Control of seed and organ size by a ubiquitin-mediated signalling cascade

Lead Research Organisation: John Innes Centre
Department Name: Cell and Develop Biology

Abstract

This research aims to understand how the characteristic size of plant organs, such as petals, leaves and seeds is determined. One of the most obvious features of any organism is its size, which is highly characteristic for a given species but can vary hugely between species. Despite being such a clear and distinguishing feature, very little is known about what determines the final size of organs and whole organisms. We know that species have a very tight size range that they grow to, and then cease growing. We know organs grow through the multiplication of cells that differentiate to perform different functions in the organ, and that cells stop multiplying in a strictly coordinated manner when the growing organ reaches its final size. For example some organs, such as the liver, exhibit a remarkable ability to grow back to their exact original size after being surgically reduced. Biologists contend that this could be due to the cells in the growing liver (or those in another organ such as a plant petal) "know" where they are relative to neighboring cells and "remember" how many times that have divided, so that when they have divided a set number of times in an organ they stop dividing. Another view is that cells grow to form an organ within a "field" or grid system that provide points of reference that establish when cells stop dividing. There is experimental evidence for both points of view in diverse organisms such as mice, flies and plants, but much more needs to be understood before we can hope to explain such a basic biological characteristic as size.

The aim of the research in this project is to discover more about the basic mechanisms that plant organs use to determine their final size. Plants offer some specific advantages compared to animals as each cell is surrounded by a cell wall that renders them immobile once they have divided. This means the "fate" of cells can be mapped as the organ grows so we can tell where each cell in the final organ originated. Also, leaves and petals are flat sheets of cells a few cell layers deep, so they can be easily observed during growth. In plants it is now possible to track the individual cells in a growing leaf, allowing many new levels of understanding to be created. Our past work has identified an interesting protein that limits cell proliferation during organ formation in the plant Arabidopsis. When mutated so it no longer functions correctly, organs such as petals and leaves grow up to 25% larger. Interestingly larger seeds also form, as the seed covering in the mother plant is much larger. There are also more seeds formed so each plant makes more, larger seeds. This discovery was interesting to industry, who are now trying to make maize and soybean plants yield more using the gene we discovered.

In this project we aim to understand more about how this protein works, and we have amassed evidence that helps to plan the next stages of our research. We know the protein, called DA1 ("DA" is "big" in Chinese, reflecting its discovery by a Chinese researcher at JIC), is able to cleave other proteins, and we have identified two of these proteins. We aim to identify more proteins that are cleaved and then work out how they may influence leaf and petal growth, and how DA1-mediated cleavage may coordinate the activities of these proteins to achieve proper leaf and petal size. We also want to understand in more detail how the cleavage activity of DA1 is controlled, and where and when it is active during leaf growth.

This work is ultimately useful because crop plant yield, for example wheat or rice grains, is determined by the number and size of seeds produced in plants. Increased crop production without making a larger environmental impact is a key goal we need to achieve in the coming years to feed the world population, and the ideas and resources produced in this project could contribute to these solutions.

Technical Summary

We have identified a set of genes involved in a ubiquitin-mediated signaling cascade that regulates the period of cell proliferation during organ formation in the model plant Arabidopsis. The aim of this research is to characterize some of the targets of this signaling cascade and determine how they influence organ and seed size in Arabidopsis. The ubiquitin cascade involves the DA1 gene that encodes a Zn metallopeptidase domain. It is initiated by ubiquitination of DA1 by the E3 ligase EOD1/BB. This is a regulatory form of ubiquitination that activates the peptidase activity. This may be through conformational changes as observed in other Zn metallopeptidases, which must be tightly regulated due to the irreversible effect of their catalytic activity.

A key part of this project aims to understand more about how DA1 activity is regulated by EOD/BB- mediated ubiquitination, and to demonstrate where and when it is active during leaf formation. We aim to make a FRET substrate that reports its activity during live cell imaging of leaf growth so we can relate its activity to changes in cell division patterns across the growing leaf primordium. We have identified ten other genes that also interact with DA1 through yeast-2-hybrid and genetic screens. Some of these may be substrates for DA1Ub peptidase activity, and DA1 may influence the period of cell proliferation during organ formation by affecting their activity through protein cleavage. We aim to assess whether they are cleaved by DA1, and what the consequences of this cleavage are for leaf, petal and seed size. Finally, two of the targets of DA1 cleavage are E3 ligases, EOD1/BB and DA2. Both have established roles in determining organ size: EOD1/BB has been shown to limit organ and seed size in Arabidopsis, and DA2 is an ortholog of GW2, which strongly influences grain size and yield in rice and wheat. We aim to develop proteomic applications to identify their substrates and understand their influence on growth.

