Physicochemical,Structural, and Proteomic Analysis of Starch Granules from Maize Landraces of Northwest Mexico

The variability in grain and starch characteristics and their relationship with the accumulation of starch granule associated proteins were investigated in five maize landraces of Northwest Mexico (Blando de Sonora, Chapalote, Elotero de Sinaloa, Reventador, and Tabloncillo). Significant differences were observed in grain hardness related traits, starch physicochemical properties, and structural properties. Blando de Sonora showed very soft grains, whereas the hardest grains were observed for Chapalote and Reventador. Starch granules isolated from landraces with hard grains contained more amylose and showed polygonal shapes, lower crystallinity and enthalpy of gelatinization, and greater retrogradation and proportion of long amylopectin chains. Proteomic analysis identified the enzymes granule‐bound starch synthase I (GBSSI), starch synthase I and IIa, starch branching enzyme IIb, sucrose synthase 1, and pyruvate phosphate dikinase 2 as granule‐associated proteins. The abundance of GBSSI correlated significantly with amylose content, consistent with the positive correlation observed between amylose and grain hardness. These results showed that the variability in the characteristics evaluated was mainly related to changes in the proportion of amylose in the starch granules, which were associated with differences in the expression of GBSSI. This information may be useful to define strategies for the exploitation and conservation of the landraces.     

Granule Bound Starch Synthase I (GBSSI) Gene Effects Related to Soft Wheat Flour/Starch Characteristics and Properties

Eight soft spring wheat (Triticum aestivum L.) genotypes representing the four granule bound starch synthase I (GBSSI) classes were evaluated with respect to flour/starch characteristics and pasting behaviors. Native starch was isolated from genotype straight‐grade flours (94.8–98.1% of starch recovered) to approximate the starch populations of the parent flours. As anticipated, amylose characteristics varied among the genotypes according to GBSSI class and accounted for the primary compositional difference between genotypes. Total (TAM), apparent (AAM), and lipid‐complexed (LAM) amylose contents ranged from 1.0–25.5 g, 0.7–20.4 g, and 0.3–5.6 g/100 g of native starch, respectively, and gradually decreased with the progressive loss of active Wx alleles. In addition, genotype flour total starch (FTS) and A‐type starch granule contents, which ranged from 81.7–87.6 g/100 g of flour (db) and 61.6–76.8 g/100 g of native starch (db), respectively, generally decreased with an increase in waxy character in parallel with amylose characteristics, as likely secondary effects of Wx gene dosage. Though amylose characteristics predominantly accounted for the majority of genotype flour pasting properties, FTS content and ratios of A‐ to B‐type granules also exhibited significant influence. Thus, loss of one or more Wx genes appeared to induce measurable secondary effects on starch characteristics and properties.     

Resistant Starch Content Is Related to Granule Size in Barley

Physical properties of resistant starch (RS) were examined in a range of barley genotypes to determine the contribution of starch and seed physical characteristics to the RS component. Thirty‐three barley genotypes were studied, which varied significantly in their RS, amylose, and starch contents and grain yield. From 33 genotypes, 13 exhibiting high RS were selected for detailed physicochemical analysis of starch. In high‐RS varieties, granule size and number were unimodal, compared with normal starches from a reference genotype, which showed a bimodal distribution. Principal component analysis (PCA) showed that a higher content of granules <15 µm was positively correlated with RS and amylose content, whereas the proportion of granules 15–45 µm was negatively correlated with the RS and amylose contents. Physical fractionation of starches by centrifugation into different population sizes demonstrated that size alone is not an accurate indicator of the population of A‐type and B‐type granules within a given genotype. PCA also showed that large 15–45 µm granules were positively correlated with seed thickness and that thousand grain weight was positively correlated with seed width. High‐RS and high‐amylose genotypes showed variation in overall yield and starch content, with some genotypes showing yield comparable to the reference genotype. Analysis of amylopectin chain length distribution showed that high amylose or RS content was not associated with a higher proportion of amylopectin long chains when compared with either waxy or reference (normal) barley genotypes. This study highlights useful markers for screening barley genotypes with favorable starch characteristics.     

