Auxentric - a hormone-based mechanism to control chromatin state

Lead Research Organisation: John Innes Centre
Department Name: Crop Genetics


The fate of all cells, whether unicellular or part of a multicellular organism, is a function of the genes they express. During the development of multicellular organisms, mobile molecules such as hormones regulate cell fate by controlling gene expression. The plant hormone auxin was one of the first hormones to be studied and the effect of auxin on light-regulated plant growth (phototropism) was investigated by Charles Darwin and his son Francis in the 1880s. It was, however, not until the 1930s that the auxin molecule was isolated and its molecular structure determined as indole 3-acetic acid (IAA). In plants, auxin plays an essential role in initiating organ formation and in patterning the organs in specific tissue types, for example, lateral roots, young leaves and the gynoecium (the female reproductive organ). In the classical auxin-signalling mechanism, the auxin molecule promotes the interaction between specific proteins thereby causing the breakdown of repressors of gene expression. It has also been established that auxin can influence its own transport by controlling the localisation of auxin transporters. Although these mechanisms of auxin signalling can explain many processes of auxin action, other transcriptional signalling pathways are likely to exist to account for the multitude of processes in which auxin plays a role.

We have recently described an alternative auxin-signalling pathway mediated by the auxin response factor, ETTIN, which plays a particularly important role during the establishment of polarity in organ development. This novel auxin-signalling mechanism (referred to here as the 'Auxentric' mechanism) is fundamentally different from established processes of auxin signalling as it involves a direct effect of the hormone molecule on ETTIN-transcription factor (TF) complexes without the involvement of protein degradation. Interestingly, our preliminary data suggest that this effect leads to changes in chromatin states in a mechanism similar to thyroid hormone signalling in animals.

In this proposal, we will reveal the mechanism by which auxin mediates its effect on ETTIN-containing complexes to control gene expression. We will, moreover, unravel the biophysical and structural characteristics of the protein and hormonal components involved. Above and beyond this specific mechanism, Auxentric may have far-reaching implications for the existence of alternative mechanisms by which hormones regulate plant growth and development. The work proposed here will thus introduce a novel gene expression-based mechanism of hormone perception in plants and draw unprecedented links between auxin dynamics and gene regulation at the chromatin level.

Technical Summary

In multicellular organisms, mobile signals such as hormones are key to regulating cell fate and organ morphology via controlling gene expression. Several animal hormones affect gene expression by modulating chromatin state via direct interaction with DNA-binding receptor proteins. In plants, the hormone auxin functions in organogenesis and patterning and we have identified an atypical auxin signalling mechanism with reminiscence to animal hormone signalling. Our preliminary evidence suggests that this mechanism involves auxin-sensitive interaction between the auxin response factor ETTIN and proteins that control chromatin dynamics.

The overall aim of this proposal is to understand how ETTIN-containing modules translate auxin levels into developmentally specific transcriptional outputs. To achieve this, we will use a combination of biophysical, proteomics and chromatin dynamics approaches to 1) identify auxin-controlled interactions between ETTIN and components of the chromatin-regulatory network and define the link between the ETTIN-dependent mechanism and regulation of chromatin state at ETT-target loci and 2) provide a mechanistic and structural understanding of this novel plant hormone signalling pathway and reveal molecular details as to how auxin affects ETTIN-containing complexes.

This proposal will reveal a novel mechanism by which cellular auxin levels are translated via a specialised transcription-factor module into precise transcriptional outputs required for specific developmental processes. Over and above the ETTIN-specific mechanisms studied here, the findings from this proposal will have wider implications for how auxin and potentially other hormones function. Moreover, the discoveries uncovered so far suggest an unprecedented flexibility by which hormones regulate development of multicellular organisms and may have implications for disciplines as far apart as cancer research and crop improvement.

Planned Impact

Who will benefit from this research and how?
The output of this research will be a new understanding of auxin mediated epigenetic regulation of gene expression. The research will be undertaken in a model species Arabidopsis, but because the mechanism is likely to be conserved it will be particularly relevant to other commercially important brassicas, e.g., oilseed rape, and more broadly, other staple crops. Moreover, since most plant developmental processes are influenced by auxin-based gene regulation, there will likely be a number of additional benefits to be exploited for crop improvement such as more efficient nutrient uptake and improved plant architecture.

The immediate beneficiaries of this work would be plant breeders and Biotech scientists who would use the research outputs in breeding programmes and genetic modification. However, the project may also identify new targets for agrochemical interventions and of benefit to the broader agrochemical industry. The results expected from this proposal will provide the first step in a model-to-crop pipe line and will point out directions for improving crop performance through regulation of auxin dynamics. In this way, this project will make a direct contribution to the BBSRC strategic priorities on Crop Science to tackle the challenge of Food Security.

