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Characterisation of selected Arabidopsis aldehyde dehydrogenase genes: role in plant stress physiology and regulation of gene expression [Elektronische Ressource] / vorgelegt von Tagnon Degbedji MIssihoun

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176 pages
Characterisation of selected Arabidopsis aldehyde dehydrogenase genes: role in plant stress physiology and regulation of gene expression Dissertation Zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Tagnon Dègbédji MISSIHOUN aus Cotonou, Benin Bonn, November 2010 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes 1. Referentin: Prof. Dr. Dorothea Bartels 2. Koreferent: Priv. Doz. Dr. Hans-Hubert Kirch Tag der Promotion: 22. Februar 2011 Erscheinungsjahr: 2011 II DECLARATION I hereby declare that the whole PhD thesis is my own work, except where explicitly stated otherwise in the text or in the bibliography. Bonn, November 2010 ------------------------------------ Tagno D.MISHOUN III DEDICATION To My wife: Fabienne TOSSOU-MISSIHOUN and our kids Floriane S. Jennifer and Sègnon Anges-Anis My parents: Lucrèce KOTOMALE and Dadjo MISSIHOUN My sister and brothers: Mariette, Marius, Ricardo, Renaud, Ulrich And my dearest aunts and uncles: Hoho, Rebecca, Cyriaque, Dominique, Alphonsine IV CONTENTS ABBREVIATIONS ..............
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Characterisation of selected Arabidopsis
aldehyde dehydrogenase genes: role in plant
stress physiology and regulation of gene
expression

Dissertation

Zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn

vorgelegt von

Tagnon Dègbédji MISSIHOUN

aus
Cotonou, Benin

Bonn, November 2010


Angefertigt mit Genehmigung der Mathematisch-
Naturwissenschaftlichen Fakultät
der Rheinischen Friedrich-Wilhelms-Universität Bonn





Gedruckt mit Unterstützung des Deutschen Akademischen
Austauschdienstes








1. Referentin: Prof. Dr. Dorothea Bartels

2. Koreferent: Priv. Doz. Dr. Hans-Hubert Kirch


Tag der Promotion: 22. Februar 2011

Erscheinungsjahr: 2011

II
DECLARATION

I hereby declare that the whole PhD thesis is my own work, except where explicitly
stated otherwise in the text or in the bibliography.






Bonn, November 2010 ------------------------------------
Tagno D.MISHOUN














III
DEDICATION

To

My wife: Fabienne TOSSOU-MISSIHOUN and our
kids Floriane S. Jennifer and Sègnon Anges-
Anis


