An investigation into the putative functions of the tobacco Annexin Ntann12

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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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Declaration

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 have 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 Ca²⁺-binding site and recombinant as well as native Ntann12 binds to negatively charged phospholipids in a Ca²⁺-dependent manner. It is mainly expressed in root differentiated cells where the protein was immunolocalized in the cytosol and in the nucleus. 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 Ca²⁺

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 salt, 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 Ca²⁺.

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 has given me over the past four years, and Prof. Mondher El Jaziri for giving me the opportunity to pursue a Thesis in his lab. I would also like to thank my thesis committee president, Prof. Fabrice Homblé, for his continued encouragement.

The daily work of this Thesis has been made infinitely easier through the efforts of Dr. Olivier Vandeputte, Dr. Billo Diallo, Adeline Mol and Sylvain Lestrade who have kept the lab running smoothly over the past few years. You have always seen the best in me and been there to lend a patient ear – thank you so much.

Thanks for Koen Goethals and Danny Vereecke for providing the BY-2 cell suspension and R. fascians strains. Thanks for Glenda Willems, Annabelle Calomme, Jean-Philippe Vandenauwe, Bertrand Chanson, David Hutin, Laurent Grumiaux, Johnny Mukoko-Bopopi and Laeticia Foritz for their contribution.

I also want to thank my parents (Florentine and Samuel Oukouomi), Patricia, Anaïs, Judith, Christèlle, Suares, William, Ghisleine and Jacob, for setting the foundations of my education from day one, for their unwavering support and for giving me the funds, the strength and courage to follow this road.

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List of figures

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

hirsutum)………...

Fig. 2 Amino acid sequence alignment of selected plant annexins………...

Fig. 3 The sulfur cluster (S3 cluster)………...

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

Fig. 5 Phylogenetic tree including plant and animal annexins………

Fig.6 Histochemical analysis of pMtAnn1-GUS expression in transgenic Medicago roots……….………..

Fig. 7 Tolerance to water stress………..

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

Fig. 9 Ntann12 gene expression analysis in tobacco BY-2 cells co-cultured for two days with R. fascians...

Fig. 10 Comparison of predicted amino acid sequence of Ntann12 with those of Fragaria x ananassa, M. truncatula Mtann1, A. thaliana AnnAt8 and Zea mays p33...

Fig. 11 Effects of biotic stresses on Ntann12 expression……….…..

Fig. 12 RT-PCR analysis of Ntann12 expression upon abiotic stresses in BY-2 cells……….

Fig. 13 Intracellular localization of Ntann12 fused to EGFP protein in tobacco BY-2 cells………..………...

Fig. 14 Histochemical analysis of GUS activity during the development of tobacco seedlings transformed with the pNtann12-GUS construct………

Fig. 15 Ntann12 promoter response to R. fascians infection of tobacco plants……….…………...

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Fig. 16 RT-qPCR analysis of Ntann12 expression during leafy gall ontogenesis……….

Fig. 17 Production of recombinant Ntann12 and its calcium dependent phospholipid binding property..………

Fig. 18 Ntann12 expression in 4-week-old plants.………..…….

Fig. 19 Subcellular distribution and Ca²⁺ response of native Ntann12 proteins………...

Fig. 20. Ntann12 immunolocalization in tobacco roots visualized by fluorescence micrographs of root cross sections...

Fig. 21 Transmission electron micrographs of Ntann12 immunogold labelling………

Fig. 22 pNtann12 responses to 48h light (16/8 light/dark photoperiod), auxin and auxin inhibitor treatments in transgenic tobacco plants………

Fig. 23 pNtann12 responses to salt stress, osmotic stress, and water stress in transgenic tobacco plants……….

