Periodic Reporting for period 1 - EDGE-CAM (Edge-based mechanisms coordinating cell wall assembly during plant morphogenesis)
Reporting period: 2021-09-01 to 2023-02-28
A fundamental question in biology is how multicellular organisms robustly produce organ shapes. The underlying process of morphogenesis involves the integration of biochemical, genetic, and mechanical factors across multiple spatio-temporal scales. In plants, morphogenesis is dominated by the rigid cell wall, which fixes cells in their position. Adjacent cells must therefore coordinate their growth patterns, which are in turn controlled by the mechanical properties of the cell wall. Cell walls are assembled by a complex intracellular trafficking machinery that delivers cell wall components and their associated biosynthetic machinery to different subcellular regions.
Based on our recent discovery that a trafficking route directed to cell edges is essential for cell wall assembly and directional growth at the cell and organ scale, we propose that morphogenesis is controlled by a signalling module at cell edges which integrates feedback from the cell wall. This hypothesis provides a mechanistic explanation for the integration of cell and tissue-level mechanical factors into coordinated cell wall assembly. We propose that a receptor-like protein recently identified as the first known cargo of edge-directed trafficking acts as a core component of a cell wall signalling pathway at edges.
This proposal aims to test our hypothesis through a combination of experimental and computational methods: (1) at the molecular level, we will identify further components of the signalling module through ligand screening, comparative proteomics, and forward genetics; (2) at the cellular level, we will functionally characterise trafficking pathways and their regulation by edge signalling through quantitative imaging, glycomics, and computational mechanics; and (3) at the organ level, we will dissect how robust growth emerges from edge-based feedback on these trafficking pathways. Collectively, these results will provide a multi-scale mechanistic model of morphogenesis in plants. This model will further our fundamental understanding of plants, and can form the basis for rational modifications in an agricultural context.
Based on our recent discovery that a trafficking route directed to cell edges is essential for cell wall assembly and directional growth at the cell and organ scale, we propose that morphogenesis is controlled by a signalling module at cell edges which integrates feedback from the cell wall. This hypothesis provides a mechanistic explanation for the integration of cell and tissue-level mechanical factors into coordinated cell wall assembly. We propose that a receptor-like protein recently identified as the first known cargo of edge-directed trafficking acts as a core component of a cell wall signalling pathway at edges.
This proposal aims to test our hypothesis through a combination of experimental and computational methods: (1) at the molecular level, we will identify further components of the signalling module through ligand screening, comparative proteomics, and forward genetics; (2) at the cellular level, we will functionally characterise trafficking pathways and their regulation by edge signalling through quantitative imaging, glycomics, and computational mechanics; and (3) at the organ level, we will dissect how robust growth emerges from edge-based feedback on these trafficking pathways. Collectively, these results will provide a multi-scale mechanistic model of morphogenesis in plants. This model will further our fundamental understanding of plants, and can form the basis for rational modifications in an agricultural context.
Overview:
During the first reporting period, we have made substantial progress on all objectives of the original proposal. Our most important achievement has been the functional characterisation of RLP4s in the context of edge-based growth control, which revealed:
-the mechanisms controlling RLP4s polarity at cell edges
-that RLP4s act as mechanosensors at cell edges
-that RLP4s are required for directional growth
-that RLP4s control edge-directed transport in a positive feed-back loop
We have published a preprint containing part of these findings (Elliott et al. 2022, bioRvix), and are currently completing an updated manuscript containing all our findings, which we are aiming to submit in May. These findings are a major milestone within the ERC project.
We have furthermore advanced other aspects of the work programme to the point where we have developed three new working hypotheses for major questions within the project:
1. We propose that edge-directed trafficking controls growth through the accessibility of xyloglucan at cell edges, which contributes to the formation of growth rate-limiting mechanical hotspots at edges.
2. We propose that cell edge polarity is an emergent property depending on local cell wall density, which is affected by three parameters: cell wall biochemical composition, cell geometry, and turgor pressure.
3. We propose that cell-based growth control has specifically emerged at the organ surface, in three-dimensional organs. We propose the unique mechanical niche at the organ surface both explains the need for and activates edge-based growth control.
We have put in place suitable methods and collaborations to test these three hypotheses outlined above, which will allow us to reach three major milestones within the project.
We have encountered some minor problems related to protein purification and have adapted our experimental timeline in response to serendipitous hiring opportunities (see detailed description below). However overall, we have not encountered any substantial problems and the research programme is well under way.
My team and I have presented our data at five international conferences (FASEB Mechanisms of Plant Development conference (USA), Plant Biomechanics conference (France), Plant Cell Biology International (Greece), BIRS-CMO workshop Multiscale Modeling of Plant Growth (online), Gordon Conference – Plant and Microbial Cytoskeleton (USA) and four invited seminars (Plant Membrane seminar series (online); and departmental seminars at the Swedish University of Agricultural Sciences, Heinrich-Heine University Düsseldorf, and University of Oxford), during the first reporting period, and have received excellent feedback from the community on these occasions.