My parents: Lucrèce KOTOMALE and Dadjo
MISSIHOUN


My sister and brothers: Mariette, Marius, Ricardo, Renaud, Ulrich


And my dearest aunts and uncles: Hoho, Rebecca, Cyriaque, Dominique,
Alphonsine







IV
CONTENTS

ABBREVIATIONS ...............................................................................................................................................X
FIGURES AND TABLESXIII
SUMMARY............................ 1
1. INTRODUCTION........ 3
1.1 Climate changes and environmental stress...................................................................................... 3
1.2 Plant stress and mechanisms of tolerance........................................................................................ 3
1.3 Gene products related to abiotic stress ............................................................................................ 4
1.3.1 Regulatory pathways of stress-related gene expression in plants............................................... 5
1.3.1.1 Osmotic/oxidative stress signalling ....................................................................................... 5
2+1.3.1.2 Ca -dependent signalling...................................................................................................... 6
1.3.2 ABA signalling........................................................................................................................... 7
1.3.2.1 ABA metabolism ................................................................................................................... 7
1.3.2.2 ABA perception..................................................................................................................... 7
1.3.2.3 ABA signal transduction........................................................................................................ 8
1.3.3 Stress inducible proteins and other compounds........................................................................ 10
1.3.3.1 LEA proteins....................................................................................................................... 10
1.3.3.2 Compatible solutes............................................................................................................... 11
1.3.3.2.1 Mannitol, D-ononitol and sorbitol ................................................................................ 11
1.3.3.2.2 Trehalose ...................................................................................................................... 12
1.3.3.2.3 Sucrose ......................................................................................................................... 12
1.3.3.2.4 Fructans ........................................................................................................................ 13
1.3.3.2.5 Proline .......................................................................................................................... 13
1.3.3.2.6 Glycine betaine............................................................................................................. 14
1.3.3.3 Small RNAs 15
1.3.3.4 Reactive Oxygen Species (ROS) ......................................................................................... 15
1.3.3.5 Aldehydes and the peroxidation of membrane lipids........................................................... 16
1.3.3.6 Aldehyde dehydrogenases (ALDHs) as ROS-detoxifying enzymes.................................... 17
1.3.3.7 Aldehyde dehydrogenase genes........................................................................................... 18
1.3.3.8 Betaine aldehyde dehydrogenases ....................................................................................... 19
1.3.3.9 Aminoaldehyde dehydrogenases and the polyamine metabolism........................................ 20
1.4 Objectives of the study..................................................................................................................... 20
2. MATERIALS AND METHODS.............................................................................................................. 22
2.1 Materials........................................................................................................................................... 22
2.1.1 Plant materials .......................................................................................................................... 22
2.1.2 Chemicals.. 22
2.1.3 DNAs, vectors and bacteria...................................................................................................... 22
2.1.3.1 cDNAs.. 22
2.1.3.2 Vectors................................................................................................................................. 23
V
2.1.3.2.1 pJET1.2......................................................................................................................... 23
2.1.3.2.2 pBT10-GUS.................................................................................................................. 23
2.1.3.2.3 pRTL2-GUS vector ...................................................................................................... 23
2.1.3.2.4 pGJ280.......................................................................................................................... 23
2.1.3.2.5 pET28a 23
2.1.3.2.6 pBIN19 and pROK2 ..................................................................................................... 24
2.1.3.2.7 pPG-Tkan ..................................................................................................................... 24
2.1.3.3 Bacteria................................................................................................................................ 24
2.1.4 Enzymes and DNA-marker 24
2.1.5 Software, programs and online tools ........................................................................................ 25
2.1.6 Machines and other devices...................................................................................................... 25
2.1.7 Membranes 26
2.1.8 Kits............ 26
2.1.9 Media, buffers and solutions .................................................................................................... 26
2.1.9.1 Media.... 26
2.1.9.2 Buffers and solutions ........................................................................................................... 27
2.2 Methods............................................................................................................................................. 28
2.2.1 Growth conditions .................................................................................................................... 28
2.2.1.1 Seed culture and plant growth ............................................................................................. 28
2.2.1.2 Growth of microorganisms .................................................................................................. 29
2.2.2 Primers...... 29
2.2.3 Extraction of nucleic acids ....................................................................................................... 31
2.2.3.1 Extraction of genomic DNA from A. thaliana..................................................................... 31
2.2.3.2 Plasmid DNA mini-prep (Birnboim and Doly 1979; Sambrook et al. 1989)....................... 31
2.2.3.3 Purification and precipitation of DNA................................................................................. 32
2.2.3.4 Extraction of DNA fragments from agarose gels................................................................. 32
