Effect of Grain Structure and Cooking on Sorghum and Maize in vitro Protein Digestibility

Uncooked and cooked sorghum showed improvement in in vitro protein digestibility as the structural complexity of the sample reduced from whole grain flour through endosperm flour to protein body-enriched samples. This was not the case for maize. Cooking reduced protein digestibility of sorghum but not maize. Treating cooked sorghum and maize whole grain and endosperm flours with alpha -amylase to reduce sample complexity before in vitro pepsin digestion slightly improved protein digestibility. The reduction in sorghum protein digestibility on cooking was not related to the total polyphenol content of samples. Pericarp components, germ, endosperm cell walls, and gelatinised starch were identified as possible factors limiting sorghum protein digestibility. Electrophoresis of uncooked and cooked protein-body-enriched samples of sorghum and maize, and prolamin fractions of sorghum under non-reducing conditions showed oligomeric proteins with molecular weights (M_r) 45, 66 and >66 kDa and monomeric kafirins and zeins. Protein-body-enriched samples of sorghum had more 45–50 kDa oligomers than those of maize. In cooked sorghum, some of these were resistant to reduction. Pepsin-indigestible residues from protein-body-enriched samples consisted mainly of α-zein (uncooked and cooked maize) or α-kafirin (uncooked sorghum), whilst cooked sorghum had in addition, β- and γ-kafirin and reduction-resistant 45–50 kDa oligomers. Cooking appears to lead to formation of disulphide-bonded oligomeric proteins that occurs to a greater extent in sorghum than in maize. This may explain the poorer protein digestibility of cooked sorghum.     

Effect of suppressing the synthesis of different kafirin sub-classes on grain endosperm texture, protein body structure and protein nutritional quality in improved sorghum lines

To improve sorghum grain protein nutritional quality, improved sorghum lines were transformed to suppress the synthesis of different kafirin sub-classes, or backcrossed into transgenic lines with improved protein quality. Co-suppression of the alpha-, gamma- and delta-kafirin sub-classes and removal of the tannin trait resulted in transgenic sorghum lines with high cooked protein digestibility (±80%), improved Amino Acid Score (0.8) and Protein Digestibility Corrected Amino Acid Score (0.7) compared to the non-transgenic null controls (±50%, 0.4 and 0.2, respectively). These high protein quality lines had a floury endosperm. They also had modified protein body structure, where the protein bodies were irregular shaped with few to numerous invaginations and were less densely packed, with a dense protein matrix visible around the protein bodies. When fewer sub-classes were suppressed, i.e. gamma 1 and delta 2, the endosperm was corneous with normal protein body structure but the improvement in cooked protein digestibility appeared to be less. Apparently, co-suppression of several kafirin sub-classes is required to obtain high protein nutritional quality sorghum lines, but this seems to result in floury-type grain endosperm texture.     

Transgenic sorghum with suppressed synthesis of kafirin subclasses: Effects on flour and dough rheological characteristics

Arising from work showing that conventionally bred high protein digestibility sorghum types have improved flour and dough functionality, the flour and dough properties of transgenic biofortified sorghum lines with increased protein digestibility and high lysine content (TG-HD) resulting from suppressed synthesis of several kafirin subclasses, especially the cysteine-rich γ-kafirin, were studied. TG-HD sorghums had higher flour water solubility at 30 °C (p < 0.05) and much higher paste viscosity (41% higher) than their null controls (NC). TG-HD doughs were twice as strong as their NC and dynamic rheological analysis indicated that the TG doughs were somewhat more elastic up to 90 °C. CLSM of doughs and pastes indicated that TG-HD had a less compact endosperm protein matrix surround the starch compared to their NC. The improved flour and dough functional properties of the TG-HD sorghums seem to be caused by reduced endosperm compactness resulting from suppression of synthesis of several kafirin subclasses which modifies protein body and protein matrix structure, and to improved protein-starch interaction through hydrogen bonding specifically caused by reduction in the level of the hydrophobic γ-kafirin. The improved flour functionality of these transgenic biofortified sorghums can increase their commercial utility by complementing their improved nutritional quality.     

