Nadine Paris

Sebastian Wolf

Kian Hématy

Magelie Uyttewaal

Katia Belcram

Gwendal Cueff

David Bouchez

Taline Elmaya

Ivan Le Masson

Véronique Pautot

Phillipe Le Bris

Aurelie Hurel

Olivier Grandjean

Qinwu, Jie, Jing, Zeyun, Yannick, Claudia Chuande

Laia Federica

Keywords:

I would like to convey my gratitude to all those who made this thesis possible, contributed to the build-up of my scientific personality

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List of Figures

Primary cell wall

Plant cells are surrounded by strong cell walls, which allow turgor pressure to be built up, determine cell and organ shape and provide protection against biotic stresses. Cell walls also must be able to expand to allow growth (Cosgrove 2005;Hofte and Voxeur 2017). How cell walls are modified to enable cellular expansion, while maintaining their integrity remains an important and fascinating scientific question.

The dual function of walls of growing cells (so-called primary walls) relies on the formation of reinforced gel structures consisting of cellulosic microfibrils, connected to matrix phase that consists mainly of hemicellulose and pectins (Somerville et al. 2004;[START_REF] Burton | Heterogeneity in the chemistry, structure and function of plant cell walls[END_REF]. Cellulose microfibrils with their extreme tensile strength are the main load-bearing elements of the cell wall. They are composed of β-1,4-linked glucose, and are synthesized by cellulose synthase complexes embedded in the plasma membrane (PM) (Somerville et al. 2004). Hemicellulose and pectin are synthesized in the Golgi apparatus and then secreted into the apoplast (Figure 1.1). Primary cell walls also contain a small amount (10%) of proteins, which can connect to the polysaccharides network and can modify the wall structure during development and in reaction to diverse environmental changes.

An important challenge is to understand how the cell wall extensibility is regulated.

Recent studies showed that cell wall loosening agents target the load-bearing cellulosexyloglucan interaction sites, also called 'biomechanical hotspots', to regulate cell wall loosening [START_REF] Cosgrove | Re-constructing our models of cellulose and primary cell wall assembly[END_REF][START_REF] Cosgrove | Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes[END_REF]. Although some evidence suggested that pectin also has a load-bearing role in expanding cells, this point remained controversial. In this Introduction, I will provide an update of the literature on the link between pectin modification and cell wall loosening, with a focus on the contradictions and challenges presented in these studies, and discuss evidence showing how pectin metabolism is intimately linked to the pH and the rheology of the cell wall.

Pectins and homogalacturonan modification

Pectins influence various cell wall properties such as porosity, surface charge, pH, and ion balance by forming networks and trapping solute molecules [START_REF] Harholt | Biosynthesis of pectin[END_REF].

Pectins are block co-polymers, characterized by the presence of galacturonic acid (GalA). They represent up to 40% of the dry mass in primary cell walls of dicots. Three types of pectin polymers can be distinguished: homogalacturonan (HG), rhamnogalacturonans (RG)-I and RG-II (Figure 1.1). HG is the most abundant form, representing around 20% of the dry cell wall in Arabidopsis leaves [START_REF] Zablackis | Characterization of the cellwall polysaccharides of Arabidopsis thaliana leaves[END_REF].

HG synthesis and secretion

The main steps of HG synthesis include polymerization, methylesterification and acetylation, all of which occur in the Golgi apparatus [START_REF] Caffall | The structure, function, and biosynthesis of plant cell wall pectic polysaccharides[END_REF][START_REF] Wolf | Homogalacturonan methyl-esterification and plant development[END_REF]. The HG backbone is a polymer of alpha-1, 4-linked GalA residues, which is synthesized from the nucleotide-sugar precursor, UDP-D-GalA, which is in turn synthesized from UDP-D-Glucose via a UDP-D-GlcA intermediate.

Galacturonosyltransferases (GAUTs) specifically catalyze the transfer of D-GalA from UDP-D-GalA to the non-reducing end of a growing stretch of HG via a luminally facing GAUT catalytic domain, in the cis-Golgi [START_REF] Sterling | The catalytic site of the pectin biosynthetic enzyme alpha-1, 4-galacturonosyltransferase is located in the lumen of the Golgi[END_REF]. So far, GalA transferase activity has been confirmed in vitro for GAUT1 and GAUT4 [START_REF] Sterling | The catalytic site of the pectin biosynthetic enzyme alpha-1, 4-galacturonosyltransferase is located in the lumen of the Golgi[END_REF][START_REF] Biswal | Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis[END_REF]. Two polypeptides of GAUT1 bind to one polypeptide of GAUT7, and this heterocomplex is responsible for retaining GAUT1 in the Golgi membranes [START_REF] Atmodjo | Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan: galacturonosyltransferase complex[END_REF]). In the medial-Golgi, the HG backbone is methylesterified at the C6 position by pectin methyl transferase (PMT), which transfers a methyl group from S-adenosyl methionine.

The QUASIMODO-2/TUMOROUS SHOOT DEVELOPMENT-2 (QUA2/TSD2) has been proposed to a pectin A B methyltransferase [START_REF] Krupkova | The tumorous shoot development gene of Arabidopsis encoding a putative methyltransferase is required for cell adhesion and coordinated plant development[END_REF]Mouille et al. 2007). The qua2 mutant showed a phenotype similar to that of qua1, another putative HG GAUT, including a 50% reduction in HG content. The latter suggests that the methylesterification of HG is a critical step for the GAUT-catalyzed polymerization reaction. Another family of HG methylesterases is encoded by cotton Golgi-related (CGR) 2/3 [START_REF] Kim | CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana[END_REF]. It is interesting to note that non-methylesterified HG accumulated in a double cgr 2/3 mutant in contrast to qua2, which had reduced HG levels without a change in the degree of methylesterification (DM). The reason for this difference is not known but it is possible that GAUTs and CGRs synthesize HG in the context of different polymers.

Alternatively, the HG produced in cgr 2/3 may be more prone to HG demethylesterification in the cell wall for some unknown reason. The acetylation of HG is catalyzed by pectin O-acetyltransferase (PAT), which transfers an acetyl group from acetyl CoA to a HG acceptor. Acetyl groups can decorate the GalA residues of HG at the O-2 or O-3 positions [START_REF] Ishii | O-acetylated oligosaccharides from pectins of potato tuber cell walls[END_REF].

The secretion process of HG remains poorly understood. HG has been clearly shown to localize in vesicular structures using immunoelectron microscopy [START_REF] Kim | The plant secretory pathway: an essential factory for building the plant cell wall[END_REF]. HG-specific antibody labeling revealed a non-uniform distribution of HG in pea parenchyma cells [START_REF] Willats | Pectin: cell biology and prospects for functional analysis[END_REF] as well as Nicotiana tabacum and Solanum chacoense pollen tubes (PTs) (Bosch and Hepler 2005;[START_REF] Parre | Pectin and the role of the physical properties of the cell wall in pollen tube growth of Solanum chacoense[END_REF]. This supports the existence of a mechanism for localized HG secretion to the cell wall, or the concentration of HG in specific cell wall regions in muro after secretion. An example of directional pectin secretion is observed during the deposition of Arabidopsis seed coat mucilage, which is mainly composed of RG-I, whereas HG is deposited specifically at the outward facing cell walls, through polarized secretion [START_REF] Haughn | Arabidopsis seed coat mucilage is a specialized cell wall that can be used as a model for genetic analysis of plant cell wall structure and function[END_REF]. The trans-Golgi network-localized ECHIDNA/YIP4a and 4b protein complex is required for correctly localized secretion of cell wall polysaccharides, including HG, in the Arabidopsis seed coat cells [START_REF] Gendre | Trans-Golgi network localized ECHIDNA/Ypt interacting protein complex is required for the secretion of cell wall polysaccharides in Arabidopsis[END_REF]. The echidna mutant shows a highly reduced accumulation of mucilage in the cell walls of the seed coat, but mucilage can be detected in the vacuole, highlighting the importance of a strict cellular control of cell wall secretion [START_REF] Mcfarlane | Cell wall polysaccharides are mislocalized to the Vacuole in echidna mutants[END_REF].

HG demethylesterification

Upon secretion, HG can be selectively demethylesterified in muro by pectin methylesterases (PMEs) [START_REF] Pelloux | New insights into pectin methylesterase structure and function[END_REF]. PMEs are essential for the gelling behavior of pectins and the rheology (see below) of the cell wall. PMEs are encoded by large multigene families (e.g. 69 members in Arabidopsis). They are classified into two groups based on the presence (type I) and absence (type II) of a PRO domain, which shares similarities with the PME inhibitor (PMEI) domain [START_REF] Pelloux | New insights into pectin methylesterase structure and function[END_REF]). For type I PMEs, two conserved cleavage motifs were identified between PRO and PME domains, which can be cleaved in a secretory compartment by AtS1P, a subtilisin-like protease [START_REF] Wolf | Homogalacturonan methyl-esterification and plant development[END_REF]). The 3D crystallographic structure of plant PME shows that mature PME has three parallel right-handed β-sheets. The pectin-binding cleft is formed by two loops that are composed of aromatic residues (Figure 1.2, Di Matteo et al. 2005;[START_REF] Pelloux | New insights into pectin methylesterase structure and function[END_REF].

Most plant PMEs studied so far are processive, remove methylester groups from stretches of GalA residues, resulting in a blockwise distribution of methylester groups, whereas fungal PMEs are mostly non-processive, they dissociate from the substrate after each enzymatic reaction, leading to random demethylesterification patterns. The terms "blockiness" (Degree of blockiness (DB)) defines the ratio of contiguous free GalA units relative to the total amount of free GalA residues in the HG. This value is calculated from the amount of monomer, dimer, and trimer of GalA, produced when pectin is incubated with endopolygalacturonase, divided by the total amount of GalA present in the pectin sample (Daas et al. 2009). The processivity of the plant PMEs, however, can be influenced by the reaction conditions. For instance, in high pectin concentrations and in the presence of Ca 2+ , a processive PME can become non-processive [START_REF] Vincent | Microrheological investigations give insights into the microstructure and functionality of pectin gels[END_REF].

In many developmental processes, PMEs are regulated by either differential transcription or posttranslational control by protein inhibitors (PMEIs). In Arabidopsis, 76 genes have been annotated as encoding putative PMEIs. PMEIs inhibit plant PMEs through interaction in a complex of 1:1 stoichiometry, in which PMEI covers the pectinbinding cleft of PME and hides its putative catalytic site, thereby impairing access to the substrate (Di Matteo et al. 2005). The structure of the PME-PMEI complex is shown in Figure 1.2. Until now, 14 PMEIs have been functionally analyzed and shown to play different functions in Arabidopsis growth and development (Table .1.1).

Pectin network and connections to cellulose, hemicellulose and proteins

HG is found in a variety of cell wall fractions, from water-or chelator-soluble, alkaline extractable to strongly cellulose-associated fractions [START_REF] Caffall | The structure, function, and biosynthesis of plant cell wall pectic polysaccharides[END_REF]. This most likely reflects the methylesterification pattern and the covalent association of HG with the other pectic polymers RG-I and RG-II [START_REF] Vincken | If homogalacturonan were a side chain of rhamnogalacturonan I. Implications for cell wall architecture[END_REF]Atmodjo et al. 2013;[START_REF] Popper | Widespread occurrence of a covalent linkage between xyloglucan and acidic polysaccharides in suspension-cultured angiosperm Cells[END_REF] and proteoglycans [START_REF] Caffall | The structure, function, and biosynthesis of plant cell wall pectic polysaccharides[END_REF], which in turn can form covalent and non-covalent cross-links with other cell wall polymers. RG-II can form dimers via borate diester linkages. More than 95% of RG-II exists as dimers, which determine strength and pore size of the cell wall and are essential for cell wall integrity as shown by the disorganized walls in plants starved for boron and the lethality of mutants lacking RG-II dimers. RG-I can bind to cellulose in vitro through its arabinan, galactan or xylan side chains and close pectin-cellulose interactions have been observed in intact, never-dried cell walls using NMR. HG also interacts with xyloglucan (XG) as shown by the release of XG fragments from the cell wall by endopolygalacturonase treatment, but no covalent crosslinks between the two polymers have been observed so far [START_REF] Talmadge | The structure of plant cell walls. I. The macromolecular components of the walls of suspension-cultured sycamore cells with a detailed analysis of the pectic polysaccharides[END_REF][START_REF] Popper | Widespread occurrence of a covalent linkage between xyloglucan and acidic polysaccharides in suspension-cultured angiosperm Cells[END_REF]. XG strongly binds to cellulose in vitro and cellulose-XG-cellulose connections are observed in intact cell walls. The latter form the so-called biomechanical hotspots, which are thought to be the targets of specialized microbial endoglucanases and expansins, which can promote wall "creep" at low pH in extensibility tests [START_REF] Cosgrove | Re-constructing our models of cellulose and primary cell wall assembly[END_REF][START_REF] Cosgrove | Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes[END_REF]. An alternative way to crosslink pectins to other cell wall polymers is via structural proteins of the cell walls.

Structural proteins make up 2 ~ 10% of wall dry weight (O'Neill et al. 2003) and are classified into four groups. Three of them are named for their uniquely enriched amino acid: the hydroxyproline-rich glycoproteins (HRGPs), the proline-rich proteins (PRPs), and the glycine-rich proteins (GRPs). Arabinogalactan proteins (AGPs) form the fourth class. AGPs are more aptly named proteoglycans because they can be more than 95% carbohydrate (Buchanan et al. 2000). Recently, linkages between pectins and HRGPs and AGPs have been identified. Extensin is one of the best-studied HRGPs of plants, which consists of repeating Serine-(Hydoxyproline)4 and Tyrosine-Lysine-Tyrosine sequences (Buchanan et al. 2000). Loss-of-function mutants for AtEXT3 are embryo lethal with incomplete cell plates, indicating the important role of extensins [START_REF] Cannon | Selfassembly of the plant cell wall requires an extensin scaffold[END_REF]. Pectin-extensin binding was suggested by an in vitro study on the physical properties of a thin film made by the successive adsorption of low methylesterifed pectin and extensin. It was observed that the positively charged extensins create an interpenetrating network with negatively charged pectin and that extensin promotes the dehydration and compaction of the pectin [START_REF] Valentin | Elaboration of extensinpectin thin film model of primary plant cell wall[END_REF]. The discovery of the remarkable ARABINOXYLAN PECTIN ARABINOGALACTAN PROTEIN1 (APAP1) from the medium and cell walls of Arabidopsis cell cultures revealed pectin-AGPs linkages (Figure 1.1). APAP1 consists of an AGP backbone, which is linked to RG-I/HG through a rhamnosyl residue in the AG and with arabinoxylan attached either to a rhamnosyl residue in the RG-I domain or directly to the arabinosyl residue in the AG domain [START_REF] Tan | An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein[END_REF]. The importance of APAP1 in the cell wall architecture is

shown by the increased extractability of pectic polysaccharides in APAP1 mutants and the observation that more than 95% of RG-I released from cell walls of Arabidopsis suspension-cultured cells was covalently attached to APAP1 [START_REF] Tan | An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein[END_REF]. In summary, in contrast to previous models in which cellulose-hemicellulose forms the load-bearing network embedded in, but independent from the pectin network, which primarily has a role in controlling the porosity of the cell wall, recent finding showed that pectic polysaccharides form one interconnected network with cellulose and hemicellulose and cell wall structural proteins in the walls through covalent and noncovalent linkages. As a result, the whole cell wall behaves as a fiber-reinforced hydrogel.

Pectin gels and rheology

Gels and phase transition

A gel is a form of matter intermediate between a solid and a liquid. It consists of polymers, or long-chain molecules, cross-linked to create a tangled network immersed in a liquid medium. The properties of the gel depend strongly on the interaction between polymers and liquid. Interestingly, around phase transition points, drastic changes in gel volume can be brought about by small changes in the external conditions. For example (Figure 1.3), the hydrophobicity of the medium, the pH, ionic strength, temperature or mechanical stresses can influence the gel volume and one can observe phase transition points were extremely small changes in any of these parameters induce abrupt volume changes. The gel volume appears to be governed by three forces that act to expand or shrink the polymer network: the rubber elasticity of the polymers, the polymer-polymer affinity and the hydrogen-ion pressure (Tanaka 1980). Interestingly, many biological gels are poised around such phase transitions, which provides a mechanism to render the associated responses exquisitely sensitive to small changes in the intra-or extra-cellular environment [START_REF] Pollack | Cells, gels and the engines of life[END_REF]. A B A B C

Pectin gels and their properties

In vitro: Pectins are typical gelling agents, traditionally used to make jams and jellies.

Pectins also play a critical role in planta in the control of the hydration and the rheology of the cell wall [START_REF] Parre | Pectin and the role of the physical properties of the cell wall in pollen tube growth of Solanum chacoense[END_REF][START_REF] Suárez | Cellular localization and levels of pectins and arabinogalactan proteins in olive (Olea europaea L.) pistil tissues during development: Implications for pollen-pistil interaction[END_REF][START_REF] Willats | Pectin: cell biology and prospects for functional analysis[END_REF]. In vitro studies showed that pectin at low concentrations in water cannot gelate but forms a solution, because the pectin molecules are too far apart to interact with one another (Guimara et al. 2009). The viscosity of pectin solutions increases with increasing pectin concentration. When the pectin concentration exceeds a certain percentage (depending on other conditions), a hydrogel is formed consisting of a 3D network of polymer chains, that entraps solvent and solutes (Loh et al. 2014;Tanaka 1980).

Depending on the DM and the presence of Ca 2+ , pectin gels can be classified into two types: Low DM (LDM, DM < 30%) and High DM (HDM, DM > 55%) gels (Figure 1.4 and 1.5).

HDM gel:

To form a HDM gel, the pH has to be adjusted to below 3.5 and a large amount of co-solute has to be added. Two speculative structure models for HDM gel were established based on fiber diffraction studies (Walkinshaw and Arnott 1981;[START_REF] Braccini | Chain-chain interactions for methyl polygalacturonate: Models for high methyl-esterified pectin junction zones[END_REF]. In these models, the polygalacturonate chains forms a 3-fold helix with a rise per residue of 4.3 Å (Figure 1.4). Walkinshaw and Arnott. (1981) reported that HDM gels condensed into uniaxially oriented fibers and that the parallel arrangement of the chains provided the most efficient packing for the postulated net on the basis of theoretical calculations. One model of the junction zones in HDM gels is shown in Figure 1.4b. [START_REF] Braccini | Chain-chain interactions for methyl polygalacturonate: Models for high methyl-esterified pectin junction zones[END_REF] instead, found that the antiparallel arrangement of methyl polygalacturonate chains is much more favorable than the parallel one, with nearly a 2-fold ratio between the interaction energies of the best associations, as shown in Figure 1.4c. 6). Hydrogen bonds between pectin molecules, favored by the conformation of adjacent GalA units, are the main interaction that sustains the HDM gel structure. Individual hydrogen bonds are relatively weak, but a large number of them confer significant thermodynamic stability to the gel. Hydrogen bonds only are insufficient to overcome the entropic barrier to gelation. HDM gels need the methylesters to coalescence, which can reduce the surface area exposed to water and therefore overall decrease the entropy of HG molecules. The contribution of hydrophobic interactions to the free energy of junction zones in HDM is half that arising from hydrogen bonding [START_REF] Oakenfull | Hydrophobic interaction in the gelation of high methoxyl pectins[END_REF]. An increase in hydrophobic interactions can make HDM more viscous (Guimara et al. 2009).

The conditions required for gelation and the properties of the gel ultimately depend on intrinsic and extrinsic factors. Intrinsic factors include the number and distribution pattern (the DB) of free carboxyl groups, the molecular weight and pectin concentration, while extrinsic factors include the types of other pectin polymers, co-solute concentration, ionic strength (Ca 2+ concentration and pH), and temperature [START_REF] Gigli | Rheological behaviour of low-methoxyl pectin gels over an extended frequency window[END_REF]. A high co-solute concentration, such as sucrose, promotes gelling through reducing water activity, which promotes chain-chain interactions [START_REF] Thibault | Physico-chemical properties of pectins in the cell walls and after extraction[END_REF]. An intermediate temperature range, from 10°C to 30°C can facilitate both hydrogen bonds and hydrophobic interactions [START_REF] Da Silva | Kinetics and thermal behaviour of the structure formation process in HMP/sucrose gelation[END_REF]. Lowering the pH below the pKa of GalA (3.24) protonates carboxylic groups, reducing electrostatic repulsions along and between pectin chains and promotes the formation of inter-and intra-molecular hydrogen bonds between non-dissociated carboxylic and alcohol groups [START_REF] Thakur | Chemistry and uses of pectin -A review[END_REF]. concentration and pH), and temperature [START_REF] Chan | Pectin as a rheology modifier: origin, structure, commercial production and rheology[END_REF]. The gel strength of LDM gels increases with increasing GalA concentration and with increasing DB [START_REF] Basak | Formation and rupture of Ca 2+ induced pectin biopolymer gels[END_REF][START_REF] Ngouémazong | Stiffness of Ca 2+ -pectin gels: Combined effects of degree and pattern of methylesterification for various Ca 2+ concentrations[END_REF][START_REF] Vincent | Microrheological investigations give insights into the microstructure and functionality of pectin gels[END_REF]. If the DM is too low, HG can precipitate out of solution instead of forming a stable gel, in which the polymers are dynamically pinned and the system mobility is reduced significantly, and extensive lateral association of the dimers is dynamically forbidden (Region c in Figure 1.7, [START_REF] Vincent | Microrheological investigations give insights into the microstructure and functionality of pectin gels[END_REF]. Similar to HDM gels, the presence of co-solute can also promote inter-chain interactions [START_REF] Fu | Rheology and structure development during gelation of low-methoxyl pectin gels: The effect of sucrose[END_REF], bivalent ions such Fe 2+ , Mg 2+ or heavy metals can alter the viscosity of LDM gels [START_REF] Mierczynska | Effect of Ca 2+ , Fe 2+ and Mg 2+ on rheological properties of new food matrix made of modified cell wall polysaccharides from apple[END_REF]. Whether these ions bind to pectin in an egg-box-like model is still unknown.

Higher ambient temperature can accelerate gelation through promoting the formation of egg-box junction zones. Low temperature facilitates hydrogen bonds, which are weaker than Ca 2+ bonds, and therefore weakens the gel [START_REF] Chan | Pectin as a rheology modifier: origin, structure, commercial production and rheology[END_REF]. HG gels undergo a conformational transition below a pH value corresponding to the pKa of GalA: carboxyl groups are protonated and the chains are essentially uncharged.

Gelation occurs by dimerization of (antiparallel) 3-fold helices as a result of the suppression of electrostatic repulsion, and the formation of hydrogen bonds between the protonated carboxylic acids [START_REF] Gilsenan | Thermally reversible acid-induced gelation of low-methoxy pectin[END_REF][START_REF] Mansel | Zooming in: Structural investigations of rheologically characterized hydrogen-bonded low-methoxyl pectin networks[END_REF][START_REF] Li | Cross-linked polysaccharide assemblies in marine gels: An atomistic simulation[END_REF] In vivo: Changes in the abiotic environment (temperature, ion concentrations, osmolarity or mechanical stresses) can affect plant growth by changing the rheology of the cell wall. For instance, high concentrations of NaCl in the medium of Arabidopsis root cells, caused a transient decrease in wall stiffness [START_REF] Feng | The FERONIA receptor kinase maintains cell-wall integrity during salt stress through Ca 2+ signaling[END_REF]. The recovery of wall stiffness did not occur in mutants for the CrRLKL1 receptor kinase FERONIA (FER), causing root cells to burst in the presence of NaCl. This bursting could be prevented by supplying extra Ca 2+ and boron (promoting crosslinks between RG-II molecules) to the medium. These observations are consistent with the idea that monovalent Na + ions interfere with load-bearing Ca 2+ -pectate bonds; that plant cells actively recover cell wall strength by promoting the formation of additional crosslinks in the cell wall and that this cell wall repair activity requires FER activity [START_REF] Feng | The FERONIA receptor kinase maintains cell-wall integrity during salt stress through Ca 2+ signaling[END_REF]. In this context, HG modification most likely plays a critical role in the homeostasis of the cell wall rheology.

HG interacts with cell surface receptors

HG also binds to many cell surface receptors, such as Catharanthus roseus RLK1 (CrRLK1) -like family [START_REF] Lin | FERONIA's sensing of cell wall pectin activates ROP GTPase signaling in Arabidopsis[END_REF][START_REF] Feng | The FERONIA receptor kinase maintains cell-wall integrity during salt stress through Ca 2+ signaling[END_REF]Sebastjen et al. 2018), extensin containing receptors [START_REF] Valentin | Elaboration of extensinpectin thin film model of primary plant cell wall[END_REF][START_REF] Mecchia | RALF4/19 peptides interact with LRX proteins to control pollen tube growth in Arabidopsis[END_REF] Frank et al. 2018;[START_REF] Li | FERONIA and her pals: functions and mechanisms[END_REF]. The Xenopus malectin domain binds to alpha-1,3-linked diglucosides on N-linked glycans and plays a role protein quality control in the ER [START_REF] Schallus | Malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein Nglycosylation[END_REF]. Unlike animal malectin, these malectin-like domains lack critical residues involved carbohydrate binding [START_REF] Du | Crystal structures of the extracellular domains of the CrRLK1L receptor-like kinases ANXUR1 and ANXUR2[END_REF]).

Nevertheless, the ectodomain of FER interacts directly with HG in vitro, although no genetic evidence exists so far for a signaling role for HG binding [START_REF] Feng | The FERONIA receptor kinase maintains cell-wall integrity during salt stress through Ca 2+ signaling[END_REF].

Likewise, in vitro pull-down assays showed that FER preferentially binds to HG LDM [START_REF] Lin | FERONIA's sensing of cell wall pectin activates ROP GTPase signaling in Arabidopsis[END_REF]. Furthermore, it is worthwhile to note that oligogalacturonides (OGs) bind to members of the WAKs family, which appear to play a role in growth regulation and defense signaling [START_REF] Kohorn | The cell wall-associated kinases, WAKs, as pectin receptors[END_REF].

