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Quantification of the role of mechanical stresses in plant cell morphogenesis.

Periodic Reporting for period 2 - PlantCellMech (Quantification of the role of mechanical stresses in plant cell morphogenesis.)

Période du rapport: 2018-04-15 au 2019-04-14

Specific cell and tissue form is essential to support many biological functions. During development, the creation of different shapes fundamentally requires the integration of genetic, biochemical and physical mechanisms.
In plants, a stiff pecto-cellulosic network encapsulates cells and counterbalances stress created by turgor pressure inside the cell, thereby controlling cell shape. It is well established that the cytoskeletal microtubules network play a key role in the morphogenesis of the plant cell wall by guiding the organisation of new cell wall material. Moreover, it has been suggested that mechanical stresses orient the microtubules along their principal direction. Nevertheless, to fully understand how plant cells are shaped and how mechanical stresses influence this process, a quantitative approach needs to be established.

In this project, we aim to provide new fundamental knowledge on the role of mechanics in plant development at the cellular scale. New experimental and imaging methods are now available to achieve this aim. We combine laboratory experimental approaches and mechanical modeling to study quantitatively how single plant cells respond to mechanical signals. The outgoing host at Caltech, and the candidate have had success developing a custom-made micro-wells device to mechanically disrupt single plant cells shape. Coupled with mechanical modeling and using a novel software developed by the returning host at the Sainsbury Laboratory, this approach will help to fully develop a computational model of plant cells and tissues morphogenesis, as they respond biologically to changes in directions and amounts of physical stress. The success of this project will have a significant societal impact on improving our understanding of how plants grow, and can grow in agricultural settings.
The past two years we have developed a technique to confine single plant protoplasts into molds of defined shapes. The principle is to confine a plant protoplast expressing fluorescent cytoskeletal reporters into micro-wells of different shapes with sizes of 10 to 30 µm. The protoplasts are then monitored with a confocal microscope to evaluate changes in cytoskeletal organization and dynamics during the process of symmetry breaking. These experiments are the basis of assessing quantitatively how different shapes control cytoskeleton organization behaviour by regulating the distribution of physical stresses (see Figure).
Our recent findings suggested that while the magnitude of local alignment of the actin and microtubules networks are not influenced by shape, the main orientation is dependent on the shape. These results are in agreement with numerical simulations of a 3D self-organizing microtubule network (Mirabet et al, Plos Computational Biology, 2018). Experiments on protoplasts treated with drugs altering the cytoskeleton and on protoplasts with genetic mutations of the cytoskeletal network were also performed. Those experiments will allow us establishing which molecular actors regulate such a response.
Polar transport of the plant hormone auxin is necessary for floral organ initiation. With the design of a new device to apply controlled mechanical forces on plant tissues, we have shown that calcium waves induced by mechanical perturbation are necessary for the correct polarization of PIN1 (one of the plant hormone transporters).
During the past two years, we participated in several international congresses to present this work and always received positive feedback.
Calcium signalling plays a key role in the development of patterning and morphogenesis in early embryos in ascidians, frogs, and zebrafish, but the link underlying calcium signalling and morphogenesis is not known. During this project, we developed a device able to apply localized forces at the shoot apical meristem of plants. We show that such mechanical perturbation is sufficient to induce a calcium wave across the meristem. Moreover we provided a new framework for the function of calcium signals in cellular polarity during organ initiation.
In addition, we designed a specific device able to stretch a single protoplast and measure the applied force under confocal observation. Using this new device, we show for the first time that single plant protoplast responds to stretching and that calcium is released in its cytoplasm at a specific threshold force.
So far, our work on the experimental technique to culture single cells into defined shapes led to new findings on how the cytoskeleton of plant cells reacts to different shapes. We now plan to couple our micro-wells design with a microfluidic device to bring a constant nutrient intake allowing the protoplasts to regenerate their cell wall inside the micro-wells. There is no doubt that the establishment of this new device will be useful for many applications and many walled cell organisms, broadening the impact of this project. We also plan to perform Atomic Force Microscopy (AFM) measurements on regenerating protoplasts inside the wells to quantify the evolution of the cell wall stiffness during this process. By correlating this quantification with microscopic observations we will assess the role of each components of the cell wall in establishing a robust shape.
By the end of the project we should be able to tell whether mechanical stresses directly control cytoskeletal organisation, thus being at the initiation of cell polarisation or whether the cell wall (and which components) is the initiator of the polarisation then creating a specific stress field during growth that influence cytoskeletal organisation.
By adding a cellular level description, these experiments will be the basis of generating models to test differential feedback mechanisms between cell wall, cytoskeleton and physical forces that determine aspects of plant morphogenesis and development.
The original experimental technique that we developed to culture single cells into defined shapes brings technical innovation and could be of use for mechanical studies in others walled cell organisms, which also must respond at the cellular level to mechanical forces, thereby broadening the impact of this project and opening up best career possibilities to the researcher.
By quantifying the role of mechanical stresses in plant cell growth and morphogenesis, we strongly believe that the approach developed in this project will bring new knowledge in plant development. The issues of this research could find numerous applications in developing new agricultural techniques without having to deal with genetically modified organism.
This particular interdisciplinary competence that the researcher will use throughout her future career will have broad scientific and technological benefits for European research with application to a broad range of fields in developmental biology. More precisely, the future research activity of the beneficiary on the role of mechanical signals in plant growth could generate technological innovation in agronomy and agriculture (crop yield, fruits size...) with an impact on the economy of the European society. During her future career, she also plans to work on the role of mechanics in the morphogenesis of other organisms (diatoms, unicellular algae or micro-organisms), which could lay a foundation for biotechnology and biomaterial innovations.
Microscopy picture of the cytoskeleton of plant cells confined in different shapes.