Fig. 24 Characterization of T2 transgenic tobacco progenies overexpressing and downregulating Ntann12………

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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 2,4-D ABA APL ATP ATPase BSA CalS cDNA Cys DAPI DMSO DNA EDTA EGF eGFP EGTA F-actin G1 G2 GA3

GFP

5’-untranslated region

2,4-Dichlorophenoxyacetic acid Abscisic acid

Acute Promyelocytic Leukemia Adenosine Triphosphate

Adenosine Triphosphatase Bovine Serum Albumin Callose Synthase

complementary DeoxyriboNucleic Acid Cysteine

4,6-DiAmidine 2-Phenyl Indole DiMethyl SulfOxide

DeoxyriboNucleic Acid

EthyleneDiamineTetraacetic Acid Epidermal Growth Factor

enhanced Green Fluorescent Proteins Ethylene Glycol Tetraacetic Acid Filamentous actin

GAP 1 GAP 2

Gibbereline A3

Green Fluorescent Protein

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GTP GTPase HEPES His IAA KDa LS M Met mRNA MM MS NAA NPA NIP

Nod factors PBST PC PCR PS PVDF qPCR RNA

Guanosine triphosphate Guanosine TriPhosphatase

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

3-Indole Acetic Acid KiloDalton

Linsmaier and Skoog medium Mitosis

Methionine

Messenger RiboNucleic Acid Molecular Mass

Murashige and Skoog medium 1-Naphtalene Acetic Acid 1-NaphthylPhthalamic Acid Non Infected Plant

Nodulation factors

Phosphate Buffered Saline - Tween L-α-PhosphatidylCholine

Polymerase Chain Reaction L-α-PhosphatidylSerine PolyVinylidene DiFluoride

Quantitative Polymerase Chain Reaction RiboNucleic Acid

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RNAi ROS RT-PCR RT-qPCR S

SDS-PAGE T-DNA TDZ TIBA WT

RiboNucleic Acid interference Reactive Oxygen Species

Real-Time Polymerase Chain Reaction

Real-Time quantitative Polymerase Chain Reaction DNA Synthesis phase

Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis Transfert DNA

ThiDiaZuron

2, 3, 5-TriIodoBenzoic Acid Wild Type

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Chapter One

Introduction

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1. Introduction

1.1- Definition

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 region (Fig.1).

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^st repeat), in light blue (2^nd repeat), in aquamarine (3^rd repeat) and green (4^th 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 molecule are drawn in red. Taken from Hofmann et al. (2003).

A B D

E C N-terminal tail

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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 consists of a four-helix bundle (A, B, D and E) where the helices are arranged in an approximately anti-parallel fashion, and a fifth helix (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 III site (Weng et al., 1993). In the type II site (to distinguish it from the type I EF-hand Ca²⁺ site, Capozzi et al., 2006), Ca²⁺ 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 present 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 (AnxGh1); A. thaliana (AnxAt1, AnxAt2); Lycopersicum esculentum (AnxLe35) and M. truncatula (AnxMt1). 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 Ca²⁺-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 methionine residue, form a cluster Met-Cys-Cys (Fig. 3). The cysteine residues of the S3 cluster are usually involved in posttranslational modifications, and are present in all 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 electron density shown was calculated as omit map and is contoured at 1.5 σ. Helices IIB and IIIE are shown as Ca traces. Inset: the distances between the individual sulfur atoms are given in Å. Taken from Hofmann et al. (2003).

Two main differences are observed between plant and animal annexins.

First, the endonexin sequence of plant annexins is present only in the first and fourth repeat regions, while it is well conserved in at least three of the four repeat regions of animal annexins (Mortimer et al., 2008). Secondly, the N- terminal region 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 slightly curved disc. The convex side contains the Ca²⁺-binding sites (described as type II and type III) and faced the membrane surface when an annexin is associated with phospholipids; Ca²⁺ 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 molecules within the cytosol (Fig. 4) (Gerke and Moss, 2002).

Closed conformation

Open conformation

N-terminal tail

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 have been identified in more than 65 species, including protists, fungi, plants and vertebrates (Moss and Morgan, 2004). Animal annexins have 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 have 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 phylogenic 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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Fig. 5 Phylogenetic tree including plant and animal annexins. A. thaliana, AnxAt1-8; Zea mays, AnxZm3, AnxZm 35; Fragaria x ananassa, AnxFa4; O.

sativa, AnxOs1; T. aestivum, AnxTa1; C. annuum, AnxCa24 ; M. truncatula, AnxMt1 ; C. elegans, Nex-1 ; H. sapiens, AnxA1, AnxA5, AnxA6; M. musculus, AnxA1, AnxA2, AnxA3, AnxA11, AnxA13 ; D. rerio, AnxA1a ; R. norvegicus AnxA1, AnxA2, AnxA3. 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 all annexins, individual members differ in their calcium-sensitivity and phospholipid headgroup specificity (e.g., phosphatidic acid, phosphatidylserine, phosphatidylinositol) (Gerke and Moss, 2002).