Apoplastic pH

Cell walls and spaces between cells make up the apoplast. The pH of the apoplast (pH Apo ) is tightly regulated and contributes to the control of the mechanical properties of the cell walls and, as a result, affect growth and development. About 50 years ago, the acid growth theory was proposed to explain this mechanism. The core of the acid growth theory is that a decrease in pH Apo as a result of the activation of the PM-localized H + -ATPase (also called proton pump) by auxin, results in the activation of cell wallloosening enzymes, which in concert with the turgor pressure, enable cell expansion [START_REF] Hocq | Connecting homogalacturonan-type pectin remodeling to acid growth[END_REF].

Regulation CW enzyme activity by pH

In vitro experiments showed that many cell wall remodeling agents and their inhibitors are regulated by pH Apo (Table 1.2). Among them, a principal role in cell wall loosening is played by expansin, which is a protein that disrupts cellulose-hemicellulose bonds with a low pH optimum (pH 4.5) (Mc-Queen-Mason and Cosgrove 1994;Mc-Queen-Mason et al. 1992;[START_REF] Cosgrove | Re-constructing our models of cellulose and primary cell wall assembly[END_REF][START_REF] Cosgrove | Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes[END_REF]. The pH-dependence has also been characterized for some PMEs and PMEIs. For instance, AtPME3 activity has a pHoptimum of 7.5 [START_REF] Sénéchal | Tuning of pectin methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER[END_REF], AtPMEI4 and AtPMEI7 showed a similar pHdependence for their inhibiting capacity [START_REF] Hocq | Combined experimental and computational approaches reveal distinct pH dependence of pectin[END_REF][START_REF] Sénéchal | Structural and dynamical characterization of the pH-dependence of the pectin methylesterasepectin methylesterase inhibitor complex[END_REF] while

AtPMEI9 is less pH-dependent [START_REF] Hocq | Combined experimental and computational approaches reveal distinct pH dependence of pectin[END_REF].

Proton motive force and turgor pressure regulation

Protons constitute one of the major energy currencies of the plant cell. H + -ATPase powers the transport of H + out of the cytosol, establishing an electrochemical potential across the membranes. This transmembrane H + potential is then used to power the transport of other ions or solutes across the membranes (Figure 1.8). The net direction of transport across a membrane is dictated by the driving force on the transport system.

Two factors contribute to the overall driving force: the solute concentration difference across the membrane and the membrane electrical potential difference (membrane potential, Vm). In the particular case of H + , a simplified expression based on the pH, can be obtained for the electrochemical potential difference. In Arabidopsis thaliana root cells, a membrane potential in excess of approximately 200 mV and a ΔpH (proton chemical concentration gradient) of approximately 2 (equivalent to approximately 120 mV) both contribute to the large proton motive force (PMF) (Buchanan et al. 2000).

Proton motive force regulates turgor pressure through mediating ion transport Turgor pressure, an essential feature of plants, is often considered as the motor of growth [START_REF] Beauzamy | Flowers under pressure: Ins and outs of turgor regulation in development[END_REF]. This force within the cell pushes the PM against the cell wall and plays a key role in plant cell growth where the cell wall undergoes irreversible expansion due to the force of turgor pressure as well as structural changes in the cell wall that alter its extensibility [START_REF] Cosgrove | Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes[END_REF]. Turgor pressure is caused by the osmotic flow of water. The three major classes of osmo-active molecules modulated in osmoregulation are ions, sugars and amino acids. Plant cell have two mechanisms to regulate turgor pressure: one is transpiration, which results in water loss and decreases turgidity in cells. The other relies on its semipermeable membrane, which only allows some solutes to travel in and out of the cell. Most PM solute transport systems in plants are H + coupled, and an ATP-coupled proton pump creates the PMF that energizes these systems [START_REF] Bush | Proton-coupled sugar and amino acid transportation in plants[END_REF][START_REF] Sussman | Molecular analysis of proteins in the plant plasma membrane[END_REF], resulting in the elevated turgor pressure that drives cell expansion (Figure 1.8).

Cation transport

K + : PMF drives K + transport through channels and transporters against its concentration gradient. According to a current model (Nieves-Cordones et al. 2014;

Wang and Wu 2017), non-selective channels are the main pathways for K + uptake at high concentrations, while at intermediate concentrations, the inward rectifying channel AKT1 dominates K + uptake [START_REF] Lagarde | Tissue-specific expression of Arabidopsis AKT1 gene is consistent with a role in K + nutrition[END_REF][START_REF] Sentenac | Cloning and expression in yeast of a plant potassium ion transport system[END_REF]. Under lower concentrations of external K + , AKT1 channels, together with the high-affinity K + uptake system HAK5 [START_REF] Rubio | Cloning of Arabidopsis and barley cDNAs encoding HAK potassium transporters in root and shoot cells[END_REF] contribute to K + acquisition, and at extremely low concentrations the only system capable of taking up K + is HAK5. As a cation, K + can be passively driven into the cell down the ΔVm, via a secondary transport system such as a uniport, AKT1. When the K + supply is reduced to 0.1 mM, the ΔVm becomes closer to K + diffusion potential and K + influx through the AKT1 complex decreases.

The H + / K + symport activity of HAK5, is up-regulated under a low K + supply, allowing K + uptake. In tomato (Solanum lycopersicum), the change in ΔVm lead to increased LeHAK5 expression as well [START_REF] Nieves-Cordones | K + uptake in plant roots: the systems involved, their regulation and parallels in other organisms[END_REF]. Moreover, 50% of reduced H + -ATPase concentration in an Arabidopsis knockdown mutant, aha2-4, caused the upregulation of the HAK5 compared with wild-type seedlings under normal growth conditions [START_REF] Haruta | The effect of a genetically reduced plasma membrane proton motive force on vegetative growth of Arabidopsis[END_REF]. Therefore, the AKT1-and HAK5-dependent K + transport is affected by the PMF.

Cation/H + exchangers (CHX) are antiporters that are involved in K + transport (Sze and Chanroj 2018). For instance, PTs express 18 genes encoding putative cation-H + antiporter CHX family members [START_REF] Sze | Expression patterns of a novel AtCHX gene family highlight potential roles in osmotic adjustment and K + homeostasis in pollen development[END_REF]. Of these genes, CHX13 and CHX14

are PM transporters involved in K + homeostasis [START_REF] Sze | Expression patterns of a novel AtCHX gene family highlight potential roles in osmotic adjustment and K + homeostasis in pollen development[END_REF][START_REF] Zhao | AtCHX13 is a plasma membrane K + transporter[END_REF][START_REF] Cosgrove | Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes[END_REF]. In the aha2-4 mutant, CHX17 was also up-regulated under normal growth conditions in the same way as HAK5 [START_REF] Haruta | The effect of a genetically reduced plasma membrane proton motive force on vegetative growth of Arabidopsis[END_REF]. In summary, both K + symporters and antiporters are coupled to the PMF. [START_REF] Amtmann | Mechanisms of Na + uptake by plant cells[END_REF].

Na + : Na + is
Moreover, the transcriptional upregulation of PM H + -ATPases in saline environments is also consistent with a role for the PMF in the regulation of Na + influx and efflux [START_REF] Amtmann | Mechanisms of Na + uptake by plant cells[END_REF].

Ca 2+ : Unlike K + and Na + , Ca 2+ indirectly affects the turgor pressure by acting as a negative regulator [START_REF] Okazaki | Calcium ion and turgor regulation in plant cells[END_REF]. In the plant Lamprothamnium, an increase in turgor pressure activated the PM Ca 2+ channels, such as cylic nucleotidegated channels (CNGC) and glutamate-like receptor families (GLR) [START_REF] González-Fontes | Understanding calcium transport and signaling, and its use efficiency in vascular plants[END_REF]. The elevated Ca 2+ influx increased the cytosolic Ca 2+ , which activates K + and/or Cl -channels in PM and/or tonoplast. Consequently, the passive effluxes of K + and Cl -leads to a decrease in turgor pressure [START_REF] Okazaki | Calcium ion and turgor regulation in plant cells[END_REF]).

In the soil, Ca 2+ entry into the root is strongly favored by PMF (González-Fontes et al. [START_REF] González-Fontes | Understanding calcium transport and signaling, and its use efficiency in vascular plants[END_REF][START_REF] Blackford | Voltage sensitivity of H + /Ca 2+ antiport in higher plant tonoplast suggests a role in vacuolar calcium accumulation[END_REF].

2017). Even though cell walls contains high concentrations of Ca

Anion transports: Anion channels/transporters are key to osmoregulation [START_REF] Barbier-Brygoo | Anion channels/transporters in plants: from molecular bases to regulatory networks[END_REF]. Anions such as chloride, nitrate, or malate constitute a major part of the plant cell osmoticum. Anion movements through membranes, when coordinated with a cation flux, are thus expected to play a key role in the regulation of cell turgor.

Plant cells contain the Rapid-type (R-type) and Slow-type (S-type) anion channels [START_REF] Hedrich | Ca 2+ and nucleotide dependent regulation of voltage dependent anion channels in the plasma membrane of guard cells[END_REF]Linder and Baschke 1990). The S-type current was proposed as the main mechanism for the depolarization, the loss of anions, and ultimately the decrease in guard cell turgor. The R-type anion channel accommodates, in the millisecond range, its gating to the membrane potential and shows a V-shaped currentvoltage relationship with a steep voltage dependency. The S-type channel shows slow activation/deactivation kinetics in the 10-s range and a weak voltage dependency.

Sugar and amino acid transports: Sugar production or breakdown can elevate turgor pressure. In sink tissues, storage polysaccharides break down to increase the osmotic potential; for example, starch split is converted into glucose, fructose and/or malic acid, increasing the concentration of osmo-active solutes [START_REF] Taiz | Plant physiology[END_REF]. The sucrose concentration can increase in the cytosol because of sucrose transport. One major way to regulate proton-coupled sugar transporters is indirectly by regulating H + -ATPase activity [START_REF] Lalonde | The dual function of sugar carriers: transport and sugar sensing[END_REF]. For instance, sucrose transporter, a high-affinity sucrose-H + symporter, which transport sucrose and protons in the same direction across a membrane is regulated by the PMF [START_REF] Lalonde | The dual function of sugar carriers: transport and sugar sensing[END_REF]. The stoichiometry of the proton-sucrose symport is 1:1 [START_REF] Bush | Proton-coupled sugar and amino acid transportation in plants[END_REF]. Furthermore, the phloem contains an The C terminus of the enzyme functions as an inhibitory domain. Phosphorylation of a serine in the C terminus and binding of two 14-3-3 proteins remove this inhibition and activate the enzyme. The enzyme is also activated by binding of fusicoccin and 14-3-3 protein. Modified from (Buchanan et al. 2000) osmotic pump whose efficacy rests primarily on the cooperative functions of three types of PM proteins: (1) H + /sucrose symporters; (2) H + -ATPases; and (3) water transporters (Sze et al. 1999;[START_REF] Lalonde | The dual function of sugar carriers: transport and sugar sensing[END_REF]. The pH Apo ranges from 5.0 to 6.0, which is of importance for the proton binding of H + /sucrose symporters which have an acidic pH optimum [START_REF] Bush | Electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport[END_REF]). Besides, the transport of hexose, such as glucose, was driven not only by the size of the imposed gradient, but also by either H + or Vm [START_REF] Bush | Proton-coupled sugar and amino acid transportation in plants[END_REF].

Amino acids are osmo-active, and their concentrations are modulated during osmoregulation. Amino acids are transported into plant cells by proton-coupled symports. Alanine accumulation against a significant concentration gradient and saturation kinetics indicated active amino acid transport [START_REF] Bush | Amino acid transport into membrane vesicles isolated from Zucchini: evidence of a proton-amino acid symport in the plasmalemma[END_REF]). The protonalanine symport is located in the PM (Bosh 1988). [START_REF] Li | pH-dependent amino acid transport into plasma membrane vesicles isolated from sugar beet leaves: I. Evidence for carrier-mediated, electrogenic flux through multiple transport systems[END_REF] investigated the proton-amino acid symport activity using purified PM vesicles isolated from sugar beet leaf tissue. In these experiments, a pH-dependent alanine, leucine, glutamine, glutamate, isoleucine, and arginine transport was demonstrated.

Regulation of apoplastic pH

The cell wall pH regulates the cell wall modifying proteins (Table 1.2), which alter the extensibility of the walls, allowing cellular expansion. A deep understanding of the pH Apo regulation is required to answer the question of how plant cells grow.

H + -ATPases: The long-standing acid growth theory postulates that the PM-localized H + -ATPase, activated by plant hormone auxin, is the primary factor to acidify the apoplast. Arabidopsis contains 11 genes encoding PM H + -ATPases (AHA1-11; [START_REF] Baxter | Genomic comparison of P-type ATPase ion pumps in Arabidopsis and rice[END_REF]. So far, AHA1 and AHA2 encode the two most highly expressed isoforms [START_REF] Baxter | Genomic comparison of P-type ATPase ion pumps in Arabidopsis and rice[END_REF][START_REF] Haruta | Molecular characterization of mutant Arabidopsis plants with reduced plasma membrane proton pump activity[END_REF]. The H + -ATPase possesses 10 transmembrane domains and an autoinhibitory domain at the C-terminus (Figure 1.9, [START_REF] Buchanan | Biochemistry and Molecular Biology of Plants[END_REF]. This autoinhibitory domain plays a pivotal role in regulating H + -ATPase activity.

Tobacco (Nicotiana tabacum) plants engineered to express a constitutively active PM H + -ATPase derivative lacking the C-terminal inhibitory domain exhibit increased cell expansion (Gévaudant et al. 2007). In addition, the autoinhibitory domain can bind to conserved regulatory molecule,14-3-3 proteins, which are expressed in all eukaryotic cells, where they regulate many cellular targets. These regulatory proteins bind to the autoinhibitory domain of H + -ATPase, forming a complex, thus activating H+ transport [START_REF] Kanczewska | Activation of the plant plasma membrane H + -ATPase by phosphorylation and binding of 14-3-3 proteins converts a dimer into a hexamer[END_REF]. The complex can be stabilized by fusicoccin, a toxic compound produced by fungus Fusicoccum amygdali (Figure 1.9) [START_REF] Morsomme | The plant plasma membrane H + -ATPase: structure, function and regulation[END_REF]. Auxin regulates H + -ATPases through phosphorylation of the autoinhibitory domain. Type 2C protein phosphatase PP2C-D dephosphorylates the penultimate threonine at the C-terminus of H + -ATPase (in Arabidopsis, Thr 948 and Thr 947 for AHA1 and AHA2, respectively) and leads to H + -ATPase activation [START_REF] Takahashi | Auxin activates the plasma membrane H + -ATPase by phosphorylation during hypocotyl elongation in Arabidopsis[END_REF]). Auxin-induced SAUR19 is able to inhibit PP2C-D activity [START_REF] Spartz | SAUR Inhibition of PP2C-D phosphatases activates plasma membrane H + -ATPases to promote cell expansion in Arabidopsis[END_REF], leading to the activation of H + -ATPase, apoplast acidification and cell expansion [START_REF] Spartz | SAUR Inhibition of PP2C-D phosphatases activates plasma membrane H + -ATPases to promote cell expansion in Arabidopsis[END_REF][START_REF] Takahashi | Auxin activates the plasma membrane H + -ATPase by phosphorylation during hypocotyl elongation in Arabidopsis[END_REF].

Besides auxin, other plant hormones, such as brassinosteroid (BR, [START_REF] Caesar | A fast brassinolide-regulated response pathway in the plasma membrane of Arabidopsis thaliana[END_REF],

ABA [START_REF] Merlot | Constitutive activation of a plasma membrane H + -ATPase prevents abscisic acid-mediated stomatal closure[END_REF][START_REF] Hayashi | Abscisic acid suppresses hypocotyl elongation by dephosphorylating plasma membrane H + -ATPase in arabidopsis thaliana[END_REF] and methyl jasmonate (MeJA) [START_REF] Zhu | Plasma membrane H + -ATPase is involved in methyl jasmonate-induced root hair formation in lettuce (Lactuca sativa L.) seedlings[END_REF], can also regulate H + -ATPase activity. The BR receptor protein (BRI1), can activate a H + -ATPase, AHA1. BRI1 interacts with AHA1 on the PM, phosphorylating its C-terminal autoinhibitory domain [START_REF] Caesar | A fast brassinolide-regulated response pathway in the plasma membrane of Arabidopsis thaliana[END_REF]. ABA inhibits PP2C protein [START_REF] Hayashi | Abscisic acid suppresses hypocotyl elongation by dephosphorylating plasma membrane H + -ATPase in arabidopsis thaliana[END_REF]. ABA binds to its receptors to inhibit PP2C-A, releasing the protein kinase SnRK2 into an active form that can directly inhibit AHA2-mediated acidification and thereby repress root growth [START_REF] Merlot | Constitutive activation of a plasma membrane H + -ATPase prevents abscisic acid-mediated stomatal closure[END_REF][START_REF] Hayashi | Abscisic acid suppresses hypocotyl elongation by dephosphorylating plasma membrane H + -ATPase in arabidopsis thaliana[END_REF].

MeJA treatment stimulated PM H + -ATPase activity in parallel with H + efflux from the root tips of lettuce seedlings and rhizosphere acidification [START_REF] Zhu | Plasma membrane H + -ATPase is involved in methyl jasmonate-induced root hair formation in lettuce (Lactuca sativa L.) seedlings[END_REF].

Rapid alkalinization factor and FERONIA: Rapid alkalinization factor (RALF) is a 49-amino-acid secreted peptide first discovered in a screen for endogenous plant peptides with the ability to increase the pH of the media of cultured cells [START_REF] Pearce | RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development[END_REF]. Silencing the NaRALF in N. attenuata caused roots to grow longer and trichoblasts that developed into abnormal root hairs (RHs) [START_REF] Wu | NaRALF, a peptide signal essential for the regulation of root hair tip apoplastic pH in Nicotiana attenuata, is required for root hair development and plant growth in native soils[END_REF]).

Arabidopsis has 36 RALFs regulating developmental and physiological processes [START_REF] Ge | Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling[END_REF][START_REF] Haruta | A peptide hormone and its receptor protein kinase regulate plant cell expansion[END_REF][START_REF] Mecchia | RALF4/19 peptides interact with LRX proteins to control pollen tube growth in Arabidopsis[END_REF][START_REF] Stegmann | The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling[END_REF].

Overexpression and silencing of AtRALF1 respectively resulted in reduced and increased root cell size and lateral root number [START_REF] Bergonci | Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation[END_REF]. All RALF peptides tested induced alkalinization [START_REF] Morato Do Canto | Biological activity of nine recombinant AtRALF peptides: Implications for their perception and function in Arabidopsis[END_REF][START_REF] Pearce | Structure-activity studies of RALF, rapid alkalinization factor, reveal an essential YISY-motif[END_REF].

A specific-YISY-motif located at positions 5 through 8 from the N-terminus, which is highly conserved within the plant kingdom, is required for productive binding of RALF to its putative receptor [START_REF] Pearce | Structure-activity studies of RALF, rapid alkalinization factor, reveal an essential YISY-motif[END_REF]. All characterized RALF receptors belong to the CrRLK1L family [START_REF] Ge | Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling[END_REF][START_REF] Haruta | A peptide hormone and its receptor protein kinase regulate plant cell expansion[END_REF][START_REF] Mecchia | RALF4/19 peptides interact with LRX proteins to control pollen tube growth in Arabidopsis[END_REF][START_REF] Stegmann | The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling[END_REF]. For instance, FER is a receptor for RALF1 and RALF23 [START_REF] Haruta | A peptide hormone and its receptor protein kinase regulate plant cell expansion[END_REF][START_REF] Stegmann | The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling[END_REF]), BUPS1/2 and ANX1/2 for RALF4, RALF19 and RALF34 [START_REF] Ge | Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling[END_REF][START_REF] Mecchia | RALF4/19 peptides interact with LRX proteins to control pollen tube growth in Arabidopsis[END_REF]) and THESEUS (THE1) for RALF34 [START_REF] Gonneau | Receptor kinase THESEUS1 is a Rapid Alkalinization Factor 34 receptor in Arabidopsis[END_REF]. Although two different peptides applied sequentially were able to induce a higher alkalinization response than that produced when the same peptide was applied (Morato do Canto et al. 2014), RALFs also showed competitive relationships, such as RALF34 competing for RALF4 and RALF19 binding to their receptors [START_REF] Ge | Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling[END_REF]. Based on the alkalinization, pollen germination and Ca 2+ mobilization assays, it is assumed that at least two or three different pathways for sensing the peptides are functioning in the same tissue [START_REF] Morato Do Canto | Biological activity of nine recombinant AtRALF peptides: Implications for their perception and function in Arabidopsis[END_REF].

Indeed, in addition to RALF binding to different CrRLK1Ls, RALF4 and RALF19 also interact with LEUCINE-RICH REPEAT EXTENSIN (LRX) in PTs [START_REF] Mecchia | RALF4/19 peptides interact with LRX proteins to control pollen tube growth in Arabidopsis[END_REF]. RALFs regulate extracellular alkalinization through the inhibition of H + transport. For example, RALF1 treatment promoted phosphorylation of Ser-899 in PM H + -ATPase 2 (AHA2) [START_REF] Haruta | A peptide hormone and its receptor protein kinase regulate plant cell expansion[END_REF], leading to inhibition of H + -ATPase activity, medium alkalinization and growth suppression. In contrast, the pump activity was constitutively up-regulated in loss-of-function fer mutant [START_REF] Haruta | A peptide hormone and its receptor protein kinase regulate plant cell expansion[END_REF].

Interestingly, the fungal pathogen Fusarium oxysporum secretes a RALF homolog, F-RALF, which also elicited a rapid alkalinization response in tomato and inhibited root elongation in both tomato and Arabidopsis [START_REF] Masachis | A fungal pathogen secretes plant alkalinizing peptides to increase infection[END_REF], indicating that pathogens can highjack this endogenous pH regulating system to their advantage.

FER is a regulator of many biological processes, ranging from fertilization to inhibition of cell elongation and growth [START_REF] Li | FERONIA and her pals: functions and mechanisms[END_REF]. Besides cell wall alkalinization, FER plays essential roles in other signaling pathways. For instance, FER acts as a RALFregulated scaffold that modulates the assembly of immune receptor kinase complex consisting of EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2) with their co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) [START_REF] Stegmann | The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling[END_REF]. FER, together with its co-receptor LORELEI (LRE), is also required for cytosolic Ca 2+ oscillations in male gametophyte (pollen) and female tissues during fertilization, PT rupture and programmed cell death of one of two synergid cells (Duan et al. 2014;[START_REF] Ngo | A calcium dialog mediated by the FERONIA signal transduction pathway controls plant sperm delivery[END_REF]. This explains the defect in fertilization in fer mutants [START_REF] Escobar-Restrepo | The FERONIA receptor-like kinase mediates male-female interactions during pollen tube reception[END_REF]. FER is required for the production of high levels of reactive oxygen species (ROS) at the entrance to the female gametophyte. ROS accumulation is thought to be responsible for PT rupture in a Ca 2+ dependent process involving Ca 2+ channel activation (Duan et al. 2014). FER is required for RALF-induced Ca 2+ spikes that further produce an inhibitory effect on root growth [START_REF] Haruta | A peptide hormone and its receptor protein kinase regulate plant cell expansion[END_REF]. The RALF-induced Ca 2+ increase observed in the wild type was absent in the fer-4 mutant, indicating that this fer4 mutant is deficient in the RALFdependent Ca 2+ signaling system. FER is required for mechanical signal transduction in Arabidopsis roots [START_REF] Shih | The receptor-like kinase FERONIA is required for mechanical signal transduction in Arabidopsis seedlings[END_REF]. A fer mutant shows severely altered cytosolic Ca 2+ signaling, pH and growth responses to different forms of mechanical perturbation.

Ca 2+ signals and pH responses are either abolished or exhibit qualitatively different signatures in fer-4 mutants exposed to local touch or bending stimulation [START_REF] Shih | The receptor-like kinase FERONIA is required for mechanical signal transduction in Arabidopsis seedlings[END_REF]. Interestingly, FER kinase activity was not essential for FER-dependent mechanical Ca 2+ signaling, suggesting either an interaction between FER and another kinase or that FER plays a role as a scaffold not only for pathogen-associated molecular patterns (PAMPs) triggered immunity complexes but also for mechanosensing protein complexes.

ROS:

Plant cell growth requires constant balancing between cell wall strength, to resist the turgor pressure, and extensibility, to allow expansion. ROS are important regulators of this balance. ROS comprise several molecules, which may contribute to wall stiffening (e.g. H2O2, through peroxidase-promoted oxidative crosslinking of polymers (extensins or in grasses, feruoylated arabinoxylans)) or wall loosening (HO • , through polymer severing, [START_REF] Fry | Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals[END_REF]. FER functions as a cell surface regulator in a FER-ROPGEF-ROP complex required for auxin-promoted to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent ROS accumulation in roots, which is also required for normal RH growth (Duan et al. 2014). FER is also required for the production of high levels of ROS at the entrance to the synergid cell through a NADPH reductase and the authors hypothesize that HO • accumulation may explain the rupture of the PT upon arrival at the synergid cell (Duan et al. 2014).

BR: FER can also promote cell growth through integrating different hormone signaling pathways (Duan et al. 2014;[START_REF] Guo | Three related receptor-like kinases are required for optimal cell elongation in Arabidopsis thaliana[END_REF]. GTP BINDING PROTEIN 10) [START_REF] Yu | FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase[END_REF]. RHO GTPase, considered as a molecular switch, shuttles between the GDP-bound inactive state and the GTP-bound activated state and its activation is predominantly mediated by a family of guanine nucleotide exchange factors (GEFs) referred to as ROPGEFs. FER activates ABI1 through regulating RHO GTPase activity via its interaction with ROPGEFs, being a negative regulator of ABA signaling [START_REF] Duan | FERONIA receptor-like kinase regulates RHO GTPase signaling of root hair development[END_REF][START_REF] Yu | FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase[END_REF]. Seedlings of loss-of-function mutants in FER or downstream interactors GEF1/4/10 or ROP11/ARAC10, respectively, are hypersensitive to ABA [START_REF] Yu | FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase[END_REF]. In response to ABA, they display higher ROS levels in guard cells, increased stomatal closure, and stronger growth inhibition of seedlings and roots. In return, ABI2 directly interacts with and dephosphorylates FER thus counteracting RALF1-mediated FER phosphorylation and activation [START_REF] Chen | FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis[END_REF].

