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

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.

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.

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.