2.2.3.5 Extraction of total RNAs from A. thaliana.......................................................................... 32
2.2.4 Qualitative and quantitative estimation of concentrations of macromolecules ........................ 33
2.2.4.1 Qualitative and quantitative estimation of DNA and RNA ................................................. 33
2.2.4.2 Quantitative estimation of protein extracts 33
2.2.5 Cloning of DNA fragments ...................................................................................................... 34
2.2.5.1 Polymerase chain reaction (PCR)........................................................................................ 34
2.2.5.2 Restriction endonuclease treatments.................................................................................... 34
2.2.5.3 Dephosphorylation............................................................................................................... 35
2.2.5.4 Ligation................................................................................................................................ 35
2.2.5.5 Transformation .................................................................................................................... 35
2.2.5.5.1 Calcium-competent E. coli ........................................................................................... 35
2.2.5.5.2 Transformation of calcium-competent E. coli .............................................................. 35
2.2.5.5.3 Preparation of electrocompetent E. coli........................................................................ 36
2.2.5.5.4 Preppetent A. tumefaciens........................................................... 36
VI
2.2.5.5.5 Transformation via electroporation (Tung and Chow 1995) ........................................ 36
2.2.5.5.6 Biolistic transformation of Arabidopsis leaves............................................................. 37
2.2.5.5.7 A. tumefaciens-mediated transient transformation of Arabidopsis seedlings: FAST
assay (Li et al. 2009).......................................................................................................................... 38
2.2.5.5.8 A. tumefaciens-mediated stable transformation of Arabidopsis plants ......................... 39
2.2.6 Screening methods ................................................................................................................... 39
2.2.6.1 Blue-white screening of bacterial colonies.......................................................................... 39
2.2.6.2 Screening for transformed bacterial clones 40
2.2.6.3 Screening for transgenic Arabidopsis seeds ........................................................................ 40
2.2.6.4 Preparation of bacterial glycerol stocks............................................................................... 40
2.2.7 Reverse transcriptase (RT)-PCR analysis................................................................................. 40
2.2.8 Electrophoresis and blotting methods....................................................................................... 41
2.2.8.1 Agarose gel electrophoresis................................................................................................. 41
2.2.8.2 DNA-blot analysis: (Sambrook et al. 1989) 41
2.2.8.3 RNA blot analysis................................................................................................................ 42
2.2.8.4 Staining of the RNA-blot membrane with Methylene Blue................................................. 42
322.2.8.5 Synthesis of α P-DNA hybridisation probes (Feinberg and Vogelstein 1983)................... 43
2.2.8.6 Semi-quantitative analyses of the gene expression from the RNA blots ............................. 43
2.2.8.7 Protein extraction from plant tissues (Laemmli 1970)......................................................... 43
2.2.8.8 Extraction and analysis of recombinant ALDH proteins from E. coli cells......................... 44
2.2.8.9 Extraction and purification of the recombinant ALDH proteins by His-tag affinity-
chromatography ...................................................................................................................................... 44
2.2.8.10 Aldehyde dehydrogenase activity of the recombinant ALDH protein............................ 45
2.2.8.11 SDS-polyacrylamide gel electrophoresis (SDS-PAGE).................................................. 45
2.2.8.12 Coomassie blue staining of SDS-PAGE ......................................................................... 46
2.2.8.13 Ponceau-Red Staining..................................................................................................... 47
2.2.8.14 Protein-blot analysis........................................................................................................ 47
2.2.8.15 Purification of ALDH-specific IgG antibodies from a crude antiserum ......................... 48
2.2.9 Stress experiments with bacterial cells..................................................................................... 48
2.2.10 Plant stress treatments .............................................................................................................. 49
2.2.10.1 Stress treatment of seedlings........................................................................................... 49
2.2.10.2 Stress treatment of soil-grown plants.............................................................................. 49
2.2.10.3 Drought stress treatment ................................................................................................. 49
2.2.10.4 Salt and Paraquat® stress treatments 50
2.2.11 Biochemical analyses and microscopy 50
2.2.11.1 Determination of chlorophyll content (Arnon 1949) ...................................................... 50
2.2.11.2 Lipid peroxidation assay 50
2.2.11.3 H O measurement.......................................................................................................... 51 2 2
2.2.11.4 Proline determination...................................................................................................... 51
2.2.11.5 GUS staining of Arabidopsis leaves ............................................................................... 52
VII
2.2.11.6 GUS-Assay with X-Gluc as substrate (Jefferson et al. 1987) ......................................... 52
2.2.11.7 Fluorometric detection of the GUS activity .................................................................... 52
2.2.11.8 In situ visualization of lipid peroxidation-derived aldehydes 53
2.2.11.9 Microscopic observation of the GFP activity in bombarded leaves................................ 53
3. RESULTS................................................................................................................................................... 54
3.1 Betaine aldehyde dehydrogenase genes from Arabidopsis with different subcellular localizations
affect stress responses..... 54
3.1.1 Comparison of BADH gene sequences .................................................................................... 54
3.1.2 Expression patterns of ALDH10A8 and ALDH10A9 genes ...................................................... 54
3.1.3 Sub-cellular localization of ALDH10A8 and ALDH10A9 proteins ........................................ 56
3.1.4 Isolation of homozygous ALDH10A8 T-DNA insertion mutants............................................ 59
3.1.5 Functional analysis of the ALDH10A8 T-DNA insertion mutant KO8-2 ................................ 61
3.1.6 Enzymatic properties of the recombinant ALDH10A9 protein................................................ 64
