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Thèse de doctorat/ PhD Thesis Citation APA:

Oukouomi Lowe, Y. (2010). An investigation into the putative functions of the tobacco Annexin Ntann12 (Unpublished doctoral dissertation). Université libre de Bruxelles, Faculté des Sciences – Sciences biologiques, Bruxelles.

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D 03712

Université Libre de Bruxeiies

An investigation into the putative functions of the tobacco Annexin Ntann12

Thèse pour obtenir le grade de Docteur en Sciences Discipline: Biologie Végétale

Présentée par : Yves Oukouomi Lowé Promoteur : Dr. Marie Baucher

Année académique 2009-2010

Laboratoire de Biotechnologie Végétale Rue Adrienne Bolland, 8

6041 Gosselies

Université Libre de Bruxelles

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Déclaration

I, Yves Oukouomi Lowé, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis.

Yves Oukouomi Lowé

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Abstract

Annexins are defined as calcium-binding proteins, and they hâve been associated in plants with different biological processes such as responses to biotic and abiotic stress. Ntann12 expression is induced upon infection of tobacco plant by R. fascians.

Ntann12 possesses the conserved annexin repeat with the sequence for type II Ca^'^-binding site and recombinant as well as native Ntann12 binds to negatively charged phospholipids in a Ca^'^-dependent manner. It is mainly expressed in root differentiated cells where the protein was immunolocalized in the cytosol and in the nucléus. Ntann12 was examined by western blot in both microsomal and cytosolic fractions from tobacco roots cells, and was detected in both the cytosol and microsome. The relative increase of Ntann12 proteins associated with the microsome is coupled with an increase in Ca^^

concentration.

At the physiological level, Ntann12 expression is induced by exogenous application of auxin, and was found to be regulated in the root System by a light- induced signal coming from plant aerial part and polar auxin transport was identified to be the cellular process required for Ntann12 expression in root cells. Furthermore, Ntann12 expression is down-regulated by sait, osmotic and water stress. These results collectively suggest that the annexin Ntann12 is implicated in auxin metabolism.

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Résumé

Les annexines sont définies comme étant des protéines qui se lient de manière calcium-dépendante aux phospholipides membranaires chargés négativement.

Elles ont été associées à différents processus biologiques tels les réponses des plantes aux stress biotiques et abiotiques. Nous avons identifié une annexine végétale, appelée Ntann12, dont l’expression est induite après infection des plantes par la bactérie Rhodococcus fascians.

Ntann12 possède les domaines caractéristiques des annexines et se lie aux phospholipides chargés négativement, de manière calcium-dépendante.

L’expression de Ntann12 est très abondante dans les cellules différentiées des racines, où la protéine a été détectée par immunolocalisation dans le cytosol et dans le noyau. Des analyses par western blot ont montré que l’accroissement relatif de la quantité de protéines liées aux membranes est positivement corrélé à l’augmentation de la concentration en Ca^"^.

Au niveau physiologique, l'expression de Ntann12 est induite par l’apport exogène d’auxine. Elle est contrôlée dans les racines par un signal induit par la lumière, et provenant des parties aériennes. Le transport polaire de l'auxine a été identifié comme étant le processus cellulaires nécessaires à l'expression de Ntann12 dans les racines. En outre, cette expression est réprimée par les

stress salin, osmotique et hydrique. Ces résultats suggèrent que l’annexine Ntann12 est impliquée dans le métabolisme de l’auxine.

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Acknowledgements

I would like to start by thanking my supervisor Dr. Marie Baucher for the guidance she bas given me over the past four years, and Prof. Mondher Ei Jaziri for giving me the opportunity to pursue a Thesis in his iab. i wouid aiso iike to thank my thesis committee president, Prof. Fabrice Hombié, for his continued encouragement.

The daiiy work of this Thesis has been made infiniteiy easier through the efforts of Dr. Oiivier Vandeputte, Dr. Biiio Diaiio, Adeiine Moi and Syivain Lestrade who hâve kept the iab running smoothiy over the past few years. You hâve aiways seen the best in me and been there to iend a patient ear - thank you so much.

Thanks for Koen Goethais and Danny Vereecke for providing the BY-2 ceii suspension and R. fascians strains. Thanks for Gienda Wiiiems, Annabeiie Caiomme, Jean-Phiiippe Vandenauwe, Bertrand Chanson, David Mutin, Laurent Grumiaux, Johnny Mukoko-Bopopi and Laeticia Foritzfor their contribution.

i aiso want to thank my parents (Fiorentine and Samuei Oukouomi), Patricia, Anaïs, Judith, Christèiie, Suares, Wiiiiam, Ghisieine and Jacob, for setting the foundations of my éducation from day one, for their unwavering support and for giving me the funds, the strength and courage to foiiow this road.

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Contents

List of figures... 9

List of tabie...11

List of Abbreviations... 12

1. Introduction... 16

1.1- Définition... 16

1.2- Structure and diversity of annexins...16

1.2.1- Structure... 16

1.2.2- Diversity of annexins...21

1.3- Biochemical properties... 23

1.3.1- Calcium-dependent phospholipid binding property... 23

1.3.2- Posttranslational modifications of annexins... 23

1.4- Fonctions of animal annexins...25

1.4.1- Annexins in membrane organization and traffic... 25

1.4.1.1- Exocytosis...25

1.4.1.2- Endocytosis... 26

1.4.1.3- Annexins and membrane domains...26

1.4.1.4- Annexin as ions channels or ions channels regulators... 27

1.4.2- Annexins in disease... 27

1.4.2.1- Diabètes, cardiovascular disease...27

1.4.2.2- Inflammation and apoptosis... 29

1.4.2.3- Cancer...30

1.5- Involvement of plant annexins in varions biological fonctions... 31

1.5.1- Growth and Development... 32

1.5.2- Exocytosis and endocytosis... 33

1.5.3- Vacuoles biogenesis...34

1.5.4- Cell Wall maturation... 34

1.5.5- Ca^"" channels formation, [Ca^'']cyt modulation... 37

1.5.6- Interaction with actin, nucléotide phosphodiesterase activity, nuclease activity... 37

1.5.7- Peroxidase activity... 38

1.5.8- Interaction with the callose synthase (CalS)... 38

1.6- Annexins involvement in the mechanisms of plant responses to environmental factors and stress... 39

1.6.1- Light / Darkness... 39

1.6.2- Température and thermal stress...39

1.6.3- Osmotic, water and sait stress... 40

1.6.4- Oxidative Stress... 42

1.6.5- Mechanical stress... 42

1.6.6- Gravitropism... 42

1.6.7- Biotic stress...43

1.7- Hormonal control of annexin expression...43

1.8- Objectives of the Thesis... 44

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2- The tobacco Ntann12 gene, encoding an annexin, is induced upon

