Many cells in diverse organisms need to acquire specific shapes in order to fulfil their functions within the organism. Cells commonly become polarised with one end being asymmetri- cally oriented in a certain direction. Moreover, the polarities of individual cells are often coordinated within the tissue layer. However, the mechanisms underlying the establishment of cell polarity and its coordination in plants are poorly understood. We have introduced the root epidermis of the model plant Arabidopsis thaliana as a system to study cell polarity and its coordination within the plane of the tissue layer (planar polarity). Here, we employ the polar localisation of Rho-of-plant (ROP) proteins to, and the polar outgrowth of root hairs from, near the basal ends of the outer epidermal membrane as polarity markers. Similarly, we use the polar localisation of the PIN2 auxin efflux carrier to apical epidermal membranes as a polarity indicator. By combining forward and reverse genetic, molecular, cell biological and physiological approaches, we have started to unravel mechanisms underlying the coordination and execution of cell polarity in root epidermal cells.

Markus Grebe 1150Planar polarity in the Arabidopsis root epidermis We have established the Arabidopsis root epidermis as a system for the functional dissection of planar polarity of root-hair po- sitioning.We have demonstrated that the combinatorial action of three genes (AUXIN RESISTANT1, AUX1, ETHYLENE INSENSITIVE2, EIN2 and GNOM) is required to coordinate root-hair positioning within the epidermal layer.These genes act to establish a concentration gradient of the plant hormone auxin in the root tip.This auxin gradient helps to instruct the coordinated polar positioning of root hairs by acting prior the polar accumulation of small Rho-of-Plant (ROP) GTPases at the polar site of hair initiation (Fischer et al. 2006).The auxin gradient is modulated by local auxin biosynthesis in the very root tip of young seedlings. Subsequent redistribution of auxin to root epidermal cells is facilitated by the AUX1 auxin influx carrier and the PIN2 auxin efflux mediator and contributes to the coordination of planar epidermal polarity at a distance from the auxin concentration maximum (Ikeda et al. 2009).Yet, the connection to downstream molecules, and the signalling cascade between auxin action and polar ROP or actin recruitment, remain a black box in terms of our understanding.

grebe 2Left panel, green fluorescence of the PIN2 protein fused to enhanced Green-Fluorescent Protein (PIN2-EGFP). Right panel, overlay with filipin-sterol fluorescence (false colour red). Upper row, arrowheads indicate polar PIN2- EGFP localisation only on the upper (apical) membranes of root epidermal cells. Middle row, epidermal cell during division. Lower row, an epidermal cell just after division. Note, both newly formed membranes show PIN2-EGFP fluorescence (Men et al. 2008).We are currently characterising novel planar polarity mu- tants, one of which we have recently shown to be defective in the SABRE (SAB) gene encoding a large, conserved protein of unknown molecular function. SABRE affects the organiza- tion of the microtubule cytoskeleton from cell division plane orientation to planar polarity formation (Pietra et al. 2013). We are currently further addressing the molecular function of SABRE in cytoskeleton organization, cell and planar polarity formation.In the Shape Systems project funded by the Wallenberg foundation, we are, moreover, taking a systems biology approach towards root epidermal cell shape formation.

Sterol function in epidermal cell polarity
In a second line of research, we address the role of membrane sterols at the end of cytokinesis (Boutteé et al. 2010) and during establishment of epidermal cell polarity (Men et al. 2008).The most abundant plant sterol sitosterol is a lipid similar to choles- terol in animals.We have established methods for the subcellular visualisation of plant sterols and their endocytic trafficking in Arabidopsis thaliana (Grebe et al. 2003). My group demonstrat- ed that the correct sterol composition of plant membranes is essential for the establishment of the specific polar localisation of the auxin transporter PIN2, just after cell division. During cell division, PIN2 is integrated into cell-plate membranes and is located at both newly formed membranes directly after cell division. In order to acquire its polar apical localisation, PIN2 needs to be removed from one end of the cell by internalisation (endocytosis) from the membrane.This can only occur when the membrane has the correct sterol composition. In a sterol biosynthesis mutant, which contains a large amount of precursor sterols that normally do not accumulate in the membrane,PIN2 is not appropriately removed from one end of the cell and apical PIN2 polarity cannot be established (Men et al.2008).Moreover, we found that sterol-dependent endocytosis is already needed to focus a major component of vesicle fusion to the cell division plane, the cell plate syntaxin KNOLLE, at the end of cytokinesis (Boutté et al. 2010). We are currently further addressing the sub-cellular mechanisms underlying these phenomena, and the role of sterols during planar polarity formation.
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Publications list

