Periodic Reporting for period 4 - CELLONGATE (Unraveling the molecular network that drives cell growth in plants)
Berichtszeitraum: 2023-07-01 bis 2024-08-31
The main aim of the CELLONGATE project was 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.
Within the CELLONGATE project, we aimed 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.
With completing the project, we have established novel methodologies how to analyze and quantify the responses of plant roots to external stimuli. We have discovered a novel perception pathway for the phytohormone auxin – a pathway that does not regulate gene expression, such as the well described nuclear auxin response pathway, but instead controls the ultra-rapid responses of root cells to auxin. This pathway is controlled by an auxin receptor called AFB1 that localizes to the cytoplasm of root cells. The ability of roots to respond to auxin very rapidly is crucial for the correct navigation of roots in the dark environment of soil. Furthermore, we have established novel methods to monitor how the roots change the acidity of the rhizosphere, and dissected the pathway that controls this process in the root tips of the model plant Arabidopsis thaliana. Finally, we have determined the gene expression changes by which the various auxin perception pathways control the elongation and zonation of root cells.
Within the CELLONGATE project, we aimed 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.
With completing the project, we have established novel methodologies how to analyze and quantify the responses of plant roots to external stimuli. We have discovered a novel perception pathway for the phytohormone auxin – a pathway that does not regulate gene expression, such as the well described nuclear auxin response pathway, but instead controls the ultra-rapid responses of root cells to auxin. This pathway is controlled by an auxin receptor called AFB1 that localizes to the cytoplasm of root cells. The ability of roots to respond to auxin very rapidly is crucial for the correct navigation of roots in the dark environment of soil. Furthermore, we have established novel methods to monitor how the roots change the acidity of the rhizosphere, and dissected the pathway that controls this process in the root tips of the model plant Arabidopsis thaliana. Finally, we have determined the gene expression changes by which the various auxin perception pathways control the elongation and zonation of root cells.
To achieve the goals of the project, we have built a microscopy setup that enabled us to analyze the dynamics of the reaction of Arabidopsis roots to stimuli in an unprecedented 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 without disturbing the growing Arabidopsis roots. In addition, we have established novel approaches to quantitatively analyze the behavior of roots using advanced image analysis methods. We have published the description of the microscopy setup as a part of a scientific publication (Serre et al., 2021, Nature Plants 7: 1229), and we have described the image analysis program called ACORBA in another publication (Serre et al., 2022, Quantitative Plant Biology 3, e9).
To visualize the dynamics of cellular process in real time, we optimized the usage of genetically-encoded fluorescent sensors that monitor cellular physiology, such as hormone levels, ion concentrations and pH of cellular compartments. This toolbox enabled us to analyze the earliest events by which Arabidopsis roots response to the phytohormone auxin. We introduced a novel tool to quantify the potassium levels in the cytoplasm (Wu et al., 2022, PLoS biology 20, e3001772). We have further visualized and quantified the ability of roots to acidify the rhizosphere and we have identified the pathway that controls the longitudinal zonation of acidification in the Arabidopsis root tip (Serre et al., 2023, eLife 12, e85193).
Further, we have focused on revealing the signaling pathway that underlies the ultra-rapid responses of Arabidopsis roots to the phytohormone auxin. We have established that one of the auxin receptors called AFB1 triggers rapid membrane depolarization in root cells, and that this response is required for an efficient response of root to the change in gravity direction (Serre et al., 2021, Nature Plants 7: 1229). In a follow-up publication, we have discovered that the AFB1 receptor functions in a fundamentally different manner from the auxin receptors described so far. The AFB1 receptor perceives auxin in the cytoplasm of root cells and triggers changes in transmembrane ion fluxes, characterized by rapid changes in cytoplasmic calcium concentrations (Dubey et al., 2023, Molecular Plant 16, 1120).
