Cross-talk between cold and light signalling pathways in Arabidopsis thaliana.

When temperatures drop, we can put more clothes on, or turn the heating up. Plants can't. They can't move, so have to face the brunt of being cold. One of the major problems plants face is one they share with all other cellular life, namely that freezing of cells is very damaging. If that weren't bad enough, before temperatures even drop as low as freezing point, plants have another more specific problem. The definition of a plant is that it produces its own food, using light, via photosynthesis. The harnessing of such potent energy (sunlight), doesn't come without risks. The energy must be very efficiently coupled to producing sugar, or else the energy can cause damage to the plant cells (this is known as photooxidative stress). When temperatures drop, the efficient coupling of light energy becomes difficult, and the energy can produce highly reactive chemicals (reactive oxygen species, which act like bleach) that can damage and kill the plants. How do plants cope with these problems? Plants have a repertoire of different genes which they can switch on and off responsively to help them cope with changes in their environment. We know that when the temperature drops, a plant will switch on many genes whose purpose is to protect it from the consequences of frost damage. We have discovered a set of genes that are switched on by the plant in response to darkness but switched off during cold temperatures. Our research is aimed at discovering how the plant does this and what the benefit is of doing so. We will be looking for key patterns (sequences) in the DNA of these genes, which act as on/off switches when light levels or the temperature drop. By introducing artificial changes into these switching mechanisms and monitoring their effects, we will learn more about how the switches work. We can then examine the effect that switching the genes on and off has on the plant's ability to cope with life at low temperatures and in frost conditions.

Low temperature affects plant growth performance by imposing stress both in the form of increased photooxidation and freezing damage. To cope with these stresses, plants alter the pattern of expression of a large number of genes, which underpins protection mechanisms. In the model plant Arabidopsis, hundreds of genes are regulated in response to cold. Our lab and others have been successful in identifying the molecular mechanisms behind the signalling and transcriptional regulation of genes up-regulated to cold, leading to cold acclimation (freezing tolerance). Almost as many genes are cold down-regulated as up-regulated, and recently published research indicates that down-regulation in cold is an essential requirement for cold acclimation. In addition, gene repression in cold is a necessary mechanism for avoiding photooxidative stress which inceases with higher light intensities under cold conditions. The interaction between cold and light therefore is of physiological importance, but the molecular basis for this interaction is unknown. The work we propose here builds upon our previous research on signalling and gene expression leading to cold acclimation. We have identified a regulon of cold down-regulated genes from our microarray experiments, which are also light- and sugar-repressed. Under our conditions this regulon accounts for most of the cold downregulated genes. We seek to use these genes as tools/markers to uncover the molecular basis of crosstalk between cold and light signals. We have identified a region of transcriptional promoter (49bp) from DIN3, taken as our model gene for this regulon, that conveys regulation via sugar, light and dark. We have within this element identified one cis acting element (TATCCA) which is required for responsive expression, and have identified one transcription factor (LDMYB) which regulates the gene, most likely via this element. Thus the general aims of this grant are to further characterize these specific cis/trans elements, identify others acting in this promoter which contribute to cold and light/sugar regulation and determine their role in vivo in low temperature physiological responses of Arabidopsis. We will also test the possibility that cold-regulation of gene expression occurs via sugar signalling. The specific objectives are: (1) Identify the promoter elements in the model cold- and light-regulated gene, DIN3, that are required for light/dark-, sugar- and cold-regulation. (2) Identify the transcription factors binding to these elements. (3) Test the contribution of these transcription factors to the enhancement of cold acclimation and growth performance at lower temperatures. The techniques employed for these 3 objectives will be: production of promoter fusion constructs with firefly luciferase (1), production of transgenic lines (1,3), measurement of luciferase activity by luminometry (1); yeast one-hybrid analysis (2); growth assays under varying low temperature light conditions, using fresh and dry weight and chlorophyll measurement (3); northern blot analysis (2). We have used yeast one-hybrid analysis previously as part of BBSRC no. P14424 and all other techniques listed above are routine in my lab. Measurement of freezing tolerance by electrolyte leakage is a technically demanding method, and our collaborator (Dr Mike Thomashow, MSU) has agreed to do this part of the work for us (see attached letter); his lab is arguably the best in the world for this technique (3).