Western Washington University Western CEDAR WWU Graduate School Collection WWU Graduate and Undergraduate Scholarship Fall 2016 Histidine Domain-Arabinogalactan Proteins (HD-AGPs) in the Solanaceae Jenna K. (Jenna Kristine) Brooks Western Washington University, [email protected] Follow this and additional works at: https://cedar.wwu.edu/wwuet Part of the Biology Commons Recommended Citation Brooks, Jenna K. (Jenna Kristine), "Histidine Domain-Arabinogalactan Proteins (HD-AGPs) in the Solanaceae" (2016). WWU Graduate School Collection. 541. https://cedar.wwu.edu/wwuet/541 This Masters Thesis is brought to you for free and open access by the WWU Graduate and Undergraduate Scholarship at Western CEDAR. It has been accepted for inclusion in WWU Graduate School Collection by an authorized administrator of Western CEDAR. For more information, please contact [email protected]. HISTIDINE DOMAIN-ARABINOGALACTAN PROTEINS (HD-AGPS) IN THE SOLANACEAE By Jenna Kristine Brooks Accepted in Partial Completion Of the Requirements for the Degree Master of Science Kathleen L. Kitto, Dean of the Graduate School ADVISORY COMMITTEE Chair, Dr. Anu Singh-Cundy, Department of Biology Dr. Lynn Pillitteri, Department of Biology Dr. Marion Brodhagen, Department of Biology MASTER’S THESIS In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Western Washington University, I grant to Western Washington University the non- exclusive royalty-free right to archive, reproduce, distribute, and display the thesis in any and all forms, including electronic format, via any digital library mechanisms maintained by WWU. I represent and warrant this is my original work, and does not infringe or violate any rights of others. 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Signature: Jenna Kristine Brooks Date: November 14th, 2016 HISTIDINE DOMAIN-ARABINOGALACTAN PROTEINS (HD-AGPS) IN THE SOLANACEAE A Thesis Presented to The Faculty of Western Washington University In Partial Fulfillment Of the Requirements for the Degree Master of Science By Jenna Kristine Brooks November 2016 ABSTRACT Arabinogalactan Proteins (AGPs) are a large family of hydroxyproline-rich glycoproteins found in the extracellular matrix of diverse cell types in plants. These glycoproteins are proposed to play a role in cell wall development, cell-cell interactions, and pattern formation. The goal of this research was to compare Histidine-Domain Arabinogalactan Proteins (HD-AGPs) in the Solanaceae and to examine the patterns of expression in vegetative and reproductive tissue. HD-AGP cDNA clones were isolated from pistils of Petunia inflata (PifPRP1), P. axillaris parodii (Pa2PRP1), and P. exserta (PePRP1). HD-AGPs from the two-interbreeding species, P. axillaris parodii and P. exserta, are 99.9% identical at the nucleotide level and identical at the amino acid level. HD- AGPs from P. inflata (PifPRP1) and P. axillaris parodii (Pa2PRP1), two reproductively isolated species, show that PifPRP1 is 98.5% identical to Pa2PRP1, with a 12 bp deletion in the hypervariable domain that is specific to the P. axillaris complex. PCR using genomic DNA yielded a single amplicon from each of these species, but two bands from Petunia hybrida and from Nicotiana tabacum. Comparison of intron sequence yielded some interesting insights about the evolutionary trajectory of sister species in these clades. Immunoblots using P. axillaris axillaris revealed discrete glycoforms of HD-AGPs in vegetative tissues contrasting with the tremendous size heterogeneity displayed by HD-AGPs expressed in the pistil. Etiolated seedlings accumulate higher levels of the protein, including significant amounts of an unglycosylated isoform. These findings suggest an important role for HD-AGPs in rapid elongation of cell types in both reproductive and vegetative tissues. iv ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Anu Singh-Cundy for her patient, nurturing attitude toward the scientific process. Her pursuit of knowledge and excitement of discovery made graduate school a more enlightening and expansive experience than I could have ever hoped for. I am thankful to the other thesis committee members, Drs. Lynn Pillitteri and Marion Brodhagan, who helped me throughout the project and ensured I was prepared to attain my research goals. Thanks to the many undergraduates for their help with my project, especially Jillaine Corey, Gavin Macklin, Amreen Toor and Kaytlyn Ly for all their hard work and time spent in the lab. I am grateful for the reagents and other lab supplies provided by the Pillitteri and Moyer labs. Thanks, go to Dr. Sandra Shultz for allowing me to use the FluorChem Imager. I thank the Hodgson Family Endowment and the WWU Office of Research and Sponsored Programs (RSP) for the grants they awarded in support of my research; without these funds my project would not have been as complete and comprehensive. I would also like to thank my family for supporting me through this time in my life and for giving me the confidence to know I can accomplish anything I put my mind to. Thank you to Ryan Antolock, for his shared pursuit of knowledge that encouraged me to expand my educational horizons; and for the support he extended over many years to get me to where I am today. I am grateful for the friendships I have forged throughout my time at Western. There are so many wonderful fellow graduate students who made my experience less stressful and downright fun. It is amazing to have spent so much time with others who have similar interests and zest for learning. I am thankful to my partner, Ryan McLaughlin, for his unwavering support and care. His positive demeanor and encouraging attitude has kept me going during the difficult times and pushed me to strive to reach my highest goals. v TABLE OF CONENTS ABSTRACT ............................................................................................................................. iv LIST OF FIGURES ............................................................................................................... viii LIST OF TABLES ................................................................................................................... xi INTRODUCTION .................................................................................................................... 1 The Genus Petunia ............................................................................................................ 2 The Genus Nicotiana ......................................................................................................... 4 Pollen-Pistil Interactions.................................................................................................... 5 Transmitting Tissue-Specific (TTS) Proteins within the Pistil ......................................... 7 Histidine-Domain Arabinogalactan Proteins (HD-AGPs) ................................................ 9 METHODS ............................................................................................................................. 12 Plant Material and Growth Conditions ............................................................................ 12 Nucleotide Sequence Analysis ............................................................................................ 13 Genomic DNA Extraction ............................................................................................... 13 RNA Extraction ............................................................................................................... 14 Reverse-Transcription (cDNA Synthesis) ....................................................................... 15 PCR Amplification with Phusion® High Fidelity Taq Polymerase ................................ 18 Purification of Amplicons (Phusion® Products) using Phenol-Chloroform Extraction . 19 Addition of 3’-A Overhangs ............................................................................................ 19 PCR Cloning into the pENTR 5’-TOPO® TA Vector .................................................... 20 Sanger Sequencing of HD-AGP Genomic and cDNA Clones ........................................ 21 Protein Analysis .................................................................................................................. 22 Protein Extraction ............................................................................................................ 22 SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) ................ 23 Protein Immunoblots ....................................................................................................... 24 RESULTS ............................................................................................................................... 27 HD-AGP Intron from P. hybrida is Nearly Identical to Intron Identified in P. inflata ... 27 HD-AGP Sequences