Physicochemical and Enzyme Characterization of Small and Large Starch Granules Isolated from Two Maize Cultivars

In this study, some morphological, physicochemical, and the initial characterization of the starch granule enzymes of blue and white maizes of small and large granules are described. Starch was isolated from blue and white maizes, and the small and large granules were separated. The efficiency of separation was higher in large granules than small of the blue maize starch. The amylose content was slightly greater (1.3%) in large granules than smaller in both starches studied. No differences in the average gelatinization temperature were found between large and small granules, but the enthalpy of gelatinization value was higher in the small granules. The electrophoretic pattern showed that the granule‐bound starch synthase (GBSSI) had higher expression in large than small granules and that explain the higher amylose content in the former granules. The differences showed in the starch biosynthesis enzymes in small and large granules might explain partially the physicochemical and functional properties of maize.     

Molecular and Chemical Characterization of a New Waxy Allele in Barley (Hordeum vulgare L.)

Barley m38 mutant was selected for its high level of mixed‐linkage (1,3),(1,4)‐β‐d ‐glucan (MLG) in the grain. This elevated level of MLG was found to be associated with decreased amylose accumulation as well as other chemical composition alterations. Molecular characterization results revealed m38 as a new allele of the Waxy gene, encoding an endosperm‐specific granule‐bound starch synthase I (GBSSI). Additional mapping data from amylose phenotype and GBSSI gene specific markers supported the conclusion of the GBSSI mutation in m38. The m38 locus contains a nucleotide alteration that would result in the substitution of glycine at position 263 with serine in the putative adenosine‐5′‐diphosphate‐glucose binding domain. This amino acid substitution alters loop structures on the exterior surface of the folded protein and may affect its enzyme activity. Characterizations of m38 in this report provide for a new allele of the Waxy gene and additional evidence of pleiotropic effects on other chemical components including increased MLG, fructans, and fats and decreased amylose and protein.     

Soft Wheat Starch Pasting Behavior in Relation to A‐ and B‐type Granule Content and Composition

Flours of two soft wheat cultivars were fractionated into native, prime, tailing, A‐, and B‐type starch fractions. Starch fractions of each cultivar were characterized with respect to A/B‐type granule ratio, amylose content, phosphorus level (lysophospholipid), and pasting properties to investigate factors related to wheat starch pasting behavior. While both cultivars exhibited similar starch characteristics, a range of A‐type (5.7– 97.9%, db) and B‐type granule (2.1–94.3%, db) contents were observed across the five starch fractions. Though starch fractions displayed only subtle mean differences (<1%) in total amylose, they exhibited a range of mean phosphorus (446–540 μg/g), apparent amylose (18.7–23%), and lipid‐complexed amylose (2.8–7.5%) values, which were significantly correlated with their respective A‐ and B‐type granule contents. A‐type (compared with B‐type) granules exhibited lower levels of phosphorus, lipid‐complexed amylose, and apparent amylose, though variability for the latter was primarily attributed to starch lipid content. While starch phosphorus and lipid‐complexed amylose contents exhibited negative correlation with fraction pasting attributes, they did not adequately account for starch fraction pasting behavior, which was best explained by the A/B‐type granule ratio. Fraction A‐type granule content was positively correlated with starch pasting attributes, which might suggest that granule size itself could contribute to wheat starch pasting behavior.     

Differences in Starch Granule Composition and Structure Influence In Vitro Enzymatic Hydrolysis of Grain Meal and Extracted Starch in Two Classes of Canadian Wheat (Triticum aestivum L.)