Rhodoccocus fascians infection and during leafy gaii development...47

2.1- Introduction... 47

2.2- Experimental procedures... 49

2.2.1- Plant material and growth conditions...49

2.2.2- Infection of BY-2 cell suspensions by R. fascians and other bacteria 49 2.2.3- Abiotic stress treatments...50

2.2.4- RNA extraction and mRNA differential display... 50

2.2.5- Cloning of the Ntann12 cDNA from BY2 cells and from tobacco plants... 51

2.2.6- RT-PCR analysis and real-time quantitative RT-PCR (RT-qPCR)...52

2.2.7- Amplification of the promoter région of Ntann12, construction of Ntann12 promoter-GUS constructs and plant transformation... 53

2.2.8- Génération of EGFP fusion constructs and transformation of BY-2 cells... 54

2.2.9- Nucléotide sequence accession numbers... 55

2.3- Results...56

2.3.1- Identification of Ntann12, an annexin gene induced in BY-2 cell suspensions following R. fascians infection... 56

2.3.2- Ntann12 gene response is not spécifie to R. fascians... 58

2.3.3- Ntann12 is induced by abiotic stress... 59

2.3.4- Ntann12 is localized in the cytoplasm of BY-2 cells...60

2.3.5- Ntann12 expression analysis in tobacco plants and in response to R. fascians infection...61

2.3.6- Ntann12 expression analysis during the leafy gall ontogenesis... 63

2.4- Conclusions...65

3- The tobacco Ntann12 annexin is regulated downstream of a signal transduction pathway involving light and polar auxin transport... 67

3.1- Introduction...67

3.2- Materials and methods... 69

3.2.1- Plant materials and growth conditions... 69

3.2.2- Production of the recombinant Ntann12 protein in Escherichia coli. 69 3.2.3- Plant protein analysis...70

3.2.4- Préparation of anti-Ntann12 antibodies... 71

3.2.5- Electrophoresis and immunoblotting...71

3.2.6- Phospholipid binding assay... 72

3.2.7- Immunolabeling... 73

3.2.8- RNA analysis... 74

3.2.9- pNtann12-GUS expression... 74

3.2.10- Production of transgenic plants overexpressing or downregulating Ntann12...75

3.3- Results...76

3.3.1- Recombinant tobacco Ntann12 is a Ca^'^-dependent phospholipid- binding protein...76

3.3.2- Ntann12 is highiy expressed in roots... 77

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3.3.3- Subcellular distribution of native Ntann12 is modulated by Ca^"^

concentration... 79

3.3.4- Ntann12 is mainly localized in the nuclei of root cortical cells...81

3.3.5- Light and polar auxin transport regulate Ntann12 expression in tobacco root System... 84

3.3.6- Ntann12 expression in plant is repressed by sait stress, osmotic stress and water stress... 87

3.3.7- Tobacco phenotype is not affected in transgenic plants altered for Ntann12 gene expression... 88

3.4- Conclusions...90

4- Discussions...92

4.1- Ntann12 is an annexin... 92

4.2- Ntann12 expression is developmentally regulated and is mainly localized in root maturation zone (differentiated cells)...93

4.3- Ntann12 expression is induced in leaves infected by R. fascians... 95

4.4- Exogenous lAA induced Ntann12 expression... 96

4.5- Ntann12 expression is modulated by light and by polar auxin transport 100 4.6- Ntann12 expression is repressed by sait, osmotic and water stress...101

4.7- Ntann12 is localized in the nucléus and in the cytoplasm... 103

4.8- Tobacco phenotype is not affected in transgenic plants altered for Ntann12 gene expression...105

Reference List...107

Annexes... 119

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List of figures

Page Fig. 1 The three-dimensional crystal structure of annexin Gh1 {Gossypium

hirsutum)... 16 Fig. 2 Amino acid sequence alignment of selected plant annexins... 18 Fig. 3 The sulfur cluster (S3 cluster)... 19 Fig. 4 Model describing the conformational change of annexin Al after a calcium-dependent binding to negatively charged membrane phospholipids... 20 Fig. 5 Phylogenetic tree including plant and animal annexins... 22 Fig.6 Histochemical analysis of pMtAnn1-GUS expression in transgenic Medicago roots... 36 Fig. 7 Tolérance to water stress... 41 Fig. 8 Leafy gall formed 60 days after infection by R. fascians of a 1 month old tobacco plant... 44 Fig. 9 Ntann12 gene expression analysis in tobacco BY-2 cells co-cultured for two days with R. fascians... 56 Fig. 10 Comparison of predicted amino acid sequence of Ntann12 with those of Fragaria x ananassa, M. truncatula Mtanni, A. thaliana AnnAtS and Zea mays p33... 57 Fig. 11 Effects of biotic stresses on Ntann12 expression... 59 Fig. 12 RT-PCR analysis of Ntann12 expression upon abiotic stresses in

BY-2 cells... 60 Fig. 13 Intracellular localization of Ntann12 fused to EGFP protein in tobacco BY-2 cells... 61 Fig. 14 Histochemical analysis of GUS activity during the development of tobacco seedlings transformed with the ç>Ntann12-GUS construct... 62 Fig. 15 Ntann12 promoter response to R. fascians infection of tobacco plants... 63

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Fig. 16 RT-qPCR analysis of Ntann12 expression during leafy gall ontogenesis... 64 Fig. 17 Production of recombinant Ntann12 and its calcium dépendent phospholipid binding property... 76 Fig. 18 Ntann12 expression in 4-week-old plants... 78 Fig. 19 Subcellular distribution and Ca^'" response of native Ntann12 proteins... 80 Fig. 20. Ntann12 immunolocalization in tobacco roots visualized by fluorescence micrographs of root cross sections... 82 Fig. 21 Transmission électron micrographs of Ntann12 immunogold labelling... 83 Fig. 22 pNtann12 responses to 48h light (16/8 light/dark photoperiod), auxin and auxin inhibitor treatments in transgenic tobacco plants... 85 Fig. 23 pNtann12 responses to sait stress, osmotic stress, and water stress in transgenic tobacco plants... 88 Fig. 24 Characterization of T2 transgenic tobacco progenies overexpressing and downregulating Ntann12... 89

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List of table

Table 1: Involvement of A. thaliana annexins in various biological functions... 31

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List of Abbreviations

5’ UTR 5’-untranslated région

2,4-D 2,4-Dichlorophenoxyacetic acid

ABA Abscisic acid

APL Acute Promyelocytic Leukemia ATP Adenosine Triphosphate ATPase Adenosine Triphosphatase

BSA Bovine Sérum Albumin

Cals Callose Synthase

cDNA complementary DeoxyriboNucleic Acid

Cys Cysteine

DAPI 4,6-DiAmidine 2-Phenyl Indole DMSO DiMethyl SulfOxide

DNA DeoxyriboNucleic Acid

EDTA EthyleneDiamineTetraacetic Acid EGF Epidermal Growth Factor

eGFP enhanced Green Fluorescent Proteins EGTA Ethylene Glycol Tetraacetic Acid F-actin Filamentous actin