Planned Impact

A. Science. The outcomes of the proposed research will have a direct and influential impact on a wide range of scientific investigations in both plants and animals. 1) our discovery of a novel ubiquitin signaling cascade in plants will increase the interest and focus of scientists studying a wide range of biological processes that may be regulated by ubiquitination. 2), by identifying new genes influencing organ growth and a mechanism that may integrate their activities to set organ and size, the proposed work will promote new research aimed at understanding what is currently a very poorly understood phenomenon. 3), the work will have a major impact on research into the regulation of protein levels by developing proteomics applications for identifying the substrates of E3 ubiquitin ligases. As they control many processes, this method will have a significant impact and will help open up the proteome to more systematic analysis.

B. Industry. The genes and mechanisms we aim to discover have significant potential to help increase crop yield. The industrial beneficiaries include Plant Biosciences Ltd who patented DA1/EOD1 technology for increasing seed yield, and the technology licensee BASF Plant Sciences. They are currently assessing the technology in maize and soybean. If promising they may adopt it, and the current project will provide further foundations for extending the scope of the patent and preparing new ones, for example, by identifying new genes controlling organ growth. Speculatively, the FRET assay for DA1 cleavage activity could be used in a screen for small molecule inhibitors of its Zn-metallopeptidase activity for promoting growth. Many inhibitors, known from pharmacology, are used to specifically inhibit Zn-metallopeptidases. If adopted by BASF, DA1/EOD1 technology will have direct impacts on the production of important global crops by increasing yields. Alternative routes to application also exist in China, where DA1/EOD1 technology is being assessed in rice.

C. Producers and Consumers. Although the impact of this proposed work downstream of the plant biotech sector remains uncertain and speculative until the crop assessments are completed, it is worthwhile noting that if the technology is adopted and developed, there is a direct route to the field in many areas of the world through the BASF-Monsanto alliance, leading to a global impact on food security. Making crops with higher yield is a key priority for them, so rapid progress can be expected if their assessment is positive.

D. Researcher. This project provides outstanding opportunities for the researcher in terms of a very promising and productive project, training in biochemistry, proteomics and bioimaging, interactions with industry, transferable skills development and working with a large cohort of other early stage career scientists. The impacts include enhanced career opportunities, increasing the skills base of the UK, and preparation for possible career in industry.

E. JIC and TSL. This project builds on background work done in the GRO ISP that has been licensed to PBL, which is partly owned by JIC. The project will have a significant impact on JIC's KEC activities due to the strategic relevance of the work, and through publication in open access and high profile journals, adding to JIC and TSL scientific standing. By developing new proteomics applications the work will have a local impact on the range of technology on NRP.

F. BBSRC and policy makers. The project, through its impact plan, directly supports BBSRC and BIS strategic priorities in food security and sustainability by creating new knowledge to increase crop yields.
 
Description In the first year of this grant we have consolidated work describing the functions of the DA1 peptidase and how it controls organ size. This will lead to a high impact publication and the potential for further patents in addition to the three we already have concerning DA1 technology. This technology is being used to assess how it influences crop yield. Update: we have published this work in a high impact journal, and are now well on the way to understanding the upstream regulation of DA1 activity by receptor-like kinases. This will result in two more smaller but higher potential impact publications

The key findings include the observation that brassinosteroid signalling leading to cell proliferation functions in part by inhibiting the activities of DA1. This is a major discovery

We are now completing the preparation of a publication describing how brassinosteroids promote growth by inhibiting DA1 growth repressive activities. This study has widened out to include other receptor proteins including a recently discovered role of TMK4 as an auxin receptor, and CLV2 as a peptide receptor (with Yunhai Li in CAS, Beijing). DA1 appears to be a key intermediary in regulating protein levels in response to external growth cues.
Exploitation Route Through academic impact by publication and presentations
Through IP and licensing DA1 technology
Through exploiting crop genome sequences and new technologies for discovering and creating alleles in the DA1 family
Sectors Agriculture, Food and Drink

 
Description This project has been running for 1 year. It builds on previous BBSRC-funded work on identifying genetic networks that control organ and seed size in Arabidopsis. Since the start of this project the main aim has been to consolidate and extend initial findings for publication. This work includes confirming the activity of DA1 peptidase and its regulation. The impacts of this work will stem from publication and the patenting of further aspects of DA1 technology WE have now published the main findings of our research in the high impact general journal Genes and Development. This completes essentially all the objectives of the grant. Current work involves: 1. Identifying the location and timing of DA1-mediated cleavage of BB using BB-VENUS fusions in transgenic plants 2. Biochemical and genetic characterisation of wheat DA1 and DA2/GW2A orthologs 3. Understanding upstream regulation of DA1 by receptor-like kinases UPDATE: we are making good progress in understanding how BRI1 and TMK1/TMK4 regulate DA1 and DAR1. It appears that they phosphorylate DA1 and DAR1 and inhibit peptidase activity, while permitting ubiquitylation. WE now have the genetic evidence for this biochemical model In the final year of the project we are completing work for a publication that will describe the phosphorylation of DA1 by BRI and BAK1 in response to brassinosteroid treatment. This inhibits DA1 activity by promoting the formation of DA1 homodimers. Phosphorylated forms of DA1 can still be ubiquitylated but they have no peptidase activity. We show that brassinosteroids function in a new pathway to promote cell proliferation by inhibiting DA1 activity, which is a negative regulator of cell proliferation This work is now essentially completed and is being prepared for publication. In summary we have described a new mechanism that integrates different external signals (hormones, peptides) and regulates the stabilities of growth promoting and repressive proteins by peptidase-mediated cleavage and N-end-rule mediated protein destabilisation.
First Year Of Impact 2019
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Collaboration with Corteva on soybean seed size 
Organisation Corteva Agriscience
Country United States 
Sector Private 
PI Contribution We showed that loss-of-function mutations in the HECT E3 ligase UPL3 leads to substantially increased seed size and lipid yields in Arabidopsis and oilseed rape. The aim of the collaboration with Corteva is to see i UPL3 has the same phenotype in soybean and to explore the mechanisms involved
Collaborator Contribution Cash and clones
Impact none yet
Start Year 2018
 
