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INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 ! EXPRESSION AND REGULATION OF THE TOBACCO ANIONIC PEROXIDASE GENE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Karen Lynn Klotz, B.A., M.S. ***** The Ohio State University 1995 Dissertation Committee: Approved by L. M. Lagrimini M. Knee P. S. Jourdan Advisor Graduate Program m K. R. Davis Horticulture UMI Number: 9612212 Copyright 1995 by Klotz, Karen Lynn All rights reserved. OMI Microform 9612212 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 Copyright by Karen Lynn Klotz 1995 To My Husband, Mark and My Parents ACKNOWLEDGEMENTS I would like to express my sincere appreciation to my advisor Dr. L. Mark Lagrimini for his guidance and insight in this research. I would also like to thank the members of my guidance committee, Drs. Michael Knee, Pablo Jourdan, and Keith Davis for their comments and suggestions. I would like to thank the Department of Horticulture and the Ohio Agricultural Research and Development Center for financial support. Thanks also go to Drs. Fernando Finger and Vicki Gingas for their encouragement and friendship. VITA September 30, 1961 Born - Toledo, OH 1983 B.A., Capital University Columbus, OH 1987 M.S., University of Minnesota, Minneapolis, MN 1985-1989 Research Chemist, The Upjohn Company, Kalamazoo, MI PUBLICATIONS Diaz-De-Leon, F., Klotz, K., Lagrimini, L. M. (1993). "Nucleotide Sequence of the Tobacco (Nicotiana tabacum) Anionic Peroxidase Gene." plant phisiol. , 101:1117-1118. Lagrimini, L. M., Vaughn, J., Finer, J., Klotz, K., Rubaihayo, P. (1992). "Expression of a Chimeric Tobacco Peroxidase Gene in Transgenic Tomato Plants." j . amer. soc. hort. sci., 117:1012-1016. FIELDS OF STUDY Major Field: Horticulture Plant Molecular Biology iv TABLE OF CONTENTS ACKNOWLEDGMENTS ..................................... iii V I T A .................................................iv LIST OF T A B L E S ...................................... vii LIST OF FIGURES....................................... ix CHAPTER PAGE I. INTRODUCTION ................................. 1 List of References......................31 II. FURTHER MOLECULAR CHARACTERIZATION OF THE TOBACCO ANIONIC PEROXIDASE GENE 3 6 Introduction ......................... 36 Materials and Methods ............... 39 Results and Discussion ............... 43 List of References......................65 III. REGULATION OF TOBACCO ANIONIC PEROXIDASE GENE EXPRESSION ............................. 71 Introduction ......................... 71 Materials and Methods ............... 73 R e s u l t s ............................... 87 Discussion............................ 125 List of References 13 0 IV. REGULATORY REGIONS OF THE TOBACCO ANIONIC PEROXIDASE PROMOTER ....................... 134 Introduction ......................... 134 Materials and Methods 13 6 R e s u l t s .............................. 143 Discussion............................ 161 List of References.....................167 v V. HISTOLOGICAL AND DEVELOPMENTAL EXPRESSION OF THE TOBACCO ANIONIC PEROXIDASE GENE .... 170 Introduction ......................... 170 Materials and Methods ................ 172 R e s u l t s .............................. 176 Discussion............................ 195 List of References.....................204 VI. DISCUSSION.............................. 208 LIST OF R E F E R E N C E S .............................. 215 LIST OF TABLES TABLE PAGE 1. Protein extraction buffers examined for their compatibility with GUS and CAT a s s a y s ..................................... 90 2. GUS and CAT activities of tobacco protoplasts transformed with pKK010593 or calf thymus DNA and extracted into different buffer systems ................. 90 3. Methods used to lyse p r o t o p l a s t s ............ 91 4. Yield of soluble protein and GUS activity from tobacco protoplasts transformed with pKK060992 and lysed by methods in Table 3 ......................... 91 5. GUS and CAT activity of tobacco protoplasts transformed with pKK060992 and pCaMVCN after 48 hrs. exposure to no hormones, IAA, BA, or ethylene............. 94 6. GUS activity of tobacco protoplasts transformed with pKK060992 and pCaMVCN after 3 6 hrs. exposure to no hormones, IAA, NAA, BA or e t h y l e n e ....................97 7. GUS activity of tobacco protoplasts transformed with pKK060992 and incubated with 0, 100, or 1000 ppm ethylene for 24 to 72 hrs....................103 8. GUS activity of tobacco protoplasts transformed with pKK060992 and pCaMVCN and incubated for 36 hrs. with PCIB, IAA, or IAA and P C I B ...................... 105 vii 9. GUS activity of tobacco protoplasts transformed with pKK060992 and pCaMVCN and incubated for 36 hrs. with GA, ABA, JA, SA, fungal cell wall elicitor, IAA, or subjected to a 41°C, 2 hr. heat shock treatment............................ 108 10. GUS activity of roots from young N. sylvestris 601-19-L plants exposed to NAA, PCIB or no hormones for 48 hrs . 112 11. GUS activity of young N. sylvestris 601-19-L plants exposed to no hormones, NAA or PCIB for 72 hrs. at 27°C in light or d a r k .............................. 115 12. GUS activity of root cultures incubated for 48 hrs. on HF medium with 3 uM IBA or no added hormone........................ 117 13. GUS activity of root cultures after 72 hrs. with no added hormones, 50 uM NAA or 100 uM P C I B ............................ 118 14. GUS activity of tobacco protoplasts transformed with methylated or unmethylated pKK060992 or unmethylated pKK092093 and their response to auxin .... 123 15. GUS, CAT and GUS/CAT activity of tobacco protoplasts transiently transformed with tobacco anionic peroxidase promoter deletions fused to GUS and an equimolar amount of the full peroxidase promoter fused to CAT as an internal standard ......................... 146 16. GUS, CAT and GUS/CAT activity of tobacco protoplasts transiently transformed with tobacco anionic peroxidase promoter deletions fused to GUS and a 0.9 equimolar amount of the full peroxidase promoter fused to CAT as an internal standard.....................153 17. GUS activity of tobacco protoplasts transiently transformed with the tobacco anionic peroxidase promoter deletions fused to GUS and incubated with or without 100 uM IAA for 36 hrs. 159 viii LIST OF FIGURES Structures of the lignin precursors p- coumaryl alcohol, coniferyl alcohol and sinapyl alcohol ........................... Formation of the free radical of p- coumaryl alcohol by peroxidase and hydrogen peroxide and its isomerization . Idealized structure of lignin showing several different types of covalent bonds .................................... Structure of isodityrosine, a compound important in the cross-linking of cell wall glycoproteins ....................... Restriction site map of the tobacco anionic peroxidase gene ................... Map of the transcription start site determined by primer extension ........... DNA sequence of the tobacco anionic peroxidase 5' regulatory region from -3146 bps to the translation start site . Comparison of the consensus sequence for the TATA element for plant genes and sequence of the TATA element of the tobacco anionic peroxidase promoter ........ Comparison of sequences containing the GATA motif from the promoter regions of the tobacco anionic peroxidase, CaMV 35S ribosome, and several chlorophyll a/b binding proteins ..................... 10. Sequence of the four TC-rich repeats and their location in the tobacco anionic peroxidase promoter relative to the transcription start si t e ................ 57 11. Sequence and location of an auxin responsive element identified in PS- IAA4/5 and found in various auxin- regulated genes and the tobacco anionic peroxidase gene ........................... 59 12. Conserved sequence elements found in auxin-responsive genes and the tobacco anionic peroxidase gene...... .............. 61 13. Sequences within the tobacco anionic peroxidase

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