G1 GAP 1

G2 GAP 2

GA3 Gibbereline A3

GFP Green Fluorescent Protein

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GTP Guanosine triphosphate GTPase Guanosine TriPhosphatase

HEPES 4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid

His Histidine

lAA 3-lndole Acetic Acid

KDa KiloDalton

LS Linsmaier and Skoog medium

M Mitosis

Met Méthionine

mRNA Messenger RiboNucleic Acid

MM Molecular Mass

MS Murashige and Skoog medium NAA 1-Naphtalene Acetic Acid N PA 1-NaphthylPhthalamic Acid NIP Non Infected Plant

Nod factors Nodulation factors

PBST Phosphate Buffered Saline - Tween PC L-a-PhosphatidylCholine

PCR Polymerase Chain Reaction PS L-a-PhosphatidylSerine PVDF PolyVinylidene DiFluohde

qPCR Quantitative Polymerase Chain Reaction

RNA RiboNucleic Acid

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RNAi RiboNucleic Acid interférence ROS Reactive Oxygen Species

RT-PCR Real-Time Polymerase Chain Reaction

RT-qPCR Real-Time quantitative Polymerase Chain Reaction

S DNA Synthesis phase

SDS-PAGE Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis T-DNA Transfert DNA

TDZ ThiDiaZuron

Tl B A 2, 3, 5-TrilodoBenzoic Acid

WT Wild Type

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Chapter One

Introduction

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1. Introduction

1.1- Définition

Annexins are defined as proteins capable of binding to negatively charged membrane phospholipids in a calcium-dependent manner, and which contain a conserved structural element called annexin repeat, a segment of some 70 to 75 amino acids (Clark and Roux, 1995; Gerke and Moss, 2002).

1.2- Structure and diversity of annexins 1.2.1- Structure

Annexins are composed of two principal domains: the conserved C-terminal protein core and the N-terminal région (Fig.1).

N-terminal

A B D

C E

Fig. 1 The three-dimensional crystal structure of annexin Gh1 {Gossypium hirsutum). The C-terminal core comprises four annexin repeats shown in dark blue (1®* repeat), in light blue (2"'^ repeat), in aquamarine (3'^'^ repeat) and green (4*^ repeat). Each of the repeats consists of a five-helix bundle (A, B, C, D and E). The N-terminal domain (N-terminal tail) is unstructured. Exposed surface residues on the convex side of the molécule are drawn in red. Taken from Hofmann et al. (2003).

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An annexin core comprises four (in the 35-40 kDa annexins) or eight (in the 68 kDa annexins) annexin repeats (Jost et al., 1994). Each of the repeats consiste of a four-helix bundle (A, B, D and E) where the helices are arranged in an approximately anti-parallel fashion, and a fifth hélix (C) oriented almost perpendicular to the bundle (Fig. 1) (Dabitz et al., 2005).

The annexins calcium binding sites are divided into high affinity type II site (K-G-X-G-T-{38}-D/E) and low affinity type NI site (Weng et al., 1993). In the type II site (to distinguish it from the type I EF-hand Ca^"" site, Capozzi et al., 2006), Ca^"" co-ordination in an individual annexin repeat is provided by carboxyl oxygens from the loop between helices A and B and by carboxyl oxygens from an acidic amino acid located 40 residues downstream of a conserved glycine présent in the interhelical loop. Type III site, on the other hand, possess a different architecture which, in most cases, involves the loop between helices D and E and one nearby acidic residue (Weng et al., 1993).

Besides the calcium binding sites, several other sequences are relatively conserved in numerous plant annexins such as the actin-binding motif (IRI), the GTP-binding motif (GXXXXGKT), the His40 key peroxidase residue and the sulfur cluster (named S3 cluster) (Fig. 2) (Hofmann et al., 2003; Mortimer et al., 2008).

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Fig. 2 Amino acid sequence alignment of selected plant annexins. Annexins of : Zea mays (AnxZm33, AnxZm 35); Capsicum annuum (AnxCa32); G. Hirsutum (AnxGhl); A. thaliana (AnxAtI, AnxAt2); Lycopersicum esculentum (AnxLe35) and M. truncatula (AnxMtl). Sequences shading: red box and white character, strict identity; red box and red character, similarity in a group; blue box, similarity across groups. Symbols; green triangle, His40 key peroxidase residue; blue square, IRI actin-binding motif; yellow star, GXXXXGKT, DXXG putative GTP-binding motif; purple square, KGXGT-38-D/E Ca^"’-binding sites;

black triangle, putative S3 cluster; turquoise circle, conserved tryptophan required for membrane binding. Sequence alignment was generated using Clustal X (Thompson et al., 1997; default settings). Features were added using ESPript (Gouet et al., 2003). Taken from Mortimer et al. (2008).

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The S3 cluster consists of two adjacent cysteine residues which, in combination with a nearby méthionine residue, form a cluster Met-Cys-Cys (Fig. 3). The cysteine residues of the S3 cluster are usually involved in posttranslational modifications, and are présent in ail eight A. thaliana annexins (Hofmann et al., 2003; Konopka-Postupolska et al., 2009).

Fig. 3 The sulfur cluster (S3 cluster). Spatial arrangement of the S3 cluster formed by Met112, Cys116, and Cys243. The électron density shown was calculated as omit map and is contoured at 1.5 a. Helices IIB and NIE are shown as Ca traces. Inset: the distances between the individuel sulfur atoms are given in A. Taken from Hofmann et al. (2003).

Two main différences are observed between plant and animal annexins.

First, the endonexin sequence of plant annexins is présent only in the first and fourth repeat régions, while it is well conserved in at least three of the four repeat régions of animal annexins (Mortimer et al., 2008). Secondly, the N- terminal région of plant annexins is short (=10 amino acids), while that of animal annexins is significantly longer (= 40 amino acids) (Gerke et al., 2005;

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Mortimer et al., 2008). According to the model proposed by Gerke and Moss (2002), animal annexin N-terminal tail is bound at its C-terminal domain when it is free (closed conformation), and when combined with phospholipids, its N- terminal tail is detached from its C-terminal domain (open conformation) (Fig.

4). Each annexin repeat forms a slightiy curved dise. The convex side contains the Ca^"^-binding sites (described as type II and type III) and faced the membrane surface when an annexin is associated with phospholipids; Ca^"^ ions forming a bridge between the annexin and negatively charged membrane phospholipids (Fig. 4). The N-terminal tail is located on the concave side. When annexin is associated with phospholipids, the concave side is oriented toward the cytosol and the N-terminal tail, released, may interact with other parts of the annexin or with other molécules within the cytosol (Fig. 4) (Gerke and Moss, 2002).

plus Ca2* •

Closed conformation

Open conformation

Fig. 4 Model describing the conformational change of annexin A1 after a calcium-dependent binding to negatively charged membrane phospholipids.