3.2 Molecular and functional characterization of the aldehyde dehydrogenase gene ALDH3H1........... 68
3.2.1 ALDH3H1 gene description ..................................................................................................... 68
3.2.2 Age-dependent accumulation of the ALDH3H1 protein.......................................................... 68
3.2.3 Generation and molecular characterisation of transgenic plants over-expressing the ALDH3H1
protein …………………………………………………………………………………………………70
3.2.4 Functional characterization of ALDH3H1 over-expressors exposed to various abiotic stressors
…………………………………………………………………………………………………72
3.2.4.1 In vitro-based stress experiments......................................................................................... 73
3.2.4.2 Soil-based stress experiments .............................................................................................. 75
3.2.5 Identification and characterisation of further T-DNA insertion mutants of the ALDH3H1 gene
…………………………………………………………………………………………………77
3.2.6 Molecular characterisation of the 3h1-A and 3h1-C mutants ................................................... 78
3.2.7 The origin of the transcript T3 in the 3h1-A line ...................................................................... 82
3.2.7.1 Generation and analysis of the 3h1-intron::GUS construct................................................. 83
3.2.7.2 Functional analysis of the 3h1-intron::GUS construct in planta ......................................... 83
3.2.8 Stress-responsive expression of the transcript T3..................................................................... 84
3.2.9 Comparative analysis of the ALDH3H1 T-DNA insertion mutants in response to stress......... 85
3.2.10 Sub-cellular localization of the putative protein derived from the transcript T3 ...................... 86
3.3 Responsiveness of the aldehyde dehydrogenase gene ALDH7B4 to aldehydes ................................... 88
3.3.1 Generation of ALDH7B4-promoter::GUS expressing plants.................................................... 88
3.3.2 Molecular characterization and segregation analysis of the 7B4-GUS and 35S-GUS lines.…89
3.3.3 Activity of the ALDH7B4 promoter in reproductive organs and seeds .................................... 91
3.3.4 Activity of the transgenic 7B4-GUS lines in response to aldehyde and abiotic stress treatments
…………………………………………………………………………………………………93
3.3.5 Comparison of the ALDH7B4 promoter activation and the MDA accumulation ..................... 96
3.3.6 Analysis of the ALDH7B4 gene promoter sequence and effects of the mutation of the DRE and
ACGT-boxes ............................................................................................................................................... 97
VIII
3.3.7 Production and strategy of screening of the EMS-derived mutant population ....................... 100
4. DISCUSSION........................................................................................................................................... 102
4.1 Functional analysis of putative betaine dehydrogenase genes from Arabidopsis.............................. 102
4.1.1 Arabidopsis BADH coding genes are stress inducible ........................................................... 103
4.1.2 BADHs are probably aminoaldehyde detoxifying enzymes............................... 103
4.1.3 The ALDH10A8 knock-out mutant is stress sensitive ............................................................ 105
4.2 Molecular and functional analyses of the ALDH3H1 gene locus........................................................ 105
4.2.1 What can one learn from over-expressing the ALDH3H1 protein? ....................................... 105
4.2.2 ALDH3H1 locus contains an alternative promoter directing the expression of an alternative
first exon (AFE) transcript......................................................................................................................... 108
4.2.3 Influence of the use of AFE-transcripts on the protein sub-cellular localization.................... 109
4.2.4 What can one understand from the differential expression of ALDH3H1 transcript isoforms?
………………………………………………………………………………………………..110
4.2.5 Is the T3 transcript variant relevant for the plant viability?.................................................... 112
4.3 Study of the ALDH7B4 gene promoter................................................................................................. 113
4.3.1 The Arabidopsis antiquitin-like protein ALDH7B4 is a good candidate to investigate aldehyde
dehydrogenase gene regulation ................................................................................................................. 113
4.3.2 Induction patterns of the ALDH7B4 gene promoter ............................................................... 115
4.3.3 Biological activities of α, β-unsaturated aldehydes and related oxilipins................................ 116
4.3.4 How could bioactive aldehydes function as signal compounds?............................................ 117
4.3.4.1.1 Functional analysis of the cis-acting elements in the ALDH7B4 promoter................... 118
4.3.5 Generation of the EMS-mutagenized seed population and screening strategy....................... 119
4.4 Conclusions and future perspectives .................................................................................................... 120
5. APPENDICES ......................................................................................................................................... 122
5.1 Accession numbers of the ALDH genes........................................................................................ 122
5.2 Gene sequences 122
5.2.1 ALDH10A8 gene sequence..................................................................................................... 123
5.2.2 9 gene sequence 126
5.2.3 ALDH3H1 gene sequence....................................................................................................... 129
5.2.4 ALDH7B4 gene promoter sequence ....................................................................................... 132
5.3 Vector maps.................................................................................................................................... 133
6. REFERENCES......... 138
7. ACKNOWLEDGEMENTS .................................................................................................................... 160
8. MEETING AND CONFERENCES ATTENDED WITH POSTER PRESENTATIONS ................ 162
9. LIST OF PUBLICATIONS 162




IX
ABBREVIATIONS

4 –MUG 4 –Methylumbelliferyl glucuronide
A Adeni
ABA Abscisic acid
ABAL 4-aminobutyraldehyde
ABRE ABA responsive element
ALDH Aldehyde dehydrogenase
AMADH Aminoaldehyde dehydrogenase
Amp Ampicillin
APAL 3-aminopropionaldehyde
APS monium persulfate
bp Nucleotide base pair
BSA Bovine serum albumin
β-ME β-mercaptoethanol
bZIP asic leucine zipper
C Cytosine
CaMV Cauliflower mosaic virus
CAO Copper Amine Oxidase
cDNA Complementary DNA
CRT C-repeat
D Dalton
DAB 3,3’-diaminobenzidine
dATP Desoxy-adenosin-triphosphate
dCxy-cytidin-triphosphate
dCTP xy-cytidin-triphosphat
dGxy-guanosin-triphosphate
DMF N,N-Dimethylformamid
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
dNTP Deoxyribonucleotide triphosphate
DRE Dehydration responsive element
DTT Dithiothreitol
dTTP Desoxy-thymidin-triphosphate
DW Dry weight
EDTA Ethylenediaminetetraacetate
fwt Fresh weight
fwd Forwad
g gram
X

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