Starch granule composition and amylopectin structure affect starch digestibility, an important factor influencing wheat grain utilization for human food consumption. Six bread wheat cultivars with four belonging to the Canada Western Red Spring (CWRS) and two Canada Prairie Spring Red (CPSR) market classes were analyzed for the relationship between their grain constituents and in vitro enzymatic hydrolysis of starch. CPSR cultivars had higher starch and amylose concentrations compared with CWRS cultivars, which had a higher protein concentration. Starch granule size distribution did not differ among the genotypes, except AC Foremost, which had significantly (P < 0.05) higher volume percent of B‐type starch granules (≈15%) and lower volume percent of A‐type starch granules (≈9%) compared with other cultivars. Fluorophore‐assisted capillary electrophoresis revealed a lower content of R‐IV (DP 15–18, ≈6%) and a higher content of R‐VII (DP 37–45, ≈7%) chains in the CPSR cultivars compared with the CWRS cultivars. Starch in vitro enzymatic hydrolysis showed that compared with CWRS cultivars, the two CPSR cultivars had reduced amounts of readily digestible starch and higher amounts of slowly digestible starch and resistant starch. Consequently, the two CPSR cultivars also showed lower hydrolysis indexes in grain meal as well as extracted starch. CPSR cultivars, with higher starch and amylose concentrations, as well as a higher content of long chains of amylopectin, showed a reduced starch in vitro enzymatic hydrolysis rate.     

Starch from Hull‐less Barley: Ultrastructure and Distribution of Granule‐Bound Proteins

Starch granules isolated from waxy, normal, and high‐amylose hullless barley grains were examined by transmission electron microscopy with cytochemical techniques. The micrographs showed two distinct regions of different sizes: 1) densely packed granule growth rings (which varied in size and number depending on the genotype), and 2) a loose filamentous network located in the central region of the granule. The granule ring width decreased with increasing amylose content. In all three genotypes, the growth rings closer to the granule surface were narrower in width than those within the granule interior. The waxy starch had wider intercrystalline amorphous growth rings, semicrystalline growth rings, and more open crystalline lamellae than normal and high‐amylose starches. Granule bound proteins (mainly integral proteins) were located in the central and peripheral (growth ring) regions of the granule.     

Association of Starch Granule Proteins with Starch Ghosts and Remnants Revealed by Confocal Laser Scanning Microscopy

Starch suspensions (0.25%) were gelatinized to 70 and 100°C, and starch ghosts (defined as gelatinized starch granule envelopes after the majority of internal starch polymers have been released) and remnants were collected by centrifugation and washed with water. Protein was revealed in isolated gelatinized normal starch ghosts using confocal laser scanning microscopy and a protein‐specific dye that fluoresces only after reaction with primary amines in protein. This technique eliminates background interference from residual dye. Observation of fluorescent‐labeled protein in the starch ghosts at different optical depths of field revealed that protein was concentrated in the envelopes of swollen, gelatinized potato, maize, and wheat starch ghosts. Only traces of protein were found in gelatinized starch granule remnants of waxy maize and amylose‐free potato starches after they were heated to 100°C, indicating that the proteins observed in gelatinized normal maize starch were largely granule‐bound starch synthase (GBSS). Moreover, fragility of the gelatinized waxy and amylose‐free starch granule remnants might be caused in part by the lack of GBSS. Gel electrophoresis of proteins in starch ghosts confirmed that GBSS in potato and maize was tightly associated with the starch ghosts. The study provides a structural explanation for a role of granule‐associated proteins in maintaining the integrity of starch ghosts and remnant structures, and their consequent effect on paste rheology.     

Comparison of Flour Starch Properties in Half‐Sib Families of Opaque‐2 Maize (Zea mays L.) from Argentina

Since the discovery of the o2 mutation in maize, many studies have reported the characterization of the protein quality of opaque‐2 genotypes. However, few have reported the properties of their starch. The objective of this study was to characterize flour starch properties of 12 half‐sib families of opaque‐2 maize from Argentina. Chemical composition and thermal and pasting properties of whole grain flour were determined. Nonopaque genotypes were used as a control. Starch content of opaque‐2 genotypes did not show significant differences compared with nonopaque genotypes, yet amylose content was significantly lower. A high variability in pasting and thermal properties was observed in genotypes. Opaque samples showed a significantly higher peak viscosity and a lower pasting temperature compared with nonopaque samples, probably owing to larger and less compact starch granules in the floury endosperm. The higher the gelatinization enthalpy of opaque‐2 genotypes was, the lower the amylose content in relation to nonopaque varieties. Two retrogradation endotherms were observed in DSC analysis: one corresponding to amylopectin crystallization and the other to melting of amylose‐lipid complex. Both enthalpies were considered total starch retrogradation (ΔH _RT). A wide range of variation was obtained in ΔH _RT in opaque‐2 genotypes, but no significant differences between opaque and nonopaque genotypes were observed. The differences in starch properties found in this study would make it possible to identify opaque‐2 families with particular characteristics for the development of starchy food items adapted to specific processing traits.     

