Geometric-edge specification in cell growth mechanics and morphogenesis

A fundamental challenge in biology is to explain how complex organisms develop the intricate anatomical forms observed in nature. The productivity and utility of crop plants is critically dependent on the development of particular anatomical forms that are often grossly altered from the wild state. The development of biological form requires chemical and mechanical information to be integrated at several scales of organisation: molecules assemble into larger assemblies; molecular assemblies organise internal cell structures; intracellular structures determine cellular properties; groups of cells assemble into tissues, tissues into organs, and organs into organisms. This highly complex process is nevertheless remarkably robust - petals on a symmetrical flower each have similar size and shape for example, or modern wheat varieties which grow to a remarkably uniform height.

A curious feature of development is that despite variability at lower scales of organisation (e.g., cell size, shape and number) biological form is typically robust at higher scales. This is rather like a dry-stone wall having a regular height and thickness despite variability in the sizes of the stones from which it is built. A recent idea, supported by our recent work, is that the variability at subcellular scales of organisation is not simply 'noise in the system' but is an essential part of the mechanism that maintains robust reproducible form at higher scales.

The overall shape of an organism is determined by the size, shape and arrangement of its component cells. Plant cells are surrounded by a rigid cell wall that resists their high internal pressure and determines their shape. Cell walls also prevent cells in plan from slipping past each other as they do during the formation of animal embryos. Consequently, the final form of the plant is determined principally by controlling the shape into which each cell grows. This requires the direction of cell growth to be controlled. Growth is driven by the cells' internal pressure, which acts equally in all directions, so the direction of growth is determined by the mechanical properties of cells' wall at the different regions of that cell. To successfully generate the final plant form, the control of cell growth must be coordinated across hundreds and thousands of cells. This requires both chemical and mechanical signalling between cells in growing organs. It also requires mechanisms that allow each individual cell to respond appropriately to these signals, adopting a shape that is appropriate to its position in the final structure. Little is known about how this happens.

This research aims primarily to increase our understanding of
- how growth and form are controlled at the level of individual plant cells
- how this is co-ordinated between cells to achieve proper form at the multicellular level
- how variability at lower scales influences the final form

We will focus on an important, newly discovered, mechanism that contributes to the control of plant cell growth. We will investigate the molecular and mechanical contribution that this mechanism makes during plant development. In short, we have recently discovered an internal transport mechanism in plants that delivers material specifically to the geometric edges of cells (i.e. where two faces meet). We have shown that when this transport mechanism is disrupted, cells and tissues become disorganised. We believe that this is part of a mechanism that allows cells to adjust their own size, shape and growth rate to produce an appropriate final form. We believe this is based on the detection of, and response to, mechanical stresses in the tissue.

To test our hypotheses we have assembled an interdisciplinary team of biologists, physicists and engineers to tackle this problem with a combination of computational models, genetic and biochemical analysis, plus 4D-light microscopy and mechanical measurements by dynamic atomic-force microscopy.

This proposal addresses a basic challenge in biology, which is to explain how multicellular organisms develop the intricate anatomical forms seen in nature. This complex process requires chemical and mechanical information to be integrated across several organisational scales but is remarkably robust despite variability at the cellular scale. Increasingly, heterogeneity in cell size and shape is seen not as simple stochastic noise but as a contributor to robust development at higher organisational scales. The cellular mechanisms involved are obscure, however.

Plant morphogenesis depends critically on growth anisotropy mediated by cell polarity and controlled relaxation of cell wall stress.
Through our study of intracellular membrane trafficking pathways that are unique to plant cells, we have recently reported (Kirchhelle et al., 2016 Dev. Cell 38:386-400) that polyhedral plant cells are polarised not just at opposing faces but also with respect to their geometric edges (where two faces meet). A previously unrecognised vesicle trafficking pathway to these edges, mediated by the small plant-specific regulatory GTPase RAB-A5c, is essential for cell growth anisotropy during morphogenesis.

Based on this work and subsequent observations, we hypothesise:
1. The existence of a regulative cellular mechanism to detect and respond to local shear stress in cell walls at subcellular scale during development.
2. That membrane trafficking to individual cell edges, mediated by RAB-A5c GTPase, is an essential part of this mechanism.
3. That RAB-A5c-controlled heterogeneity in local growth rate at the subcellular scale promotes regular morphogenesis at the multicellular scale.

We propose an interdisciplinary set of computational and empirical tests of these hypotheses, involving 3D mechanical models of cell growth informed by 4D confocal and 3D EM imaging, dynamic AFM, plus mechanical and genetic interventions in Arabidopsis with normal or inhibited RAB-A5c function.

We envisage beneficiaries of the output from this research in academia, stakeholders within the agricultural industry and the general public.

Industrial relevance: Key challenges in 21st century agriculture are to ensure a secure supply of food for the rising world population and to add market value to crops. Estimates are that even with optimal varieties and best available crop-protection regimes yields are still typically only ca. 75% of the theoretical maximum. Without protection against weeds, pests and pathogens yields would be approximately 20% of theoretical maximum.
The plant cell wall is a major contributor to determining the harvested yield and to the quality of food, fuel, and fibre crops. Examples for mechanical properties that influence the quality of an agricultural product are fibre quality in flax, texture before and after cooking of vegetables, and consistency and resistance to bruising of fruit. Mechanical properties are also an important feature of crop performance, for example its resistance to lodging and root growth in drying soils.
Cell walls are also an herbicide (and safener) target owing to their importance to plant survival and unique composition (HRAC Group L herbicides and, via action on microtubules and cell division, Group K2). Most current herbicides have a Mechanism of Action (MoA) that dates to the 1970s and early '80s with no new MoA successfully implemented since then. In a situation that parallels the decline in antibiotic efficacy, weed resistance to these compounds is an increasing cause of yield loss, particularly in European non-GM agriculture (http://www.ewrs.org/weed_mapping.asp). At the same time regulatory and economic hurdles have increased the commercial pressures on new development.

We have described new cellular mechanisms of control of cell wall architecture and mechanics. Controlled manipulation of these mechanisms could conceivably be used in the rational modification of crop traits for improved yield or quality. Validated models of the sort we will develop could also help modern agriculture optimise the mechanical properties of a crop to inform breeding and evaluate the likely performance of different varieties in the same setting. For example, the model could be parameterised using an experimental dataset, predictions evaluated in silico, followed by design of resource-efficient validation studies enabling new plant varieties or traits to be selected. Although we are working with an exerimentally tractable laboratory model species rather than a crop plant, the molecular mechanism we are studying is clearly identifiable in crops so findings should be readily translatable.
The University of Oxford has an extensive network of mechanisms for outreach and interaction with external groups from the commercial, translational and public sectors. We will make full use of these, as appropriate, throughout the project to ensure that the output is relevant, informed and understood.

Engaging the Public: We will work through the University of Oxford's Engaging the Public through science communication events provides an opportunity to educate the general public about cutting-edge science and can provide interesting feedback. Several aspects of our project lend themselves to a public engagement effort and will be designed and pursued in collaboration with the Botanical Gardens in Oxford. Firstly, we will communicate the flexibility of plant morphogenesis and how is has been utilised to breed the vast variety of modern vegetables. Secondly, the display will demonstrate the importance of mechanical properties of crops, using an interactive display. Thirdly, we will relate how scientists from different disciplines (plant sciences, engineering, computer sciences) interact to solve complex problems. We will develop an exhibit for public engagement events at the Harcourt Arboretum in Oxford and the Festival of Nature in Bristol during the second and third year of the project