Characterization of the proteins PCH1 and PCHL and their influence on PHYB dark reversion
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| 016 | 7 | |a 1170198139 |2 DE-101 | |
| 020 | |c Broschur | ||
| 035 | |a (OCoLC)1084669348 | ||
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| 100 | 1 | |a Enderle, Beatrix |e Verfasser |0 (DE-588)1164261819 |4 aut | |
| 245 | 1 | 0 | |a Characterization of the proteins PCH1 and PCHL and their influence on PHYB dark reversion |c Autor: Beatrix Enderle |
| 264 | 1 | |a Freiburg im Breisgau |c Juni 2018 | |
| 300 | |a xvii, 144 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Enthält Zeitschriftenaufsätze | ||
| 502 | |b Dissertation |c Albert-Ludwigs-Universität Freiburg im Breisgau |d 2018 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u http://d-nb.info/1170198139/04 |3 Inhaltsverzeichnis |
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| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-031062955 | |
Datensatz im Suchindex
| DE-BY-UBR_call_number | 24/19.0176 |
|---|---|
| DE-BY-UBR_katkey | 6124775 |
| DE-BY-UBR_location | 00 |
| DE-BY-UBR_media_number | 069041085815 |
| _version_ | 1822675857558208512 |
| adam_text | CONTENTS
DECLARATION OF AUTHORSHIP III
ABSTRACT VII
1 INTRODUCTION 1
1.1 THE SIGNIFICANCE OF LIGHT IN PLANT L I F E
..............................................................
1
1.1.1 PLANT
PHOTORECEPTORS............................................................................
1
1.1.2 EARLY SEEDLING
DEVELOPMENT.................................................................
2
1.2 THE PHYTOCHROME PHOTORECEPTOR SYSTEM
......................................................... 2
1.2.1 PHOTOBIOLOGICAL PROPERTIES OF PHYTOCHROM
ES...................................... 3
1.2.2 BIOCHEMICAL PROPERTIES OF PHYTOCHROM ES
...........................................
4
1.2.3 THE PHYTOCHROME FAMILY IN ARABIDOPSIS THALIANA
...................................
6
1.2.4 PHYTOCHROME DOWNSTREAM SIG N ALLIN G
................................................. 7
1.3 PHYTOCHROME B
...................................................................... 8
1.3.1 PHYTOCHROME B
LOCALIZATION.................................................................
9
1.3.2 THE PHYB MODEL
......................................................................
10
1.3.3 PHYB DARK
REVERSION............................................................................
11
1.4 PCH1 AS A NOVEL INTERACTOR OF PHYTOCHROM
ES................................................. 12
1.5 AIM OF THIS TH E S IS
................................................
12
2 METHODS 13
2.1 MOLECULAR C LO N IN G
................................................................... 13
2.1.1 POLYMERASE CHAIN
REACTION.................................................................
13
2.1.2 OVERLAP-EXTENSION POLYMERASE CHAIN REACTION
...................................
13
2.1.3 AGAROSE GEL
ELECTROPHORESIS.................................................................
13
2.1.4 DNA PURIFICTION FROM AGAROSE G E LS
...................................................
13
2.1.5 PREPARATIVE PLASMID D IGESTION
...........................................................
14
2.1.6 CLASSIC
LIGATION....................................................................................
14
2.1.7 LIGATION USING GIBSON
ASSEMBLY........................................... 14
2.1.8 E.COLI TRANSFORMATION
.............................
14
2.1.9 DNA PLASMID PREPARATION AND
SEQUENCING......................................... 14
2.1.10 AGROBACTERIUM TUMEFACIENS TRANSFORMATION
...........................................
14
2.1.11 IN SILICO
CLONING....................................................................................
15
2.2 RNA RELATED
TECHNIQUES.................................................................................
15
2.2.1 RNA EXTRACTION FROM SEEDLINGS .
.
........................................................
15
2.2.2 REVERSE
TRANSCRIPTION.........................................................................
15
2.2.3 QUANTITAVE REAL-TIME POLYMERASE CHAIN
REACTION.............................. 15
2.2.4 DATA AN A LY
SIS......................................................................................
16
2.3 PROTEIN EXTRACTION AND
DETECTION......................................................................
16
2.3.1 DENATURING PROTEIN
EXTRACTION..............................................................
16
2.3.2 NON DENATURING PROTEIN EXTRACTION AND CO-IMMUNOPRECIPITATION ....
16
2.3.3 PROTEIN QUANTIFICATION USING AMIDOBLACK
..............................................