Novel ssIIa Alleles Produce Specific Seed Amylose Levels in Hexaploid Wheat

Novel starch synthase II (ssIIa ) alleles were created in a soft wheat (Triticum aestivum L.) via ethyl methanesulfonate mutagenesis and combined by crossing to create four unique ssIIa triple mutant haplotypes. A range of starch amylose content was obtained from the ssIIa triple mutant lines (35.6–46.8%), with moderate increases in amylose content found in lines carrying two ssIIa null alleles and one partially functional ssIIa allele. Seeds from all ssIIa triple mutants had significantly higher amylose and protein contents but also lower starch content, kernel weight, and flour swelling power compared with their wild‐type sister lines. Seed starch amylose content was negatively correlated with individual kernel weight (r = –0.54), starch content (r = –0.85), and flour swelling power (r = –0.91) but positively correlated with grain protein (r = 0.78), demonstrating that unique ssIIa triple mutant null combinations confer defined levels of seed starch amylose, protein content, and kernel size. The ssIIa mutant lines also had a hard grain texture (≈86 single kernel characterization system units), whereas all parental material and wild‐type sister lines had soft grain texture (≈35). This change in grain texture was independent of Ha locus genotype, because all lines carried a functional (soft type) Ha locus. The ssIIa alleles and allelic combinations reported here demonstrate the ability to create defined levels of seed starch, amylose, and protein.     

Influence of Starch and Gluten Characteristics on Rheological Properties of Wheat Flour Gel at Small and Large Deformation

Starch and gluten were isolated from 10 wheat cultivars or lines with varied amylose content. The rheological properties of 30% wheat flour gel, starch gel, and the gel of isolated gluten mixed with common starch were determined in dynamic mechanical testing under shear deformation, creep‐recovery, and compression tests under uniaxial compression. Variation of wheat samples measured as storage shear modulus (G′), loss shear modulus (G″), and loss tangent (tan δ = G″/G′) was similar between flour and starch gels and correlated significantly between flour and starch gel. The proportion of acetic acid soluble glutenin exhibited a significant relationship with tan δ of gluten‐starch mixture gel. The small difference in amylose content strongly affected the rheological parameters of flour gels in creep‐recovery measurement. Wheat flour gel with lower amylose content showed higher creep and recovery compliance that corresponded to the trend in starch gel. Compressive force of flour gel at 50 and 95% strain correlated significantly with that of starch gel. Gel mixed with the isolated gluten from waxy wheat lines appeared to have a weaker gel structure in dynamic viscoelasticity, creep‐recovery, and compression tests. Starch properties of were primarily responsible for rheological changes in wheat flour gel.     

Noodle Quality as Related to Sorghum Starch Properties

Starch was extracted from 10 sorghum genotypes and physicochemical properties (amylose content and pasting, textural, and thermal properties) were evaluated. The amylose content was 24–30%. DC‐75 starch had the highest peak viscosity (380 Rapid Visco Analyser units). Gelatinization peak temperature occurred over a narrow range (67–69°C). Genotypes Kasvikisire and SV2 produced white starches. Starches from other genotypes were different shades of pink. The starch noodles prepared were, accordingly, either white or pink. Cooking enhanced the pink coloration of noodles. Cooking loss, noodle rehydration, and elasticity were evaluated. Cooking loss was low (mean 2.4%). Noodle elasticity was highly correlated with starch pasting properties of hot paste viscosity (HPV) (r = 0.81, P < 0.01) and cold paste viscosity (CPV) (r = 0.75, P < 0.01). Noodle rehydration was significantly correlated to the initial swelling temperature of starch (T_i) (r = ‐0.91, P < 0.001) and gelatinization peak temperature (T_p) (r = 0.69, P < 0.05). The findings suggest a potential area of food application for sorghum genotypes of different grain colors. Evaluation of starch properties could be a good starting point for selecting sorghum genotypes with superior noodle‐making properties.     