Description Collaboration with VIB Gent on organ size control in Arabidopsis 
Organisation Flemish Institute for Biotechnology
Country Belgium 
Sector Academic/University 
PI Contribution WE work with VIB Gent on understanding mechanisms of growth control in Arabidopsis. We are the lead organisation in this collaboration, and the collaboration involves the exchange of genetic material and data so we can work together on joint publications. So far the collaboration has been productive with one joint paper, and another paper is in production
Collaborator Contribution VIB provides genetic material for testing. WE exchange different transgenic lines and mutations in order to make more rapid progress and expand the scope of our work
Impact publication: Dong H., Dumenil J., Lu F. H., Na L., Vanhaeren H., Naumann C., Klecker M., Prior R., Smith C., McKenzie N., Saalbach G., Chen L., Xia T., Gonzalez N., Seguela M., Inze D., Dissmeyer N., Li Y., Bevan M. W.(2017) Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis. Genes & Development 31 197-208
Start Year 2016
 
Description DA1 and organ size control 
Organisation Chinese Academy of Sciences
Department Institute of Genetics & Developmental Biology
Country China 
Sector Academic/University 
PI Contribution We collaborate scientifically. we exchange information and methods, provide training and advice, write publications together
Collaborator Contribution Yunhai Li has conducted extensive genetic screens to identify suppressors and enhancers of the da1-1 mutant, which has a large organ and seed phenotype in Arabidopsis. He initiated this work at JIC and took it with him when he went to CAS. Our work has complemented his approach by conducting biochemical analyses and protein studies. These identified a novel mechanism controlling organ growth
Impact several high impact papers three patents several PhD students trained Excellent research destinations of post-docs good training opportunities
Start Year 2013
 
Description Segmentation and cell tracking to understand the role of DA1 and brassinosteroids on leaf growth 
Organisation University of Cambridge
Department The Sainsbury Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated a detailed dataset of segmented images of cells during leaf growth- work done by PhD student Rachel Prior. New computational methods for segmentation, cell shape analyses and cell tracking were developed by Matthew Hartley at JIC. We are collaborating with Ross Carter and Henrik Jonsson at SLCU in analysing these datasets to understand the cellular basis of DA1 and brassinosteroid action during early stages of leaf growth
Collaborator Contribution SLCU- expertise in cell tracking and computational analyses of cell growth
Impact A publication is being prepared
Start Year 2018
 
Title DA1 technology 
Description The patents are concerned with protecting the use of mutations in DA1 and DA2, which are negative regulators of seed and organ size. In crop plants induced mutations or natural variation in these genes increases seed size and overall seed yield 
IP Reference WO2009/047525 & US2011-0004962, WO2015/067943 
Protection Patent granted
Year Protection Granted 2009
Licensed Commercial In Confidence
Impact In wheat the DA2 gene was shown recently to be an ortholog of GWA2, underlying a major QTL for thousand grain weight
 
Description ABCEED 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Prof Bevan participated in the annual ABCEED meeting in Berlin to discuss progress with the project and plan future work.
Year(s) Of Engagement Activity 2015
 
Description Attendance at ERA-CAPs coordination meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Prof Bevan attended the ERA-CAPs coordination meeting in Lisbon for discussions on how to address major challenges to secure future food supplies and a viable bio-economy in Europe.
Year(s) Of Engagement Activity 2015
 
Description Collaboration with Bayer Crop Sciences 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact JIC and Bayer discussed joint research project aimed at implementing DA1 technology to control seed size in oilseed rape.
Year(s) Of Engagement Activity 2016
 
Description Public Lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I presented a lecture on plant research in my group at a Science, Chemistry and Innovation event in London on 28 Nov 2018. The audience was mixed, comprising students, the general public and industrialists
Year(s) Of Engagement Activity 2018
 
Description TV interview with BBC Look East 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Mike Bevan participated in a television interview with BBC Look East.
Year(s) Of Engagement Activity 2013