Dynamic re-programming of the cold transcriptome in Arabidopsis

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Genes are the repositories of hereditary information; proteins are the machines that carry out the functions of living cells. Gene expression usually refers to the process by which a gene gives rise to a protein. In eukaryotes, gene expression is complex and when such protein-coding genes are expressed, the DNA sequence is first copied into a precursor messenger RNA (pre-mRNA) by transcription. The pre-mRNA undergoes several processing steps to form the mature messenger RNAs (mRNAs) which direct synthesis of the corresponding protein (translation). In this project we focus on an extremely important RNA processing step called alternative splicing (AS). AS generates different mRNA transcripts from the same gene and thereby can modulate transcript and protein levels and functions. Plants experience continual changes in environmental conditions and have evolved systems to cope with stress-causing conditions and to survive the stress. In this project we are focussed on the response of plants (Arabidopsis) to low temperature and the role of temperature-dependent AS in the plant response to cold. The overall expression of a plant at any particular time is called the transcriptome and it the collection of all of the gene transcripts being expressed. It is determined by 1) transcription (turning genes on or off, up or down) and 2) AS (making >1 transcript from a gene). The high quality of our data allows networks of transcription and AS to be constructed. This will identify key factors which regulate transcriptional and AS responses and the re-programming of the transcriptome when plants are exposed to low temperatures. We will characterise such genes for their effect on sensitivity or tolerance to low temperature thereby identifying candidate genes for improving cold tolerance in crop species.In this research we will address five main objectives by exploiting our expertise in alternative splicing and circadian clock analyses. (1) In the dynamic transcript expression profiles that we have already obtained, we observe changes in the rhythmic expression/AS of many genes at low temperature, including genes that lose or gain rhythmicity. This suggests that the circadian clock may be involved in the regulation of these genes and by analysing the transcriptomes of plants grown at 20C and 4C we will identify those genes whose altered expression/AS is controlled by the clock. (2) We see some AS responses that occur very rapidly after the onset of cooling, suggesting activation of pre-existing splicing factors (SFs) rather than formation of new ones, with phosphorylation as a likely mechanism. We will investigate immediate/early AS and phosphorylation of SFs to identify candidate SFs that may be involved in these rapid responses to cooling. (3) A major part of the research will be the characterisation of novel cold response genes which show significant AS changes in response to low temperature. Mutant, over-expression and complementation lines will be assessed for cold sensitivity/tolerance and acclimation.(4) We are now able to generate high resolution data enabling us to build transcription and splicing factor networks and identify and validate key SFs (and transcription factors) that regulate cold-induced AS. The data also gives the opportunity to integrate transcription and AS networks providing a much better understanding to how the transcriptome is dynamically altered.(5) For future network modelling, knowledge of RNA-binding sites of SFs is essential. We will characterise the RNA-binding sites of key SFs identified here as a first step to developing a splicing code.