DSC Characteristics of Gelatinization of Starches of Single-, Double-, and Triple-Mutants and Their Normal Counterpart in the Inbred Oh43 Maize (Zea mays L.) Background

Starch granules were isolated from mature kernels of single-, double-, and triple-mutants of endosperm starch modifying genes [waxy(wx), dull(du), amylose extender(ae), sugary-2(su₂)] and their normal counterpart in the inbred Oh43 maize (Zea mays L.) background. Gelatinization characteristics of the starch granules were investigated by means of differential scanning calorimetry (DSC). The onset, peak, and conclusion temperatures of gelatinization (To, Tp, and Tc, respectively) and the heat of gelatinization (ΔH) of starch were determined from the DSC thermograms. The Tc of wx was higher than normal starch, and the ΔH of wx was larger than normal starch. The Tp and Tc values of ae starch were the highest. The To, Tp, and Tc values of su₂ starch were low. The noticeable effects of each of the wx, ae, and su₂ genes on To, Tp, Tc and ΔH were observed in starches of their respective mutant combinations. Apparent epistatic effects of the genes for gelatinization temperature were observed. Pronounced effect of the heating rate on gelatinization temperature of su₂ mutant were lower than other mutant starches and were affected by the heating rate.     

A Novel Type of Corn Starch from a Strain of Maize. Part 3. The Properties of the Starch from Amylo-waxy Maize and Its Hybrids with Waxy Maize

A pair of hybrids were produced by alternative crossbreeding of waxy maize with amylo-waxy maize which had double recessive genes, amylose extender (ae) and waxy (wx). Investigation of the starches proved that they did not coincide with waxy starches. Hidden amylose extender genes also participated in the starch produced in the endosperm of hybrid maize. Amylo-waxy starch looks promising as a new material of food industry.     

Structure and Physicochemical Properties of Endosperm Starches of a Waxy Allelic Series and Their Respective Normal Counterparts in the Inbred Oh43 Maize Background

Research over the past three decades has greatly increased our understanding of the biochemical genetics of various waxy (wx) alleles of maize, but our knowledge about the structure and physicochemical properties of endosperm starches obtained from the wx alleles is still incomplete. We further investigated the structure and physicochemical properties of endosperm starches from a wx allelic series and their normal counterparts in the Oh43 inbred background. Starch granules were prepared from mature kernels of wx mutant alleles; wx-C31, wx-R, wx-90, wx-a, wx-B3, wx-m-1, wx-m-8, and wx-S5, and their respective normal counterparts in the inbred Oh43 maize background. Measurements of absorption spectra of starch-iodine complexes and by gel permeation chromatography of Pseudomonasisoamylase-debranched starches showed that all of the starches from the wx allelic series were uniquely waxy type in characteristics, and their normal counterparts were characteristically normal type. Pasting characteristics of starch granules and retrograded starches of the wx allelic series and their respective normal counterparts were investigated by differential scanning calorimetry (DSC). The peak temperature (Tp) and conclusion temperature (Tc) values from starches of the wx allelic series were slightly higher than those of their respective normal counterparts and the ΔHs of starches of the wx allelic series were greater than those of their respective normal counterparts. The onset temperature (To), Tp, and Tc of all retrograded starches were very similar, however, the ΔHs of the retrograded starches of the wx allelic series were greate than those of their respective normal counterpart. Starch granules of the wx allelic series were hydrolyzed more rapidly than those of their respective normal counterparts.     

Gelatinization Characteristics of Starch from du,wx, ae,and ae wx Endosperm of Sweet Corn Inbred la5125

Starch gelatinization in excess water was studied by differential scanning calorimetry for the mutants dull (du), waxy (wx), and amylose extender (ae), and the double mutant amylose-extender, waxy (ae wx) from the Ia5125 sweet corn inbred background. Onset temperature (T_o), peak temperature (T_max), and enthalpy (ΔH) were determined. For du and wx starches T_max was within 1°C of the value for the normal starch (T_max = 69.4°C). For starches from ae and ae wx mutants, T_max was 7–8°C higher than the normal starch. The highest enthalpies were observed for wx (3.26 cal/g) and ae wx (3.39 cal/g) starches; ae (2.93 cal/g) and normal (2.52 cal/g) starches were intermediate, and du (2.32 cal/g) starch had the lowest enthalpy. The endotherm of the ae starch was completed at just above 100°C, in distinction to reports for ae mutants from dent lines. Although the ae wx endotherm occurred at a higher temperature than the wx, the endotherm was sharpened relative to the ae endotherm, and was complete at 90°C.     

