Branching Enzyme I

Developing maize kernels contain three starch branching enzymes. Homogeneous branching enzyme I has now been shown to be capable of branching amylose as well as amylopectin but not glycogens. The formation of glycogen-like molecules by the further branching of amylopectin suggests that branching enzyme I plays a role in phytoglycogen formation. Its presence in nonmutant kernels which do not accumulate phytoglycogen, however, indicates that amylopectin per se is not accessible to branching enzyme I for phytoglycogen formation. Treatment of sugary (su) starch granules with enzyme I resulted in soluble phytoglycogen-like glucan. No glucan was released by treatment of nonmutant starch granules. In addition, nonsolublized amylopectin and amylose fractions of the su starch were branched during the incubations. These observations are consistent with plastid changes which show a conversion of starch granules to phytoglycogen in sugary endosperm cells.