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Role of the vacuolar SV/TPC1 channel in plant Ca signalling

Reference	BB/D52316X/1
Principal Investigator / Supervisor	Professor Dale Sanders
Co-Investigators / Co-Supervisors	Professor Alistair Hetherington, Professor Frans Maathuis
Institution	University of York
Department	Biology
Funding type	Research
Value (£)	280,039
Status	Completed
Type	Research Grant
Start date	01/09/2005
End date	31/08/2008
Duration	36 months

Abstract

A wide variety of signals in plants is transduced through changes in cytosolic free Ca2+ ([Ca2+]c). Accordingly, a similarly diverse range of calcium channels has been characterised electrophysiologically in the plasma and endomembranes of plants. These channels are gated by depolarising or hyperpolarising voltages, or by ligands. Until now, it has not been possible to correlate the activities of these channels with activation by specific stimuli because the genetic identity of the channels has remained obscure. This in turn, has precluded our finding answers to important questions in calcium signalling, such as how temporal and spatial diversity in calcium signals is generated and how in turn these various patterns of calcium signals encode stimulus specificity. We have recently identified for the first time at a molecular level a Ca2+-permeable channel that resides on the vacuolar membrane. Electrophysiologically, this channel has been extremely well-characterised: it is known as the SV channel. It is outward rectifying and activated by phosphorylation, as well as by [Ca2+]c. This latter property is strongly suggestive that the channel is involved in calcium-induced Ca2+ release. All or part of the SV channel is encoded by the TPC1 gene, present as a single copy in Arabidopsis. tpc1 mutants have no detectable SV channel activity in their vacuoles, are defective in their ability to close stomata in response to elevated extracellular Ca2+ and in suppression of germination by abscisic acid. Further aspects of the phenotype have not been extensively investigated. We now have a unique opportunity to investigate the role of Ca2+ channels in signal transduction from a molecular perspective. We will extend the range of conditions for which a mutant phenotype is screened and analysed. We will further image, using cameleon indicator, the calcium signalling properties of tpc1 mutants to establish the contribution of SV channels to calcium signals. The TPC1 protein contains regions that are clearly indicative of activation by factors established as activating SV channels: basic residues in the S4 domains (voltage gating); EF hands (Ca2+ activation); phosphorylatable serine residues. We will mutate each of these regions and analyse the electrophysiological properties of the resultant channel in a tpc1 mutant background after transient expression in protoplasts. The Ca2+ signalling properties of the mutants will be imaged after stable transformation to determine upstream elements responsible for channel activation. Interaction of calcium signalling will be addressed by examining the phenotypes of mutant plants that are also defective in activation of selected ligand-gated channels.

Summary

All living organisms must respond to signals. Such signals can relate to stress (e.g. cold, heat, infection), can be important in the development of the organism (e.g. hormones) or can stimulate short-term behaviour (e.g. light). After a signal is sensed by an organism, a complex series of events must occur within cells to enable the appropriate response. In plants, one common event that occurs soon after the sensing of many different types of signal is that the calcium level within cells increases. It is thought that this increase in calcium level is essential for enabling cells to formulate the correct response to a particular signal. The increase in calcium level triggers a train of biochemical events that result in the response. But what causes the increase in calcium level? Membranes that surround the cell contain proteins that are known as calcium channels. When these channels open they allow calcium to flow in to the cell, either from the outside, or from special calcium stores within the cell. Although we know that these calcium channels exist, we do not know how they are activated by particular signals. We have recently made a major discovery. We have identified a gene that encodes a calcium channel called the SV channel. The SV channel is located in the membrane that surrounds the vacuole, a very big store of calcium within plant cells. Mutant plants that do not have this gene are not able to respond to one type of hormonal signal and do not close their stomata (the small pores in a leaf that regulate gas and water exchange) in some conditions. The discovery of the gene that encodes the SV channel now enables us to answer a range of important questions. First we will be able to ask what contribution the SV channel makes to calcium signals in plant cells. We are able to do this because we have mutant plants that do not possess the SV channel, and because we can image the calcium within cells. Second, by looking at the structure of the channel protein and then producing protein with altered characteristics, we can ask how the SV channel becomes activated by signals. For example, some regions of the protein indicate that it will respond to changes in membrane voltage; some parts bind calcium itself; other parts can be activated by enzymes called kinases. By producing SV channels that are changed in one of these regions and examining the effects on calcium levels within cells, we will be able to tell how important these regions are in allowing calcium to flow through the channel. This work is important because it will allow us for the first time to see how these important calcium channels are involved in signalling.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Plant 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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