Post-translational regulation of cell physiology by the circadian clock

Lead Research Organisation: University of Bristol
Department Name: Biological Sciences

Abstract

Plant cells contain a biological clock that is fundamental to plant growth and survival. I discovered that seedlings in which the clock is stopped are half the size of those with a normal clock, and that if the clock is not synchronized with the environment, seedlings are stunted with reduced photosynthesis (Dodd et al. 2005). Maximum seed production also depends on the biological clock. Since these findings are of enormous agricultural importance, understanding the functions of the circadian clock is a timely and essential part of ensuring sustainable and secure food production in the future. Critically, our knowledge of the mechanisms by which the circadian clock enhances plant performance is very limited. The experiments that we propose will provide new knowledge to address this deficit. They will investigate two unresolved questions in plant biology; (i) what aspects of cell function are controlled by the circadian clock? (ii) what processes communicate timing information from the clock to circadian-regulated aspects of cell function? (i) What aspects of cell function are controlled by the circadian clock? Our preliminary experiments indicate that current understanding of the circadian organization of plant cell function requires extensive revision with information concerning protein abundance. We will identify membrane and soluble proteins that have circadian rhythms of abundance to discover biochemical mechanisms that are optimized by circadian regulation. (ii) How does reversible phosphorylation communicate timing information from the clock to circadian-regulated proteins within the cell? Reversible phosphorylation of proteins forms an important part of cell signalling that regulates the activity of enzymes directly, controls gene expression, and controls protein degradation. Genes encoding a large number of protein kinases are circadian regulated, and examples of circadian-regulated protein kinases are present in both plants and animals. This suggests that reversible phosphorylation has the potential to signal circadian timing information within plant cells. We will use two strategies to investigate the involvement of reversible phosphorylation in circadian signalling. First, we will identify proteins that undergo circadian rhythms of phosphorylation using a technique called phosphoproteomics. Second, we will identify the protein kinases and phosphatases that underlie the rhythms of phosphorylation for the proteins we identify that are of known importance to the cell. This information will be used to understand the signalling pathway between the circadian clock and the protein that is regulated by reversible phosphorylation. By combining my expertise (PI, Dr Dodd) in the circadian regulation of cell physiology and signalling with (a) specialist technology, expertise and methods within the Proteomics Laboratory in the Department of Biology's Technology Facility and (b) expertise in discovering novel protein-protein interactions in Arabidopsis (Hybrigenics S.A.) and protein kinase signalling (Prof. Jörg Kudla, Universität Münster), this research will advance significantly our understanding of the integration of the circadian clock in plants with cell function.

Technical Summary

There is an urgent need to establish the cellular bases for the large increases in productivity that are conferred by the circadian clock. This requires knowledge of both the cellular targets of circadian regulation and of the signalling pathways that communicate timing information from the circadian oscillator to these mechanisms. Surprising differences exist between the circadian timing of transcript and protein abundance in the liver and SCN of the mouse. Our new data reveal this is also the case in Arabidopsis. Proteome information is therefore required to understand the integrated circadian regulation of cell physiology. I propose to (a) investigate the circadian organization of the abundance and phosphorylation state of the membrane and cytosolic sub-proteomes, and (b) test the hypothesis that reversible phosphorylation functions as a output timing signal from the clock. Inclusion of membrane sub-proteomes in these experiments is important because membrane transport is pivotal to abiotic stress tolerance, cell signalling, growth regulation and mineral nutrition, and these mechanisms are commonly regulated by reversible phosphorylation. We have developed strategies for quantitative label-free LC/MS-based analysis of plant sub-proteomes over circadian time-courses and request funds to capitalize on our exciting preliminary findings. We will quantify circadian variation in protein and phosphoprotein abundance within the a) cytosolic and b) plasma and vacuolar membrane-enriched sub-proteomes from Arabidopsis. Using defined criteria, we will select from these data a small number of proteins involved in core aspects of metabolism and stress tolerance and identify candidate interacting protein kinases and phosphatases using yeast-two-hybrid screening. Potential interactions will be investigated in vivo using bimolecular fluorescence complementation. Finally, we will investigate the functioning of newly-identified putative circadian signalling pathways.

