Final Report Summary - RLK NEGREG (Mechanisms and functions of receptor-like kinase (RLK) negative regulation in plant development.)
In plants, receptor-like kinases (RLKs) form the largest family of membrane receptors (>400) and they are involved in virtually all aspects of the plant life cycle, including development, immunity and reproduction. Although a few model RLKs, such as the steroid receptor BRI1, have been studied extensively, there are still many with unknown function. Besides, we still know very little on how those receptors are activated. We previously proposed a “double lock” activation model for BRI1 that involves the specific recognition of this receptor with a coreceptor at both its extracellular and kinase domain in a ligand dependent manner (Jaillais et al., 2011 PNAS). Critical in this model, is the role in the activation mechanism of a negative regulator called BKI1 that inhibits the interaction between the receptor and co-receptor kinase domain in the absence of ligand (Jaillais et al., 2011 Genes & Dev). Notably, the functions and mechanisms of RLK negative regulation have virtually not been addressed, while it is established that receptor inhibition play a critical role in signalling and diseases in metazoan. During my postdoctoral work, I characterized a negative regulator of BRI1 called BKI1 (for BRI1 KINASE INHIBITOR) and identified a small family of related proteins of unknown function, called MAKRs for MEMBRANE ASSOCIATED KINASE REGULATORs. Our recent results suggest that some MAKR proteins regulate BRI1, while others control different developmental pathways. Additionally, our preliminary data indicate that this protein family is targeted to membranes by electrostatic interactions with anionic phospholipids (e.g. phosphoinositides).
Objectives of the project: In this project we are combining biochemistry, genetics and cell biology to address the mechanisms by which BKI1/MAKRs regulate RLK activity as well as to identify the developmental pathways and receptors targeted by these proteins. We are also investigating the role of anionic phospholipids in the spatial compartmentalization of signalling pathways.
Work performed since the beginning of the project: Since the beginning of the project, I have hired one post-doc, Mar Marques-Bueno, to work on the MAKR2 protein. Mar studied the localization and expression pattern of MAKR2, as well as the phenotype of MAKR2 loss- and gain-of-function. She has also identified candidates MAKR2 interacting proteins by a yeast-two hybrid screening.
I also trained two Ph.D student, Mathilde Simon and Matthieu Platre, who worked on the role of anionic phospholipids in RLK signalling. Mathilde and Matthieu raised a collection of transgenic Arabidopsis lines that express genetically encoded biosensors for a wide variety of anionic lipids (Simon, Platre et al., 2014 Plant Journal). They then used this resource to address the role of these lipids in brassinosteroid signalling and BKI1/MAKR function.
Main results:
Phosphatidylinositolphosphates (PIPs) are phospholipids that contain a phosphorylated inositol head group. PIPs represent a minor fraction of the total phospholipids, yet they are involved in many regulatory processes such as cell signalling and intracellular trafficking. Membrane compartments are enriched or depleted in specific PIPs, which constitute a signature for these compartments and contribute to their identity. The precise subcellular localisation and dynamics of most PIP species is not fully understood in plants. We designed genetically encoded biosensors with distinct relative affinities and expressed them stably in Arabidopsis thaliana (Simon, Platre et al., 2014 Plant Journal). Analysis of this multi-affinity “PIPline” marker set revealed previously unrecognized localisation for various PIPs in root epidermis (http://www.ens-lyon.fr/RDP/SiCE/PIPline.html(se abrirá en una nueva ventana)). Notably, we found that PI(4,5)P2 is able to drive PIP2-interacting protein domains to the plasma membrane in non-stressed root epidermal cells. Our analysis further revealed that there is a gradient of PI4P, with the highest concentration at the plasma membrane, intermediate concentration in post-Golgi/endosomal compartments and lowest concentration in the Golgi. Finally, we also uncovered that there is a similar gradient of PI3P from high in late endosomes to low in the tonoplast. All together our library extends the palette of available PIP biosensors and should promote rapid progress in our understanding of PIP dynamics in plants.
Many signaling proteins permanently or transiently localize to specific organelles for function. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. We found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PI4P) (Simon, Platre et al., In revision). Our results further revealed that, contrarily to other eukaryotes, PI4P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID, as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) family, which are involved in brassinosteroid and receptor-like kinase signaling. We anticipate that this PI4P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition.