Auxin: FER seems to be positively influencing auxin signaling. fer seedlings are insensitive to auxin-stimulated RH elongation and ROS accumulation; however, not all FER dependent functions (such as precocious RH bursting) can be explained by auxinsignaling insensitivity (Duan et al. 2014;[START_REF] Li | Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis[END_REF]. In addition, FER is required for auxin-induced transient alkalinization of the extracellular matrix and inhibition of cell elongation in roots, for instance during the gravitropic response [START_REF] Barbez | Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana[END_REF].

Ethylene: FER might negatively regulate S-adenosylmethionine (SAM) synthesis by interacting with SAM1 and SAM2. fer plants are also hypersensitive to ethylene,

showing severe hypocotyl shortening in the presence of saturating ethylene levels in the dark [START_REF] Deslauriers | FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in Arabidopsis hypocotyls[END_REF]. Additionally, fer mutants contain higher levels of SAM and ethylene, with SAM being part of the ethylene biosynthetic pathway [START_REF] Mao | FERONIA receptor kinase interacts with S-adenosylmethionine synthetase and suppresses S-adenosylmethionine production and ethylene biosynthesis in Arabidopsis[END_REF]. Interestingly, overexpression of SAM SYNTHASE1 (SAM1) or SAM2 trigger growth-related phenotypes similar to those of the fer mutant.

Matrix charge

Due to the presence of HG in the plant cell wall, the apoplast is negatively charged and behaves as an insoluble polyanion under most pH conditions [START_REF] Ricard | Ionic control of immobilized enzymes. Kinetics of acid phophatase bound to plant cell walls[END_REF]1984). The fixed negative charges create an electrostatic potential difference (Δψ)

between inside and outside of the wall and contribute to the local pH by sequestering H + . This in turn affects the cell expansion rate by controlling the pH-dependent activity of cell wall modifying agents [START_REF] Moustacas | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF], Table 1.2), where acidic (pH 4 ~ 5) and alkaline pH (> 6) promote and inhibit growth, respectively. With the cell wall volume increase in growing cells, and the arrival of newly synthesized HG HDM , the matrix charge density is expected to decline and the pH to increase [START_REF] Moustacas | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF][START_REF] Ricard | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF]. Therefore, the cell wall must have subtle molecular mechanisms to maintain the acidic milieu for cell wall extension. Ricard and co-workers [START_REF] Crasnier | Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls[END_REF][START_REF] Moustacas | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF]1991;[START_REF] Nari | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF]1991;[START_REF] Ricard | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF] combined in vitro enzymological approaches on isolated walls and mathematical modeling to study this mechanism and reported an exquisite relationship among the Δψ, PME, metal ions and cell wall extension rate (Figure 1.10).

The results show:

(1) The decrease or increase in Δψ controls the increase or decrease in pH, respectively [START_REF] Moustacas | Pectin methylesterase, metal ions and plant cell-wall extension. The role of metal ions in plant cell-wall extension[END_REF][START_REF] Nari | Pectin methylesterase, metal ions and plant cell-wall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase[END_REF].

(2) PME catalyzed the HG demethylesterification with a high pH optimum (pH 7.5),

which produces H + and additional anionic sites, causing a decrease in pH Apo [START_REF] Nari | Pectin methylesterase, metal ions and plant cell-wall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase[END_REF] and its own inhibition. The subsequent decrease in fixed charge density in growing cells leads to a pH increase, the activation of PME and the restoration of the charge density and so forth [START_REF] Moustacas | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF][START_REF] Nari | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF][START_REF] Ricard | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF].

(3) This process is amplified by the attraction of metal ions (such as Ca 2+ ) in the polyanionic cell-wall matrix. Ca 2+ , which tightly binds to cell wall pectate affects the net charge of the cell wall and therefore the amplitude of electrostatic repulsion effects [START_REF] Crasnier | Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls[END_REF][START_REF] Moustacas | Pectin methylesterase, metal ions and plant cell-wall extension. The role of metal ions in plant cell-wall extension[END_REF]. In addition, whereas metal ions do not intrinsically activate PME, they tend to dissociate enzyme molecules from 'blocks' of carboxy groups of the cell wall [START_REF] Nari | Pectin methylesterase, metal ions and plant cell-wall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase[END_REF]. As a result, metal ions increase the number of enzyme molecules that are available for the HG demethylesterification.

In this context it is interesting to consider a role for RALF/CrRLK1L signaling as an amplifier of the cell wall-associated pH homeostatic mechanism, through the recruitment of the H + -ATPase in the system. Indeed, most RALFs are poly-cationic peptides that are thought to segregate between their CrRLK1L receptor complex and negatively charged HG. Upon co-secretion with HG HDM , RALF peptides would be [START_REF] Crasnier | Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls[END_REF][START_REF] Moustacas | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF]1991;[START_REF] Nari | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF]1991;[START_REF] Ricard | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF] available for CrRLK1L binding. This would amplify the cell wall alkalinization response, associated with the decay in cell wall charge density, through the inhibition of the H + -ATPase. Subsequent activation of PME and the increase in negative charge density of HG would cause not only passive acidification through the sequestering of H + , but also the amplification of this response through the sequestering of the polycationic RALF and the derepression of the H + -ATPase [START_REF] Doblas | Cell wall integrity signaling in plants: Malectindomain kinases and lessons from other kingdoms[END_REF].

It is worth noting that these studies of Ricard and colleagues were carried out in vitro on the extracted cell wall of soybean and that the cell wall pH was estimated from the H + release in the bulk phase. Very little information exists however on the cell wall pH in vivo and its effect on plant cell growth.

How to measure apoplastic pH

Given the key role for pH in cell wall metabolism and in growth, its measurement has been an important goal of plant biologists. The pH difference between cellular compartments, however, can reach as much as 6 pH units, posing a challenge to imaging approaches to visualize the pH of the various regions in these compartments [START_REF] Swanson | In vivo Imaging of Ca 2+ , pH, and reactive oxygen species using fluorescent probes in plants[END_REF]. Intracellular pH measurement methods were first developed to study animal systems and were adapted later on for cytoplasmic and vacuolar pH measurements in plants [START_REF] Kurkdjian | Intracellular pH: measurement and importance in cell activity[END_REF]. Recently, methods have emerged for the measurement of the pH Apo of plant cells based on pH-sensitive fluorescent dyes or proteins in addition to the older methods based on H + -selective microelectrodes, the isolation of apoplastic fluid, bromocerol purple in agar [START_REF] Geilfus | The pH of the apoplast: dynamic factor with functional impact under stress[END_REF][START_REF] Martinière | Development and properties of genetically encoded pH sensors in plants[END_REF][START_REF] Swanson | In vivo Imaging of Ca 2+ , pH, and reactive oxygen species using fluorescent probes in plants[END_REF]). I will focus here on the first three, most commonly used, approaches (Table 1.3).

H + -selective microelectrodes

H + -selective microelectrodes allow pH measurements over time in vivo, enabling the temporal mapping of pH kinetics from milliseconds to hours. The most common type are H + -sensitive glass microelectrodes, which cover the entire physiological pH range that may prevail in any plant tissues or cellular compartments [START_REF] Ammann | Ion-selective Microelectrodes: Principles, Design and Application[END_REF]). An estimate of pH Apo can be recorded by placing the electrode on the cell wall of leaf mesophyll or root cortex cells using a micromanipulator to position the microelectrode with micrometer accuracy [START_REF] Felle | The mycorrhiza fungus Piriformospora indica induces fast root-surface pH signaling and primes systemic alkalinization of the leaf apoplast upon powdery mildew infection[END_REF]). To measure pH Apo dynamics that are under the influence of the mesophyll cells, it may be necessary to remove the epidermis to position the electrode on top of the apoplastic region [START_REF] Rubio | Direct uptake of HCO3in the marine angiosperm Posidonia oceanica (L.) Delile driven by a plasma membrane H + economy[END_REF]. By placing the microelectrode on the surface of growing Arabidopsis RHs, it was found that ROSrelated RH elongation is coupled to spatially distinct regulation of extracellular pH [START_REF] Monshausen | Oscillations in extracellular pH and reactive oxygen species modulate tip growth of Arabidopsis root hairs[END_REF]. Using this approach, [START_REF] Felle | The mycorrhiza fungus Piriformospora indica induces fast root-surface pH signaling and primes systemic alkalinization of the leaf apoplast upon powdery mildew infection[END_REF] [START_REF] Pitann | The apoplastic pH and its significance in adaptation to salinity in maize (Zea mays L.): comparison of fluorescence microscopy and pH-sensitive microelectrodes[END_REF][START_REF] Zimmermann | System potentials, a novel electrical long-distance apoplastic signal in plants, induced by wounding[END_REF]. This application has led to many significant findings that have improved the mechanistic understanding of the role of the sub-stomatal cavity pH in stress responses [START_REF] Felle | The apoplastic pH of the substomatal cavity of Vicia faba leaves and its regulation responding to different stress factors[END_REF]. The drawback of this approach is that the inserted microelectrodes present a physical barrier that inhibits full stomatal closure, affecting gas exchange of closed stomates. Moreover, the fabrication of microelectrodes and their insertion into the cavity are labor intensive and not every attempt is successful. The pH recordings yield information about the pH in the close vicinity of the microelectrode with low spatial resolution. However, the accuracy of the microelectrode-based acquired data is high and reliable [START_REF] Felle | The apoplastic pH of the Zea mays root cortex as measured with pH-sensitive microelectrodes: aspects of regulation[END_REF][START_REF] Ammann | Ion-selective Microelectrodes: Principles, Design and Application[END_REF]).

pH-sensitive fluorescent proteins

Genetically encoded pH sensors that have been designed to overcome the limitations of pH sensitive dyes, such as the leakage into the symplast or the impermeability of certain tissues (Table 1.3). Green fluorescent protein (GFP) absorbs light at two maxima: 395 nm when protonated and 475 nm when deprotonated [START_REF] Bizzarri | Green fluorescent protein based pH indicators for in vivo use: a review[END_REF]).

This shift in absorption has been extensively used to record pH in living organisms [START_REF] Kneen | Green fluorescent protein as a noninvasive intracellular pH indicator[END_REF] With this approach, however, the big advantage of genetically encoded sensors is lost in that they cannot be targeted to specific microdomain in the apoplast. Care must be taken, however, because at pH < 5, GFPs exhibit irreversible conformational changes leading to protein instability [START_REF] Kneen | Green fluorescent protein as a noninvasive intracellular pH indicator[END_REF]. This problem must be taken into account since the pH Apo often reaches values is below pH 5 [START_REF] Grignon | pH and ionic conditions in the apoplast[END_REF]. No matter the type of genetically-encoded pH sensor, estimation of the local pH value from the fluorescence data is not straightforward. It requires in vitro or in vivo calibration to characterize the relationship between the fluorescence properties of the sensor and pH Apo [START_REF] Gao | Self-reporting Arabidopsis expressing pH and Ca 2+ indicators unveil ion dynamics in the cytoplasm and in the apoplast under abiotic stress[END_REF][START_REF] Schulte | A novel fluorescent pH probe for expression in plants[END_REF][START_REF] Shen | Organelle pH in the Arabidopsis endomembrane system[END_REF]. In vitro calibration uses heterologously expressed sensors in buffers of different pH. Such a calibration does not take into account the ionic strength and buffering capacity of the cell and thus can lead to substantial artifacts [START_REF] Schulte | A novel fluorescent pH probe for expression in plants[END_REF]. In vivo calibration uses sensor-expressing plant cells that are treated with buffer solution at certain pH points. This approach should be favored whenever possible, but also has its limitation.

First, there is no objective way to ensure that the equilibrium has indeed been reached between the bathing media and the cell compartment where the sensor is located.

Second, the buffer solution can modify the ion content of the cell and consequently native buffering capacity and ionic strength [START_REF] Martinière | Development and properties of genetically encoded pH sensors in plants[END_REF].

In summary, among the discussed methods for the measurement of pH apo , H + -selective microelectrodes are relatively precise in the entire physiological pH range but the setup of instruments and fabrication microelectrodes is labor-intensive and its spatial resolution is low. pH-sensitive fluorescence dyes have higher spatiotemporal resolution but a limited pH range of responsiveness. pH-sensitive genetically encoded florescence proteins can be used in different organs and can be targeted to distinct domains in the apoplast, but they require the generation of transgenic plants, may emit light from subcellular compartments during sorting and also have a limited pH range.

Relationships between growth, apoplastic pH, and HG demethylesterification

As above-mentioned, pH Apo is an essential factor to control cell growth. The mechanism that underlies this regulation has been studied in great detail in tip-growing PTs and RHs. The rapid, directed growth of the PTs is a central event in the sexual reproduction of higher plants. RHs play critical roles in water and nutrient uptake, and in anchoring the plant in the soil. PT and RH provide important models for study of the cellular processes underlying growth. Here I focused on the relationship between pH Apo , pectin metabolism and tip growth in these contexts.

pH

Since mid-1990s, the role of cytoplasmic and surface pH in RH growth was studied in Sinapis alba RHs (Herrmann and Felle 1995). It was shown that an active PM H + -ATPase is required for growth. pH Apo is associated with RH development in

Arabidopsis and the processes of tip growth and initiation of RHs show differences in their pH requirements: pH Apo decreased during the initiation of RH, while it increased during the RH growth [START_REF] Bibikova | Localized changes in apoplastic and cytoplasmic pH are associated with root hair development in Arabidopsis thaliana[END_REF]. Artificially preventing the wall acidification with pH buffers arrested the initiation process but growth resumed when the wall was returned to an acidic pH (Monshausen at al. 2007). These results highlight the role of the spatio-temporal control of pH Apo in cell expansion and morphogenesis in plants [START_REF] Bibikova | Localized changes in apoplastic and cytoplasmic pH are associated with root hair development in Arabidopsis thaliana[END_REF]. Moreover, the increase in pH Apo of Medicago RH could be a signal for the sensing of rhizosphere-associated signaling molecules such as Nod factors [START_REF] Felle | Rapid alkalinization in alfalfa root hairs in response to rhizobial lipochitooligosaccharide signals[END_REF]. RHs show oscillations in extracellular pH associated with oscillating growth, where the decrease in growth rate shows lags around 7 s behind the pH increase (Monshausen at al. 2007).

PTs show both a longitudinal cytosolic pH gradient [START_REF] Feijó | Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip[END_REF]) and pH and growth oscillations [START_REF] Messerli | Cytoplasmic acidification and current influx follow growth pulses of Lilium longiflorum pollen tubes[END_REF]. Lily PTs possess a constitutive alkaline band at the base of the "clear zone", and an acidic domain at the extreme apex [START_REF] Feijó | Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip[END_REF]), which may be explained by the localization of the PM H + -ATPase along the shank but not at the apex [START_REF] Lefebvre | Identification of a Nicotiana plumbaginifolia plasma membrane H + -ATPase gene expressed in the pollen tube[END_REF]). The "clear zone" is a region just near the apex (so named due to the amyloplasts with highly refractile starch grains) in which vesicles accumulate and fuse, secreting wall material and membrane necessary for tube elongation (Hepler et al. 2013). Intracellular pH oscillations occur several hours after germination and lag the growth rate oscillations by more than 7.5 s [START_REF] Messerli | Cytoplasmic acidification and current influx follow growth pulses of Lilium longiflorum pollen tubes[END_REF][START_REF] Lovy-Wheeler | Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of Lily[END_REF]. Spatially, the pH decreases at RH tip and travels as far as 60 µm down the shaft of the tube [START_REF] Messerli | Cytoplasmic acidification and current influx follow growth pulses of Lilium longiflorum pollen tubes[END_REF][START_REF] Feijó | Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip[END_REF][START_REF] Messerli | Pulsatile influxes of H + , K + and Ca 2+ lag growth pulses of Lilium longiflorum pollen tubes[END_REF]. The changes in intracellular pH were associated with a H + influx at the extreme apex of the PT [START_REF] Feijó | Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip[END_REF][START_REF] Messerli | Pulsatile influxes of H + , K + and Ca 2+ lag growth pulses of Lilium longiflorum pollen tubes[END_REF] and an efflux in the region that corresponds to the position of the alkaline band [START_REF] Feijó | Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip[END_REF]. Obviously, the H + influx and efflux change pH Apo . The average fluxes at the cell surface during the peaks of the H + pulse was 489 ± 81 pmol cm -2 s -1 . The H + pulse has a phase lag of 103 ± 9 degrees with respect to the growth pulses (Figure 1.11) [START_REF] Messerli | Pulsatile influxes of H + , K + and Ca 2+ lag growth pulses of Lilium longiflorum pollen tubes[END_REF][START_REF] Lovy-Wheeler | Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of Lily[END_REF]. Without the distribution of PM H + -ATPase [START_REF] Lefebvre | Identification of a Nicotiana plumbaginifolia plasma membrane H + -ATPase gene expressed in the pollen tube[END_REF], how can H + influx and its oscillation occur at the PT apex? Given that the cell wall of PT apex is rich in HG HDM (Bosch et al. 2005) Besides pH oscillations in tip growth, there are other factors that may directly or indirectly interplay with pH in the regulation of tip growth, including Ca 2+ , K + , PME, ROS, actin dynamics and cell wall deposition (Figure 1.11 and 1.12).

Ca 2+

It has been known for decades that Ca 2+ is essential for PT growth [START_REF] Brewbaker | The essential role of calcium ion in pollen germination and pollen tube growth[END_REF]. Only growing PTs possess Ca 2+ channel activity and tip-focused Ca 2+

gradients [START_REF] Malhó | Calcium channel activity during pollen tube growth and reorientation[END_REF]. The Ca 2+ gradient is restricted to a small area of PM at the extreme tube apex in PTs of Lilium longiflorum, Nicotiana sylvestris, and Tradescantia virginiana. Both Ca 2+ channel activity and Ca 2+ gradients were involved in PT reorientation [START_REF] Malhó | Calcium channel activity during pollen tube growth and reorientation[END_REF]. Additionally, recovery from mild temperature shock or caffeine is associated with a global swelling of the PT tip, concomitant with a high transient entry of Ca 2+ into the tip [START_REF] Pierson | Tiplocalized calcium entry fluctuates during pollen tube growth[END_REF]. As in other tip-growing cells, the cytosolic Ca 2+ gradient and an elevated Ca 2+ in the tip is essential for tip growth in Sinapis alba RHs [START_REF] Felle | The cytosolic Ca 2+ concentration gradient of Sinapis alba root hairs as revealed by CA 2+ -selective microelectrode tests and fura-dextran imaging[END_REF]. The inwardly directed Ca 2+current, localized elevated cytosolic Ca 2+ in the tip, and constant Ca 2+ -circulation are pivotal for RHs tip growth [START_REF] Herrman | Tip growth in root hair cells of Sinapis alba L: significance of internal and external Ca 2+ and pH[END_REF].

Cytosolic Ca 2+ and extracellular influx Ca analysis of the oscillatory growth rate and intracellular Ca 2+ data indicates that the increase in Ca 2+ slightly follows (+ 10° ~ 40°), rather than leads, the increase in growth rate [START_REF] Messerli | Periodic increases in elongation rate precede increases in cytosolic Ca 2+ during pollen tube growth[END_REF][START_REF] Cárdenas | Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization[END_REF], while the extracellular influx Ca 2+ oscillations lags as much as ~ 140° [START_REF] Messerli | Pulsatile influxes of H + , K + and Ca 2+ lag growth pulses of Lilium longiflorum pollen tubes[END_REF]; Figure 1.11 and 1.12).

Therefore, Ca 2+ oscillations do not control the oscillations in secretion or in growth rate.

It is worthwhile to note that as described above cytosolic Ca 2+ and extracellular influx Ca 2+ oscillations lack agreement in the phase relationship [START_REF] Messerli | Periodic increases in elongation rate precede increases in cytosolic Ca 2+ during pollen tube growth[END_REF][START_REF] Cárdenas | Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization[END_REF][START_REF] Messerli | Pulsatile influxes of H + , K + and Ca 2+ lag growth pulses of Lilium longiflorum pollen tubes[END_REF]. One reasonable explanation is that Ca 2+ of extracellular influx binds to the cell wall, instead of the direct movement into the cytoplasm and equivalently that cell wall could be a "Ca 2+ sink", delaying Ca 2+ intracellular influx. Because the calculation of the influx needed to sustain in the cytosolic Ca 2+ yields values of 0.5 ~ 2.0 pmol cm -2 s -1 , which is a small fraction of the measured amount, 15 ~ 20 pmol cm -2 s -1 . By contrast, the predict influx of influx, and even more out of phase with the process of exocytosis. A series of events including the increase in NAD(P) + , F-actin, exocytosis, build-up of wall material at the tip, and alkalinity all precede and thus anticipate the increase in growth rate. These events deserve further attention as potential stimulators of the growth rate. Modified from (Hepler et al. 2013) 35 pmol cm -2 s -1 , based on the calculations of the amount of wall material and the HG demethylesterification is much closer to the measured amount (Holdway-Clarke et al.

1997; Hepler and Winship 2010

). Here again it is possible that Ca 2+ influx reflects the binding to HG LDM in the cell wall.

K +

K + influx is a component of the current pulses peaks 7.5 s after the peaks of the growth pulses [START_REF] Messerli | Cytoplasmic acidification and current influx follow growth pulses of Lilium longiflorum pollen tubes[END_REF][START_REF] Messerli | Pulsatile influxes of H + , K + and Ca 2+ lag growth pulses of Lilium longiflorum pollen tubes[END_REF]. The average fluxes at the cell surface during the peaks of the K + pulse was 688 ± 144 pmol cm -2 second -1 . The K + pulses have a phase lag of 100 ± 11 degrees with respect to the growth pulses (Figure 1.12) [START_REF] Messerli | Pulsatile influxes of H + , K + and Ca 2+ lag growth pulses of Lilium longiflorum pollen tubes[END_REF]. The K + and H + pulses are remarkably similar with their timing, average peak amplitude and proportional relationship to the preceding growth pulse. The close correspondence in timing and magnitude of the H + and K + influx pulses suggests the possibility that a H + /K + cotransporter is responsible for the measured fluxes. The H + /K + cotransporter is thought to aid K + uptake under low extracellular K + conditions. K + influx acts to restore either the total ionic concentration and/or osmotic concentration after the increase in cell volume. The K + influx is helping the cell to rapidly recover turgor pressure for the increased volume of the tube during the tip growth [START_REF] Messerli | Pulsatile influxes of H + , K + and Ca 2+ lag growth pulses of Lilium longiflorum pollen tubes[END_REF]).

PME

Given the central importance of pectin and its methyesterification state in the mechanics of the cell wall, it can be expected that PME levels also have a profound effect on PT growth (Bosch and Hepler 2005;[START_REF] Mckenna | Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes[END_REF]. That this is indeed the case is supported by a number of observations: Exogenous orange PME treatment triggered a dissipation of the Ca 2+ gradient, the thickening of the apical cell wall and subsequent inhibition of cell elongation. Overexpression of NtPPME1 also inhibited PT growth (Bosch et al. 2005). Solanum PTs treated with orange peel PME exhibited a dramatic increase in stiffness and reduction of viscosity at the apex [START_REF] Parre | Pectin and the role of the physical properties of the cell wall in pollen tube growth of Solanum chacoense[END_REF].

In Arabidopsis, a relatively small reduction of the total PME activity (in the atpme1 A B mutant) inhibited PT growth and caused irregular and curved morphology [START_REF] Tian | Pollen-specific pectin methylesterase involved in pollen tube growth[END_REF]). Higher PME activity may also explain the inhibition of RH growth in a loss of function mutant for CrRLK1L ERULUS (ERU). Indeed, higher amount of demethylesterified HG accumulated at the RH tip in eru relative to the wild type (WT) and this could be prevented, together with the rescue of RH growth, by the chemical inhibition of PME activity [START_REF] Schoenaers | The auxin-regulated CrRLK1L kinase ERULUS controls cell wall composition during root hair tip growth[END_REF].

ROS

ROS also play a key signaling role in controlling growth of RHs and PTs. In RHs, oscillations in ROS levels are also associated with growth oscillations, which lag growth by ~ 7 s (Monshauson et al. 2007). RHs of the root hair-defective rhd2-1 mutant, which lack a functional version of the NADPH oxidase ATRBOH C, burst at the transition to tip growth, suggesting a role for ROS in cell wall cross linking. This phenotype could be rescued by elevating the pH of the growth medium to > 6.0, suggesting that PME activity and Ca 2+ -pectate crosslinks can compensate for the lack of oxidative crosslinks [START_REF] Schopfer | Hydroxyl radical-induced cell-wall loosening in vitro and in vivo: implications for the control of elongation growth[END_REF].

Actin dynamics

The actin cytoskeleton is a major component of the PT, where it participates in cytoplasmic streaming. The cortical actin fringe is dynamic, with rapid turnover being for Ca 2+ to over-turn the load-bearing egg-box [START_REF] Rojas | Chemically mediated mechanical expansion of the pollen tube cell wall[END_REF]. This suggested that exocytosis and wall expansion are tightly coupled in the tip growth.

Two paradoxes related to HG modification

HG demethylesterification can have opposing effects on cell wall stiffness

HG LDM binds to Ca 2+ and cooperatively forms Ca 2+ -crosslinked stretches, so called egg-boxes in vitro [START_REF] Parre | Pectin and the role of the physical properties of the cell wall in pollen tube growth of Solanum chacoense[END_REF]. Also in the cell wall, HG demethylesterification is commonly thought to result in stiffer walls. For instance, in growing PTs, HG LDM is associated with stiffer walls and cessation of wall expansion [START_REF] Geitmann | The local cytomechanical properties of growing pollen tubes correspond to the axial distribution of structural cellular elements[END_REF]. Similarly, HG demethylesterification is associated with the decline in the growth rate and increased wall stiffness along the apical-to-basal gradient of growing stems (Phyo et al. 2017). However, a series of elegant experiments with clear but perplexing results show that HG demethylesterification that localized in the shoot apical meristem of Arabidopsis results in elastically softer regions of the meristem surface, as measured by AFM [START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF]2011). Also in the hypocotyl, HG demethylesterification leads to increase in cell wall elasticity and growth rate [START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF]: the transverse walls of slowly-growing cells in hypocotyl contained primarily HG HDM , while rapidly-growing longitudinal walls had more HG LDM .

