New signalling network in plant abiotic stress discovered through a genetic approach [Elektronische Ressource] / vorgelegt von Andrés Peñalosa Barbero

Zellbio

Sujets

• Biologie

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 24
• Poids de l'ouvrage: 7 Mo

Extrait

New signalling network in plant abiotic stress
discovered through a genetic approach



Inauguraldissertation zur Erlangung der Doktorwürde der Fakultät
für Biologie der Albert-Ludwig-Universität Freiburg im Breisgau


vorgelegt von

Andrés Peñalosa Barbero

aus
Madrid, Spanien

Freiburg im Breisgau, Mai 2004
Dekan: Prof. Dr. G. Fuchs

Promotionsvorsitzender: Prof. Dr. K. F. Fischbach

Leiter der Arbeit: Prof. Dr. G. Neuhaus



Referent: Prof. Dr. G. Neuhaus
Koreferent: Prof. Dr. J. Weckesser
3. Prüfer: Prof. Dr. E. Wellmann
Tag der Verkündigung des Prüfungsergebnisses: 21/06/2004 Publication related to this work:


Medina J, Rodríguez-Franco M, Peñalosa A, Carrascosa MJ,
Neuhaus G, Salinas J. (2004) Arabidopsis mutants deregulated in
RCI2A expression reveal new signalling pathways in abiotic stress
responses. Submitted to Plant Journal.










A mi abuelo Eduardo
To my grandfather Eduardo









“ La ciencia es una estrategia, es una forma de amar la verdad, es algo más
que materia, pues el misterio se oculta detrás” (L.E. Aute)
“Science is a strategy, is a way to love the truth, is something else than
matter, because the mystery hides behind” (L.E. Aute) TABLE OF CONTENTS


1. INTRODUCTION ......................................................................................... 1

1.1 ABIOTIC STRESS................................................................................................ 1
1.3 DEHYDRATION STRESS.................................................................................. 6
1.4 SALT STRESS....................................................................................................... 9
1.5 ROLE OF ABA IN ABIOTIC STRESS............................................................. 11
1.6 HORMONES INTERACTIONS ...................................................................... 14
1.7 EFFECTS OF SUGARS ..................................................................................... 16
1.8 RARE COLD INDUCIBLE GENES ................................................................ 18
1.9 GENETIC APPROACH TO DISSECT ABIOTIC STRESS SIGNALLING
PATHWAYS ............................................................................................................ 19
1.10 AIM OF THE WORK...................................................................................... 22

2. MATERIALS AND METHODS ........................................................ 23

2.1 MATERIALS ..................................................................................................... 23
2.1.1 Chemicals ........................................................................................................ 23
2.1.2 Instrumentation.............................................................................................. 23
2.1.3 Enzymes and molecular biology kits .......................................................... 24
2.1.4 General buffers and solutions ...................................................................... 24
2.1.5 Hybridisation probes..................................................................................... 25
2.1.6 Molecular markers ......................................................................................... 26
2.1.7 Informatic tools 28
2.1.8 Culture media................................................................................................ 28
2.1.9 Plant material.................................................................................................. 29
2.2 METHODS......................................................................................................... 30
2.2.1 Molecular biology methods.......................................................................... 30
2.2.1.1 Isolation and purification of plasmid DNA from E. coli ................... 30
2.2.1.2 Amplification of plasmid DNA fragments by PCR ........................... 31
2.2.1.3 Electrophoresis, isolation, and extraction of DNA fragments.......... 31
2.2.1.4 Plant genomic DNA isolation ............................................................... 31
2.2.1.5 Mapping ................................................................................................... 32
2.2.1.6 Northern Blot analysis............................................................................ 33
2.2.2 Plant methods................................................................................................. 35
2.2.2.1 Arabidopsis in vitro culture...................................................................... 35
2.2.2.2 Arabidopsis growth conditions on soil .................................................. 35
2.2.2.3 Stress treatments of seedlings ............................................................... 35
2.2.2.4 Luciferase measurements 36
2.2.2.5 Stress treatments of plants for Northern analysis.............................. 37
2.2.2.6 Phenotypic analysis ................................................................................ 38
2.2.2.7 Cross-fertilization of Arabidopsis plants............................................... 41 2.2.2.8 Segregation analysis ............................................................................... 42
2.2.2.9 Mapping mutations using molecular markers ................................... 42

3. RESULTS........................................................................................................ 43

3.1 ISOLATION OF DEREGULATED MUTANTS IN A.THALIANA STRESS
SIGNALLING PATHWAYS...................................................................................... 43
3.1.1 Primary screening .......................................................................................... 43
3.1.2 Screening of the M3 and following generations......................................... 44
3.2 CHARACTERISATION OF THE LUCIFERASE EXPRESSION IN THE
SELECTED MUTANT LINES ................................................................................... 45
3.3 EXPRESSION ANALYSIS OF ENDOGENOUS RCI2A AND OTHER
STRESS-INDUCIBLE GENES BY NORTHERN BLOTTING ............................... 50
3.4 SEGREGATION ANALYSIS OF THE MUTATED LINES ............................ 56
3.5 STRESS TOLERANCE PHENOTYPIC CHARACTERISATION ................... 58
3.5.1 Phenotypes after freezing temperatures..................................................... 58
3.5.2 Phenotypes after ABA treatments ............................................................... 59
3.5.3 Phenotypes after salt stress........................................................................... 64
3.5.4 Phenotypes after ethylene treatment .......................................................... 68
3.5.5 Flowering phenotypes................................................................................... 70
3.5.6 Additional phenotypes of mutant lor19 ..................................................... 71
3. 6. GENETIC MAPPING OF THE MUTATIONS................................................ 82
3.6.1 Selection of mapping populations............................................................... 82
3.6.2 PCR-based molecular markers analysis 85
3.6.3 Mapping locations ......................................................................................... 86

4. DISCUSSION............................................................................................. 92

5. SUMMARY................................................................................................ 110

6. REFERENCES ......................................................................................... 111
Introduction 1

1. INTRODUCTION
1.1 ABIOTIC STRESS

During a typical life cycle, plants are exposed to a wide range of environmental
changes that may disturb the normal growth and development they accomplish in
optimal growth conditions.
Evolution has led to the development of mechanisms aimed to increase their tolerance
to these negative factors, including both physical adaptations and several complex
mechanisms of interactive cellular and molecular changes triggered after the onset of
various stresses.
In general terms, the first step for this process consists of the perception of the adverse
situation. Then, through a release or activation of second messengers, different
signalling cascades are set in motion in order to relay the information. Transcription
effectors can be induced and activated in that way, bringing about the expression of
specific stress-responsive genes, which encode proteins involved in the protection of
the plant cell against the effects of the damaging situation and/or the repair of the
injuries already caused.
The current approaches to elucidate the molecular mechanisms modulating the stress
signalling networks are based on the control of the expression of specific stress-related
genes. In order to easily interpret the