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Leghemoglobins in the Genus Phaseolus and Their Significance for Nitrogen Fixation

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Leghemoglobins in the Genus Phaseolus and Their Significance for Nitrogen Fixation LEGHEMOGLOBINS IN THE GENUS PHASEOLUS AND THEIR SIGNIFICANCE FOR NITROGEN FIXATION by Monika Magdalena Liilsdorf Ing. Agrar., Universitat Bonn, Germany, F.R., 1982 M.Sc, The University of Manitoba, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS. FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF PLANT SCIENCE) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA APRIL 1989 (c)Monika Magdalena Lulsdorf, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Leghemoglobins (Lbs) are important soluble proteins in the legume root nodule formed in response to infection by soil Rhizobium bacteria. With the exception of Phaseolus vulgar is, Lbs generally show structural and functional heterogeneity for their oxygen-binding function in the nodule. P_. vulgar is seeds 'Contender' were mutagenized with gamma-rays or ethyl methane sulfonate. Among 1400 M2 off• spring derived from the treated plants screened for Lb va• riability, no mutation in Lb was detected. These observa• tions are consistent with a single active gene for Lb in Phaseolus. Fifty accessions from the genus Phaseolus were screened for Lb variation. P_. acutifolius ssp. contained a single Lb component which had a different electrophoretic mobility compared to that in P_. vulgar is. P_. f ilif ormis showed two Lb bands, both different from P_. vulgaris, while P. lunatus was found to contain two major and probably three minor Lb components. These data represent the first report of intra-generic Lb variability in Phaseolus. Using embryo rescue technique, hybrids were produced from _P. vulgar is x P. acutif ol ius ssp. and P. vulgar is x P.. filiformis crosses. Rooted cuttings from each hybrid and i i its parents were used to estimate the nitrogen fixation (acetylene reduction) rate and to determine the total amount of nitrogen accumulated during the growth period. The P_. vulgaris x P_. filiformis hybrids had a significantly higher nitrogen fixation rate and accumulated more nitrogen than either parent. The P_. filiformis parent performed better than the P_. vulgar is parent but only in the acetylene reduction test. One of the P_. vulgaris x P_. acutifolius hybrids also fixed nitrogen at a higher rate than either parent. Leghemoglobin components from a P_. filiformis hybrid and from P.. lunatus 'Lima Hendersons* were isolated by cellulose ion-exchange chromatography. The N-terminal amino acid sequence for peak II of the P. filiformis hybrid and peaks I and II from P_. lunatus nodules was determined. The first 37 amino acids of each component were identical to the published sequence for P_. vulgaris. Mobility and/or functional differences are likely to be found in the se• quence nearer the location Of the heme cavity. The data.of this investigation support the contention that Lb hetero• geneity is functional and constitutes an adaptation for more effective nitrogen fixation. TABLES OF CONTENTS ABSTRACT . ii LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS viii GLOSSARY ix ACKNOWLEDGEMENTS x Chapter Page I. GENERAL INTRODUCTION 1 II. GENERAL LITERATURE REVIEW 13 2.1. HEME 13 2.2. GLOBIN 15 2.3. BIOSYNTHESIS, ASSEMBLY AND DEGRADATION 17 2.4. INTRACELLULAR LOCATION 20 2.5. LEGHEMOGLOBIN FUNCTION 22 2.6. QUANTITATIVE RELATIONSHIP BETWEEN LEGHEMO• GLOBIN AND NITROGEN FIXATION 31 2.7. LEGHEMOGLOBIN HETEROGENEITY 3 4 2.8. EVOLUTION OF LEGHEMOGLOBIN GENES 40 III. INDUCED MUTAGENESIS FOR LEGHEMOGLOBIN VARIABILITY 50 3.1. INTRODUCTION 5 0 3.2. LITERATURE REVIEW 53 3.3. MATERIALS AND METHODS 6 0 3.4. RESULTS AND DISCUSSION 63 IV. LEGHEMOGLOBIN VARIABILITY IN THE GENUS PHASEOLUS 68 4.1. INTRODUCTION 68 4.2. LITERATURE REVIEW 7 0 iv 4.3. MATERIALS AND METHODS 76 4.3.1. PLANT MATERIAL 7 6 4.3.2. GROWING CONDITIONS 79 4.3.3. LEGHEMOGLOBIN TESTING 81 4.3.4. INTERSPECIFIC HYBRIDIZATION 82 4.3.5. EMBRYO RESCUE AND HYBRID GROWTH 8 3 4.3.6. NITROGEN FIXATION EXPERIMENT 85 4.3.7. PROTEIN SEQUENCING 8 7 4.4. RESULTS AND DISCUSSION 88 4.4.1. LEGHEMOGLOBIN VARIABILITY IN THE GENUS PHASEOLUS 88 4.4.2. INTERSPECIFIC HYBRIDIZATION 96 4.4.3. NITROGEN FIXATION EXPERIMENTS 98 4.4.4. PROTEIN SEQUENCING 112 V. GENERAL DICUSSION AND CONCLUSIONS 117 VI. SUMMARY AND CONCLUSIONS 124 LIST OF REFERENCES ' 125 .V LIST OF TABLES Table Page I. Phaseolus germplasm accessions 77 II. , Grouping of 'Plant Species' for analysis 99 III. Analysis of variance for ARA of P. vulgaris, P_. acutifolius, P_. filiforalis and inter• specific hybrids. Additional analysis for the partitioned sums of