17
2.3.4 SDS-POLYACRYLAMIDE- GELELECTROPHORESIS (SDS-PAGE)
........................
17
2.3.5 PROTEIN
TRANSFER....................................................................................
17
2.3.6 IMMUNODETECTION OF PROTEINS
..............................................................
18
2.3.7 QUANTIFICATION OF WESTERN BLOT SIG N A
LS................................................. 18
2.4 YEAST
TECHNIQUES...............................................................................................
18
2.4.1 TRANSFORMATION OF YEAST C E L L S ........................... 18
2.4.2 YEAST-2-HYBRID FILTER LIFT ASSA Y
..............................................................
18
2.4.3 QUANTITATIVE YEAST-2-HYBRID ASSAY / MILLERS A S S A Y
.............................. 19
2.5 PLANT RELATED
TECHNIQUES.................................................................................
19
2.5.1 PLANT CULTIVATION
.................................................................................
19
PLANT CULTIVATION ON S O I L
......................................................................
19
PLANT CULTIVATION IN PETRI
DISHES............................................................ 20
2.5.2 CROSSING OF PLANTS
..............................................................................
20
2.5.3 PLANT
TRANSFORMATION............................................................................
20
2.5.4 GENERATION OF STABLE TRANSGENIC P LA N
TS................................................. 20
2.5.5
GENOTYPING.........................................................................................
21
2.6 PHYSIOLOGICAL AN ALY
SES..............................................................................
21
2.6.1 PHOTON-FLUENCE-RATE RESPONSE C U RV E S
.........................................
..
. 21
2.6.2 LIGHT-PULSE EXPERIMENTS
......................................................................
22
2.6.3 FLOWERING TIME
EXPERIMENTS.................................................................
22
FLOWERING TIME IN LONG DAY
CONDITIONS................................................. 22
FLOWERING TIME IN SHORT DAY C ONDITIONS
...........................................
22
2.7 VISUALIZATION OF PROMOTER
ACTIVITY....................................................................
23
2.7.1 PROMOTER LUC
ACTIVITY.........................................................................
23
2.7.2 PROMOTER GUS
ACTIVITY.........................................................................
23
2.8
MICROSCOPY......................................................................................................
23
2.8.1 MICROSCOPE USED IN THIS
STUDY.............................................................. 23
2.8.2 PREPARATION AND FIXATION OF SEEDLINGS FOR M ICROSCOPY
........................
23
2.9 SPECTROSCOPIC ANALYSES OF PHYB PFR AND PTOT
IN VIVO ....................................... 24
2.9.1 OPERATION MODE OF THE RATIO-SPECTROPHOTOMETER
................................
24
2.9.2 MEASUREMENT OF PHYB PFR INITIALLY PRESENT IN SEEDLINGS
................
24
2.9.3 DATA ANALYSIS
......................................................................................
25
2.10
STATISTICS.........................................................................................................
25
3 RESULTS 27
3.1 IDENTIFICATION OF PC H 1
....................................................................................
27
3.1.1 GENE MODEL OF PCH1 AND P C H L
......................................................... 27
3.1.2 PCH1 AND PCHL INTERACT WITH PHYB IN Y2H AND C
O-IP...................... 28
3.2 PHENOTYPE OF THE
PCHL AND
PCHL M U TA N
TS......................................................... 32
3.2.1 THE LACK OF PCH1 RESULTS IN RED LIGHT HYPOSENSITIVITY
........................
33
3.3 PHENOTYPE OF THE PCH1 AND PCHL OVEREXPRESSION LINES
................................
35
3.3.1 THE OVEREXPRESSION OF PCH1 AND PCHL RESULTS IN A STRONG RED LIGHT
HYPERSENSITIVITY....................................................................................
35
3.3.2 THE OVEREXPRESSION OF PCH1 AND PCHL DOES NOT ALTER PHYB PROTEIN
LEVELS....................................................................................................
39
3.3.3 PCHLOX AND PCHLOX SEEDLINGS ARE SENSITIVE TO RED LIGHT PULSES ....
40
3.3.4 SEEDLINGS OVEREXPRESSING PCH1 OR PCHL RETAINS ACTIVE PHYB DURING
N IG H
T....................................................................................................
43
3.4 ENDOGENOUS PCH1 AND PCHL ARE NECESSARY TO MAINTAIN ACTIVE PHYB
DURING
N I G H T
..............................................................................................................
45
3.4.1 THE LACK OF FUNCTIONAL PCH1 AND PCHL ACCELERATES THE LOSS OF
ACTIVE
PHYB AFTER
LIGHT-OFF..............................................................................
