UC Riverside UC Riverside Electronic Theses and Dissertations Title Elucidation of the Role of Leucine Aminopeptidase A in the Wound Response Pathway in Tomato Permalink https://escholarship.org/uc/item/2nn0x0f1 Author Scranton, Melissa Ann Publication Date 2013 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA RIVERSIDE Elucidation of the Role of Leucine Aminopeptidase A in the Wound Response Pathway in Tomato A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Plant Biology by Melissa Ann Scranton March 2013 Dissertation Committee: Dr. Linda L. Walling, Chairperson Dr. Thomas Eulgem Dr. Isgouhi Kaloshian Copyright by Melissa Ann Scranton 2013 The Dissertation of Melissa Ann Scranton is approved: Committee Chairperson University of California, Riverside ACKNOWLEDGEMENTS I would like to thank all the members past and present of the Walling world for their support and encouragement. In particular, I thank my advisor Linda for all her patience and for pushing me to be the best researcher possible. I would also like to thank Fran for being the emotional support for the whole lab and undergraduate researchers Rebekah Silva and Ashley Yee for making the work just a little more fun. I would like to thank all the members of UCR. I would like to especially thank all my committee members for all their support and advice. In particular I would like to thank Dr. Thomas Eulgem and Isgouhi Kaloshian for their time and commitment to my dissertation. Dr. Li Fan and Kevin Du Prez generated the first crystal structures of the tomato LAP protein. Dr. Thomas Girke helped train me in R code writing and in the analysis of the microarray studies. Dr. Glenn Hicks helped troubleshoot many different assays throughout the years. Dr. Mike Stanghellini diagnosed many plant pathogen infections. Dr. Elizabeth Vierling and Eman Basha from the University of Massachusetts, Amherst provided controls and advice for the chaperone assays. Dr. Gregg Howe from Michigan State University provided lab support during an internship to learn LC-MS. I thank the UCR botany and Plant Sciences department for their financial, technical, and emotional support. In particular, I thank Rob Lennox, Bob Bergeron, and Eric McCullough for their help fixing and maintaining our plant growth chambers. Financial support was provided by NSF grants IOB 0645230 and MCB 0313579 to Linda Walling and an NSF-funded ChemIGERT fellowship (DGE 0504249) and IOB 064523 to Melissa Scranton. Chapter 1 of this dissertation was originally published in The Journal of Biological Chemistry. Scranton MA, Yee A, Park S-Y, Walling LL. Plant leucine aminopeptidases moonlight as molecular chaperones to alleviate stress-induced damage. The Journal of Biological Chemistry 2012; 287: 18408-18417. © the American Society for Biochemistry and Molecular Biology. iv DEDICATION To all the graduate students who put in more hours than anyone knows and to the family and friends who support them v ABSTRACT OF THE DISSERTATION Elucidation of the Role of Leucine Aminopeptidase A in the Wound Response Pathway in Tomato by Melissa Ann Scranton Doctor of Philosophy, Graduate Program in Plant Biology University of California, Riverside, March 2013 Dr. Linda L. Walling, Chairperson Historically, peptidases have been considered as housekeeping proteins involved in protein degradation and amino acid turnover. However, recent work has highlighted the fact that peptidases, including aminopeptidases, have critical regulatory roles in the cell including modulation of growth, development and stress responses. Recently, plant leucine aminopeptidases (LAPs) have been recognized for their roles in modulation of late wound- responses and insect defense. While no known mechanism of action has been identified, LAPs were presumed to act through their aminopeptidase function to affect the stability, activity, or localization of a peptide and protein involved in defense. In my dissertation studies, I have identified two new molecular functions for plant LAPs as molecular chaperones and Cys-Gly dipeptidases. In addition, microarray analyses has demonstrated the tomato LAP-A regulates early and late wound responses both positively and negatively. In particular, microarray analyses identified two new sets of genes modulated by LAP-A: late wound dehydrins and Pathogenesis- Related 1. This study also provides evidence LAP-A may act though the negative regulator salicylic acid or the positive regulator hydrogen peroxide to modulate wound signaling. The role of LAP-A in glutathione metabolism is also discussed. Together this study has provided evidence that LAP-A’s role in wound signaling and insect defense is more complex than initially anticipated vi and may be the result of LAP-A utilizing multiple functions (aminopeptidase, chaperone and/or Cys-Gly dipeptidase activities) in different environments and in response to different stresses. vii TABLE OF CONTENTS INTRODUCTION .............................................................................................................................. 1 REFERENCES .......................................................................................................................... 31 CHAPTER 1 ................................................................................................................................... 61 ABSTRACT ................................................................................................................................ 61 INTRODUCTION ....................................................................................................................... 62 EXPERIMENTAL PROCEDURES ............................................................................................ 64 RESULTS .................................................................................................................................. 69 DISCUSSION ............................................................................................................................ 74 REFERENCES .......................................................................................................................... 80 CHAPTER 2 ................................................................................................................................. 106 ABSTRACT .............................................................................................................................. 106 INTRODUCTION ..................................................................................................................... 107 RESULTS AND DISCUSSION ................................................................................................ 111 CONCLUSION ......................................................................................................................... 136 EXPERIMENTAL PROCEDURES .......................................................................................... 138 REFERENCES ........................................................................................................................ 143 CHAPTER 3 ................................................................................................................................. 201 ABSTRACT .............................................................................................................................. 201 INTRODUCTION ..................................................................................................................... 202 RESULTS ................................................................................................................................ 207 DISCUSSION .......................................................................................................................... 218 EXPERIMENTAL PROCEDURES .......................................................................................... 224 REFERENCES ........................................................................................................................ 231 DISCUSSION ............................................................................................................................... 263 viii LIST OF FIGURES Figure 1.1 ................................................................................................................................... 85 Figure 1.2 ................................................................................................................................... 86 Figure 1.3 ................................................................................................................................... 87 Figure 1.4 ................................................................................................................................... 88 Figure 1.5 ................................................................................................................................... 89 Figure 1.6 ................................................................................................................................... 90 Figure 1.7 ................................................................................................................................... 91 Figure 1.8 ................................................................................................................................... 92 Figure 1.9 ..................................................................................................................................