South Dakota State University Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange Electronic Theses and Dissertations 2018 Stability Analysis of Immunogenic Gliadin Accumulation in Hard Red Spring Wheat (Triticum Aestivum L.) Stacy Lindblom-Dreis South Dakota State University Follow this and additional works at: https://openprairie.sdstate.edu/etd Part of the Agronomy and Crop Sciences Commons, and the Food Science Commons Recommended Citation Lindblom-Dreis, Stacy, "Stability Analysis of Immunogenic Gliadin Accumulation in Hard Red Spring Wheat (Triticum Aestivum L.)" (2018). Electronic Theses and Dissertations. 2430. https://openprairie.sdstate.edu/etd/2430 This Dissertation - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. STABILITY ANALYSIS OF IMMUNOGENIC GLIADIN ACCUMULATION IN HARD RED SPRING WHEAT (TRITICUM AESTIVUM L.) BY STACY LINDBLOM-DREIS A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy. Major in Plant Science South Dakota State University 2018 iii ACKNOWLEDGEMENTS Recognition and gratitude go to all the individuals and groups who supported this dissertation work: Dr. William Berzonsky, who gave me the first paper I read about celiac epitopes in gliadin and became a committee member; Dr. Karl Glover, who chose to be my advisor and gave me the opportunity to conduct this work; Dr. Padmanaban Krishnan, committee member and reference regarding wheat quality; Dr. Jai Rohila, a committee member whose protein chemistry knowledge was invaluable in my experimental setup; committee member Dr. Brent Turnipseed whose advice on seed quality and germination was invaluable; the SDSU Spring Wheat Breeding Program staff and students; Dr. David Knudsen, with whom I have had many interesting and educational discussions regarding immunohistochemistry, antibody production, and animal models of disease; my former colleagues at the SDSU Veterinary Science Department and Connie Tande of the SDSU Extension Plant Pathology Laboratory, who gave me advice on ELISA development; Dr. Sunish Sehgal, who generously provided the used of his plate reader; and my family and friends who had unending patience with me during my intense work efforts. This work was funded by The South Dakota Wheat Commission. The GRIN accessions were provided by the National Small Grains Collection (Aberdeen, ID) with the assistance of curator Harold Bockelman. Part of this project was conducted using the South Dakota State University Functional Genomics Core Facility, supported in part by the National Science Foundation/EPSCoR Grant No. 0091948 and by the State of South Dakota. iv CONTENTS ABBREVIATIONS ............................................................................................................ x LIST OF FIGURES .......................................................................................................... xii LIST OF TABLES ........................................................................................................... xiv ABSTRACT .................................................................................................................... xvii CHAPTER 1. INTRODUCTION ....................................................................................... 1 Background ......................................................................................................................... 1 Statement of the problem .................................................................................................... 4 Aims and objectives ............................................................................................................ 5 Aim 1: To quantify gliadin accumulation in 200 wheat cultivars. ................................. 5 Aim 2: To characterize genetic polymorphism in the selected cultivars. ....................... 6 Aim 3: To examine genotype-by-environment interactions ........................................... 6 Rationale and significance .................................................................................................. 6 Expected outcomes ............................................................................................................. 8 Context ................................................................................................................................ 9 Literature Cited ................................................................................................................. 10 CHAPTER 2. LITERATURE REVIEW .......................................................................... 21 Abstract ............................................................................................................................. 21 Gluten and human health .................................................................................................. 21 Historical milestones in diagnosis of celiac disease ..................................................... 22 Incidence of celiac disease and causal factors .............................................................. 23 The immune response underlies celiac disease pathology ............................................ 27 Celiac disease differs from other wheat sensitivities .................................................... 34 v Celiac disease differs from allergies and food intolerances ......................................... 37 Misconceptions regarding gluten and human diseases ..................................................... 40 Historical plant breeding targets ................................................................................... 41 Genetic engineering versus transgenic introgression and selection .............................. 44 Dwarfing genes and protein accumulation ................................................................... 45 Older cultivars are “healthier” than new cultivars of wheat ......................................... 47 Wheat has been implicated in non-celiac health conditions ............................................. 48 Claims supported by evidence ...................................................................................... 48 Claims unsupported by evidence .................................................................................. 51 Gluten and wheat health.................................................................................................... 51 Seed storage proteins of grains ..................................................................................... 52 The origin and fate of seed storage proteins in wheat .................................................. 54 Gene expression ............................................................................................................ 55 Transport and storage .................................................................................................... 55 Physiological functions of gliadin in wheat .................................................................. 56 The pathway is robust and dynamic ............................................................................. 57 Seed storage proteins and end-use quality of wheat ......................................................... 58 Conclusion ........................................................................................................................ 59 Literature Cited ................................................................................................................. 61 CHAPTER 3. PHENOTYPIC ASSAY OF GLIADIN ACCUMULATION ................... 90 Abstract ............................................................................................................................. 90 Introduction ....................................................................................................................... 90 vi Detection of target molecules ....................................................................................... 91 ELISA formats .............................................................................................................. 95 Special considerations ................................................................................................... 99 Methods........................................................................................................................... 100 Plant materials ............................................................................................................. 100 ELISA development ................................................................................................... 101 Sample preparation ..................................................................................................... 102 Indirect ELISA for quantifying total gliadin .............................................................. 103 Direct ELISA for quantifying total gliadin ................................................................. 103 Assay parameters and criteria ..................................................................................... 103 Commercial ELISA ...................................................................................................