Final Report Summary - GREENLATPOL (Mechanisms underlying lateral polarity establishment in plant cells)
Plants like other multicellular organisms establish elaborate body plans with one end of the organism being different from another. Such asymmetric organization along an axis, termed polarity, is already of utmost importance at the single-cell level. Therefore, understanding the mechanisms underlying the establishment of cell polarity is a central theme of biological research. Yet, how cell polarity is established at inner and outer membrane domains of plant cells remains largely unknown. Here, we aimed to identify components needed for polarity at inner and at outer domains of plant cells.
We examined the outermost tissue layer of the root (root epidermis) of the genetic model plant Arabidopsis thaliana to uncover components that contribute to the polarization towards the inner and towards the outer root epidermal membrane domain. The outer membrane domain faces the soil, while the inner membrane domain faces the internal tissue layer. We investigated polar placement of the nucleus towards the inner and its polar migration towards the outer membrane domain as well as the polar localization of the PENETRATION3 (PEN3) protein at the outer membrane domain.
As a first step, we performed a forward-genetic screen to uncover genes required for polar nuclear migration and for outer lateral membrane polarity of the PEN3 protein. We also examined the effects of mutations in candidate genes on these processes. As a result, we identified nine genes contributing to polar nuclear positioning as well as six genes needed for membrane trafficking and/or for polar localization of the PEN3 protein.
As a second step, we examined cytoskeletal and signalling requirements of nuclear migration. This led to the identification of an actin isoform needed for inner and for outer nuclear placement and to the description of the contribution of positive and negative regulators of Rho-of-plant (ROP) small GTPases, that mediate actin organisation, to inner and to outer polar nuclear positioning. We discovered that signalling by the plant hormones ethylene and auxin regulates polar nuclear placement and we established a genetic framework of the factors involved based on multiple mutant analyses. Furthermore, we monitored the velocity and the directionality of polar nuclear migration by live-cell imaging and determined their dependence on the actin cytoskeleton, on the activity of ROP regulators, on ethylene as well as on auxin signalling.
As a step towards an understanding of PEN3 outer membrane domain polarity, we employed reverse genetics to unravel cytoskeletal and membrane trafficking requirements. This revealed the contribution of a regulator of small ARF-GTPases to endoplasmic reticulum-to-Golgi trafficking of the PEN3 protein, the requirement of a regulatory protein of small Rab GTPases and of a factor interacting with Rab-associated proteins for correct routing of the PEN3 protein via the trans-Golgi network (TGN), the requirement of then ACTIN7 protein for PEN3 trafficking from the TGN to the plasma membrane, and the need of the EXO84b exocyst complex tethering factor for polar localization of the PEN3 protein. We generated plants expressing a green-to-red photo-convertible fluorescent protein fusion of the PEN3 protein, allowing us to trace its trafficking in root epidermal cells after a population of PEN3 molecules had been photo-converted to exert red fluorescence. The genetic screen identified a gene of previously unknown function specifically required for trafficking of the PEN3 protein.
Thus, the project established an initial molecular and genetic framework for trafficking of the PEN3 outer membrane domain protein, it revealed a new protein specifically contributing to PEN3 trafficking from the endoplasmic reticulum and it identified a factor required for polar tethering of PEN3 at the outer membrane domain. Moreover, it established an initial genetic framework for hormonal and cytoskeletal control of polar nuclear migration.
We examined the outermost tissue layer of the root (root epidermis) of the genetic model plant Arabidopsis thaliana to uncover components that contribute to the polarization towards the inner and towards the outer root epidermal membrane domain. The outer membrane domain faces the soil, while the inner membrane domain faces the internal tissue layer. We investigated polar placement of the nucleus towards the inner and its polar migration towards the outer membrane domain as well as the polar localization of the PENETRATION3 (PEN3) protein at the outer membrane domain.
As a first step, we performed a forward-genetic screen to uncover genes required for polar nuclear migration and for outer lateral membrane polarity of the PEN3 protein. We also examined the effects of mutations in candidate genes on these processes. As a result, we identified nine genes contributing to polar nuclear positioning as well as six genes needed for membrane trafficking and/or for polar localization of the PEN3 protein.
As a second step, we examined cytoskeletal and signalling requirements of nuclear migration. This led to the identification of an actin isoform needed for inner and for outer nuclear placement and to the description of the contribution of positive and negative regulators of Rho-of-plant (ROP) small GTPases, that mediate actin organisation, to inner and to outer polar nuclear positioning. We discovered that signalling by the plant hormones ethylene and auxin regulates polar nuclear placement and we established a genetic framework of the factors involved based on multiple mutant analyses. Furthermore, we monitored the velocity and the directionality of polar nuclear migration by live-cell imaging and determined their dependence on the actin cytoskeleton, on the activity of ROP regulators, on ethylene as well as on auxin signalling.
As a step towards an understanding of PEN3 outer membrane domain polarity, we employed reverse genetics to unravel cytoskeletal and membrane trafficking requirements. This revealed the contribution of a regulator of small ARF-GTPases to endoplasmic reticulum-to-Golgi trafficking of the PEN3 protein, the requirement of a regulatory protein of small Rab GTPases and of a factor interacting with Rab-associated proteins for correct routing of the PEN3 protein via the trans-Golgi network (TGN), the requirement of then ACTIN7 protein for PEN3 trafficking from the TGN to the plasma membrane, and the need of the EXO84b exocyst complex tethering factor for polar localization of the PEN3 protein. We generated plants expressing a green-to-red photo-convertible fluorescent protein fusion of the PEN3 protein, allowing us to trace its trafficking in root epidermal cells after a population of PEN3 molecules had been photo-converted to exert red fluorescence. The genetic screen identified a gene of previously unknown function specifically required for trafficking of the PEN3 protein.
Thus, the project established an initial molecular and genetic framework for trafficking of the PEN3 outer membrane domain protein, it revealed a new protein specifically contributing to PEN3 trafficking from the endoplasmic reticulum and it identified a factor required for polar tethering of PEN3 at the outer membrane domain. Moreover, it established an initial genetic framework for hormonal and cytoskeletal control of polar nuclear migration.