Developmental Changes in Starch Properties of Several Endosperm Mutants of Maize

The properties of starches during development of several endosperm mutants of maize were investigated. Starch contents of normal and waxy (wx) kernels were higher than those of dull (du), sugary-2 (su₂), and amylose-extender (ae) kernels, and in general the greater increase in starch contents occurred earlier in kernel development. The normal, du, su₂, and ae mutants in that order, respectively, increased the apparent amylose concentration of the starches as measured by the “blue value” method. Within each of these genotypes, absorption intensity increased and λ max. of absorption curves shifted to a longer wavelength region with increasing maturity. Waxy starches did not change in either of these properties at any of the developmental stages. Few detectable differences were observed in X-ray diffraction intensity, gelatinization temperature, heat of gelatinization, and the photopastegrams within these genotypes during development. Exhaustive degradation of starch granules by glucoamylase showed that ae starches were more resistant to amylase than the other starches. Except for wx, the starches tended to be more resistant to amylase with increasing maturity.     

Chain Length Distribution of Amylopectins of Double- and Triple-Mutants Containing the Waxy Gene in the Inbred Oh43 Maize Background

Starch granules were isolated from mature kernels of double- and triple-mutant combinations of the waxy (wx) gene with other starchmodifying genes in the inbred Oh43 maize background. The amylose content and the distribution of unit chain-length of amylopectin were determined by enzymatic-chromatographic methods. Starches of the mutants containing the wx gene comprised 100% amylopectin. Amylopectin of the amylose-extender;waxy (ae wx) mutant had an increased proportion of long B chains and decreased proportion of short B chains, compared with wx amylopectin, whereas amylopectin of the dull;waxy (du wx) mutant had a decreased proportion of long B chains and an increased proportion of short B chains. Therefore, the ae wx and du wx mutants amylopectins were novel. The A:B chain ratios for amylopectins examined, namely for ae wx, amylose-extender;waxy;floury-2 (ae wx fl₂), amylose-extender;waxy;sugary-1 (ae wx su₁), amylose-extender;waxy;sugary-2 (ae wx su₂), brittle-1;waxy (bt₁wx). dull;waxy (du wx), and sugary-2;waxy (su₂wx) amylopectins, were in a range of 1.1 to 1.4 and similar to the wx amylopectin. Thus, starches of double-mutant combinations of the wx gene with other starch-modifying genes are good sources for elucidating the fine structure of amylopectin, in regards to long- and short-B chains, which is affected by a single recessive gene coupled with the wx gene.     

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.     

The Interaction of the Amylose-Extender and Waxy Mutants of Maize (Zea Mays L.) Fine Structure of Amylose-Extender Waxy Starch

Investigations into the nature of the effect of the amylose-extender (ae) mutant of maize (Zea mays L.) on amylopectin structure were conducted by studying the fine structure of amylose-extender waxy (ae wx) starch. Approximately 59.6% of the starch from ae wx endosperms was converted to maltose by β-amylase. This starch contained 21% apparent amylose and had a λmax of 580 for the iodine-starch complex. Fractionation of ae wx starch on Sepharose 4B-200 gave an elution profile with a single peak characteristic of amylopectin. From these observations we concluded that ae wx starch consisted of an altered amylopectin, with iodine binding properties such that an apparent amylose content of 21% was measured. The fine structures of ae wx and waxy (wx) starches were determined. Pullulanase-debranched chains of whole starches and β-limit dextrins were fractionated by gel permeation. The amylopectin of ae wx was shown to be a loosely branched amylopectin with an average internal chain length of 52 glucose units compared with a length of 30 glucose units for wx. The ae wx outer chains were longer than those of wx and fewer in number per mg of starch. These characterizations demonstrate that ae wx starch has a unique structure which is similar to the anomalous amylopectin reported in ae starch.     