Annexins bind to virtually all cell membranes, including plasma membrane, vacuoles, nucleus, mitochondria, peroxisome, chloroplast, endoplasmic reticulum, Golgi 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 have to describe how

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

The N-terminal region, called N-terminal tail, appears to be the main site of posttranslational modifications, including phosphorylation, S-glutathiolation, S- nitrosylation and N-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 have been described (Glenney et al., 1985; Bellagamba et al., 1997; Muimo et al., 2000; Biener et al., 1996; Sarafian et al., 1991). Plant annexins AnnAt1, AnnGh2 and p33 possess phosphorylation sites that are similar to those observed in human annexins A1 and A2 (Delmer and Potikha, 1997).

The specific 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 mediation 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 have been described

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(Sullivan et al., 2000). The S-glutathiolation of A. thaliana annexin AnnAt1, observed in vitro and in vivo, is induced after treatment with ABA and reduced by 50% their Ca²⁺ affinity (Konopka-Postupolska et al., 2009). The S- nitrosylation of AnnAt1 was also described (Lindermayr et al., 2005).

Protein N-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). N-Myristoylation promotes weak and reversible protein-membrane and protein-protein interactions (Farazi et al., 2001). The N- myristoylation of human annexins A13a and A13b have 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 A1, A2, A3, A6, A7, A11, A13b, and B7, have 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 A13b (Gerke et Moss, 2002). Annexin A2 is involved in Ca²⁺-regulated exocytosis in permeabilized chromaffin cells: the time-dependent loss of

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

In polarized epithelial cells, annexin A13b associates specifically with sphingolipid- and cholesterol-rich membrane domains of the trans-Golgi network, and myristoylated annexin A13b 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 A1, A2 and A6 are present on endosomal compartments, and unique endosome targeting sequences have been identified in the N-terminal domain of annexins A1 and A2 (Emans et al., 1993; Seemann et al., 1996). In fibroblasts from annexin A1-knockout mice, multivesicular endosomes are formed in the absence of annexin A1, but these endosomes contain fewer internal vesicles (Emans et al., 1993; Seemann et al., 1996).

1.4.1.3- Annexins and membrane domains

In the sarcolemma of smooth muscle cells, changes in intracellular Ca²⁺

concentrations occurring during smooth muscle contraction appear to regulate the dynamics of rafts, their lateral assembly, and association with the actin cytoskeleton. These changes correlate with the Ca²⁺-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 Ca²⁺ rise in smooth muscle cells triggers the binding of annexin A2 to lipid rafts and a clustering of these rafts which is promoted by lateral annexin assembly (Babiychuk and Draeger, 2000).

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

1.4.1.4- Annexin as ions channels regulators

Theoretical calculations predict that human annexin A5 could sufficiently perturb the organization of lipids in the bilayer at the site of Ca²⁺-dependent attachment to effectively electroporate the membrane and therefore permit Ca²⁺ entry (Demange et al., 1994).

Human annexins A2, A4 and A6 modulate plasma membrane Cl^--channels and sarcoplasmic reticulum Ca²⁺-release channels (Gerke and Moss, 2002).

1.4.2- Annexins in disease

1.4.2.1- Diabetes, cardiovascular disease

Annexin A2 has been implicated in the pathology of both type I and type II diabetes, due to its role in vascular endothelial biology and hypercoagulation which occurs in both forms of diabetes (Ishii et al., 2001). Cell-surface annexin A2 that functions as a co-receptor for tissue plasminogen and plasminogen activator promotes the production of plasmin, which dissolves blood clots (through fibrin degradation 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 deposition of fibrin in their tissues (Ling et al., 2004). Plasmin production is reduced in high glucose and insulin conditions. This reduction was partially prevented upon addition of annexin A2 (Ishii et al., 2001).