Selecting and Sorting Waxy Wheat Kernels Using Near‐Infrared Spectroscopy

An automated single kernel near‐infrared (NIR) sorting system was used to separate single wheat (Triticum aestivum L.) kernels with amylose‐free (waxy) starch from reduced‐amylose (partial waxy) or wild‐type wheat kernels. Waxy kernels of hexaploid wheat are null for the granule‐bound starch synthase alleles at all three Wx gene loci; partial waxy kernels have at least one null and one functional allele. Wild‐type kernels have three functional alleles. Our results demonstrate that automated single kernel NIR technology can be used to select waxy kernels from segregating breeding lines or to purify advanced breeding lines for the low‐amylose kernel trait. Calibrations based on either amylose content or the waxy trait performed similarly. Also, a calibration developed using the amylose content of waxy, partial waxy, and wild‐type durum (T. turgidum L. var durum) wheat enabled adequate sorting for hard red winter and hard red spring wheat with no modifications. Regression coefficients indicated that absorption by starch in the NIR region contributed to the classification models. Single kernel NIR technology offers significant benefits to breeding programs that are developing wheat with amylose‐free starches.     

Comparison of Physical Properties of Wheat Starch Gels with Different Amylose Content

The effects of amylose content and other starch properties on concentrated starch gel properties were evaluated using 10 wheat cultivars with different amylose content. Starches were isolated from grains of two waxy and eight nonwaxy wheat lines. The amylose content of waxy wheat lines was 1.4–1.7% and that of nonwaxy lines was 18.5–28.6%. Starch gels were prepared from a concentrated starch suspension (30 and 40%). Gelatinized starch was cooled and stored at 5°C for 1, 8, 16, 24, and 48 hr. The rheological properties of starch gels were studied by measuring dynamic viscoelasticity with parallel plate geometry. The low‐amylose starch showed a significantly lower storage shear modulus (G′) than starches with higher amylose content during storage. Waxy starch gel had a higher frequency dependence of G′ and properties clearly different from nonwaxy starches. In 40% starch gels, the starch with lower amylose showed a faster increase in G′ during 48 hr of storage, and waxy starch showed an extremely steep increase in G′. The amylose content and concentration of starch suspension markedly affected starch gel properties.     

Genetic Diversity in Properties of Starch from Zimbabwean Sorghum Landraces

Starch was isolated from 95 sorghum landraces from Zimbabwe using an alkali steep and wet‐milling procedure. The physicochemical properties of sorghum starch were examined for potential use in Southern Africa. All the landraces evaluated had a normal endosperm indicated by the amylose content of the starches. Starch properties were not correlated to most of the physical grain quality traits evaluated. Grain hardness was weakly correlated to starch gel adhesiveness (r = 0.36) and amylose content (r = 0.38) (P < 0.001). The mean peak viscosity (PV) of the sorghum starches was 324 Rapid Visco Analyser units (RVU) compared with 238 RVU in a commercial corn starch sample; PV was 244–377 RVU. Some landraces had low shear‐thinning starches, implying good paste stability under hot conditions. Pasting properties were highly correlated among the sorghum starches. The starch gel hardness showed considerable variation (44–71 g) among the landraces. Gelatinization peak temperatures were 66–70°C. The thermal properties of starches were not correlated with starch swelling and pasting properties. Genotype grouping by highest and lowest values in each category would allow selection of sorghums based on a specific attribute depending on the desired end use.     

Quality of Spaghetti Made from Full and Partial Waxy Durum Wheat

The waxy character is achieved in durum wheat (Triticum turgidum L. var. durum) when the granule‐bound starch synthase activity is eliminated. The result is a crop that produces kernels with no amylose in the starch. The presence of two Waxy loci in tetraploid wheat permits the production of two partial waxy wheat genotypes. Advanced full and partial waxy durum wheat genotypes were used to study the effect of waxy null alleles on pasta quality. Semolina from full and partial waxy durum wheats was processed into spaghetti with a semicommercial‐scale extruder, and pasta quality was evaluated. Cooked waxy pasta was softer and exhibited more cooking loss than pasta made from traditional durum cultivars. These features were attributed to lower setback of waxy starch as measured with the Rapid Visco Analyser. High cooking loss may be due to the lack of amylose‐protein interaction, preventing the formation of a strong protein network and permitting exudates to escape. Waxy pasta cooked faster but was less resistant to overcooking than normal pasta. Partial waxy pasta properties were similar to results obtained from wild‐type pasta. This indicates that the presence of a single pair of functional waxy genes in durum wheat was sufficient to generate durum grain with normal properties for pasta production. Waxy durum wheat is not suitable for pasta production because of its softening effect. However, this property may offer an advantage in other applications.     

