Calcium-regulation of nuclear proteins involved in stress tolerance

Stress tolerance in crops and plants relies on perception of the primary signal, and transduction to the cellular machinery to produce a tolerance response. In plants and crops the major line of defence is changes in gene expression to mediate tolerance. Increases in cellular calcium concentration are key intermediates in this process, brokering exchange of information between the environment and the nucleus.
Our previous work has provided profound knowledge on which genes are regulated by calcium, and by inference, which transcription factors are used. However, we do not yet have any understanding of other protein components in the nucleus which are required for mediating stress induced tolerance, via calcium signalling. This project will adopt a whole system proteomics approach in Arabidopsis to identify these proteins, characterise them genetically and mechanistically, and exploit this information to produce (in the future) crop lines with enhanced reactive stress tolerance. The key hypothesis for this work is that to mediate gene expression, calcium needs to trigger changes in the levels of specific regulatory nuclear proteins. Therefore calcium is likely to control the levels of those proteins through expression (including by regulating translation), or by regulating transport of those proteins into the nucleus, or by regulating the stability of those proteins through mechanisms such as ubiquitination. To test this hypothesis nuclear proteomics will be performed on samples extracted from plants treated with chemicals leading to changes in cellular calcium, as well as natural signal such as low temperature and drought stress, compared to controls. By making quantitative comparisons between samples this will allow identification of proteins whose abundance changes in a calcium-dependent manner. Once proteins are identified, the mechanism of calcium-dependent regulation of abundance will be determined, as will their role in stress tolerance, and information used to engineer wheat to better tolerate stress.
Aims:
(1) To answer the following biological questions:
(i) Which nuclear proteins are regulated in abundance by calcium in response to stress?
(ii) What mechanism controls the abundance of individual nuclear proteins?
(iii) What are the roles of these proteins in stress tolerance?
(2) To in the future exploit findings from Arabidopsis towards adapting wheat to perform better in response to stress.
Methodology: To answer question (i) from the first aim quantitative proteomics will be performed on Arabidopsis treated with calcium agonists, and stresses which induce calcium responses in the presence and absence of calcium antagonists. Question (ii) will be answered by testing the movement of proteins in response to calcium using fluorescent protein tagging and confocal microscopy, immunoprecipitation and Western blot analysis. Question (iii) will be achieved by performing genetic gain of function and loss of function analysis to test the role of these proteins in stress tolerance and stress gene expression. To achieve aim 2, transgenic wheat lines will be created using the wheat transformation facility in the Durham Centre for Crop Improvement Technology (DCCIT) to overexpress and knock down chosen genes and test effects upon stress gene expression and stress tolerance.
Fit with BBSRC priorities: This work fits within BBSRC Strategic research priority 1: "agriculture and food security", specifically, developing fundamental knowledge and applied solutions to producing more drought-tolerant crops fits into the aim "generating crops adapted to the challenges of future environments", important due to decreasing water availability for agriculture worldwide.