Size Matters: A systems approach to understanding cell size control in a developing multicellular tissue

Cells are the building blocks of tissues, but how 3D structures are built from cells is a fundamental unsolved problem in biology. It is even more complex when we consider a growing tissue, in which the physical properties of the building blocks (cells) change constantly as they grow and divide.

We study the growing shoot tip or shoot meristem of the model plant Arabidopsis. This structure is required to produce new leaves and flowers as the plant grows. The early stages of organ development require efficient tissue growth and an increase in cell size is normally observed at this time. Over several years, we have developed techniques allowing us to image the meristem over extended periods in the confocal microscope. We can follow individual cells over time and determine their growth and division. In order to divide, a cell goes through a defined series of processes known as the cell cycle. Using fluorescent reporters we have developed, we can for the first time simultaneously determine the position of all cells in the cell cycle during imaging time courses of growing plant tissues.

Our recently published study showed that the size cells reach when they divide is on average consistent for a given tissue and set of environmental conditions, but is highly plastic when these change. For example we found that cells were smaller, and tissue growth slower, when plants were grown under environmental conditions that restrict photosynthesis. Variation in cell size arises through unequal division and has to be removed. This is done by establishing a balance between growth and division on a cell by cell basis. The plasticity of the system leads us to consider that cell size at division is not determined by a direct "cellular ruler" but is instead determined as a consequence (or "emergent property") of the contributing processes of growth and division. This mechanism appears to be conserved from unicellular organisms which can also achieve a larger cell size and higher absolute growth rate under plentiful conditions. Regulation of cell size in these simple cells is achieved by balancing cell growth and division via the protein synthetic capacity of the cell.

We have developed a model that can predict accurately the size of plant cells based on the rate of growth and the accumulation of activity of two regulatory proteins required for cell division (called CDKs) as the cell grows. We tested this extensively using different mutants and growth conditions and identified the key processes that lead to cell size control. These processes appear to be the production and thresholding of CDK activity. In this proposal we will identify the "sizer" molecules involved in these processes that establish the link between growth and division of cells and analyse their function using our state-of-the-art imaging and analysis techniques. We have a number of candidate sizers, with known roles in CDK regulation, but we will also carry out experiments to identify new candidates in an unbiased manner using a genome-wide approach based on identifying the rate at which proteins are being synthesized under different conditions. We will use a combination of experiments and mathematics to develop a model that will allow us to understand how these sizer molecules are regulated and what effect this has on cell size control.

The shoot apical meristem (SAM) is the source of all above-ground plant growth and hence central to plant development and productivity. It is a shallow dome of continuously dividing cells that initiates rapidly growing organ primordia on its flanks that develop into flowers or leaves. Cell size is tightly regulated in all organisms, and in mitotically active tissues is determined by the opposing forces of cell growth and division. However, until recently was cell size control was poorly investigated in the important SAM tissue due to technical challenges. By combining time-lapse confocal imaging of live SAMs carrying a new reporter of cell cycle phase, we have recently shown that cell size in the SAM is dependent on developmental stage, genotype and environmental signals (Jones et al., Nature Comms 8:15060; 2017). Notably we found that low light conditions, which limit photosynthesis, led to a reduction in cell size across the SAM. We furthermore demonstrated that cell size at division could be accurately predicted by a minimal two-stage cell cycle model in which either the production or threshold of CYCLIN DEPENDENT KINASE (CDK) is cell size dependent. Here we seek to identify the "sizer" molecules that link cell growth to division and determine cell size. We have identified candidates which we will test, and use to build predictive models of cell size at division based on ordinary differential equations. We will test these predictions by manipulating sizer levels and changing environmental conditions. We will also use ribosomal footprinting to identify potential new sizers in an unbiased way and use cell level determination of protein synthesis rates to understand the link to cell growth parameters.

Key beneficiaries are identified as:
- Academic researchers and scientists, particularly plant scientists, but also the broader community of cell and developmental biologists, as well as systems biologists interested in predictive modelling of biological systems
- Agronomists and crop breeders
- Industrial researchers, including life scientists, mathematicians and computer scientists
- School pupils
- Members of the public.
Beneficiaries will be engaged throughout the project in order to deliver a range of economic and societal impacts.

1. Economic Impacts
Underpinning Knowledge: This project will advance understanding of plant growth and underpin the development of strategies allowing fuller exploitation of plant metabolism for sustainable agriculture. This impact is likely to be realised beyond the grant period through the integration of cell growth and division into models of plant development at different scales. To ensure this takes place, we will actively engage with life scientists, mathematicians and computer scientists from academia and industry and use knowledge gained from such interactions to inform implementation of our models with future compatibility in mind. These interactions will also generate awareness of our work and allow us to explore future collaborations to apply our models. Professional interactions will primarily be through academic conferences and publications, including regional and national meetings at which commercial researchers are brought together (e.g. meetings of UK Plant Sciences Federation, and those organised by Innovate UK and Welsh Government).
Enhancing research capacity: The fundamental nature of the question addressed means that we do not anticipate that there will be any immediate opportunities to commercialise our work. We will however develop techniques and tools that will contribute to the knowledge and skills of public and private research groups. For example, we will develop a protocol for measuring protein synthesis in plants using a proprietary product of ThermoScientific with their support, which will allow a broader range of researchers to access this tool. Models, transgenic lines and gene constructs generated by the work may similarly be of use to third parties and upon publication we will be freely available.
Skilled staff: Staff employed in this project will receive valuable interdisciplinary training contributing to the highly skilled workforce required to build and sustain the knowledge based economy. Notably the project will use advanced mathematical modelling, live cell imaging and next generation sequencing techniques, all skills in high demand in the biosciences sector. Furthermore all the researchers will gain experience of collaborative, multidisciplinary research and will be encouraged to develop project management and communication skills valuable for work in a variety of settings.

2. Societal Impacts
Improved teaching and learning: We will use the interdisciplinary nature of the team as an opportunity to develop an activity that can be used to promote both plant biology and mathematics/computer programming in secondary schools with the aim of increasing uptake of these subjects at university and filling skills gaps. We will target schools where fewer than average students go on to further education.
Public awareness and understanding of science: Improved public understanding of plant biology is necessary to allow members of the public to participate in an informed manner in ongoing debates including the use of genetically modified plants. Public opinion regarding genetic modification is likely to determine how fundamental knowledge is translated into improved agricultural sustainability. We will use the project as a means to engage with the general public and promote a greater awareness of current plant research. All team members will participate in public engagement events and so aid with the dissemination of scientific knowledge.