  1. Auxin and ROP GTPase signaling of polar nuclear migration in root epidermal hair cells
    Plant Physiol. 2018,176(1):378-391
  2. Arabidopsis BTB/POZ protein-dependent PENETRATION3 trafficking and disease susceptibility
    Nat Plants. 2017, 3 (11):854-858
  3. Outer, inner and planar polarity in the Arabidopsis root
    Curr Opin Plant Biol. 2018, 41:46-53
  4. Regulating plant physiology with organic electronics
    2017, 114(18):4597-4602
  5. A Model Analysis of Mechanisms for Radial Microtubular Patterns at Root Hair Initiation Sites
    Front Plant Sci. 2016, 7:1560
  6. A framework for lateral membrane trafficking and polar tethering of the PEN3 ATP-binding cassette transporter
    Plant Physiol. 2016, 172 (4):2245-2260
  7. Ratiometric Fluorescence Live Imaging Analysis of Membrane Lipid Order in Arabidopsis Mitotic Cells Using a Lipid Order-Sensitive Probe
    Methods Mol Biol. 2016; 1370:227-39
  8. Arabidopsis D6PK is a lipid domain-dependent
    mediator of root epidermal planar polarity
    Nature Plants 1, (2015), 15162
  9. Arabidopsis AIP1-2 restricted by WER-mediated patterning modulates planar polarity
    Development 2015, 142(1):151-161
  10. Immunocytochemical fluorescent in situ visualization of proteins in Arabidopsis
    Methods Mol Biol. 2014, 1062:453-72
  11. High Lipid Order of Arabidopsis Cell Plate Membranes Mediated by Sterol and DYNAMIN-RELATED PROTEIN1A Function
    Plant J. 2014, 80(5):745-757
  12. SABRE is required for stabilization of root hair patterning in Arabidopsis thaliana
    Physiol Plant. 2015, 153(3):440-453

  13. Sterol dynamics during endocytic trafficking in Arabidopsis
    Methods Mol Biol. 2014, 1209:13-29

  14. Arabidopsis SABRE and CLASP interact to stabilize cell division plane orientation and planar polarity
    Nat. Commun. 2013 15 october (published online)

  15. The Endoplasmic Reticulum Is the Main Membrane Source for Biogenesis of the Lytic Vacuole in Arabidopsis
    Plant Cell. 2013; 25(9):3434-3449

  16. Nakamura M, Kiefer CS, Grebe M
    Planar polarity, tissue polarity and planar morphogenesis in plants
    Curr Opin Plant Biol. 2012 Aug 17. [Epub ahead of print]
  17. Grebe M
    The patterning of epidermal hairs in Arabidopsis-updated
    Curr Opin Plant Biol. 2012 Feb;15(1):31-37
  18. Grebe M
    Plant biology: Unveiling the Casparian strip
    Nature: 2011 473:294-295
  19. Gendre D, Oh J, Boutté Y, Best JG, Samuels L, Nilsson R, Uemura T, Marchant A, Bennett MJ, Grebe M, Bhalerao RP
    Conserved Arabidopsis ECHIDNA protein mediates trans-Golgi-network trafficking and cell elongation
    Proceedings of the National Academy of Sciences of the United States of America: 2011 108:8048-8053
  20. Grebe M
    Out of the shade and into the light
    Nature Cell Biology: 2011 13:347-349
  21. Boutté Y, Men S, Grebe M
    Fluorescent in situ visualization of sterols in Arabidopsis roots
    Nature Protocols: 2011 6:446-456
  22. Pietra S, Grebe M
    Auxin paves the way for planar morphogenesis
    Cell: 2010 143:29-31
  23. Grebe M
    Cell polarity: Lateral perspectives
    Current Biology: 2010 20:R446-R448
  24. Boutté Y, Frescatada-Rosa M, Men S, Chow C-M, Ebine K, Gustavsson A, Johansson L, Ueda T, Moore I, Jürgens G, Grebe M
    Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis

