ADVANCED MICROSCOPY IMAGING OF PHLOEM PROTEINS IN ARABIDOPSIS REVEAL INSIGHTS INTO MUNCH’S PRESSURE FLOW HYPOTHESIS By DANIEL ROBERT FROELICH A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY School of Biological Sciences MAY 2014 © Copyright by DANIEL ROBERT FROELICH, 2014 All Rights Reserved © Copyright by Daniel Robert Froelich, 2014 All Rights Reserved To the faculty of Washington State University: The members of the Committee appointed to examine the dissertation of DANIEL ROBERT FROELICH find it satisfactory and recommend that it be accepted. Michael Knoblauch, Ph.D., Chair Hanjo A. Hellmann, Ph.D. Winfried S. Peters, Ph.D. Raymond W. Lee, Ph.D. ii Acknowledgments I would not have been able to complete this Ph.D. without the tremendous support of so many people. Firstly, I want to thank Michael Knoblauch: my committee chair, boss and soccer buddy for coming out to Washington and inviting me into his lab. I thank Winifried Peters at Indiana Purdue Ft. Wayne for being on my committee despite having to put up all the logistical issues every time we meet or I needed a form signed. Hanjo Hellman has been a great help, always asking the right questions when I have become too myopic, focusing too closely on the details at the expense of the whole. Ray Lee has been invaluable. He taught me the importance of knowing my audience and how to cater my presentations to them. In the lab, I especially thank Dan Mullendore. We have worked together from the beginning and having such a talented peer has been invaluable. I hope I have helped him as much as he did for me. Tim Ross Elliott is a great guy to share an office with and he ensures that we will come back from every conference will have some fantastic stories. Hélène Pellissier and Ray Collier taught me how to clone. Under their guidance, I have created new organisms! Sierra Beecher is always such a sweet person and so nice to talk to. She has helped me both in the lab and out. Other past lab members: Jamie Watts, Adelina Petrova and Hannah Merley made this program a special adventure! Valerie Lynch-Holm and Christine Davitt are truly microscopy wizards. They have inspired my career in microscopy and taught me so much. Chuck Cody has always kept my plants green despite my best efforts to kill them. iii Without my favorite group of runners, the Beer Chasers, I would have surely lost my mind long ago. Wednesday nights are always a highlight of the week. Even the quarterly Beer Miles, so terrible during the event but amazing shortly after, are a hilarious way to break up the graduate student life. Scott, Annie, Graham, Steffie, Buzz are Aaron are all my closest, greatest friends. Most of all, I owe all this work to Nicole, my lovely wife. She has put up with me and helped celebrate the successes, and bore more than her fair of the weight from my failures. Without her, I would have burned out long ago, but with her support, I can flourish. This research was supported by the NSF. I personally received funding from the Herbert Eastlick fellowship, Betty Higinbotham travel grants, NASA and the Vincent Franceschi Graduate Research Fellowship. Science is expensive, but these groups have recognized our merit and paved the way for world class research. Thanks to everyone I missed and always: Go Cougs! iv ADVANCED MICROSCOPY IMAGING OF PHLOEM PROTEINS IN ARABIDOPSIS REVEAL INSIGHTS INTO MUNCH’S PRESSURE FLOW HYPOTHESIS Abstract By Daniel Froelich, Ph.D. Washington State University May 2014 Chair: Michael Knoblauch Phloem proteins have been widely regarded as a wound response mechanism. All imagery showing apparent occlusions of this protein at a sieve plate have been dismissed as preparation artifact and ignored. Unfortunately, these images only show one still frame of a movie and so all conclusions are susceptible to misinterpretation. Presented here is a combination of high resolution still images with complete context from a dynamic in vivo reference. This new perspective shows that not only are the Sieve Element Occluding Related (SEOR) phloem protein agglomerations in Arabidopsis common in healthy, translocating, uninjured plants, but that they do not appear to occlude the phloem at all. The previously known purpose of this very common family of proteins is once again obscure. v Table of Contents Acknowledgments.......................................................................................................................... iii Abstract ........................................................................................................................................... v Table of Contents ........................................................................................................................... vi List of Figures ................................................................................................................................ xi List of Tables ............................................................................................................................... xiii Chapter 1 - Introduction .................................................................................................................. 1 1.1 Phloem anatomy ................................................................................................................................. 2 1.1.1 Phloem fibers ............................................................................................................................... 2 1.1.2 Phloem Parenchyma .................................................................................................................... 3 1.1.3 Sieve elements ............................................................................................................................. 3 1.1.4 Companion cells ........................................................................................................................... 4 1.2 Phloem loading ................................................................................................................................... 4 1.3 Phloem Unloading ............................................................................................................................... 7 1.4 Phloem Transport ............................................................................................................................... 8 1.4.1 Sieve element plastids ............................................................................................................... 10 1.5 Endoplasmic reticulum ..................................................................................................................... 11 1.6 Mitochondria .................................................................................................................................... 12 1.7 Phloem proteins ................................................................................................................................ 12 1.7.1 Dispersive phloem proteins ....................................................................................................... 13 1.7.2 Non-dispersive phloem proteins ................................................................................................ 13 1.8 Microscopy ........................................................................................................................................ 14 1.8.1 Optical microscopy ..................................................................................................................... 15 1.8.2 Epi-fluorescent microscopy........................................................................................................ 18 vi 1.8.3 Confocal laser scanning microscopy .......................................................................................... 19 1.8.4 Electron microscopy ................................................................................................................... 20 1.8.5 Scanning electron microscopy ................................................................................................... 22 1.8.6 Transmission electron microscopy ............................................................................................ 24 1.9 References ........................................................................................................................................ 28 Chapter 2 - Phloem Ultrastructure and Pressure Flow: Sieve-Element-Occlusion-Related Agglomerations Do Not Affect Translocation .............................................................................. 32 2.0 Author contributions ......................................................................................................................... 32 2.1 Abstract ............................................................................................................................................. 33 2.2 Introduction ...................................................................................................................................... 34 2.3 Results ............................................................................................................................................... 37 2.3.1 Development and Structure of SEOR1 ....................................................................................... 39 2.3.2 TEM of Sieve Tubes ...................................................................................................................