Planned Impact

Non-academic beneficiaries of this research will be (a) plant breeders and agricultural biotechnologists, (b) recipients of research training, (c) the public, (d) the UK economy overall. This research will provide information to address the large question of how the circadian clock benefits living organisms and how timing information is communicated within cells, and so has implications beyond the plant sciences. The agricultural biotechnology and plant breeding sectors will benefit from this research. A key challenge for 21st century plant sciences is to identify genetic targets that can be manipulated to optimize productivity and nutritional content. I previously showed that circadian regulation is essential for maximum productivity. My preliminary proteomics experiments have revealed circadian regulation of proteins required for the agronomically-essential mechanisms of photosynthesis, water use and stress tolerance. Our findings will therefore be important for plant breeders and agricultural biotechnologists developing high-performance plants because deliberate or inadvertent alterations to the circadian regulation of agronomically-important proteins is likely to have substantial productivity implications. However, we do not know the identity or understand the circadian regulation of these proteins. This research seeks to address this deficit. University-sector outputs that are most frequently considered to be important by commercial organizations are (in order of ranking) publications, informal interactions and interactions at public meetings/conferences (Cohen et al. 2002). These strategies form part of this proposal and we will use them to ensure that agricultural biotechnologists and plant breeders can benefit from our findings. Research training will be received by the PDRA employed on this grant. Training will cover experimental and analytical techniques in functional proteomics, bioluminescence and fluorescence imaging of gene expression and cell signalling, molecular cloning, analysis, presentation and sharing of large and complex data-sets, and communication of research by oral and written presentation. The training will therefore contribute to both capacity-building in specific research skills and also incorporate training in broad-based skills. A full-time Skills and Training Co-ordinator is employed by the Department of Biology, who facilitates career development of PDRAs and graduate students in order to maximise the impact of the key university-sector output of highly trained staff. Masters-level proteomics courses in the Centre for Excellence in Mass Spectrometry (Univ. of York) draw upon frontier technologies and methods in proteomics and so development and findings from this research will feed directly into this future training. The PI has a track record of participation in Science Week open days and will during 2010 tutor for the Gatsby Summer School, which is an important opportunity to enthuse the very best undergraduates with plant sciences and so retain the best researchers within the UK's knowledge-based economy. It is important that research findings are communicated to the public because curiosity-driven research has enriching quality of life benefits. To ensure this occurs, we will exploit the Departmental BioLog magazine, Biology Matters schools outreach magazine, University of York newsletter, Department of Biology Outreach Committee and Research Open Days, and press releases when appropriate. The presence of research of the highest international quality within the UK makes the UK an attractive host for knowledge-based and biotechnology companies. This is for two reasons: (a) the university sector output of highly informed, knowledgeable, bioscience literate and technically-skilled individuals; (b) the potential for interactions between industry and university research laboratories. This project incorporates training and conferences that will enable these benefits to be realized.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/I005811/1 01/09/2011 30/09/2012 £422,716
BB/I005811/2 Transfer BB/I005811/1 01/10/2012 28/02/2015 £265,155
 
Description A core aim of the grant has been to better understand the circadian regulation of cell physiology using a combination of proteomics and cell signalling studies. Preliminary proteomics work established the existence of circadian rhythms in the abundance of chloroplast-encoded photosynthesis proteins. We investigated the basis for this and discovered a novel type of signalling pathway that connects the nuclear-encoded circadian clock with chloroplasts (Noordally et al. Science 2013). Furthermore, the other key findings from this study have been published in three more articles; Belbin et al. New Phytol. 2017; Simon et al. Plant Physiol. 2018; Frank et al. Curr. Biol. 2018, with a side project paper published as Cano-Ramirez et al. Curr. Biol. 2018.
Exploitation Route The findings may be of use to those wishing to develop new crop varieties that use fewer resources by identifying new loci for selective breeding/marker assisted selection (e.g. plant breeders). The findings may be of use to organisations wishing to increase biofuel plant feedstock production through the optimisation of photosynthesis.
Sectors Agriculture, Food and Drink,Chemicals,Environment,Manufacturing, including Industrial Biotechology

 
Description Findings so far on these funding sources have given rise to a number of publications, including one very high impact one that was associated with several press releases. The press releases took the information into the public domain and were reported by the media, which provides enriching quality of life benefits to the general public by providing information concerning publically-funded science. Research findings have been disseminated to the public, opinion formers, and politicians orally through an event within the Bristol Life Sciences Building during October 2014. Certain findings were also discussed in detail with Syngenta during February 2013, which has since led to a collaboration between Dodd and Syngenta that has the potential to provide new information that will increase crop peformance. Furthermore, research supported by this grant has led to Dr Dodd being awarded a Royal Society Industry Fellowship to collaborate with a company (LettUs Grow) that is developing new technologies for vertical agriculture.
Sector Agriculture, Food and Drink
Impact Types Cultural,Societal

 
Description Adaptive significance of circadian gating
Amount £204,663 (GBP)
Funding ID 74705 
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 01/2019 
End 01/2022
 
Description BBSRC CASE studentship
Amount £68,000 (GBP)
Funding ID BB/M016900/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2019
 
Description Daiwa Anglo Japanese Foundation International Exchanges
Amount £10,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 01/2015 
End 12/2016
 
Description Increasing the productivity of vertical farms using novel aeroponic technologies
Amount £30,700 (GBP)
Funding ID SIF\R2\182028 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 03/2019 
End 02/2020
 
