Functional characterization of 2-hydroxy acid oxidases, dehydrogenases in Arabidopsis thaliana
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| 245 | 1 | 0 | |a Functional characterization of 2-hydroxy acid oxidases, dehydrogenases in Arabidopsis thaliana |c vorgelegt von Anke Kuhn |
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Datensatz im Suchindex
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| adam_text | ILL
CONTENTS
CONTENTS III
ABBREVIATIONS VIII
1. INTRODUCTION 1
1.1. 2-HYDROXY ACIDS AND THE CORRESPONDING OXIDIZING ENZYMES 1
1.2. GLYCOLATE METABOLISM DURING PHOTORESPIRATION 2
1.2.1. PHOTORESPIRATION, THE OXIDATIVE PHASE OF PHOTOSYNTHESIS ... 2
1.2.2. THE PHOTORESPIRATORY PATHWAY IN HIGHER PLANTS 4
1.3. LACTATE METABOLISM DURING THE DETOXIFICATION OF METHYLGLYOXAL AND
HYPOXIA 6
1.4. 2-HYDROXYGLUTARATE: A RECENTLY DISCOVERED 2-HYDROXY ACID IN PLANT
METABOLISM 8
1.5. AIMS OF THIS STUDY 10
2. MATERIALS AND METHODS 11
2.1. PRIMERS 11
2.2. PLANTS 12
2.2.1. PLANT MATERIAL 12
2.2.2. PLANT GROWTH CONDITIONS 12
2.2.2.1. PLANT GROWTH ON SOIL 12
2.2.2.2. GROWTH OF A. THALIANA IN STERILE CULTURE 13
2.2.2.3. GROWTH OF A. THALIANA ROOT CULTURES 13
2.2.2.4. GREENHOUSE CONDITIONS 13
2.2.2.5. GROWTH CHAMBER CONDITIONS 14
2.2.2.6. GROWTH IN HIGH C0
2
CONDITIONS 14
2.2.2.7. PLANT PEST CONTROL 14
2.2.3. SAMPLING OF PLANT TISSUE 14
2.2.4. SEED STERILIZATION WITH ETHANOL 15
2.3. MICROBIOLOGICAL METHODS 15
2.3.1. E. COLI STRAINS 15
HTTP://D-NB.INFO/1033648590
CONTENTS IV
2.3.2. E. COLI GROWTH CONDITIONS 15
2.3.3. GENERATION OF CHEMICALLY COMPETENT E. COLI 16
2.3.4. TRANSFORMATION OF CHEMICALLY COMPETENT E. COLI BY HEAT SHOCK 17
2.3.5. A. TUMEFACIENS STRAINS 17
2.3.6. A. TUMEFACIENS GROWTH CONDITIONS 17
2.3.7. GENERATION OF CHEMICALLY COMPETENT A. TUMEFACIENS 18
2.3.8. TRANSFORMATION OF CHEMICALLY COMPETENT A. TUMEFACIENS ...
18
2.4. MOLECULAR BIOLOGICAL METHODS 19
2.4.1. EXTRACTION OF GENOMIC DNA FROM ARABIDOPSIS THALIANA LEAVES . 19
2.4.2. PCR (POLYMERASE CHAIN REACTION) 19
2.4.3. AGAROSE GEL ELECTROPHORESIS 20
2.4.4. DNA QUANTIFICATION 21
2.4.5. EXTRACTION OF RNA FROM ARABIDOPSIS THALIANA 21
2.4.6. CDNA-SYNTHESIS 21
2.4.6.1. DNASE TREATMENT 22
2.4.6.2. REVERSE TRANSCRIPTION 22
2.4.6.3. RT-PCR 22
2.4.7. QUANTITATIVE REAL TIME PCR (Q-RT-PCR) 23
2.4.8. MINIPREP FOR E. COLI PLASMID ISOLATION 23
2.4.9. ELUTION OF DNA FROM AGAROSE GELS 24
2.4.10. RESTRICTION DIGESTION 24
2.4.11. DEPHOSPHORYLATION OF DNA 24
2.4.12. LIGATION OF DNA FRAGMENTS 24
2.4.13. LR REACTION 25
2.4.14. TOPO CLONING 25
2.4.15. DNA SEQUENCING 26
2.5. VECTORS AND VECTOR GENERATION 26
2.5.1. VECTORS 26
2.5.2. VECTOR GENERATION 27
2.6. BIOCHEMICAL METHODS 28
2.6.1. DETERMINATION OF PROTEIN CONCENTRATION 28
2.6.2. SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE) 28
2.6.3. COOMASSIE STAINING 29