Taken from Gerke and Moss (2002).

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1.2.2- Diversity of annexins

Annexins hâve been identified in more than 65 species, including protists, fungi, plants and vertebrates (Moss and Morgan, 2004). Animal annexins hâve been discovered over the years 1970 (Creutz et al., 1978). The first plant annexin was found in tomato, using antibodies directed against animal annexins (Boustead et al., 1989). Since then, annexins hâve been identified in many plant species (Smallwood et al., 1992; Blackbourn et al., 1991; Randall, 1992;

Seals et al., 1994; Proust et al., 1996; Thonat et al., 1997; Kovacs et al., 1998;

Lim et al., 1998; Hofmann et al., 2000; Seigneurin-Berny et al., 1999; Clark et al., 2001; de Carvalho-Niebel et al., 2002; Lee et al., 2004; Dabitz et al., 2005;

Cantero et al., 2006; Vandeputte et al., 2007).

A phylogénie tree common between animal and plant annexins show that plant annexins form a separate monophyletic group (Fig. 5) (Mortimer et al., 2008).

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AnnAx4 (AnxD16) ---AnxAtl (AnxD26)

--- AnxCa24

--- AnxAt2 (AnxD27) --- AnxAt6 (AnxD29) --- AnxAt? (AnxD28) --- AnxZin33 ---AnxZm35 --- AnxAtS (AnxDI9)

--- AnxFa4 ---AnxMtl

--- AnxAt3 (AnxD14) --- AnxOsl

--- AnxAtS (AnxDIO) --- AnxTa I

--- C. elegans Nexl M. nmsculns AnxA13

— D. rerio AnxAla

— //. sapiens AnxAl

— /Vf. ntusculus AnxAl R. norvégiens AnxAl

“ R. norvégiens AnxA2

\f. mnsenlns AnxA2 M. inusenlns AnxAll

~ R. norvégiens AnxA3

■ ;W. mnsenlns AnxA3 H. sapiens AnxA6 ' H. sapiens AnxAS

Fig. 5 Phylogenetic tree including plant and animal annexins. A. thaliana, AnxAtl-8; Zea mays, AnxZmS, AnxZm 35; Fragaria x ananassa, AnxFa4; O.

sativa, AnxOsl; T. aestivum, AnxTa 1; C. annuum, AnxCa24 ; M. truncatula, AnxMtl ; C. elegans, Nex-1 ; H. sapiens, AnxAl, AnxAS, AnxA6; M. musculus, AnxAl, AnxA2, AnxAS, AnxA11, AnxAl3 ; D. rerio, AnxAla; R. norvégiens AnxAl, AnxA2, AnxAS. Sequence alignment was done by the Cluster X program (Thompson et al., 1997) and the tree was constructed by the program TreeView. Taken from Mortimer et al. (2008).

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1.3- Biochemical properties

1.3.1- Calcium-dependent phospholipid binding property

Biochemically, annexins are defined as soluble, hydrophilic proteins that bind to negatively charged membrane phospholipids in a calcium-dependent manner (Gerke and Moss, 2002). This phospholipid binding property is retained within the annexin core (Fig.1) (Gerke and Moss, 2002). Although calcium-dependent phospholipid binding is shared by ail annexins, individuel members differ in their calcium-sensitivity and phospholipid headgroup specificity (e.g., phosphatidic acid, phosphatidylserine, phosphatidylinositol) (Gerke and Moss, 2002).

Annexins bind to virtually ail cell membranes, including plasma membrane, vacuoles, nucléus, mitochondria, peroxisome, chloroplast, endoplasmic réticulum, Goigi apparatus (Breton et al., 2000; Seals et al., 1994; Seals and Randall, 1997; Eubel et al., 2008; Seigneurin-Berny et al., 1999; Lee et al., 2004; Mortimer et al., 2008). Annexin-membrane binding is mainly calcium- dependent. However, calcium-independent binding, although negligible, was observed in vitro with some annexins (Dabitz et al., 2005).

1.3.2- Posttranslational modifications of annexins

Posttranslational modifications of annexins affect their conformations, their location and their activities (Gerke and Moss, 2002; O’Brian and Chu, 2005;

Konopka-Postupolska et al., 2009). Future analyses hâve to describe how

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these modifications are mechanistically linked to the different annexin functions (Gerke and Moss, 2002; Konopka-Postupolska étal., 2009).

The N-terminal région, called N-terminal tail, appears to be the main site of posttranslational modifications, including phosphorylation, S-glutathiolation, S- nitrosylation and A/-myristoylation (Gerke and Moss, 2002; Gerke et al., 2005;

Mortimer et al., 2008). These modifications are commonly observed in protein- effector involved in cell signalling (O’Brian and Chu, 2005), and underscore the regulatory importance of N-terminal tail (Gerke and Moss, 2002). A number of tyrosine, histidine and serine/threonine kinases that phosphorylate human annexins A1 and A2 hâve been described (Glenney et al., 1985; Bellagamba et al., 1997; Muimo étal., 2000; Biener et al., 1996; Sarafian étal., 1991). Plant

annexins AnnAtI, AnnGh2 and p33 possess phosphorylation sites that are similar to those observed in human annexins Al and A2 (Delmer and Potikha, 1997).

The spécifie posttranslational modification of protein cysteine-sulfur by the addition of the tripeptide glutathione is termed S-glutathiolation, and the addition of nitric oxide (NO) to cysteine-sulfur in proteins is termed S-nitrosylation (Hao et al., 2006; Gow et al., 2002). in plants, the S-glutathiolation and S-

nitrosylation (consisting of the cysteine residues oxidation), are induced by abiotic stress through the médiation of ROS System (reactive oxygen species) (Gould et al., 2003; Apel and Hirt, 2004). The cysteine residues of S3 cluster (Fig. 3) are usually involved in these mechanisms (Konopka-Postupolska et al., 2009). The S-glutathiolation of animal annexins A2 hâve been described

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(Sullivan et al., 2000). The S-glutathiolation of A. thaliana annexin AnnAtI, observed in vitro and in vivo, is induced after treatment with ABA and reduced by 50% their Ca^* affinity (Konopka-Postupolska et al., 2009). The S- nitrosylation of AnnAtI was aiso described (Lindermayr étal., 2005).

Protein A/-myristoylation refers to the covalent attachment of myristate, a 14- carbon saturated fatty acid, to the N-terminal glycine of eukaryotic and viral proteins (Farazi et al., 2001). A/-Myristoylation promotes weak and réversible protein-membrane and protein-protein interactions (Farazi et al., 2001). The N- myristoylation of human annexins Al3a and Al3b hâve been observed (Wice and Gordon, 1992).