45
3.4.2 PHYB NUCLEAR BODY STABILITY IN PCHL P CH L
..............................................
48
3.5 PHYB PFR STABILIZATION COINCIDES WITH THE FORMATION AND STABILITY OF
NUCLEAR
BODIES
....................................................................................................
51
3.5.1 NUCLEAR BODY FORMATION OF YFP-PCH1 AND YFP-PCHL IN RED LIGHT . .
51
3.5.2 NUCLEAR BODY FORMATION OF YFP-PCH1 AND YFP-PCHL IN FAR-RED LIGHT
55
3.5.3 PCH1 AND PCHL COLOCALIZE WITH PHYB TO NUCLEAR BO D IE S
...................
59
3.5.4 PCH1 EFFECTIVELY STABILIZES PHYB NUCLEAR BO D IE S
................................
63
3.5.5 OVEREXPRESSION OF PCH1 OR PCHL INHIBIT PHYB DARK REVERSION, THUS
MAINTAINING HIGH PHYB PFR LEVELS IN
DARKNESS...................................... 66
3.5.6 PCH1 AND PCHL INFLUENCE PHYB SIGNALLING UNDER LOW LIGHT CONDITIONS
68
3.6 INTEGRATION OF LIGHT SIGNALLING PATHW
AYS......................................................... 71
3.6.1 PHYA REGULATES EXPRESSION OF PCH1 AND PCHL IN RESPONSE TO LIGHT .
. 71
3.6.2 SPATIO-TEMPORAL ANALYSIS OF THE PCH1 AND PCHL PROMOTER ACTIVITY .
. 74
3.6.3 RESPONSIVENESS
AMPLIFICATION..............................................................
79
3.6.4 CONTROL OF FLOWERING TI M E
.................................................................... 83
4 DISCUSSION 87
4.1 PCH1 AS A NOVEL INTERACTOR OF PHYTOCHROME B
................................................. 87
4.1.1 LIGHT SPECIFICITY OF THE PCH1/PCHL INTERACTION WITH PHYB
................
87
4.2 THE INFLUENCE OF PCH1 AND PCHL ON PHYB DARK REVERSION
...........................
88
4.2.1 DARK REVERSION OF P H Y B
......................................................................
88
4.2.2 PCH1 AND PCHL EFFECTIVELY INHIBIT PHYB DARK REVERSION
...................
88
4.2.3 ENDOGENOUS LEVELS OF PCH1 AND PCHL ARE SUFFICIENT TO STABILIZE
PHYB 89
4.2.4 PCH1 AND PCHL HAVE DISCRETE STABILIZATION CAPACITIES FOR PHYB IN
NU
CLEAR B O D IE S
.........................................................................................
90
4.3 A MECHANISTIC VIEW ON PHYB PFR STABILIZATION BY PCH1 AND PCHL
................
91
4.3.1 DARK REVERSION AS A CENTRAL REGULATOR IN THE PHYB THREE STATE
DIMER MODEL 91
4.3.2 PCH1 AND PCHL INTERACT WITH BOTH DIMER FORMS: THE PFR-PR
HETERODIMER
AND THE PFR-PFR HOM ODIM
ER................................................................. 92
4.3.3 HYPOTHESIS I: STABILIZATION BY INTERACTION WITH THE PFR
CONFORMATION . 94
4.3.4 HYPOTHESIS II: STABILIZATION OF PHYB PFR OCCURS IN NUCLEAR
BODIES. . . . 94
4.3.5 FUTURE EXPERIMENTS TO CLARIFY THE MECHANISM BY WHICH PCH1 AND PCHL
STABILIZE PHYB IN P F R
............................................................................
96
4.4 PHYSIOLOGICAL RELEVANCE OF PCH1 AND PCHL
...................................................
97
PCH1 AND PCHL MIGHT BE FINE TUNING PHYB SENSITIVITY IN A DIURNAL
M A N N E
R.................................................................................
97
PCH1 MIGHT INFLUENCE FLOWERING BY THE LIGHT INPUT INTO THE CIRCADIAN
CLOCK
......................................................................................
98
PCH1 AND PCHL WORK AS SIGNAL INTEGRATORS
...................................... 99
POTENTIAL INFLUENCE OF PCH1 AND PCHL ON PHYB MEDIATED THERMOSENS
ING
.........................................................................................
99
4.5 FUTURE BIOCHEMICAL APPLICATIONS OF PCH1 AND
PCHL.........................................100
CO-CRYSTALLIZATION OF PCH1 AND P H Y B
....................................................100
5 MATERIALS 103
5.1 CHEMICALS AND
MATERIALS....................................................................................
