Auxin in transcription factor complex controls polarity in plant organogenesis

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

Abstract

Multicellular organisms including plants and animals develop specialised organs, which are composed of different types of tissues. The structure - or pattern - of organs is determined by the polarity within tissues along axes of symmetry. In order to coordinate polarity across a tissue or organ, multicellular organisms use mobile substances such as hormones.
The plant hormone auxin was one of the first hormones ever 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, including for example lateral roots, young leaves and those of the female reproductive organ, the gynoecium. Control of auxin dynamics is achieved at the levels of biosynthesis, transport and signalling. The auxin molecule was previously shown to mediate the interactions between specific proteins thereby causing the degradation of repressors of gene expression. It has also been established that auxin can influence its own transport via inhibiting internalisation of PIN 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 plethora of processes in which auxin plays a role.
We have identified an interaction between two proteins found in the model plant Arabidopsis, which both are key regulators of polarity in the gynoecium of the flower. They have been named ETTIN and IND and both act as transcription factors (i.e. directly control the expression of genes). From experiments carried out in yeast we know that together these proteins can physically bind auxin in a so-called receptor complex, and our preliminary data suggest that the target gene set of ETT and IND changes when they bind auxin. This suggests the existence of an alternative signalling pathway for auxin. ETTIN controls the initiation and patterning of other plant organs, and in accordance with this, we identified other transcription factors that ETT can interact with in a similar auxin-sensitive manner. It is therefore likely that this new pathway is conserved in plant development.
Through experiments described in this proposal, we aim to reach a mechanistic and developmental understanding of this newly discovered auxin-signalling module, which may be particularly well suited to facilitate precise switches in polarity throughout plant development.

Technical Summary

Auxin comprises the most important intercellular signal in plant development and functions in organogenesis and patterning through biosynthesis, transport and signalling. Auxin signalling occurs through binding of the auxin molecule to a TIR1/AFB F-box protein allowing interaction with Aux/IAA transcriptional repressor proteins. These are subsequently degraded via the 26S proteasome leading to derepression of auxin response factors (ARFs).
Different combinations between members of the ARF, Aux/IAA and AFB families are believed to provide cell and tissue specificity to the auxin response. Moreover, auxin signalling also occurs through the auxin-binding protein 1 (ABP1), which - in the presence of auxin - inhibits endocytosis of PIN auxin efflux carriers to regulate directional transport.
From experiments carried out in yeast and plants, we have identified a protein-protein interaction between two key regulators of polarity establishment during Arabidopsis gynoecium development. These are the auxin response factor ETTIN (ETT) and the bHLH protein IND. Interestingly, this protein complex can bind the natural auxin, indole-3-acetic acid (IAA) and our preliminary data suggest that binding of IAA changes the ETT/IND target gene set. This suggests the existence of an alternative signalling pathway for auxin, and since ETTIN is involved in the initiation and patterning of other organs, this pathway may be widespread throughout plant development.
Here we will carry out studies to reveal the significance, mechanism and conservation of this novel IAA-controlled module, which is distinctly different from previously established modes of auxin action.

Planned Impact

Who will benefit from this research and how?
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. Indeed such a model-to-crop pipeline has already been successfully achieved in the Ostergaard laboratory through the transfer of knowledge on fruit opening in Arabidopsis to address seed loss related to pod shatter in oilseed rape.
The results expected from this proposal provides the first step in a similar pipe line and will point out directions for improving crop performance through regulation of auxin dynamics. In this way, the work directly addresses the BBSRC strategic priorities on Crop Science to tackle the challenge of Food Security.

The agricultural industry: The industry will benefit from technology development to improve crop performance with respect to yield and sustainability. Given the wide function of auxin and demonstrated role of the transcription factor ETT in root development and leaf initiation, it is likely that the results can also be used in aspects of plant development in addition to gynoecium development to improve e.g. seedling establishment, plant architecture and nutrient uptake. These processes are not restricted to oilseed rape, and the results may therefore be transferable to other crops.