    The EMBO Journal: 2010 29:546-558
  25. Boutté Y, Grebe M
    Cellular processes relying on sterol function in plants
    Current Opinion in Plant Biology: 2009 12:705-713
  26. Petersson SV, Johansson AI, Kowalczyk M, Makoveychuk A, Wang JY, Moritz T, Grebe M, Benfey PN, Sandberg G, Ljung K
    An auxin gradient and maximum in the Arabidopsis root apex shown by high-resolution cell-specific analysis of IAA distribution and synthesis
    The Plant Cell: 2009 21:1659-1668
  27. Ikeda Y, Men S, Fischer U, Stepanova AN, Alonso JM, Ljung K, Grebe M
    Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis
    Nature Cell Biology: 2009 11:731-738
  28. Singh SK, Fischer U, Kumar M, Grebe M, Marchant A
    Insight into the early steps of root hair formation revealed by the procuste1 cellulose synthase mutant of Arabidopsis thaliana
    BMC Plant Biology: 2008 8:57
  29. Men S, BouttéY, Ikeda Y, Li X, Palme K, Stierhof YD, Hartmann MA, Moritz T, Grebe M
    Sterol-dependent endocytosis mediates post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity
    Nature Cell Biology: 2008 10:237-244
  30. BouttéY, Ikeda Y, Grebe M
    Mechanisms of auxin-dependent cell and tissue polarity
    Current Opinion in Plant Biology: 2007 10:616-623
  31. Fischer U, Ikeda Y, Grebe M
    Planar polarity of root hair positioning in Arabidopsis
    Biochemical Society Transaction: 2007 35:149-151
  32. Fischer U, Ikeda Y, Ljung K, Serralbo O, Singh M, Heidstra R, Palme K, Scheres B, Grebe M
    Vectorial information for Arabidopsis planar polarity is mediated by combined AUX1, EIN2, and GNOM activity
    Current Biology: 2006 16:2143-2149
  33. Grebe M
    Acid growth and plant development - Response
    Science: 2006 311:953-954
  34. Friml J, Benfey P, Benkova E, Bennett M, Berleth T, Geldner N, Grebe M, Heisler M, Hejatko J, Jurgens G, Laux T, Lindsey K, Lukowitz W, Luschnig C, Offringa R, Scheres B, Swarup R, Torres-Ruiz R, Weijers D, Zazimalova E
    Apical-basal polarity: why plant cells don't stand on their heads
    Trends in Plant Science: 2006 11:12-14
  35. Villarejo A, Buren S, Larsson S, Dejardin A, Monne M, Rudhe C, Karlsson J, Jansson S, Lerouge P, Rollands N, von Heijne G, Grebe M, Bako L, Samuelsson G
    Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast
    Nature Cell Biology: 2005 7:1224-1231
  36. Grebe M
    Growth by auxin: When a weed needs acid
    Science: 2005 310:60-61
  37. Fischer U, Men S, Grebe M
    Lipid function in plant cell polarity
    Curr Opin Plant Biol: 2004 7:670-676
  38. Grebe M
    Ups and downs of tissue and planar polarity in plants
    Bioessays: 2004 26:719-729
  39. Grebe M, Xu J, Mobius W, Ueda T, Nakano A, Geuze HJ, Rook MB, Scheres B
    Arabidopsis endocytic sterol trafficking involves actin-dependent transport via Rab5-positive early endosomes
    European Journal of Cell Biology: 2003 82:112-112
  40. Grebe M, Xu J, Mobius W, Ueda T, Nakano A, Geuze HJ, Rook MB, Scheres B
    Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes
    Curr Biol: 2003 13:1378-1387
  41. Willemsen V, Friml J, Grebe M, van den Toorn A, Palme K, Scheres B
    Cell polarity and PIN protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1 function
    Plant Cell: 2003 15:612-625
  42. Grebe M, Friml J, Swarup R, Ljung K, Sandberg G, Terlou M, Palme K, Bennett MJ, Scheres B
    Cell polarity signaling in Arabidopsis involves a BFA-sensitive auxin influx pathway
    Curr Biol: 2002 12:329-334
  43. Grebe M, Xu J, Scheres B
    Cell axiality and polarity in plants - adding pieces to the puzzle
    Current Opinion in Plant Biology: 2001 4:520-526
  44. Heese M, Gansel X, Sticher L, Wick P, Grebe M, Granier F, Jurgens G
    Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis
    J Cell Biol: 2001 155:239-249
  45. Grebe M, Gadea J, Steinmann T, Kientz M, Rahfeld JU, Salchert K, Koncz C, Jurgens G
    A conserved domain of the arabidopsis GNOM protein mediates subunit interaction and cyclophilin 5 binding
    Plant Cell: 2000 12:343-356
  46. Steinmann T, Geldner N, Grebe M, Mangold S, Jackson CL, Paris S, Galweiler L, Palme K, Jurgens G
    Coordinated polar localization of auxin efflux carrier PIN1 by GNOM ARF GEF
    Science: 1999 286:316-318
  47. Jurgens G, Grebe M, Steinmann T
    Establishment of cell polarity during early plant development
    Current Opinion in Cell Biology: 1997 9:849-852