1.4- Functions of animal annexins

1.4.1- Annexins in membrane organization and traffic 1.4.1.1- Exocytosis

A number of annexin proteins, including annexins Al, A2, A3, A6, A7, Ail, Al 3b, and B7, hâve been linked to exocytotic processes (Gerke et Moss, 2002;

Gerke et al., 2005). The most convincing evidence for such an involvement which go beyond the localization of the protein to secretory organelle membranes and/or the plasma membrane has been reported for annexins A2 and Al3b (Gerke et Moss, 2002). Annexin A2 is involved in Ca^'^-regulated exocytosis in permeabilized chromaffin cells: the time-dependent loss of

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secretot7 capacity could be blocked by the addition of annexin A2 to the chromaffin cells culture medium (Ali et al., 1989).

In polarized épithélial cells, annexin Al3b associâtes specifically with sphingolipid- and cholesterol-rich membrane domains of the frans-GoIgi network, and myristoylated annexin Al 3b is required for the budding of these domains, which are subsequently delivered to the apical plasma membrane (Lafont et al., 1998).

1.4.1.2- Endocytosis

Annexins Al, A2 and A6 are présent on endosomal compartments, and unique endosome targeting sequences hâve been identified in the N-terminal domain of annexins Al and A2 (Emans et al., 1993; Seemann et al., 1996). In fibroblasts from annexin Al-knockout mice, multivesicular endosomes are formed in the absence of annexin Al, but these endosomes contain fewer internai vesicles (Emans étal., 1993; Seemann étal., 1996).

1.4.1.3- Annexins and membrane domains

In the sarcolemma of smooth muscle cells, changes in intracellular Ca^""

concentrations occurring during smooth muscle contraction appear to regulate the dynamics of rafts, their latéral assembly, and association with the actin cytoskeleton. These changes correlate with the Ca^'"-dependent association of annexin A2 with membrane rafts and the translocation of annexin A6 to a membrane-cytoskeleton complex (Babiychuk and Draeger, 2000). It was

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proposed that an initial Ca^"^ rise in smooth muscle cells triggers the binding of annexin A2 to lipid rafts and a clustering of these rafts which is promoted by latéral annexin assembly (Babiychuk and Draeger, 2000).

Annexin A2 is an F-actin binding protein itself and therefore could aiso participate more directiy in the formation of membrane-cytoskeleton links (Gerke and Moss, 2002).

1.4.1.4- Annexin as ions channeis regulators

Theoretical calculations predict that human annexin A5 could sufficiently perturb the organization of lipids in the bilayer at the site of Ca^‘"-dependent attachment to effectively electroporate the membrane and therefore permit Ca^"" entry (Démangé et al., 1994).

Human annexins A2, A4 and A6 modulate plasma membrane Cl’-channels and sarcoplasmic réticulum Ca^'^-release channeis (Gerke and Moss, 2002).

1.4.2-Annexins in disease

1.4.2.1- Diabètes, cardiovascuiar disease

Annexin A2 has been implicated in the pathology of both type I and type II diabètes, due to its rôle in vascular endothélial biology and hypercoagulation which occurs in both forms of diabètes (Ishii étal., 2001). Cell-surface annexin A2 that fonctions as a co-receptor for tissue plasminogen and plasminogen activator promotes the production of plasmin, which dissolves blood dots (through fibrin dégradation and therefore fibrinolytic homeostasis maintenance)

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so, preventing excessive coagulation (Hajjar et al., 1994; Kim et al., 2002).

Consistent with this, histopathological examination of mice that lack annexin A2 reveals extensive déposition of fibrin in their tissues (Ling et al., 2004). Plasmin production is reduced in high glucose and insulin conditions. This réduction was partially prevented upon addition of annexin A2 (Ishii et al., 2001).

Annexin A2 is highiy affected by several risk factors that are linked with diabètes and cardiovascular diseases, and the stress-associated modifications of annexin A2 considerably altered the properties of the protein. For example, oxidative stress is associated with elevated levels of cellular glutathione and the activation of nitric oxide synthase. Annexin A2 is glutathionylated in HeLa cells (Sullivan et al., 2000) and nitrosylated in lung épithélial cells (Rowan et al., 2002). Annexin A2 may aiso contribute to diabètes pathology through its phosphorylation. Insulin-dependent tyrosine phosphorylation of annexin A2 is known to resuit in changes in the actin cytoskeleton. This remodelling significantly alters the cell morphology, such that actin dômes are formed, and aIso diminishes cell adhesion (Rescher étal., 2008).

More direct evidence for the involvement of annexin A2 in disease pathology emerged from studies on leukemic cells from patients with acute promyelocytic leukemia (APL). Patients with APL exhibit an increased tendency to hémorrhagie diathesis and respond well to treatment with all-trans-retinoic acid.

APL leukocytes were found to strongly overexpress annexin A2 at the cell surface and aiso to stimulate the génération of plasmin from tPA twice as efficiently as other leukemic cells (Menell et al., 1999). Plasmin génération was

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blocked by anti-annexin A2 antibodies and could be induced in non-APL cells by ectopic expression of annexin A2. Moreover, exposure of APL cells to all- trans-retinoic acid led to a marked réduction in annexin A2 mRNA and protein which correlated with diminished tPA binding (Menell et al., 1999).

1.4.2.2- Inflammation and apoptosis

In vitro and in vivo models both show that exogenousiy administered annexin

Al inhibits neutrophil extravasations and thereby limits the degree of inflammation (Perretti et al., 2003). This activity is retained in N-terminal annexin Al peptides, which are probably generated by proteolysis at sites of inflammation and interact with spécifie receptors on leukocytes (Perretti et al., 2003) .

Annexin Al has been implicated in apoptosis. For neutrophils, it can trigger pro-apoptotic responses (Solito et al., 2003), whereas in Jurkat-T-Lymphocytes, it can function as an engulfment ligand that is presented on the surface when cells become apoptotic (Arur et al., 2003). Annexin Al has aiso been identified as a sélective surface marker of the vascular endothélium in several solid tumours, and radioimmunotherapy using anti-annexin Al antibodies has been shown to destroy such annexin Al positive tumours specifically (Oh et al., 2004) .

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1.4.2.3- Cancer

Annexin A7 is expressed at low levels in the most metastatic malignant melanomas (Kataoka et al., 2000). Interestingly, ectopic expression of annexin A7 in two prostate tumor cell lines reduced cell prolifération and that heterozygous annexin A7 knock-out mice hâve a more cancer-prone phenotype (Srivastava et al., 2001). Annexin A6 has tumor suppressor activity in human A431 cells (Chetcuti et al., 2001). Annexin Al and annexin A2 appear to be dovymregulated in prostate cancer. Annexin A5 is upregulated in melanomas and downregulated in leukemias cancers. Annexin A9 is upregulated in prostate and colon cancers (Chetcuti et al., 2001). Nevertheless, evidence in support of causative rôles for any annexin in the development of cancer or in cell transformation is still mainly circumstantial (Gerke and Moss, 2002).