Electrophoretic analysis of malting degradability of major sorghum reserve proteins

Proteolysis is vital to the generation of amino acids and short peptides during malting. The qualitative and quantitative effects of malting on proteolytic digestion were investigated for 11 Botswana sorghum cultivars. Protein hydrolysis was influenced by sorghum grain cultivar. All protein fractions were degraded, although the extents of their digestion appeared cultivar-dependent. The most significant changes in total and free kafirins occurred among the HMW aggregates while the 45 kDa dimer appeared the most recalcitrant. Free kafirin monomers were digested in variety-defined manner; α-kafirin faded the most (ca. 82%) in Phofu, but appeared least digested (≤10%) in Lekgeberwa; β-kafirin faded the most in Phofu and Segaolane (90–94%) but only 46% in Lekgeberwa. Overall, α-kafirin appeared the least digested of the free monomers. The proportion of free kafirin accounted for by the 45 kDa and monomeric kafirins increased in all malts (except Lars Vyt and Mafia), probably due to protein depolymerisation. Grain variety significantly influenced (p < 0.001) malt FAN levels. Mean FAN values were highest in Segaolane (225 mg/100 g) followed by Sefofu, Town and BSH-1, but lowest in Mafia (96.9 mg/100 g). Results indicate wide variability in the proteolytic malting digestibility of the eleven sorghum cultivars.     

Preferential binding of sorghum tannins with γ-kafirin and the influence of tannin binding on kafirin digestibility and biodegradation

Kafirins, sorghum prolamins bind with sorghum condensed tannins (CTs). The binding of different kafirin species with sorghum CTs was investigated. Analysis by chemical assay and by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), reversed-phase high-performance liquid chromatography (RP-HPLC), and free zone capillary electrophoresis (FZCE), showed that γ-kafirin bound more CTs than the other kafirin species. SDS–PAGE suggested that the γ-kafirin-bound tannins were in the form of aggregates of molecular size >200k. RP-HPLC and FZCE revealed that sample preparation and drying the kafirins prior to the binding assays had a significant impact on γ-kafirin solubility. The effect of tannin binding on kafirin and kafirin film digestibility and film biodegradation was determined. Kafirins bound to tannins had lower digestibilities than unbound kafirins. Films made from tannin-bound kafirin had much lower digestibility and were less biodegradable than films made from unbound kafirin. The increase in kafirin film life by tannin modification appears to be due to a decrease in protein digestibility caused by kafirin–tannin binding. These findings suggest that γ-kafirin content in sorghum may be manipulated to either reduce or increase tannin binding in order to change the functionality of the kafirin in food, feed or film applications.     

Digestibility of protein and starch from sorghum (Sorghum bicolor) is linked to biochemical and structural features of grain endosperm

Although a principal source of energy and protein for millions of the world's poorest people, the nutritional value of sorghum (Sorghum bicolor L. Moench) is diminished because of low digestibility of grain protein and starch. To address this problem, we analyzed the properties of two sorghum lines that have a common pedigree but differ in digestibility. Consistent with results based on a ruminal fluid assay, the protein and starch of one line (KS48) was more thoroughly digested than that of the other (KS51) using in vitro assays based on pepsin and α-amylase. The indigestibility of KS51 relative to KS48 was shown to be due to (i) a greater abundance of disulfide-bonded proteins; (ii) presence in KS51 of non-waxy starch and the accompanying granule-bound starch synthase; and (iii) the differing nature of the protein matrix and its interaction with starch. The current findings suggest that each of these factors should be considered in efforts to enhance the nutritional value of sorghum grain.     