103
5.2 BUFFERS AND SOLUTION COM
POSITION....................................................................108
5.3 TECHNICAL EQUIPM
ENT.........................................................................................
113
5.4 PLASMID CONSTRUCTION AND
SOURCES....................................................................114
5.4.1 PLASMIDS FOR TRANSFORMATION OF ARABIDOPSIS THALIANA .
.
..............................114
5.4.2 PLASMIDS FOR EXPRESSION IN MAMMALIAN CELL C U LTU R E
..............................
116
5.4.3 PLASMIDS FOR EXPRESSION IN Y E A S T
.........................................................116
5.5 PLANT
LINES..........................................................................................................118
5.6 OLIGONUCLEOTIDES
...............................................................................................
120
5.7 SOFTWARE AND ONLINE
RESOURCES............................................................................
122
5.7.1 ONLINE
RESOURCES............................................................................
..
. 122
5.7.2
SOFTWARE..................................................................................................122
6 EXPERIMENTS PERFORMED BY COLLEAGUES AND COLLABORATORS 123
6.1 EXPERIMENTS FROM OTH ERS
...............................................
123
6.1.1 INITIAL Y2H SCREEN, THAT IDENTIFIED PC H 1
.................................................
123
6.1.2 CO-IMMUNOPRECIPITATION FROM MAMMALIAN CELL CU LTU RE
......................123
6.1.3 CO-IMMUNOPRECIPITATION FROM PLANT EXTRACTS
.........................................
123
6.1.4 WESTERN BLOT DETECTING ENDOGENOUS P H Y B
...........................................
123
6.1.5 PROTEIN DEGRADATION EXPERIMENT USING A BORTEZOMIB TREATMENT
...........
123
7 ZUSAMMENFASSUNG 125
8 ACKNOWLEDGEMENT - DIE DANKSAGUNG 127
A APPENDIX A 129
A. L RIGHTS AND
PERMISSIONS......................................................................................
129
A.1.1 FIGURE 1.1 PHYTOCHROME ABSORBANCE
SPECTRA............................................129
A.L.2 FIGURE 1.2 PHYTOCHROME STRUCTURE
.........................................................
130
A.L.3 FIGURE 1.3 THE PHYB DIMER M O D E L
......................................................
131
B APPENDIX B 133
B. L CALCULATION OF THE PFR/PTOT STEADY STATES USING PHYBASICALX02DREV
..........
133
|
| any_adam_object | 1 |
| author | Enderle, Beatrix |
| author_GND | (DE-588)1164261819 |
| author_facet | Enderle, Beatrix |
| author_role | aut |
| author_sort | Enderle, Beatrix |
| author_variant | b e be |
| building | Verbundindex |
| bvnumber | BV045679454 |
| ctrlnum | (OCoLC)1084669348 (DE-599)BSZ508481953 |
| discipline | Biologie |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV045679454 |
| illustrated | Illustrated |
| indexdate | 2024-12-24T07:10:58Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-031062955 |
| oclc_num | 1084669348 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | xvii, 144 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2018 |
| publishDateSearch | 2018 |
| publishDateSort | 2018 |
| record_format | marc |
| spellingShingle | Enderle, Beatrix Characterization of the proteins PCH1 and PCHL and their influence on PHYB dark reversion |
| subject_GND | (DE-588)4113937-9 |
| title | Characterization of the proteins PCH1 and PCHL and their influence on PHYB dark reversion |
| title_auth | Characterization of the proteins PCH1 and PCHL and their influence on PHYB dark reversion |
| title_exact_search | Characterization of the proteins PCH1 and PCHL and their influence on PHYB dark reversion |
| title_full | Characterization of the proteins PCH1 and PCHL and their influence on PHYB dark reversion Autor: Beatrix Enderle |
| title_fullStr | Characterization of the proteins PCH1 and PCHL and their influence on PHYB dark reversion Autor: Beatrix Enderle |
| title_full_unstemmed | Characterization of the proteins PCH1 and PCHL and their influence on PHYB dark reversion Autor: Beatrix Enderle |
| title_short | Characterization of the proteins PCH1 and PCHL and their influence on PHYB dark reversion |
| title_sort | characterization of the proteins pch1 and pchl and their influence on phyb dark reversion |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1170198139/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031062955&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT enderlebeatrix characterizationoftheproteinspch1andpchlandtheirinfluenceonphybdarkreversion |