Abstract :
[en] Plant oxylipins including free and esterified oxylipins are structurally diverse molecules produced following fatty acid oxidation. Free oxylipins regroup various compounds with important functions in plant defence and developmental processes. Notably, jasmonic acid as well as its derivatives and some of its precursors are a family of phytohormones largely studied for their biological activities. On the other hand, esterified oxylipins have been much less studied but recent research showed their possible ubiquity in plants, as well as their probable involvement in plant defence and developmental mechanisms. Arabidopsides are a particular class of esterified oxylipins, and more precisely of oxidized galactolipids. Those were firstly described in A. thaliana but were also highlighted in other plant species and families. Interestingly, the oxidized fatty acyl chain of those molecules is either 12-oxo-phytodienoic acid or dinor-12-oxo-phytodienoic acid, both being intermediates in jasmonic acid synthesis. 12-oxo-phytodienoic acid is also a signal molecule implied in the modulation of plant defence gene expression. It has therefore been hypothesized that esterified oxylipins may either have direct functions or be used as a pool of free oxylipins. The latter may then act directly or be used for the synthesis of jasmonic acid. In the first part of the present thesis, a literature review discusses esterified oxylipin diversity, biosynthesis and potential functions.
The second part of this thesis describes the extraction and purification of the 5 main arabidopside molecules (A, B, D, E and G) produced by A. thaliana in stress conditions. In that way, following the induction of arabidopside production and the extraction of lipids, the latter were fractionated on silica column. Arabidopsides were finally purified by preparative-HPLC. An analytical method was then developed and validated to detect and quantify those molecules in leaf samples. Lipids were extracted and the polar lipid fraction was purified by solid phase extraction before being analysed by HPLC-MS. External calibration with purified arabidopside standards was chosen for arabidopside quantification. The method selectivity, limits of detection and quantification, intra-day and inter-day precision and accuracy, matrix effects and recoveries were determined. As arabidopsides are not commercially available, the obtention of purified molecules and the validation of the quantification method by HPLC-MS were used in the next part of this work to more deeply explore the functions of those understudied molecules.
As arabidopsides are biosynthesized from chloroplast membrane galactolipids, their precise localization among the different chloroplast compartments was determined in the third part of the present thesis. Results showed that arabidopsides are mainly found in thylakoid membranes but that the more polar forms are also present in the chloroplast stroma. As chloroplasts are largely implied in plant stress responses, and notably through the synthesis of reactive oxygen species and phytohormone precursors, we studied the impact of biotic stress induced by the recognition of the avirulence protein AvrRpm1 from Pseudomonas syringae on the structure of the chloroplasts of A. thaliana. Electron microscopy results showed that chloroplast structure is largely affected in stress conditions. In addition, thylakoid membrane fluidity was increased in such conditions, which could be due to arabidopside formation in those membranes. To better understand how arabidopside formation modifies thylakoid membrane properties upon stress, complementary biophysics and bioinformatics methods were used. Results showed that arabidopsides display very different interfacial properties than non-oxidized galactolipids which are found in thylakoid membranes in physiological conditions. Moreover, it was highlighted that arabidopsides A, B and D can interact with model membranes, leading to their destabilization. Results then suggest that arabidopside formation in stress conditions probably modify thylakoid membrane properties, which could affect their functions.
Globally, this work has contributed to a better knowledge of arabidopsides produced by A. thaliana in terms of quantification, localization in planta and effects on thylakoid membranes. It therefore provides new elements for a better understanding of the potential functions of these molecules, notably in the field of plant immunity.
