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Unraveling the molecular network that drives cell growth in plants

Periodic Reporting for period 2 - CELLONGATE (Unraveling the molecular network that drives cell growth in plants)

Reporting period: 2020-07-01 to 2021-12-31

The main aim of the CELLONGATE project is to reveal the cellular and molecular mechanisms of how plants control the growth of their cells. Plant cells build plant bodies using a hydrostatic skeleton – pressurized cells surrounded by cell walls, composite structures of high tensile strength that counterbalance the high intracellular turgor pressure. As a consequence, plant cells cannot move inside plant bodies. Therefore, the formation of the astonishing spectrum of plant shapes and patterns relies on precise control of how and where cells divide and how much and in what direction the cells grow. Cell elongation also underlies movements, by which plants optimize the positions of their organs towards gravity, light, water and nutrients. The phytohormone auxin is a central regulator of plant development, growth and plant movements. The application of auxin to plant cells triggers very rapid changes in cell elongation that are caused by modification of cell wall properties, fluxes of ions across membranes and cellular dynamics. We currently do not fully understand how auxin achieves all of its roles.
Within the CELLONGATE project, we aim to reveal the molecular and cellular mechanisms by which auxin controls cellular elongation in the roots of the model plant Arabidopsis thaliana. Understanding these mechanisms will reveal the core machinery that plant cells use for controlling cellular expansion, which in turn will help us understand how plants shape their bodies and move their organs. Mechanistic understanding of plant growth and development is a crucial prerequisite for the ability to customize plant phenotypes for the need of agriculture, which stands on the shoulders of plants as primary producers.
To achieve the goals of the project, we have assembled a microscopy setup that enables us to analyze the dynamics of the reaction of Arabidopsis roots to stimuli, such as auxin treatments, gravitropic stimulus or stresses, in a high spatio-temporal resolution. This setup consists of a spinning disk fluorescence microscope with a vertical stage – where the roots can grow downwards, combined with a microfluidic chip platform – where we can perform various treatments in liquid media without disturbing the growing Arabidopsis roots.
In order to be able to visualize the dynamics of cellular process in real time, we are optimizing the usage of existing fluorescent sensors and we are preparing novel genetically-encoded fluorescent sensors that monitor cellular physiology, such as hormone levels, ion concentrations and pH of cellular compartments. This toolbox, together with the microscopy setup, enabled us to analyze the earliest events by which Arabidopsis roots response to the phytohormone auxin.
In parallel, we use protein-protein interaction methods, proteomic and transcriptomic approaches to identify the molecular chain of events that connects the perception of the phytohormone auxin by the TIR1/AFB1 receptors with the early cellular responses such as membrane depolarization and cell wall pH changes. By this approach, we obtained numerous potentially interesting proteins and we are further testing their involvement in cell growth regulation and auxin signaling. We also analyze the role of the known molecular players in cell growth regulation and auxin responses using the methodological approaches that we have established.
This unique combination of approaches allowed us to discover that the phytohormone auxin causes very rapid depolarization of membranes of Arabidopsis root cells that is controlled specifically by one of the known receptors called AFB1, and that similar events occur during gravitropic bending of Arabidopsis roots; we have published the results in 2021 in the Nature Plants journal.
We expect that we will uncover the nature of the signaling that underlies rapid auxin responses, we want to understand the subcellular and tissue localization of auxin signaling hotspots. We expect to reveal unknown proteins that regulate growth processes downstream of auxin signaling. We also plan to shed more light onto the spatial and temporal dynamics of cell wall and cell surface pH regulation and ion fluxes dynamics that steers the elongation of plant root cells.
Elongating cells of Arabidopsis root highlighted by membrane potential dye