Resumen de Abiotic stress in plants: Late Embryogenesis Abundant proteins
Imen Amara
In order to improve our understanding on LEA proteins and their molecular functions in drought tolerance, the present work analyzes in the first place, the composition of LEA subproteomes from Arabidopsis seeds and maize embryos; second, three maize embryo LEA proteins from groups 1, 2, and 3 are analyzed in order to detect functional differences among them and finally, transgenic maize plants over-expressing group 5 “rab28” lea gene are characterized. The following results are presented: - Chapter 1. Proteomic approach to analyze the composition of LEA subproteomes from Arabidopsis seeds by mass spectrometry. The main objective was the development and isolation method to obtain enriched LEA populations from Arabidopsis seeds. LEA subproteomes were obtained using an extraction procedure that combines heat stability and acid solubility of LEA proteins. To identify the protein content, we followed two approaches: first, a classical 1D (SDSPAGE) gel-based procedure associated with MS analysis using an electrospray ionization source coupled on-line to liquid chromatography (LC-ESI-MSMS) and second, a gel-free protocol associated with an off-line HPLC and analysis via matrix assisted laser desorption/ionization (LC-MALDI-MSMS). - Chapter 2. Proteomic analysis of LEA proteins accumulated in maize mature seeds. Identification of LEA protein content by mass spectrometry and selection of three LEA proteins, Emb564, Rab17 and Mlg3, as representatives of groups 1, 2 and 3 for further study. Comparative functional analysis covering different aspects of maize Emb564, Rab17 and Mlg3 proteins, posttranslational modifications, subcellular localization and their properties in in-vitro and in- vivo assays. - Chapter 3. Characterization of transgenic maize plants expressing maize group 5 rab28 LEA gene under the ubiquitin promoter. Evaluation of Rab28 transcripts and protein levels, phenotype and stress tolerance traits of transgenic plants under drought stress. Investigation of the subcellular localization of transgenic and wild-type Rab28 protein using immunocytochemical approaches.
