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

Roya Janeen Ndimba (2017-12)

Thesis (PhD)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Cereals are globally recognised as a cornerstone of human nutrition, and as a result play a pivotal role in efforts to address food insecurity and malnutrition. In Africa, the highest rates of hunger and malnutrition are evident, which is often due to an over-reliance on cereals as a principal source of nutrition. To address this problem, biofortification strategies are currently underway which aim to produce improved cereal crops, particularly with enhanced grain protein and mineral nutritional profiles. Two important African cereals that have been included in such biofortification programmes are 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 on by 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 mineral qualities, 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 pearl millet grains (produced via conventional plant breeding) (Part 2) were examined in order to assess the effect that biofortification process has had on the composition and other important quality characteristics of the grain. In the case of the genetically engineered sorghum, several independent transgenic lines, produced using RNA interference (RNAi) to suppress different subsets of kafirins were assessed in comparison to the wild-type progenitor to reveal if any unwanted changes occurred in the physico-chemical characteristics of the grain, apart from the intended change in the targeted protein profile. To carry out this comparison, an assessment of several key physical and biochemical parameters of the transgenic versus the wild-type grain were carried 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 protein bodies of all the sorghum genotypes, using transmission electron microscopy (TEM), revealed some important differences in morphology. Kafirin suppression was confirmed in all the transgenic lines using one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDSPAGE), as well as a compensatory synthesis of other grain proteins in the fractionated protein profile. Identification of some of the compensatory proteins was done using nanoflow liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry (Nano-LC/MALDIMS). Lastly, an analysis of the mineral content in bulk (by Inductively Coupled Plasma – Atomic Emission 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 endosperm region. In conclusion, the results of these experiments have been instrumental in highlighting important similarities and differences between the transgenic and non-transgenic sorghum, which have implications for the further development of these protein biofortified lines for enhanced nutrition. In the second section of the work, papers are presented on work done on the elemental mapping of 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 minerals in pearl millet is presented. Micro-PIXE was used to map the distribution of several nutritionally important 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 within the 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 low concentrations of the surveyed minerals. Within the germ, the scutellum was revealed as a major storage tissue for phosphorus (P) and potassium (K), whilst calcium (Ca), manganese (Mn) and zinc (Zn) were more prominent within the embryo. Iron (Fe) was revealed to have a distinctive distribution pattern, confined to the dorsal end of the scutellum; but was also highly concentrated in the outer grain layers. Interestingly, the hilar region was also revealed as a site of high accumulation of minerals, particularly for sulphur (S), Ca, Mn, Fe and Zn, which may be part of a defensive strategy against infection or damage. In the second paper, the use of micro-PIXE to study differential mineral accumulation in two contrasting pearl millet genotypes is presented. Using micro-PIXE fully elemental maps were generated for each of the contrasting grain types, allowing for a comparison of the spatial distribution 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 an approximate two-fold increase in Fe and Zn levels in both the grain endosperm and seed coat region, in comparison to the low Fe/Zn phenotype. These studies serve to highlight the utility of the micro-PIXE technique for localising and quantifying in-tissue concentration levels of important dietary minerals, such as Fe and Zn. The presented work therefore gives several new insights into the intended and perhaps nonintended differences that can result from the biofortification of cereals grains. This information can be of some benefit to the continued effort by plant scientists to improve the nutritional quality of the important staple foods that sustain millions of the world’s most poor and marginalised people.