Heritability and quantitative trait loci of composition and structural characteristics in sorghum grain

Breeding efforts in cereal crops directed toward developing or improving end-use products of grain require assessment of existing phenotypic variance and an understanding of the genetic control of grain quality traits. To this end, a grain sorghum [Sorghum bicolor (L.) Moench] mapping population consisting of 113 F_2:7 recombinant inbred lines (RILs) derived from a cross between Sureño and RTx430 was evaluated in multiple environments for grain composition (fat, fiber, protein, starch) using near-infrared reflectance spectroscopy (NIRS), and size estimates of grain parts (embryo, vitreous endosperm, floury endosperm, kernel area) using an image-based phenotyping software system. Estimates of broad-sense heritability of grain compositional traits ranged from 0.11 to 0.90, whereas those of grain size ranged from 0.16 to 0.72. Composite interval mapping (CIM) was applied to a single nucleotide polymorphism (SNP)-based linkage map to identify marker-trait associations, and through these efforts, a total of 37 quantitative trait loci (QTL) for grain quality were identified across environments. Each QTL explained between 7 and 23% of the phenotypic variation for a given grain trait. Three of the five QTL that colocalized were for traits with significant negative correlation, which included grain protein content that was negatively correlated with grain starch content. In addition, several traits that were positively correlated (e.g. fat and fiber content) also revealed colocalized QTL. Finally, we compared the present study with previous studies identifying grain composition trait loci in an effort to identify genomic regions controlling grain traits across a diversity of environments and sorghum genotypes.     

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.     

A comparison of inbred line and F1 testers for evaluating sorghum experimental lines in testcrosses

Selecting an effective tester for evaluating the hybrid performance and heterosis of new inbred lines is crucial to ensure their accurate evaluation. Most sorghum breeding programs use at least two elite inbred line testers when evaluating new inbred lines. These testcrosses provide valuable information, but the specificity of inbred line testers may incorrectly characterize experimental lines that would combine well with other genetic backgrounds. Reducing this risk by increasing the number of preliminary testcrosses is limited in breeding programs by resource availability. Efficiently diversifying testcrosses may be accomplished through the use of F₁ testers. Testcrosses made with F₁ testers simultaneously evaluate inbred lines against two genetic backgrounds. In preliminary testcrosses, this may reduce the probability of incorrectly discarding new lines or improve the efficiency of preliminary testcrossing. The objective of this research was to compare two sorghum inbred line testers to their respective F₁ as a tester for evaluating sorghum inbred lines (R-lines) in testcrosses for grain yield. The testers used were A.Tx623, A.Tx2752 and their F₁ hybrid, A.Tx623/B.Tx2752. Data from testcross hybrids from 33 different inbred lines grown in three environments indicated that the F₁ tester was comparable in accuracy and discriminatory efficiency to the inbred line testers. The results indicated that a single cross tester in sorghum can serve to effectively evaluate new germplasm.     

The Gsp-1 genes encode the wheat arabinogalactan peptide

Western blotting, ELISA and ¹H-NMR spectroscopy showed that RNAi down-regulation of the wheat Gsp-1 gene resulted in reduced contents of both arabinogalactan peptide (AGP) and grain softness protein (GSP-1) in mature wheat grains confirming that these components are encoded by the same gene. A small increase in grain hardness and decrease in the viscosity of aqueous extracts of the transgenic lines also indicated small effects on functional properties. Immunolocalisation using a novel wheat AGP monoclonal antibody in conjunction with confocal microscopy showed that the major form of AGP which was eliminated in knockout lines is located within the cell, probably in the vacuole, and not in the plasma membrane or cell wall. However, clear localisation of the AGP epitope to the plasma membrane was observed in both control and transgenic lines and probably resulted from the presence of one or more separate forms of arabinogalactan protein. The existence of such additional form(s) was also indicated by ¹H-NMR spectroscopy which showed that the ratio of arabinose to galactose differed between the control and transgenic lines.     