Annexin A2 is highly affected by several risk factors that are linked with diabetes 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 epithelial cells (Rowan et al., 2002). Annexin A2 may also contribute to diabetes pathology through its phosphorylation. Insulin-dependent tyrosine phosphorylation of annexin A2 is known to result in changes in the actin cytoskeleton. This remodelling significantly alters the cell morphology, such that actin domes are formed, and also diminishes cell adhesion (Rescher et al., 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 hemorrhagic 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 also to stimulate the generation of plasmin from tPA twice as efficiently as other leukemic cells (Menell et al., 1999). Plasmin generation 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 reduction 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 exogenously administered annexin A1 inhibits neutrophil extravasations and thereby limits the degree of inflammation (Perretti et al., 2003). This activity is retained in N-terminal annexin A1 peptides, which are probably generated by proteolysis at sites of inflammation and interact with specific receptors on leukocytes (Perretti et al., 2003).

Annexin A1 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 A1 has also been identified as a selective surface marker of the vascular endothelium in several solid tumours, and radioimmunotherapy using anti-annexin A1 antibodies has been shown to destroy such annexin A1 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 proliferation and that heterozygous annexin A7 knock-out mice have a more cancer-prone phenotype (Srivastava et al., 2001). Annexin A6 has tumor suppressor activity in human A431 cells (Chetcuti et al., 2001). Annexin A1 and annexin A2 appear to be downregulated 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 roles 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

AnnAt1 All tissues,

abundant in roots / Cytoplasm

Exocytosis, Peroxydase activity, Temperature responses, Salt 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, Temperature responses, Salt stress, Water stress

AnnAt3 All tissues, most abundant in roots and flowers / -

Growth and development, Temperature responses, Water stress

AnnAt4 Most abundant in roots and flowers / Cytoplasm

Growth and development, Temperature responses, Salt stress

AnnAt5 All tissues, most abundant in flowers and roots / -

Growth and development, Interaction with actin, Salt stress, Light responses AnnAt6 Mainly in flower /- Growth and development,

Light responses,

Temperature responses, Salt stress, Water stress

AnnAt7 Mainly in flower /- Growth and development, Temperature responses, Salt stress

AnnAt8 All tissues /- Growth and development, Salt stress, Water stress Tobacco Sp32 All tissues /

Cytoplasm

Cell wall maturation (cell cycle)

Proust et al., 1999 Celery,

Tobacco

VCaB42 All tissues / Cytoplasm

Vacuole biogenesis Seals et al., 1994;

Seals and

Randall, 1997 M.

truncatula

MtAnn1 All tissues / Cytoplasm

Cell division, Nodule organogenesis, Biotic stress

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

1992 Tomato p34, p35 All tissues /

Cytoplasm

Growth and development, F- actin binding, Nucleotide phosphatase activity

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

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

Wheat p39, All tissues / Sensors or transducers of Breton et al.,

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Plant Gene Localization / Subcellular localization

Implication in various biological function

Authors

Maize p33, p34, p35

All tissues / Cytoplasm

Exocytosis, Ca²⁺ transport and [Ca²⁺]cyt regulation, ATPase activity, Peroxidase activity

Blackbourn and Battey, 1993;

Carroll 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 All tissues / Nucleus

Cell division, Osmotic stress, Water stress, Hormonal control

Kovács et al., 1998

M. pudica p35 All tissues / Cytoplasm

Light responses (nyctinastic movements)

Hoshino et al., 2004

C.

annuum

annexin 24 (Ca32)

All tissues / - Ca²⁺ channel, Hormonal control

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

Plant annexins functions remain to be determined. However, plant annexins have 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; Kovács et al., 1998) and on their biochemical properties (Mc Clung et al., 1994; Calvert et al., 1996; Hofmann et al., 2000;

Laohavisit et al., 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 age 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 all tissues examined (hypocotyl, cotyledons, 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 cotyledons (Clark et al., 2001; 2005a; Cantero et al., 2006).

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

1.5.2- Exocytosis and endocytosis

Exocytosis is a fundamental process for the secretion 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 et al., 1998).

