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

De
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...........................................................................................
Publié le : jeudi 1 janvier 2004
Lecture(s) : 24
Tags :
Source : FREIDOK.UB.UNI-FREIBURG.DE/FREIDOK/VOLLTEXTE/2004/1358/PDF/PHDANDRESPENALOSA.PDF
Nombre de pages : 131
Voir plus Voir moins






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 analysis of stress signalling pathways in the
laboratory, plants need to be isolated from other stresses. However, the studies carried
out to date have already demonstrated a high complexity of interactions occurring
between different abiotic stress factors.
It is therefore likely that one single stress factor triggers the activation of several
signalling pathways (divergence). In contrast, different stimuli might provoke a
similar cellular effect, thus certain shared signalling mechanisms, and the subsequent
cell responses can be active under different stress conditions (convergence). Introduction 2
1.2 LOW TEMPERATURE STRESS

Membrane systems are primary sites of cell injury under freezing temperatures (Levitt,
1980; Steponkus, 1984). Under these conditions, the formation of ice crystals primarily
in the extracellular spaces, due to the low concentration of solute in this compartment,
results in a drop of water potential outside the cell. This phenomenon provokes the
water outflow from inside the cell, and the subsequent dehydration. Stabilisation of
the membranes is a key factor in the resistance of the freezing situation. Changes in
lipid composition of the membrane (Steponkus et al., 1993; Uemura et al., 1997), the
accumulation of sugars (Anchordoguy et al., 1987; Strauss et al., 1986) and other
osmoprotectants such as proline (Nanjo et al., 1999), as well as the expression of some
stress-inducible genes encoding proteins with protective functions seem to be related
to this effect.
In addition, secondary lesions may be provoked by the freezing-induced production of
reactive oxygen species (ROS) (McKersie and Bowley, 1997), by the adhesion of
intracellular ice crystals causing cell rupture (Olien and Smith, 1977), and by the
eventual protein denaturation (Guy and Li, 1998) occurred under these conditions.
In Arabidopsis, no cold sensor has been described to date, and the start of the signal
transduction has been hypothesized to be closely related with the alterations in the
fluidity of the plasma membrane probably affecting some calcium channels that would
switch on the signal (Orvar et al., 2000; Zhu, 2001a). Calcium acts in plant cells as a
second messenger during the abiotic stress signalling (Sanders et al., 1999; Knight et al.,
2+2000). Characteristic cytosolic oscillations (Ca signatures) can be detected upon
application of different stimuli, and they have been shown to be specific of the stress
applied (Plieth et al., 1999). Furthermore, different calcium signatures are obtained in
various tissues, during developmental stages (Kiegle et al., 2000), or if the plant is
previously exposed to certain stress conditions (Knight et al., 1997).
Studies carried out in other species such as alfalfa and Brassica napus, revealed that the
cold-dependent calcium oscillations require changes in the fluidity of the membrane
and reorganisation of the cytoskeleton (Orvar et al., 2000; Sangwan et al., 2001). In
Arabidopsis, the locus FRY1 (FIERY1) encodes an inositol polyphosphatase 1-
phosphatase, which catabolizes IP. A mutation in this gene, fry1, yields 3Introduction 3
hypersensitivity to cold, ABA and salt stresses. Probably, sustained IP levels in this 3
mutant disturb the specific calcium signatures under these stress situations (Xiong et
al., 2001a).
The transduction of the signal is proposed to be mediated by different calcium sensing
protein kinases and protein phosphatases belonging to families such as CDPKs
(Calcium-Dependent Protein Kinases) or complexes formed by CBL (Calcineurin B-
Like) and CIPK (CBL-Interacting Protein Kinases). Additionally, many MAPK
(Mitogen-Activated Protein Kinases) have been cloned and proposed to be involved in
triggering regulatory cascades after environmental stress (Mizoguchi et al., 1998).
Arabidopsis, as many species from temperate regions, has the ability to increase its
freezing tolerance through a pre-exposure to low non-freezing temperatures, by a
process known as cold-acclimation. A variety of mutants have been isolated based on
their differences in the capacity to withstand freezing temperatures after an
acclimation period. Several genes have been shown to be up regulated under cold
stress (for review, see Thomashow, 1999) and many of them are involved in the cold
acclimation process.
Three members of the CBF (C-repeated Binding Factor)/DREB1 (Drought Responsive
Element Binding 1) subfamily of transcription factors (CBF1/DREB1B, CBF2/DREB1C
and CBF3/DREB1A) belonging to the APETALA2 (AP2)/ethylene-responsive-element-
binding protein (EREBP) superfamily have been cloned (Stockinger et al., 1997;
Shinwari et al., 1998) and revealed to be induced by low temperatures, but not by
ABA, dehydration, or salt stresses. These CBF proteins have defined a regulatory
module, in which they control the expression of the majority of the cold-responsive
genes through binding to the cis-element termed C-repeated/DRE present in their
promoter regions (Stockinger et al., 1997; Gilmour et al., 1998).
Although none of them contains in its promoter region DRE elements, they are known
to be autorregulated by their own expression or the downstream target gene products
(Guo et al., 2002). An activator for the CBF3 gene expression has recently been cloned;
the ICE1 (inducer of CBF expression 1) gene encodes a MYC-like/bHLH transcription
factor (Chinnusamy, 2003) that is expressed at basal levels under normal conditions
but it is super-induced by low temperatures, ABA and salt stresses, but not by
dehydration. A hypothetical intermediate is proposed to activate ICE1 under these

Soyez le premier à déposer un commentaire !

17/1000 caractères maximum.