Effect of the use of dilute alkaline prior to Bacillus subtilis-based biocontrol steeping and germination conditions on red sorghum malt β-glucanase activities and residual β-glucans

Malting is the ideal stage to deal with β-glucans. Their hydrolysis is very important as the diffusion of both hormones and hydrolytic enzymes in the endosperm of germinated grain depend on it. A high malt β-glucanase activity is not a guarantee of an extensive hydrolysis of β-glucans. When Bacillus subtilis is used to control mould growth, red sorghum malt β-glucanase activity (measured using carboxymethylcellulose as the substrate) was improved without significantly affecting the hydrolysis of malt β-glucans. Thus, in order to reduce the residual β-glucans content, soaking in 0.2% NaOH was combined with a biocontrol. Soaking in 0.2% NaOH is recognized as capable of improving grain hydration by opening-up the endosperm cell walls. The combined use of 0.2% NaOH with B. subtilis-based biocontrol treatments during red sorghum malting, leads to malt with increased β-glucanase activity and a significant reduction of residual β-glucans when compared with the 16 h biocontrol steeping without prior steeping in 0.2% NaOH. β-glucanase activity increases with increased germination temperature and time while, conversely, the residual β-glucans content of the malts decreases. Indeed, while the level of β-glucanase was not vastly different between the malts obtained after steeping in distilled water and those obtained after 8 h steeping in 0.2% NaOH followed by 8 h resteeping in distilled water (NaOH + H₂O treatment), their residual β-glucans levels differ significantly. B. subtilis-based treatment leads to malt with improved β-(1-3)- and β-(1-4)-glucanase activities without significantly improved malt β-(1-3),(1-4)-glucanase activity. While malts obtained after 84 h germination weren't significantly different in terms of malt β-(1-3),(1-4)-glucanase activities for all steeping treatments, the use of 0.2% NaOH steeping prior to resteeping led to malts with improved β-glucans content. Combining the steeping in dilute alkaline and biocontrol enables taking advantage of the dilute alkaline effect on residual β-glucans content, due probably to the opening-up of the cell walls and the improvement of water uptake, and that of the biocontrol (improvement of β-glucanase synthesis).     

Specific expression of a sesame storage protein in transgenic rice bran

Rice oil bodies enclosed by unique structural proteins, oleosins, are found in the embryo and the aleurone layer, but not the starchy endosperm where starch and storage proteins are accumulated. To examine oleosin promoter specificity, a sesame storage protein, 2S albumin, was expressed in transgenic rice seeds under the control of a rice oleosin promoter. In all transgenic rice seeds, the sesame 2S albumin was found exclusively in the bran fraction after milling. Immunological staining revealed that the sesame 2S albumin was also located in the embryo and the outermost cells of the starchy endosperm. Furthermore, immunogold labeling showed that the transgenic 2S albumin was deposited in both type-I and type-II protein bodies of the outermost cells of the endosperm as well as in the type-II protein bodies of the embryo. The methionine and cysteine contents in the bran from four homozygous transgenic lines were elevated by 24–38 and 50–62%, respectively, compared with those of wild-type plants. The results suggest that the rice oleosin promoter is bran-specific and could be used to add value to rice bran, an abundant by-product of rice polishing, by genetic engineering.     

Protein quality and endosperm modification of quality protein maize (Zea mays L.) under two contrasting soil nitrogen environments

Quality protein maize (QPM) breeding involves the combined use of the opaque-2 (o2) gene and the genetic modifiers of the o2 locus to develop cultivars with modified kernel endosperm, and increased concentrations of lysine and tryptophan. This study was designed to assess grain yield performance, endosperm modification, and protein quality and quantity under two contrasting soil nitrogen environments. A 15-parent diallel cross was evaluated under one low nitrogen stress and one optimal nitrogen environment each at Harare (Zimbabwe) and Bako (Ethiopia). Most QPM hybrids showed higher protein quality levels than the best non-QPM check under both conditions. Protein concentration tended to vary across nitrogen levels, but not endosperm type. Significant differences were found for the test of main effect (nitrogen-level) for endosperm modification and tryptophan concentration. This indicated that QPM maintains quality even under low soil nitrogen, a widespread condition in Africa. General combining ability (GCA) mean squares were highly significant for most protein quality traits for each environment and across environments whereas specific combining ability (SCA) mean squares were not significant in most cases. This indicated that additive gene effects were primarily responsible for variation of most traits evaluated and hence progeny performance can adequately be predicted on the basis of parental performance. Inbred lines P2, P4 and P12 had desirable GCA effects for endosperm modification while P1 and P3 had the best GCA for tryptophan concentration in grain. The current study suggests that hybrids with desirable endosperm modification, protein quality and stable performance under low nitrogen stress and optimal conditions can be produced with careful selection.     

