Effect of homogalacturonan demethylesterification on apoplastic pH in Arabidopsis root

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HG demethylesterification

Upon secretion, HG can be selectively demethylesterified in muro by pectin methylesterases (PMEs) (Pelloux et al. 2007). 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 (Pelloux et al. 2007). 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 (Wolf et al. 2009). 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; Pelloux et al. 2007).
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 Ca2+, a processive PME can become non processive (Vincent and Williams 2009).
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 pectin-binding 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 (Caffall and Mohnen 2009). This most likely reflects the methylesterification pattern and the covalent association of HG with the other pectic polymers RG-I and RG-II (Vincken et al. 2003; Atmodjo et al. 2013; Popper and Fry 2005) and proteoglycans (Caffall and Mohnen 2009), 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 endo-polygalacturonase treatment, but no covalent crosslinks between the two polymers have been observed so far (Talmadge et al. 1973; Popper and Fry 2005). 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 (Cosgrove 2014 and 2015). 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 (Cannon et al. 2008). 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 (Valentin et al. 2010). 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 (Tan et al. 2013). 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 (Tan et al. 2013).

HG interacts with cell surface receptors

HG also binds to many cell surface receptors, such as Catharanthus roseus RLK1 (CrRLK1) -like family (Lin et al. 2018; Feng et al. 2018; Sebastjen et al. 2018), extensin containing receptors (Valentin et al. 2010; Mecchia et al. 2017), LRR receptor-like protein 44 (RLP44) (S. Wolf, personal communication) and Wall Associated Kinases (WAKs, Kohorn and Kohorn 2012). CrRLK1L proteins have a signal peptide, an extracellular domain consisting of two tandem malectin-like domains A and B (MALA, MALB), an extracellular juxtamembrane region, a transmembrane domain, and a kinase domain (Frank et al. 2018; Li et al. 2016). 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 (Schallus et al. 2008). Unlike animal malectin, these malectin-like domains lack critical residues involved carbohydrate binding (Du et al. 2018).

Regulation CW enzyme activity by pH

In vitro experiments showed that many cell wall remodeling agents and their inhibitors are regulated by pHApo (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; Cosgrove 2014 and 2015). The pH-dependence has also been characterized for some PMEs and PMEIs. For instance, AtPME3 activity has a pH-optimum of 7.5 (Sénéchal et al. 2015), AtPMEI4 and AtPMEI7 showed a similar pH-dependence for their inhibiting capacity (Hocq et al. 2017; Sénéchal et al. 2017) while AtPMEI9 is less pH-dependent (Hocq et al. 2017).

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 (Ammann 2013). An estimate of pHApo 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 (Felle et al. 2009). To measure pHApo 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 (Rubio et al. 2017). By placing the microelectrode on the surface of growing Arabidopsis RHs, it was found that ROS-related RH elongation is coupled to spatially distinct regulation of extracellular pH (Monshausen et al. 2007). Using this approach, Felle et al (2009) found that the mycorrhiza fungus Piriformospora indica induces root surface alkalinization that was hypothesized to support the systemic stress response of barley (Hordeum vulgare) to B. graminis f. sp. hordei. Furthermore, in planta measurements are available with microelectrodes to investigate the stomatal cavity, because the microelectrodes can be inserted through the opened stomata (Guzel Deger et al. 2015; Zimmermann et al. 2016), allowing the pH in the sub-stomatal cavity to be quantified (Hanstein and Felle 1999; Pitann et al. 2009; Zimmermann et al. 2009). 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 (Felle and Hanstein 2002). 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 (Felle 1998; Ammann 2013).

