Investigations into extracellular ATP signalling and FB1-induced cell death in Arabidopsis thaliana.

Abstract

Extracellular ATP (eATP) is an important signalling molecule involved in regulation of plant growth and development, interactions with other organisms and responses to several environmental stimuli. The molecular targets mediating the physiological effects of eATP in plants remain to be identified. The work presented in this thesis focuses on identifying the signalling components that underlie the physiological roles of eATP in plants, with a strong emphasis on cell death control.
An Arabidopsis thaliana (Arabidopsis) cell culture system combined with 2-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry was used to identify proteins differentially expressed following ATP treatment. Twenty four putative cell death proteins were identified using the cell death-inducing toxin Fumonisin B1 (FB1) in combination with an ATP reversal filter. The potential role of these candidates in eATP- regulated cell death was tested using a variety of cell death assays on Arabidopsis T-DNA insertion KO mutants. The mitochondrial ATP synthase β-subunit, AT5G08690, was shown to be a novel cell death gene.
The early effects of eATP on global protein and transcript abundance were also investigated. 2D-DiGE identified 53 proteins differentially regulated by ATP and bioinformatic analysis revealed new effects of eATP on general metabolism. Re- examination of a previously acquired DNA chip experiment that used ATP and FB1 treatments identified 10 genes that are differentially expressed within minutes by eATP that can be used as molecular markers.
2D-DiGE proteomics was also used to investigate the specific toxic effects of FB1 on Arabidopsis. A subset of proteins that were specifically regulated by FB1 treatment were tested for a role in FB1-induced cell death using cell death assays on Arabidopsis T-DNA insertion KO mutants. The UDP-glucose pyrophosphorylase, AT3G03250, was identified as a cell death gene responsive to FB1.