Public: The public would benefit from greater predictability of yields, through greater stability in production costs, which would impact on prices in the shops. There are also obvious environmental benefits if yield could be improved through the discoveries that will come out of this proposal. Oilseed rape has emerged as the second largest oilseed crop with an annual worldwide production of 38 million tons of oil and demand is increasing. For this to be sustainable, seed yield needs to be dramatically increased through more efficient breeding programmes while at the same time minimising the amount of fertiliser input in order to protect the environment. I believe that the data obtained here has the potential to set out strategies to optimise fertility, plant architecture and nutrient uptake and thus contributing significantly towards such a goal.

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 the BBSRC-funded Crop Improvement and research Club (CIRC) and the Defra-funded Oilseed Rape Genetic Improvement Network (OREGIN). Both networks bring together academic researchers and breeding companies to discuss ongoing projects as well as to establish new interdisciplinary collaborations.

Commercialisation: 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 annual meetings, the OREGIN meetings and the 6-monthly dissemination events for grant holders within the BBSRC CIRC initiative.

Publications

10 25 50
 
Description This grant allowed the identification and characterisation of a new signalling pathway for the plant hormone auxin. Whereas the canonical auxin-signalling pathway is based on degradation of transcriptional repressors that inhibit auxin response factors (ARFs) from regulating the downstream target genes, the pathway identified here works directly on at least one specific ARF called ETTIN (ETT). Our work has established that ETT directly interacts with a number of transcription factors (TFs) from divergent families. We also demonstrated that these interactions are sensitive to auxin. This effect is specific to the naturally occurring auxin indole acetic acid (IAA) and not synthetic auxin such as NAA and 2.4-D that are known to affect the canonical pathway. In a paper published in Genes & Development (Simonini et al 2016), we revealed that this mechanism is important for a range of developmental processes throughout plant development including lateral root formation, ovule development, phylotaxi as well as gynoecium development. In a subsequent paper (Simonini et al 2017 Plant Cell), we identified direct targets of ETT and interestingly a subset of targets whose expression were dependent on ETT in an auxin-sensitive manner. We validated these targets genetically and found that they were involved/required for processes for which ETT was required as well. Finally, in a heavily genetics and developmentally-focussed paper, we showed that ETT interacts in the same protein complexes as several TFs and transcriptional repressors to regulate style formation in both Arabidopsis and Brassica (Simonini et al 2018 Development). Results in this paper shows that the ETT-mediated auxin signalling pathway is conserved at least within the Brassicaceae. Unpublished data has since confirmed that this mechanism also functions in formation of the style in gynoecia from tomato.
At the end of the project, the postdoc developed a system to recombinantly produce the part of ETT protein responsible mediating the auxin sensitivity (Simonini et al 2018 Scientific Reports). Work is currently ongoing to characterise the effect of IAA on ETT biophysically.
Exploitation Route The ETTIN-mediated auxin signalling pathway seems particularly important in establishment of the female reproductive structure, the gynoecium. We have established that the pathway is conserved at least between Arabidopsis and Brassica. Although early days, it is therefore likely that our discoveries in this area will lead to increased fertility and hence seed production. Scientifically, the results we are getting directly from this work is leading us in a number of directions that has led to additional funding (BB/S002901/1). These new directions range from the development of roots over leaves to fruits and will explore molecular mechanisms such as biochemical condensation/phase separation and auxin-regulated transcription factor complex composition.
Sectors Agriculture, Food and Drink

 
Description This grant was very fundamental in nature, but has led to a number of follow-on projects and grants including one responsive mode grants and a number of important collaborations both within the UK and abroad. A number of important publications from the lab came out of work associated with this grant and this work forms the basis of several projects going forward in the lab at the moment and will continue to do so in the years to come. Moreover, the auxin community is increasingly taking this non-canonical auxin signalling pathway to hear and I expect the number of collaborators to increase significantly in the near future with benefit for the lab, BBSRC and UK science.
First Year Of Impact 2015
Sector Education
Impact Types Societal