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1.5- Involvement of plant annexins in various biological functions

Table 1: Plant annexins in various biological functions

Plant Gene Localization / Subcellular localization

Implication in various biological function

Authors

A.

thaliana

AnnAtI AH tissues,

abundant in roots / Cytoplasm

Exocytosis, Peroxydase activity. Température responses. Sait stress, Water stress, Oxidative stress.

Hormonal control

Clark et al., 2001;

2005a; Cantero et al., 2006; Lee et al., 2004

AnnAt2 Mainly in roots and flowers / -

Growth and development, Exocytosis, Température responses. Sait stress, Water stress

AnnAtS AH tissues, most abundant in roots and flowers / -

Growth and development.

Température responses, Water stress

AnnAt4 Most abundant in roots and flowers / Cytoplasm

Growth and development.

Température responses, Sait stress

AnnAtS AH tissues, most abundant in flowers and roots / -

Growth and development.

Interaction with actin. Sait stress, Light responses AnnAtô Mainly in flower /- Growth and development,

Light responses.

Température responses. Sait stress, Water stress

AnnAt? Mainly in flower /- Growth and development.

Température responses. Sait stress

AnnAtS AH tissues /- Growth and development.

Sait stress, Water stress Tobacco Sp32 AH tissues /

Cytoplasm

Cell Wall maturation (cell cycle)

Proust et al., 1999 Celery,

Tobacco

VCaB42 AH tissues / Cytoplasm

Vacuole biogenesis Seals et al., 1994;

Seals and

Randall, 1997 M.

truncatula

MtAnnI AH tissues / Cytoplasm

Cell division. Nodule organogenesis. Biotic stress

de Carvalho- Niebel et al., 2002 Potato p34, p35 AH tissues / - Growth and development Smallwood et al.,

1992 Tomato p34, p35 AH tissues /

Cytoplasm Growth and development, F- actin binding. Nucléotide phosphatase activity

Smallwood et al., 1992; Calvert et al., 1996

Wheat p34, p35, AH tissues /- Growth and development Smallwood et al., 1992

Wheat P39, p22.5

AH tissues / Cytoplasm

Sensors or transducers of

calcium signal linked to cold Breton et al., 2000

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Plant Gene Localization / Subceilular iocalization

Implication in various biological fonction

Authors

Maize p33, p34, p35

AH tissues / Cytoplasm

Exocytosis, Ca"^* transport and [Ca^*]cyt régulation, ATPase activity, Peroxidase activity

Blackbourn and Battey, 1993;

CarroH et al., 1998; Mc Clung et

al., 1994;

Laohavisit et al., 2009

B. dioica p33, p35 Internodes / Cytoplasm

Mechanical stress Thonat et al., 1997

M. sativa AnnMs2 AH tissues / Nucléus

Cell division, Osmotic stress, Water stress. Hormonal control

Kovâcs et al., 1998

M. pudica p35 AH tissues /

Cytoplasm

Light responses (nyctinastic movements)

Hoshino et al., 2004

C.

annuum

annexin 24 (Ca32)

AH tissues / - Ca'^'" channel, Hormonal control

Hofmann et al., 2000; Proust et al., 1996

Plant annexins functions remain to be determined. However, plant annexins hâve been implicated in various biological functions (table 1), based on the variability of their expressions (Clark et al., 2001; 2005a; Cantero et al., 2006;

Lee et al., 2004), on their subcellular localization (Seals and Randall, 1997; de Carvalho-Niebel et al., 2002; Kovâcs et al., 1998) and on their biochemical properties (Mc Clung et al., 1994; Calvert et al., 1996; Hofmann et al., 2000;

Laohavisit étal., 2009; Konopka-Postupolska et al., 2009).

1.5.1- Growth and Development

The expression patterns of eight annexins in A. thaliana were analyzed (Clark et al., 2001; 2005a; Cantero et al., 2006). The expression of these annexins varies by âge and tissue specificity. This variability suggests that annexins are involved specifically in various developmental stages (Clark et al., 2001; 2005a;

Cantero et al., 2006). A. thaliana annexins are expressed from germination to

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flowering, and in ail tissues examined (hypocotyl, cotylédons, leaves, stems, roots, flowers). The levels of expression in a given tissue vary from one gene to another, and vary during development. The eight genes are transcriptionally active in normal growth conditions, their expression levels decreased 26 hours after seedling (except AnnAt4), then increases again after seven days of growth. The highest expression levels were observed in roots and hypocotyls, while the lowest expression levels were observed in cotylédons (Clark et al., 2001; 2005a; Cantero étal., 2006).

The expression of annexins p34 and p35 of tomato, potato and wheat, aiso vary by âge and tissue specificity (Smallwood et al., 1992).

1.5.2- Exocytosis and endocytosis

Exocytosis is a fundamental process for the sécrétion of polysaccharides required for the development of the wall. Annexins p33 and p35 stimulate calcium-dependent fusion of vesicles with plasma membrane in maize roots protoplasts (Blackbourn et al., 1991; 1992; Blackbourn and Battey, 1993;

Carroll étal., 1998).

Annexins AnnAtI and AnnAt2 were detected by immunofluorescence in secretory cells, including peripheral cells of root cap, root hair cells, cells near the apical meristem and companion cells of maize (Clark et al., 1992; 2005a).

Authors suggest that annexins AnnAtI and AnnAt2 could be involved in sécrétion mediated by the Goigi apparatus.

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It has been shown that tobacco annexins Sp32 are mainly localized at the intercellular junctions. It was assumed that these annexins are involved in the formation of the cell wall (Proust et al., 1999).

1.5.3- Vacuoles biogenesis

Vacuoles biogenesis is a key component of cell expansion. Celery and tobacco annexin VCaB42 binds to vacuolar membranes and VCaB42 gene expression is correlated with vacuoles biogenesis in growing cells (Randall, 1992; Seals et al., 1994; Seals and Randall, 1997).

1.5.4- Cell wall maturation

In plants, some annexins hâve a differential expression or a variable distribution in successive phases of the cell cycle. The tobacco annexin Sp32 is expressed during transition phases G2/M and G1/S and during mitosis. The study of Sp32 expression in different organs showed a more pronounced expression in tissues composed of dividing cells: the transcripts were detected at high levels in flower buds and young stems, a weaker expression was observed in roots and young leaves, and no expression was detected in older leaves (Proust et al., 1999).

Immunolocalization shows that the majority of Sp32 proteins is présent in intercellular junctions, forming a ring structure under the plasma membrane.

