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

Many plants from the world's cooler regions, including the UK, can survive winter conditions due to a process known as cold acclimation. Gardeners may be familiar with this as "cold hardening", the phenomenon whereby plants grown in cold but not frosty conditions in the autumn become better prepared for the subsequent freezing temperatures of winter and are more likely to survive. Plants that are not able to do this are usually killed by winter frosts. For crop plants this causes major yield losses.

With an ever growing world population, it is estimated that food production will have to increase by 70% over the next 40 years. To achieve this we need both better yielding crops and crops that can survive the onslaughts of our environment. Freezing conditions during winter are responsible for severe crop losses in many regions of the world, including the UK. Enabling crop plants to improve their tolerance of adverse environmental conditions such as frost would provide benefits to agriculture, meaning that crops can be grown during parts of the year that would otherwise be too cold for them to survive, and allowing crops to be grown in areas that can currently not be used, for instance at higher altitudes and in colder areas.

Like humans, plants have thousands of genes that determine their characteristics and what they can and cannot do. Unlike us, plants cannot move away from unfavourable environments so many of their genes are involved in helping them defend themselves against the potentially damaging conditions they experience. Every gene, whether human or plant, is "switched on" when needed. When genes are switched on they make useful proteins, natural chemicals each with a unique function. Plants that are capable of cold acclimation can survive frosts because they have genes that, when switched on, produce proteins whose role is to protect against freezing conditions. Interestingly, these proteins are also often powerful protectants against drought. Therefore, discovering the identity of such genes is the first step in helping to make crops more tolerant of cold and drought.

We will discover new genes for freezing tolerance by comparing the genetic make-up of plants that can tolerate freezing with that of plants that cannot. Exciting new technology of the type used in the human genome project now allows us to examine the entire genetic code (all of the DNA that makes up the genes) of a plant so that we can look for differences in the genetic code of tolerant and susceptible plants. Finding differences in the DNA will show us the genes that are responsible for the survival of tolerant plants. When these genes are switched on they will make protective proteins; testing where in the plant these proteins are found will give us information as to the role they play in protecting it. If the proteins are important for protection, we might expect that plants producing them in larger amounts would be more tolerant, therefore we will test to see if this is indeed the case.

We will perform these experiments in a model lab plant that grows quickly and is easy to study, allowing us to make more progress in a shorter time. When we have discovered which genes are most important for freezing and drought tolerance, we will apply this knowledge to a crop plant: we will produce wheat plants that make these proteins in greater abundance and we will test whether or not the plants we have produced can better withstand freezing and drought conditions. If this is successful, the information we have generated will be of benefit to crop biotechnologists and traditional breeders attempting to make more frost- and drought-resistant varieties of food crops.

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. The economic importance of producing more freezing tolerant spring wheat is detailed in our case for support, and is further supported by a statement from Syngenta (see attached letter of support). Importantly, better alleles for SFR orthologues could be mined in any crop species in which 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 these 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 the "Stress and Responses to the Environment" and "Crops for the Future" modules on which Professor Knight teaches.
(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.