squares for 'Plant Species' is also shown 100 IV. Orthogonal contrasts among groups for the ARA experiment 101 i V. Mean acetylene reduction (nm/plant/hour) for each 'Plant Species' and group measured in the ARA experiment 102 VI. Orthogonal contrasts within groups for the ARA experiment 103 VII. Analysis of variance for Total N in P_. vulgaris, P. acutifolius, P. filiformis and interspecific hybrids. Additional analysis of the partitioned sums of squares for 'Plant Species' is also shown 105 VIII. Orthogonal contrasts among groups for the Total N experiment 106 IX. Means (mg total nitrogen) for each 'Plant Species' and group of the Total N experiment 107 X. Orthogonal contrasts within groups for the Total N experiment involving P_. vulgar is, P_. acutifolius, .P. filiformis and inter• specific hybrids 109 vi LIST OF FIGURES Figure Page 1. CAE Lb profile showing P_. vulgaris (C) , P.. acutif olius N1602 (A^ ) , and P.. acutifol ius ssp. latif olius (A|_) 89 2. CAE Lb profile showing P_. vulgar is wild (P^) , P_. f iliformis (P p) , P. mi cr ocarpus (P^ ) and P. vulgar is 'Gallatin1 (G) 91 3. CAE Lb profile showing a P_. vulgaris x P_. filiformis hybrid (1), P. acutifolius (2), P. vulgaris x P_. acutifolius hybrid (3) and P_. vulgaris (4) 92 4. CAE Lb profile showing P_. vulgaris (C) , P.. lunatus 'Lima Hendersons' (L), £. lunatus 'Market Sample' (M) and P_. acutifolius (A).. 93 5. Elution profile for P. vulgar is x P. filiformis hybrid Lb components on DE5 2-cellulose columns 113 6. Elution profile for P_. lunatus 'Lima Henderson' Lb components on DE52-cellulose columns .... 115 vii LIST OF ABBREVIATIONS ADP adenosine diphosphate ALA aminolevulinic acid ARA acetylene reduction assay ATP adenosine triphosphate CAE cellulose acetate electrophoresis CDNA complementary DNA CI AT Centro Internacional de Agricultura Tropicale DE diethylaminoethyl- DF degrees of freedom EMS ethyl methane sulfonate Hb hemoglobin IAEA International Atomic Energy Agency IEF isoelectric focusing Lb leghemoglobin Ml mutant generation one MS mean squares RCBD randomized complete block design TDM total dry matter Ti tumor inducing Tn transposon .vi i i GLOSSARY Rhizobium is a generic name and refers to the genus of bacteria known as Rhizobium. The plural Rhizobia refers to more than one cell of Rhizobium, and rhizobial is an adjective describing some attribute of Rhizobium. Bacteroid This term is used to describe the nitrogen fixing form of Rhizobia found in root nodules. Peribacteroid Membrane or membrane envelope refers to the plant membrane surrounding each bacteroid or group of bacteroids in nitrogen fixing root nodules. Peribacteroid Space refers to the space between the peri- bacteroid membrane and its contained bacteroids. Periplasmic Space refers to the space between the bacteroid inner (plasma) and outer (cell wall) membrane. Leghemoglobin (Lb) refers to the oxygen-carrying hemepro- tein found in legume root nodules. Oxy-Lb is the oxygenated form, deoxy-Lb refers to the deoxygenated form. Hemoglobin (Hb) is a general term for hemeproteins regardless of animal or plant origin. Accession refers to a sample in a gene bank; its number is unique. Cultivar refers to a cultivated variety. Ml generation refers to the first mutated generation, i.e. plants grown from the treated seeds. Nitrogen refers to the gaseous element N2; should be correctly termed 'dinitrogen'. Total N refers to the total amount of nitrogen fixed. (References: Appleby 1984, Date 1978, Summerfield 1988.) .ix ACKNOWLEDGEMENT I would like to thank Dr. Galway from Cambridge University, U.K., and Dr. Michaels from the University of Guelph, Canada, for the plant material. Furthermore, I wish to thank my thesis supervisor Dr. F.B. Holl for his guidance and support throughout this study. Thanks are extended to my committee members, Dr. A.J. Griffiths, Dr. V.C. Runeckles, Dr. M. Upadhyaya, and my external examiner Dr. D.P.S. Verma for their time and effort for reviewing this research. I also wish to thank Dr. G. Eaton for his assistance with the statistical analysis of this experiment. Special thanks to my husband, Claude Lapointe, for his support and encouragement without which I might never have finished this project. Finally, thanks to all those friends who made me laugh and thus made the situation a bit more bearable. x I. GENERAL INTRODUCTION The successful establishment of nitrogen fixing no• dules relies on a complex sequence of physiological pro• cesses, many of which involve interaction between the as• sociated Rhizobium bacterium and its host plant. A cha• racteristic of this symbiotic process is the presence of a red pigment which was first shown to be a hemeprotein by Kubo in 1939.
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