Authors suggest that Sp32 could be involved in cell wall maturation (Proust et al., 1999).

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The subcellular localization of M. sativa annexin AnnMs2 varies during different phases of the cell cycle. In interphase cells, AnnMs2 is localized in the nucleolar cortex, the perinuclear région and cytoplasm. In mitotic cells, it is mainly detected at chromosomes. At the end of mitosis and after nuclear membrane formation, AnnMs2 is again détectable in perinuclear région (Kovécs étal,, 1998).

Nodulation factors (or Nod factors) induce the expression of M. truncatula annexin MtAnnI, and studies of co-location with a construction MtAnn1-GFP suggest that the gene MtAnnI is involved in the early stages of cell division necessary for nodules formation. MtAnnI is a cytosolic protein that accumulâtes specifically at the nuclear periphery of cells in the cortex during the early stages of nodule organogenesis (Fig. 6) (de Carvalho-Niebel et al., 2002).

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Fîg. 6 Histochemical analysis of pMtAnn1-GUS expression in transgenic Medicago roots. Localization of GUS activity in transgenic roots in response to S. meliloti (a-e), to purified Nod factors (f-g), and in non-symbiotic conditions (h- k). (a) Sites of GUS activity in whole root segment close to the root tip, 48 h post-inoculation (hpi) with S. meliloti. (b) 80 pm-thick transversal sections of agarose-embedded roots 48 hpi. GUS activity is mainly in the outer cortex (oc) and the endodermis (arrowhead). (c,d,e) 80 pm-longitudinal sections of S.

me//7of/-inoculated roots (48 hpi). Tissues hâve been stained both for GUS and P-galactosidase. The latter localizes S. meliloti expressing the constitutive lacZ fusion in infection threads (arrows); d and e correspond to different focal planes at the same site showing GUS activity associated with infected and neighbouring cortical cells. (f) root segment of transgenic M. varia 48 h after the addition of 10'® M Nod factors, (g) 80 pm-thick transverse section of agarose- embedded M. truncatula root 48 h after 10 ® M Nod factor addition. GUS staining is indicated in the outer cortex (oc) and endodermal tissues (arrowhead). (h-k) sequential stages of latéral root development in transgenic M. truncatula roots, (h) GUS activity is localised in dividing cells of the latéral root primordium; (i) at the base of an emerging latéral root; (j) the GUS staining pattern of the latéral appears ring-like when viewed from above, (k) GUS activity is présent in the subapical région of the root tip referred to as the distal élongation zone. Bars=50 pm. Taken from de Carvalho-Niebel et al. (2002).

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1.5.5- channels formation, [Ca^*]cyt modulation

Crystallographic studies hâve indicated that some annexins possess a hydrophilic pore located at the intersection of four domains, forming a prominent ion channel covered with highiy conserved charged residues (Kourie and Wood, 2000). The function of calcium channel has been demonstrated in vitro for C.

annuum annexin 24 (Hofmann et al., 2000), and has aiso been suggested for

wheat annexin p39 (Breton étal., 2000).

Laohavisit et al. (2009) hâve shown that Z. mays annexins p33 and p35 are involved in Ca^'^ transport and [Ca^'^Jcyt régulation.

1.5.6- Interaction with actin, nucléotide phosphodiesterase activity, nuclease activity

Actin filaments are involved in maintaining cell shape and are involved in cell signalling (Drobak et al., 2004). AnnAtS has the capacity to link actin filaments in vitro in calcium-dependent manner. A motif susceptible to explain the link

with the F-actin, named the IRI motif, has been localized in five of the eight A.

thaliana annexins. For AnnAtS, this motif overlaps that of Ca^"" binding, which

could hâve structural implications in terms of Ca^'^-annexin-actin interaction (Clark et al, 2001).

Calvert et al. (1996) hâve purified annexins p34 and p35 in tomato and were characterized as being capable of binding to F-actin in calcium-dependent manner. Annexin p35 has nucléotide phosphatase activity with substrate as

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ATP and GTP, and this enzyme activity is not affected by binding to F-actin, but is inhibited when the protein is bound to phospholipids (Calvert et al., 1996; Lim étal., 1998).

In maize, annexins p33 and p35 hâve ATPase activity in vitro (Mc Clung et al., 1994). Shin and Brown (1999) hâve isolated annexin p35.5 from cotton fiber

cells. The recombinant protein produced in E. coli shows nuclease activity preferentially turned toward the GTPase activity than ATPase. This activity requires the presence of Mg^"" and is inhibited by Ca^"" (Shin and Brown, 1999).

1.5.7- Peroxidase activity

In examining the primary structure of annexins AnnAtI {A. thaliana), p33 and p35 (Z. mays), several functional areas can be identified, including areas similar to those encountered in peroxidases (Gidrol et al., 1996 ; Laohavisit et al., 2009). AnnAtI, p33 and p35 hâve peroxidase activity in vitro (Gidrol et al., 1996; Laohavisit et al., 2009). A post-translational modification of AnnAtI would be necessary to establish the peroxidase activity (Gorecka et al., 2005;

Konopka-Postupolska et al., 2009).

1.5.8- Interaction with the callose synthase (CalS)

Andrawis et al. (1993) showed that the CalS interact with annexins purified from cotton fiber. A study of the CalS in A. thaliana suggests an interaction between annexins and callose (Verma and Hong, 2001).

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1.6- Annexins involvement in the mechanisms of plant responses to environmental factors and stress

1.6.1- Light/ Darkness

Light affects annexin expression in Arabidopsis. In the hypocotyl, AnnAtS expression increases with red light, and it decreases reversibly with far-red light. AnnAtô has the same behavior in the cotylédons (Cantero et al., 2006).

In Mimosa pudica, the expression and localization of annexin p35 are linked to nyctinastic movements of the pulvinus. The p35 protein is abundant at night and mainly cytosolic, and the day, it is less abundant and is redistributed in the outermost periphery of motor cells (Hoshino et al., 2004). Authors suggest that annexin p35 contributes to the nyctinastic movements of the pulvinus.

1.6.2- Température and thermal stress

A température of 4°C significantly induced AnnAtI and AnnAt3 expression, and significantly represses AnnAt2 expression (Cantero et al., 2006). A température of 37°C significantly induced AnnAt2, AnnAtG and AnnAtJ expression and significantly suppresses AnnAt3 and AnnAt4 expression (Cantero et al., 2006).

Breton et al. (2000) hâve highiighted four annexins in a particular variety of wheat résistant to cold. They observed the accumulation of two of these (p39 and p22.5) in membranes during wheat acclimation to cold (4°C), with a peak of accumulation observed after one day of exposure. Being aiso présent in a wheat variety little résistant to cold, the researchers concluded that these

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annexins may play a rôle as sensors or transducers of calcium signal linked to cold (Breton et al., 2000).

