Mechanisms and function of alternative splicing in the plant circadian clock

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Molecular clock circuits comprise a set of interlocked transcriptional feedback loops with imposed delays that generate a characteristic ~24h rhythm. Its machinery is controlled by several mechanisms, including gene expression, chromatin remodelling, protein phosphorylation and protein turnover. Our recent work has added a new dimension by showing that alternative splicing (AS) can play a significant role in determining the level of functional transcripts/proteins of key clock genes, particularly in rapid or long-term responses to temperature change.

We will identify AS in clock-associated genes which input signals to the clock and in selected regulatory genes. This will generate a panel of primers covering key AS events which will be used in our sensitive HR RT-PCR system to address different aspects of the project. The functional relevance of AS will be tested at the promoter, transcript, protein and whole plant levels using clock mutant lines complemented with gene versions where AS is compromised or limited. These lines will be analysed for physiological and molecular phenotypes. To address what factors regulate AS of the core clock and clock-associated genes, and identify the downstream effects of reduced clock protein levels at lower temperatures, we need a genome-wide approach. We will perform deep RNA-sequencing across multiple time-points in the diurnal cycle before and after transfer to low temperature. This will generate expression and AS information on genes expressed under these conditions. Gene expression and splicing network analysis will identify candidate genes which may regulate or be regulated by the clock. Studies of natural variation may pinpoint other regulatory genes. We will characterise selected regulatory candidate genes (e.g. splicing factors) using overexpressing lines or lines that carry mutations and testing for effects on AS in clock genes.

Background
The research in this proposal is basic science on how alternative splicing (AS) regulates gene expression and thereby function in the circadian clock and its responses to external cues. Although at this stage the research is relatively far removed from direct application, the clock is so important for optimising plant growth and development in a changing environment that it is directly relevant to crop performance in the field. We have already made significant progress towards addressing similar questions in crop plants (potato and barley) in collaboration with scientists at the James Hutton Institute.

Who will benefit?
Understanding the molecular mechanisms that regulate the clock in a constantly changing environment could lead to new crop improvement strategies that mitigate the impact of predicted medium-term changes in seasonal temperatures, rainfall etc. The circadian clock is important to agricultural crops as it influences a range of processes that are important for productivity such as flowering time, starch production, disease resistance, stomatal movements, responses to stress and lignification. Understanding the basic molecular regulation of the clock will allow us to establish whether crop growth and productivity can be enhanced by controlling clock function. The work will therefore be of interest to crop scientists and plant biotechnology companies working on phenotypic traits and the underpinning genetics in crop species, and to government bodies responsible for future-proofing food production. Many of the principles governing the function of circadian clocks are broadly applicable across species; furthermore, there is an interactive chronobiology community which discusses ideas from many organisms. Hence our work is of potential interest to diverse communities such as human sleep researchers. The work will also be of interest to individuals (e.g. authors of textbooks) and organisations (e.g. Glasgow Science Centre) involved in science communication with schools and the general public. Our experience (e.g. at outreach activities such as the Glasgow Science Festival) is that the public can engage with such questions as 'can plants tell the time?' and that this can lead to discussion of why it is important to understand the timing mechanism.

How will they benefit?
Our work will be brought to the attention of industry and government through our interactions with crop geneticists and breeders at the James Hutton Institute. For example, JHI scientists are engaged in high throughput mapping of QTLs for traits in potato and barley. These scientists have interactions with the barley and potato breeding and processing communities in the UK, Europe and world-wide. Two key areas of interest are maturity determinants in barley and dormancy in potato both of which are influenced by the circadian clock. These areas of research are supported by the Scottish Government and policy groups within the government. We already have interactive research collaborations and have access to expertise, genetic resources and genomic information to catalyse translation of our basic research into the crop arena, so the long-term benefit will be in the broad area of food security.

We will disseminate the outcomes of our research at national and international plant, chronobiology and RNA meetings, and also present our work and publicise our research achievements e.g. to industry representatives who visit JHI or GU. Both universities seek to engage postdocs and PhDs to take part in the public engagement and impact agenda, for example by running Generic Skills programmes which also covers publicity activities. They also have Corporate Communications offices that regularly publicise research and promote engagement with the local media.