HG demethylesterification

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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