Transition from vitreous to floury endosperm in maize (Zea mays L.) kernels is related to protein and starch gradients

Relationships between kernel vitreousness and proteins and starch partitioning to the floury and vitreous regions of the endosperm were monitored in a set of 13 maize inbred lines. Decrease of protein contents from the vitreous to the floury endosperms were mainly assigned to α-zeins. Using Raman microspectroscopy, we observed a protein gradient from the periphery to the center of endosperms that well fitted with the inverse relationships between vitreousness and protein content of the vitreous and floury regions. In addition, Raman microspectroscopy highlighted an increase of starch crystallinity from the periphery to the center of the maize endosperms. This agrees with the higher amylose and associated lipid contents within starches of vitreous than in those of floury endosperms. Finally, starch granules from vitreous regions displayed more channels than the floury ones. These channels contain proteins that might favor adhesion of proteins to starch granules or granule–granule contacts to form the close packing of the vitreous endosperm. Therefore transition from vitreous to floury endosperm is at least the result of both protein and starch gradients. These gradients are probably associated with metabolic gradients that have been observed during endosperm development.     

Starch granule morphology in oat endosperm

Mature and developing oat (Avena sativa) grains were sectioned and image analysis methods used to estimate the starch granule-size distribution and morphology in endosperm cells. This showed that oat endosperm cells contain two types of starch granule: compound starch granules such as those seen in rice endosperm and in most other grasses; and simple granules similar to the B-type starch granules seen in the endosperm of Triticeae species such as wheat (Triticum aestivum). The simple granules in oats are similar in size and relative abundance to B-type granules in Triticeae suggesting that they may share a common evolutionary origin. However, there is a fundamental difference between oats and Triticeae in the timing of granule initiation during grain development. In Triticeae, the B-type granules initiate several days after the A-type granules whereas in oats, both the simple and compound granule types initiate at the same time, in early grain development.     

Biochemical characterization of QTLs associated with endosperm modification in quality protein maize

Genetic analysis using quality protein maize (QPM) recombinant inbred lines derived from K0326Y QPM and W64Ao2 identified three quantitative trait loci (QTL) in bins 1.06, 7.02 and 9.03 associated with opaque2 endosperm modification. We evaluated the effects of these QTLs on protein accumulation and starch physicochemical properties. The QTL in bin 1.06 is close to α-zein genes, and vitreous individuals with this QTL had increased accumulation of 19-kDa α-zein, 27-kDa γ-zein and legumin-1. The QTL in bin 7.02 corresponds to the γ-zein locus, and greater accumulation of this protein was found in vitreous individuals. The QTL in bin 9.03 is close to starch biosynthetic genes; greater accumulation of granule-bound starch synthase and amylose was observed in vitreous kernel samples with this locus and that in bin 1.06, as well as less gelatinization enthalpy and crystallinity. Vitreous kernels contained angular-shaped/compact starch granules and more short-intermediate length chains of amylopectin. These results support that endosperm modification in QPM is associated with increased accumulation of γ-zein and other storage proteins, but also show that synthesis of less crystalline starch with more amorphous regions at the periphery of granules, which favor their packing and association with endosperm proteins, may also be an important factor.     

Enhancement of the pasting properties of teff and maize starches through wet–heat processing with added stearic acid

This study determined the effects of stearic acid on the functional properties of teff starch, a compound granule starch in comparison to maize, a simple type granule starch. Stearic acid was incorporated into teff and maize starches and pasted (held for 5 or 120 min at 91 °C) with an RVA (Rapid Visco Analyser). Teff starch with added stearic acid (0.25 and 1.5% starch basis) did not produce a pasting peak viscosity within short holding time (5 min) compared to maize starch. The paste viscosity of both teff and maize starches with stearic acid increased by about three times with long pasting (120 min). This increase in paste viscosity occurred earlier for teff starch than maize starch. Teff starch with stearic acid was more viscous and was non-gelling. Confocal laser scanning microscopy showed that stearic acid did not diffuse in teff starch granules, but seemed to coat them. However, stearic acid diffused inside maize starch granules through channels. This microstructural difference may explain the different pasting behavior. The early high paste viscosity and non-gelling properties of the teff starch modified with stearic acid could have promising applications in foods, for example better mouthfeel with lower starch concentration.     

