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University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 A Xerox Education Company SOMMER, Harry Edward, 1941- INFLUENCE OF 2[4 DICHLOROPHENOXYACETIC ACID ON NITRATE REDUCTASE AND PROTEIN IN WILD CARROT L.) T ISSU E CU LTURE.(DAUCUS CAROTA L.) TISSUE CULTURE.(DAUCUS The Ohio State University, Ph.D., 1972 Botany University Microfilms, A XEROX Company, Ann Arbor, Michigan THIS DISSERTATION HAS BpEN MICROFILMED EXACTLY AS RECEIVED. INFLUENCE OF 2,4 DICHLOROPHENOXYACETIC ACID ON NITRATE REDUCTASE AND PROTEIN IN WILD CARROT fDAUCUS CAROTA L.) TISSUE CULTURE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Harry E. Sommer, 3,S. in Agriculture, M.S. ★ * * * * The Ohio State University 1972 Adviser Dept, of Botany PLEASE NOTE: Some pages may have indistinct print. Filmed as received. University Microfilms, A Xerox Education Company ACKNOWLEDGMENTS I wish to thank the Graduate Committee of the Botany Department for their support and advice. I wish to thank the following for their support, ad­ vice, and aid: Dr. R. W. Sharp, Dr. C. A. Swanson, Dr. F. E. Deather- age, Dr. J. A. Schmitt, Dr. R. A. Popham, Dr. M. L. Evans, Dr. David Lee, Dr. Ruy de Araujo Caldas, Dr. Linda S. Caldas, Dr. Henrique Amorim, Alta Scrimsher, Sheila Long, Jane Etta Holiday, Sara Hill, Cheryl Sands, Anna Bezruczko, and Roy Postle. I wish to thank the Department of Biochemistry, De­ partment of Microbiology, and AEC for financial support. Finally, I wish to thank my wife for bringing supper to the laboratory during the longer experiments and her other aids to the completion of this work. VITA 25 July, 19 4 1 .......... Born-Chatham, New York 1963............. .. B.S. in Agriculture, University of Vermont, Burlington 1966....................M.S., University of Maine, Orono 1966-1968 ............. Officer, U.S. Army iii TABLE OP CONTENTS Page ACKNOWLEDGMENTS............................ ii VITA ................................................. iii LIST OF T AB L E S ............. v LIST OP FIGURES . ...............................vi INTRODUCTION . ................................. I LITERATURE REVIEW ................................... 6 MATERIALS AND METHODS ................. ...............51 RESULTS . .......................................... 60 DISCUSSION ................. 87 SUMMARY ..................................................105 BIBLIOGRAPHY ................................. .109 iv LIST OF TABLES Table Page 1. Activity of Nitrate Reductase and Nitrite Disappearance in Various Wild Carrot Preparations ....................... ..... 61 2. Ammonium Sulfate Fractionation of Wild Carrot Extract ............................. 62 3. Effect of Breakage Buffer Components .... 63 4. .Assay Mixture Components and Nitrate Reductase Activity ......................... 65 5. Effect of Molybdate on Assay of Nitrate R e d u c t a s e ........... 6 6 6 . n moles Nitrite/ml Nutrient ................ 76 7. n moles Nitrite/ml Nutrient ................ 8 6 v LIST OP FIGURES Figure Page 1. Activity of Nitrate Reductase vs Time and vs Amount of Enzyme . ........... 67 2. Effects of pH and Substrate Concentration on Nitrate Reductase Activity .... 68 3. Fresh weight/culture ............. 70 4. Protein/culture .................. ; • 71 5. n moles NOg Reduced/min/Culture . , 72 6 . n moles NO^ Redueed/min/mg Protein 73 7. [NO^J in Nutrient 74 8. [P0|] in Nutrient 77 9. [P0=] in Nutrient (High Phosphate Nutrient) 78 10. Fresh Weight/Culture (High Phosphate Nutrient) ......................... , 79 11. Protein/Culture (High Phosphate Nutrient) 81 12. n moles NOr Reduced/min/Culture (High Phos­ phate Nutrient) .................... 