COST-STSM-FA0605 8132 Anne-Sophie Leprince
STSM research title: Analysis of phosphoinositides involved in the regulation of proline
metabolism in response to salt stress.
This project was carried out by Anne-Sophie Leprince, PCMP laboratory, Pierre and Marie
Curie University, Paris, France from 04/07/2011 to 29/07/2011 in Prof T. Munnik laboratory,
Swammerdam Institute for Life Science, University of Amsterdam, Netherland.
The aims of this STSM project
Recently using a pharmacological approach, we observed that LY294002, a
phosphatidylinositol 3-kinase (PI3K) inhibitor, decreases in a dose-response manner proline
accumulation in response to salt stress. Plant PI3K belongs to the VPS34 family, which is
known to phosphorylate phosphatidylinositol (PI) as a single substrate at the D-3 position of
the inositol ring. The goal of this mission was to study the impact of salt stress and of
LY294002 on PI3K activity in Arabidopsis thaliana seedlings by measuring PI3P levels.
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Description of the experiments and main results obtained
For all the experiments the protocol developed by Prof Munnik was followed. After an
overnight labelling with
32
Pi, six day-old Arabidopsis seedlings were exposed to salt
treatment with or without LY294002 during various period of time. Lipids were then
extracted and separated on TLC plates. Spots corresponding to phosphatidylinositol
monophosphates (PIPs) were scraped off from TLC plates. PIPs were then deacetylated and
the corresponding isomers were separated by anion exchange HPLC. These experiments
allowed us to determine the PI3P ratio among the PIP pool. Interestingly, a 50% decrease of
PI3P levels was observed when LY294002 is present in the medium.
The results obtained during this month support our hypothesis concerning a key role of PI3K
on the regulation of proline accumulation. This biochemical approach was essential to
validate the pharmacological approach that we have previously developed and is important for
a further publication. I would like to thank the COST program for giving me this opportunity
and Prof Munnik for his availability during this month and the helpful discussions concerning
this project.