In another line of research, we aimed to dissect the genetic network that underlies the growth decisions that condition the ability of the root to navigate in the heterogenous environments of the soil. To achieve this, we have established a specific system to speed up and slow down root elongation using a genetical trick to modulate the auxin gene transcription pathway. We harnessed this system to identify genes responsible for steering the cell expansion dynamics in the root elongation zone of Arabidopsis thaliana (Kubalova et al., 2024, New Phytologist 241, 2448).
Finally, we contributed to a major study that demonstrated that an ultra-rapid auxin response pathway is deeply conserved over the plant kingdom (Kuhn et al., 2024, Cell 187, 130). This finding underscores the importance of studying the dynamics of auxin responses. In addition to peer-reviewed scientific publications, the results were presented at numerous international conferences and invited seminars at scientific institutions. The results were also communicated towards the general public using press releases and interviews for media in Czechia.
To visualize the dynamics of cellular process in real time, we optimized the usage of genetically-encoded fluorescent sensors that monitor cellular physiology, such as hormone levels, ion concentrations and pH of cellular compartments. This toolbox enabled us to analyze the earliest events by which Arabidopsis roots response to the phytohormone auxin. We introduced a novel tool to quantify the potassium levels in the cytoplasm (Wu et al., 2022, PLoS biology 20, e3001772). We have further visualized and quantified the ability of roots to acidify the rhizosphere and we have identified the pathway that controls the longitudinal zonation of acidification in the Arabidopsis root tip (Serre et al., 2023, eLife 12, e85193).
Further, we have focused on revealing the signaling pathway that underlies the ultra-rapid responses of Arabidopsis roots to the phytohormone auxin. We have established that one of the auxin receptors called AFB1 triggers rapid membrane depolarization in root cells, and that this response is required for an efficient response of root to the change in gravity direction (Serre et al., 2021, Nature Plants 7: 1229). In a follow-up publication, we have discovered that the AFB1 receptor functions in a fundamentally different manner from the auxin receptors described so far. The AFB1 receptor perceives auxin in the cytoplasm of root cells and triggers changes in transmembrane ion fluxes, characterized by rapid changes in cytoplasmic calcium concentrations (Dubey et al., 2023, Molecular Plant 16, 1120).
In another line of research, we aimed to dissect the genetic network that underlies the growth decisions that condition the ability of the root to navigate in the heterogenous environments of the soil. To achieve this, we have established a specific system to speed up and slow down root elongation using a genetical trick to modulate the auxin gene transcription pathway. We harnessed this system to identify genes responsible for steering the cell expansion dynamics in the root elongation zone of Arabidopsis thaliana (Kubalova et al., 2024, New Phytologist 241, 2448).
Finally, we contributed to a major study that demonstrated that an ultra-rapid auxin response pathway is deeply conserved over the plant kingdom (Kuhn et al., 2024, Cell 187, 130). This finding underscores the importance of studying the dynamics of auxin responses. In addition to peer-reviewed scientific publications, the results were presented at numerous international conferences and invited seminars at scientific institutions. The results were also communicated towards the general public using press releases and interviews for media in Czechia.
We established a unique combination of advanced microscopy methods which allowed us to monitor the behavior of roots and the reaction of root cells to external stimuli. We harnessed this system to discover a novel signaling pathway that the plant Arabidopsis thaliana uses to perceive and respond to the phytohormone auxin in an ultra-rapid manner. We have shown that this auxin signaling involves the change of ion fluxes across the plasma membrane of root cells. We have demonstrated that the ability to respond to auxin in a rapid manner is required for the efficient gravitropic reaction of root cells. We further clarified a signaling pathway that controls how the root tips acidify the rhizosphere, which is important for both the nutrient acquisition from soil, and the efficient navigation of the root through the complex soil environment. Finally, we have revealed the gene network that the auxin signaling machinery uses to control the rate of cell expansion in growing roots. The results of the CELLONGATE project significantly advanced the understanding of how plant roots regulate the growth of their cells, and added a novel perception pathway for one of the most important growth regulators of plants – the phytohormone auxin.