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

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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.

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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 Transformation of competent cells ....................................................................... 43
2.2.2.3 Transformation of Agrobacterium tumefaciens ................................................... 43
2.2.2.4 Agrobacterium tumefaciens2mediated 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 RT2PCR .................................................................... 46
2.2.3.5 Isolation of total RNA using Qiagen RNeasy Plant Mini Kit .............................. 47
2.2.3.6 Determination of nucleic acids concentration ...................................................... 47
2.2.4 Molecular biology methods .................................................................................. 47
2.2.4.1 PCR (Polymerase Chain Reaction) ...................................................................... 47
2.2.4.2 Reverse Transcription Polymerase Chain Reaction (RT2PCR) ........................... 48
2.2.4.3 Purification of PCR product and DNA gel extraction .......................................... 49
2.2.4.4 Digestion by restriction endonucleases ................................................................ 49
2.2.4.5 Separation and visualization of nucleic acids on agarose gel electrophoresis ..... 49
2.2.4.6 Molecular cloning using the Gateway recombination technology ....................... 50
2.2.4.7 DNA sequencing .................................................................................................. 52
2.2.4.8 Southern blotting .................................................................................................. 52
2.2.5 Isolation and molecular characterization of pip2 T2DNA insertional mutants .... 54
2.2.6 Protein methods .................................................................................................... 55
2.2.6.1 Preparation of microsomal fractions from Arabidopsis thaliana tissues ............. 55
2.2.6.2 Protein isolation from E. coli ............................................................................... 56
2.2.6.3 Purification of GST2fusion protein ..................................................................... 56
2.2.6.6 ESEN test for determination of protein concentration ......................................... 57
2.2.6.7 SDS polyacrylamide Gel Electrophoresis (SDS2PAGE) .................................... 58
2.2.6.8 Staining of SDS2PAG with Coomassie Brilliant blue ......................................... 60
2.2.6.9 Western blot ......................................................................................................... 60
2.2.7 GUS staining of Arabidopsis thaliana plants ....................................................... 62
2.2.8 Custom2made DNA array harboring transport2related genes .............................. 63
2.2.8.1 Construction of target DNA sequences and array production. ............................. 63
2.2.8.2 Hybridization with T7 reference probes ............................................................... 63
2.2.8.3 Isolation of mRNA using Oligo(dT)25 Dynabeads and preparation of complex
probes ................................................................................................................... 65
2.2.8.4 Hybridization with complex probes and data acquisition .................................... 67
2.2.8.5 Microarray buffers ................................................................................................ 67
2.2.8.6 Data evaluation and analysis ................................................................................ 69
2.2.9 Genome2wide microarray analyses using Affymetrix ATH1 GeneChip ............. 70
2.2.9.1 RNA preparation and Affymetrix GeneChip hybridization ................................. 70
2.2.9.2 Normalization, statistical processing and data analysis ....................................... 70
2.2.10 Co2expression analyses ....................................................................................... 72
2.2.11 Principal component analysis (PCA) of PIP transcriptional responses to diverse
stimuli using publicly available microarray data ................................................. 73
2.2.12 Microscopy ........................................................................................................... 74
2.2.12 Internet addresses ................................................................................................. 74
3 RESULTS ............................................................................................................ 76
3.1 PIP2 expression in Arabidopsis plants ............................................................. 76
3.1.1 Analysis of PIP2 promoter::GUS expression profiles in Arabidopsis plants ...... 76 Contents 6
3.1.2 PIP gene expression profile based on microarray data ........................................ 80
3.1.3 PIP2;4 cellular and subcellular localization ......................................................... 82
3.2 Isolation and molecular characterization of pip2 insertional mutants .......... 85
3.2.1 Isolation of pip2 single and double mutants ......................................................... 85
3.2.2 Molecular characterization of pip2 insertional mutants ....................................... 86
3.3 Phenotypical analysis of pip2 knockout mutants ............................................. 91
3.3.1 Growth responses of pip2 mutants in standard and water stress conditions ........ 91
3.3.2 Morphological analyses of pip2 mutants ............................................................. 97
3.3.3 Leaf water relations .............................................................................................. 98
3.3.3.1 Loss of water from detached rosettes ................................................................... 98
3.3.3.2 Transpiration, stomatal conductance, net photosynthesis and water use efficiency
measurements ..................................................................................................... 101
3.3.4 Isotope tracing in plant water relations – deuterium content in leaf water ........ 103
3.3.4.1 Theory: Modeling leaf water isotope content .................................................... 103
3.3.4.2 Evaluation of the leaf water isotope model ........................................................ 108
3.3.4.3 Comparison of deuterium content in rosette leaves water of wild2type and pip2
mutants ............................................................................................................... 114
3.3.5 Root gravitropism of pip2 mutants..................................................................... 116
3.4 Transcriptional analyses of pip2 knockout mutants ..................................... 118
3.4.1 Custom2made array covering target genes related to membrane transport ........ 118
3.4.2 Whole genome transcriptional analysis using Affymetrix ATH1 GeneChip .... 121
3.5 PIP2s coexpression and stress responsiveness analyses using publicly
available microarray data ............................................................................... 125
3.5.1 Co2expression analyses revealed transcriptional interrelation of PIP2 genes with
MIP members and other processes. .................................................................... 126
3.5.2 Principal component analysis of PIP2 transcriptional responses to various stimuli
revealed differential reactions. ........................................................................... 133
3.6 Interaction between PIP2 and PIP1 isoforms ................................................ 137
3.6.1 Specificity of antisera against AtPIP1 and AtPIP2 ............................................. 139
3.3.1 Expression of PIP2 and PIP1 proteins in pip2 knockout mutants ...................... 141
4 DISCUSSIO1 ........................................ ........................................................... 145
4.1 Distinct PIP2 localizations allow for differential roles in water relation .......... 145
4.2 Establishment of a method for assessing in vivo plant water transport ............. 147
4.3 Non2invasive analyses of the impact of PIP2 on plant water translocation using
pip2 knockout mutants ....................................................................................... 149
4.4 PIP2s differentially respond to stress ................................................................. 151
4.5 PIP2;3 specifically required for NaCl resistance ............................................... 152
4.6 PIP2 genes are embedded in different functional context ................................. 154
4.7 Differential PIP22MIP interactions ................................................................... 155
4.8 Conclusion .......................................................................................................... 157
5 REFERE1CES ........................................ ......................................................... 160
6 SUPPLEME1TARY MATERIAL A1D A11EXES ................ ................... 174

