Identifying components in the SFR6 pathway controlling cold acclimation and drought tolerance

Research in our laboratory is directed towards learning more about how plants tolerate freezing temperatures and drought conditions. Both of these are very important attributes for crop plants in this country as well as worldwide, and crops with the ability to withstand such unfavourable conditions can produce higher yields and can be grown in a wider range of locations improving their value to agriculture. Like humans, plants possess thousands of genes that determine their attributes, including their ability to cope with the environment in which they find themselves. We have discovered a gene in the model plant Arabidopsis which enables this plant to tolerate freezing temperatures and dehydrating conditions that would otherwise lead to death. We have called this gene SFR6. We have shown that 'sfr6 mutant plants' (plants with a defective SFR6 gene) lose their tolerance of freezing and drought conditions (environmental stress), a result that demonstrates how important the SFR6 gene is. Our previous work with these defective mutant plants has identified the reason for their inability to deal with cold and drought: SFR6 has an effect on numerous other genes whose roles in a normal plant are to protect it against these unfavourable environmental conditions. When SFR6 is missing /or simply defective- these other protective genes are unable to carry out their usual function. The question addressed in this new research proposal, is 'how does SFR6 influence this large number of protective genes associated with environmental stress tolerance?' Every gene (be it from a plant or an animal) facilitates the production of a specific protein. The SFR6 gene instructs the plant to produce the SFR6 protein. By knowing the genetic code that makes up the SFR6 gene (we discovered this last year), we can predict what the SFR6 protein will look like, however, we cannot predict how it will work. In this project we aim to discover whereabouts in the plant cell the SFR6 protein can be found, and what it is doing. Different parts of a plant cell have their own specialised function, and if we find SFR6 in a particular place, this will direct us towards identifying the type of job that the SFR6 protein is doing. In order to have any effect at all on the plant, the SFR6 protein must interact with other molecules in the cell in order to make them work. We propose here a selection of techniques which involve extracting SFR6 protein from a plant cell and examining what it is attached to. Any molecules found attached to SFR6 are most likely to be the molecules whose behaviour it needs to influence, in order to achieve its effects on cold and drought tolerance. Finally, we know that a defect in the SFR6 gene leads to a loss of environmental stress tolerance in Arabidopsis plants. We aim to ask 2 further questions related to this observation. Firstly, can we INCREASE stress tolerance in Arabidopsis by elevating the production of SFR6 protein and secondly, if this is possible, can we transfer this attribute to economically relevant crop species grown in the UK and throughout the world? The experiments described in the final part of our proposal will address these questions.

During cold acclimation to freezing temperatures some plant species (including Arabidopsis) greatly increase their expression of COR genes encoding proteins with protective functions. The majority of these are also expressed in response to dehydration stress and effect improved drought tolerance. Our lab has demonstrated that the sfr6 mutant of Arabidopsis shows severely reduced COR expression, and lowered drought/frost tolerance (Knight et al 1999, Plant Cell 11: 875; Boyce et al 2003, Plant Journal 34: 395). The sfr6 lesion is associated specifically with a failure in the normal operation of the DREB1 (CBF)/ DREB2 transcription factors that control COR gene expression. We have only recently identified the SFR6 gene; this proposal addresses how it achieves its function. Confocal microscopy of GFP-tagged SFR6 in planta will identify the subcellular location of SFR6 and provide a starting point for elucidating whether SFR6 acts directly upon transcription in the nucleus (Section 1). As no recognisable domains occur in the predicted SFR6 protein, we propose to reveal its function by identifying the macromolecules with which it interacts. Y2H analysis will be used to screen for potential SFR6-interacting proteins whilst TAP-tagging will identify proteins and/or DNA associated with SFR6 in planta. STrEP-tagging and Biacore analysis will be used to confirm that any putative interactions are genuine (Section 2). Finally, we will assess the potential for SFR6 to be used in future crop improvement programs, first by testing the effect of SFR6 overexpression in Arabidopsis and later, in crop species. We will use a homologous/heterologous transient expression system (using biolistic bombardment) to assess the potential for SFR6 to increase COR gene expression in wheat and potato tissues (Section 3). Information derived may indicate the value of testing the effects of SFR6 overexpression in stably transformed crop plants.