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Open Thesis.Pdf The Pennsylvania State University The Graduate School Intercollege Graduate Degree Program in Plant Physiology ON THE ROLE OF THE ENZYME PEPTIDE METHIONINE SULFOXIDE REDUCTASE IN THE RESPONSE OF ARABIDOPSIS PLANTS TO OXIDATIVE STRESS A Thesis in Plant Physiology by Hernán Mauricio Romero-Angulo © 2005 Hernán Mauricio Romero-Angulo Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2005 The thesis of Hernán Mauricio Romero-Angulo was reviewed and approved* by the following: Ming Tien Professor of Biochemistry and Molecular Biology Thesis Co-Advisor Co-Chair of Committee Eva J. Pell John and Nancy Steimer Professor of Agricultural Sciences Thesis Co-Advisor Co-Chair of Committee Hong Ma Professor of Biology B. Tracy Nixon Professor of Biochemistry and Molecular Biology Teh-hui Kao Professor of Biochemistry and Molecular Biology Chair, Intercollege Program in Plant Physiology *Signatures are on file in the Graduate School ABSTRACT Reactive oxygen species (ROS) are produced during cellular metabolism. Low steady state levels of ROS are maintained by enzymatic and non-enzymatic mechanisms. Under extreme environmental conditions, ROS production can overwhelm the detoxification systems and oxidative stress results. ROS can oxidize methionine (Met) residues to methionine sulfoxide (MetSO) thereby inactivating enzymes or tagging proteins for hydrolysis. The enzyme peptide methionine sulfoxide reductase (PMSR) reduces MetSO to Met, repairing damaged proteins. The MetSO-PMSR-Met system has been referred to as the “last chance” defense mechanism against ROS. The role of PMSR in protection against oxidative stress is well recognized in bacteria, yeast and animal cells; however the function of PMSR in plants is yet to be fully established. The objective of this research was to determine the role of PMSR in the response of plants to oxidative stress. One family of PMSR proteins, PMSRA of Arabidopsis was used as a model system. Arabidopsis has five PMSRA genes being expressed in different organs. PMSRA1 to PMSRA3 are cytosolic proteins, PMSRA4 is plastidic and PMSR5 is secreted. PMSRA4 transcript is the most abundant; PMSRA2, PMSRA3 and PMSRA5 are highly expressed in roots and stems; PMSRA1 is abundant in flower buds; and PMSRA4 is up-regulated by ozone, methyl viologen, cercosporin and high light. Kinetic analysis of recombinant enzyme showed that PMSRA4 had twice the kcat and was four times more efficient (kcat/Km) than PMSR3. Transgenic plants over-expressing PMSRA3 and PMSRA4 were more resistant to methyl viologen and ozone. PMSRA3 over-expressing plants were more resistant to cercosporin and PMSRA4 over-expressing plants more resistant to high light. Transgenic plants with low levels of PMSRA4 were susceptible to oxidative stress in the chloroplast. Treatments that lower PMSRA4 levels in wild-type plants, such as Mn2+ excess, increased the susceptibility of plants to oxidative stress damage (e.g. a modest 2- day water deprivation in plants grown under Mn2+ excess induces toxicity). PMSRA activity is essential for plant survival to oxidative stress. It appears that specific isozymes protect discrete cellular compartments. PMSRA4 is the most abundant and most efficient PMSRA in Arabidopsis and protects against oxidative stress derived from different cell compartments. iii TABLE OF CONTENTS LIST OF FIGURES .............................................................................................................vii LIST OF TABLES ...............................................................................................................ix ACKNOWLEDGEMENTS .................................................................................................x Chapter 1 LITERATURE REVIEW...................................................................................1 REACTIVE OXYGEN SPECIES ................................................................................1 OXIDATIVE STRESS .................................................................................................4 CELLULAR PROTECTION AGAINST OXIDATIVE STRESS...............................5 Superoxide Dismutase ...........................................................................................7 Ascorbate Peroxidase.............................................................................................7 Glutathione Reductase...........................................................................................8 Catalase..................................................................................................................8 PEPTIDE METHIONINE SULFOXIDE REDUCTASE.............................................9 Discovery of PMSR Activity.................................................................................11 PMSR and Oxidative Stress...................................................................................12 Possible Role of PMSR in Protein Regulation ......................................................13 PMSR and Aging...................................................................................................14 Biochemical Properties of PMSR..........................................................................15 PMSR in Plants......................................................................................................18 REFERENCES .............................................................................................................20 Chapter 2 EXPRESSION PROFILE ANALYSIS AND BIOCHEMICAL PROPERTIES OF THE PEPTIDE METHIONINE SULFOXIDE REDUCTASE A (PMSRA) GENE FAMILY IN ARABIDOPSIS.......................................................................................29 ABSTRACT..................................................................................................................29 INTRODUCTION ........................................................................................................30 MATERIALS AND METHODS..................................................................................31 In silico Analysis of PMSRA Genes ......................................................................31 Plant Material.........................................................................................................32 RNA Isolation and RT-PCR ..................................................................................32 PMSRA Expression in E. coli ...............................................................................34 RESULTS .....................................................................................................................36 The Plant PMSRA Family.....................................................................................36 Expression of PMSRAs in the Organs of Arabidopsis...........................................42 Response of Arabidopsis PMSRA Genes to Stress Conditions .............................42 Biochemical Properties of PMSRA3 and PMSRA4..............................................47 DISCUSSION...............................................................................................................50 REFERENCES .............................................................................................................54 iv Chapter 3 OVER-EXPRESSION OF A CYTOSOLIC PEPTIDE METHIONINE SULFOXIDE REDUCTASE (PMSRA3) CONFERS INCREASED TOLERANCE TO OXIDATIVE STRESS IN ARABIDOPSIS.................................................................58 ABSTRACT..................................................................................................................58 INTRODUCTION ........................................................................................................59 MATERIALS AND METHODS..................................................................................60 Construction of Transgenic Plants.........................................................................60 Analysis of Gene Expression.................................................................................61 Oxidative Stress Treatments..................................................................................62 Electrolyte Leakage ...............................................................................................63 Protein Analysis.....................................................................................................63 Statistical Analysis.................................................................................................64 RESULTS .....................................................................................................................64 Expression of PMSRA3 in Transgenic and WT Plants.........................................64 Leaves of Transgenic and WT Plants After Oxidative Stress. ..............................67 Electrolyte Leakage After Oxidative Stress ..........................................................68 Gas Exchange After Oxidative Stress....................................................................70 Chlorophyll a Fluorescence After Oxidative Stress ..............................................72 MetSO Content After Oxidative Stress .................................................................72 Rubisco and Antioxidant Enzymes levels After Oxidative Stress.........................74 DISCUSSION...............................................................................................................75 REFERENCES .............................................................................................................80
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