Phone: +41 32 718 23 62

E-mail: sarah.rottet@unine.ch

NAP13 and NAP14, two Non-intrinsic ABC Proteins. Implication in lipid transport between thylakoids and plastoglobules?

Since my Master’s thesis, I have worked on the characterisation of two chloroplast proteins of the ABC (ATP-Binding Cassette) superfamily, atNAP13 (Non-intrinsic ABC Protein 13 or ABCI10) and atNAP14 (or ABCI11). Depending on their localisation and predicted ability to provide energy to transport processes as ATP-hydrolyzing subunits, we hypothesize that they are involved in the lipid exchange between thylakoid membranes and PG, doing so in association with channel and substrate recognition subunits. In order to understand their involvement in chloroplast and PG metabolism, I isolated Arabidopsis thaliana knockout mutants, which for both genes have a seedling lethal albino phenotype. In parallel, I engineered stable overexpression lines that rescue the albino phenotype of the homozygous mutant background. The recombinant, complementing proteins are tagged with a combination of the CBP (calmoduling-binding peptide) and IgG binding domain or HA (hemagglutinin) in order to purify interacting partners by TAP-tag pull-down or co-immunoprecipitation. I also generated additional complemented lines in which the NAP proteins are tagged with YFP for localisation purposes. I characterised all of these lines using, transmission electron microscopy and confocal microscopy for the ultrastructural analysis and localisation, SDS-PAGE and Western blotting for protein analysis and UPHPLC-QTOFMS for lipid profiling.

Plastoglobule carotenoid cleavage dioxygenase 4 (CCD4) acts in carotenoid degradation during chloroplast to gerontoplast differentiation

In parallel, I work on another highly promising project. A predicted carotenoid cleavage dioxygenase was previously identified in the PG proteome by our and other groups and has been hypothesized to be involved in carotenoid cleavage in senescent leaves. As expected, I observed a strong “stay yellow” phenotype in the senescent Arabidopsis knockout mutant. Failure in carotenoid cleavage was confirmed by UHPLC-QTOFMS-based carotenoid profiling. Currently I am developing an enzymatic in vitro test to demonstrate carotenoid cleavage by CCD4. Applications to crop species are easily conceivable in this case because the carotenoid cleavage dioxygenase family plays an important role in numerous metabolic pathways such as flower scent/colour development, fruit flavour production, hormone signalling and others. Nutritional aspects cannot be ignored, when it comes to the accumulation of β-carotene (provitamin A) in crop plants.

Biochemistry

Recombinant protein expression, extraction and purification. Analysis by SDS-PAGE and Western blotting. Prenylquinone and carotenoid extraction for UPHPLC-QTOFMS analysis . Chloroplast extraction and membrane fractionation on sucrose gradient (Plastoglobule isolation).

Molecular and Cellular Biology

Genomic DNA (EDWARDS, CTAB) and plasmid extraction, respectively from Arabidopsis thaliana and Escherichia coli. Cloning technique (traditional and Gateway). Plant RNA extraction and cDNA synthesis. Standard PCR and Thermal Asymmetric InterLaced  PCR (TAIL-PCR). Restriction enzyme digests. Agarose  gel electrophoresis. Bacterial transformation (heat shock and electroporation). Yeast transformation and yeast two hybrid system. Arabidopsis thaliana stable and Nicotiana benthamiana transient transformation mediated by Agrobacterium (floral dipping/agroinfiltration of leaves)

Microscopy

Optical microscopy. Epi fluorescence binocular and confocal microscopy (Leica SP5). Sample preparation and observation by transmission electron microscopy (TEM Philips CM200).