2.6.4. WESTERN BLOT 30
2.6.4.1. PROTEIN TRANSFER ONTO A PVDF MEMBRANE BY A SEMI-
DRY BLOT SYSTEM 30
2.6.4.2. IMMUNOLOGICAL DETECTION OF PROTEINS 30
CONTENTS V
2.6.5. PROTEIN EXTRACTION FOR NATIVE PAGE 31
2.6.6. NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS (NATIVE PAGE) ...
31
2.6.7. IN-GEL ACTIVITY ASSAY 32
2.6.8. EXPRESSION OF RECOMBINANT PROTEINS 32
2.6.9. PURIFICATION OF HETEROLOGOUSLY EXPRESSED, HIS-TAGGED PROTEINS 33
2.6.10. ANALYSIS OF THE PROSTHETIC GROUP 34
2.6.11. SPECTROPHOTOMETRIC ENZYME ASSAYS 35
2.6.11.1. ANALYSIS OF COFACTORS 36
2.6.11.2. DETERMINATION OF THE OPTIMAL BUFFER 36
2.6.11.3. DETERMINATION OF .THE PH OPTIMUM 36
2.6.11.4. DETERMINATION OF POSSIBLE SUBSTRATES 36
2.6.11.5. DETERMINATION OF KINETIC CONSTANTS 37
2.6.12. ENZYME ASSAYS USING AN OXYGEN ELECTRODE - K
M
DETERMINA
TION OF 0
2
37
2.7. METHODS FOR CO-LOCALIZATION STUDIES OF PROTEINS 38
2.7.1. INFILTRATION OF N. BENTHAMIANA WITH A. TUMEFACIENS 38
2.7.2. PROTOPLAST ISOLATION OF N. BENTHAMIANA LEAVES 39
2.7.3. CONFOCAL FLUORESCENCE MICROSCOPY 40
2.8. METABOLOMIC ANALYSES 40
2.8.1. GAS CHROMATOGRAPHY MASS SPECTROMETRY (GC-MS) ANALYSIS . 40
2.8.2. STABLE ISOTOPE DILUTION LIQUID CHROMATOGRAPHY MASS SPECTROM-
ETRY/MASS SPECTROMETRY (LC-MS/MS) 41
2.9. CHLOROPHYLL FLUORESCENCE PARAMETERS 41
2.9.1. PULSE AMPLITUDE MODULATION FLUOROMETRY (PAM) 41
3. RESULTS 43
3.1. CHARACTERIZATION OF ATD-2HGDH 43
3.1.1. SUBCELLULAR LOCALIZATION OF ATD-2HGDH 43
3.1.2. ATD-2HGDH PROTEIN AND TRANSCRIPT ACCUMULATION DURING DE
VELOPMENTAL SENESCENCE 44
3.1.3. IMAGING PAM MEASUREMENT AS INDICATOR OF PLANT FITNESS ...
45
3.1.4. METABOLITE PROFILING OF AGEING WILD-TYPE AND D-2HGDH 46
3.1.5. PLANT CULTIVATION ON MEDIUM SUPPLEMENTED WITH DIFFERENT PU
TATIVE SUBSTRATES 48
3.1.6. ACCUMULATION OF D-2HG IN THE PHOTORESPIRATORY MUTANT SHM
1 -
1 50
3.1.7. D-2HGDH ACTIVITY IN SHML-1 MUTANTS 53
3.1.8. INHIBITION STUDIES OF ATD-2HGDH 56
CONTENTS
VI
3.2. CHARACTERIZATION OF THE A. THALIANA GLYCOLATE OXIDASE (GOX) FAMILY
. . 59
3.2.1. THE EVOLUTION OF THE GOX FAMILY 59
3.2.2. SUBCELLULAR LOCALIZATION OF ATGOX3 61
3.2.3. BIOCHEMICAL ANALYSIS OF ATGOXI, ATGOX2 AND ATGOX3 ...
62
3.2.3.1. EXPRESSION AND PURIFICATION OF ATGOXI AND ATGOX2 62
3.2.3.2. ANALYSIS OF THE
PROSTHETIC GROUP OF ATGOXI,
ATGOX2
AND ATGOX3 62
3.2.3.3. ANALYSIS OF POSSIBLE ELECTRON ACCEPTORS 64
3.2.3.4. DETERMINATION OF THE PH OPTIMA 65
3.2.3.5. SUBSTRATE SCREEN 65
3.2.3.6. DETERMINATION OF THE KINETIC CONSTANTS 67
3.2.4. ON THE IN VIVO FUNCTION OF ATGOX3 71
3.2.4.1. ISOLATION OF GOX3 LOSS-OF-FUNCTION MUTANTS 71
3.2.4.2. GOX3 TRANSCRIPT ACCUMULATION DURING DEVELOPMEN
TAL AND DARK-INDUCED SENESCENCE 72
3.2.4.3. GOX3 PHENOTYPE
DURING DEVELOPMENTAL AND DARK-INDUCED
SENESCENCE 73
3.2.4.4. DETERMINATION OF LACTATE AND GLYCOLATE LEVELS DURING
DEVELOPMENTAL AND DARK-INDUCED SENESCENCE ....