1.8.2 HG demethylesterification, wall mechanics and growth: controversial results in the literature.

Two recent studies support the observation of [START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF][START_REF] Peaucelle | Report Pectininduced changes in cell wall mechanics underlie organ initiation in Arabidopsis[END_REF][START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF] that HG demethylesterification also can lead to an increase in wall elasticity and cell expansion rate in context of asymmetrical leaf growth in tomato [START_REF] Qi | Mechanical regulation of organ asymmetry in leaves[END_REF]) and gynoecium morphogenesis [START_REF] Andres-Robin | Evidence for the regulation of gynoecium morphogenesis by ETTIN via cell wall dynamics[END_REF]. This raises the question how the same process (HG demethylesterification) can have opposite effects on cell wall mechanics and cell expansion rate.

One point of view challenges the correctness of the in vivo data supporting a cell wall loosening effect of HG demethylesterification. For instance, the model proposed by [START_REF] Qi | Mechanical regulation of organ asymmetry in leaves[END_REF], which correlates the appearance of HG LDM (revealed using anti-glycan antibody 2F4 recognizing HG LDM ), in inner abaxial cell layers, with wall loosening, was challenged with an alternative model by [START_REF] Coen | Early shaping of a leaf[END_REF]. This model in which the epidermis has a constraining role and HG LDM -rich inner wall layers that are stiffer, also can predict the observed changes in leaf shape. [START_REF] Qi | Mechanical regulation of organ asymmetry in leaves[END_REF] reported indeed a PME-induced increase in the elasticity of cell walls in the epidermis but did not measure the stiffness in inner layers (This is difficult using AFM). Additional Functional studies have been carried out in Arabidopsis thaliana for 14 distinct PMEIs (Table 1.1). Interestingly, previous studies showed antagonistic effects of the overexpression of PMEI3 and PMEI5, causing a reduction and an increase in HG LDM respectively [START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF][START_REF] Peaucelle | Report Pectininduced changes in cell wall mechanics underlie organ initiation in Arabidopsis[END_REF][START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF][START_REF] Wolf | A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling[END_REF], where PMEI5 overexpression enhanced BRI1 signaling with a resultant increase in HG demethylesterification [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signalling[END_REF][START_REF] Cosgrove | Re-constructing our models of cellulose and primary cell wall assembly[END_REF]. Likewise, manipulation of PMEI4 and PMEI9 expression led to opposite root phenotypes. Indeed PMEI4 overexpression and knock-out lines showed a decreased and increased root length respectively and PMEI9 overexpression and knock-down alleles showed an increased and decreased root length respectively [START_REF] Hocq | Combined experimental and computational approaches reveal distinct pH dependence of pectin[END_REF]). These results raise the question how different PMEIs can have antagonistic roles in vivo?

To address these two questions, more research is needed. As shown in this introduction there are many interdependent factors that affect the properties of pectins gels and many pathways regulate the expression and activities of PMEs and PMEIs. The study of the control pH Apo may shed some new light this complex issue. (TAIR, https://www.arabidopsis.org). We used the MUSCLE algorithm for the amino acid multiple sequence alignment, and a maximum likelihood tree was constructed using PYHML (Figure 2.1). A similar tree topology was also obtained when a different alignment algorithm was used (t-coffee, ClustalW2) (Figure 2.1). We investigated the expression patterns of the PMEI genes using Genevestigator (data not shown). Twentythree PMEI genes that showed the relatively higher expression levels, including PMEI1-10, PMEI12-13, At4g12390, At5g62350, At1g14890, At2g01610, At1g23205, At1g70720, At1g23350, At4g15750, At1g47960, At5g64620 and At5g46950. Among those, six genes (PMEI3, 4, 5, 7, 8 and 10) were selected for inducible overexpression based upon their more ubiquitous expression patterns and/or higher expression levels under stress conditions. The work on PMEI3 and PMEI5 is shown in this study, while sequences of 76 PMEIs was used the MUSCLE algorithm, and a maximum likelihood tree was constructed using PYHML (A). A similar tree topology was also obtained when a different alignment algorithm was used (t-coffee, B).

Objectives of the thesis

PMEIs were divided into 5 groups (G) in left tree. Identified PMEI1-14 are in red boxes, see also Table 1.1. studies in our laboratory showed that the overexpression of PMEI3 caused, as expected for a PME inhibitor, an increase in pectin methylesterification (as shown in the shoot apical meristem or the hypocotyl epidermis, [START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF][START_REF] Peaucelle | Report Pectininduced changes in cell wall mechanics underlie organ initiation in Arabidopsis[END_REF][START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF].

Unexpectedly however, the overexpression of PMEI5 caused a decrease in pectin methylesterification (observed in root cells, [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signalling[END_REF][START_REF] Cosgrove | Re-constructing our models of cellulose and primary cell wall assembly[END_REF]. The increased PME activity in the PMEI5 overexpression lines was the result of the compensatory overexpression of other PME isoforms, which was mediated by the BRI1 and the receptor-like protein RLP44. These observations suggest that overexpression of PMEI5 but not of PMEI3 triggered compensatory BR signaling. One way to explain this is by assuming that the two proteins inhibit different sets of PMEs, which generate different HG demethylesterification patterns, only one of which is recognized as a signal for RLP44/BRI1 signaling. It should be noted however, that previously reported PMEI3 and PMEI5 inducible overexpression lines used two different induction systems, the ethanol-inducible pAlcA and the estradiol-inducible pMDC7 respectively [START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF][START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signalling[END_REF]. To rule out an effect of the induction system on the different response phenotypes, we decided to express the two PMEIs in the same dexamethasone (DEX)-inducible binary transactivation system in a GreenGate cloning system [START_REF] Lampropoulos | GreenGate -A novel, versatile, and efficient cloning system for plant transgenesis[END_REF]) (Figure 7.6 and 7.7). In this system (Figure 7.6), part 2 is the activator construct and part 1 and 3 are acceptor constructs. LhGR encodes a fusion protein between the LacI transcription factor and a glucocorticoid receptor. The latter binds the heat shock protein HSP70. Upon binding to the inducer DEX, LhGR is released from HSP70 and migrates into the nucleus, where it binds to the p4Op promoter driving the transcription of PMEI3, PMEI5 and the "mTURQOUISE (mTUR)" reporter gene. The "REDSEED" reporter gene is a DsRed expressed from the seedspecific promoter (the promoter of the napin gene of Brassica napus) and can be used to screen for transformants in T1 seeds. This allows experiments to be carried out one generation earlier than when using resistance genes as selectable markers. background. Ten to 256 red seeds (Figure 2.4) of each group were collected, with transformation efficiencies ranging from 1.7/10,000 to 15.5/10,000 (Table 2.1). This result showed the REDSEED gene was functional in the expression cassette. To identify plants with unique insertion, a Chi-squared test was performed with T2 red seeds of 16

individual Col-FX0, Col-FX3 and Col-FX5 plants and 5, 8 and 8 unique insertionplants were selected (data not shown). Interestingly, the fluorescence intensity levels allowed homozygote and heterozygote seeds to be distinguished (Figure 2.4). The transformants also showed the sulfadiazine-resistance, indicating that the antibiotic resistance gene was also functional (Figure 2.5).

UBQ10-mTURQUOISE

Col-FX3

Col-FX0

Col-FX5

After amplification of seeds, we looked for phenotypes segregating among the T2 plants. Previous studies reported that the inducible and constitutive PMEI5 overexpression lines showed obvious phenotypes including dwarfism, twisted organ growth, waving roots and abnormal siliques [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signalling[END_REF][START_REF] Cosgrove | Re-constructing our models of cellulose and primary cell wall assembly[END_REF] and that PMEI3 overexpression lines showed PIN-like apical meristem and smaller plants as compared with WT [START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF]). We did not observe such phenotypes among the Col-FX3 and Col-FX5 plants under non-induction conditions. To test the inductivity of transgenic plants, T2 seedlings grown in a growth chamber and in the green house were induced with 50 µM DEX. 24 h after first spraying with DEX, we looked for the growth and developmental phenotypes. Un-induced transgenic plants and induced WT were used as negative controls. Unfortunately, no twisted organ, waving roots or abnormal siliques were observed (Figure 2.6 and 2.7).

We next tested the inducibility of the fluorescent reporter mTUR, which is present the three constructs. To test its signal in our transgenic plants, we induced 4-d old T2

seedlings with 50 µM DEX for 1 h, 4 h, 8 h and 24 h and observed the seedlings using confocal microscopy. No fluorescent mTUR signal could be observed after DEX induction, in contrast to a positive control line containing a DEX-inducible mTUR gene (kindly provided by Sebastian Wolf) (Figure 2.8). Quantative (q)-Real time (RT) PCR was used to quantify PMEI3, PMEI5 and mTUR transcripts after DEX induction for 1.5h. We failed to observe an increase in transcript levels for any of the 3 genes. We also measured the PME activity of the cell wall-enriched extracts from the induced T2 transgenic seedlings by 50 µM DEX using a gel diffusion assay (GDA). The PME activity was not altered in transformants relative to the wild type and non-induced controls (Figure 2.9). In conclusion, we successfully obtained transformants for PMEI3-F, PMEI5-F and Final control, but the transformed plants did not show DEX-inducibility of any of the transgenes, including the mTUR reporter. We do not know for the moment why this is the case, it may reflect co-suppression of the repeated promoter fragments in the constructs. Additional studies are required to sort this out. Given the limited time to finish my PhD thesis, I decided not to pursue the analysis of my transgenic lines and to remain focused on the previously defined objectives of my thesis using different tools.

Results

Chapter 3 Biochemical characterization of PMEI3 and PMEI5

PMEI3 and PMEI5 expression in Pichia pastoris.

To investigate the difference in biochemical properties between PMEI3 and PMEI5, they were heterologously expressed in Pichia pastoris. The coding sequences and codon-optimized sequences of PMEI3 and PMEI5 minus their signal peptides were cloned downstream and in frame with the poly-Histidine sequence present in an inducible expression vector, pPICZαB. Three types of constructs of PMEI3 and PMEI5

were created (Figure 3.1), named PMEI3/5, PMEI3/5t, and CO-PMEI3/5, respectively.

In PMEI3/5t, a poly-Histidine tag and a stop codon were cloned upstream and downstream of gene of interest, respectively. In CO-PMEI3/5, PMEI3 and PMEI5 minus their signal peptides were condon-optimized according to the codon usage frequency of Pichia pastoris. All final constructs were checked for the presence of the desired insert using colony PCR and restriction enzyme digestion (Figure 3. complex reveals that both inhibitors directly bind to the substrate-binding cleft of their respective target enzymes. Sequence comparison showed that PMEI5 is more similar to a CIF than to a PMEI [START_REF] Lionetti | Three pectin methylesterase inhibitors protect cell wall integrity for Arabidopsis immunity to Botrytis[END_REF]. We therefore carried out an invertase activity assay on the supernatant of PMEI5, PMEI5t and CO-PMEI5, but no CIF activity could be observed (Figure 3.5).

Using Mass spectrometry (MS), PMEI3 bands and the "putative" PMEI5 bands were further analyzed (with the INRA Platform PAPSSO). Nineteen peptides corresponded to the AtPMEI3 primary sequence, which confirmed the successful heterologous expression of PMEI3 in Pichia. Form the "putative" PMEI5 bands, 21 peptides corresponded to the sequence of formate dehydrogenase (AB472090) in Pichia cells (Figure 3.6), indicating that the bands did not correspond to PMEI5. To rule out the possibility that PMEI5 was retained inside yeast cells, we lysed Pichia cells containing CO-PMEI3 and CO-PMEI5 constructs. We did not observe a signal inside cells for neither of the PMEIs (Figure 3.7). This showed that the Pichia expression system has high secretion efficiency for PMEI3 and that PMEI5 is not expressed in these cells, despite the use of a codon optimized sequence. It is not known why PMEI5 is not expressed, but it is possible that PMEI5 protein accumulation requires the presence of (an) accessory protein(s).

PMEI3 characterization

PMEI3 recombinant protein was purified subsequently using cOmplete Hig-tag purification Resin and in vitro characterized by GDA on six different PME preparation (Table 3.1). In addition, we quantified the binding of PMEI3 to PME3 using MicroScale Thermophoresis (MST).

PMEI3 activity measurement.

Using a colorimetric methanol quantification assay, we first determined the activity of the six PME preparations (Orange PME, citrus thermal-tolerant (TT) PME, AtPME2, AtPME3, Arabidopsis flower PMEs and root PMEs) on a reference substrate (citrus pectin) at pH 7.5 and selected a non-saturating enzyme concentration to be used in the inhibition assays (rectangle in Figure 3.8). For the inhibition assays we used a GDA with three concentration (0.025, 0.05 and 0.25 µg) of recombinant PMEI3 along with mock and boiled control samples, incubated with the PME preparation for 1h at pH 5.0, 6.3 or 7.5. The enzyme activity was deduced from the halo diameter after calibration with samples of known PME activity determined by the colorimetric assay.

As shown in Figure 3.9, the PME activity for all preparations was inhibited by PMEI3 in a pH-dependent manner, with strong inhibition at pH 5.0 and no inhibition at pH 7.5.

The concentration-and pH-dependence, however, varied between the samples.

Concerning the concentration dependence, PME3 and root PMEs showed the highest sensitivity to PMEI3 at pH 5, followed by flower PMEs, AtPME2 and orange PME.

Citrus TT PME was the least sensitive to PMEI3. Concerning the pH sensitivity, PME activity in all samples was also inhibited, to a lesser extent, at pH 6.3, except for AtPME2, which did not show any inhibition at this pH. Interestingly, boiling did not affect, or only slightly reduced, the PMEI3 activity indicating a very stable 3D structure for the inhibitor. The pH sensitivity of the inhibiting activity of PMEI3 appeared very similar to that of PMEI4 [START_REF] Hocq | Combined experimental and computational approaches reveal distinct pH dependence of pectin[END_REF]) and PMEI7 [START_REF] Sénéchal | Tuning of pectin methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER[END_REF] tested on AtPME3, but different from recombinant PMEI9 [START_REF] Hocq | Combined experimental and computational approaches reveal distinct pH dependence of pectin[END_REF], the activity of which did not show any pH dependence in the pH range tested. In AtPMEI7, E75 played an essential role in the pH-dependence of its interaction with AtPME3. Mutating this residue caused a 20-fold decrease in binding affinity. The deprotonation of E75 would shift the conformation so that intramolecular salt bridges are formed with positively charged residues sitting in the vicinity of titratable residue [START_REF] Sénéchal | Structural and dynamical characterization of the pH-dependence of the pectin methylesterasepectin methylesterase inhibitor complex[END_REF]). The same mechanism may govern the AtPMEI3-PME interactions given the strong sequence similarity between AtPMEI3 and AtPMEI7 (Figure 2.1).

PMEI3/PME3 interaction

Given the strong pH-dependent inhibition of AtPME3 activity by PMEI3, we analyzed the physical AtPME3/PMEI3 interaction in vitro. The binding affinities of purified AtPME3 with different concentrations of recombinant AtPMEI3 were determined using MST. This sensitive method assesses the protein-protein interaction in vitro by measuring the motion of molecules along microscopic temperature gradients and detects changes in their hydration shell, charge, or size (Wienken et al. 2010;Jerabek-Willemsen et al. 2014). Recombinant AtPME3 was labeled with a blue fluorescent dye, NHS amine. A constant concentration of labeled AtPME3 was added in capillaries with serial dilutions of recombinant unlabeled AtPMEI3. MST curves at pH 5, 6.3 and 7.5

showed a low affinity (Kd ≈ 45 µM) interaction between recombinant AtPME3 and AtPMEI3 (Figure 3.10a), lower affinity than that previously reported for PMEI4-, PMEI7-or PMEI9 [START_REF] Sénéchal | Tuning of pectin methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER[END_REF][START_REF] Hocq | Combined experimental and computational approaches reveal distinct pH dependence of pectin[END_REF]. To mimic in vivo conditions the interaction in the conditions of the GDA, purified proteins in acidic buffer were mixed with 0.1% pectin at pH 5. Interestingly, PMEI3-PME3 binding affinity increase 45x in the presence of pectin (Kd ≈ 1 µM) (Figure 3 and 20.3% relative to close to 100% for non-red seeds. LuPME1-pHusion sensor transgenic plant showed a higher germination rate, 60.5% (Figure 4.3). To test if the sensor constructs were functional in the transgenic plants, 6 d old seedlings selected for their red seeds were observed using the Axiozoom. Unfortunately, no fluorescent plants were found for any of the constructs. These results suggested that the cell wall-localized pH sensors interfered with seed germination and that the selection of viable seeds yielded plants in which the expression of the pH sensor was repressed.

Given these disappointing results, we focused on the use of two previously reported pH sensors, one targeted to the apoplast (apopHusion) and one to the apoplastic side of the PM (PM-Apo) to measure pH Apo in Arabidopsis roots. PM-Apo comprises pHluorin anchored to the PM by a transmembrane domain. This allows the pH to be measured in a < 10nm range close to the PM [START_REF] Martinière | Uncovering pH at both sides of the root plasma membrane interface using noninvasive imaging[END_REF]. The signal of this pH sensor showed a patchy expression in roots, suggesting stochastic co-suppression of the reporter (Figure 4.4) and in the cells that showed high fluorescence intensity, the longitudinal cell walls had less pHluorin signal than transverse cell walls (Figure 4.4), suggesting a selective subcellular targeting. The apopHusion [START_REF] Gjetting | Live imaging of intra-and extracellular pH in plants using pHusion, a novel genetically encoded biosensor[END_REF] combines EGFP in a tandem fusion to monomeric mRFP1 fused to the secretory signal peptide, [START_REF] Gjetting | Live imaging of intra-and extracellular pH in plants using pHusion, a novel genetically encoded biosensor[END_REF] were able to constitutively target the apopHusion to the apoplast in the leaf epidermis, the guard cells, the mesophyll, or the root apex of Arabidopsis. This genetically encoded sensor helped to visualize a fast alkalinization response in the epidermal apoplast of elongating roots in response to external addition of indole-3-acetic acid [START_REF] Gjetting | Live imaging of intra-and extracellular pH in plants using pHusion, a novel genetically encoded biosensor[END_REF]. We also investigated the use of a new pH indicator, HPTS (8-hydroxypyrene-1,3,6trisulfonic acid trisodium salt) to measure the pH Apo . HPTS is a water-soluble fluorescent dye displaying pH-dependent spectral characteristics [START_REF] Barbez | Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana[END_REF]Amali et al. 2011). In plants, HPTS has been used to define the pH of extracted apoplastic fluid from plant tissues (Kasik et al. 2013;Villiers et al. 2013). To test whether HPTS can be used to directly assess pH Apo with a cellular resolution in planta, we immersed 4-day-old seedlings in HPTS-containing liquid growth medium at 6 different pH levels for 30 min and imaged roots using confocal microscopy. A ratiometric image was obtained using 458/405 nm the signal intensity ratio for each pixel, which was calibrated using a standard curve for the 6 pH values of the medium (Figure 4.5). The dye showed a decrease in pH Apo induced by the H + -ATPase activator fusicoccin (Figure 4.5). To test the capacity of HPTS to dynamically detect changes in pH Apo , we added pH 4.85 medium to roots in a medium of pH 5.77 and observed an almost instantaneous drop in the 458/405 nm intensity ratio (Figure 4.5). These results indicate that HPTS is suitable to detect exogenously-imposed pH Apo changes.

The cell wall pH is a key regulator of the cell expansion rate. According to the textbook view, the PM-localized H + -ATPase directly regulates the pH Apo , which in turn controls the activity of the cell wall loosening agents that affect the cell wall extensibility. As discussed in the introduction, HG demethylesterification is a key element controlling the rheology and extensibility of the cell wall. This reaction increases the negative charge density of the HG, which may contribute to the formation of Ca 2+ -pectate that is thought to stiffen cell wall [START_REF] Parre | Pectin and the role of the physical properties of the cell wall in pollen tube growth of Solanum chacoense[END_REF]. The reaction also releases H + into cell wall. It has been theorized that the released H + and the PME-induced increased negative charge density may contribute to the acidification of the cell wall and an increase in the cell wall expansion rate. In vitro studies on isolated plant cell walls suggested that PME-catalyzed HG demethylesterification altered the electrical potential and the H + concentration between the inside and outside of the wall [START_REF] Ricard | Ionic control of immobilized enzymes. Kinetics of acid phophatase bound to plant cell walls[END_REF]1984). However, no in vivo experimental evidence exists so far to support this view. In this study, we addressed this issue by using pharmacological and genetic manipulation of the PME activity to investigate the impact of HG demethylesterification on pH Apo and growth in Arabidopsis roots.

Dose-dependent inhibition of Arabidopsis root growth by PME inhibitor EGCG.

To analyze the effect of the inhibition of HG demethylesterification on root growth, we employed a systemic chemical inhibitor of PMEs, (-)-epigallocatechin gallate (EGCG), which inhibits PME activity by binding to PME or to pectin (Lewis et al. 2008;[START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signalling[END_REF]. Arabidopsis Col-0 seedlings were grown under light or dark conditions on solid 1/2 MS medium supplemented 200 µM EGCG and hypocotyl length, primary root length and RH density were measured 2 d after germination (Figure 5.1). The primary root length of EGCG-treated seedlings was significantly shorter than that of mock- treated control seedlings both in light-and dark-grown conditions. The hypocotyl length however, was unaffected by the EGCG treatment, suggesting that either pectin metabolism is not important for hypocotyl growth or that the EGCG is not transported to the growing zone in the hypocotyl. The second possibility is the most likely one given the fact that EGCG binds to the cell walls of the root cells. We therefore focused on the root in all subsequent experiments. To confirm the EGCG effect, Col-0 seedlings were grown on solid 1/2 MS solid medium supplemented EGCG at concentrations from 0 to 100 µM. As shown in Figure 5.1, the root length of 4-d-old seedlings decreased with increasing EGCG concentration. 50 µM EGCG-treated plants showed a root waving phenotype (Figure 5.1), which is consistent with previous observations [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signalling[END_REF]) using EGCG or constitutive and inducible PMEI5 overexpressing lines.

PME activity is also involved in RH growth [START_REF] Schoenaers | The auxin-regulated CrRLK1L kinase ERULUS controls cell wall composition during root hair tip growth[END_REF]. RHs show oscillations in growth rate, the peaks of which lag the peaks in the accumulation of demethylesterified HG at the root tip [START_REF] Rounds | Propidium iodide competes with Ca 2+ to label pectin in pollen tubes and Arabidopsis root hairs[END_REF]). In the present study, EGCGtreated seedling showed more and longer RHs in relative to mock-treated seedlings (Figure 5.1). The same phenotype was observed with polyphenon 60, a catechin extract that also inhibits PME activity in a concentration-dependent manner [START_REF] Schoenaers | The auxin-regulated CrRLK1L kinase ERULUS controls cell wall composition during root hair tip growth[END_REF]. In conclusion, EGCG treatment causes the inhibition and the promotion of root and RH growth respectively. We assume that this effect reflects the inhibition of PME activity.

EGCG treatment alters cell shape in root

In the EGCG-treated roots, cells in the differentiation zone (DZ) tended to bulge out (Figure 5.2). To determine at which stage the EGCG treatment affects root development, we analyzed the 3D cell shape from the root tip to the elongation zone (EZ). Four-dold seedlings, grown in solid 1/2 MS medium supplemented with 50 µM EGCG were stained by calcofluor and imaged using confocal microscopy. Images were The L/W ratio became significantly higher in EGCG-treated vs mock-treated cells, from cell 27 and 22 (starting for the QC) in trichoblast and atrichoblast respectively indicating that growth symmetry breaking occurred earlier in EGCG-treated relative to mock-treated roots. Similarly, the cell volume became significant higher in EGCGtreated vs control cells, from cell 26 and 19 in trichoblast and atrichoblast respectively (Figure 5.2), indicating that 50 µM EGCG had the first visible effects on cells in the TZ. We therefore focused primarily on the TZ for the analysis of the pH Apo .

EGCG treatment promotes an increase in pH Apo in root cells

To monitor changes in pH Apo after EGCG treatment, the genetically encoded pH sensor, apopHusion and the pH sensitive fluorescence dye HPTS, were used to measure pH Apo in growing roots. Four-d-old apopHusion expressing seedlings vertically grown on 1/2 MS solid medium were gently mounted onto a home-made root growth chip and fixed by medical adhesive or by covering the seedlings with cotton and a small plastic grid (See Method and materials). To let the mounted seedlings adapt to the new growth conditions, they were grown in unbuffered1/2 MS liquid medium (pH = 5.8) for 15 min.

Then the same volume of 1/2 medium supplied with various drugs was inserted into the channel and the seedlings were grown for 45 more min. During this 1 h growth period, EGFP and mRFP1 fluorescence intensity values were acquired in the root within 1 min intervals. Regions of interest (ROIs) in the MT, TZ and EZ were analyzed respectively.

We first pharmacologically modulated the activity of the PM H + -ATPases and analyzed the EGFP/mRFP1 ratio after imaging. Fusicoccin treatment, which activates the PM H + -ATPases [START_REF] Barbez | Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana[END_REF], resulted in decreased EGFP/mRFP1 ratio values, indicating a decrease in pH Apo (Figure 5.3). In contrast, treatment with vanadate, which inhibits the PM H + -ATPases, led to an increased EGFP/mRFP1 ratio values (Figure 5.3).