Annexins AnnAt1 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 AnnAt1 and AnnAt2 could be involved in secretion mediated by the Golgi 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 have 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 present 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 region and cytoplasm. In mitotic cells, it is mainly detected at chromosomes. At the end of mitosis and after nuclear membrane formation, AnnMs2 is again detectable in perinuclear region (Kovács et al., 1998).

Nodulation factors (or Nod factors) induce the expression of M. truncatula annexin MtAnn1, and studies of co-location with a construction MtAnn1-GFP suggest that the gene MtAnn1 is involved in the early stages of cell division necessary for nodules formation. MtAnn1 is a cytosolic protein that accumulates 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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Fig. 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 µm-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 µm-longitudinal sections of S.

meliloti-inoculated roots (48 hpi). Tissues have been stained both for GUS and β-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^-8 M Nod factors. (g) 80 µm-thick transverse section of agarose- embedded M. truncatula root 48 h after 10^-9M Nod factor addition. GUS staining is indicated in the outer cortex (oc) and endodermal tissues (arrowhead). (h-k) sequential stages of lateral root development in transgenic M. truncatula roots. (h) GUS activity is localised in dividing cells of the lateral root primordium; (i) at the base of an emerging lateral root; (j) the GUS staining pattern of the lateral appears ring-like when viewed from above, (k) GUS activity is present in the subapical region of the root tip referred to as the distal elongation zone. Bars=50 µm. Taken from de Carvalho-Niebel et al. (2002).

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1.5.5- Ca²⁺ channels formation, [Ca²⁺]cyt modulation

Crystallographic studies have indicated that some annexins possess a hydrophilic pore located at the intersection of four domains, forming a prominent ion channel covered with highly 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 also been suggested for wheat annexin p39 (Breton et al., 2000).

Laohavisit et al. (2009) have shown that Z. mays annexins p33 and p35 are involved in Ca²⁺ transport and [Ca²⁺]cyt regulation.

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

Actin filaments are involved in maintaining cell shape and are involved in cell signalling (Drøbak et al., 2004). AnnAt5 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 AnnAt5, this motif overlaps that of Ca²⁺ binding, which could have structural implications in terms of Ca²⁺-annexin-actin interaction (Clark et al, 2001).

Calvert et al. (1996) have 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 nucleotide 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 et al., 1998).

In maize, annexins p33 and p35 have ATPase activity in vitro (Mc Clung et al., 1994). Shin and Brown (1999) have 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 Mg²⁺ and is inhibited by Ca²⁺ (Shin and Brown, 1999).

1.5.7- Peroxidase activity

In examining the primary structure of annexins AnnAt1 (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). AnnAt1, p33 and p35 have peroxidase activity in vitro (Gidrol et al., 1996; Laohavisit et al., 2009). A post-translational modification of AnnAt1 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, AnnAt5 expression increases with red light, and it decreases reversibly with far-red light. AnnAt6 has the same behavior in the cotyledons (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- Temperature and thermal stress

A temperature of 4°C significantly induced AnnAt1 and AnnAt3 expression, and significantly represses AnnAt2 expression (Cantero et al., 2006). A temperature of 37°C significantly induced AnnAt2, AnnAt6 and AnnAt7 expression and significantly suppresses AnnAt3 and AnnAt4 expression (Cantero et al., 2006).

Breton et al. (2000) have highlighted four annexins in a particular variety of wheat resistant 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 also present in a wheat variety little resistant to cold, the researchers concluded that these

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

1.6.3- Osmotic, water and salt stress

In A. thaliana, salt stress significantly induced expression of AnnAt1, AnnAt4, AnnAt5, AnnAt6, AnnAt7 and AnnAt8, and significantly represses AnnAt2 expression (Lee et al., 2004). Salt stress induces translocation of AnnAt1 and AnnAt4 proteins from cytosol to membranes (Lee et al., 2004).