Mapping Quantitative Trait Loci for Starch Granule Traits in Barley

Starch accumulates in barley (Hordeum vulgareL.) endosperm in large (type A) and small (type B) granules. The sizes, shapes and relative proportions of A and B granules may affect the quality of barley malt for brewing. The objective of this study was to use genetic markers to map quantitative trait loci (QTL) affecting starch granule traits in a cross between a malting barley cultivar, Morex, and a feed barley cultivar, Steptoe. Data on starch granule dimensions were obtained using digital image analysis. With simple interval mapping, a region of chromosome 2 (2H) was found to contain QTL affecting the overall mean granule volume, the proportion of A granules, the mean volume of A granules, the mean maximum diameter of A granules and the mean F-shape of B granules. This region also affected days to heading and plant height, but contained no QTL for grain or malt quality traits. With composite interval mapping, QTL affecting starch granule traits were detected in two additional regions, one on chromosome 4 (4H) affecting the mean F-shape of B granules and one on chromosome 7 (5H) affecting the mean maximum diameter of A granules.     

Effects of endosperm texture and cooking conditions on the in vitro starch digestibility of sorghum and maize flours

The effects of endosperm vitreousness, cooking time and temperature on sorghum and maize starch digestion in vitro were studied using floury and vitreous endosperm flours. Starch digestion was significantly higher in floury sorghum endosperm than vitreous endosperm, but similar floury and vitreous endosperm of maize. Cooking with 2-mercaptoethanol increased starch digestion in both sorghum and maize, but more with sorghum, and more with vitreous endosperm flours. Increasing cooking time progressively reduced starch digestion in vitreous sorghum endosperm but improved digestibility in the other flours. Pressure-cooking increased starch digestion in all flours, but markedly more in vitreous sorghum flour; probably through physical disruption of the protein matrix enveloping the starch. Irrespective of vitreousness or cooking condition, the alpha-amylase kinetic constant (k) for both sorghum and maize flours remained similar, indicating that differences in their starch digestion were due to factors extrinsic to the starches. SDS-PAGE indicated that the higher proportion of disulphide bond-cross-linked prolamin proteins and more extensive polymerisation of the prolamins on cooking, resulting in polymers of M_r>100k, were responsible for the lower starch digestibility of the vitreous sorghum endosperm flour.     

Channels within soft wheat starch A- and B-type granules

The nature of channels within wheat starch granules was investigated using scanning electron and confocal laser scanning microscopy. A-type granules stained with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA, protein-specific probe) revealed a network of radially oriented, channel-like protein structures similar to those previously reported. However, treatment of the same starch granules with methanolic merbromin (fluorescent dye) solution, which is used to highlight external granule surfaces (including those of channels) under non-swelling conditions, revealed few, if any, channels extending into the granule interior. This discrepancy suggested that channels within wheat starch granules were filled at least in part with protein. Removal of protein with protease facilitated greater access of methanolic merbromin to channels and/or cavities for both granule types. For A-type starch granules, relatively large channels were observed in the equatorial groove region, while finer channels originated from other regions of the granule. This work reports the first visualization of B-type granule channels, which most frequently occurred as less-defined voids (as opposed to the fine, discrete channels of A-type granules) extending to granule surfaces. Channels of A- and B-type starch granules appeared to facilitate transfer of chemical reagent into the granule matrix, though this effect was aided by granule swelling (hydration) and/or removal of channel-associated protein.     

Investigation of the high-amylose maize starch gelatinization behaviours in glycerol-water systems

The effect of glycerol on gelatinization behaviours of high-amylose maize starch was evaluated by confocal laser scanning microscopy (CLSM), scanning electronic microscope (SEM), differential scanning calorimetry (DSC), texture analyzer (TPA) and rheometer. Gelatinization of the high-amylose maize starches with glycerol content of 10% (w/w) began at 95.4 °C (T_o), peaked at 110.3 °C (T_p), and completed at 118.9 °C (T_c). The birefringence began to disappear at around 100 °C and finished at 120 °C which corresponded well to the onset and conclusion temperatures obtained by DSC. The high-amylose maize starch granules maintained original morphological structure at 100 °C and swelled to a great degree at 110 °C. The high-amylose maize starch paste formed at 100 °C showed the lowest hardness (39.92 g), while at 120 and 130 °C, showed the highest hardness (610.89 g and 635.43 g, respectively). It should be noted that in going from 100 °C to 110 °C there is a significant increase in the viscosity of the slurry solution. The identical apparent viscosity was observed when the shear rate exceed 100 s⁻¹, resulting from the high-amylose maize starch granules were completely gelatinized at 120 °C, which was consistent with DSC analysis.     

