Research objectives and content
Calcium fluxes control many biological processes and are an essential step in many signaling pathways in plants and I animals. Although there exists a lot of information on calcium increase due to different biotic and abiotic stimuli, the information on the molecular level of calcium channels, channel regulators or associated proteins is still missing. In this proposal I present a novel approach based on established methods to done such proteins in plants. The project is based on T-DNA insertional mutagenesis (tagging) and subsequent selection during protoplast regeneration under non-permissive calcium concentration in the medium. It has been shown already that protoplasts can not continuously divide under very low and high calcium concentrations although they are able to survive these conditions. In a first approach we will use this optimized selection system (low and high calcium) to regenerate mutants created by activation and inactivation tagging. In all experiments protoplasts from aequorin expressing transgenic lines will be used for direct visualization of the calcium fluctuations in these plants. The regenerated mutants subsequently characterized and the tagged genes will be cloned and further analyzed. A second more focused approach, will be the selection of tagged mutants related to cold acclimation and cold stress in Arabidospsis thaliana. A similar protoplast approach for activation and inactivation tagging will be used. Also this Arabidopsis plants carry the constitutively expressed aequorin gene. This novel strategy should give new and additional information of the molecular level of induced calcium fluctuations in plants.
Training content (objective, benefit and expected impact)
This project involves a high degree of scientific training. Beside the conventional molecular and biochemical methods, the techniques established in the laboratory of Prof. Neuhaus, calcium imaging by a liquid nitrogen cooled slow scan CCD camera system, as well as different transformation techniques (Agrobacterium mediated transformation, particle bombardment for transient assays, microinjection for functional analysis of cloned proteins) will be used. Furthermore the attempt to establish the tagging technique in Arabidopsis, will have a high impact for other research areas. The scientific training I can get by working on this project will substantially increase my technical and scientific knowledge. Links with industry / industrial relevance (22)