Project details – University of Copenhagen

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Uncovering new membrane bending functions in plants

Main area:Molecular Biology
Target group:Biology-Biotechnology, Natural Resources, Other Education
Educational level:Master
Project description:

Background                                                                                                                                    

The recently discovered CURT1 (CURvature Thylakoid 1) proteins are small structural proteins responsible for bending thylakoid membranes to create their unique ultrastructure (Armbruster et al., 2013). Arabidopsis thaliana has four CURT1 proteins (CURT1A-D), which localise to the thylakoid membrane. CURT1A is the most abundant CURT1 protein in the leaf and exerts the greatest effect on membrane bending, and CURT1D expression is minimal. It is not clear whether CURT1s B, C, and D represent functional redundancy or whether they have additional specialised roles.                                              

To date, studies of CURT1 proteins have focused on total thylakoid extractions from chloroplasts of whole leaves. It is not known whether expression levels of the four CURT1 isoforms are uniform across different leaf cell types, meaning that data collected from the average of a whole leaf is representative of all cells, or whether specialisations exist in different cell types. Furthermore, nothing is known about whether CURT1 proteins are expressed in other green tissues such as stems, and whether they occur in non-photosynthetic tissues such as roots and flowers.

Aim                                                                                                                                                  

This masters project aims to understand expression of the four A. thaliana CURT1 isoforms in a whole plant context and at a greater level of detail than is currently available. Additionally, the experiments conducted will probe the effects of overexpression of the individual CURT1 proteins in a curt1abcd mutant background.

Approach                                                                                                                           

You will use promoters for the four A. thaliana CURT1 genes to drive the expression of a marker gene, β-glucuronidase (GUS) in transgenic plants. Expression of GUS in different plant tissues can be quantified using a histochemical stain. This method allows you to qualitatively identify CURT1 promoter activity throughout the plant at a macro level (e.g. whole organs such as roots and leaves) at different growth stages. In organs where expression is identified, staining will be quantified in specific cell types using microscopy. Promoter strength can also be quantified by comparing GUS expression with the expression of a green fluorescent protein under the control of a constitutive systemic promoter.

You will further develop transgenic A. thaliana lines where each of the CURT1 genes is individually overexpressed in a curt1abcd mutant. The resulting transgenic Arabidopsis lines will be an important resource for studying the individual roles of CURT1 proteins A-D.

Methods used:Molecular cloning and basic microbiology (Escherichia coli and Agrobacterium tumefaciens), Stable genetic transformation of A. thaliana, Histochemical staining and enzymatic reporter assays, Microscopy (light microscopy and confocal laser scanning microscopy), Protein extraction, electrophoresis, and protein immunoblotting (western blotting)
Keywords:chloroplast, thylakoid protein complexes, membrane curvature
Project home page: http://cpsc.ku.dk/meet-the-scientists-page/mathias-pribils-group/
Supervisor(s): Mathias Pribil
Email:pribil@plen.ku.dk