The MT, TZ and EZ in vanadate-treated root showed similar patterns of EGFP/mRFP1 ratio changes, whereas the EZ was less sensitive to fusicoccin treatment compared with MT and TZ. EGCG treatment caused also an increase in EGFP/mRFP1 ratio value in MT, TZ and EZ (Figure 5.3), with the strongest effect in the TZ. To confirm the effect of EGCG on the pH Apo , we also used HPTS. HPTS calibration and the effect of fusicoccin on pH Apo was shown in Chapter 4. Four-d-old seedlings were incubated in 1/2 MS liquid medium with EGCG for 30 min and with HPTS plus EGCG for 30 more min. We again observed an increase in 458/405 ratio in EGCG-treated, relative to mock-treated roots in both longitudinal and transverse cell walls in the TZ (Figure 5.4). We also studied the kinetics of the response and observed a transient pH increase within 300 sec after the addition of EGCG. The response peaked more slowly in larger than in smaller cells.

Together, combining the use of apopHusion and HPTS, we observed that EGCG treatment led to an increase in pH Apo in the Arabidopsis root.

To test whether the EGCG-induced pH increase depended on the inhibition of the H + -ATPase, we treated the seedlings with a combination of EGCG and fusicoccin.

Interestingly, EGCG overruled the effect of fusicoccin on the pH Apo as shown by the increase in the EGFP/mRFP1 ratio in MT, TZ and EZ (Figure 5.3). These results suggest that, irrespective the H + -ATPase activity, the inhibition of HG demethylesterification triggered an increase in pH Apo , although it cannot be ruled out that EGCG shut down the H + -ATPase activity downstream of the fusicoccin effect.

EGCG-triggered increase in pH

Apo correlates with root growth inhibition.

According to the acid growth theory, the decrease in pH Apo , triggered by the H + -ATPase, promotes cell expansion, though the activation of pH-dependent cell wall-loosening proteins [START_REF] Hocq | Connecting homogalacturonan-type pectin remodeling to acid growth[END_REF]. To clarify the relationship between the EGCG-triggered increase in pH Apo and root growth, we monitored pH Apo in parallel to root growth in In conclusion, these preliminary results show that EGCG-induced pH Apo increase coincided with the inhibition of root growth and that these responses were fully reversible.

Exogenous PME treatment decreased pH Apo in root cells

The EGCG results suggest that the inhibition of PME was responsible for the observed apoplast alkalinization. To confirm this, we measured pH Apo using HPTS in roots after 45min treatment with orange PME (1.0 unit/root). We observed a significant decrease in the 458/405 ratio in both longitudinal, transversal and global cell walls in the TZ in PME-treated roots relative to mock-treated controls (Figure 5.6). We next used the apopHusion to confirm the HPTS data and to study the kinetics of the acidification response. It should be noted that citrus PME is kept in a PBS buffer at pH 7.5. As a result, we observed an initial increase in pH after PME or mock buffer control treatment.

However, after the initial pH increase we observed a significant pH decrease in PMEbut not in mock-treated roots (Figure 5.7). These data confirm that exogenous PME can promote a decrease in pH Apo in root cells.

Effect of dexamethasone-induced expression of PMEI5 on pH Apo

Previous studies reported that the overexpression of the putative PME inhibitor PMEI5, promoted a compensatory upregulation of PMEs mediated by activation of BR signaling through a feedback mechanism [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signalling[END_REF][START_REF] Cosgrove | Re-constructing our models of cellulose and primary cell wall assembly[END_REF]. We hypothesized that the elevated PME activity could also lead to a decrease in pH Apo . To test this, we used the dexamethasone-inducible PMEI5 overexpressing line to induce PMEI5 expression for 24 h (sufficient to induce the BRI1-mediated compensatory response, [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signalling[END_REF]) after which the pH Apo in root was measured in roots using HPTS. As expected, dexamethasone-induced PMEI5 roots showed a lower 458/405 ratio relative to mock-induced seedlings (Figure 5.8).

The effect of exogenous PME and PMEI3 on root growth

Given that exogenous PME treatment caused a decrease in pH Apo , we asked if exogenous PME and PMEI3 treatments could affect root growth in long-term. To test this, we treated 2-d-old seedlings for 2 days with 10 U/mL orange PME or 14 µg/mL recombinant PMEI3 using the corresponding buffers as mock control. PME-treated roots were longer than those of mock-treated seedlings. whereas PMEI3-treated roots were shorter than those of mock-treated controls (Figure 5.9). Together, these results

show a correlation between PME activity, cell wall pH and root growth.

Summary

In this study, we investigated the link between pectin modification, cell wall pH and cell expansion rate. To this end, we used genetic and pharmacological approaches to manipulate the level of HG demethylesterification in living Arabidopsis roots combined with the monitoring of the pH Apo using genetically encoded ratiometric pH sensors and a pH-sensitive fluorescent dye.

We first attempted to construct new tools (heterologously expressed purified proteins and inducible overexpression lines) for the in vivo manipulation of PME activity. We focused on two PMEIs (PMEI3 and PMEI5), which, based on previous in vivo data, might have different biochemical specificities. PMEI3 could be produced in Pichia pastoris, whereas PMEI5 was not expressed in this host. Recombinant PMEI3 showed inhibitory activity, at low pH, of a broad range of PME activities. We also used a DEX inducible binary system in GreenGate vectors for expression of the two PMEIs.

Unfortunately, none of the transgenic lines showed inducible expression of the PMEIs or the reporter gene. This may be due to the co-suppression of the repeated pOp promoter sequences in the constructs. Next, for the measurement of the cell wall pH, we attempted to generate new genetically-encoded cell wall bound pH sensors.

Unfortunately, the constructs appeared to be lethal in transgenic plants and no transformants with stable fluorescence signals were obtained. We therefore relied for our further experiments on the previously reported pH-sensitive dye HPTS and apoplastic ratiometic sensor apopHusion.

Our major finding is that the cell wall pH is not only determined by the activity of the PM H + -ATPase but also by the PME activity in the cell wall. This conclusion is supported by the following observations: (i) the PME inhibitor EGCG promoted root growth inhibition in parallel with the alkalinization of the apoplast, (ii) EGCG overruled the effect of fusiccocin on the pH Apo , suggesting an effect independent from the H + -ATPase, (iii) citrus PME promoted an increase in root length and apoplast acidification, (iiii) enhance endogenous PME activity upon long term induction of PMEI5 was also associated with apoplast acidification. The effect of PMEI3 overexpression and exogenous PMEI3 on the pH Apo remains to be determined.

If confirmed, the results are consistent with the theory of Ricard and colleagues [START_REF] Crasnier | Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls[END_REF][START_REF] Moustacas | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF]1991;[START_REF] Nari | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF]1991;[START_REF] Ricard | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF], that the cell wall pH reflects the ability of cell wall polymers to sequester H + . A recent publication also comforts this view. The authors used the PManchored pH sensors PM-Apo to show that in a zone < 10 nm above the PM the pH can be up to 2 pH units more basic than in the cell wall (Martinière et al. 2018 and N. Paris personal communication). Given the high diffusion rate of H + in solution, such strong H + gradients can only be maintained by the local sequestering of H + by anionic cell wall polymers. These observations are a valuable addition to the acid growth theory, which stated that cell wall acidification through the activation of the proton pump promotes the activity of cell wall loosening agents (such as expansins) and hence wall relaxation.

We propose that PME activity plays a key role in this process by controlling the buffering capacity of the cell wall. This, combined with the high pH optimum of most PMEs, an optimum sharpened by the inhibition of the activity at low pH by PMEs, may explain the oscillations in pH and growth rate observed in tip growing cells (roots and PTs) and possibly also in diffusely growing cells. PME activity at high pH would create anionic sites, leading to the local acidification, the inhibition of PME and wall relaxation. Cell wall expansion and the arrival of new uncharged HG would lead again to cell wall alkalinization, and so forth.

Perspectives

1. The effect of PME activity on the cell wall pH and growth rate will have to be confirmed using purified PMEI3 and the ethanol-inducible PMEI3 line using the RootChips. It will also be interesting to test PMEI9, which has a pH-independent PMEinhibitory activity [START_REF] Hocq | Combined experimental and computational approaches reveal distinct pH dependence of pectin[END_REF] and is expected to shut down PME activity completely, in contrast to PMEI3, which does not inhibit PME at high pH.

2. Other cell wall-bound and non-lethal pH sensors need to be made to measure the pH in specific cell wall domains.

3. Other expression systems must be tested for the expression PMEI5. It is possible that PMEI5 expression requires the presence of a binding partner such as a PME. In this context it might be interesting to try to express tagged PMEI5 in N. benthamiana and isolate the protein complex.

4. An important challenge will be to understand the coordination between pectin metabolism and the activity of the PM H + -ATPase. In this context it will be interesting to study the role of Rapid Alkaliniztion Factor (RALFs) and their CrRLK1L receptors.

RALFs are polycationic peptides that trigger cell surface alkalinization upon binding to their receptors, through the repression of the H + -ATPase activity. An interesting possibility is that the sequestering of the polycationic RALF peptides away from their receptors by polyanionic HG domains may amplify the cell wall acidifying effect of PME by triggering the derepression of the H + -ATPase. Using the tools developed in this work, it will be possible to study the in vivo interaction between RALF signaling and PME activity in the control of cell wall pH and cell expansion.

5. The results of our study suggest that HG demethylesterification may also control the ion balance between apoplast and symplast. This hypothesis is among my future research interests.

pH measurement using apopHusion

Four-d-old seedlings were mounted on the home-made devices as described in Figure 7.1. Seedling were immobilized using the grid and cotton as described or, if indicated, medical adhesive, which was spread evenly and air dried for 30 sec after which the The Rootchip carrier was covered with a transparent plastic board. 5 ml of distilled water was added into reservoir before covering the carrier in order to keep the inside humid. The whole device was placed in the same growth chamber for 3 d until imaging.

The fluid speed was controlled at 1 µL/min/channel by a home-made pump (Figure 7.2).

Drug concentration used were 200 µM EGCG-, 50 nM IAA-and 2 µM RALF34containing dissolved in 1/2 MS medium at 4 µL/min/channel fluid speed.

Imaging and image processing: Roots tips that had reached the channels were imaged on inverted Zeiss 710 microscope (Figure 7.3). Images were taken with a 10× objective.

EGFP was excited at 488 nm using a white light laser and detected between 500 nm and 550 nm. mRFP was excited at either 558 nm with a white light laser and detected between 600 nm and 630 nm. Six Z-stacks were acquired for both fluorescent proteins with 5 min intervals. During the acquisition, the seedlings were illuminated with white incandescent light.

Image processing and analysis were performed in FIJI. We first made average Z projections of both EGFP and mRFP1 and then substracted the background from each signal. The noise background outside of root was cut manually. The ratio of XZ orthogonal views of EGFP to that of mRFP were calculated. If the root was bent or did not grow along the root axis, the ratiometric images were straightened (Figure 7.3).

3D imaging EGCG treated roots

Calcuofluor staining: Seedlings were grown vertically on 1/2 MS medium with 0, 50 µM, or 200 µM EGCG, respectively, at pH 5.8. Four-d-old seedlings were fixed overnight in an ethanol/acetic anhydride solution (75/25 v/v). Seedlings were then incubated in ethanol for 15 minutes (80% ethanol at 80°C), rehydrated in 50% then 30% ethanol for 10 minutes, then incubated in sodium hydroxyde 0.2N/SDS% for two hours at room temperature. Samples were then rinsed in water and stained for 30 minutes in calcofluor white 0.25% (Fluorescent Brightener 28) to which a few drops of 10 N NaOH were added to obtain complete dissolution. Seedlings were then rinsed in water and mounted in Citifluor.

Imaging and images processing: Roots were imaged on a Zeiss LSM 710 confocal laserscanning microscope. Samples were excited at 405 nm with an emission band of 410~550 nm. The distance interval of root Z-stacks was less than 0.45 um.

MorphlibJ analysis of 3D image

3D images of the root organization were analyzed using the Fiji software. We pretreated 3D images with Gaussian blur 3D (x, y and z = 1) and then performed "Attenuation correction". A marker-based watershed segmentation of the images was applied with the "Morphological Segmentation" plugin of the MorpholibJ package [START_REF] Legland | MorphoLibJ: Integrated library and plugins for mathematical morphology with ImageJ[END_REF], http://imagej.net/MorphoLibJ), under tolerance < 5, connectivity = 6 and "Advanced option" mode. Over-segmentation (one cell split in several parts) and undersegmentation (merged cells) errors were corrected by manually modifying markers before rerunning the watershed transformation. The process was repeated until no segmentation error remained. On the obtained labeled image from the Catchment basin file, we then used in-house developed tools (https://github.com/L-EL/labeledImg_tools) to manually extract cells of interest from cell files. To obtain precise cell files, we used "orthogonal view" and the « 3D Viewer » plugin from Fiji on each segmented cell file.

The volume and cell surface were quantified using the "MorpholibJ>>Analyze>>Particle Analysis3D" plugin of Fiji. To map the volume or the surface area, we labeled images with "Assign measure to label". The transversal width and longitudinal length of root cells were measured manually on the "selected labels" cell files.

Determination of protein content

The concentration of proteins was determined by the method described by Bradford (1976) using bovine serum albumin (BSA, Sigma, 05479) as the standard in phosphate buffer pH 7.5. Bradford 5X was diluted by double distilled water. 200 µL of Bradford 1X was allocated per well of 96-well plate. The first two columns were added BSA (0.5 µg/µL) in a range of 1 µL to 6 µL, whose values were used to make a standard curve.

Wells B1/2, C1/2, D1/2, E1/2, F1/2, G1/2 and H1/2 contained 0-6 µL BSA, respectively.

5-10 µL of protein samples were added in other wells of the same plate and mixed well.

After reaction for 5-45 mins, the blue intensity of each well was read using a Biotek microplate reader at 595 nm.

Cell wall-enriched protein extraction

Fifty mg of frozen material of 10-d-old roots and 4-week-old flowers were ground with liquid nitrogen. 1 ml buffer (containing 50 mM Na2HPO4, 20 mM citric acid, 1 M NaCl, 0.01% Tween 20 at pH 7.0) was added. The solution was shaken at 250 rpm for 1 h.

The extracts were clarified by centrifugation at 10000 rpm for 30 min at 4°C. The supernatant was filtered using an Amicon ultracentrifuge filter 0.5 ml/10000 MWCO (Millipore, catalog no. UFC5010BK) in order to remove salts. Protein concentration was determined by the Bradford method (details see above).

Methanol colorimetric assay

Inhibition of AtPME2, AtPME3, citrus PME, citrus thermal-tolerance PME and PMEs from flower and root were determined using a colorimetric microassay adapted from [START_REF] Klavons | Determination of methanol using alcohol oxidase and its application to methyl ester content of pectins[END_REF]. The reaction solution contained 5 µL of purified protein extract, 90% methylesterfied Citrus pectin (Sigma P9561) at 1mg/ml, 0.008 U of Pichia pastoris alcohol oxidase (Sigma A2404) and 50 mM sodium phosphate buffer (pH = 7.5) to a final volume of 100 µl. AtPME2, AtPME3, citrus PME, and citrus thermaltolerance PME were prepared at 3 concentrations (1, 1/10 and 1/100 of purified proteins)

and PMEs from flowers and roots at 2 concentration (1 and 1/10). The mixture was incubated at 28°C for 30 min. A first OD reading 420 nm was done on a BioTek PowerWaveXS2 spectrometer. 100 µL of developing solution (2 mM ammonium acetate, 0.02 M pentane-2,4-dione, 0.05 M glacial acetic acid) was added and the solution was incubated at 68°C for 15 min after which a second reading was done. A standard dilution series from 0 to 10 nmol of methanol was included in each batch.

Results were expressed as nmol MeOH/min/µL of protein using the methanol standard curve.

Gel diffusion assay

Citrus pectin (Sigma P9561) that was a 90% methylesterfied was dissolved in 0.1 M citric acid and 0.2 M Na2HPO4 buffer at pH 5.0, 6.3 and 7.5 [START_REF] Hocq | Combined experimental and computational approaches reveal distinct pH dependence of pectin[END_REF][START_REF] Sénéchal | Tuning of pectin methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER[END_REF][START_REF] Geilfus | The pH of the apoplast: dynamic factor with functional impact under stress[END_REF][START_REF] Downie | A gel diffusion assay for quantification of pectin methylesterase activity[END_REF]. One percent (w/v) agarose was added to the pectin buffer and heated until the agarose had dissolved. The solution was cooled to 60°C and transferred to a petri dish (50 ml per dish) and allowed to solidify. Wells were punched in the gel with a 2-mm-diameter plastic sucker and the gel plug removed by aspiration. The gel was nicked and the orientation of the nick marked on the outside of the petri dish with a marker. Ten µL of enzyme mixture was pipetted in to each well.

Once loaded, the lids were placed on the dishes, the wells were incubated at 37°C for 16 h. After incubation, the gels were rinsed briefly with water and stained with 0.02% Ruthenium Red (Sigma) for 1h at room temperature. The dye was then poured off and the gel was rinsed with water again. Diameters of the red halos around each well were measured using the Fiji software.

Medium pH measurement assay

Ten 4-d old seedlings were transferred to a well containing 1 mL 1/2 MS salts, 1.0% sucrose, pH 5.8, and 14.0 µg/mL PMEI3 protein or the same volume of MES buffer.

After 48h incubation, we added a pH indicator, fluorescein-dextran conjugate (MW, 10,000, Sigma, 30 µg/mL) in 200 µL of medium was added. Final fluorescein-dextran concentration is was 30 µL/mL. The fluorescent intensity of the medium (excitation at 485 nm, emission 535 nm) was recorded with a Fluoroscan Ascent (Thermo Scientific).

A standard curve for pH was obtained using the 1⁄2 MS media adjusted to pH 6.2, 6.4, and 6.6.

MicroScale Thermophoresis (MST)

Molecular interaction between purified AtPME3 and AtPMEI3 was analyzed using MST, according to the method previously described [START_REF] Sénéchal | Tuning of pectin methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER[END_REF][START_REF] Geilfus | The pH of the apoplast: dynamic factor with functional impact under stress[END_REF][START_REF] Hocq | Combined experimental and computational approaches reveal distinct pH dependence of pectin[END_REF]. For each experiment, lysine residues of AtPME3 were labeled. 30 µL of 100% DMSO was mixed thoroughly with the solid fluorescent dye, Monolith protein labelling kit blue NHS amine reactive (Nano Temper, catalog no. MO-L003).

The concentration of the blue dye was adjusted to a 3-fold concentration of the AtPME3 (333 nM) by using the labeling buffer. 100 µL of AtPME3 protein was mixed with 100 µL dye solution in and incubated 30 min at room temperature in the dark. In the meantime, a PD SpinTrap G-25 column was prepared for removing the free dye. Eight ml of final storage buffer (50 mM Na2HPO4/NaH2PO4 buffer at pH 5.0, 6.3 and 7.5) was added to equilibrate the column. 200 µL of labeling reaction solution was loaded onto the column. The volume was adjusted to 500 µL using the final storage buffer after the sample has entered the bed completely. The flow-through was discarded. 600 µL of storage buffer was added and the eluate was collected after discarding the first few µL.

The eluate was stored at 4°C in the dark until use. After the fluorescence intensity and standard capillary (NanoTemper, catalog no. MO-K002) were pretested, the binding affinity test was performed on MST. For each experiment, a constant concentration of labeled AtPME3 was titrated with decreasing concentrations of non-labeled recombinant AtPMEI3 from 100 µM to 24 nM. The resulting mixtures were incubated 15 min at room temperature and then loaded into the capillary. Thermophoresis experiments were performed with 40% of MST power and 90% of LED power for fluorescence acquisition.

Mass spectrometry

SDS-PAGE samples preparation:

Each SDS-PAGE band was manually excised from the gels to be hydrolysed according to [START_REF] Shevchenko | Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels[END_REF]. Each sample was destained in a 40% ethanol-10% acetic acid solution (v/v) for 15 min and progressively dehydrated with 15 min incubations in first a 50% acetonitrile-25 mM ammonium bicarbonate solution and then in 100% acetonitrile. The dehydrated gel pieces were then reduced for 30 min with a 10 mM solution of DTT at 56°C and alkylated for 45 min with 55 mM iodoacetamide. The samples were then dehydrated as above. For digestion, the samples were incubated overnight at 37°C with 0.01 µg/µL of trypsin (Promega, Madison, WI) and dissolved in 50 mM ammonium bicarbonate, pH 8. The tryptic fragments were extracted from the gel by centrifugation after incubation of 15 min in a 0.5% trifluoroacetic acid -50% acetonitrile (v/v) and in 100% acetonitrile solution, dried in a speed-vac, and reconstituted with 25 µL of a 2% acetonitrile -0.1% formic acid (v/v) buffer.

LC-MS/MS analysis:

HPLC was performed on a NanoLC-Ultra system (Eksigent). A 4 µL sample of the peptide solution was loaded at 7.5 µL min -1 on a precolumn cartridge (stationary phase: C18 Biosphere, 5 µm; column: 100 µm inner diameter, 2 cm; Nanoseparations) and desalted with 0.1% AF and 2% ACN. After 3 min, the precolumn cartridge was connected to the separating PepMap C18 column (stationary phase: C18 Biosphere, 3 µm; column: 75 µm inner diameter, 150 mm; Nanoseparations). Buffers

A and B respectively were prepared with 0.1% HCOOH in water, and with 0.1% HCOOH in acetonitrile. The peptide separation was achieved at 300 nL min -1 with a linear gradient: from minute 0 to 10, at 5 to 30% of B; from minute 10 to 12, at 30 to 95% of B; from minute 12 to 18, at 95% of B (the regeneration step); from minute 18

to 19, at 95 to 5% of B; from minute 19 to 20, at 5% of B (the equilibration step).

Remaining percentages are those of buffer A. One run took 45 mins. Eluted peptides were online analyzed with an LTQ XL ion trap (Thermo Electron) using a nanoelectrospray interface. Ionization (1.5 kV ionization potential) was performed with liquid junction and a non-coated capillary probe (10 µm inner diameter; New Objective).

Peptide ions were analysed using Xcalibur 2.07 with the following data-dependent acquisition steps: (1) full MS scan (mass-to-charge ratio (m/z) 350-1 400, centroid mode) and ( 2) MS/MS (isolation width 1m/z, qz = 0.25, activation time = 30 ms, and Saccharomyces cerevisiae α-factor secretion signal (Figure 7.4). The vector allows high-level, methanol-inducible expression of the gene of interest in Pichia, and can be used in Pichia strain X-33. The coding sequences of PMEI3 and PMEI5 were amplified by using PCR using the primers shown in table (Table 7.1 Recombinant plasmids (at a concentration > 500 ng/µL) were transformed into Pichia pastoris strain X33. Transformed Pichia was plated onto YPDS medium containing 50 µg/mL of Zeocin.

Expression and induction: Transformants were selected and then cultivated in BMGY medium (Table 7.2). After 12-h incubation at 37°C with 250 rpm shaking, the OD600 of the culture was measured. The recombinant Pichia was transferred into BMMY medium in a bigger volume (Table 7.2) for a large-scale expression. The OD600 were methanol was added to the medium to keep its final concentration at 0.5% (v/v). To make sure the recombinant proteins expressed correctly, the concentrated supernatant of the culture was tested using SDS-PAGE and/or Immunoblotting (details see above).

Purification: After 4-d expression, Pichia were removed by centrifugation at 5000 rpm for 5 min. To the protein-containing supernatant was added PBS buffer 10X (Eurobio, GAUPBS00-01), 400 mM NaCl, 5 mM imidazole and 500 µL cOmplete Hig-tag purification Resin (obtained from Roche, ref 05893682001). The mixture was gently shaken at 4°C for 4 h in dark. The supernatant was removed by centrifugation at 8000 rpm for 5 min at 4°C. The resin was washed by 5 ml of wash buffer 1 (PBS 1X with 400 mM NaCl and 5 mM imidazole), 5 ml of wash buffer 2 (PBS 1X with 400 mM NaCl and 30 mM imidazole) and eluted by elution buffer (PBS 1X with 400 mM NaCl and 400 mM imidazole) for 3 times (800 µL for each). The 3 elutes were collected and centrifuged at 8000 rpm for 30 min by using 3-kDa filter (Amicon Ultra-2 mL, UFC200324). The buffer change was performed using PD SpinTrap G-25 column.

Final protein concentration was measured with Bradford assay. The purified PMEI3 was stored at 4°C in the dark until use.

Cell lysis and immunoblotting

During the purification of PMEI proteins, the cells pellet was collected, rapidly frozen by liquid nitrogen and then stored at -80°C before use. PMEI3 and PMEI5 Pichia cells were thawed at room temperature. We resuspended 1 mL of pellet in 2 mL of phosphate buffer pH 7.0. 300 µL of protease inhibitor 1x was (Constant systems Ltd. United Kingdom). The lysate was centrifuged at 5000 rpm for 15 min at 4°C. Both supernatant and pellet were analyzed using immunoblotting.

SDS-PAGE electrophoresis and Immunoblot:

The proteins were resolved using a 15% acrylamide/bisacrylamide SDS-PAGE ( 7.5. The transfer "sandwich" was exposed to 25 V and 1 A for 30 mins by a

Trans-Blot TURBO transfer system (Bio-Rad, catalog no. 170-4155). The membrane was incubated in 4.0% commercial milk for 30 mins and then incubated for 1 h at room temperature under shaking (50 rpm) with 4.0% commercial milk containing 1/5000 of commercial primary antibody raised against poly-His tag directly coupled with peroxidase (Sigma, catalog no. A7058). After washed 3x by PBS 1x, 10 mins for each, the membrane was added 500 µL of DAB substrate for peroxidase was added (Thermo Scientific, catalog no. 34002) before imaging. Imaging was performed with LAS-4000 according to the user manual.

Fourier Transform InfraRed Spectroscopy

Four-day-old seedlings were collected and immerged in 100% ethanol until use. Before acquisition, the seedlings were squashed between two BaF2 windows and abundantly rinsed in distilled water for 2 min. The samples were then dried on the window at 37°C for 20 min. An area of 30 mm x 30 mm halfway the root, on the side of the central cylinder, was selected for spectra collection. Spectra were collected using a ThermoNicolet Nexus spectrometer with a Continuum microscope accessory.

Greengate cloning of PMEI3/5 inducible constructs and transformation

All constructs are based on the GreenGate cloning system [START_REF] Lampropoulos | GreenGate -A novel, versatile, and efficient cloning system for plant transgenesis[END_REF].

