Deciphering the Symbiotic Calcium Code: The activation of CCaMK

Plants must acquire their nutrition from their environment and this has led to the increased use of fertilizers. This approach is not a sustainable route for agriculture and alternative methods need to be developed to meet future crop demands. A large number of plants, however, have already developed strategies for obtaining their nutrition via symbioses with bacteria and fungi. Symbioses of legumes with nitrogen fixing bacteria provide the plant with nitrogen in the form of ammonia. Symbioses with fungi provide the plant with phosphorous, other elements, and water.
A holy grail of plant research would be to exploit our understanding of how plants coordinate and promote these beneficial interactions to produce crops that are less reliant on fertilizers. A key part of this process is the generation and decoding of signals that tell the plant what to do. These messages are hidden in calcium oscillations. The aim of this project is to understand how these calcium oscillations lead to the activation of proteins that determine how the plant should develop. Previous studies suggest that an enzyme, protein kinase, may decode these calcium oscillations, thus raising the question how can the same messenger robustly determine different responses? A key question is therefore how calcium oscillations can activate this plant-specific protein kinase. In this proposal we will determine the mechanisms by which the kinase of a model legume (Medicago truncatula) decodes the oscillatory calcium responses. We will achieve this through a multidisciplinary approach using a combination of high resolution imaging of calcium within a root hair with mathematical modelling and detailed biochemical studies of the relevant components.

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.

Increasing populations, limited energy resources, and global climate change are placing unprecedented challenges on food production. Improved plant nutrition has led to crop yield increases and the continued use of inorganic fertilisers is essential to maintain existing production levels. It is often the essential nutrients nitrogen and phosphorus that limit plant growth, and several plant species have established symbiotic relationships with microorganisms to overcome such limitations. In addition to the symbiotic relationship with arbuscular mycorrhizal fungi that many plants enter into to secure their phosphorus (and water and other nutrients) uptake, legumes also establish interactions with rhizobial bacteria that result in fixed atmospheric nitrogen being transferred to the plant.

Understanding how these processes are governed and the intrinsic signalling pathways that give rise to specificity is a first major step towards the exploitation of this knowledge for engineering crops for the future that have reduced requirements on inorganic fertilisers. Although this project is fundamental by nature, it will provide important insights into the mechanisms by which perinuclear symbiotic calcium oscillations are generated, transmitted and decoded. Exploitation of these results will lead to a more targeted approach to manipulating these pathways in future crops.

Another area of impact of the proposed work lies in training. The postholders will work very closely together and receive well rounded interdisciplinary training in mathematical modelling of spatiotemporal problems, confocal imaging, image processing software, biochemical techniques, binding kinetics, and legume biology. These are all highly valuable skills that can be transferred to a wide range of problems.

The results of this research will be communicated to other researchers through the standard channels of publications, seminars, posters and conference presentations. We will also actively engage in explaining our research to the general public and established mechanisms are in place for facilitating this communication process (Teacher Scientist Network, Friends of John Innes events, Science Art Writing school days, etc).