AFRIKAANSE OPSOMMING: Graangewasse word wêreldwyd erken as ’n hoeksteen van menslike voeding en speel gevolglik ’n sentrale rol wanneer dit kom by die aanspreek van voedselonsekerheid en wanvoeding. Die hoogste vlakke van honger en wanvoeding kom in Afrika voor, in baie gevalle as gevolg van ’n oor-afhanklikheid op graangewasse as vernaamste bron van voeding. Om hierdie probleem aan te spreek, word biofortifiseringstrategieë tans onderneem met die doel om verbeterde graangewasse te produseer, veral met verhoogde graanproteïen- en mineraalvoedingsprofiele. Twee belangrike Afrika-grane wat in sulke biofortifiseringsprogramme ingesluit is, is sorghum (Sorghum bicolor (L.) Moench) en pêrelmanna (Pennisetum glaucum (L.) R. Br.). Sorghum en manna dien reeds vir eeue as belangrike stapels en word deur miljoene van die wêreld se armes gebruik as kos, veral in gebiede wat geneig is tot droogte. Hierdie grane skiet egter in baie gevalle tekort in terme van hulle proteïen- en of mineraalgehalte, en dus is daar ’n behoefte aan die produksie van biogefortifiseerde sorghum en pêrelmanna. In hierdie studie is biogefortifiseerde sorghum (geproduseer deur genetiese manipulasie) (Deel 1) en biogefortifiseerde pêrelmannagrane (geproduseer deur konvensionele planteteelt) (Deel 2) ondersoek om die effek van die biofortifiseringsproses op die samestelling en ander belangrike gehaltekenmerke van die graan te assesseer. In die geval van geneties gemodifiseerde sorghum is verskeie onafhanklike transgeniese lyne wat deur die gebruik van RNA steuring (RNA interference – RNAi) geproduseer is om verskillende substelle van kafiriene te onderdruk, geassesseer in vergelyking met die wilde tipe stamvader om uit te vind of enige ongewenste veranderinge in die fisies-chemiese kenmerke van die graan plaasgevind het, buiten die bedoelde verandering in die geteikende proteïenprofiel. Om hierdie vergelyking uit te voer, is ’n assessering van verskeie belangrike fisiese en biochemiese parameters van die transgeniese teenoor die wilde tipe graan uitgevoer. Met gebruik van eenrigting variansie-analise (ANOVA) is belangrike verskille in graangewig, -digtheid, endospermtekstuur en lisiengehalte gevind. Ultrastrukturele analise van die proteïenliggaampies van al die sorghum-genotipes m.b.v. TEM het ’n paar belangrike verskille in morfologie getoon. Kafirien-onderdrukking is in al die transgeniese lyne met behulp van eendimensionele SDS PAGE bevestig, asook ’n kompensatoriese sintese van ander graanproteïene in die gefraksioneerde proteïenprofiel. Die identifisering van sommige van die kompenserende proteïene is gedoen met nano-LC MALDI massa spektrometrie. Laastens is ’n analise van die mineraalinhoud in grootmaat (deur ICP) en binne die graanweefsel deur mikro-PIXE uitgevoer. Elementale kartering van die graanweefsel, met gebruik van mikro-PIXE, het ’n noemenswaardige afname in Zn (> 75%) getoon wat in die buitenste endospermstreek gelokaliseer is. Ten slotte, die resultate van hierdie twee eksperimente was instrumenteel in die uitlig van belangrike ooreenkomste en verskille tussen die transgeniese en nie-transgeniese sorghum wat belangrike implikasies het vir die verdere ontwikkeling van hierdie proteïenbiogefortifiseerde lyne vir verhoogde voeding. In die tweede deel van die werk is voorleggings gedoen oor werk op die elementale kartering van pêrelmannakultivars betrokke in pogings tot minerale biofortifisering. In die eerste voorlegging word ’n algemene oorsig aangebied van die gebruik van mikro- PIXE om die verspreiding van minerale in pêrelmanna te bestudeer. Mikro-proton geïnduseerde X-straal uitstraling (mikro-PIXE) is gebruik om die verspreiding van verskeie minerale van voedingsbelang te karteer wat in die graanweefsel van die twee kultivars van pêrelmanna (Pennisetum glaucum (L.) R. Br.) gevind is. Die verspreidingskaarte toon dat die oorheersende lokalisering van minerale binne die kiem (bestaande uit die saadlob en vrug) en die buitenste graanlae (spesifiek die perikarp en aleuroon) was; terwyl die meeste van die endospermweefsel redelike lae konsentrasies van die ondersoekte minerale bevat het. Binne die kiem is die saadlob gevind om die vernaamste stoorweefsel vir P en K te wees, terwyl Ca, Mn en Zn meer prominent in die vrug was. Fe het ’n kenmerkende verspreidingspatroon gehad, en is beperk tot die dorsale kant van die saadlob, maar dit was ook baie gekonsentreerd in die buitenste lae van die graan. Van belang is dat dit na vore gekom het dat die omgewing van die naeltjie (hilar region) ’n ligging was vir ’n groot akkumulasie van minerale, veral S, Ca, Mn, Fe en Zn, wat moontlik deel is van ’n verdedigingstrategie teen besmetting of skade. In die tweede voorlegging word die gebruik van mikro-PIXE om differensiële mineraalophoping in twee kontrasterende pêrelmanna-genotipes te bestudeer, aangebied. Met gebruik van mikro-PIXE is volledig kwantitatiewe elementkaarte vir elk van die kontrasterende graantipes gegenereer, wat dit moontlik gemaak het om die ruimtelike verspreidingspatrone en weefselspesifieke konsentrasies van verskeie belangrike minerale, soos K, Ca, Fe en Zn, te vergelyk. In die geval van die hoë Fe/Zn fenotipe het kwantitatiewe mikro-PIXE analises ’n ongeveer tweevoudige verhoging in Fe- en Znvlakke in beide die endosperm en saadhuid gebied bevestig, in vergelyking met die lae Fe/Zn fenotipe. Hierdie studies dien om die bruikbaarheid van die mikro-PIXE tegniek vir die lokalisering en kwantifisering van in-weefsel konsentrasievlakke van belangrike dieetminerale, soos Fe en Zn, te beklemtoon. Die werk wat hier aangebied word, verskaf verskeie nuwe insigte in die bedoelde en dalk onbedoelde verskille wat kan voortspruit uit die biofortifisering van graankorrels. Hierdie inligting kan van waarde wees vir die voortgesette poging deur plantwetenskaplikes om die voedingswaarde te verbeter van belangrike soorte stapelvoedsel wat miljoene van die wêreld se armste en mees gemarginaliseerde mense onderhou.

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