The structures of final construct were shown in Figure 7.6 and 7.7. (obtained from Fisher Scientific -Germany GmbH, Schwerte, Germany) and ligated with T4 DNA ligase (Fisher Scientific -Germany GmbH, Schwerte, Germany). For complex PCR products, the single elements were amplified separately with primers shown in Table 7.5. Bsa I restriction enzyme sites were added to the primers, the single PCR products were digested with the respective enzymes, ligated and the reaction products used as templates for subsequent PCRs. pGGF000 entry plasmid was used to directly mutagenesis, changing the G site (GTAT) into a H site (TAGG). The new entry plasmid was called pGGFH000. p4Op DNA and the expression cassette 2 were a gift from the laboratory of Sebastian Wolf (University of Heidelberg, Germany). E. coli cells electro-transformed with the resulting plasmids were grown in medium with 50 mg/mL spectinomycin or 50 mg/mL kanamycin. For Agrobacterium. tumefaciens, ampicillin and spectinomycin were used at 100 mg/mL and kanamycin at 50 mg/mL, were mixed in a 1.5 ml eppendorf tube. The tube was heated at 95°C for 5 mins and cooled at room temperature for 1 h. The oligonucleotide duplex was stored on ice until use.

GreenGate Reaction: Plasmids were purified from the bacterial hosts using the innuPREP Plasmid Mini Kit (Analytik Jena AG, Jena, Germany) according to the user manual. DNA was eluted with 100~125 µL elution buffer. The DNA concentration was usually not determined exactly but estimated after gel electrophoresis estimated to be around 100 ng/µL judging from earlier preparations done with the same kit. For the GreenGate reaction itself 1.5 µL plasmid of each of the six modules were mixed with 1 µL of the destination vector, 1.5 µL CutSmart Buffer, 1.5 µL ATP (10 mM), 1 µL T4 DNA ligase (30 U/µL) and 1 µL BsaI-HF in a total volume of 15 µL. We performed 50 cycles of 37°C for 5 minutes and 16°C for 5 minutes each, followed by 50°C for 5 minutes and 80°C for 5 minutes. Later, we reduced the incubation time to 2 min each Bayer CropScience Deutschland GmbH, Langenfeld, Germany) was used at a concentration of 20 mg/L, both for spraying and watering. The concentrations of sulfadiazine, BastaTM, and kanamycin for selection on MS plates were 0.75-7.5 mg/mL, 10 mg/mL, and 50 mg/mL, respectively.

Greengate cloning of pH sensor constructs and Arabidopsis and tobacco transformation

The main steps of pH sensor construct building and Arabidopsis thaliana transformation and plant selection are similar to those of the inducible PMEIs lines (see above), with tiny modifications. The map of constructs was shown in Figure 7.8.

Primers used for pH sensor constructs are shown in Table 7.6.

Nicotiana Benthamiana Infiltration: Agrobacterium. tumefaciens strain ASE was transformed with the respective constructs. Transgenic clones were cultured in liquid selective LB medium for 2 nights at 28°C, resuspended in infiltration medium (10 mM MgCl2, 10 mM MES, 150 mM acetosyringone, pH 5.7) and pressed with a blunt syringe through the stomata at the abaxial site of leaves of approximately 4-week-old plants.

The plants were put back in the incubators for 48 or 72 h before analyzing the fluorescence levels. GUS activity test: Fresh or frozen samples were ground in GUS buffer. The mixture was centrifuged at 10000 rpm for 30 min at 4°C. Supernatant was collected for the activity test. Total protein concentration was measured using Bradford assay (details see above). One or 0.5 µg total protein was added to the well. 150 µL 2 mM MUG was mixed in each well just before the acquisition. The kinetics of fluorescent intensity (excitation at 355 nm, emission 460 nm,) was recorded with a Fluoroscan Ascent (Thermo Scientific) at 37°C for 30 min. J'ai produit PMEI3 dans une levure ce qui m'a permis de montrer que la protéine a une activité inhibitrice pH-dépendante sur un large éventail de PMEs. Par ailleurs, j'ai utilisé et développé des senseurs ratiométriques pour la mesure du pH à la surface de la cellule. En dehors des senseurs existants, j'ai aussi essayé d'adresser les mêmes biosenseurs à la paroi. A l'aide de ces outils, j'ai ensuite étudié l'impact de la modification de l'activité PME sur le pH et la croissance cellulaire. J'ai pu observer qu'un inhibiteur chimique de la PME, l'(-)epigallocatechin gallate (EGCG) entrainait une augmentation du pH apoplastique (pH Apo ) dans la racine, et ceci indépendamment de la H + -ATPase ; une inhibition de la croissance et une perte de l'anisotropie des cellules. Par ailleurs, un traitement avec PMEI3 ralentissait la croissance et un apport de PME entrainait une réduction du pH Apo et une augmentation de la croissance. Enfin, une induction de 24h de PMEI5 causait une réduction du pH Apo , ce qui est en accord avec l'activation compensatoire d'autres PMEs suite à une signalisation liée eux brassinostéroide accrue décrit précédemment. En conclusion, nos résultats suggèrent que la démethylesterification des HG crée domaines anioniques qui peuvent séquestrer des protons ce qui pourrait activer localement des protéines qui stimulent le relâchement de la paroi avec un pH optimum acide entrainant une augmentation de la vitesse de croissance cellulaire.

Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Title : The role of pectin metabolism in the control of cell wall pH and rheology Keywords : pectin, cell wall, pH, rheology, growth Abstract : Pectin, a matrix component in the primary cell wall, plays a role in controlling cell wall porosity, cell elongation and cell adhesion and constitutes an important factor in plant development. The demethylesterification of homogalacturonan (HG), the most abundant pectic polymer, has vast consequences on the mechanical properties of the cell wall, and affects developmental processes such as stomata opening, organ initiation and anisotropic cell growth. HG is selectively demethylesterified in muro by pectin methylesterases (PME), which in turn can be inhibited by endogenous PME inhibitor proteins (PMEIs). Demethylesterified HG is thought to form Ca 2+ -pectate complexes, which contribute to wall stiffening, but recent evidence suggest that it can also promote cell wall loosening and expansion, through a so far unknown mechanism. In this study I addressed this paradox by investigating the link between pectin metabolism, cell wall pH and extensibility. To this end I developed and used genetic and pharmacological tools for the in vivo manipulation of PME activity. I generated inducible overexpression lines for two distinct PMEIs, which unfortunately were not functional. I also produced PMEI3 from Arabidopsis in a yeast and showed that the protein displayed an inhibiting activity on a broad range of PMEs.

In addition, I developed and used tools to monitor the cell surface pH. In addition to using existing genetically-encoded ratiometric apoplastic pH sensors, I also tried to generate similar sensors targeted to the cell wall. Using these tools I then studied the impact of changes in pectin methylesterification on the cell wall pH and cell expansion. I discovered that a chemical inhibitor of PME, (-)-epigallocatechin gallate (EGCG), promoted an increase in apoplastic pH (pH Apo ) in root cells, independently from the inhibition of the H + -ATPase, and triggered root growth inhibition and abnormal cell shape. Exogenous PMEI3 application also inhibited root growth. In addition, PME application caused a decrease in pH Apo and enhanced root growth. Interestingly, long-term induction of PMEI5 could reduce pH Apo , consistent with the previously described activation of brassinosteroid signaling causing a compensatory increase in PME activity. Together, my study provides evidence that HG demethylesterification leads to a decrease in pH Apo and an increase in cell growth in the Arabidopsis root. Our results support the view that the negatively charged pectate can sequester protons and thus may contribute to the activation of cell wall loosening proteins and cell growth.

Figure 1 . 1 Figure 1 . 2 Figure 1 . 3 Figure 1 . 4 Figure 1 . 5 Figure 1 . 7 Figure 1 . 8 Figure 1 . 9 Figure 2 . 1 Figure 2 . 2 Figure 2 . 3 Figure 2 . 4

 111213141517181921222324 Figure 1.1 Model of primary cell wall and structure of cell wall polysaccharides and proteoglycan APAP1……………………………………………………………….4 Figure 1.2 Structure of the PME-PMEI complex…………………… ……………6 Figure 1.3 Gel phase transition……………………………………………………14 Figure 1.4 Model for methyl polygalacturonate structure…………………...……14 Figure 1.5 Model for methyl Ca 2+ -pectate…………………………….…………..16 Figure 1.6 HDM and LDM gelation………………………………………………16 Figure 1.7 Different types of LDM pectin gel depending on Ca 2+ concentration, consecutive demethylesterified residues and HG polymer concentration………....18 Figure 1.8 Proton efflux by H + -ATPase affects cation and anion transportation cross plasma membrane through elevating proton motive force………………………...26 Figure 1.9 Models for the activation of plasma membrane H + -ATPase by phosphorylation and fusicoccin……………………………………………………32 Figure 1.10 Theoretical mode of the electrostatic and ionic control of the cell wall loosening proteins system……………………………………………………….…40 Figure 1.11 Phase Relationships in oscillatory growth in pollen tube…………….52 Figure 1.12 Pollen tube tip growth………………………………………………...54 Figure 1.13 Ethanol treatment caused a significant reduction in the ester bonds in the cell wall……………………………………………………………………….……60 Figure 2.1 Phylogeny of PMEIs in Arabidopsis…………………………..…..…..66 Figure 2.2 Representative agarose gel image showing colony PCR of the intermediate vector………………………………………………………………………………68 Figure 2.3 Representative agarose gel image showing colony PCR and common PCR of the designed destination vector………………………………………………... 70 Figure 2.4 Representative red seeds of inducible PMEI3 and PMEI5 overexpressing lines…………………………………………………………………………….….73

Figure 2 . 5

 25 Figure 2.5 Sulfadiazine resistance test of T2 transformants of inducible PMEI3 and PMEI5 overexpression and vector control lines…………………………………..73

Figure 2 . 6 Figure 2 . 7 XIFigure 2 .Figure 2 .Figure 3 . 1 Figure 3 . 2 Figure 3 . 3 Figure 3 . 4 Figure 3 . 5 Figure 3 . 6 Figure 3 . 7 Figure 3 . 8 Figure 3 . 9 Figure 3 .Figure 4 . 1 Figure 4 . 2 Figure 4 . 3

 2627223132333435363738393414243 Figure 2.6 T2 transformant phenotyping of inducible PMEI3 and PMEI5 overexpressing and vector control lines…………………………………………..74 Figure 2.7 T2 transformant phenotype of inducible PMEI3 and PMEI5 overexpression lines……………………………………………………………….75

Figure 4 . 4 Figure 5 . 1 112 Figure 5 . 2 Figure 5 . 3 XIIFigure 5 . 7 Figure 5 . 9 Figure 7 . 1 Figure 7 . 2 Figure 7 . 3 Figure 7 . 4 Figure 7 . 5 Figure 7 . 6 Figure 7 . 7 Figure 7 . 8

 4451112525357597172737475767778 Figure 4.4 PM-Apo showing a patchy expression in root cells…………………..106 Figure 4.5 HPTS calibration and its robustness test in vivo………………………106 Figure 5.1 Dose-dependent inhibition of Arabidopsis root growth by PME inhibitor EGCG…………………………………………………………………………….112 Figure 5.2 EGCG treatment altered cell shape in root………………………......115 Figure 5.3 EGCG treatment promotes an increase in pH Apo in root cells………..118 Figure 5.4 EGCG treatment promotes an increase in pH Apo in transition zone….120 Figure 5.5 EGCG-triggered increase in pH Apo correlates with root growth inhibition…………………………………………………………………….…....121 Figure 5.6 Exogenous PME treatment reduced pH Apo in transition zone………..122

Figure 1 . 1 :

 11 Figure 1.1: Model of primary cell wall (A) and structure of cell wall polysaccharides (B) and proteoglycan APAP1 (C). Modified from[START_REF] Lampugnani | Building a plant cell wall at a glance[END_REF]

Figure 1 . 2 :

 12 Figure 1.2: Structure of the PME-PMEI complex. (A) Ribbon representation illustrating the relative positions of PMEI and PME in the complex. PME is shown in green-blue on the left side. PMEI is represented in yellow-red on the right side; the α -helices of the four-helix bundle are indicated as α1 to α4, whereas helices of the N-terminal region are named αa, αb, and αc. (B) Molecular surface of the PME-PMEI complex. Representation of the molecular surface of PME (violet) and PMEI (yellow) in the complex. Modified from (Di Matteo et al. 2005).

Figure 1 . 3 :

 13 Figure 1.3: Gel phase transition (A) Acrylamide gel strips are incubated in a medium of increasing hydrophobicity (increasing aceton concentrations). The dramatic volume decrease between 47% and 48% aceton reflects a phase transition. (B) Small change in temperature elicits a phase transition in a gel in an acetone-water mixture of fixed composition. Modified from (Tanaka 1980).

Figure 1 . 4 :

 14 Figure 1.4: Model for methyl polygalacturonate structure. (A) Parallel chains are packed in a hexagonal lattice. (B) and (C) Best associations of three interacting methyl polygalacturonate chains for (B) parallel and (C) antiparallel arrangements. Distances are given in Å. For the sake of simplicity, the hydroxylic hydrogen atoms are not represented. Modified from (Walkinshaw and Arnott. 1981; Braccini et al. 2005).

Figure 1 . 5 :

 15 Figure 1.5: Model for methyl Ca 2+ -pectate. (A) Molecular conformation of fully methylesterified HG showing atom labeling for the helical repeating unit. (B) The Ca 2+ -pectate molecular conformation. Coordination of Ca 2+(striped circles) to polymer oxygen functions is denoted by thin unbroken lines. (C) The Ca 2+ -pectate unit cell viewed parallel to the helix axis. Ca 2+ coordination to the polyanion oxygen atoms is denoted by thin unbroken lines. At least 2 water molecules would be required to complete the Ca 2+ coordination shells. Modified from (Walkinshaw and Arnott 1981).

Figure 1 . 6 :

 16 Figure 1.6: High degree-of-methylesterification (HDM) and LDM gelation. (A) HDM gelation is governed by intermolecular hydrogen bonds and hydrophobic interactions between methylesters. (B) LDM gelation mechanism is governed by ionic cross-linking via Ca 2+ between two carboxyl groups from two different chains in close proximity.Modified from[START_REF] Chan | Pectin as a rheology modifier: origin, structure, commercial production and rheology[END_REF]).

Figure 1 . 7 :

 17 Figure 1.7: Different types of LDM pectin gel depending on Ca 2+ concentration, consecutive demethylesterified residues and HG polymer concentration. (A) Gel state diagram. (B) Schematic of gel state diagram corresponding to (A). Reff value: the ratio of Ca 2+ concentration to the quantity contained in blocks of consecutive demethylesterified residues longer than the amount required to bind Ca 2+ into an egg-box. Cp: HG polymer concentration. Modified from (Vincent and Williams 2009).

Figure 1 . 8 :

 18 Figure 1.8: Proton efflux by H + -ATPase affects cation and anion transportation cross plasma membrane through elevating proton motive force. The cytosolic ion concentrations determine turgor pressure. PM: plasma membrane.

Figure 1 . 9 :

 19 Figure 1.9: Models for the activation of plasma membrane H + -ATPase by phosphorylation and fusicoccin.

  FER is transcriptionally induced by BRs, and are down-regulated in the loss-of-function BR mutant bri1 and up-regulated in the constitutive BR-response mutant bes1-D. Besides FER, the CrRLK1Ls THE1 and HERK1 are also transcriptionally upregulated by BR through BRI1 and BES1. herk1/the1 double mutants enhance loss of function bri1 and suppress bes1-D mutant phenotypes. The two pathways regulate a common set of 127 target genes, including those involved in cell elongation (Deslauriers and Larsen 2010; Duan et al. 2014; Guo et al. 2009). Abscisic acid: Phosphatase ABA INSENSITIVE 2 (ABI2) negatively regulates abscisic acid (ABA) signaling (Yu et al. 2012), which physically interacts with and be activated by RHO GTPase, such as ROP11/ARAC10 (RHO OF PLANTS 11/RAC-LIKE

Figure 1 . 10 :

 110 Figure 1.10: Theoretical model of the electrostatic and ionic control of cell wall loosening. PME activity catalyzing HG demethylesterification has a high pH optimum. The H + released from this reaction increases the electrostatic potential Δψ of the cell wall relative to the medium and reduces the pH. This in turn activates cell wall loosening proteins with low pH optimum, leading to cell wall extension. Cell wall extension combined with the deposition of new wall material leads to the dilution of the H + and the alkalination of the cell wall. The yellow "lightning" refers to the active version of the protein. (Adapted from[START_REF] Crasnier | Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls[END_REF][START_REF] Moustacas | Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells[END_REF]

1. 6

 6 .2 pH-sensitive fluorescent dyesA second method combines pH-sensitive fluorophores and confocal or fluorescence microscopy. This fluorophore should fulfill several requirements. (i) It must not penetrate into the symplast. This can be prevented by conjugating the dye to a 10-kDasized dextran[START_REF] Monshausen | Oscillations in extracellular pH and reactive oxygen species modulate tip growth of Arabidopsis root hairs[END_REF] 2009). (ii) The probe must change a physicochemical attribute, such as emission intensity, in a strict dependency to the free H + concentration. (iii) It should not be cross-sensitive to other ions, metabolites, or chemical parameters such as the redox state of the biological system. Methods based on the pH dependency of the emitted light intensity, have to correct for variations in the probe concentration itself. To this end, the protonated form of the fluorophore is excited by light of a specific wavelength, while a different wavelength is used to measure a pH-independent signal. The pH-sensitive/pH-insensitive channel ratio is then determined for each pixel[START_REF] Gilroy | Fluorescence ratio imaging and photometry of calcium in living plant cells[END_REF]. Calibration in vivo is then required to convert this ratio into pH[START_REF] Geilfus | Bacterially produced Pt-GFP as ratiometric dual-excitation sensor for in planta mapping of leaf apoplastic pH in intact Avena sativa and Vicia faba[END_REF]. The fluorescein derivatives fluorescein isothiocyanate, Oregon green 488, or 2', 7'-bis-(2-carboxyethyl)-5-(6)carboxy fluorescein (BCECF) have been frequently used as dyes to monitor the pH of the leaf apoplast via the dual-excitation technique[START_REF] Pitann | The apoplastic pH and its significance in adaptation to salinity in maize (Zea mays L.): comparison of fluorescence microscopy and pH-sensitive microelectrodes[END_REF][START_REF] Mclachlan | The breakdown of stored triacylglycerols is required during light-induced stomatal opening[END_REF][START_REF] Hoffmann | FITC-dextran for measuring apoplast pH and apoplastic pH gradients between various cell types in sunflower leaves[END_REF]. An alternative method is based upon the excitation of the fluorophore at one wavelength and the emission at 2 different wavelengths only one which is correlated with pH. To measure the surface pH of roots,Monshausen et al. (2009) bathed Arabidopsis roots in a medium containing the ratiometric pH indicator fluorescein dextran (10 kDa). By observing changes in the emission intensity of the dye, which is dissolved in the bathing solution that harbors the roots, it was possible to analyze the root surface pH. This revealed that the root surface pH is regulated by calcium signals during mechanosensing in Arabidopsis roots.[START_REF] Barbez | Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana[END_REF] were able to load the ratiometric pH indicator 8-hydroxypyrene-1,3,6trisulfonic acid trisodium salt (HPTS) into the root apoplast by immerging seedlings of Arabidopsis for 30 min in liquid growth medium that contained the dye. By using this loading technique in combination with two-excitation-wavelength-ratio imaging, this research revealed that auxin signaling triggered cell-wall acidification, leading to increased cellular expansion.

  and optimized versions of GFP for pH measurement have been generated. Ecliptic and superecliptic pHluorins display almost no fluorescence when protonated, allowing sensitive pH measurements.[START_REF] Gao | Self-reporting Arabidopsis expressing pH and Ca 2+ indicators unveil ion dynamics in the cytoplasm and in the apoplast under abiotic stress[END_REF] fused the pHlourin to a chitinase signal sequence to target this pH indicator directly to the cell wall. This study revealed that the salt stress-induced pH apo signature differed from that induced by osmotic stress.[START_REF] Martinière | Uncovering pH at both sides of the root plasma membrane interface using noninvasive imaging[END_REF] fused pHlourin to a modified transmembrane domain containing 26 amino acids of the PM-localized TM23. This pH Apo sensor, briefly called PM-Apo, is able to record the pH value at a position of a few nanometer away from the PM. The results with PM-Apo revealed pH of 6.5 to 6.7 close to the PM, suggesting a large difference in pH between PM and cell wall (pH ~ 4.0). Combined PM-Apo and an interface-localized pH sensor, PM-cyto, which consists of pHlourin and a farnesylation sequence of Ras, the transmembrane H + gradient (= pHcyto -pHApo) was precisely measured in mature root cells from the epidermis to stele[START_REF] Martinière | Uncovering pH at both sides of the root plasma membrane interface using noninvasive imaging[END_REF]. Although pHluorin and its derivative sensors have proven their utility[START_REF] Martinière | Development and properties of genetically encoded pH sensors in plants[END_REF] 2018), they also have a number of disadvantages, such as (1) the equilibrium between the protonated and deprotonated states of these proteins is affected by temperature and ionic strength. (2) the light emitted by sensor is dependent on its own concentration. (3) the fluorescence sensor is easily degraded or quenched in acidic milieu. To circumvent these issues, other sensors are based on the measure of pH by Förster resonance energy transfert (FRET) between two fluorescent proteins or by the emission intensity ratio of two fluorescent proteins. In particular, the tandem of enhanced GFP (EGFP) and pH insensitive monomeric red FP (mRFP1) is suitable for measuring pH in the apoplast[START_REF] Gjetting | Live imaging of intra-and extracellular pH in plants using pHusion, a novel genetically encoded biosensor[END_REF]. apopHusion is targeted to the apoplast thanks to a chitinase signal peptide[START_REF] Gjetting | Live imaging of intra-and extracellular pH in plants using pHusion, a novel genetically encoded biosensor[END_REF], as confirmed in leaf epidermis, the guard cells, the mesophyll, and root apex in Arabidopsis. Using this genetically encoded sensor,[START_REF] Gjetting | Live imaging of intra-and extracellular pH in plants using pHusion, a novel genetically encoded biosensor[END_REF] discovered a fast alkalinization response in the epidermal apoplast of elongating roots to the external addition of indole-3-acetic acid.[START_REF] Fendrych | TIR1/AFB-Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls[END_REF] found that apoplastic acidification, elongation, and auxin transcriptional responses all happen ~20 min after auxin application in the etiolated Arabidopsis hypocotyl. The power of the apopHusion approach was especially evident when auxin-induced pH changes in the apoplast were examined in vivo during gravitropic responses.Genetically encoded pH probes have the drawback, however, that they may accumulate in an active form within the secretory system. As a result, quantification of the pH Apo requires correction for the intracellular signal[START_REF] Swanson | In vivo Imaging of Ca 2+ , pH, and reactive oxygen species using fluorescent probes in plants[END_REF]. A second drawback is that it is not known to what extent other physico-chemical parameters can influence the behavior of sensors. A further disadvantage is that genetically-encoded probes require plants that are readily transformable, complicating their use in nonmodel plants. In order to circumvent the problem, Geilfus et al. (2014) purified ratiometric dual-excitation pH-sensor GFPs from a bacterial expression system and loaded them non-invasively through the open stomata into the apoplast of intact plants.

  2+ oscillations were extensively studied in Lily PTs. The former is often measured by fluorescent dyes, such as fura-2-dextran and the latter is recorded by non-invasive extracellular, ion-selective Ca 2+ electrode (Hepler et al. 2013). When pulsatile growth began, the basal Ca 2+ influx decreased and a pulsatile component appeared, superimposed on the reduced basal Ca 2+ flux. The peaks of the Ca 2+ pulses at the cell surface averaged 38.4 ± 2.5 pmol cm -2 second -1 . Longer tubes had large pulsatile Ca 2+ fluxes with smaller baseline fluxes. Cross-correlation

Figure 1 .

 1 Figure1.11: Phase Relationships in oscillatory growth in pollen tube. The positive sign (+) indicates that the increase in the particular event follows the increase in growth rate, whereas the negative sign (-) indicates that the increase in the event precedes the increase in growth rate. The increase in the intracellular Ca 2+ follows the increase in growth rate. In addition, the increase in intracellular Ca 2+ is out of phase with the increase in extracellular Ca 2+

Figure 1 .