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.     

Chain Length Distribution of Amylopectins of Several Single Mutants and the Normal Counterpart,and Sugary-1 Phytoglycogen in Maize (Zea mays L.)

β-Limit dextrins of starches of normal (nonmutant), amylose-extender (ae), dull (du), sugary-2 (su₂) and waxy (wx) maize, and phytoglycogen of sugar-1 (su₁) maize were prepared. The β-limit dextrins were successively debranched by isoamylase and pullulanase, and followed by quantitative gel-filtration. The ratio of A to B chains for the ae starch appeared to be high and that for su₁ phytoglycogen was low. The ratio of short B to long B chains for the du starch was high and that for the ae starch appeared to be low. The phytoglycogen did not contain long B chains. The unit chain-length distribution of amylopectins of the normal, wx, and su₂ starches were similar. Fine structures of maize amylopectins and su₁ phytoglycogen were modeled and described.     

Physicochemical Characteristics of Starches of Two Sets of Near‐isogenic Wheat Lines with Different Amylose Content

Wheat lines with modified amylose content were produced by controlling the null alleles of three homoeologous Wx loci, Wx‐A1, Wx‐B1 and Wx‐D1 and those with triple‐null Wx alleles were found to yield waxy, amylose‐free starch. In this study, the new near‐isogenic lines developed by the recurrent backcross method between cv. Kanto 107 and the waxy mutant lines, K107Wx1 or K107Wx2, were cultivated and the physicochemical characteristics of their starches were investigated. The apparent amylose contents of the waxy near‐isogenic lines were 1.6 – 3.8%, whereas those of the non‐waxy near‐isogenic lines were 23.2 – 25.4%. The waxy lines had higher gelatinization temperature and enthalpy than the non‐waxy lines and no peak of amylose‐lipid complex as determined by DSC. The X‐ray diffraction patterns from both waxy and non‐waxy near‐isogenic lines corresponded to A‐type crystallization, like their parent lines. The starches isolated from the waxy lines had lower pasting onset and peak temperatures, higher peak viscosities and breakdown and lower final viscosity than those of the non‐waxy lines in the Viscoamylograph. The starch of non‐waxy near‐isogenic lines had lower peak and final viscosities and higher breakdown than did the recurrent parent line, cv. Kanto 107. The characteristics of the new wheat starches were well understood and classified, which will contribute to the wide application of these starches in food processing.     

Development Changes in Fine Structure of Starches of Several Endosperm Mutants of Maize

Distribution of components of maize starches from several endosperm mutants sampled at three stages of development were investigated by gel filtration after debranching by isoamylase. The waxy (wx) starches consisted of only amylopectin, and the distribution of components did not change during development. In other starches: normal, amylose-extender (ae), dull (du), sugary-1 (su₁) and sugary-2 (su₂) starches, amylose contents increased during the 20–35 days after pollination (DAP) period of development. The ae, du, su₁ and su₂ mutants produced high-amylose starches, while amylopectin of su₂ was normal type and those of ae, du and su₁ were novel type, and ae starches possessed longer average chain-lengths of amylopectin. Phytoglycogens were prepared from su₁ kernels during development and their characteristics were compared with those of su₁ starches. The characteristics of phytoglycogens did not change during the developmental phase studied and the average chain-lengths of phytoglycogens were shorter than those of amylopectin of su₁ starches at each stage of development.     

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     

Effects of the Barley amo1 and wax Genes on Starch Structure and Physicochemical Properties

Starch structural mutants showing abnormal endosperm characteristics have been used for investigating the effects of the mutation on structure and physicochemical properties of starches. Inbred lines of barley cultivars ‘Shikoku Hadaka 97’ and ‘Glacier AC38’ were used to investigate the impact of amo1 and waxy genes on starch properties. The amo1 type starch had high apparent amylose content and low starch content. The amo1+waxy type starch contained very little amylose. The content of long chains of amylopectin as detected with high‐performance size‐exclusion chromatography (HPSEC) was decreased, and that of amylopectin chains with the degree of polymerization (DP) of 12‐36 was increased in amo1 and amo1+waxy type starches. The amo1 and amo1+waxy type starches exhibited high gelatinization temperatures and low gelatinization enthalpies.     