 
Description Biochemical and structural characterisation of ETT-mediated auxin signalling 
Organisation University of Grenoble
Country France 
Sector Academic/University 
PI Contribution Development of tools for recombinant production of ETTIN protein.
Collaborator Contribution Development of tools for recombinant production of TPL protein and providing expertise in biochemical and structural characterisation of protein-protein-ligand interactions..
Impact None yet.
Start Year 2017
 
Description Production of recombinant ETT for biochemical analysis 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Centre National de la Recherche Scientifique Grenoble
Country France 
Sector Public 
PI Contribution Postdoc went to the Laboratory of Dr Darren Hart at CNRS in Grenoble to make an expression plasmid that would allow us to produce recombinant protein of the auxin response factor ETTIN for further structural and biochemical analyses.
Collaborator Contribution Provided lab space, equipment and advice on the experiments.
Impact Publication in Scientific Reports (Simonini et al. 2018) Production of expression plasmid to allow structural and biochemical analysis of the ETT protein and its interaction with auxin.
Start Year 2016
 
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 Understanding the effect of N6A-methylation of mRNA in meristem size control 
Organisation University of Copenhagen
Country Denmark 
Sector Academic/University 
PI Contribution We performed in situ hybridisation on ECT genes and analysis of fruit shape in eat mutants.
Collaborator Contribution The laboratory of Professor Peter Brodersen have characterised a subclass of the ECT family in Arabidopsis both genetically and molecularly.
Impact A manuscript led by Prof Brodersen was submitted to the journal Development including our contribution.
Start Year 2017
 
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
URL https://pages.wustl.edu/ipgsa
 
Description Hosted a Nuffield student 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact In July 2014 Lars Østergaard hosted a Nuffield Student to provide them with training and lab experience.
Year(s) Of Engagement Activity 2014
 
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
 
Description Presentation at ETH, Z├╝rich, Switzerland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact An audience of 40-50 members of staff and students at ETH (and neighbouring university) attended my talk on a novel signalling pathway for the plant hormone auxin.
Year(s) Of Engagement Activity 2018
 
Description Presentation at Institute of Botany, Chinese Academy of Sciences, China 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 40-50 members of the Institute of Botany, Chinese Academy of Sciences attended my presentation on a novel auxin signalling pathway. The audience consisted of professors, postdoc, PhD students and technicians.
Year(s) Of Engagement Activity 2018
URL http://english.ib.cas.cn
 
Description Presentation at Rutgers University, New Jersey, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 40-50 members of the Waksman Institute at Rutgers University attended my presentation on a novel auxin signalling pathway. The audience consisted of professors, postdoc, PhD students and technicians.
Year(s) Of Engagement Activity 2018
URL https://www.waksman.rutgers.edu
 
Description Presentation at University of Lausanne 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presentation at University of Lausanne
Year(s) Of Engagement Activity 2019
 
Description Presentation at University of Lausanne 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited presentation at the University of Lausanne, Department of Plant Molecular Biology
Year(s) Of Engagement Activity 2017
 
Description Presentation at the "Auxin and Cytokinin in Plant Development" conference in Prague 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact As part of the 'Auxin and Cytokinin in Plant Development' conference in Prague I was an invited speaker and gave a presentation on a novel auxin signalling pathway.
Year(s) Of Engagement Activity 2018
URL https://www.acpd2018.org
 
Description Seventh Symposium on Plant Molecular Genetics (Sao Paolo, Brazil) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The Brazilian Plant Genetics Scientific Community is hosting its Seventh Symposium on Plant Molecular Genetics, which will be held in Campos do Jordão, São Paulo, Brazil, from April 29 to May 3, 2019.

This is a joint effort between the Brazilian Genetics Society and the plant genetics scientific community to reinforce the need of Brazil to motivate the establishment of a strong research foundation in plant biology, given the importance of agriculture for the Brazilian economy.
Year(s) Of Engagement Activity 2019
URL https://www.sbg.org.br/en/events/vii-simposio-brasileiro-de-genetica-molecular-de-plantas/welcome
 
Description Workshop on Flower development in Padova, Italy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk on genetic control of fruit-shape establishment
Year(s) Of Engagement Activity 2017