Functional analysis of Nt-Hypo1 and NtPLC3, two novel pollen-specific proteins from Nicotiana tabacum [Elektronische Ressource] / presented by Diana Helling

Dhelling

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2005
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences


















presented by
Diana Helling
born in: Bielefeld, Germany
thOral-examination: December 7 , 2005






Functional Analysis of Nt-Hypo1 and NtPLC3,
Two Novel Pollen-Specific Proteins
From Nicotiana tabacum





















Referees: Prof. Dr. Thomas Rausch
Prof. Dr. David G. Robinson
CONTENTS
Contents.................................................................................................................................I
Summary .......................................................................................................................... VII
Zusammenfassung .............................................................................................................IX
1 Introduction ................................................................................................................. 1
1.1 Pollen tube growth: a model system for polarized plant cell growth .................... 1
1.1.1 Ion gradients and fluxes involved in polarized pollen tube growth .............. 3
1.1.1.1 Calcium...................................................................................................... 3
1.1.1.2 Protons....................................................................................................... 4
1.1.1.3 Potassium and chloride.............................................................................. 4
1.1.2 Cytoskeleton and polarized pollen tube growth ............................................ 4
1.1.2.1 Microfilaments........................................................................................... 4
1.1.2.2 Microtubules.............................................................................................. 5
1.2 The role of small GTPases in the control of pollen tube tip growth ..................... 6
1.2.1 The small GTPase superfamily Ras............................................................... 6
1.2.2 The regulation of Rho GTPases..................................................................... 7
1.2.2.1 Rho GTPases and their regulation in plants .............................................. 8
1.2.2.2 The role of Rho GTPases in the regulation of pollen tube growth............ 9
1.2.3 Guanine nucleotide exchange factors.......................................................... 11
1.2.3.1 ange factors in plants ....................................... 13
1.2.3.1.1 Plant-specific Rho GEFs do exist in plants 15
st1.2.4 Aims of the dissertation (1 project) ........................................................... 16
1.3 Phospholipid signaling in pollen tubes................................................................ 17
1.3.1 Phosphoinositides ........................................................................................ 17
1.3.2 Phosphatidylinositol 4,5-bisphosphate synthesis and functions.................. 18
1.3.3 Phospoinositide-specific phospholipases C................................................. 20
1.3.3.1 General overview..................................................................................... 20
1.3.3.2 Molecular domain structure..................................................................... 21
1.3.3.3 Membrane binding................................................................................... 22
1.3.3.4 Phosphoinositide hydrolysis.................................................................... 22
1.3.3.5 Cloning and cellular functions of PI-PLCs in plants............................... 23
1.3.3.5.1 PI-PLCs and their role in pollen tube growth.................................... 25
nd1.3.4 Aims of the dissertation (2 project) .......................................................... 27
2 Results......................................................................................................................... 28
ICONTENTS
2.1 Identification and functional characterization of a new interactor of pollen tube Nt-
Rac5 ......................................................................................................................... 28
2.1.1 Sequence analysis of Nt-Hypo1 and comparison with Arabidopsis thaliana
and Oryza sativa proteins of unknown function .......................................... 31
2.1.2 Analysis of Nt-Hypo1 expression by Northern blotting.............................. 32
2.1.3 Effects of transient expression of Nt-Hypo1 in pollen tubes of Nicotiana
tabacum........................................................................................................ 33
2.1.3.1 Transient over-expression of YFP and Nt-Hypo1................................... 33
2.1.3.2 Subcellular localization of Nt-Hypo1...................................................... 34
2.1.3.3 In vivo interaction of Nt-Hypo1 and Nt-Rac5 ......................................... 35
2.1.4 Over-expression of recombinant Nt-Hypo1 and purification from E. coli.. 36
2.1.4.1 In vitro interaction of Nt-Hypo1 with wild-type and mutant Nt-Rac5.... 36
2.1.4.2 Nt-Hypo1 – antibody production............................................................. 39
2.1.5 Nt-Hypo1 gene-knockout by antisense oligodeoxynucleotides, T-DNA
insertions and RNA interference.................................................................. 39
2.1.5.1 Antisense oligodeoxynucleotides ............................................................ 39
2.1.5.2 T-DNA insertion...................................................................................... 40
2.1.5.3 RNA interference..................................................................................... 40
2.1.6 Identification of novel Nt-Hypo1 interactors by yeast two-hybrid screening .
.....................................................................................................................43
2.2 Identification of novel phosphoinositide-specific phospholipase C isoforms from
Nicotiana tabacum................................................................................................... 47
2.2.1 Identification and sequence analysis of NtPLC3 and NtPLC4.................... 47
2.2.1.1 Phylogenetic analysis of NtPLC3 and NtPLC4....................................... 50
2.2.2 Analysis of NtPLC3 expression by Northern blotting................................. 51
2.2.3 Determination of the enzymatic activity of wild-type and mutant NtPLC3 52
2.2.3.1 Purification of recombinant NtPLC3 over-expressed in E. coli.............. 52
32.2.3.2 In vitro enzyme activity assay using [ H] phosphatidylinositol 4,5-
bisphosphate ............................................................................................ 54
2.2.4 Effects of transient expression of NtPLC3 in pollen tubes of Nicotiana
tabacum........................................................................................................ 56
2.2.4.1 Transient expression of wild-type and mutant NtPLC3 .......................... 56
2.2.4.2 Subcellular localization of NtPLC3......................................................... 58
2.2.4.3 lar localization of NtPLC3 domain-deletion mutants................ 59
IICONTENTS
2.2.4.4 Determination of the specific function of NtPLC3´s catalytic core in
membrane association.............................................................................. 61
2.2.5 Inhibition of PI-PLC activity by U-73122 treatment in vivo....................... 63
2.2.5.1 Complementation of PI-PLC inhibition by NtPLC3 over-expression in
vivo........................................................................................................... 64
2.2.6 Visualization of PI-4,5-P and its implication in pollen tube polarity......... 65 2
2.2.7 Visualization of diacylglycerol and its dependence on PI-PLC activity..... 67
3 Discussion ................................................................................................................... 72
3.1 Identification of a novel pollen-specific protein that interacts with Nt-Rac5 ..... 72
3.1.1 Transient over-expression of Nt-Hypo1 does not affect tobacco pollen tube
growth or morphology ................................................................................. 73
3.1.2 Nt-Hypo1 accumulates in the cytosol of growing pollen tubes................... 73
3.1.3 Interaction of Nt-Hypo1 with Nt-Rac5 cannot be visualized in vivo .......... 73
3.1.4 Nt-Hypo1 interacts with Nt-Rac5 in vitro ................................................... 74
3.1.5 Effects of Nt-Hypo1 gene-knockdown........................................................ 74
3.1.6 Nt-Hypo1 interacts with several pollen tube proteins ................................. 76
3.1.7 Note added in proof ..................................................................................... 79
3.2 Identification of two novel phosphoinositide-specific phospholipases C from
Nicotiana tabacum.............................................................