Calcium oscillations are known to play a key role in nodulation. However, it is not known how calcium changes activate
downstream events that give rise to this important developmental programme. Mycorrhization shares many components of
the nodulation pathway, in fact, the nodulation pathway likely evolved from the former. The activation of a calcium and
calmodulin dependent protein kinase, CCaMK, is necessary for both processes and genetic studies suggest that the pathways diverge at this point. Within the proposed research programme, we wish to exploit recent advances in location specific cameleon lines that we have engineered into M. truncatula, developments in high resolution confocal imaging, and stochastic spatio-temporal modelling to build 3D models of signal generation on realistic geometries in order to determine the calcium concentration profiles that CCaMK is exposed to. We will reconstruct the spatiotemporal patterns observed in and around the nucleus solving a number of plausible models (CICR, voltage-gated, ligand-gated channels) on realistic geometries using the fire-diffuse-fire framework. These models will be parameterised by values derived from a statistical
analysis of confocal images of fine slices of the nuclear membrane. Proteomic approaches will be employed to determine the concentrations of CCaMK and calmodulin (Cam). We will use label-free quantification as our primary method of measuring the abundance of CCaMK and CaM in root hairs. This information will be combined with detailed studies on the phosphorylation dynamics of key residues within CCaMK as a function of calcium concentration and the kinetics of calcium and calmodulin binding to unravel the mode of activation of CCaMK and the decoding strategy.