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Juliette Bardel-Lavaux

Plant mitochondria: Proteomics study and characterization of the first steps of the fatty acid and lipoate biosynthetic pathway

Published on 12 September 2003


Thesis presented September 12, 2003

Abstract:
In plant mitochondria, site of various biosynthetic reactions, relatively few mitochondrial proteins has been characterised. In the first part, we have undertaken an analysis of plant mitochondria by building the proteome of mitochondria isolated from different tissues: green and etiolated leaves and organs (seeds and roots) from pea. A systematic identification of major soluble proteins was undertaken and a comparison of the 2D-gel maps of soluble proteins from these different tissues highlights the presence of some proteins that were specifically expressed in these tissues. By this analysis, 433 spots were detected in pea leaf mitochondria. Using three different approaches (Edman degradation, MALDI-TOF and ESI-MS/MS), approximately 73 % of proteins are identified of total soluble proteins. We used traditional 2D polyacrylamide gel electrophoresis, in combination with size exclusion chromatography as a third dimension, to identify the major protein and further resolve their macromolecular complexity. A serie of expected major proteins (glycine cleavage system, and serine hydroxymethyl transferase) previously described involved in the photorespiratory pathway in higher plant were identified and represent about 37 % of total soluble proteins. Interestingly, the prominence of ALDH (7.5 %) in leaves suggests a detoxification function of aldehydes. The soluble mitochondrial proteome was also studied in a developmental perspective in order to analyze the effects of the tissue differentiation on the mitochondrial proteome from etiolated leaves, roots and seeds. The comparative study has led to analysis of 45 others spots and to identification of proteins which were specifically present in root (several multiforms of FDH) or in seed (LEA, HSP 22) mitochondrias.
In the second part, we have undertaken a biochemical study of fatty acids and lipoic acid biosynthesis in plant mitochondria. We characterized the enzymes implicated in the step of malonate activation into malonyl-ACP and acetyl-ACP, essential precursors for initiation of fatty acids elongation. This activation is initiated by at least two metabolic pathways: one catalysed by malonyl-ACP synthetase (MAS) and the second by malonyl-CoA synthetase (MCS) coupled to malonyl-CoA: ACP transferase (MCAT). By a method based on a complementary use of mass spectrometry (MALDI-TOF) and radioactivity assay, we characterized and purified the malonyl-ACP synthetase (MAS) and the malonyl-CoA: ACP transferase (MCAT). In pea, we demonstrated two MCAT activities: one located in plastid and the other in mitochondria. As there is only one putative gene coding for the MCAT in Arabidopsis, we started GFP (green fluorescent protein) experiments in order to specify the relation between subcellular localization and existence of at least two isoformes produced by one gene.

Keywords:
Plant mitochondria, Proteomic, Tissue differentiation, Mitochondrial biosynthesis of fatty acids, Malonate, ACP, CoA, Mass spectrometry

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