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Functional Analysis of PIP2 Aquaporins in Arabidopsis Thaliana

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Functional Analysis of PIP2 Aquaporins in Arabidopsis Thaliana Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität 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. Karl-Peter 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 gene-specific and non-redundant 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 root-specific member and preferentially 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 stress-responsive expression patterns revealed distinct transcriptional responsiveness of PIP2 genes. No visible phenotypes were observed for pip2 knock-out lines under normal growth conditions. However, pip2;3 mutants specifically showed salt-sensitivity, 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 custom-made DNA array focusing on membrane proteins indicated that loss-of-function 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 co-expression 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 non-invasively 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 D-enriched 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 T E T S ABSTRACT .............................................................................................................................. 3 ABBREVIATIOS .................................................................................................................. 7 FIGURES AD TABLES ........................................................................................................ 8 1 ITRODUCTIO .............................................................................................. 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 AD 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 detached-rosette 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 Transformation of competent cells ....................................................................... 43 2.2.2.3 Transformation of Agrobacterium tumefaciens ................................................... 43 2.2.2.4 Agrobacterium tumefaciens -mediated plant transformation ................................ 44 2.2.3 Nucleic acid isolation ........................................................................................... 45 2.2.3.1 CTAB DNA Minipreparation from plant tissue ................................................... 45 2.2.3.2 Genomic DNA preparation for Southern blotting using DNeasy Plant Mini Kit 45 2.2.3.3 Plasmid DNA preparation .................................................................................... 45 2.2.3.4 Isolation of total RNA for RT-PCR ....................................................................
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