THE EXPORT OF POLYAMINES IN PLANTS IS MEDIATED BY A NOVEL CLADE OF BIDIRECTIONAL TRANSPORTERS Lingxiao Ge A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2015 Committee: Paul Morris, Advisor Nancy Ann Orel, Graduate Faculty Representative Vipa Phuntumart Ray Larsen Scott Rogers ii ABSTRACT Paul Morris, Advisor Ubiquitously existent in nature, polyamines play an important role as signaling compounds in plant responses to both biotic and abiotic stresses. My research has shown that the clade of Arabidopsis thaliana and rice BIDIRECTIONAL AMINO ACID TRANSPORTERS (BAT) function as antiporters of polyamines and amino acids. The method used in this study takes advantage of the genetic resources of the model organism Escherichia coli. A double knockout E. coli strain that is deficient in all polyamine antiporters was created and then used to heterologously express candidate plant transporters AtBAT1.1, AtBAT1.2, and OsBAT1. Inside- out membrane vesicles of these transgenic E. coli cells were generated by ultrasound sonication or French press. A radioisotope assay was then performed using these vesicles to confirm the specificity of the target proteins as polyamine antiporters. To determine the role of BATs in polyamine homeostasis we tested their GFP fusions by transient expression in Nicotiana benthamiana. The GFP-tagged AtBAT1 and OsBAT1 displayed plastid localization in tobacco leaves by using confocal microscopy. Furthermore, the overexpression of OsBAT1 in A. thaliana wild-type results in novel phenotypes that could have potentially economical usage. This indicates that by altering the expression of a single gene of polyamine transporter, we can manipulate the plants phenotypes similar to natural variations. Metabolic pathways can be localized to one organelle or distributed across several cellular compartments. Previous work has identified only a single cytosolic pathway for putrescine synthesis in Arabidopsis. Here we show that both A. thaliana and soybeans have a plastid-localized putrescine pathway consisting of an arginine decarboxylase and an agmatinase that combine to synthesize putrescine from arginine. iii I dedicate my dissertation work to my parents, Ling Li and Ge Jizhong. iv ACKNOWLEDGMENTS I would like to express my sincere gratitude to my mentor Dr. Paul Morris for his support, guidance, and advisory that benefited me much in the completion of this study. In addition to mentoring in school work and lab tasks, Paul offered me the best opportunities to attend national and international conferences and workshops. His intelligence and passion in research always inspired me on the way to become a successful scientist. I have a distinct appreciation for Dr. Vipa Phuntumart on my Dissertation Committee. Vipa always treated me like her own student, welcomed me to use her lab space and equipment, and provided valuable comments on my research. I would like to thank my other committee members, Dr. Ray Larsen for providing the P1 bacteria phage strain and permitting me to use the ultrasound sonicator, and Dr. Scott Rogers for training me to use the ultracentrifuge, in addition to their service on the committee. I also thank Dr. Nancy Orel for being the graduate faculty representative on my committee. I would like to acknowledge Dr. Andrea Kalinoski of the University of Toledo for helping us take great pictures on the confocal laser microscope. In addition, many thanks to my labmates, Jigar Patel and Sheaza Ahmed, who often gave assistance and advice to my projects. A special thank you to Menaka Ariyaratne, who did all the HPLC analyses for this study. At last but not least, I thank my family and all my friends in the US and China for their support and encouragement. v TABLE OF CONTENTS Page CHAPTER I GENERAL INTRODUCTION .................................................................... 1 1.1 What Are Polyamines? ..................................................................................... 1 1.2 Importance of Polyamines in Plants ................................................................... 2 1.3 Polyamine Metabolism in Plants ........................................................................ 4 1.4 Polyamine Transporters ..................................................................................... 7 1.5 Project Overview ............................................................................................... 9 1.6 References .......................................................................................................... 10 CHAPTER II A NOVEL METHOD TO IDENTIFY PLANT ANTIPORTERS OF AMINO ACIDS AND POLYAMINES ............................................................................................... 16 2.1 Introduction ......................................................................................................... 16 2.2 Materials and Methods ........................................................................................ 20 2.2.1 Bioinformatics ...................................................................................... 20 2.2.2 DNA Cloning and Constructs .............................................................. 20 2.2.3 E. coli DKO Mutant with P1 Transduction ......................................... 21 2.2.4 Expression of Target Genes in E. coli DKO Mutant ........................... 22 2.2.5 Generation of Inside-Out Membrane Vesicles .................................... 22 2.2.6 Isotope Assay for Polyamine Antiporters ............................................ 23 2.2.7 HPLC Analysis of Polyamine Levels in E. coli Cells ......................... 24 2.3 Results ................................................................................................................. 25 2.3.1 Identification of Candidate Plant Polyamine/Amino Acids Antiporters ........................................................................................................................ 25 vi 2.3.2 Functional Characterization of BAT Transporters in E. coli mutant ... 30 2.3.2.1 OsBAT1 is an antiporter of putrescine and arginine. ........... 30 2.3.2.2 AtBAT1.1 is an antiporter of spermidine and arginine. ........ 31 2.3.2.3 AtBAT1.2 is an antiporter of spermidine and arginine. ........ 33 2.3.3 HPLC Analysis of Polyamine Levels in E. coli Mutant Expressing BAT Transporters ................................................................................................... 34 2.4 Discussion ........................................................................................................... 35 2.4.1 The first plant antiporters of polyamines and amino acids .................. 35 2.4.2 Substrate specificity of BATs .............................................................. 36 2.4.3 Codon optimization is a useful tool for characterizing heterologous proteins in E. coli ......................................................................................................... 38 2.4.4 E. coli inside-out membrane vesicles as a model system for characterizing plant antiproters ............................................................................................. 38 2.5 References ........................................................................................................... 41 CHAPTER III OVEREXPRESSION OF BAT AFFECTS PLANT DEVELOPMENT ..... 46 3.1 Introduction ......................................................................................................... 46 3.2 Materials and Methods ........................................................................................ 48 3.2.1 Organ/Tissue expression of OsBAT1 and AtBAT1 ............................. 48 3.2.2 Generation of transgenic plants ........................................................... 48 3.2.3 Characterization of transgenic plants ................................................... 49 3.2.4 Subcellular localization ........................................................................ 49 3.3 Results ................................................................................................................. 52 vii 3.3.1 The tissue/organ expression of AtBAT1 and OsBAT1 by microarray analysis ........................................................................................................... 52 3.3.2 OsBAT1 overexpression results in delayed flowering and thicken stems due to elevated polyamine levels .......................................................................... 53 3.3.3 OsBAT1, AtBAT1.1, and AtBAT1.2 are localized to plastids ............ 57 3.4 Discussion ........................................................................................................... 60 3.4.1 Subcellular localization of BATs ......................................................... 60 3.4.2 Overexpression of OsBAT1 in Arabidopsis and results in phenotypic changes ........................................................................................................... 61 3.5 References ........................................................................................................... 64 CHAPTER IV HIDING IN PLAIN SIGHT: A THIRD ROUTE FOR PUTRESCINE BIOSYNTHESIS IN PLANTS .............................................................................................. 70 4.1 Abstract ................................................................................................................ 71 4.2 References ...........................................................................................................