Influence of Amylose Content on Cookie and Sponge Cake Quality and Solvent Retention Capacities in Wheat Flour

Reduced amylose wheat (Triticum æstivum L.) produces better quality noodles and bread less prone to going stale, while little is known about the relationships between amylose content and the quality of soft wheat baking products such as sugar snap cookies (SSC) and Japanese sponge cakes (JSC). Near‐isogenic lines developed from wheat cultivar Norin 61, differing in their level of granule‐bound starch synthase (Wx protein) activity, were used to produce wheat grains and ultimately flours of different amylose contents. These were tested with regard to their effect on soft wheat baking quality and solvent retention capacities (SRC). Amylose content was strongly correlated to cookie diameter (r = 0.969, P < 0.001) and cake volume (r = 0.976, P < 0.001), indicating that the soft wheat baking quality associated with SSC diameter and JSC volume were improved by an incremental increases in amylose content. Among the four kinds of SRC tests (water, sodium carbonate, sucrose and lactic acid), the water SRC test showed the highest correlation with amylose content, SSC diameter, and JSC volume. When the regression analysis was conducted between the nonwaxy and partial waxy isogenic lines that are available in commercial markets, only water SRC was significantly correlated to amylose content (r = –0.982, P < 0.001) among of four SRC tests. This suggests that, unlike udon noodle quality, high‐amylose content is indispensable in improving soft wheat baking quality, a process requiring less water retention capacity.     

Effects of Growing Environment on Properties of Starch from Five Australian Wheat Varieties

The effects of growing conditions on properties of starch from wheat grain were examined. Growing conditions affected starch and amylose content, granule size distribution, protein associated with starch granules, and starch swelling power in grains from five commercial Australian milling wheat varieties grown at multiple locations in two years in crop production systems. Soil nitrogen and meteorological conditions were major contributors to variability in grain yield and grain protein and starch contents. The volume proportion of B‐granules was positively affected by warmer temperatures before flowering but negatively correlated with high temperatures during grain filling. Genotype was the main source of variability in the proportion of B‐granules and granule dimensions, starch‐granule proteins, and starch swelling power, although there were also significant contributions to variability from the growing conditions. Seasonal effects and interactions between genotype and season and location were significant sources of variability in amylose content, proportion of short chains of amylopectin, and flour swelling power. The positive relationships between starch content and the number of clear days and atmospheric temperatures before flowering indicate that conditions that enhance accumulation of assimilates before anthesis influence the deposition of reserves in developing grain.     

Grain qualities and their genetic derivation of 7 new rice for Africa (NERICA) varieties

NERICA are interspecific rice varieties from crossing between the high-yielding Asian rice ( Oryza sativa spp. Japonica) with locally adapted African rice ( Oryza glaberrima). In this study, we analyzed grain qualities of 7 NERICA varieties (NERICA 1 to 7) and genetic derivation of quality-related genes. Quality analyses of NERICA grains showed that 7 NERICA varieties were clearly classified into two groups based on the difference of amylose content, and the difference influenced the pasting and physical properties of grains. Genetic analysis of the gene encoding granule-bound starch synthase I (GBSSI), which is known as a key enzyme on amylose synthesis in rice grain, revealed that varieties with higher amylose content ( approximately 29%) have the gene derived from O. glaberrima parent, and group 2 with lower amylose content ( approximately 22%) have the gene from O. sativa parent. These results indicated that the difference in amylose content as well as grain properties among 7 NERICA varieties is mainly determined by the genetic derivation of GBSSI. Further genetic analysis of starch synthesis-related genes suggested that the genetic derivation of SSIIa also influences the chain length of amylopectin in 7 NERICA varieties.