Functional analysis of PIP2 aquaporins in Arabidopsis thaliana [Elektronische Ressource] / Olivier Da Ines

Olivier Da Ines

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der LudwigMaximiliansUniversität München









Functional analysis of PIP2 aquaporins in Arabidopsis thaliana











Olivier Da Ines


aus


Mâcon, France









2008




Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 von PD Dr. Anton R. Schäffner betreut.


Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.



München, am 11.03.2008








Olivier Da Ines









Dissertation eingereicht am 11.03.2008

1. Gutacher PD Dr. Anton Schäffner

2. Gutachter Prof. Karl2Peter Hopfner

Mündliche Prüfung am 08.05.2008



3
ABSTRACT
Plants harbor about 30 genes encoding major intrinsic proteins (MIP) which were named
aquaporins because of the frequently observed water channel activity and their putative
involvement in water relations. Plasma membrane intrinsic proteins (PIP) are subdivided into
two groups with five PIP1 and eight PIP2 members in the model plant Arabidopsis thaliana.
PIP2 genes are highly homologous to each other, which might indicate a redundant function.
However, several lines of evidence argue for gene2specific and non2redundant functions. PIP2
genes exhibit overlapping, but differential expression patterns when assessed by
promoter::GUS transgenic lines. Most PIP2 genes were expressed in the region of the
vascular tissue, however with differential cellular patterns. PIP2;5 and PIP2;7 were more
uniformly found in leaves. PIP2;4 was the only root2specific member and preferentialy
expressed in outer cell layers, whereas PIP2;6 and PIP2;8 were mostly found in young leaves
with a distinct expression. PCA analyses based on publicly available stress2responsive
expression patterns revealed distinct transcriptional responsiveness of PIP2 genes. No visible
phenotypes were observed for pip2 knock2out lines under normal growth conditions.
However, pip2;3 mutants specifically showed salt2sensitivity, although the duplicated gene
PIP2;2 has only eight amino acids different from PIP2;3 that mostly cluster in the region of
the extracellular loop C. Transcriptional analysis using a custom2made DNA array focusing
on membrane proteins indicated that loss2of2function of distinct PIP2 genes did not interfere
with major transport processes, at least at the transcriptional level. For several mutants of
abundantly expressed PIP2 isoforms, root transcriptome analysis was extended to the whole
genome using Affymetrix ATH1 GeneChip. Furthermore, PIP2 co2expression analyses
revealed enrichment for different functional categories. Only PIP2;1 and PIP2;2 showed
significant correlations with other PIP genes, however most PIP2 genes were also correlated
to TIP isoforms, suggesting an functional relationship of plasma membrane and tonoplast
permeabilities. Surprisingly, at the protein level, the loss of PIP2;1 or PIP2;2 specifically
provoked a decrease in PIP1 proteins indicating a dependence of PIP1 stability on these PIP2
members.
So far, direct evidence for a participation of single PIP aquaporins in water transport in planta
is scarce. To study non2invasively the impact of PIP2 on water uptake, the kinetics of water
translocation was analyzed by mass spectrometry of water extracted from in pip2;1 and
pip2;2 mutants grown in D2enriched medium. Retarded deuterium transfer into leaves of the
single mutants indicated a direct involvement of both PIP proteins in water relations, whereas
surprisingly the corresponding double mutant had compensated this slower uptake. Contents 4
C O 1 T E 1 T S
ABSTRACT .............................................................................................................................. 3
ABBREVIATIO1S ..................................... ............................................................................. 7
FIGURES A1D TABLES ................................ ........................................................................ 8
1 I1TRODUCTIO1 ...................................... ........................................................ 10
1.1 Water uptake and transport across plant tissues ............................................ 10
1.2 Aquaporin water channels ................................................................................. 14
1.2.1 History and discovery of aquaporins .................................................................... 14
1.2.2 Classification of the plant MIP superfamily of proteins ...................................... 15
1.2.3 Structural features of aquaporins and transport selectivity .................................. 16
1.2.3.1 Common structural features of aquaporins .......................................................... 16
1.2.3.2 Water transport selectivity ................................................................................... 18
1.2.3.3 Additional transport specificities ......................................................................... 19
1.2.4 Plant aquaporins expression and localization ....................................................... 21
1.2.5 Regulation of plant aquaporins ............................................................................ 24
1.2.5.1 Phosphorylation .................................................................................................... 24
2+1.2.5.2 Regulation by pH and Ca .................................................................................. 25
1.2.5.3 Aquaporin interaction and trafficking .................................................................. 26
1.2.6 Integrated function of plant aquaporins inferred from transgenic plants ............. 28
1.3 Goals of the project ............................................................................................ 31
2 MATERIALS A1D METHODS.............................. ......................................... 32
2.1 Materials ............................................................................................................. 32
2.1.1 Plant materials ...................................................................................................... 32
2.1.2 Vectors and bacteria ............................................................................................. 33
2.1.3 Antibiotics ............................................................................................................ 34
2.1.4 Restriction enzymes and Modifying enzymes ..................................................... 34
2.1.5 Antibodies ............................................................................................................ 34
2.1.6 Isotopically labeled compounds ........................................................................... 35
2.1.7 Oligonucleotides and sequencing ......................................................................... 35
2.1.8 Chemicals ............................................................................................................. 35
2.1.9 Medium and solutions .......................................................................................... 35
2.1.10 Apparatus ............................................................................................................. 36
2.2 Methods ............................................................................................................... 36
2.2.1 Culture of Arabidopsis thaliana plants ................................................................ 36
2.2.1.1 Growth conditions ................................................................................................ 36
2.2.1.2 Growth in soil, crossing, seeds harvesting and storage ........................................ 36
2.2.1.3 Seed sterilization .................................................................................................. 37
2.2.1.4 In vitro culture on solid medium .......................................................................... 37
2.2.1.5 Hydroponic culture ............................................................................................... 38
2.2.1.6 Growth measurements on in vitro culture ............................................................ 39
2.2.1.7 Leaf water loss measurement using detached2rosette assay ................................. 39
2.2.1.8 Root bending assay (gravitropism response) ....................................................... 40
2.2.1.9 Analysis of deuterium (D) translocation in plant ................................................. 40
2.2.2 Microbiological methods ...................................................................................... 42 Contents 5
2.2.2.1 Preparation of competent cells ............................................................................. 42
2.2.2.2 T