Water stress significantly induces expression of genes AnnAt1, AnnAt3, AnnAt6 and AnnAt8 and significantly represses AnnAt2 expression (Cantero et al., 2006). Konopka-Postupolska et al. (2009) have regenerated insertion mutants in which AnnAt1 transcripts were not detected (ΔAnnAt1 plants) and other transgenic plants which overexpress the gene AnnAt1 (35S:: AnnAt1 plants). Two tests were performed. First, one month old seedlings have been subjected to water stress for two weeks. Five days after stress, the first signs of drying have been observed in ΔAnnAt1 plants, whereas normal plants (Col-0) and 35S::AnnAt1 plants remained turgid and green. After prolonged stress (two weeks), ΔAnnAt1 plants completely lose their turgidity and the 35S::AnnAt1 plants are more resistant than Col-0 plants (Fig. 7A).

Secondly, one month old seedlings have been subjected to water stress for three weeks to complete loss of turgid, and irrigation was restored for two weeks. ΔAnnAt1 plants did not survive drying while the 35S::AnnAt1 plants have all found their turgidity (Fig. 7B). These results indicate that drought

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tolerance is positively correlated with AnnAt1 expression level (Konopka- Postupolska et al., 2009).

Treatment of M. sativa cells by osmotic stress, salt stress and water stress allowed demonstrating overexpression of annexin AnnMs2 during these stress (Kovács et al., 1998).

Fig. 7 Tolerance to water stress. A (short-term drought): Col-0, ΔAnnAt1 and 35S::AnnAt1 plants were grown; after 4 weeks of culture, watering was suspended; the photo shows the differences between the reactions of plants after 5 days without watering. B (long-term drought): after 8 weeks of culture (Col-0, ΔAnnAt1 and 35S:: AnnAt1 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).

35S::AnnAt1 Col-0 ∆AnnAt1

Col-0

∆AnnAt1

35S::AnnAt1

A

B

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1.6.4- Oxidative Stress

Superoxide radicals are a common cause of damage at the cellular level in all aerobic organisms. AnnAt1 (also called Oxy5), isolated from A. thaliana during oxidative stress, complement an E. coli mutant ΔoxyR, suggesting a role for this protein in response to oxidative stress (Gidrol et al., 1996 ; Konopka- Postupolska et al., 2009). H2O2 accumulation in stomata cells is negatively correlated with AnnAt1 gene expression and this accumulation was higher in ΔAnnAt1 plants (which downregulate AnnAt1), and is low in 35S::AnnAt1 plants (that overexpress AnnAt1) (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 cells of the internodes and accumulate at the plasma membrane of these cells 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 nucleus of epidermal cells of plumules. Redistribution of this annexin to the cells 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 AnxMt1 in M.

truncatula. AnxMt1 is involved in early stages of nodule formation (de Carvalho- Niebel et al., 2002).

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

1.7- Hormonal control of annexin expression

Phytohormones direct the processes of growth and development in plants (Paciorek and Friml, 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 control of expression of these genes (reviewed by Mortimer et al., 2008). Ethylene is the hormone that initiates the physiological processes involved in fruit ripening (Johnson and Ecker, 1997). Auxin plays a central role in many physiological and developmental processes, including fruit development (Kepinski and Leyser, 2005a).

Osmotic stress, salt stress and water stress act through the ABA, which also causes an increased expression of genes involved in these stresses (Kovács et al., 1998; Lee et al., 2004; Hoshino et al., 2004; Cantero et al., 2006). ABA induced overexpression and S-glutathiolation of annexin AnnAt1, 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, subsequent 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 differentiation, cell elongation, inhibition of buds growth) are similar to those observed after treatment with auxin, and others (deformed leaves, proliferation of buds, slowing senescence) are typical effects of cytokinins (Vandeputte et al., 2005).

Indeed, R. fascians produces and secretes IAA (Vandeputte et al., 2005) and several cytokinins (Eason et al., 1996).

The study of differential gene expression before and after infection of tobacco BY-2 cells by R. fascians has identified a highly 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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role of Ntann12 gene. This thesis aims to investigate the role 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 downregulation of Ntann12 into transgenic tobacco plants have been done with the view to study the consequences 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.

