Mechanisms and function of alternative splicing in the plant circadian clock

Circadian rhythms are ubiquitous in nature: most organisms exhibit robust daily rhythms in biological processes, growth or behaviour. The most familiar rhythm to us is our sleep/wake cycle which is set to local time and gives rise to jet lag when time-zones are traversed. Rhythms are driven by 'circadian clocks' located in every living cell that continue to run even in the absence of external signals. These biological devices allow an organism to anticipate predictable environmental changes such as the regular day to night transitions and to adjust its behaviour accordingly. Circadian clocks comprise molecular circuits organised into feedback loops that generate the oscillatory behaviour of clock components. By linking to different molecular pathways through the cycle, the clock is able to regulate the timing of key pathways (e.g. metabolic, signalling, growth and development) through the day. Clockwork failure can have a large fitness costs to the organism; in humans it contributes to several disease states.

Circadian clocks can operate over a temperature range. This is particularly important for plants that are exposed to the ever-changing environment. Chemical reaction rates are very sensitive to temperature change, yet the clock remains invariant in the face of daily temperature changes and a range of weather patterns through the seasons. Yet how this is achieved is not known. A key objective for this project is therefore to understand molecular processes that buffer the plant clock from temperature changes. This is important as maintaining clock function is essential for optimal photosynthesis, growth, and the timing of reproduction - factors that influence seed abundance (a yield output for crop plants).

When genes are expressed the DNA sequence is first copied into RNA (transcription), the RNA is processed and then it directs synthesis of the corresponding protein (translation). In this project we focus on deciphering the role of post-transcriptional RNA processing (alternative splicing: AS) in temperature-dependent clock function in the model plant Arabidopsis. AS generates different transcripts from the same gene and thereby can modulate transcript and protein levels and functions. We have shown that AS is important in controlling clock gene expression. We will establish the extent of AS in clock input genes, how this is affected at low temperature and their influence expression/AS of clock genes. Plants experience continued and variable temperature changes throughout the day/night cycle - we will investigate the degree and timing of temperature change which is able to elicit a temperature-dependent AS response. Light also has a major influence on the clock and we will identify which AS events respond to light intensity changes and whether they are different from temperature-dependent events.

A major question is how the different types of AS in different clock genes are regulated and how this mechanism contributes to temperature buffering of the circadian clockwork. We will use RNA sequencing to assess AS events during cooling. Co-expression and co-splicing network analysis will identify genes whose expression/splicing profiles correlate with those of the core clock and clock-associated genes to identify putative regulatory genes. We will also examine the natural genetic variation in this process and how this might aid our understanding.

The role of AS in regulation of clock gene expression is highly relevant to crop plants and yield. To utilise the knowledge and approaches from our Arabidopsis research, we have started to examine AS control in potato and barley to begin the translation toward application. We anticipate that this work will provide new insights into what controls phenotypes such as earliness in barley and endodormancy in potato and ultimately lead to strategies for the generation of new genotypes that have increased robustness to temperature and other stresses that can affect plants.

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.