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Investigating the role of pectin methylesterases in regulating root development in Arabidopsis thaliana

Investigating the role of pectin methylesterases in regulating root development in Arabidopsis thaliana
Investigating the role of pectin methylesterases in regulating root development in Arabidopsis thaliana
The growth, development, and the final shape of plants depend on modifications to the structure and elasticity of the primary cell wall which consists of cellulose, hemicellulose and pectin. The level of methylesterification of homogalacturonan (HG) component of pectin in the cell wall is one of the major factors that could determine cell wall rigidity, cell expansion, root bending, and other plant developmental processes. Demethylesterified HG backbones have the ability to crosslink via calcium making the cell wall more rigid or to be hydrolysed and make it weaker. However, it is still poor understanding of how pectin methylesterification could affect developmental processes like root growth. The methylesterification state of pectin is controlled by pectin methylesterases (PMEs) and their inhibitors (PMEIs). Arabidopsis thaliana genome contains a large number of PME and PMEI isoforms making gene knockout approaches difficult due to potential redundancy. Therefore, an alternative approach is needed that provides the means to simultaneously affect the activity of multiple PMEs. Polyphenon-60 (PP60) was used as the main tool in this study to investigate PMEs and PMEIs activity on Arabidopsis root development. PP60 is a green tea catechin extract believed to act in a similar way as PMEI by binding to the active site of PME as shown by in vitro analysis. Here it has been discovered that PP60 caused overexpression of some PME genes and enhanced PME activity in vivo, contrary to what has been speculated in previous studies. The addition of PP60 to the growing media caused inhibition of primary root elongation and reduced the number of lateral roots and root hairs probably due to increased PME activity. Phytohormones, auxin and brassinosteroids are involved in root development and it has been shown that the manipulation of phytohormone levels together with PME activity influenced primary root growth. Finally, border-like cells contain a high amount of HG and experiments showed that addition of PP60 made the cells dispersed in a slightly acidic environment. The activity of cell wall modifying enzymes and PMEs can change under different pH regimes. More basic pH 8 caused cells to cluster together but they were surrounded by a vast amount of mucilage rich in de-esterified homogalacturonan. Together, these results suggest different actions of PMEs and PP60 depending on the media pH. Moreover, pectins are involved in cell wall integrity maintenance mechanism and are overproduced when the cell wall is subjected to damage by cellulose biosynthesis inhibition. The inhibition of cellulose biosynthesis by isoxaben caused changes in metabolism and increased production of nucleotides suggesting monosaccharides being incorporated to compensate lack of cellulose microfibrils. In summary, the results showed that disruption in either pectin or cellulose components of the primary cell wall caused by chemical treatment trigger mechanisms responsible for maintenance of cell wall integrity and therefore plant growth and development.
University of Southampton
Ociepa, Przemyslaw
e2ec21ec-db42-4146-af29-24b254837698
Ociepa, Przemyslaw
e2ec21ec-db42-4146-af29-24b254837698
Marchant, Alan
3e54d51c-53b0-4df0-b428-2e73b071ee8e

Ociepa, Przemyslaw (2017) Investigating the role of pectin methylesterases in regulating root development in Arabidopsis thaliana. University of Southampton, Biological Sciences, Doctoral Thesis, 236pp.

Record type: Thesis (Doctoral)

Abstract

The growth, development, and the final shape of plants depend on modifications to the structure and elasticity of the primary cell wall which consists of cellulose, hemicellulose and pectin. The level of methylesterification of homogalacturonan (HG) component of pectin in the cell wall is one of the major factors that could determine cell wall rigidity, cell expansion, root bending, and other plant developmental processes. Demethylesterified HG backbones have the ability to crosslink via calcium making the cell wall more rigid or to be hydrolysed and make it weaker. However, it is still poor understanding of how pectin methylesterification could affect developmental processes like root growth. The methylesterification state of pectin is controlled by pectin methylesterases (PMEs) and their inhibitors (PMEIs). Arabidopsis thaliana genome contains a large number of PME and PMEI isoforms making gene knockout approaches difficult due to potential redundancy. Therefore, an alternative approach is needed that provides the means to simultaneously affect the activity of multiple PMEs. Polyphenon-60 (PP60) was used as the main tool in this study to investigate PMEs and PMEIs activity on Arabidopsis root development. PP60 is a green tea catechin extract believed to act in a similar way as PMEI by binding to the active site of PME as shown by in vitro analysis. Here it has been discovered that PP60 caused overexpression of some PME genes and enhanced PME activity in vivo, contrary to what has been speculated in previous studies. The addition of PP60 to the growing media caused inhibition of primary root elongation and reduced the number of lateral roots and root hairs probably due to increased PME activity. Phytohormones, auxin and brassinosteroids are involved in root development and it has been shown that the manipulation of phytohormone levels together with PME activity influenced primary root growth. Finally, border-like cells contain a high amount of HG and experiments showed that addition of PP60 made the cells dispersed in a slightly acidic environment. The activity of cell wall modifying enzymes and PMEs can change under different pH regimes. More basic pH 8 caused cells to cluster together but they were surrounded by a vast amount of mucilage rich in de-esterified homogalacturonan. Together, these results suggest different actions of PMEs and PP60 depending on the media pH. Moreover, pectins are involved in cell wall integrity maintenance mechanism and are overproduced when the cell wall is subjected to damage by cellulose biosynthesis inhibition. The inhibition of cellulose biosynthesis by isoxaben caused changes in metabolism and increased production of nucleotides suggesting monosaccharides being incorporated to compensate lack of cellulose microfibrils. In summary, the results showed that disruption in either pectin or cellulose components of the primary cell wall caused by chemical treatment trigger mechanisms responsible for maintenance of cell wall integrity and therefore plant growth and development.

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Published date: 30 January 2017
Organisations: University of Southampton, Centre for Biological Sciences

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Local EPrints ID: 405820
URI: http://eprints.soton.ac.uk/id/eprint/405820
PURE UUID: 9a01b5cd-9c7f-48dd-8400-c39b156a5e8c

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Date deposited: 18 Feb 2017 00:20
Last modified: 14 Mar 2019 06:11

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Contributors

Author: Przemyslaw Ociepa
Thesis advisor: Alan Marchant

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