Longer term beneficiaries will be farmers and other producers through access to new varieties and agrochemicals which increase productivity through improved crop performance, thus enhancing surety of income. Such a development would provide the wider society with greater stability in crop production which will help stabilise food prices. It would also indirectly contribute to reducing the environmental impact of food production through more targeted interventions.

What will be done to ensure that they have the opportunity to benefit from this research?
Publications: Results will be published in high-impact scientific journals and the breeding/farming press in a timely fashion. It will also be presented at national and international conferences and trade shows.
Collaborations: The PI has strong connections to the international auxin research community. This is reflected in the access to the novel and unique resources described in Case for Support, part 2. We also have strong links to the breeding industry and Brassica crop improvement programmes. The data that we obtain will be of immediate use to these interest groups, for example via stakeholder meetings in the BBSRC sLoLa project BRAVO, and the Defra-funded Oilseed Rape Genetic Improvement Network (OREGIN). Both groups bring together academic researchers and breeding companies to discuss ongoing projects as well as to establish new interdisciplinary collaborations.
The Brassica genus includes important crop plants such as oilseed rape (B. napus). The close evolutionary relation between members of the Brassica genus and the model plant Arabidopsis provides a potential goldmine for exploiting fundamental discoveries to improve crop performance. To identify and illustrate the potential application of the new level of developmental control, the PI will use the Brassica genus as a crop model in partnership with the BRAVO Consortium of UK researchers and industry partners.
We are dedicated to promote the use of our results for crop improvement purposes. Informal contacts with industrialists, biotechnologists and related stakeholders will be made as soon as any exploitable results are generated. We have tight links with relevant industries and will present results to them either when they visit JIC, at joint meetings or when visiting the companies. Such events and opportunities include the UK Brassica Research Community, the OREGIN and BRAVO meetings.


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Description In this grant, we are studying the molecular and structural basis for a newly discovered auxin-signalling pathway involving the auxin response factor, ETTIN (ETT). We have mapped a domain at the C-terminal part of ETT that directly interacts with the auxin molecule (indole-3-acetic acid, IAA). Using a recently developed E. coli-based expression system, we are able to obtain sufficient soluble amounts of this domain for structural and biochemical studies. A first publication of this work was achieved in the reporting year (Kuhn et al., 2020 eLife). Moreover, an invited methods paper on histone ChIP in inflorescence tissue was also published (Kuhn et al. 2020 Bioprotocols).
Exploitation Route We are keen to obtain ever higher molecular resolution of the components involved in this non-canonical auxin-signalling pathway. To this end, we collaborated with Prof Mariann Bienz at LMB. They carried out NMR on small peptides of ETT to further localise the auxin binding site. Unfortunately, this has not been successful. We are now aiming towards an X-ray crystallographical approach using a plant-based expression system to get enough protein. Moreover, we have submitted a responsive mode application with Prof Stefan Kepinski at Univ Leeds (BB/W016818/1 - outcome pending) to use HSQC-NMR to reveal the amino acid residues responsible for ETT dynamics and to understand the evolutionary significance of the ETT/IAA pathway.
Sectors Agriculture, Food and Drink

Description Biochemical analysis of auxin in complex with the auxin response factor ETTIN 
Organisation University of Leeds
Department School of Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of expression system to produce a domain of ETT recombinantly in E. coli.
Collaborator Contribution Production of ETT domain in E. coli followed by NMR on the 950MHz machine at Univ Leeds and analysis or results.
Impact None yet. Collaboration just started in Jan 2019.
Start Year 2019
Description Receptor-Hormone interaction assay 
Organisation Institute of Science and Technology Austria
Country Austria 
Sector Academic/University 
PI Contribution We have identified a potential interaction between the ETTIN (ETT) auxin response factor and the hormone auxin.
Collaborator Contribution The laboratory of Jiri Friml used DARTS assay to show that ETTIN interacts specifically with auxin.
Impact We have submitted a manuscript to the journal eLife, which is curretnly under review.
Start Year 2019
Description 23rd IPGSA Conference (Paris) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact 23rd IPGSA Conference (Paris)
Year(s) Of Engagement Activity 2019
Description Lecture at the University of Copenhagen, Denmark 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presentation entitled "Auxin at the heart of fruit shape diversity" attended by students and staff at the Institute of Biology, University of Copenhagen.
Year(s) Of Engagement Activity 2022
Description Plant Genomics Conference (Nanchang, China) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Plant Genomics Conference (Nanchang, China)
Year(s) Of Engagement Activity 2019