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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 (Bizzarri et al. 2009). This shift in absorption has been extensively used to record pH in living organisms (Kneen et al. 1998) 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. Gao et al. (2004) 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 pHapo signature differed from that induced by osmotic stress. Martinière et al. (2018) fused pHlourin to a modified transmembrane domain containing 26 amino acids of the PM-localized TM23. This pHApo 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 Martinière et al. (2018). Although pHluorin and its derivative sensors have proven their utility (Martinière et al. 2013 and 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 (Gjetting et al. 2012). apopHusion is targeted to the apoplast thanks to a chitinase signal peptide (Gjetting et al. 2012), as confirmed in leaf epidermis, the guard cells, the mesophyll, and root apex in Arabidopsis. Using this genetically encoded sensor, Gjetting et al. ( 2012) discovered a fast alkalinization response in the epidermal apoplast of elongating roots to the external addition of indole-3-acetic acid. Fendrych et al. (2016) 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.

Table of contents :

Chapter 1 Introduction
1.1. Primary cell wall
1.2. Pectins and homogalacturonan modification
1.2.1 HG synthesis and secretion
1.2.2 HG demethylesterification
1.2.3 Pectin network and connections to cellulose, hemicellulose and proteins
1.3. Pectin gels and rheology
1.3.1 Gels and phase transition
1.3.2 Pectin gels and their properties.
1.4. HG interacts with cell surface receptors
1.5. Apoplastic pH
1.5.1 Regulation CW enzyme activity by pH
1.5.2 Proton motive force and turgor pressure regulation
1.5.3 Regulation of apoplastic pH
1.5.4 Matrix charge
1.6. How to measure apoplastic pH
1.6.1 H+-selective microelectrodes
1.6.2 pH-sensitive fluorescent dyes
1.6.3 pH-sensitive fluorescent proteins
1.7. Relationships between growth, apoplastic pH, and HG demethylesterification
1.7.1 pH
1.7.2 Ca2+
1.7.3 K+
1.7.4 PME
1.7.5 ROS
1.7.6 Actin dynamics
1.7.7 Exocytosis
1.8. Two paradoxes related to HG modification
1.8.1 HG demethylesterification can have opposing effects on cell wall stiffness
1.8.2 HG demethylesterification, wall mechanics and growth: controversial results in the literature
1.8.3 Distinct PMEIs showed antagonistic effects on plant growth
1.9. Objectives of the thesis
Chapter 2 Generation of new inducible PMEI3 and PMEI5 overexpressing lines
Chapter 3 Biochemical characterization of PMEI3 and PMEI5 
3.1 PMEI3 and PMEI5 expression in Pichia pastoris.
3.2 PMEI3 characterization
3.2.1 PMEI3 activity measurement.
3.2.2 PMEI3/PME3 interaction
Chapter 4 Comparison of apoplastic pH measurement tools 
Chapter 5 Effect of homogalacturonan demethylesterification on apoplastic pH in Arabidopsis root
5.1. Dose-dependent inhibition of Arabidopsis root growth by PME inhibitor EGCG.
5.2. EGCG treatment alters cell shape in root
5.3. EGCG treatment promotes an increase in pHApo in root cells
5.4. EGCG-triggered increase in pHApo correlates with root growth inhibition.
5.5. Exogenous PME treatment decreased pHApo in root cells
5.6. Effect of dexamethasone-induced expression of PMEI5 on pHApo
5.7. The effect of exogenous PME and PMEI3 on root growth
Chapter 6 Summary and perspectives
6.1 Summary
6.2 Perspectives
Chapter 7 Materials and Methods
7.1. Apoplastic pH measurement using HPTS
7.2. pH measurement using apopHusion
7.3. Rootchip assay
7.4. 3D imaging EGCG treated roots
7.5. MorphlibJ analysis of 3D image
7.6. Determination of protein content
7.7. Cell wall-enriched protein extraction
7.8. Methanol colorimetric assay
7.9. Gel diffusion assay
7.10. Medium pH measurement assay
7.11. MicroScale Thermophoresis (MST)
7.12. Mass spectrometry
7.13. PMEI3/5 heterologous expression and purification
7.14. Cell lysis and immunoblotting
7.15. Fourier Transform InfraRed Spectroscopy
7.16. Greengate cloning of PMEI3/5 inducible constructs and transformation
7.17. Greengate cloning of pH sensor constructs and Arabidopsis and tobacco transformation
7.18. q-RT-qPCR
7.19. GUS staining and GUS activity measurement
References

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