1.6.3- Osmotic, water and sait stress

In A. thaliana, sait stress significantly induced expression of AnnAtI, AnnAt4, AnnAtS, AnnAtô, AnnAtT and AnnAtB, and significantly represses AnnAt2

expression (Lee et al., 2004). Sait stress induces translocation of AnnAtI and AnnAt4 proteins from cytosol to membranes (Lee et al., 2004).

Water stress significantly induces expression of genes AnnAtI, AnnAtB, AnnAtô and AnnAtS and significantly represses AnnAt2 expression (Cantero et

al., 2006). Konopka-Postupolska et al. (2009) hâve regenerated insertion

mutants in which AnnAtI transcripts were not detected {LAnnAtI plants) and other transgenic plants which overexpress the gene AnnAtI (35S:: AnnAtI plants). Two tests were performed. First, one month old seedlings hâve been subjected to water stress for two weeks. Five days after stress, the first signs of drying hâve been observed in AAnnAtl plants, whereas normal plants (Col-0) and 35S::AnnAt1 plants remained turgid and green. After prolonged stress (two weeks), LAnnAtI plants completely lose their turgidity and the 35S::AnnAt1 plants are more résistant than Col-0 plants (Fig. 7A).

Secondly, one month old seedlings hâve been subjected to water stress for three weeks to complété loss of turgid, and irrigation was restored for two weeks. LAnnAtI plants did not survive drying while the 35S::AnnAt1 plants hâve ail found their turgidity (Fig. 7B). These results indicate that drought

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tolérance is positively correlated with AnnAtI expression level (Konopka- Postupolska et al., 2009).

Treatment of M. sativa cells by osmotic stress, sait stress and water stress allowed demonstrating overexpression of annexin AnnMs2 during these stress (Kovâcs étal., 1998).

\ 35S::AnnAtl Col-0 SAnnAtl

B Col-0

AAnnAtl

35S::AnnAtl

Fig. 7 Tolérance to water stress. A (short-term drought): Col-0, AAnnAtl and 35S::AnnAt1 plants were grown; after 4 weeks of culture, watering was suspended; the photo shows the différences between the reactions of plants after 5 days without watering. B (long-term drought): after 8 weeks of culture (Col-0, AAnnAtl and 35S:: AnnAtI plants), watering was suspended for 3 weeks, then restored for 2 weeks. The photo shows the capacity of each plant to survive. Taken from Konopka-Postupolska et al. (2009).

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1.6.4- Oxidative Stress

Superoxide radicals are a common cause of damage at the cellular level in ail aérobic organisme. AnnAtI (aiso called Oxy5), isolated from A. thaliana during oxidative stress, complément an E. coli mutant àoxyR, suggesting a rôle for this protein in response to oxidative stress (Gidrol et al., 1996; Konopka- Postupolska et al., 2009). H2O2 accumulation in stomata celle is negatively correlated with AnnAtI gene expression and this accumulation vjas higher in àAnnAtI plants (which downregulate AnnAtI), and is low in 35S::AnnAt1 plants

(that overexpress AnnAtI) (Konopka-Postupolska et al., 2009).

1.6.5- Mechanical stress

Thonat et al. (1997) observed change in the localization of Bryonica dioica annexins p33 and p35 in response to mechanical stress (injury). These annexins are localized in the cytoplasm of parenchymal celle of the internodes and accumulate at the plasma membrane of these celle following an injury. The relocation of these annexins could govern the radial expansion of the cell after stress or preparing the plasma membrane to undergo further stress (reviewed by Mortimer et al., 2008).

1.6.6- Gravitropism

Pea annexin p35 was detected by immunolocalisation in the nucléus of epidermal celle of plumules. Redistribution of this annexin to the celle periphery was observed after gravistimulation (Clark et al., 2000).

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1.6.7- Biotic stress

Nodulation factors (Nod factors) induce the expression of annexin AnxMtl in M.

truncatula. AnxMtl is involved in early stages of nodule formation (de Carvalho-

Niebel et a/., 2002).

The Ntann12 gene expression is highiy induced after infection of tobacco BY-2 cells by Rhodococcus fascians (Vandeputte et al., 2007).

1.7- Hormonal controi of annexin expression

Phytohormones direct the processes of growth and development in plants (Paciorek and FrimI, 2006). The expression of pepper annexin Ca32 and that of strawberry annexin RJ4 increase during fruit ripening (Proust et al., 1996;

Wilkinson et al., 1995), implying a hormonal controi of expression of these genes (reviewed by Mortimer et al., 2008). Ethylene is the hormone that initiâtes the physiological processes involved in fruit ripening (Johnson and Ecker, 1997). Auxin plays a central rôle in many physiological and developmental processes, including fruit development (Kepinski and Leyser, 2005a).

Osmotic stress, sait stress and water stress act through the ABA, which aiso causes an increased expression of genes involved in these stresses (Kovàcs et al., 1998; Lee et al., 2004; Hoshino et al., 2004; Cantero et al., 2006). ABA

induced overexpression and S-glutathiolation of annexin AnnAtI, in vitro and in vivo (Konopka-Postupolska et al., 2009).

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1.8- Objectives of the Thesis

R. fascians provokes in tobacco plants altered morphology called leafy gall.

Leafy gall consists of small deformed leaves and many buds whose growth is inhibited (Goethals et al., 2001) (Fig. 8). It results mainly from the alteration of the endogenous hormone balance of host plant, subséquent to infection (Goethals et al., 2001 ).

Fig. 8 Leafy gall formed 60 days after infection by R. fascians of a 1 month old tobacco plant. Scale bar = 1 cm.

Some characteristics of the leafy gall (vascular tissue différentiation, cell élongation, inhibition of buds growth) are similar to those observed after treatment with auxin, and others (deformed leaves, prolifération of buds, slowing senescence) are typical effects of cytokinins (Vandeputte et al., 2005).

Indeed, R. fascians produces and sécrétés lAA (Vandeputte et al., 2005) and several cytokinins (Eason étal., 1996).

The study of differential gene expression before and after infection of tobacco BY-2 cells by R. fascians has identified a highiy expressed annexin-like gene named Ntann12 (Van Raemdonck, 1999). The importance of annexins in plants growth, development and adaptation has led naturally to our attention the

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rôle of Ntann12 gene. This thesis aims to investigate the rôle of Ntann12 in plant development and adaptation, with regards to the distribution and modulation of its expression, and to its biochemical properties. In additions, the overexpression and downrégulation of Ntann12 into transgenic tobacco plants hâve been done with the view to study the conséquences of altered expression.

The results of this thesis are presented into two parts:

1- The tobacco Ntann12 gene, encoding an annexin, is induced upon Rhodoccocus fascians infection and during leafy gall development.

2- The tobacco Ntann12 annexin is regulated downstream of a signal transduction pathway involving light and polar auxin transport.

Figure

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