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

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

Interaction of granular maize starch with lysophosphatidylcholine evaluated by calorimetry, mechanical and microscopy analysis

In this study we evaluated the thermo-mechanical properties of maize starch pastes (80% wt/wt) under the effect of exogenous lysophosphatidylcholine (LPC) using differential scanning calorimetry (DSC), dynamic mechanical spectrometry (DMS), and scanning electron microscopy (SEM). Particular attention was paid to the development of the amylose-LPC inclusion complex. Results from SEM and DSC showed that with no exogenous LPC, granular maize starch developed the amylose network structure for starch gelling at 80–95 °C. In comparison, at 1.86 and 3.35% of LPC, heating up to 130 °C was needed to develop the three-dimensional network required for starch gelling. Results showed that at these LPC concentrations LPC interacted mainly with amylose within the starch granule. At concentrations ≥8.26% the LPC interacted with amylose both inside the granule and on the granule's surface. At such LPC concentrations heating to 130 °C did not fully develop the starch network structure for gelling. These results suggested that a higher thermal stability was achieved by starch granules because of LPC inclusion complex formation. DSC or DMS did not detect the development of this complex, probably because its formation took place below the onset of gelatinization under conditions of limited molecular mobility. Subsequently, a lower level of organization (i.e. complex in form I) was achieved than in the complex developed at high temperature and water excess (i.e. complex in form II). On the other hand, the changes in the starch granule structure observed by SEM as a function of the time–temperature variable were well described by the phase shift angle (δ) rheograms for starch pastes with and without addition of LPC.     

Pest resistance to Cry1Ab Bt maize: Field resistance,contributing factors and lessons from South Africa

This paper documents the historical development of resistance of the African maize stem borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) to Bt maize (Zea mays L.). This pest was one of the first to evolve resistance to Bt maize expressing Cry1Ab protein. A time-line of events and contributing factors are presented, from the commencement of efficacy testing through to the present situation, where the Cry1Ab toxin has lost its efficacy against B. fusca at many localities throughout the maize producing region, and single-gene Bt maize events often require insecticide treatments for which farmers are compensated. Significant levels of pest survival on Bt maize was observed in the first season after commercial release in 1998 and confirmed seven years later. Reduced selection pressure on the target pest is the objective of insect resistance management (IRM), and strategies to accomplish this should receive highest priority. Where resistance is prevalent, the only viable options to reduce selection pressure are withdrawal of the product and/or enforcement of high-dose/refuge requirements. The latter action may however be of no value under conditions where resistance is prevalent, since the value of refugia to an IRM strategy may be compromised. Remedial actions taken in South Africa included the propagation and enforcement of refuge compliance followed by the release of pyramided maize hybrids in 2011. These pyramids combine Cry1A.105 and Cry2Ab2 toxin-producing transgenes, replacing the ineffective single-transgene. However, it remains uncertain if cross-resistance occurs between Cry1A.105/Cry2Ab2 and the closely related Cry1Ab toxin, and for how long this pyramided event will endure. Cultivation of Cry1Ab-expressing hybrids continues in areas where resistance levels have been confirmed to be high. In retrospect, this case provides lessons regarding IRM, not only in South Africa, but wherever Bt crops are being introduced.     

Sequential sampling plan for assessing corn rootworm (Coleoptera: Chrysomelidae) larval injury to Bt maize

In-field product performance assessments are an essential component of corn rootworm (Diabrotica spp.; CRW) resistance management plans for transgenic maize (Zea mays L.) products expressing proteins derived from the bacterium Bacillus thuringiensis (Bt). The goal of a successful field sampling program is to accurately characterize in-field product performance while also minimizing resource demand, as collection of maize root samples to evaluate CRW injury can present resource challenges such as labor intensiveness, potential safety issues, and a limited time window available for sampling. A resource-efficient sequential sampling plan was developed that utilizes data-driven root injury threshold values derived from benchmark product performance data for both single and pyramided Bt maize traits for CRW control. This sequential sampling methodology incorporates unbiased sampling and controlled false positive and false negative error rates, enabling accurate assessment decisions to be made with efficient resource use. Our proposed approach enables systematic and effective classification of in-field Bt maize product performance, with applications to other CRW control technologies besides Bt maize products.     

