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Calcium signal transduction during nodulation

Reference	BB/D521749/1
Principal Investigator / Supervisor	Professor Giles Oldroyd
Co-Investigators / Co-Supervisors	Professor J Downie, Dr Alfonso Munoz-Gutierez
Institution	John Innes Centre
Department	Disease and Stress Biology
Funding type	Research
Value (£)	278,336
Status	Completed
Type	Research Grant
Start date	01/09/2005
End date	31/01/2009
Duration	41 months

Abstract

Calcium is a critical secondary messenger in diverse signalling pathways and can be presented to the cell in a number of different ways. One of the most complex calcium responses are oscillations in cytosotic calcium termed calcium spiking. This cellular response is induced in legume root hair cells following treatment with the bacterial signalling molecule Nod factor. Genetic dissection in the model legume has identified a number of genes that are required for Nod factor signalling and function both upstream and downstream of calcium spiking. We have recently cloned two genes that function downstream of calcium spiking: a calcium/calmodulin dependent protein kinase (CCaMK) and NSP2, a putative transcriptional regulator in the GRAS family. Both of these proteins are unique to plants and indicate a novel mechanism for calcium signal transduction. The genetic position of CCaMK immediately downstream of calcium spiking and its genetic identity implicate it in decoding the calcium spiking signal. By generating gain of function mutations of CCaMK we have shown that this protein can activate nodulation gene expression and this activity requires NSP2. This work indicated that CCaMk is sufficient for Nod factor signalling and that NSP2 functions downstream of CCaMK. GFP fusions indicate tht CCaMK is nuclear localised, while NSP2 is initially localised to the ER and nuclear envelope but moves to the nucleus following Nod factor treatment. This protein re-localisation may be critical for the regulation of its activity. In this proposal we will address aspects of Nod factor signal transduction downstream of calcium spiking. We will further characterise the functional domains of CCaMK, screening for complementation, localisation and induction of nodulation genes. We will characterise the re-localisation of NSP2 in more detail and assess the relevance of nuclear localisation for GRAS protein function. Evidence in the field indicates that GRAS proteins are phosphorylated and thisphosphorylation affects protein stability. We will test whether NSP2 is phosphorylated and whether it is a direct target of CCaMK. The facts that CCaMK and NSP2 function at similar positions in the signalling pathway and are both nuclear localised following Nod factor treatment makes them likely candidates for interacting with each other. We will test for such interactions both in vitro and in vivo. We will also screen for additional interactors and downstream components of both CCaMK and NSP2. This work will dissect the specific roles that CCaMK and NSP2 play in calcium signal transduction and identify additional players in this pathway.

Summary

Calcium is vital for living organisms and fulfils many roles within cells. However, calcium is quite toxic and therefore cells actively move calcium outside or into compartments within the cell where toxicity is reduced. For this reason calcium levels are maintained at a low concentration in the body of the cell, the cytoplasm. Calcium has evolved as a signal inside the cell that responds to the perception of many different molecules including human hormones. Calcium is either released into the cell from the outside or released from the cellular compartments where calcium levels are high. This is achieved by the opening of calcium channels on membranes that separate the cytoplasm from the outside or that surround the internal compartments. This release of calcium into the cytoplasm has many different patterns, with the most complex pattern being repetitive oscillations in the cytoplasmic calcium, called calcium spiking. This calcium spiking has been observed in both plant and animal cells in response to a variety of signalling molecules. A major question that is currently being addressed by scientists is how calcium can be activated by many signalling molecules and yet have very different effects in different cells at different times in their development. This question of specificity is most likely explained by the proteins that are able to perceive the calcium changes and then activate the downstream events. A family of plants called legumes, that include peas and beans, form a beneficial interaction with a class of bacteria called rhizobia. This interaction is useful to the plant since the bacteria provide nitrogen an important nutrient for protein production. In return the plant provides the bacteria with sugars that are generated in the plant by photosynthesis. This symbiotic interaction is particularly important in agriculture as it helps to enrich the soil with nitrogen, which is important for growth of crop plants. An increase in the use of legumes to enrichthe soil instead of fertilisers will greatly reduce the levels of pollution caused by fertilisers washing into our rivers and streams. In order to establish the symbiotic interaction the legume plant must recognise the presence of the appropriate rhizobial bacteria. This is achieved through a signalling molecule released from the bacteria into the soil, where it is perceived by specialised cells on the surface of the legume root, called root hair cells. A major feature of perception of the bacterial signalling molecule is the activation of responses in the plant that are important for establishing the interaction with the bacteria. We have recently discovered two genes that are involved in the activation of these plant responses by calcium spiking. In this proposal we will study how these genes function in perceiving calcium spiking and how they are then able to induce the plant responses.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Microbiology, Plant Science, Soil Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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