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Chapter Two

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

(Molecular Plant Pathology 8(2), 185-194, 2007)

Olivier Vandeputte¹, Yves Oukouomi Lowe¹, Sylvia Burssens², Damien van Raemdonck¹, David Hutin¹, Delphine Boniver¹, Danny Geelen³, Mondher El Jaziri¹ and Marie Baucher¹ (My contributions in this work are studies made with pNtann12-GUS plants and RT-qPCR analysis of Ntann12 expression during leafy gall ontogenesis with WT plants.)

1Laboratoire de Biotechnologie Végétale, Université Libre de Bruxelles, Rue Adrienne Bolland 8, B-6041 Gosselies, Belgium

2The Institute of Plant Biotechnology for Developing Countries, Department of Molecular Genetics, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

3Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologie Park 927, B-9052 Gent, Belgium

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

2.1- Introduction

The phytopathogenic Gram-positive bacterium R. fascians infects a wide range of monocotyledonous and dicotyledonous plants causing several types of malformations (Goethals et al., 2001). The most severe symptom is the leafy gall, a particular hyperplasia comprising small leaves and numerous buds that are inhibited in their further outgrowth (Vereecke et al., 2000). The pathogenicity of R. fascians has been linked to the presence of a linear plasmid, pFiD188, which carries several virulence genes including an isopentenyl transferase (ipt) homologue that is indispensable for leafy gall formation (Crespi et al., 1992). Indeed, mutation in this ipt homologue and plasmid-free strains, such as the avirulent strain D188-5, are unable to induce leafy gall development (Crespi et al., 1992; 1994).

At present, no molecular clue about the recognition and/or signalling processes involved during the R. fascians – plant interaction has been reported.

Studying these processes is hampered by the difficulty to localize plant cells responding to R. fascians and by the unpredictable nature of leafy gall emergence (Simón-Mateo et al., 2006). In that context, the use of a cell suspension model provides a suitable alternative to investigate molecular

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processes occurring during the early cross-talks between plants and R.

fascians. Indeed, a more homogeneous plant cell response can be obtained due to the increased number of plant cells that are in contact with the bacteria in comparison to plants infected at localized sites where reacting cells are surrounded by numerous non-infected cells. Therefore, gene expression in non- infected and in R. fascians infected BY-2 tobacco cell suspension cultures was compared using differential display. This transcript profiling resulted in the identification of Ntann12, a gene coding for a novel putative annexin in tobacco.

Annexins are viewed as potential links between Ca²⁺ as an intracellular signal, and the regulation of membrane functions (Gerke et al., 2005). In this study, a focus is made on the analysis of Ntann12 expression in BY-2 cells grown under biotic and abiotic stress conditions, and in tobacco plants during seedling development and leafy gall ontogenesis.

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2.2- Experimental procedures

2.2.1- Plant material and growth conditions

N. tabacum L. cv. Bright Yellow 2 (BY-2) cell suspension was cultivated and maintained in modified liquid Linsmaier and Skoog (LS) medium, containing auxin (IAA or 2,4-D) (Nagata et al., 1992).

Non-transgenic and transgenic tobacco plants (Nicotiana tabacum cv.

Havana) were grown aseptically on half-strength MS medium (Duchefa) supplemented with appropriate antibiotics when needed, or in a greenhouse at 25°C with a 16h day/8h night photoperiod.

Developing leafy galls used for RT-qPCR experiments were induced as described previously (de O. Manes et al., 2001) on in vitro four-weeks-old tobacco plants following spot-inoculation of axillary buds. Control plants were spot-inoculated with drops of the avirulent strain D188-5 (Desomer et al., 1988;

Crespi et al., 1992) and non-infected plants (NIP) were mock-inoculated with drops of YEB medium. For RNA extraction, infected tissues were sampled at regular intervals and immediately frozen in liquid nitrogen.

2.2.2- Infection of BY-2 cell suspensions by R. fascians and other bacteria BY-2 cell suspension was incubated for 2 days in the dark at 28°C under shaking (130 rpm) before infection. For the differential display, the virulent strain D188 of R. fascians was used. After 2 days incubation in liquid YEB medium (OD600nm ~ 2), 500 µl of bacteria suspension were inoculated into 100 ml of BY-

An investigation into the putative functions of the tobacco Annexin Ntann12