IDENTIFICATION OF GENES ESSENTIAL FOR FREEZING TOLERANCE AS TARGETS FOR MANIPULATION IN CROPS

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During cold acclimation exposure to low positive temperatures increases frost tolerance in many plant species (including Arabidopsis). This process involves major reconfiguration of the transcriptome and metabolome, effecting tolerance though a variety of mechanisms including gross morphological alterations, membrane stabilisation and the accumulation of protective proteins. Previous work led to the isolation of freezing-sensitive (sfr) mutants in Arabidopsis; we have recently cloned the gene for one of these, SFR6, and demonstrated it plays a key role in freezing and drought protection (Knight et al. 2009, Plant J. 58; 97). We propose to clone the remaining 4 SFR genes from the EMS mutants isolated in this screen and test the potential for their use in crop protection. We will use next generation sequencing for bulk segregant analysis of F2 crosses between each mutant and Landsberg erecta. Bioinformatic analysis of the frequency of mutations in pooled F2 DNA will lead to rapid identification of candidate loci for each SFR gene (Objective 1). Linkage will be confirmed and non-linked genes eliminated by testing complementation and loss of function lines (mutants and RNAi) for freezing tolerance (Objective 2). SFR gene expression profiles analysed by qRT-PCR, and subcellular localisation of GFP-tagged SFR proteins will suggest modes of action. Many proteins that confer freezing tolerance increase resistance to drought also. We will overexpress the 4 new and 2 previously cloned SFR genes in Arabidopsis and test these lines for improved tolerance of either stress. Together this information will reveal possible routes to exploitation (Objective 3). The 2 most promising genes will be overexpressed in spring wheat, which exhibits lower levels of freezing tolerance than winter varieties. Spring wheat overexpressing SFR genes will be monitored for improved freezing and drought tolerance and tissue damage assessed quantitatively by electrolyte leakage tests (Objective 4).

Who will benefit?
(1) Research staff trained.
(2) Company scientists interested in the cellular and molecular basis of freezing and desiccation tolerance in crops.
(3) Academic scientists interested in the fundamental basis for plant abiotic stress tolerance.
(4) Interested sectors of the general public, botanic gardens, biology school teachers/pupils.
(5) Postgraduate and undergraduate students at Durham University.
(6) Agricultural policy makers e.g. DEFRA

How will they benefit?
(1) The PDRA will be trained in plant molecular genetics, stress physiology, cell biology and imaging, experiencing work with crops as well as Arabidopsis. Interaction with Liverpool and NIAB will provide experience in next generation sequencing, genetic mapping and wheat transformation. The technician will be trained in aspects of this work, in particular wheat stress physiology. The PDRA will benefit from improved employment prospects and progression in academic or industrial research. The technician will benefit from professional development and gain insight into applied work.
(2) Our project will identify novel genetic loci controlling the traits of desiccation and freezing tolerance. Upon successful demonstration that these genes are linked to quantitative improvements in these traits in wheat, we would seek to engage companies with TILLING platforms and an interest in developing crop varieties with improved drought/freezing tolerance. A recent example of improvement of cold tolerance in spring wheat has been calculated to save just one country (Canada) multimillions of dollars per year (http://www.nwrage.org/content/frost-tolerant-wheat-save-farmers-multi-millions), evidence of the importance of this objective. Importantly, better alleles for SFR orthologues could be mined for in any crop species where freezing or desiccation stress is an issue. DCCIT has signed an alliance for 5 years with NIAB Innovation Farm (a demonstration and networking facility for businesses, innovators and stakeholders in the agricultural and horticultural sectors) This will showcase the specific work.
(3) Besides the potential exploitation for agricultural benefit, significant new information on the cellular/biochemical/molecular basis of cold acclimation and desiccation tolerance will be derived from the identification of the 4 genes. This will benefit academic researchers in this area, and interest all those working with crop/plant systems.
(4) There is great interest amongst the general public in topics pertaining to gardening (the no. 1 leisure pursuit of the UK in terms of people involved and financial turnover), and we find that speaking to the public about the ways in which plants deal with lack of water and cold is a particularly good vector for communication with this sector. Equally, the topic appeals to farmers and others interested in arable farming (e.g. members of agricultural societies).
(5) The work proposed will be linked to at least 2 postgraduate projects in the lab. Whilst the primary focus of our proposal is the strategic goal of crop improvement; many very important and interesting academic questions will arise during this work, particularly regarding the mechanisms by which the gene products mediate tolerance. These more fundamental questions will be pursued by the graduate students. At Durham University there is strong emphasis on research-led teaching. At level 3, we teach cutting-edge, contemporary research findings. Fundamental findings, applied goals, techniques and principles from this work will be communicated via "Stress and Responses to the Environment" and "Crops for the Future" modules.
(6) It is quite possible that the knowledge gained from the plant tolerance mechanisms uncovered by our work could be used to alter how crops are managed to optimize yield in the face of freezing and desiccation stresses, and therefore may be of use to organizations e.g. DEFRA, who co-ordinate and inform farmers on best practice.