Research objectives and content
Plant hormones, such as cytokinin and auxin, play an important role in a wide range of developmental processes, but little is known of their synthesis and mode of action. One of the methods of studying this is a genetic approach involving activation gene tagging where the tagged genes, being equivalent of cellular proto-oncogenes, are likely to be involved in either hormome biosynthesis, or action. several genes have been isolated in this manner including: axi 1, a gene influencing auxin responsive gene expression; cyi 1 which encodes a small peptide growth factor: axi 4 encoding an ion co-transporter and cki 1 a two component receptor kinase. The research program that I propose to carry out in Cologne falls into two:
tobacco mutants modified in internal levels of auxin by activation tagging and the further analysis of the products of the tagged genes axi 4 and cyi 1.
Levels of active auxin in the cell are maintained by the balance of de novo biosynthesis and uptake and the formation of inactive conjugates and breakdown. to date plant enzymes involved in auxin breakdown, or conjugate formation have yet to be characterized. To isolate genes involved in these processes I will carry out activation tagging and select for growth of cells in normally toxic levels of auxin. Calli growing under selective conditione will be cultivated further and ultimately plants regenerated. Following southern analysis, plant genomic sequences flanking the T-DNA insert will be recovered by plasmid rescue in s. coli. Functional testing of rescued sequences will be carried out by PEG mediated protoplast DNA uptake followed by selection under the initial selection conditione and gene sequences responsible will be determined.
Protein partners of axi 4 and cyi 1 will be isolated by using the yeast 2-hybrid system. The aim here is to determine elements of the auxin signal transduction system upstream or downstream of these proteins. Training content (objective, benefit and expected impact)
Until now my research experience has centered on the techniques of tissue culture and the effects of expression of genes in plant cells which modify the cells response to phytohormones. I am interested in extending my experience into the area of recombinant DNA technology in general. The program outlined couples tissue culture with molecular biology, thus I think I can build on my previous experience and extend my capabilities to include work with recombinant DNA.
Links with industry / industrial relevance (22)
If the program is successful, it will shed light on the molecular basis of phytohormone action. This is particularly important because phytohormones control many aspects of plant growth and development, impacting many areas of agronomic relevance. The work with the small peptide growth factor is of particular significance because, in an analogous manner to animal systems, peptide growth factors might be used in plants, in vitro or in vivo to manipulate patterns of growth.