Biophysical characterization of the mineral composition of seeds with varying genetic background including transgenic sorghum with reduced amounts of the storage protein kafirin

[摘要] ENGLISH ABSTRACT: Cereals are globally recognised as a cornerstone of human nutrition, and as a result play a pivotalrole in efforts to address food insecurity and malnutrition. In Africa, the highest rates of hunger andmalnutrition are evident, which is often due to an over-reliance on cereals as a principal source ofnutrition. To address this problem, biofortification strategies are currently underway which aim toproduce improved cereal crops, particularly with enhanced grain protein and mineral nutritionalprofiles. Two important African cereals that have been included in such biofortification programmesare sorghum (Sorghum bicolor (L.) Moench) and pearl millet (Pennisetum glaucum (L.) R. Br.).Sorghum and millets have served as important staples for centuries, and are extensively relied onby millions of the world's poor, for nutritional sustenance, particularly in drought prone areas.Unfortunately, these grains are often nutritionally deficient in terms of their protein and/or mineralqualities, thus there is a need to produce biofortified sorghum and pearl millet.In this study, biofortified sorghum (produced via genetic engineering) (Part 1) and biofortified pearlmillet grains (produced via conventional plant breeding) (Part 2) were examined in order to assessthe effect that biofortification process has had on the composition and other important qualitycharacteristics of the grain. In the case of the genetically engineered sorghum, severalindependent transgenic lines, produced using RNA interference (RNAi) to suppress differentsubsets of kafirins were assessed in comparison to the wild-type progenitor to reveal if anyunwanted changes occurred in the physico-chemical characteristics of the grain, apart from theintended change in the targeted protein profile. To carry out this comparison, an assessment ofseveral key physical and biochemical parameters of the transgenic versus the wild-type grain werecarried out. Using one way analysis of variance (ANOVA) important differences in grain weight,density, endosperm texture and lysine content were found. Ultrastructural analysis of the proteinbodies of all the sorghum genotypes, using transmission electron microscopy (TEM), revealedsome important differences in morphology. Kafirin suppression was confirmed in all the transgeniclines using one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDSPAGE),as well as a compensatory synthesis of other grain proteins in the fractionated proteinprofile. Identification of some of the compensatory proteins was done using nanoflow liquidchromatography matrix-assisted laser desorption/ionization mass spectrometry (Nano-LC/MALDIMS).Lastly, an analysis of the mineral content in bulk (by Inductively Coupled Plasma – AtomicEmission Spectrometry (ICP-AES) and by Inductively Coupled Plasma – Mass Spectrometry (ICPMS);and within the grain tissue, by particle induced X-ray emission with a microfocused beam(micro-PIXE), was carried out. Elemental mapping of the grain tissue, using micro-PIXE,demonstrated a significant decrease in Zn (>75%), which was localised to the outer endospermregion. In conclusion, the results of these experiments have been instrumental in highlightingimportant similarities and differences between the transgenic and non-transgenic sorghum, whichhave implications for the further development of these protein biofortified lines for enhancednutrition.In the second section of the work, papers are presented on work done on the elemental mappingof pearl millet cultivars involved in mineral biofortification efforts.In the first paper, a general overview of the use of micro-PIXE to study the distribution of mineralsin pearl millet is presented. Micro-PIXE was used to map the distribution of several nutritionallyimportant minerals found in the grain tissue of two cultivars of pearl millet (Pennisetum glaucum(L.) R. Br.). The distribution maps revealed that the predominant localisation of minerals was withinthe germ (consisting of the scutellum and embryo) and the outer grain layers (specifically the pericarp and aleurone); whilst the bulk of the endosperm tissue featured relatively lowconcentrations of the surveyed minerals. Within the germ, the scutellum was revealed as a majorstorage tissue for phosphorus (P) and potassium (K), whilst calcium (Ca), manganese (Mn) andzinc (Zn) were more prominent within the embryo. Iron (Fe) was revealed to have a distinctivedistribution pattern, confined to the dorsal end of the scutellum; but was also highly concentrated inthe outer grain layers. Interestingly, the hilar region was also revealed as a site of highaccumulation of minerals, particularly for sulphur (S), Ca, Mn, Fe and Zn, which may be part of adefensive strategy against infection or damage.In the second paper, the use of micro-PIXE to study differential mineral accumulation in twocontrasting pearl millet genotypes is presented. Using micro-PIXE fully elemental maps weregenerated for each of the contrasting grain types, allowing for a comparison of the spatialdistribution patterns and tissue-specific concentrations of several important minerals such as K,Ca, Fe and Zn. In the case of the high Fe/Zn phenotype, micro-PIXE analysis confirmed anapproximate two-fold increase in Fe and Zn levels in both the grain endosperm and seed coatregion, in comparison to the low Fe/Zn phenotype. These studies serve to highlight the utility of themicro-PIXE technique for localising and quantifying in-tissue concentration levels of importantdietary minerals, such as Fe and Zn.The presented work therefore gives several new insights into the intended and perhaps nonintendeddifferences that can result from the biofortification of cereals grains. This information canbe of some benefit to the continued effort by plant scientists to improve the nutritional quality of theimportant staple foods that sustain millions of the world's most poor and marginalised people.

[发布机构] Stellenbosch University