In vitro Digestibility of Hydroxypropyl Maize,Waxy Maize and High Amylose Maize Starches

Hydroxypropyl derivatives of maize, waxy maize and high amylose maize starches were prepared and characterized. The in vitro digestibility of the raw and gelatinized starches and their derivatives was compared using porcine pancreatic α-amylase. Digestibility of the unmodified starches decreased in the order waxy maize > maize > high amylose maize. Increasing molar substitution (MS) caused a decrease in digestibility for all starch types after gelatinization. Raw maize and high amylose maize starch derivatives showed an initial decrease in digestibility followed by increases at higher MS levels. The digestibility of the raw waxy maize starch derivatives showed a continuing drop as MS increased.     

Effect of Gene Dosage at High Amylose Loci on the Properties of the Amylopectin Fractions of the Starches

The effect of gene dosage at the amylose-extender (ae) locus of maize and the rigosus-a (r_a) locus of peas on the amylopectin fraction of endosperm and cotyledon starch were examined. Amylopectin fractions were isolated by gel filtration, butanol complexing and a combination of the two methods. In general, increasing doses of the recessive allele at either locus resulted in amylopectin fractions with iodine spectra with absorption maxima at higher wavelengths and greater absorptivity. Starches from endosperm of differing ae dosage but homozygous waxy were found to contain no amylose by gel filtration. By debranching with pullulanase, these starches were shown to have increasing average chain length with increasing ae dosage. Additional data suggested that increasing dosage at the ae locus (regardless of the genotype at the waxy locus) or the r_a locus resulted in amylopectin with increasing linearity. In addition, short chained amylose (approximately 100 glucose units) was observed in all ae genotypes in a homozygous Waxy background and all r_a genotypes.     

Some Structural Characteristics of Starches of Maize Having a Specific Genetic Background

Distribution of starch components of maize (Zea mays L.) and their properties were investigated by gel filtration after debranching and by exhaustive hydrolysis of starch-granules with glucoamylase. Based on the results, relationship between genotypes and properties of starch components were discussed. The sugary-1 (su₁), sugary-2 (su₂), and dull (du) mutants produced high amylose maize, while amylopectin of su₂ was normal type and those of su₁ and du were novel type. In the double-mutant combinations, the waxy (wx) gene decreased amylose content. Starches of du su₁ or du su₂ possessed greatest amount of amylose among starches examined. Contents of amylose, intermediate fraction (Fr.) and longer branches of amylopectin increased in most cases when the amylose-extender (ae) gene was introduced. Starches of ae possessed longer average chain-lengths of amylopectin than that of normal. In ae du starches, however, elongation of the amylopectin chain and increase of the intermediate Fr. did not occur. Exhaustive degradation of starch-granules by glucoamylase showed that ae starches were more resistant to amylase.     

Gene dosage effect on starch structure studied using maize polygenic model containing ae and su1 mutant alleles

The aim of this work was to enhance our understanding on the gene dosage effect on starch structure. Two mutant alleles, ae and su1, were selected for this study. With regard to their genetic functions, ae and su1 mutations are at two opposite extremes; therefore ae and su1 constituted an ideal pair of mutant alleles for manipulating starch structures. A full set of dosage combinations of ae and su1 were constructed in the maize W64A inbred line, and matured kernels were subjected to starch structure analysis. Scanning electron microscopy showed that starch granule morphology was affected by high doses of ae and su1 alleles. X-ray powder diffraction showed that 3 doses of su1 were not able to change the crystalline pattern of starch, whereas 3 doses of ae transformed A-type pattern to B-type. Size-exclusion chromatography showed that homozygous ae, su1, and ae su1 led to drastic change of starch structure from that of non-mutant. In addition, starches from certain ae and su1 dosage combinations were different from those of homozygous genotypes. This study will lay further foundation towards genetic modification of starch for desirable functional properties in food.     

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.     

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.