82 13. n moles N0^ Reduced/min/mg Protein 83 14. [NO^J in Nutrient (High Phosphate Nutrient) 84 vi INTRODUCTION The research described in this dissertation is part of a series of studies on the control of embryogenesis in wild carrot (Daucus carota L.) tissue cultures that were initiated by Dr. D. K. Dougall while he was at the Ohio State University. We were concerned primarily with discover­ ing the biochemical differences which existed between those cultures giving rise to embryos and those not doing so. The wild carrot tissue culture system was first de­ scribed by Wetherell and Halperin (1) and later further grown on a nutrient^- that included ammonium and 2,4 D (2,4 dichlorophenoxyacetic acid.). When these cultures were transferred to a nutrient without 2,4 D, embryos formed. When ammonium was omitted from the nutrient containing 2,4 D. transfer of the culture to a nutrient without 2,4 D did not result in embryogenesis. Growth also was slow in the absence of ammonium. Based on these reports, we con­ cluded that an investigation of the ammonium requirement provided a logical point to attack the metabolic processes connected with embryogenesis. The initial steps of nitrate utilization and the effects of 2,4 D on this process appeared l Nutrient and nutrient media are used as synonymous terms in plant tissue culture. Abbreviations conform to those recommended for use in the Journal of Biological Chemistry. 1 to be the most likely avenue of approach. An initial check of the literature led to a paper by Beevers et al. (3) on the effects of 2,4 D on nitrate metabolism in corn and cucumber. C o m was chosen as a plant relatively resistant to the phytotoxic effects of 2,4 D, while cucumber, like many dicotyledons, is sensitive to 2,4 D. For corn, an increase in nitrate reductase ac­ tivity was found for all treatments. In the case of cucum­ ber, the application of 2,4 0 to the plants resulted in a lower specific activity for nitrate reductase during the period 4-12 hours after spraying. Control levels of nitrate reductase were regained at later sampling times. Since wild carrot is a dicotyledon, we might expect it to show a lower specific activity for nitrate reductase when treated with 2,4 D. Further checks of the literature were made concerning inorganic nitrogen utilization by embryos in culture. This showed .that in culture embryos differ greatly in their abil- * ity to utilize nitrate (4). Embryos from ripe seeds of oats could utilize either ammonium or nitrate for growth (5). Rijven (6 ) reported excised torpedo stage embryos of Ama- gallis arvensis L., Arabidopsis thaliana (L.) Heynh., Cap- sella bursa-patoris (L). Moech, and Sisymbrium orientale L. did not grow with nitrate as a nitrogen source. Amagalli3 arvensis L. did show significant growth with ammoni.um. The only inorganic source of nitrogen that gave significant i growth with the other three species was nitrite. In longer term experiments designed to determine if the em­ bryos could adapt to*utilize nitrate, no evidence, based on growth, could be obtained for an adaptive response in freshly excised embryos. However, while no nitrate re­ ductase was detected in freshly excised capsella embryos, nitrate reductase was detected after 2 0 hours incubation of the embryos with nitrate. For quantitative studies of nitrate reductase Rijven used 16 day old wheat embryos. These embryos showed a tenfold increase in nitrate reduc­ tase after 24 hours on nitrate supplemented sucrose. Raghavan and Torrey (7) pursued the problem of nitrogen nutrition of embryos in orchids. The embryo in a mature orchid seed resembles that of the globular stage in di­ cotyledons. The embryos were grown until plantlets developed. During the first 14 weeks on nitrate the orchids showed an increase in ng N/mg dry weight of seedlings only slightly exceeding those on nutrient without a nitrogen source. The orchids on nitrite showed even less of an increase than those without nitrogen. However the embryos on ammonium salts in some instances showed almost a doubling of ng N/mg dry weight. Ammonium nitrate supported growth appeared to follow the same pattern as those on other ammonium salts. The orchids on ammonium salts also showed a greater degree of development than those on nitrate. The superiority of ammonium as a nitrogen source for growth continued until 60 day old plantlets were transferred from ammonium nitrate nutrient to fresh ammonium nitrate or sodium nitrate nu­ trient. Growth was comparable on both nutrients 6 days after transfer. Determinations of nitrate reductase done on seed­ lings grown on ammonium nitrate gave negative results.