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MAPK-associated proteins in Arabidopsis thaliana

Sörensson, Carolin LU (2010)
Abstract
Abstract



Mitogen-Activated Protein Kinases (MAPKs) are signalling proteins that are highly conserved among eukaryotes. They are involved in many biological pathways including cell differentiation and response to different stress situations. The MAPK cascade is composed of three signalling kinases, MAP3K, MAP2K and MAPK, which phosphorylate each other in a sequential manner. The different MAPK substrates are regulated by phosphorylation on serine or threonine. This thesis presents a new method for purification of phosphoproteins from a complex protein extract. Subsequent studies using this method identified AtPHOS34 to be phosphorylated upon biotic stress. The phosphorylation site of AtPHOS34 was also ... (More)
Abstract



Mitogen-Activated Protein Kinases (MAPKs) are signalling proteins that are highly conserved among eukaryotes. They are involved in many biological pathways including cell differentiation and response to different stress situations. The MAPK cascade is composed of three signalling kinases, MAP3K, MAP2K and MAPK, which phosphorylate each other in a sequential manner. The different MAPK substrates are regulated by phosphorylation on serine or threonine. This thesis presents a new method for purification of phosphoproteins from a complex protein extract. Subsequent studies using this method identified AtPHOS34 to be phosphorylated upon biotic stress. The phosphorylation site of AtPHOS34 was also determined.



Several Arabidopsis thaliana MPK3/6 in vitro substrates were identified by a peptide and bioinformatics screen and the predicted phosphorylation sites were confirmed with kinase assays using mutated and wild-type proteins. MAPK Substrate 80 (here denoted MKS80) was characterised as an in planta MAPK substrate and in detail analyses of MKS80 linked its function to stomatal patterning and regulation of carbohydrate balance. The MKS80 interaction partner α-glucan phosphorylase (PHS2) was established by a yeast two-hybrid screen, in vitro pull-down and in planta co-immunoprecipitation assays. PHS2 catalyses the reversible reaction between glucose-1-phosphate and highly branched soluble heteroglycans and was shown to be important in senescence. Biochemical analyses revealed a stable level of PHS2 and light-dependent accumulation of MKS80. Arabidopsis over-expressing wild-type MKS80 had a lower starch content, whereas plants over-expressing the phosphomimetic form of MKS80 died at the seedling stage, clearly demonstrating the importance of this phosphorylation. In plants over-expressing MKS80, the anthocyanin level was lowered in different stress situations. Slower senescence was detected in detached leaves over-expressing MKS80, indicating a change in metabolism. A system for delayed senescence can be of interest for the agricultural industry. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Dr. Assistant Professor Ekengren, Sophia, Department of Botany, Stockholm University, 106 91 Stockholm , Sweden, 08-163754, Sophia.Ekengren@botan.su.se
organization
publishing date
type
Thesis
publication status
published
subject
keywords
PHS2, stomata, signalling, Arabidopsis thaliana, MKS80, MAPK
pages
157 pages
defense location
the Biology Lecture Hall, Sölvegatan 35, 223 62Lund
defense date
2010-11-25 10:00:00
ISBN
978-91-7473-004-3
project
Biology education
language
English
LU publication?
yes
additional info
The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Department of Cell and Organism Biology (Closed 2011.) (011002100)
id
a1c16147-9c39-4c30-a0aa-139c64a04a5f (old id 1710501)
date added to LUP
2016-04-04 13:21:37
date last changed
2018-11-21 21:13:27
@phdthesis{a1c16147-9c39-4c30-a0aa-139c64a04a5f,
  abstract     = {{Abstract<br/><br>
<br/><br>
Mitogen-Activated Protein Kinases (MAPKs) are signalling proteins that are highly conserved among eukaryotes. They are involved in many biological pathways including cell differentiation and response to different stress situations. The MAPK cascade is composed of three signalling kinases, MAP3K, MAP2K and MAPK, which phosphorylate each other in a sequential manner. The different MAPK substrates are regulated by phosphorylation on serine or threonine. This thesis presents a new method for purification of phosphoproteins from a complex protein extract. Subsequent studies using this method identified AtPHOS34 to be phosphorylated upon biotic stress. The phosphorylation site of AtPHOS34 was also determined.<br/><br>
<br/><br>
Several Arabidopsis thaliana MPK3/6 in vitro substrates were identified by a peptide and bioinformatics screen and the predicted phosphorylation sites were confirmed with kinase assays using mutated and wild-type proteins. MAPK Substrate 80 (here denoted MKS80) was characterised as an in planta MAPK substrate and in detail analyses of MKS80 linked its function to stomatal patterning and regulation of carbohydrate balance. The MKS80 interaction partner α-glucan phosphorylase (PHS2) was established by a yeast two-hybrid screen, in vitro pull-down and in planta co-immunoprecipitation assays. PHS2 catalyses the reversible reaction between glucose-1-phosphate and highly branched soluble heteroglycans and was shown to be important in senescence. Biochemical analyses revealed a stable level of PHS2 and light-dependent accumulation of MKS80. Arabidopsis over-expressing wild-type MKS80 had a lower starch content, whereas plants over-expressing the phosphomimetic form of MKS80 died at the seedling stage, clearly demonstrating the importance of this phosphorylation. In plants over-expressing MKS80, the anthocyanin level was lowered in different stress situations. Slower senescence was detected in detached leaves over-expressing MKS80, indicating a change in metabolism. A system for delayed senescence can be of interest for the agricultural industry.}},
  author       = {{Sörensson, Carolin}},
  isbn         = {{978-91-7473-004-3}},
  keywords     = {{PHS2; stomata; signalling; Arabidopsis thaliana; MKS80; MAPK}},
  language     = {{eng}},
  school       = {{Lund University}},
  title        = {{MAPK-associated proteins in Arabidopsis thaliana}},
  year         = {{2010}},
}