In parallel to our work on anionic phospholipids, we established a series of tools for tissue-specific functional genomic in Arabidopsis root (Marques-Bueno et al., 2015 Plant J). This work include the cloning and validation of 30 tissue specific promoters in the root and the building of vectors for cell type specific nucleus purification (INTACT), tissue specific gene induction and bi-cistronic gene expression using Internal Ribosomal Entry Site (IRES). This resource was a collaboration with two laboratories: the group of Gregory Vert at I2BC and François Roudier at IBENS. We have deposited all the plasmids and transgenic lines to the Arabidopsis stock center (amounting to 216 individual stocks in total, http://www.ens- lyon.fr/RDP/SiCE/SWELLINE.html and http://Arabidopsis.info/CollectionInfo?id=156(se abrirá en una nueva ventana)) for fast distribution to the community.
Objectives of the project: In this project we are combining biochemistry, genetics and cell biology to address the mechanisms by which BKI1/MAKRs regulate RLK activity as well as to identify the developmental pathways and receptors targeted by these proteins. We are also investigating the role of anionic phospholipids in the spatial compartmentalization of signalling pathways.
Work performed since the beginning of the project: Since the beginning of the project, I have hired one post-doc, Mar Marques-Bueno, to work on the MAKR2 protein. Mar studied the localization and expression pattern of MAKR2, as well as the phenotype of MAKR2 loss- and gain-of-function. She has also identified candidates MAKR2 interacting proteins by a yeast-two hybrid screening.
I also trained two Ph.D student, Mathilde Simon and Matthieu Platre, who worked on the role of anionic phospholipids in RLK signalling. Mathilde and Matthieu raised a collection of transgenic Arabidopsis lines that express genetically encoded biosensors for a wide variety of anionic lipids (Simon, Platre et al., 2014 Plant Journal). They then used this resource to address the role of these lipids in brassinosteroid signalling and BKI1/MAKR function.
Main results:
Phosphatidylinositolphosphates (PIPs) are phospholipids that contain a phosphorylated inositol head group. PIPs represent a minor fraction of the total phospholipids, yet they are involved in many regulatory processes such as cell signalling and intracellular trafficking. Membrane compartments are enriched or depleted in specific PIPs, which constitute a signature for these compartments and contribute to their identity. The precise subcellular localisation and dynamics of most PIP species is not fully understood in plants. We designed genetically encoded biosensors with distinct relative affinities and expressed them stably in Arabidopsis thaliana (Simon, Platre et al., 2014 Plant Journal). Analysis of this multi-affinity “PIPline” marker set revealed previously unrecognized localisation for various PIPs in root epidermis (http://www.ens-lyon.fr/RDP/SiCE/PIPline.html(se abrirá en una nueva ventana)). Notably, we found that PI(4,5)P2 is able to drive PIP2-interacting protein domains to the plasma membrane in non-stressed root epidermal cells. Our analysis further revealed that there is a gradient of PI4P, with the highest concentration at the plasma membrane, intermediate concentration in post-Golgi/endosomal compartments and lowest concentration in the Golgi. Finally, we also uncovered that there is a similar gradient of PI3P from high in late endosomes to low in the tonoplast. All together our library extends the palette of available PIP biosensors and should promote rapid progress in our understanding of PIP dynamics in plants.
Many signaling proteins permanently or transiently localize to specific organelles for function. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. We found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PI4P) (Simon, Platre et al., In revision). Our results further revealed that, contrarily to other eukaryotes, PI4P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID, as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) family, which are involved in brassinosteroid and receptor-like kinase signaling. We anticipate that this PI4P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition.
In parallel to our work on anionic phospholipids, we established a series of tools for tissue-specific functional genomic in Arabidopsis root (Marques-Bueno et al., 2015 Plant J). This work include the cloning and validation of 30 tissue specific promoters in the root and the building of vectors for cell type specific nucleus purification (INTACT), tissue specific gene induction and bi-cistronic gene expression using Internal Ribosomal Entry Site (IRES). This resource was a collaboration with two laboratories: the group of Gregory Vert at I2BC and François Roudier at IBENS. We have deposited all the plasmids and transgenic lines to the Arabidopsis stock center (amounting to 216 individual stocks in total, http://www.ens- lyon.fr/RDP/SiCE/SWELLINE.html and http://Arabidopsis.info/CollectionInfo?id=156(se abrirá en una nueva ventana)) for fast distribution to the community.