ACK1OWLEDGEME1TS .................................. ............................................................... 249

CURRICULUM VITAE .............................................................................................. 251 Abbreviations 7
ABBREVIATIO1S

ABA Abscisic Acid
ABC ATP2binding cassette
BSA Bovine Serum Albumin
CTAB Cetyltrimethylammoniumbromide
d day
ddH O double distilled water 2
DEPC Diethylpyrocarbonate
DMSO Dimethyl sulfoxide
dNTPs Deoxynucleotide25’2triphosphates
DTT Dithiothreitol
EDTA Ethylene Diamine Tetra2acetic Acid
GFP Green Fluorescent Protein
GUS β2Glucuronidase
GST Glutathione2S2transferase
kb Kilo base pair
kDa KiloDalton
MES 22(N2Morpholino)2ethanesulfonic acid
MIP Major Intrinsic Protein
MIPS Munich information center for protein sequences (Helmholtz
Zentrum München)
MS Murashige and Skoog
NASC Nottingham Arabidopsis Stock Center
NIP NOD262like Intrinsic Protein
PCR Polymerase chain reaction
PIP Plasma membrane Intrinsic Protein
PM Plasma membrane
RNA Ribonucleic Acid
rpm rotations per minute
RT2PCR Reverse Transcription2PCR
SD Standard Deviation
SDS Sodium Dodecyl Sulfate
SIP Small and basic Intrinsic Protein
TAE Tris2Acetate2EDTA
TE Tris2EDTA
TEMED N,N,N’,N’2 Tetramethylethylenediamine
TIP Tonoplast Intrinsic Protein
T2DNA Transfer DNA
UTR untranslated region
v/v volume per volume
Vol Volume
w/v weight per volume
wt wild2type
X2Gluc 52Bromo242chloro232indolyl2β2D2glucuronicid a c
Figures and Tables 8
FIGURES A1D TABLES