During the first reporting period, we have made substantial progress on all objectives of the original proposal. Our most important achievement has been the functional characterisation of RLP4s in the context of edge-based growth control, which revealed:
-the mechanisms controlling RLP4s polarity at cell edges
-that RLP4s act as mechanosensors at cell edges
-that RLP4s are required for directional growth
-that RLP4s control edge-directed transport in a positive feed-back loop
We have published a preprint containing part of these findings (Elliott et al. 2022, bioRvix), and are currently completing an updated manuscript containing all our findings, which we are aiming to submit in May. These findings are a major milestone within the ERC project.
We have furthermore advanced other aspects of the work programme to the point where we have developed three new working hypotheses for major questions within the project:
1. We propose that edge-directed trafficking controls growth through the accessibility of xyloglucan at cell edges, which contributes to the formation of growth rate-limiting mechanical hotspots at edges.
2. We propose that cell edge polarity is an emergent property depending on local cell wall density, which is affected by three parameters: cell wall biochemical composition, cell geometry, and turgor pressure.
3. We propose that cell-based growth control has specifically emerged at the organ surface, in three-dimensional organs. We propose the unique mechanical niche at the organ surface both explains the need for and activates edge-based growth control.
We have put in place suitable methods and collaborations to test these three hypotheses outlined above, which will allow us to reach three major milestones within the project.
We have encountered some minor problems related to protein purification and have adapted our experimental timeline in response to serendipitous hiring opportunities (see detailed description below). However overall, we have not encountered any substantial problems and the research programme is well under way.
My team and I have presented our data at five international conferences (FASEB Mechanisms of Plant Development conference (USA), Plant Biomechanics conference (France), Plant Cell Biology International (Greece), BIRS-CMO workshop Multiscale Modeling of Plant Growth (online), Gordon Conference – Plant and Microbial Cytoskeleton (USA) and four invited seminars (Plant Membrane seminar series (online); and departmental seminars at the Swedish University of Agricultural Sciences, Heinrich-Heine University Düsseldorf, and University of Oxford), during the first reporting period, and have received excellent feedback from the community on these occasions.
Based on the data collected during reporting period one, we have developed two hypotheses which go beyond the rationale laid out in the original proposal:
Firstly, we developed a new concept for how edge polarity may emerge through self-organisation of the cell wall in polyhedral cells which are turgoid. This model is based on our observations of mechanisms that disrupt RAB-A5c localisation as well as preliminary data showing that cell walls are less crowded at edges. This hypothesis can also explain the observation that many soluble proteins accumulate specifically at cell edges. This hypothesis proposes a mechanisms for edge polarity generation that does not imply a specific molecular signature but instead focusses on emergent physical properties, and could this explain why fundamental features of plant growth can be achieved with cell walls of radically different composition (one of the core challenges identified in the original DoA). Testing this hypothesis will allow us to gain a better understanding of the mechanisms regulating edge trafficking in particular, but also to develop a much broader view of pattern formation in multicellular systems.
Secondly, we developed a hypothesis rationalising the activity of edge-directed trafficking in the context of 3D organ development. Based on localisation and phenotypic data, we believe RAB-A5c activity is required and activated specifically in cells at the organ surface due to their specific mechanical niche. This idea adresses one of the core questions of the proposal, but has been expanded because we now believe it may be the difference in pressure in different cellular environments that way act as a core regulator of RAB-A5c activity, thus providing an alternative interpretation of identity in cell surface layers.
Firstly, we developed a new concept for how edge polarity may emerge through self-organisation of the cell wall in polyhedral cells which are turgoid. This model is based on our observations of mechanisms that disrupt RAB-A5c localisation as well as preliminary data showing that cell walls are less crowded at edges. This hypothesis can also explain the observation that many soluble proteins accumulate specifically at cell edges. This hypothesis proposes a mechanisms for edge polarity generation that does not imply a specific molecular signature but instead focusses on emergent physical properties, and could this explain why fundamental features of plant growth can be achieved with cell walls of radically different composition (one of the core challenges identified in the original DoA). Testing this hypothesis will allow us to gain a better understanding of the mechanisms regulating edge trafficking in particular, but also to develop a much broader view of pattern formation in multicellular systems.
Secondly, we developed a hypothesis rationalising the activity of edge-directed trafficking in the context of 3D organ development. Based on localisation and phenotypic data, we believe RAB-A5c activity is required and activated specifically in cells at the organ surface due to their specific mechanical niche. This idea adresses one of the core questions of the proposal, but has been expanded because we now believe it may be the difference in pressure in different cellular environments that way act as a core regulator of RAB-A5c activity, thus providing an alternative interpretation of identity in cell surface layers.