74
3.2.5. BIOCHEMICAL ANALYSIS OF ATHAOXI AND ATHAOX2 76
3.2.5.1. EXPRESSION AND PURIFICATION OF ATHAOXI AND ATHAOX2 76
3.2.5.2. OPTIMIZING THE ASSAY CONDITIONS FOR THE BIOCHEMICAL
CHARACTERIZATION OF ATHAOX ENZYMES 77
3.2.5.3. SUBSTRATE SCREEN 77
4. DISCUSSION 81
4.1. THE ROLE OF ATD-2HGDH IN PLANT METABOLISM 81
4.1.1. ATD-2HGDH LOCALIZES TO MITOCHONDRIA 81
4.1.2. ATD-2HGDH ACTIVITY IS INDUCED DURING DEVELOPMENTAL SENES
CENCE 81
4.1.3. D-2HG ACCUMULATES IN D-2HGDH MUTANTS DURING DEVELOPMEN
TAL SENESCENCE - INDICATIONS FOR THE INVOLVEMENT OF ATD-2HGDH
IN THE MOBILIZATION OF ALTERNATE SUBSTRATES 82
4.1.4. ATD-2HGDH IS INVOLVED IN LYSINE DEGRADATION 83
4.1.5. D-2HG ACCUMULATES IN THE PHOTORESPIRATORY SHM1-1 MUTANT
AFTER TRANSITION FROM ELEVATED TO AMBIENT C0
2
CONDITIONS - A
LINK BETWEEN ATD-2HGDH AND PHOTORESPIRATION 84
4.1.6. OUTLOOK 86
CONTENTS
VII
4.2. CHARACTERIZATION OF THE A. THALIANA GOX FAMILY 87
4.2.1. EVOLUTION OF THE A. THALIANA GOX FAMILY 87
4.2.2. ATGOX3 LOCALIZES TO PEROXISOMES 87
4.2.3. ATGOXI, ATGOX2 AND ATGOX3 BIND FMN AS PROSTHETIC GROUP 88
4.2.4. THE DIFFERENT MEMBERS OF THE ATGOX PROTEIN FAMILY POSSESS
DIFFERENT SUBSTRATE SPECIFICITIES 88
4.2.5. ATGOX3 IS LIKELY TO FUNCTION AS OXIDASE AND NOT AS DEHYDRO
GENASE BASED ON ITS KINETIC CONSTANTS FOR 0
2
93
4.2.6. INDICATIONS FOR POSSIBLE ATGOX3 IN VIVO FUNCTIONS 93
4.2.7. OUTLOOK 95
BIBLIOGRAPHY 96
ABSTRACT 106
KURZZUSAMMENFASSUNG 107
A. SUPPLEMENTARY DATA OF THE PHYLOGENETIC ANALYSIS OF THE GOX FAMILY 109
A.1. LIST OF SPECIES 109
A.2. LIST OF ACCESSION NUMBERS 110
ACKNOWLEDGEMENTS 112
ERKLARUNG 114
LEBENSLAUF 115
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| institution | BVB |
| language | English |
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| spellingShingle | Kuhn, Anke 1984- Functional characterization of 2-hydroxy acid oxidases, dehydrogenases in Arabidopsis thaliana |
| subject_GND | (DE-588)4113937-9 |
| title | Functional characterization of 2-hydroxy acid oxidases, dehydrogenases in Arabidopsis thaliana |
| title_auth | Functional characterization of 2-hydroxy acid oxidases, dehydrogenases in Arabidopsis thaliana |
| title_exact_search | Functional characterization of 2-hydroxy acid oxidases, dehydrogenases in Arabidopsis thaliana |
| title_full | Functional characterization of 2-hydroxy acid oxidases, dehydrogenases in Arabidopsis thaliana vorgelegt von Anke Kuhn |
| title_fullStr | Functional characterization of 2-hydroxy acid oxidases, dehydrogenases in Arabidopsis thaliana vorgelegt von Anke Kuhn |
| title_full_unstemmed | Functional characterization of 2-hydroxy acid oxidases, dehydrogenases in Arabidopsis thaliana vorgelegt von Anke Kuhn |
| title_short | Functional characterization of 2-hydroxy acid oxidases, dehydrogenases in Arabidopsis thaliana |
| title_sort | functional characterization of 2 hydroxy acid oxidases dehydrogenases in arabidopsis thaliana |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1033648590/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026022797&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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