 1 Figure 1.12: Pollen tube tip growth. (A) A model of pollen tube tip growth. The cell wall of flank and apical dome in pollen tube poss LDM (low degree of methylesterification) and HDM (high DM) HG, respectively. Turgor pressure (P) maintains tip growth. Exocytosis deposits cell wall components and controls the growth direction. pH and Ca 2+ gradients exist and are associated with growth rate oscillations. Color bar, blue to red represents low to high concentration. (B) A simplified diagram for tip growth regulation. Many factors are involved in pollen tube tip growth. During growth rate oscillations, exocytosis, pH, Ca 2+ , ROS, K + and P concentrations display oscillatory variations (violet wave).

  necessary to allow it to keep pace with growth. A study ofFéijo et al (1999) supposed that the alkaline band correlates with the position of the reverse fountain streaming at the base of the "clear zone", and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. Indeed, modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain and a pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Acidification causes actin filament destabilization and inhibits growth by 80%. Increased actin polymerization supports faster growth rates and a H + influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH increases, the activity of ADF/AIP again increases, repeating the cycle of events[START_REF] Lovy-Wheeler | Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of Lily[END_REF]. The protein villin/gelsolin may participate in the degradation of the actin fringe. When stimulated by elevated levels of Ca 2+ , this protein promotes the fragmentation of actin microfilaments[START_REF] Zhang | Arabidopsis VILLIN5, an actin filament bundling and severing protein, is necessary for normal pollen tube growth[END_REF]. In this view, the Ca 2+ level and the presence of villin/gelsolin at the PT apex could explain the absence of actin microfilaments at this position.1.7.7 ExocytosisCell wall polymer-containing secretory vesicles are transported along actin cables.Exocytosis has been analyzed by using fused PME-YFP, DIC images and propidium iodide (PI) staining. The wall thickness shown by the latter two methods can indirectly reflect the secretion rate. Exocytosis in PT oscillates and leads the increase in growth rate[START_REF] Rojas | Chemically mediated mechanical expansion of the pollen tube cell wall[END_REF]; the mean phase difference between exocytosis and growth is -98 ± 3 degrees in lily and -124 ± 4 degrees in tobacco (Figure1.11). The oscillation in cell wall thickness precedes the Ca 2+ oscillation[START_REF] Mckenna | Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes[END_REF], and the cell wall thickness is correlated with the extent of the subsequent growth response, suggesting that cell wall deposition rate is the main parameter controlling growth rate[START_REF] Mckenna | Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes[END_REF]. In RHs, PI staining of the apical cell wall also shows that the oscillatory increase in fluorescence precedes the changes in the growth rate by an advance of -112°[START_REF] Rounds | Propidium iodide competes with Ca 2+ to label pectin in pollen tubes and Arabidopsis root hairs[END_REF]. The deposition could actively induce dissociation of Ca 2+ -pectate cross-links in PT apex, which allows turgor force to stretch the pectic wall. The nascent HG is immediately demethylesterified and then competed

  experiments are therefore needed to see who is right.Along the same lines, a recent study[START_REF] Bou Daher | Anisotropic growth is achieved through the additive mechanical effect of material anisotropy and elastic asymmetry[END_REF]) repeated the experiments of[START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF], using the same genetic material, and came to the opposite conclusion that HG demethylesterification stiffens the wall of Arabidopsis hypocotyl epidermal cells and inhibits cell expansion. Given the importance of the findings of Peaucelle et al for my PhD thesis, I will here discuss in more detail these contradicting results.[START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF] observed in dark grown Arabidopsis hypocotyls that the cells that leave the apical hook undergo a transition from isotropic to anisotropic growth and that this symmetry breaking is associated with an increase in the elasticity of the longitudinal anticlinal cell walls as shown using AFM. They also showed increased staining for HG LDM (using 2F4 antibody) in the longitudinal relative to the transverse walls. Finally, inhibition or promotion of PME activity by the inducible overexpression of PMEI3 and PME5, respectively, inhibited and promoted an increase in cell wall elasticity and cell expansion rate. Bou Daher et al. (2018) focused on the growth acceleration of the cells at the base of the hypocotyl that takes place 24 h after germination and also observed an increase in elasticity of the longitudinal cell walls. In contrast to[START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF], however, the authors observed preferential demethylesterification in transverse and not in longitudinal walls. To this end, Bou[START_REF] Bou Daher | Anisotropic growth is achieved through the additive mechanical effect of material anisotropy and elastic asymmetry[END_REF] carried out an immunolabeling using antibodies LM20, LM19 or 2F4, recognizing HG HDM , HG LDM and Ca 2+ -crosslinked HG LDM 1 respectively, on longitudinal hypocotyl sections at 24 h post germination (hpg). They observed stronger LM20 (HG HDM ) labeling of longitudinal walls relative to transverse walls and stronger 2F4 (Ca 2+ -crosslinked HG LDM ) labeling of transverse relative to longitudinal walls, whereas LM19 labeling was symmetric. The images however, are not really convincing and no quantification of the immuno-fluorescence is provided. In addition, our own results on 2F4-labeled longitudinal sections of hypocotyls of the same age, showed a large variability in the labeling of the different cells, perhaps related to the growth stage, as well as higher labeling of longitudinal walls in zones that were expected to elongate. This supports the results of[START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF]. The other discrepancy was that Bou Daher et al. (2018) observed growth inhibition and growth promotion upon ethanol induction of PME5 and PMEI3 respectively, associated with higher and lower wall stiffness respectively. These are responses completely opposite to those observed by[START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF]. We do not understand why the results are so different, but inspection of the materials and methods section showed that Bou[START_REF] Bou Daher | Anisotropic growth is achieved through the additive mechanical effect of material anisotropy and elastic asymmetry[END_REF] used very high concentrations of ethanol (100%) to induce the constructs (which may explain why they observed 100% penetrance of the induction as opposed to the 10 -20% penetrance obtained by[START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF]. We hypothesize that the induction of PME and PMEI in the presence of such high ethanol levels might be responsible for the phenotypes, especially when considering that a low ethanol concentration itself already leads to the induction of endogenous PME activity[START_REF] Andres-Robin | Evidence for the regulation of gynoecium morphogenesis by ETTIN via cell wall dynamics[END_REF] or a significant reduction in the ester bonds in the cell wall as shown by FTIR (Figure1.13).Further work is needed to clarify this issue.In conclusion, the contradicting results of Bou[START_REF] Bou Daher | Anisotropic growth is achieved through the additive mechanical effect of material anisotropy and elastic asymmetry[END_REF] are to my opinion not sufficiently convincing to invalidate the results of[START_REF] Peaucelle | The control of growth symmetry breaking in the Arabidopsis hypocotyl[END_REF].Furthermore, the results of[START_REF] Qi | Mechanical regulation of organ asymmetry in leaves[END_REF] and Andres-Robin et al. (2018) also support a wall-loosening and growth promoting effect of PME activity in two different contexts.Alternative explanations for the dual effects of PME activity on cell wall mechanics depending on the context, may be that it depends on the nature of the PME activity (e.g. pH-or Ca 2+ -dependence, blockwise or random activity, preferences for certain HG substrates), free Ca 2+ concentrations in the apoplast, the extent of the demethylesterification or the presence of polygalacturonases or pectate lyases in the cell wall.

Figure 1 .

 1 Figure 1.13: Ethanol treatment caused a significant reduction in the amount of ester bonds in the cell wall.(A) Average Fourier Transform Infrared (FTIR) spectra of 5-d-old water-and ethanol-treated Col seedlings (colwat and coleth respectively). (B) Student's t-test: t-value for the comparison between water-and ethanol-treated seedlings (y-axis) is plotted against the wave numbers (x-axis). Horizontal lines refer to the P = 0.05 significancy threshold.Equally significant minima correspond to ester linkages (red arrows) of pectin. n > 20. This suggests that low ethanol concentrations promote de-esterification activity in the cell wall.

  At the start of my PhD, it was already known that pectin modification and pH Apo are involved in the control of cell wall extensibility and growth. What remained unclear, however, was the mechanistic link between HG demethylesterification, pH Apo and growth. In my PhD thesis, I have tried to address this issue through a set of linked but different basic questions: (i) To what extent does the pH Apo depend on HG demethylesterification? Does this also involve the PM H + -ATPase? (ii) How does the change in pH Apo triggered by HG demethylesterification in turn affect plant growth in vivo? What is the effect of this pH Apo change at the cellular level? (iii) What is the difference in biochemical properties between PMEI3 and PMEI5 in vitro? Do PMEI3 and PMEI5 have different pH optima or target different PMEs? In this study, I focused on PMEI3 and PMEI5, which, based on previous in vivo studies, may have different biochemical specificities. Firstly, I attempted to construct new inducible PMEI3 and PMEI5 overexpression lines and three cell wall-targeted genetically encoded pH sensors in parallel. Secondly, I tried to express these two PMEIs in Pichia pastoris and characterized PMEI3 activity in vitro. Lastly, I used genetic and pharmacological approaches to manipulate the level of HG demethylesterification in living Arabidopsis roots combined with the monitoring of the pH Apo using genetically encoded ratiometric pH sensors and a pH-sensitive fluorescent dye. PMEs are encoded by remarkably large gene families (69 members in Arabidopsis thaliana) and most loss of function mutants analyzed so far did not show obvious changes in PME activity or phenotype suggesting high functional redundancy. To manipulate the PME activity in vivo, we therefore explored the use of PMEI overexpressing lines. To identify PMEI gene candidates that inhibit a broad range of PMEs, we analyzed the phylogenetic relationships and expression patterns for the 76 PMEI isoforms of Arabidopsis thaliana. The predicted full-length amino acid sequences of the PMEIs were obtained from The Arabidopsis Information Resource

Figure 2 . 1 :

 21 Figure 2.1: Phylogeny of PMEIs in Arabidopsis. Phylogenetic analysis of the predicted full-length amino acid

Figure 2 . 2 :

 22 Figure 2.2: Representative agarose gel image showing colony PCR of the intermediate vector. (A and B) Driver cassette intermediate vector. (C) Effector cassette intermediate vector (P2). Five colonies from each group were tested. NC: negative control. Primers: pGGM00-F and pGGM00-R (A), UBQ10-F and pGGM00-R (B), pGGZ001-R and PMEI3-GG-R, PMEI5-GG-R (C).

Figure 2 . 3 :

 23 Figure 2.3: Representative agarose gel image showing colony PCR (A, B, and C) and common PCR (D) of the designed destination vector. Five or 6 E. coli colonies from Final control, PMEI3-F and PMEI5-F were tested (A, B, and C). Plasmids of two Agrobacteria colonies of each group (D). PC: positive control from Final control in (A). Primers: RedSeed-ter-F and Turquoise-R (A, B, C, and D)

Figure 2 . 4 :

 24 Figure 2.4: Representative red seeds of inducible PMEI3 and PMEI5 overexpressing lines. (A) Red seed of T1 transformant. (B) Heterozygote and homozygote showed different DsRed fluorescence intensity. Black-white image showed the different fluorescence intensities.

Figure 2 . 5 :

 25 Figure 2.5: Sulfadiazine resistance test of T2 transformants of inducible PMEI3 (FX3) and PMEI5 (FX5) overexpression and vector control (FX0) lines in Col and xxt1xxt2 background (A, B, C, D, and E). Plants were grown for 7 days in solid 1/2 MS medium complemented with 100 µM sulfadiazine.

Figure 2 . 6 :

 26 Figure 2.6: T2 transformant phenotyps of inducible PMEI3 (FX3) and PMEI5 (FX5) overexpressing and vector control (FX0) lines grown in Col and xxt1xxt2 background in the growth chamber (A and B). Plant were

Figure 2 .Figure 2 . 8 :

 228 Figure 2.7: T2 transformant phenotype of inducible PMEI3 (FX3) and PMEI5 (FX5) overexpression lines in Col background grown in the green house for 4 weeks (A, B and C). Plants were sprayed with 50 µM dexamethasone (DEX). Induced Col-FX3 and Col-FX5 overexpression lines did not show typical phenotypes associated with PMEI3 or PMEI5 overexpression, such as dwarfism, twisted organ growth, waving roots and abnormal siliques.

Figure 2 .

 2 Figure 2.9: qRT-PCR quantification of PMEI3, PMEI5, and mTURQUOISE transcript levels (A) and PME activity measurement (B and C) of inducible PMEI3 (FX3) and PMEI5 (FX5) overexpressing and vector control (FX0) lines in Col background, after1.5-h (A) or 24-h (B and C) 50 µM dexamethasone induction. Representative image of PME activity measurement using a gel diffusion assay (B). Error bar: STD. n > 6 in C.

  1) and confirmed by Sanger sequencing of a single clone for each construct. Precise constructs were transformed into the Pichia strain X33. To check if the recombinant protein expressed, supernatants were isolated and concentrated after 4-d cultivation with 0.5% (v/v) methanol induction, and then the recombinants PMEI3 and PMEI5 were analyzed by Western Blotting (WB) using antibodies raised against the poly-Histidine or C-Myc tag (Figure3.2 and 3.3). Induced cultures harboring the PMEI3 constructs displayed a clear band at the expected molecular mass of about 19.9 kDa (Figure3.2 and 3.3). In contrast, cultures harboring PMEI5 and CO-PMEI5 constructs showed one or two bands of 35 kDa and 55 kDa, bigger than expected size (17.9 kDa) in WB with the poly-Histidine antibody (Figure3.2 and 3.3), but not with the c-Myc antibody (Figure3.3). To assess the inhibiting activity of PMEI3 and PMEI5, concentrated supernatants were tested with 3 types of PME in GDA with a pectin of DM 90% at pH 6.3 (Figure3.4). The supernatants from cultures harboring PMEI3, PMEI3t and CO-PMEI3 showed clear inhibiting activity on orange PME, Arabidopsis flower PMEs and root PMEs, while none of supernatants from PMEI5, PMEI5t and CO-PMEI5 cultures showed inhibiting activity on these PMEs (Figure3.4).

Figure 3 .Figure 3 . 2 :Figure 3 . 3 :

 33233 Figure 3.1: constructs for heterologous expression of PMEI3 and PMEI5 in Pichia pastoris. (A) Structure of 3 PMEI3 and PMEI5 constructs. See Methods and Materials for vector map. (B) and (C) Representative agarose gel image showing colony PCR of the designed vectors (B) and restriction digestion by Sac I (C). Two or three colonies of each group were tested (B and C). PC: Positive control. Primers: 3'-AOX1 and 5'-AOX1 (B).

Figure 3 . 4 :Figure 3 . 5 :Figure 3 . 6 :Figure 3 . 7 :Figure 3

 343536373 Figure 3.4: PMEI activity measurements using gel diffusion assays. Inhibition assays on orange PME (A) and PME preparations from Arabidopsis flowers (B) and roots (C) using the supernatant of cultures harboring PMEI3 or PMEI5 constructs. Two or 4 colonies from each group were tested in (A, B, and C). Construct structures are shown in Figure 3.1. Water was used as negative control.

Figure 3 .Figure 3 . 9 :

 339 Figure 3.8: PME activity measurement using a methanol colorimetric assay. 1/10, 1/100: fold dilution. TT PME: citrus thermal tolerant PME. The concentrations corresponding to bars surrounded by rectangles were used for the inhibition assays of recombinant PMEI3. Error bar: STD. n=3.

Figure 3 .

 3 Figure 3.10: Determination of the interaction between PME3 and PMEI3 using microscale thermophoresis. (A) Interaction in binding solution at pH 5.0, 6.3 and 7.5. (B) Interaction in binding solution with or without 0.1 % pectin at pH 5.0. For the binding assay, PME3 was labeled and titrated with decreasing concentrations of PMEI3. The fitted curves and the resulting dissociation constant (Kd) values were calculated by averaging replicates. Kd values represent the mean of 3 replicates. Concentrations on the X axis are nM.

  .10b). It will be interesting to see what the underlying mechanism is of this pectin-dependent increase in affinity. As discussed in the Introduction, three main standard methods can be used to measure pH Apo in living plants. Among these methods, the genetically encoded fluorescent sensors are reliable, have a high spatiotemporal resolution and are nontoxic. To generate new genetically encoded fluorescent pH sensors in Arabidopsis to measure the pH in the cell wall, we built, using the GreenGate cloning system, 3 constructs that fused apoplast targeting signal peptide to the tandem concatenation of a pH-insensitive and a pH-sensitive fluorescent protein (Figure 4.1). Two cell wall targeting domains were chosen from Formin 1 in Arabidopsis (AtFH1), which forms a connection between the cell wall and the actin cytoskeleton, and a PME in Linum usitatissimum (LuPME1), which localizes to cell wall (plasmid was kindly provided by N. Paris). The pH-sensing moieties comprised either the pH-insensitive mRFP1 and a pH sensitive EGFP used for the ApopHusion (Gjetting et al. 2013), or the pH-insensitive mRFP1 and a pH sensitive BFP (Figure 4.1). REDSEED gene was used as a reporter gene in the three pH sensor constructs. The constructs were verified for the presence of the desired insert using colony PCR and restriction enzyme digestion (Figure 4.1) and a single clone for each construct was confirmed by Sanger sequencing of a single clone for each construct. The constructs were first tested in a transient expression assay in Nicotiana benthamiana leaves. 72 hours after infiltration, both EGFP and mRFP1 fluorescence were clearly visible in the cell wall in leaves harboring the sensors AtFH1-pHusion and LuPME1-pHusion (Figure 4.2). For AtFH1-mRFP-BFP, only the mRFP1 signal could be detected (Figure 4.2). These results confirmed that the pH sensor constructs were functional. We next generated stable Arabidopsis transformants with the 3 constructs. Red seeds from three T1 plants were sown on 1/2 MS medium. Unfortunately, germination rate of red

Figure 4 . 1 :

 41 Figure 4.1: Constructs for the expression of cell wall-targeted pH sensors. (A) Scheme of construct structure. See Material and Method for construct map. (B, C, and D): Representative agarose gel images showing colony PCR of the designed vectors. Six colonies for each group were tested. NC: negative control. Primers: pGGZ001-F and pGGZ001-R (B, C, and D).

  -pHusion and AtFH1-mRFP-BFP (Figure4.3) were only 12.3%

Figure 4 . 2 :Figure 4 . 3 :

 4243 Figure 4.2: Transient expression assay of cell wall targeted pH sensors in tobacco leaves. Confocal images of EGFP, mRFP1 and BFP of AtFH1-pHusion (A), AtFH1-mRFP-BFP2 (B), and LuPME1-pHusion (C). The experiments were repeated at least 3 times. Construct structures are shown also in Figure 4.1.

Figure 4 . 5 :

 45 Figure 4.5: HPTS calibration and robustness test in vivo. (A): HPTS 458/405 values in transition zone of the

Figure 5 . 1 : 2 C

 512 Figure 5.1: Dose-dependent inhibition of Arabidopsis root growth by PME inhibitor EGCG. (A) and (B) Col-0 dark-or light-grown seedlings grown for 2 days on solid 1/2 MS medium supplemented with 200 µM EGCG. (C) Hypocotyl length is not affected by ECGC treatment. (D) Primary root (PR) length was inhibited by EGCG treatment under both conditions. (E) Root hair density (Root system density) was reduced by EGCG treatment under dark condition. (C, D, and E) Statistical significance (0.05%) was tested using Kruskal-Wallis non-parametric H test followed by a Dunn's Multiple Comparison posthoc test. n > 70. P < 0.01. (F). Col-0 seedlings grown on solid 1/2

Figure 5 . 2 :

 52 Figure 5.2: EGCG treatment altered cell shape in root. (A) Cells in the differentiation zone (DZ) tended to bulge out (arrows) in EGCG-treated seedlings. (B) Endodermal cells in EGCG-treated seedlings showed irregular cell shapes (arrows). (C) Radial area of epidermis, cortex and endodermis cells at quiescent center (QC) in EGCG-treated seedlings was higher than that in Mock-treated seedlings. False color represents the cell area value. Wilcoxon rank sum test; ***p < 0.001. n (cell number) = 91 (Mock) and 290 (EGCG). n = 4 (Mock) and 6 (EGCG) biological

Figure 5 . 3 :

 53 Figure 5.3: EGCG treatment promotes an increase in pH Apo in root cells. pH Apo represented in false color (A-D) and quantification in meristem (MT, left), transition zone (TZ, middle), and elongation zone (EZ, right) (E-H).

Figure 5 . 7 :

 57 Figure 5.7: Exogenous PME treatment reduced pH Apo in root cells. pH Apo visualized with the apopHusion marker line. (A,B) : pH Apo represented in false colors. (C,F) : mean pH Apo ratios in whole roots (C,D); in meristem (MT),transition zone (TZ), and elongation zone (EZ) (E,F), after application of 0.5 mM PBS buffer pH 7.5 (E) of Orange PME in the same buffer (F). n=6 (mock), n=9 (Orange PME). (E,F) : After the initial pH increase, a significant decrease in pH Apo in PME-but not in mock-treated roots was observed. Significance was determined by two-way

  RootChips can combine live-cell imaging of growth and metabolism of Arabidopsis roots with rapid and gentle modulation of environmental conditions[START_REF] Grossmann | The RootChip : An Integrated Microfluidic Chip for Plant Science[END_REF]. Ten-d-old seedlings were treated with 1/2 MS liquid medium without and with EGCG in the following order: Mock-EGCG-Mock.Six to nine Z-stacks of both EGFP and mRFP1 signals were acquired in a 5 min interval.Image processing is described in Method and materials. The kymograph of EGFP/mRFP1 ratio and root growth was showed in Figure5.5. EGFP/mRFP1 ratio increased within 5 min by EGCG treatment (Figure5.5), which is consistent with the results above (Figure5.3). Simultaneously, the root growth rate diminished, shown by the flatter slope of root tip trace (Figure5.5). After removal of EGCG, the EGFP/mRFP1 ratio and root growth recovered to lower level within 40 min (Figure5.5).

Figure 5

 5 Figure 5.8: pH Apo in transition zone of inducible PMEI5 overexpressing line and WT Col0 was decreased after 24-h induction with 50 µM dexamethasone (DEX). Wilcoxon rank sum test; *p < 0.05. 21 < n < 26.

Figure 5 . 9 :

 59 Figure 5.9: Exogenous PME and PMEI3 applications promoted and inhibited root growth, respectively. Twod-old Col0 seedlings were grown in liquid ½ MS medium containing 10 U/mL orange PME (A) and recombinant PMEI3 14 µg/mL (B) for 2 days. Photographs of the roots (left panels) and root length increase (right panels). n > 35 (A), n > 30 (B). Significance was determined by two-way ANOVA followed by Tukey's test *p < 0.05, ** p < 0.01. Error bar: STD.

Figure

  Figure 7.1: Scheme of 3D-printed homemade chip mounted on a microscope stage in which seedlings are immobilized by a grid (A) or by medical-adhesive (B).

  seedlings were gently placed on the adhesive. 150 µL of 1/2 MS medium with or without chemicals (EGCG, fusicoccin and vanadate) or proteins (orange PME and PMEI3) was pipetted into the channel of the chip. The whole device was placed on the platform of Leica SP5 confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany). Treatment solution was pipetted during the interval of acquisition at appropriate time points (see Results).

Figure 7 . 2 :Figure 7 . 3 :

 7273 Figure 7.2: Microfluidic RootChip system. Image of microfluidic RootChip mounted on a plastic carrier (A). Image of microfluidic RootChip system in the growth chamber (B) and under a Zeiss 710 confocal microscope (C).

Figure 7 . 4 :

 74 Figure 7.4: Map of pPICZα B vector.

  ). The PCR products were purified using NucleoSpin Gel and a PCR Clean-up kit (obtained from Machery-Nagel) according to the user manuals. pPICZαB plasmid was propagated by transformation into E. coli strain TOP10. The purified PMEI3 and PMEI5 and pPICZαB plasmid were mixed and cleaved with restriction enzymes Pst I and Xba I at 37°C for 1 h. The reaction was terminated by heating at 75°C and the product was cleaned up. T4 ligase was used to ligate the purified DNA at 28°C for 1 h. Two µL of ligated solution was transformed into E. coli TOP 10. Transformants was selected on Low Salt LB plates containing 50 µg/mL Zeocin. Eight transformants for each were analyzed by restriction mapping or sequencing to confirm in-frame fusion of PMEI3 and PMEI5 with the αfactor secretion signal and the C-terminal tag. The appropriate transformants were cultivated to collect more plasmid. Plasmids were purified using a DNA purification kit (NucleoBond PC 100/BAC kit, obtained from Machery-Nagel). The recombinant plasmids were purified and then linearized by the Pme I restriction enzyme.

  and the cycle number to 30. 6 µL of the reaction were used for heat-shock transformation of ccdB-sensitive E. coli TOP10. The products of the first GreenGate reactions were analyzed by both restriction endonuclease test digestions and sequencing of the ligation sites. When creating the intermediate supermodules for two

Figure 7 . 5 :

 75 Figure 7.5: Scheme of protein transfer onto membrane in for immune-blotting.

Figure 7 . 6 :

 76 Figure 7.6: Cartoon of the binary activated dexamethasone-inducible system. In the absence of dexamethasone,the GR-lac-HSP70 complex is maintained in cytoplasm without activating the expression cassette. In the presence of inducer, the complex releases HSP70 and drives expression in nucleus. 1: activator cassette, 2 and 3: expression cassette. HSP70: heat shock protein 70, GR: glucocorticoid receptor. lac: lac transcription. UBQ10: ubiquitin 10 promoter. pOp: p4Op promoter. mTUR: mTURQUOISE. Sulfa R: sulfadiazine resistance. Termi: terminator.

