A Study of Seed Storage Protein Accumulation by Ectopic Expression in Arabidopsis A Thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements of the Degree of Doctor of Philosophy in the Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK By Gordon M. Gropp © Copyright Gordon M. Gropp, November, 2013. All rights reserved. PERMISSION TO USE In presenting this thesis/dissertation in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis/dissertation work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. 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Requests for permission to copy or to make other uses of materials in this thesis/dissertation in whole or part should be addressed to: Head of the Department of Plant Sciences University of Saskatchewan Saskatoon, Saskatchewan S7N 5A8 Canada ABSTRACT Understanding the mechanisms plants utilize for seed storage protein (SSP) synthesis, transport and deposition have the potential rewards of enabling high yields of modified or foreign proteins. Hayashi et al. (1999) indicated that the machinery devoted to the synthesis of protein storage vacuoles in cotyledon cells can be induced in vegetative tissue by the constitutive expression of a pumpkin 2S albumin phosphinothricin-acetyl-transferase gene fusion (pumpkin 2S-PAT) resulting in the biogenesis of precursor-accumulating (PAC) vesicles in Arabidopsis leaves. This discovery was the impetus behind the work described which sought to examine this phenomenon further by ectopically evoking SSP trafficking and vesicle biogenesis machinery in leaves. With the aim of elucidating the mechanisms necessary to evoke PAC vesicle biogenesis, a suite of constructs including the pumpkin 2S-PAT and analogous napin-PAT and napin-GFP variants were synthesized. Analysis of these transgenes in Arabidopsis revealed that the pumpkin 2S albumin has a capacity unique from napin peptides to result in fusion protein accumulation. Further, the truncated pumpkin 2S albumin peptide and the pumpkin 2S albumin C-terminus were found to direct deposition to vesicles; however, the C-terminus alone was not enough to direct deposition to vesicles unless combined with a significantly shortened napin peptide. An increased ER protein throughput was correlated to trafficking of the fusion protein by Golgi-independent mechanisms resulting in stable accumulation of the unprocessed protein whereas less ER throughput indicated passage through the Golgi-dependent pathway resulting in accumulation of a processed variant. At the level of gene expression, as examined by a microarray study, both inducible and constitutive ectopic expression of pumpkin 2S-PAT resulted in substantial perturbations of the endomembrane system affecting protein folding, flowering time and ER- associated biosynthetic functions which indicated that modulation of flowering time and photoperiodism are highly dependent on protein trafficking and vacuolar biogenesis mechanisms and that high ER protein throughput occurs at the expense of biosynthesis and cessation of ER functioning. ii ACKNOWLEDGEMENTS This thesis is the culmination of several years of labor that began with only an idea. The Hayashi et al. (1999) paper was the impetus for this idea. Not long after beginning work within the Seed Protein Group at AAFC, a large team interested in investigating the repression of seed storage protein expression in non-seed tissues, the Hayashi paper provided the impetus for examining ectopic expression more closely so as to discover the nuances of SSP sorting and trafficking. The idea was a slight digression of discovering SSP repression mechanisms in vegetative tissue, but it was thought that examining the perturbations caused by intentional ectopic expression in leaf tissue may provide insight into these mechanisms. I am eternally grateful to Dr. Derek Lydiate for embracing this idea and who championed me to become a Ph. D. graduate student within the AAFC organization. There are several individuals I would like to acknowledge for their contributions to the completion of this work: namely, Mrs. Cathy Coutu for her expert assistance with Zeiss Apotome and confocal microscopy, Dr. Dwayne Hegedus for his valuable insight and encouragement, and my colleagues at AAFC whose camaraderie offered me inspiration. I sincerely thank the members of my committee for all their numerous suggestions which made my research and thesis more sound: Dr. Yuguang Bai, Dr. Kirstin Bett, Dr. Peta Bonham-Smith, Dr. Bruce Coulman, Dr. Gordon Gray and Dr. Sean Hemmingsen. I am especially grateful to Dr. Bonham-Smith whose devotion to my thesis allowed the document, and my own understanding of the science involved, to evolve considerably. Above all, I am eternally grateful to Dr. Kirstin Bett, who was, in addition to being a member of my committee, also my co-supervisor and whose efforts resulted in the completion of this work. Kirstin, thank you for your persistence and encouragement: Derek got me started and you got me finished. Thank you. iii TABLE OF CONTENTS page PERMISSION TO USE ...................................................................................................... i ABSTRACT ....................................................................................................................... ii ACKNOWLEDGMENTS ................................................................................................ iii TABLE OF CONTENTS .................................................................................................. iv LIST OF TABLES ........................................................................................................... vii LIST OF FIGURES ........................................................................................................ viii LIST OF ABBREVIATIONS ........................................................................................... xi CHAPTER 1 INTRODUCTION ....................................................................................... 1 1.1 Hypothesis and Objectives ....................................................................................... 3 CHAPTER 2 LITERATURE REVIEW 2.1 Overview of the Plant Endomembrane System ........................................................ 4 2.2 Seed Storage Proteins and the Protein Storage Vacuole ........................................... 8 2.2.1 SSP Classification ............................................................................................ 8 2.2.2 PSV Architecture and Biogenesis .................................................................... 9 2.3 Sorting and Trafficking Mechanisms of SSP.......................................................... 12 2.3.1 Vacuolar Sorting Determinants ...................................................................... 12 2.3.2 Targeting via VSR and RMR ......................................................................... 15 2.3.3 Trafficking Machinery.................................................................................... 18 2.4 Processing Events of SSP Maturation..................................................................... 24 2.4.1 Chaperones and Foldases ............................................................................... 24 2.4.2 Glycosylation .................................................................................................. 25 2.4.3 Proteolytic Processing .................................................................................... 27 CHAPTER 3 MATERIALS AND METHODS 3.1 Informatics .................................................................................................................. 29 3.1.1 Sequence Analysis .......................................................................................... 29 3.1.2 Secondary Structure Predictions ..................................................................... 29 3.2 Construct Design ..................................................................................................... 30 iv 3.2.1 PCR-based Cloning ........................................................................................ 30 3.2.2 Recombinant PCR for Synthesis of Gene Fusions ......................................... 31 3.2.3 Site-directed Mutagenesis .............................................................................. 36