FIGURES

Figure 1. Schematic representation of the three different pathways involved in radial water
transport across plant living tissues. ..................................................................... 10
Figure 2. Schematic representation of whole plant water transport within and between
tissues. .................................................................................................................. 13
Figure 3. Phylogenetic tree of the 35 MIP proteins in Arabidopsis thaliana and their
grouping in four subfamilies. ............................................................................... 16
Figure 4. Hourglass model of AQP showing its membrane topology. ................................ 17
Figure 5. Schematic representation of PIP2 promoter::GUS constructs using the Gateway
technology. ........................................................................................................... 51
Figure 6. DNA array spotting scheme. ................................................................................ 64
Figure 7. Scatter plot of hybridizations of the different wild type biological replicates
obtained from two different Affymetrix laboratory facilities. ............................. 71
Figure 8. Histochemical localization of PIP2::GUS activity in vegetative tissues.............. 77
Figure 9. GUS expression in different cell types. ................................................................ 79
Figure 10. Hierarchical clustering of the 13 Arabidopsis PIP isoforms (Genevestigator, May
2007)..................................................................................................................... 81
Figure 11. Cellular localization of PIP2 fused to GFP in Arabidopsis root seedlings. ......... 82
Figure 12. Plasma membrane localization of PIP2;4............................................................. 83
Figure 13. Screening of pip2 T2DNA insertion mutants. ..................................................... 86
Figure 14. Agarose gel separation of PCR products amplified from cDNA of wild2type and
pip2 mutant lines. ................................................................................................. 87
Figure 15. Southern blot strategy for analyzing T2DNA insertion in pip2 mutant genomes. 88
Figure 16. DNA blots of pip2 knockout mutants. ................................................................. 89
Figure 17. Collection of pip2 insertional mutants in Arabidopis thaliana. ........................... 90
Figure 18. Four2day2old seedlings of wt andp ip2 mutant..................................................... 92
Figure 19. Relative root growth of Arabidopsis wild2type and pip2 mutants under various
water stresses. ....................................................................................................... 93
Figure 20. Growth of pip2 single mutants under various water stresses show differential
sensitivity. ............................................................................................................ 95
Figure 21. Growth of pip2;3 mutants is sensitive to salt stress. ............................................ 96
Figure 22. Stereomicroscopy images of WT and pip2 mutant root seedlings. ...................... 97
Figure 23. Rosette leaves fresh weight. ................................................................................. 98
Figure 24. Water loss using detached rosette assay. ............................................................ 100
Figure 25. Modeling of isotopic content in leaf water of hydroponically grown Arabidopsis
plants. ................................................................................................................. 110
Figure 26. Relationship between the δD of modeled and measured leaf water in Arabidopsis
plants. ................................................................................................................. 111
Figure 27. Modeling of isotopic content in leaf water of hydroponically grown Arabidopsis
plants. ................................................................................................................. 112
Figure 28. Influence of leaf water volume or root fresh weight on the isotopic content of
Arabidopsis rosette leaves water. ....................................................................... 113
Figure 29. Comparison of deuterium translocation into rosette leaves water of wild2type and
pip2 mutants. ...................................................................................................... 114
Figure 30. pip2 single mutants exhibit a reduced leaf water translocation. ........................ 115
Figure 31. Gravitropism directs downward root growth in Arabidopsis thaliana. ............. 117
Figure 32. Venn diagram of genes deregulated in various pip2 knockout mutants. ........... 124 Figures and Tables 9
Figure 33. Co2correlation scatter plot of pairs of genes with different r2values.................. 127
Figure 34. Venn diagram showing overlap for the positively correlated genes with the
individual PIP2. ................................................................................................. 131
Figure 35. Principal component analyses of PIP transcriptional responses to diverse stimuli.
............................................................................................................................ 134
Figure 36. Sequence comparison of loop E of ZmPIP and AtPIP. ....................................... 138
Figure 37. Sequence alignment of AtPIP1 and AtPIP2 with the corresponding epitope used
to raise anti2PIP1;1 or anti2PIP2;2 antisera. ....................................................... 139
Figure 38. Identification of GST2PIP fusion proteins. ........................................................ 140
Figure 39. Specificity of anti2PIP1;1 and anti2PIP2;2 antisera............................................ 141
Figure 40. Anti2PIP1;1 and anti2PIP2;2 antisera specifically recognized PIP proteins. ..... 141
Figure 41. PIP protein expression level in pip2 knockout mutants in roots. ....................... 143
Figure 42. PIP protein expression level in pip2 knockout mutants in leaves. ..................... 144
Figure 43. Structural models of PIP2;2 and PIP2;3 showing their different amino acids. .. 154