Figure 7 . 8 :

 78 Figure 7.8: Map of GreenGate construct of cell wall-targeted pH sensor. (A) pH sensor constructs consist of an apoplast targeting signal peptide (Apo-SP) and a fluorescent sensor. AtFH1: N terminal of Arabidopsis Formin1. LuPME1: mutated and non-functional flax PME. (B) GreenGate construct structures. See also Figure 6.

  des polysaccharides abondants dans la paroi primaire et caractérisés par la présence d'acide galacturonique (GalA). Les pectines jouent un rôle clé dans le contrôle de la porosité de la paroi et aussi dans les mécanismes de régulation de la croissance et du développement de la plante. L'homogalacturonane (HG) constituant majeur des pectines, est un homopolymère a-1,4-GalA linéaire qui représente 20% des polysaccharides pariétaux. Ce polymère est sécrété sous forme méthylestérifiée et ne porte donc pas ou peu de charges. La déméthylestérification d'HG par des pectine méthylestérases (PMEs) fait apparaître des charges négatives ce qui a des conséquences importantes sur les propriétés physico-chimiques de la paroi, affectant des processus physiologiques et développementaux comme l'ouverture des stomates, l'initiation d'organes et la croissance cellulaire anisotrope. De nombreuses PME existent (69 isoformes chez Arabidopsis thaliana) qui peuvent être inhibées par des inhibiteurs protéiques (les « PME Inhibitors » ou PMEI) endogènes également représentés par de multiples isoformes (76 chez Arabidopsis thaliana). Cette abondance d'isoformes illustre la complexité de la régulation de la densité de charges dans la paroi. L'HG déméthylestérifiée peut former des liaisons Ca 2+ -pectate qui peuvent rigidifier la paroi, mais des études récentes montrent que la déméthylestérification de l'HG peut également promouvoir une augmentation de l'élasticité de la paroi ainsi que la vitesse de l'expansion cellulaire à travers un mécanisme inconnu. Au cours de ma thèse je me suis interrogé sur ce paradoxe en étudiant le lien entre le métabolisme des pectines, le pH et l'extensibilité de la paroi. A cette fin, j'ai développé et utilisé des outils génétiques et pharmacologiques pour la manipulation in vivo de l'activité PME. J'ai généré des lignées permettant la surexpression inductible de deux PMEIs différents, mais qui malheureusement se sont avérés non fonctionnels. J'ai également produit une PMEI dans la levure Pichia pastoris afin d'étudier son activité in vitro et in vivo. Ainsi, j'ai pu observer que la protéine a une activité inhibitrice pH-dépendante sur un large éventail de PMEs in vitro. Par ailleurs, j'ai utilisé des senseurs ratiométriques pour la mesure du pH dans l'apoplaste de la cellule et j'ai contribué à générer des senseurs permettant de mesurer le pH dans des microdomaines au sein de la paroi. Enfin j'ai participé au développement de nouveaux outils permettant de maintenir les plantes dans des conditions optimales pour l'observation simultanée de la croissance et du pH apoplastique dans la racine suite à un changement du milieu de culture. A l'aide de tous ces outils, j'ai ensuite étudié l'impact de la modification de l'activité PME sur le pH et la croissance cellulaire. D'abord, j'ai pu observer que le traitement avec un inhibiteur chimique de la PME, l'epigallocatechin gallate (EGCG) provoquait une inhibition de la croissance et une perte de l'anisotropie des cellules de la racine et que ces réponses étaient accompagnées d'une augmentation du pH apoplastique (pH Apo ) à priori indépendamment de l'activité de l'H + -ATPase dans le plasmalemme. Par ailleurs, l'addition d'une PME dans le milieu de culture augmentait la croissance racinaire et réduisait le pH Apo , tandis que l'addition d'un PMEI ralentissait la croissance et augmentait le pH Apo . Enfin, l'induction de l'activité PME dans des plantes transgéniques provoquait également une diminution du pH Apo dans la racine. En conclusion, mes résultats montrent que la déméthylesterification de l'HG peut induire une acidification de l'apoplaste et une augmentation de la croissance. Pour expliquer ce phénomène, je propose que des domaines anioniques générés par l'activité PME dans la paroi aient la capacité de séquestrer des protons. Cette acidification locale pourrait ensuite activer des facteurs de relâchement de la paroi, comme les expansines, entraînant ainsi une augmentation de la vitesse de croissance. En revanche, les résultats ne permettent pas d'exclure que la modification de l'HG active également des voies de signalisation affectant le pH Apo et l'expansion cellulaire. Cette question fera l'objet d'études ultérieures. Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Titre : Le rôle du métabolisme des pectines dans le contrôle du pH et de la rhéologie de la paroi Mots clés : pectine, paroi, pH, rhéologie, croissance Résumé : La pectine, un composant de la matrice de la paroi primaire, joue un rôle dans le contrôle de la porosité de la paroi, l'élongation et l'adhésion cellulaire et est un facteur important dans le développement de la plante. Homogalacturonane (HG), le polymère pectique le plus abondant, est sécrété sous forme methylestérifiée et ne porte donc peu ou pas de charges négatives. La déméthylestérification d'HG par des pectine méthylestérases (PMEs) expose ensuite des charges négatives et a des conséquences importantes sur les propriétés mécaniques de la paroi, affectant des processus physiologiques et développementaux comme l'ouverture des stomates, l'initiation d'organes et la croissance cellulaire anisotropique. De multiples PME existent (chez Arabidopsis thaliana) qui peuvent être inhibées par des « PME inhibitors » (PMEIs) endogènes. L'HG déméthylestérifiée peut former des liaisons Ca 2+ -pectate qui peuvent rigidifier la paroi, mais des études récentes montrent que la déméthylestérification de HG peut également promouvoir le relâchement de la paroi et l'expansion cellulaire à travers un mécanisme inconnu. Dans ma thèse j'ai adressé ce paradoxe en étudiant le lien entre le métabolisme des pectines, le pH et l'extensibilité de la paroi. A cette fin j'ai dévelopé et utilisé des outils génétiques et pharmacologiques pour la manipulation in vivo de l'activité PME. J'ai généré des lignées permettant la surexpression inductible de deux PMEIs différents, mais qui malheureusement s'avéraient non fonctionnelles.

Table 1 .1

 1 Overview of characterized pectin methylesterase inhibitors (PMEIs) in

	Arabidopsis…………………………………………………………………….……11

Table 1 . 2

 12 Cell wall proteins sensitive to pH Apo ……………………………………..23

Table 1 . 3

 13 Fluorescent pH indicators used in plant cell walls……………………….43

Table 2 .

 2 1 T1 red seeds of inducible PMEI3 and PMEI5 overexpressing lines…….72

Table 3 .1

 3 PMEs used in this study………………………………………………….88

Table 7 .1

 7 Sequences of oligonucleotide primer used for construct designed for the heterologous expression of PMEI3 and PMEI5…………………………………..153

Table 7 .

 7 2 BMGY and BMMY components…………………………………..….158

Table 7 .

 7

Table 7 .4

 7 GreenGate vectors and modules used in this study……………..……..163

Table 7 .5

 7 Sequences of oligonucleotide primers used for inducible PMEI overexpressing constructs……………………………………………………….165

Table 7 .6

 7 Sequences of oligonucleotide primers used for pH sensors………….168

Table 7 .7

 7 Sequences of oligonucleotide primer used for qRT-PCR…………….170

Table 7 .

 7 8 GUS staining solution components…………………………………..170

Table 1 .1 Overview of characterized pectin methylesterase inhibitors (PMEIs) in Arabidopsis.

 1

		Gene ID	Genotype	Expression	Phenotype	DM*	Publication
	Gene			pattern
	name
	PMEI1	AT1G48020 Overexpressi	Pollen	Increased root growth, promote resistance to the necrotrophic fungus Botrytis	Increase	Wolf et al.2003;
			on		cinerea; expression restricted to pollen		Lionetti et al. 2007
			35S::PMEI1
	PMEI2	AT3G17220 Overexpressi	Pollen, seed Increase root growth, promote resistance to the necrotrophic fungus Botrytis	Increase	Wolf et al.2003;
			on		cinerea; expression restricted to pollen; promote growth of tobacco pollen		Lionetti et al.2007;
			35S::PMEI2		tubes		Rockel et al.2008
	PMEI3	AT5G20740 Overexpressi	Developing	Decreased primordia growth; perturbed phyllotaxis and organ initiation,	Decrease Peaucelle et al.
			on	meristemoids	increased stiffness, shorter cells		2008, 2011 and
			pAlcA::PME	zone, flower			2015
			I3
	PMEI4	AT4G25250 Overexpressi	Root, hypocotyl Dark-grown hypocotyls, controls the initiation of the acceleration but not the	Increase	Pelletier et al. 2010
			on		growth process itself
			35S::PMEI4
	PMEI5	AT2G31430 Overexpressi	Flower, bud,	Irregular root waving; faster seed germination, bigger seeds and bigger	Increase	Wolf et al. 2012 and
			on	seed	embryos; drastic reduction in dark-grown hypocotyl length, no reduced root		2015; Muller et al.
			pMDC7::PM		length; overexpression promotes BR signaling		2013; Levesque-
			EI5				Tremblay et al. 2015
	PMEI6	AT2G47670	Mutant	Stem, seed	pmei6: mucilage release was delayed and outer cell walls of epidermal cells	Decrease or	Saez-Aguayo et al.
			pmei6 and		did not fragment; 35S::PMEI6: increased labeling of cell wall fragments.	increase	2013
			Overexpressi			resp.
			on
			35S::PMEI6
	PMEI7	AT4G25260	no mutant	Leaf, root, seed Binding to PME3 is pH dependent	Increase	Senechal et al. 2015
			reported

Less resistance to B. cinerea. no evident defects in rosette leaves, floral stems, flowers, seeds, and all developmental stages, Higher resistance to the necrotrophic fungus B. cinerea, a mediators of CW integrity maintenance in plant immunity Decrease Lionetti et al. Less resistance to B. cinerea. no evident defects in rosette leaves, floral stems, flowers, seeds, and all developmental stages, Higher resistance to the necrotrophic fungus B. cinerea, a mediators of CW integrity maintenance in plant immunity Decrease Lionetti et al.

*DM: Degree of methylesterification

Table 1 .2 Cell wall proteins sensitive to apoplastic pH. Protein name Gene ID Functions pH optimum Assay Species Comment Reference

 1

	PttCel9A	CA926262	Endo 1,4-glucanase	5.5-6.0	In vitro, expressed in	Populus tremula	Plasma	membrane	Master et al.
				(30°C)	Pichia pastoris		localized,	cellulose	2004
							synthesis, Arabidopsis
							KOR homologous
	AtXTH12	At5g57530	Xyloglucan	5.0-6.0	In vitro, expressed in	Arabidopsis thaliana	Trichoblast		Maris et al.
			endotransglucosylase/hydrolase	(28°C)	Pichia pastoris				2011
			(XTH)
	AtXTH13	At5g57540	XTH	5.0-6.0	In vitro, expressed in	Arabidopsis thaliana	Trichoblast		Maris et al.
				(28°C)	Pichia pastoris				2011
	AtXTH17	At1g65310	XTH	7.0 (20°C)	In vitro, expressed in	Arabidopsis thaliana	Root		Maris et al.
					Pichia pastoris				2011
	AtXTH18	At4g30280	XTH	7.0 (28°C)	In vitro, expressed in	Arabidopsis thaliana	Root		Maris et al.
					Pichia pastoris				2011
	AtXTH19	At4g30290	XTH	7.0 (28°C)	In vitro, expressed in	Arabidopsis thaliana	Root		Maris et al.
					Pichia pastoris				2011
	Cel16	AJ242807	Endo 1,4-glucanase	6.0	In vitro, expressed in	Brassica napus	Strong dependence on	Mølhøj et al.
					Pichia pastoris		Calcium		2001
	TCH4	AC004512.2	XTH	6.0-6.5	In vitro, expressed in	Arabidopsis thaliana	Acid-induced and cold-	Campbell and
				(18°C)	baculovirus/ insect cell		tolerant enzyme	Braam. 1999;
					system				Purugganan
									et al.1997
	AtXTH24	AT4G30270	XTH	6.0-6.5	In vitro, expressed in	Arabidopsis thaliana	-		Campbell and
	(Meri-5)			(28°C)	baculovirus/insect cell				Braam. 1999
					system
	EXGT	AT2G06850	XTH	6.0-6.5	In vitro, expressed in	Arabidopsis thaliana	-		Campbell and
				(37°C)	baculovirus/insect cell				Braam. 1999
					system
	XTG14	AT4G25820	XTH	6.0-6.5	In vitro, expressed in	Arabidopsis thaliana	-		Campbell and
	(XTR9)			(18°C)	baculovirus/insect cell				Braam. 1999
					system

Table 1 .3 Fluorescent pH indicators used in plant cell walls.

 1

	Probes	Type	Excitation	Emission	pH range Usage	Advantages / disadvantages	Reference
			(nm)	(nm)		range
	SNARF	Ratiometric 488 or 514	580/640	6.0-8.0	Leaf	Simple and cheap, in vivo measurement. Imprecise	Jia and Davis 2007
							estimation of pH
	BCECF	Ratiometric 508	530	6.0-8.0	Leaf, root,	Simple and cheap, in vivo measurement. Imprecise	Jia and Davis 2007; Feijó et
						pollen tube,	estimation of pH	al. 1999
	Fluorescein	Ratiometric 490/450	515	6.0-7.2	Root, root	Simple and cheap, in vivo measurement. Imprecise	Monshausen et al. 2007 and
						hair, leaf	estimation of pH	2009;	Hoffmann	and
								Kosegarten 1995
	Oregon	Ratiometric 480/440	520	4.2-5.7	Root, leaf	Simple and cheap, in vivo measurement. Imprecise	Fasano et al. 2001 ; Javed et
	Green						estimation of pH	al. 2014
	Bromocerol	-	-	-	5.2-6.8	Root, root	Simple and cheap, in vivo measurement. Imprecise	Pacheco-Villalobos et al.
	purple					hair	estimation of pH	2016 ; Zhu et al. 2015 ;
								Masachis et al. 2016
	HPTS	Ratiometric 405/458(470) 514/514	4.5-6.5	Root	Reliable, in vivo and with high spatiotemporal	Barbez et al. 2017
							resolution. Confocal or fluorescence microscopy
							needed, calibration might be inaccurate due to
							unknown effects of the apoplastic milieu on the dye,
							limited pH range of responsiveness, cross-sensitivity
							to other ions is merely possible

Table 2 .1 T1 red seeds of inducible PMEI3 and PMEI5 overexpressing lines.

 2

	Number of seed or plant Col-	Col-	Col-	xxt1/xxt2-	xxt1/xxt2-	xxt1/xxt2-
		FX0	FX3	FX5	FX0	FX3	FX5
	Red seeds (T1)	102	256	78	19	10	22
	Amplified	16	16	16	-	-	-
	Plant with red seeds (T2)	16	16	15	-	-	-
	Unique insertion lines	5	8	8	-	-	-
	Transformed seeds ratio	7.6	15.5	4.8	3.2	1.7	4.4
	(1/10000)
	Note: FX0: vector control, FX3: PMEI3, FX5: PMEI5.

Table 3 .1 PMEs used in this study.

 3

	Name	Express or extract	Origin	Reference
		from
	Orange PME	Orange peel	Orange	Sigma P5400
	Citrus thermo-	Pichia pastoris	Citrus	J. Pelloux et al.
	tolerant PME			Unpublished
	AtPME2	Nicotiana tabacum	Arabidopsis thaliana	J. Pelloux et al.
				Unpublished
	AtPME3	Nicotiana tabacum	Arabidopsis thaliana	Sénéchal et al. 2015
				and 2017 ; Hocq et al.
				2017
	Flower PME	Arabidopsis flower	Arabidopsis thaliana	This study
	Root PME	Arabidopsis root	Arabidopsis thaliana	This study

  Apo in PME-but not in mock-treated roots was observed. Significance was determined by two-way ANOVA followed by Tukey's test. * p < 0 .05. Error bar: STD.

					PBS buffer			Orange PME
								high
								low
		2.9
		2.8
		2.7
		2.6
	Ratio	2.5
		2.4
		2.3
		2.2
		2.1
		0	10	20	30	40	50	60
					Time (min)

Table 7 .1 Sequences of oligonucleotide primer used for construct designed for the heterologous expression of PMEI3 and PMEI5.

 7

	Primer name	Sequence

  Table7.3), using a running buffer composed of 25 mM Tris-base, 192 mM glycine, and 0.1% SDS at pH 8.7. 13 µL of protein was added to 15 µL Laemmli buffer 2X (4% SDS, 10% 2-mercaptoethanol, 20% glycol, 0.004% bromophenol blue and 0.125 M Tris-HCl) and 2 µL of 1 M imidazole. The protein mix was heated at 95°C for 20 mins. 15 µL of protein mix was loaded into a

	well of SDS-PAGE gel. After electrophoresis at 100 mA for 100 mins, the gel was
	stained with Pageblue protein staining solution (Thermo Scientific, catalog no. 24620)
	and destained with distilled water for 6 h. After migration of proteins, the gel SDS-
	PAGE was immerged in cathode buffer (25 mM of Tris pH 9.4, 40mM of Glycine, 10%
	of ethanol) for 15 min. A piece of Hybond-P PVDF membrane (GE Healthcare, catalog
	no. RPN303F) was placed in 100% ethanol for 15 seconds and rinsed by water for 2
	mins. Then the membrane was incubated in anode buffer II (25 mM of Tris pH 10.4,
	10% of ethanol) for 5 mins. The membrane, gel, and 7 same-sized Whatmann papers
	(previously immerged in anode I, anode II and cathode buffers) were placed according
	to Figure

Table 7 .2 BMGY and BMMY components

 7

	Components	Buffered	Glycerol-complex	Buffered	Methanol-complex
		medium (BMGY)	medium (BMMY)
	Yeast extract		1.0%		1.0%
	Peptone		2.0%		2.0%
	Potassium phophate		100 mM		100 mM
	Yeast nitrate base		1.34%		1.34%
	Biotin		4 × 10 -5 %		4×10 -5 %
	Glycerol		1.0 %		-
	Methanol		-		0.5%

Table 7 .3 SDS-PAGE gel components

 7

	Components	Resolving Gel (10 mL)	Stacking Gel (8 mL)
	H20	1.0 mL	5.0 mL
	30% Acryl/Bis	5 mL	1.0 mL
	1 M Tris pH 8.8	3.75 mL	1.0 mL
	10% SDS	100 µL	80 µL
	10% APS	100 µL	80 µL
	TEMED	10 µL	8 µl

Table 7 .4 GreenGate vectors and modules used in this study.

 7

	Modules	Name	Type	Overhangs	Resistance	Addgene ID
	Empty entry vectors	pGGC000	CDS	C-D	Carbenicillin
		pGGE000	Plant terminator	E-F	Carbenicillin
		pGGF000	Plant resistance cassette	F-G	Carbenicillin
		pGGH000	N-tag + CDS + C-tag + terminator	B-F	Carbenicillin
	Empty intermediate vectors	pGGM000	Assembly of expression cassette # 1	A-H	Kanamycin
		pGGN000	Assembly of expression cassette # 2	H-G	Kanamycin
	Empty destination vectors	pGGZ001	Plant resistance at RB	A-G	Spectinomycin
	Plant promoters	pGGA006	UBQ10 (UBIQUITIN10) promoter	A-B	Carbenicillin
	N-tags	pGGB003	B-dummy	B-C	Carbenicillin
	C-tags	pGGD002	D-dummy	D-E	Carbenicillin
	Plant terminators	pGGE003	At1g04880 terminator	E-F	Carbenicillin
	Adapters	pGGG001	FH-adapter	F-H	Carbenicillin
	Adapters	pGGG002	HA-adapter	H-A	Carbenicillin

Table 7 .6 Sequences of oligonucleotide primers used for pH sensors

 7

	Primer name	Sequence

Table 7 .7 Sequences of oligonucleotide primer used for qRT-PCR

 7

	Primer name	Sequence	Use
	PMEI3-20740_F	AAACGGAGGTGAAGCAGTGGATG	PMEI3
	PMEI3-20740_R	GACCATGTAAAGCGCGTTGCTC	PMEI3
	PMEI5-31430_F	AAACGCGATTCCAGCCAGAGAC	PMEI5
	PMEI5-31430_R	AGGCAGAAGGGCACATCAGTTG	PMEI5
	mTurquoise-F	ACGTAAACGGCCACAAGTTC	mTURQUOISE
	mTurquoise-R	CTTCATGTGGTCGGGGTAGC	mTURQUOISE
	At1g13440-F	GGTACGACAACGAATGGGGT	Standard gene
	At1g13440-R	TGACTGCGCATGGAATCAGT	Standard gene

Table 7 .8 GUS staining solution components

 7

2F4 indeed labels Ca

2+ -crosslinked HG, but Ca 2+ is supplied in the immunolabeling buffer. Therefore, in the absence of information on the in vivo Ca 2+ concentration, the assay does not indicate whether the pectate is also crosslinked in vivo. The conclusion of the authors: « We concluded that slowly growing transverse walls had more de-methylated pectin which was calcium cross-linked, while faster growing axial walls had more methylated pectin. » is therefore erroneous.

Acknowledgements

, Aline, Veronica, and Ying. I learned a lot while working with all of you. This thesis would not have been possible were it not for collaboration with the Biologie des plantes I am greatly thankful to the China Scholarship Council, for funding me to pursue my scientific dream. I also thank Institut Jean-Pierre Bourgin, INRA

Results

Murashige and Skoog medium (perliter: 2.16 g MS salts (Duchefa), 2.0% plant agar) at approximately pH 5.8, were stratified at 4°C for 48 h in the dark and seedlings were vertically under long-day conditions at 22°C.

For calibration experiments, 4.7 mM MES in pH between 4.6 and 6.4 in steps of 0.2 or 0.4 units was added the 1/2 MS medium. Plants were grown under long-day conditions at 22°C. HPTS (8-hydroxypyrene-1, 3, 6-trisulfonic acid trisodium salt), EGCG ((-)-Epigallocatechin gallate) and fusicoccin were supplied by Sigma. HPTS stock was made by dissolving 52 mg of HPTS powder in 1 mL distilled water (pH 5.8). EGCG and fusicoccin were dissolved in water and methanol, respectively. These stocks were stored at 4°C. Four-d-old seedlings were moved gently from 1/2 MS solid medium into 1/2 MS liquid medium supplemented by 1 mM HPTS and incubated for 30 mins. For the treatments, chemicals were added together with HPTS to this medium. Solid medium with the same HPTS concentration was cut into small and thin pieces (length (5 mm) × width (5 mm) × height (1 mm)). For static experiments, such as the calibration, 3 seedlings were mounted flatly and gently on the chip 1 (Figure 7.1 right), soaked with 10 µl of liquid medium and covered by a slice of the solid medium. For dynamic experiments, 1 or 2 seedlings were mounted in the channel of chip 2 (Figure 7.1 left) and gently fixed by a piece of plastic grid covered by cotton. 800 µL or 150 µL of liquid medium (for chip 1 and chip 2, respectively), supplies or not with drugs, was slowly injected into the channel, during the acquisition under the confocal microscopy.

Confocal data acquisition was performed on SP5. For the apopHusion, data were acquired in xyzt mode using multi-positional and sequential scanning of EGFP and mRFP1, respectively. EGFP was excited at 488 nm using a white light laser and detected between 500 nm and 550 nm. mRFP was excited at either 558 nm or 585 nm with a white light laser and detected between 600 nm and 630 nm. 4 to 6 z-stacks were acquired for both channels.

The image analysis was performed using the Fiji software using the macro language (Supplementary data 1), and data were statistically evaluated with Microsoft Excel 2011. The experiments described were performed in at least 3 biological repetitions.

Rootchip assay

Plant growth and device preparation: apopHusion seeds [START_REF] Gjetting | Live imaging of intra-and extracellular pH in plants using pHusion, a novel genetically encoded biosensor[END_REF] were sterilized, plated on solid 1/2 MS medium and stratified as described above. Next, seeds were transferred to solid MS medium pH 5.8, with 0.8% phytoagar (Duchefa) and 1.0% sucrose, that had been poured into pipette tips (Sorenson CAT#35020), from which the base was removed leaving a 5-mm long apical piece. The tips with the seeds on top of the medium were positioned in an upright position in the agar of a Petri dish with solid 1/2 MS medium. After 5 d of incubation in a growth chamber under 16 h light at 22°C, when the root apex had almost reached the lower tip outlet, and the tips were plugged into the Rootchip device (9 or 6 channels, 100 µm) [START_REF] Grossmann | The RootChip : An Integrated Microfluidic Chip for Plant Science[END_REF]). The Rootchip device was assemblied according to Figure 7.2. The device was sterilized with 70% ethanol and rinsed with distilled water and then with liquid 1/2 MS medium (1% sucrose, pH 5.8). The device was then transferred onto a homemade 3D-printed rectangular chip carrier (Figure 7.2), fixed with adhesive tape, and connected to the pressure lines (line 1: TYGON AAD04103 and line2: Adtech PTFE Tubing 0.56 mm ID ×1.07 mm OD).

normalized collision energy = 35%; centroid mode). Step 2 was repeated for the three major ions detected in step 1. Dynamic exclusion was set to 45s.

Protein identification: A database search was performed with XTandem (version 10-12-01-1) (http://www.thegpm.org/TANDEM/). Enzymatic cleavage was defined as a trypsin digestion with one possible miscleavage. Regarding protein modifications, Cys carboxyamidomethylation was set to static. As potential modifications, Met oxidation, Nter acetylation, Nter deamidation on Gln, Nter deamidation on carbamidomethylated Cys, Nter neutral loss on Glu were set. Precursor mass and fragment mass tolerance were 2.0 and 0.8, respectively. A refinement search was added with similar parameters. The Araport11_genes.201606 database and a contaminant database (trypsin, keratins) were used for peptides identification.

Identified peptides were filtered and grouped using Xtandem according to: (1) A minimum of two different peptides required with an E-value smaller than 0.05, (2) a protein log(E-value) (calculated as the product of unique peptide E-values) smaller than -4.

PMEI3/5 heterologous expression and purification

Cloning: The coding sequences and codon-optimized sequences (ProteoGenix cn) of PMEI3 and PMEI5 were cloned into the pPICZα B (Invitrogen. Catalog no. V195-20).

For codon-optimized constructs, PMEI3 and PMEI5 CDS rare codon analysis was performed on the GenScript platform using the host organism Pichia pastoris. Codonoptimized genes (Supplementary data 2) were synthesized by ProteoGenix inc. pPICZα B is a 3.6 kb vectors used to express and secrete recombinant proteins in Pichia pastoris.

Recombinant proteins are expressed as fusions to an N-terminal peptide encoding the Confocal acquisition was performed on SP5. For apopHusion, EGFP was excited at 488 nm using a white light laser and detected between 500 nm and 550 nm. mRFP1 was excited at either 558 nm or 585 nm with a white light laser and detected between 600 nm and 630 nm. Fluorescence intensities were measured with the Fiji software. BFP in AtFH1-mRFP1-BFP sensor was excited at 405 nm and emitted at 515 nm.

q-RT-qPCR

Leaves or roots of each line were collected and rapidly frozen using liquid nitrogen.

Samples were stored at -80°C until grinding. Grinding was performed in a mortar with freezing by liquid nitrogen. The extraction of total RNA was performed using RNeasy Plant Mini kit (QIAGEN,Cat No. 74904) following the manufacturer's recommendations and treated with Room Temperature Stable (RTS) DNase (QIAGEN Cat No 79254) only when RNA was prepared for quantitative q-RT-PCR (Mobio).

Degradation and genomic DNA contamination were analyzed before cDNA synthesis.

The following cycle was used: 95°C for 3 min, 40 cycles of 95°C for 5 s and 60°C for 20s, followed by 65°C for 5s and 95°C for 30s. Specific probes were amplified by PCR using primers indicated in Table 7.7.

GUS staining and GUS activity measurement

Ten 4-d-old seedlings were immerged in GUS staining solution (Table 7.8). The well plate was placed in a container that can be vacuumed. The plate was kept in halfvacuum condition over night at room temperature. The seedlings were immerged by 100% ethanol for 2 h to remove chlorophyll and rinsed by water for 3 times.

ANNEXES

Supplementary Data 1. Macro used in this study.