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Florent Villiers

Proteomic analysis of Arabidopsis vacuoles in order to identify proteins involved in cadmium detoxification

Published on 31 October 2008

Thesis presented October 31, 2008

The vacuole is an organelle playing an important role in several plant cellular processes including protection against toxins. Among these, cadmium is a major pollutant, affecting plant physiology, and the vacuole is known for its capacity to store it. However, proteins involved in the transport across the tonoplast and in the chelation of cadmium inside the vacuoles are not well known. To better understand the function of this organelle we initiated a proteomics study to characterize vacuoles from Arabidopsis thaliana cells. This has allowed setting up tools and methods useful to study the dynamics of this proteome as a function of a cadmium exposure.
In the first part of my work, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis thaliana cell cultures using Ficoll density gradients. Absence of significant contamination by other cellular compartments was validated by Western-blot and enzymatic measurements. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, the protein components present in both membrane and soluble fractions of the Arabidopsis cell vacuoles were analyzed, leading to the identification of 689 proteins including 110 transporters. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein-tagged fusion proteins.
This first work was completed by a more extensive study of the vacuole proteome, in order to analyse it at a quantitative and organizational levels. IEF / SDS-PAGE were performed with the soluble proteins of vacuoles, resolved spots were quantified using PDQuest software and identified by mass spectrometry (collaboration with LEDyP, CEA-​Grenoble). Some proteins that were not identified in the previous study were found, leading to a list of 709 vacuolar proteins, among which some cytosolic proteins were identified. A map of a cytosolic protein-enriched fraction was made and compared with that of the soluble vacuolar proteome in order to understand the origin of the presence of the cytosolic proteins in our samples. These data combined with those from partial digestion experiments of intact vacuoles (performed with proteinase K) suggest that the main part of these cytosolic proteins are localized outside the vacuoles and are co-purified because of specific interactions of these proteins with the tonoplast. Some of them are also inside the vacuole, probably in the process of being degraded. Finally, supramolecular organization of the vacuolar proteome was evaluated using native electrophoresis (BN-PAGE), and putative isolated complexes were identified by mass spectrometry. These experiments confirmed the presence of some known complexes (vacuolar ATPase for example) and suggested new complexes containing proteases and/or glycosyl hydrolases.
A last aspect of my work was the development of an informatics tool, designed to store and exploit data from high throughput analyses. Such a structure has allowed cross-analysis of data from vacuolar proteomics work together with those from microarray transcriptomics experiments. This led to the identification of several vacuolar proteins transcriptionally regulated during cadmium stress. Among them, DWARF1, which catalyzes a key step in the brassinosteroids pathway, was selected to further analyze its possible implication in the mechanisms of cadmium response. We observed that brassinosteroids modulate Arabidopsis thaliana seedlings’ response to cadmium, and that this mechanism could not be linked to any known tolerance process, suggesting the existence of a new tolerance / sensitivity factor.
This work is a basis for the study of the changes occurring in the vacuolar proteome under cadmium stress, which should lead to a better comprehension of this organelle’s involvement in plant response to heavy metals. We set up new methods and tools, and obtained several results that highlight new aspects of plant vacuole physiology.

Cadmium, Arabidopsis, detoxification, heavy metal, vacuole

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