Description Leverhulme Trust research grants
Amount £459,064 (GBP)
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 07/2015 
End 06/2018
 
Description Research funding for early-career workshop
Amount £6,000 (GBP)
Organisation University of Bristol 
Sector Academic/University
Country United Kingdom
Start 01/2016 
End 04/2016
 
Description Collaboration 
Organisation Chiba University
Country Japan 
Sector Academic/University 
PI Contribution Collaboration with Chiba University
Start Year 2009
 
Description Collaboration with Ohio State University 
Organisation Ohio State University
Country United States 
Sector Academic/University 
PI Contribution We synthesized amiRNA constructs involved in sugar signalling and passed them to our partners for analysis.
Collaborator Contribution The partners provided access to techniques not currently in my lab, which have added mechanistic detail to the processes under investigation.
Impact None yet
Start Year 2013
 
Description Collaboration with Syngenta 
Organisation Syngenta International AG
Department Syngenta Ltd (Bracknell)
Country United Kingdom 
Sector Private 
PI Contribution I brought ideas to the collaboration and experiments have been performed in my lab by two team members.
Collaborator Contribution Syngenta have donated new herbicide formulations for specific physiological testing by two members of my team.
Impact No outcomes yet.
Start Year 2013
 
Description Collaboration with Tokyo Institute of Technology 
Organisation Tokyo Institute of Technology
Country Japan 
Sector Academic/University 
PI Contribution Collaboration with Tokyo Institute of Technology
Start Year 2009
 
Description Kyoto University CER 
Organisation University of Kyoto
Country Japan 
Sector Academic/University 
PI Contribution I am leading the collaboration and a PhD student in my lab is performing all experimental work in collaboration with a PhD student in the lab of Prof. Hiroshi Kudoh (Kyoto University).
Collaborator Contribution The collaborator provides access to field sites, facilities for experimentation, bioinformatics support and local facilities.
Impact Main outcome so far is obtaining funding for the collaboration. No publications yet because experimental work has just begun. The study is multi-disciplinary in that I am a molecular biologist and the collaborator is an ecologist.
Start Year 2014
 
Description Tokyo Univ collaboration (Prof Kita) 
Organisation University of Tokyo
Country Japan 
Sector Academic/University 
PI Contribution I have advised on design and implementation and assisted with interpretation of data.
Collaborator Contribution The collaborator has contributed by performing experiments with model experimental systems that cannot be used in my lab due to different facilities.
Impact No outputs to date. The collaboration is multi-disciplinary because it involves biomedical scientists.
Start Year 2014
 
Description Circadian control of the cell 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Departmental Seminar based on findings given at the University of Birmingham

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Circadian control of the cell 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited seminar based on outcomes given at Tokyo Institute of Technology

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description Departmental Seminar in Cambridge, 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I gave an invited departmental seminar, and had two days of discussion with academics in the Department of Plant Sciences

I made plans for several collaborations.
Year(s) Of Engagement Activity 2014
 
Description IBS Conference Korea 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I presented one aspect of my BBSRC funded research at a networking conference between members of the Royal Society and of the Institute for Basic Science of Korea. The conference also included discussion/brainstorming sessions and was held at Seoul National University.

plans were made for a further conference in the UK in 2015 in order to firm up academic collaborations.
Year(s) Of Engagement Activity 2014
 
Description Invited Seminar at CER, Kyoto University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Around 50 PhD students from the Center for Ecological Research at Kyoto University attended my seminar.

I wrote a grant with a collaborator in Kyoto.
Year(s) Of Engagement Activity 2014
 
Description Invited Seminar at Kyoto University's Botany Department 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I gave a departmental seminar in the Botany Department at Kyoto University.

I wrote a grant with a potential collaborator and invited 15 academics to Bristol for a workshop on plant environmental signalling.
Year(s) Of Engagement Activity 2014
 
Description Invited Seminar at Umea Plant Science Center, Sweden 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I had two days of exciting scientific discussion with academics in Umea.

Not sure what to write here.
Year(s) Of Engagement Activity 2014
 
Description Media coverage of research 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact An article associated with this grant that we published (Cano-Ramirez et al. Curr. Biol. 2018) attracted significant media coverage, including articles/interviews in the New York Times, Observer and Forbes. Dr Dodd is currently also working with BBC on this story for integration into the new so-called "Green Planet" flagship series.
Year(s) Of Engagement Activity 2018
URL https://www.nytimes.com/2018/08/24/science/marimo-floating-algae-balls.html
 
Description Science Cafe talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I led a British Science Association science cafe evening talk and discussion about circadian rhythms for the general public, consisting of a talk of about 40 minutes followed by about an hour of Q&A. I think about 35 people attended.
Year(s) Of Engagement Activity 2019