TABLES

Table 1. AtPIP2 insertional mutants................................................................................... 33
Table 2. Antibiotic stock and working solutions. Stock solutions were stored at 220°C. .. 34
Table 3. Apparatus and equipment ..................................................................................... 36
Table 4. Hydroponic medium composition (Gibeaut et al., 1997) ..................................... 39
Table 5. T2DNA or transposon flanking restriction fragments of pip2 mutants. ............... 89
Table 6. Transpiration, stomatal conductance, net photosynthesis and water use efficiency
measurements. .................................................................................................... 102
Table 7. Isotopic composition of leaf water (δD, ‰) of hydroponically grown Arabidopis
thaliana plants. ................................................................................................... 109
Table 8. MIPs expression fold2change in roots of various pip2 insertional mutants. ...... 120
Table 9. Classification of deregulated genes obtained by Affymetrix ATH1 expression
analyses of pip2 mutants. ................................................................................... 123
Table 10. Functional categories assignment of PIP2 correlated genes. ............................. 129
Table 11. Twenty most highly co2expressed genes with PIP2;6 among a dataset
(AtGenExpress biotic stress dataset) covering pathogen responses in shoot. .... 130
Table 12. Correlation of PIP2 members with PIP and TIP genes according to ACT co2
expression analyses. ........................................................................................... 132
Table 13. Arabidopsis PIP2 isoform classification. ........................................................... 159 Introduction 10
1 I1TRODUCTIO1
1.1 Water uptake and transport across plant tissues
Water, often regarded as the “solvent of life”, is the single most abundant molecules in all
cells and organisms and the maintenance of water status in suitable range is fundamental to
life. In addition to the use of water in biochemical reactions and as a solvent, plants maintain
their cellular turgor via sufficient water uptake. During development a plant can absorb water
up to 500 times its fresh weight. However, only 2 to 5% of the water absorbed is retained by
tissues for their growth, the remaining water is being lost by the leaves through
evapotranspiration (Passioura, 1980). The passage of water through the whole plant is usually
starting from the roots, the site of absorption, up to the aerial parts.
Water is first entering the roots due to water potential difference with the source water (e.g.
soil, hydroponic medium) which is encompassing osmotic potential, pressure potential as well
as gravity. The root is the fundamental organ for uptake and root water uptake in plants is
related to processes such as transpiration and stomatal conductance, leaf water relations, leaf
growth, and photosynthesis.
In the first place, to move into the roots, water has to flow radially across several cell layers
(epidermis, cortex, endodermis and pericycle) to finally reach the vascular tissues
constituting the xylem for long2distance transport (Fig. 1).

VacuolarCell
membrane wallPlasma
Plasmodesmatamembrane

AAppooppllaassttiicc ppaatthhwwaayy


Transcelullar
pathway

SSyymmppllaassttiicc
ppaatthhwwaayy
AAxxiiaall
flow

Epidermis cortex endodermis xylem

Figure 1. Schematic representation of the three different pathways involved in radial water transport
across plant living tissues.

Three paths, for which relative contributions can vary depending upon growth conditions and
species, co2exist for water transport across living tissues in plants (Fig. 1). The apoplastic
path, i.e. within the extracellular cell wall space, is complemented by a cell2to2cell path,