The effects of puroindoline b on the ultrastructure of endosperm cells and physicochemical properties of transgenic rice plant

Endosperm texture is an important factor governing the end-product quality of cereals. The texture of wheat (Triticum aestivum L.) endosperm is controlled by puroindoline a and b genes which are both absent in rice (Oryza sativa L.). It has been reported that the endosperm texture of rice can be modified by puroindoline genes. The mechanism, however, by which puroindolines affect the ultrastructure of rice endosperm cells remains to be investigated. In this study, we observed the ultrastructure of endosperm cells and the morphology of isolated starch granules of the transgenic rice expressing the puroindoline b gene. SEM and TEM observations indicated that compound starch granules were embedded within the matrix material in non-transgenic rice, Nipponbare, whereas they were surrounded by spaces in the transgenic rice. The morphology and size of each starch granule were not different between non-transgenic and the transgenic rice. However, the transgenic rice flour showed smaller particle size, higher starch damage, and lower viscosity during gelatinization than that of non-transgenic rice. These results confirm that puroindoline b reduces the grain hardness in rice. Moreover, the results also suggest that puroindoline b functions at the surface of compound starch granules, and not on polygonal starch granules in rice endosperm.     

Blue Maize: Morphology and Starch Synthase Characterization of Starch Granule

The use of pigmented maize varieties has increased due to their high anthocyanins content, but very few studies are reported about the starch properties of these grains. The aim of this work was to isolate the starch granules from pigmented blue maize and carry out the morphological, physicochemical, and biochemical characterization studies. The proximate composition of starch granules showed high protein contents, after purification, the blue maize starch presented lower protein amount than starch from white maize (control). Although the purity of starch granules was increased, the damaged starch (determined for the Maltase cross absence) was also increased. Scanning electron microscopy showed the presence of some pores and channels in the blue maize starch. The electrophoretic protein profiles showed differences in the bands that correspond to the enzymes involved in the starch biosynthesis; these differences could explain the variation in morphological characteristics of blue maize starches against starch from white maize.     

Investigation on morphological structure and crystal transition of maize starch gelatinized in pure glycerol

The gelatinization phenomena and crystalline structure of maize starch gelatinized in pure glycerol were investigated using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). Starch granules were firstly treated in water system, CLSM and SEM micrographs displayed that they were completely broken and the characteristic birefringence of the starch granules disappeared at 70 °C. As for pure glycerol system, the starch granules swelled but maintained granular shape with the increasing of temperature. The crystalline structure of starch granules was partially destroyed at 130 °C and completely destroyed at 140 °C. The DSC thermogram showed that the gelatinization temperature of starch in glycerol started at 123.7 °C, peaked at 128.4 °C, and concluded at 135.2 °C. The X-ray diffractograms indicated that the crystalline structure of maize starch was partially destroyed at 130 °C and completely destroyed at 140 °C. Thus, glycerol served an alternative solvent to destroy crystalline structure of maize starch, which may be helpful for hydrolysis of starch granules by amylase in food industry.     

Performance of Cry1A.105-selected fall armyworm (Lepidoptera: Noctuidae) on transgenic maize plants containing single or pyramided Bt genes

Cry1A.105 is a Cry protein expressed in some transgenic Bacillus thuringiensis (Bt) maize products. In this study, performance of five populations of fall armyworm, Spodoptera frugiperda (J.E. Smith), were evaluated on four non-Bt and eight commercial and experimental Bt maize hybrids/lines (hereafter referred as maize products). The five insect populations included one Cry1A.105-susceptible strain, two Cry1A.105-resistant strains, and two F₁ heterozygous genotypes. The eight Bt maize hybrids/lines consisted of five single-gene Bt maize products containing Cry1A.105, Cry2Ab2, Cry1F, or Cry1Ab protein, and three pyramided Bt maize products expressing Cry1A.105/Cry2Ab2, Cry1A.105/Cry2Ab2/Cry1F, or Cry1Ab/Vip3A for targeting aboveground lepidopteran maize pests. In the study, neonates of each population were tested on leaf tissues in the laboratory and whole plants in the greenhouse. Cry1A.105 and Cry1F maize killed 92.2–100% susceptible larvae in both test methods, while resistant larvae survived well on these two maize products. Performance of the two F₁ populations on Cry1A.105 and Cry1F maize varied between the two test methods. In leaf tissue bioassay, Cry1Ab maize was marginally effective against the susceptible population. In contrast, few live larvae and little leaf injury from any of the five populations were observed on Cry2Ab2 and the three pyramided Bt maize products. The results of this study showed evidence of cross resistance of the Cry1A.105-resistant S. frugiperda to Cry1F and Cry1Ab maize, but not to the Bt maize products containing Cry2Ab2 or Vip3A. Data generated from this study will be useful in developing resistance management strategies for the sustainable use of Bt maize technology.     