Disease and pest resistance in grains of sorghum and millets

In this review available information on the mechanisms of resistance to insect pests and fungal pathogens in sorghum and millets is discussed. The primary source of resistance lies in the chemical and physical make up of the grain. Phenolic compounds such as ferulic acid and tannins present in some sorghums are potent inhibitors of pests and pathogens. Grain hardness is a major deterrent to infection and infestation in low tannin grains. The prolamins, the grain storage proteins of sorghum, are organized into protein bodies and provide a physical and a nutritional barrier since they are resistant to digestion by insect and fungal proteases. A plethora of proteins that belong to the ‘pathogenesis related protein’ group are distributed in various parts of the grain. Some of them are located in protein bodies. Notwithstanding, sorghum is still susceptible to insect pests and fungal pathogens. An understanding of the natural mechanisms of resistance in the grain is paramount for the development of durable resistance against pests and pathogens. The pyramiding of resistance genes and the development of transgenic lines based on this understanding are two sources of hope for the future protection of sorghum and millets.     

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.     

Characterisation of oat (Avena sativa L.) oil bodies and intrinsically associated E-vitamers

Transmission electron microscopy of sections of oat (Avena sativa L.) grain suggested that the highest concentrations oil bodies were in the aleurone and germ rather than the starchy endosperm. Oil bodies recovered from homogenized tissues by centrifugation and washed in (9 M) urea were significantly (P<0.05) enriched in lipid (93.3±1.4% dry wt) and low in protein (1.4±0.2%) compared with unwashed (40.2±1.9% lipid; 23.0±1.8% protein), water-washed (78.6±1.2% lipid; 7.8±0.5% protein), and salt-washed (1 M NaCl) oil bodies (89.9±0.4% lipid; 5.1±0.4% protein). Washing significantly reduced (P<0.05) the total phenolic content of the oil bodies but significantly increased concentrations of E-vitamers, on a dry weight basis, suggesting an intrinsic association between the E-vitamers and oat oil bodies. The profile of E-vitamers in the oil bodies reflected that in oat grain with α-tocotrienol accounting for ca. 66% of the total E-vitamers. These E-vitamers may provide oxidative stability to the membrane and/or oil of oat oil bodies     

Quality characteristics and field performance of selectable marker-free transgenic rice with antisense Wx gene and improved quality derived from the elite parents of hybrid indica rice

In rice grains, high amylose content (AC) is correlated with poor grain quality, particularly in indica hybrid rice. To obtain indica hybrid rice with improved cooking and eating qualities, we introduced the antisense Waxy (Wx) gene into 2 elite parental lines of indica hybrid rice by using co-transformation methods. Subsequently, we selected several elite homozygous transgenic lines that did not contain the selectable marker. The expression of the endogenous Wx gene of the selected transgenic lines was significantly downregulated, resulting in low AC in the mature seeds; moreover, the AC in some lines reduced to the level observed in glutinous rice. With the decrease in AC, the gel consistency of the transgenic rice became softer, and the gelatinization temperature tended to be higher than those of the wild types, especially in the case of the Longtefu-derived transformants. We also analyzed the pasting properties of the selected transgenic low-AC lines, and we noted an improvement in the pasting properties of the transgenic rice lines. The results from a field trial indicated that the grain weights of the transgenic lines with lower AC exhibit remarkable reduction compared with those of the wild types.