The manipulation of signal transduction pathways controlling responses to adverse environmental conditions is likely to be the most effective way to generate stress tolerant crop plants. Before this "new" generation of resistance strategies can be realised, a considerable investment in basic research will be required, to identify the signalling molecules involved, to understand how they interact with each other and with other pathways (cross-talk), and to determine how specificity in response is achieved. CAST proposes to address these questions by systematically dissecting the signal transduction pathways involved in activating pathogen defence responses and cold tolerance. In particular, we will focus on the functions of calcium and activated oxygen species (AOS), as both have been proposed to play essential signalling roles in each response. Our consortium architecture is such that we are uniquely able to combine well characterised simplified experimental systems with highly advanced technologies that allow the dynamic visualization of individual signal transduction events at the cellular level. It is important to study both stress responses within the same project because the methodology required is common to both but is available in only a few laboratories within Europe, and because our combined studies will allow us to address the enigma of specificity, ie., to understand how common signalling intermediates such as calcium and AOS can control different responses.
A combination of cell biological and genetic approaches will be developed. For basic cold tolerance studies, we will focus exclusively on Arabidopsis. Well characterised tomato and parsley based model systems will be used to study plant:pathogen interactions, due to the lack of equivalent Arabidopsis systems. These interactions will, however, also be transferred to Arabidopsis to exploit the powerful genetic tools available.
Cell biological studies will be centered around the use of new single cell imaging techniques in cell cultures. Subcellular calcium and AOS concentration changes during cold acclimation and in response to pathogens or elicitors will be observed using, respectively, aequorin and redox-sensitive fluorescent dyes. Gene regulation will be studied using chimelic non-invasive reporter genes such as green fluorescent protein and luciferase, which provide sensitive systems for real time analysis of gene expression. We will focus on the regulation of cellular protectant genes such as those encoding AOS modulator enzymes. Observed regulatory patterns of calcium, AOS, and gene expression will then be pharmacologically manipulated using agonists and antagonists in order to link gene activation with signal transduction components. Additional cell biological studies will include AOS identification, direct microinjection of signalling intermediates, and PCR-based approaches to isolate kinases and phosphatases transcriptionally induced as part of the defence responses.
Genetic studies will utilise powerful transgenic-based approaches in Al abidopsis. A range of different classes of mutants affected in cold and pathogen response signal transduction will be isolated by chemical, transposon and T-DNA mutagenesis. In addition to stress sensitivity and gene expression, these mutants will be extensively analysed for calcium, AOS, and cross-talk phenotypes.
Stress resistance strategies are likely to be identifed from the above studies. Furthermore, the effects on stress responses of modulating AOS levels by over- and underexpression of a range of AOS-modulator gene constructs will be assessed in model plant systems. The two industrial partners of CAST will provide assistance in identifying resistance traits and will utilise the knowledge obtained during the course of this project for the production of stress tolerant target crops.
Funding SchemeCSC - Cost-sharing contracts
4421 AJ Kapelle
06120 Halle (Saale)
1600 AA Enkhuizen
NR4 7UH Norwich