Properties of maize starch modified by ball milling in ethanol medium and low field NMR determination of the water molecular mobility in their gels

Ethanol as moisturizing agent and ball-milling treatment, has been combined in order to determine their impacts on the improvement of the properties of physically modified maize (Zea mays) starch granules. The content of ethanol has been set respecting a ratio of starch to ethanol varying from 1:0 to 1:3 (w:v), and the ball-milling time varied between 0 and 72 h. We observed that the increase of the amylose content varied in a more effective way with increase of the milling time (p < 0.05) than with the variation of the starch to ethanol ratios. As expected, modified starches were more transparent, more soluble, less crystalline, and presented damaged structures. In all cases, the starch granule sizes were better distributed at ratios of starch to ethanol of 1:0 and 1:3 (w:v) respectively. In addition, the impact of the combination of these treatments on the mobility of water molecules in starch gels characterized by the transverse relaxation time (T₂), as well as the abundance of protons (¹H T₂) in each populations were determined by low field NMR. Mobility of water molecules within starch gels increased at high temperature. Nonetheless, the proton population at T₂ > 10 ms (characterized by T₂₂) for the modified starch (starch/ethanol, 1:3 w:v) was fundamental in the different water concentrations, and accounts for 70 to 90% of total protons, at temperatures >60 °C.     

A Multi-stages Biosynthetic Pathway in Starch Granules Revealed by the Ultrastructure of Maize Mutant Starches

Our previous work indicated that starches containing B-type crystallites show low susceptibility to amylolysis and suggested that B-type crystallites have an effect on starch granule organisation. To elucidate granular ultrastructure, double wxae and aedu maize mutant starches containing A- (30 and 50% respectively) and B-type (70 and 50% respectively) crystallites were treated with porcine pancreatic alpha -amylase. The surface structure of the native and degraded starches was studied by scanning electron microscopy, and the internal ultrastructure by transmission electron microscopy after staining with PATAg reagents. The results confirm the influence of B-type crystallites on granule organisation and indicate that starches containing B-type crystallites show an amylolysis attack pattern with minor exocorrosion and major endocorrosion. The granule organisation of A- and B-type starches proposed is not consistent with an onion ring model¹and may account for the different behaviour of these starches to amylolysis. Transmission electron microscopy showed that most native wxae and aedu starch granules are composed of a core with a classical alternating structure and a peripheral ring. The peripheral ring in wxae starch was ordered and resistant to amylolysis, whereas that of aedu was disordered and degradable. It is proposed that these two specific forms of granule organisation are attributable to variations in the enzymatic activities of specific starch synthesising enzymes during the course of starch biosynthesis.     

Starch isolation method impacts soft wheat (Triticum aestivum L. cv. Claire) starch puroindoline and lipid levels as well as its functional properties

Wheat (Triticum aestivum L.) kernel hardness is a major quality characteristic, which has been ascribed to the presence of puroindolines a and b. These proteins occur in higher levels at the surface of water-washed starch granules from soft wheat cultivars than at that of starch from hard wheat cultivars. In the present study, prime starch was isolated from flour from soft wheat (cultivar Claire) using a dough ball or batter based separation method. Starch isolated with the dough ball method contained lower levels of puroindolines, as well as of other starch granule associated proteins and lipids than that isolated with the batter method. Similar patterns of puroindoline and lipid levels after starch isolation can presumably be related to (polar) lipid binding by puroindolines. Both isolated starch fractions showed comparable differential scanning calorimetry thermograms, whereas higher levels of starch surface associated components restricted starch swelling. Necessary controls demonstrated that the observed differences did not arise from artefacts associated with hydration, fractionation or freeze-drying in the experimental protocols. Apparently, proteins and lipids at the starch granule surface impact water absorption and, as such, starch swelling, but they do not affect starch granule internal phenomena such as melting of the crystalline amylopectin chains.     

The tryptophan-rich domain of puroindoline is directly associated with the starch granule surface as judged by tryptic shaving and mass spectrometry

The starch granule surface is a frontline of microbial attack and defence, operating in the background of normal starch granule metabolism. Puroindoline, a wheat protein which binds starch granule surfaces, contains a unique tryptophan-rich domain likely responsible for this property, though direct evidence is lacking. To test puroindoline’s tight association, prime starch granule extracts were water-washed 8 or 20 times and residual puroindoline removed using a solution of 50% isopropanol/50 mM NaCl. We found that this solvent was consistent in the amount of protein extracted from wheat flour and washed starch, regardless of initial protein content. Relative quantification of puroindoline following water-washing was performed using dot blot. Washing more than 8 times did not further reduce puroindoline content of starch granules suggesting a strong association with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, a combination of in situ tryptic digestion and mass spectrometry was used. Following digestion and water-washing, 50% isopropanol/50 mM NaCl was used to remove tightly-associated peptides for identification by mass spectrometry. Using this method, we identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat.