Defining the symbiotic calcium channel of legumes

Cells have the capacity to recognise a wide array of signalling molecules and such recognition is essential to allow coordinated development in multicellular organisms. Calcium is commonly used as a secondary messenger that allows the recognition of a signal, for instance on the cell surface, to be transmitted to other regions of the cell, for instance the nucleus where gene regulation occurs. The potency of calcium as a signalling molecule and the fact that sustained calcium levels in the cytoplasm are toxic, means that calcium levels are tightly regulated. Calcium is either pumped outside the cell or into intracellular compartments such as the endoplasmic reticulum, the vacuole and vesicles. The release of calcium into the cytosol to act in signalling is the function of calcium channels that reside on the membranes of these compartments and allow the flow of calcium with its concentration gradient into the cytoplasm or nucleoplasm.

Proteins that function as calcium channels have been extensively studied in animal systems and a number of channel complexes have been shown to regulate calcium. Many of the animal calcium channels are absent in plants and this has hindered the characterisation of calcium signalling in plant systems. The proteins responsible for regulation of calcium in plant cells are only just beginning to be discovered. In this proposal we will define the calcium channels responsible for calcium oscillations that occur in the symbiosis signalling pathway of legumes. This signalling pathway is responsible for the establishment of nitrogen-fixing bacterial associations as well as beneficial fungal associations that occur in legume roots. We have already identified a strong candidate for the symbiotic calcium channel and in this proposal we will further validate the role of this candidate calcium channel in symbiosis signalling, as well as define the components of the channel complex that is likely to reside on the nuclear membranes.

Symbiosis signalling of legumes involves nuclear associated calcium oscillations that function as a secondary messenger linking the recognition of Nod factor and Myc factor, from the bacterial and fungal symbionts, to gene expression changes. While the genetic dissection of symbiosis signalling has revealed many components of this pathway, including a calcium responsive kinase, it has not led to the identification of the calcium channels and pumps directly responsible for the symbiotic calcium oscillations. We and others have shown that the calcium response is strongly associated with the nucleus and that calcium release is likely to occur from the nuclear membranes. Using this knowledge we have screened for candidate calcium channels and pumps that are targeted to the nuclear membrane. This approach allowed us to identify a calcium ATPase that sits on the inner and outer nuclear membranes and is responsible for reuptake of calcium during symbiotic calcium signalling. We have also identified a cyclic nucleotide gated (CNG) channel containing a nuclear-localisation signal (NLS), that when mutated greatly reduces symbiotic calcium signalling. CNGs function in tetrameric complexes and we have defined a further three CNGs in Medicago truncatula that contain putative NLS. We have already generated stable mutants for all four CNGs and this proposal will involve defining the role of these putatively nuclear localised CNGs in symbiosis signalling. We will use the mutants, in single, double and triple mutant lines to test the significance of these CNGs for symbiosis signalling. We will define the CNG complex residing on the nuclear envelope of M. truncatula and assess the ability of this complex to regulate calcium levels. The work validates the exciting discovery that a CNGC is likely to be responsible for symbiotic calcium signalling and the work in this proposal will greatly advance our understanding of calcium signalling in plant systems and specifically in the nucleus.

The availability of nitrogen is central to global food security and the application of nitrate fertilisers is currently a major driver in agricultural energy consumption and production costs. If we are to increase food production in a sustainable manner we must reduce our reliance on inorganic fertilisers, but this cannot come at the detriment of yield. Biological nitrogen fixation holds the key to breaking this dependence, but significant investment is required before it becomes a viable solution. While there is a clear challenge to deal with agricultural dependence on nitrogen fertilizers, there are no easy solutions. Engineering nitrogen-fixing cereals is a long term and challenging goal. It requires a detailed understanding of biological nitrogen fixation in legumes. It is now well established that cereals contain the symbiosis signalling pathway and that this pathway functions in cereals to allow mycorrhizal colonization. Importantly the evolution of nodulation recruited this signalling pathway for recognition of Nod factor, and this involved modifications to the signalling pathway that allowed both mycorrhizal and rhizobial recognition. We have shown that mycorrhizal fungi and rhizobial bacteria activate differential calcium signals and this must be the function of differential activation of the calcium channels. Engineering nitrogen-fixation in cereals will require the modification of the symbiosis signalling pathway to allow cereal recognition of rhizobia. In order to achieve this we will need a detailed understanding of this signalling pathway and in particular how this signalling pathway is differentially activated by mycorrhizal fungi and rhizobial bacteria, to allow specific signalling, leading to specific developmental consequences. This proposal will attempt to define the as yet unknown symbiotic calcium channel that must be central to the specificity of symbiosis signalling. This will provide the foundations for dissecting how this pathway is specifically activated during the rhizobial symbiosis and this knowledge is vital if we are to engineer this signalling pathway in cereals. It is expected that these broader implications arising from this proposal will result from further research at JIC and researchers internationally following the dissemination of the knowledge generated. Giles Oldroyd is currently leading discussions with charities and industry to define a long-term international programme of research focused on engineering nitrogen fixation in cereals. These discussions are still in their early days, but the programme of research proposed here will provide a foundation to our understanding of biological nitrogen fixation that will underpin these international efforts.