- Email: [email protected]
Plant Cell Biology: How to Give Root Hairs Enough ROPs?
Current Biology
Dispatches another colour to the visual image? That is highly unlikely. Snakes express three opsin genes in their retina, UV-sensitive and long wavelength-sensitive cone opsins and rod opsin [14]. Interactions between cone and rod signals occur as early as the retina in vertebrates, and a nocturnal snake uses colour-blind rod vision at night, when the pit organ is of most use. Therefore, it seems much more likely that the images generated by the two sensory modalities — the thermal image and the visual image — are overlaid as two separate spatial representations of the snake’s prey. Which of the two is more important seems to differ between different species: snakes with larger eyes have smaller pit organs and vice versa [15], but the combination of both sensory modalities improves the hunting success [16]. While the visual and infrared signals are likely analyzed separately, the neural processing of the thermal image seems to have evolved to be surprisingly similar to retinal mechanisms. The new findings by Bothe et al. [3] have made a big step towards understanding the neural mechanisms underlying the amazing ability of the pit vipers to strike accurately even in total darkness.
REFERENCES 1. Newman, E.A., and Hartline, P.H. (1982). The infrared ‘‘vision’’ of snakes. Sci. Am. 246, 116–127. 2. Hartline, P.H., Kass, L., and Loop, M.S. (1978). Merging of modalities in the optic tectum: infrared and visual integration in rattlesnakes. Science 199, 1225–1229. 3. Bothe, M.S., Luksch, H., Straka, H., and Kohl, T. (2019). Neuronal substrates for infrared contrast enhancement and motion detection in rattlesnakes. Curr. Biol. 29, 1827– 1832. 4. Ros, M. (1935). Die Lippengruben der Pythonen als Temperaturorgane. Jenaische Zeitschrift fuer Naturwissenschaft 70, 1–32. 5. Noble, G.K., and Schmidt, A. (1937). The structure and function of the facial and labial pits of snakes. Proc. Am. Philos. Soc. 77, 263–288. 6. Krochmal, A.R., and Bakken, G.S. (2003). Thermoregulation is the pits- use of thermal radiation for retreat site selection by rattlesnakes. J. Exp. Biol. 206, 2539–2545. 7. Chen, Q., Liu, Y., Brauth, S.E., Fang, G., and Tang, Y. (2017). The thermal background determines how the infrared and visual systems interact in pit vipers. J. Exp. Biol. 220, 3103–3109. 8. Bothe, M.S., Luksch, H., Straka, H., and Kohl, T. (2018). Synaptic convergence of afferent inputs in primary infrared-sensitive nucleus (LTTD) neurons of rattlesnakes (Crotalinae) as the origin for sensory contrast enhancement. J. Exp. Biol. 221, jeb185611.
9. Hartline, H.K., Wagner, H.G., and Ratliff, F. (1956). Inhibition in the eye of Limulus. J. Gen. Psychol. 39, 651–671. 10. Barlow, H.B., Fitzhugh, R., and Kuffler, S.W. (1957). Change in organisation in the receptive fields of the cat’s retina during dark adaptation. J. Physiol. 137, 338–354. ke sy, G. (1960). Neural inhibitory units of the 11. Be eye and skin. Quantitative description of contrast phenomena. J. Opt. Soc. Am. 50, 1060–1070. 12. Hassenstein, B., and Reichardt, W. (1956). Systemtheoretische Analyse der Zeit, Reihenfolgen, und Vorzeichenauswertung bei €fers. der Bewegungsperzepion des Ru¨sselka Chlorophanus. Naturforsch. 11b, 513–524. 13. Borst, A., and Helmstaedter, M. (2015). Common circuit design in fly and mammalian motion vision. Nat. Neurosci. 18, 1067–1076. 14. Simo˜es, B.F., Sampaio, F.L., Douglas, R.H., Kodandaramaiah, U., Casewell, N.R., Harrison, R.A., Hart, N.S., Partridge, J.C., Hunt, D.M., and Gower, D.J. (2016). Visual pigments, ocular filters and the evolution of snake vision. Mol. Biol. Evol. 33, 2483–2495. 15. Liu, Y., Chen, Q., Papenfuss, T.J., Lu, F., and Tang, Y. (2016). Eye and pit size are inversely correlated in Crotalinae: implications for selection pressure relaxation. J. Morphol. 277, 107–117. 16. Chen, Q., Liu, Y., Brauth, S.E., Fang, G., and Tang, Y. (2017). The thermal background determines how the infrared and visual systems interact in pit vipers. J. Exp. Biol. 220, 3103–3109.
Plant Cell Biology: How to Give Root Hairs Enough ROPs? Thomas Stanislas1 and Yvon Jaillais2,* 1Center
for Plant Molecular Biology (ZMBP), University of Tu¨bingen, Auf der Morgenstelle 32, 72076, Tu¨bingen, Germany veloppement des Plantes, Universite de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, Lyon 69342, France Reproduction et De *Correspondence: [email protected] https://doi.org/10.1016/j.cub.2019.04.023
2Laboratoire
Root hairs are precisely positioned close to the rootward end of epidermal cells. A new study shows that the successful production of root hairs is a two-step process with different molecular players driving the initial cell polarization and subsequent hair outgrowth.
Planar polarity is the coordination of cell polarity across a two-dimensional tissue layer [1]. Two classical examples of planar polarity are the polar emergence of hairs on epidermal cells of the
Drosophila wing and the Arabidopsis root [1,2]. However, the establishment of hair planar polarity is regulated by distinct mechanisms in animals and plants. In animals, the core planar
polarity pathway (also known as the Frizzled pathway) interprets positional cues and then positions and regulates Rho GTPases, which execute downstream cellular effects [1]. Plant
Current Biology 29, R403–R424, June 3, 2019 ª 2019 Elsevier Ltd. R405
Current Biology
Dispatches Polar growth
Epidermis
+3
Bulging
+3 +2 RLK (FER) Exocyst (SEC3A)
FAB1
Exocytosis (SYP123) +1 Actin (ARPC2) GEF4 Calcium
Initiation
-1
PIP5K3 / PI(4,5)P2 ROS (RBOHC)
PI(3,5)P2
ROP2 PI(4,5)P2
PIP5K3
Root hair
ROP
Synthesis
ER
-2 ROP
Prenylation (lipid anchor) GDP (inactive) ROP ROP
Prenylation TGN
-3 -4
Golgi
-4
Root tip
GEF4
ROP10
ROP2 ROP4 ROP6 -5 GEF3 -6 -7 First recruitment at the RHID
ROP
YIP4
ROP
ROPGTP (active)
GEF3 Root hair initiation ROP domain (RHID)
Current Biology
Figure 1. Successive phases in root hair initiation and outgrowth. Left, schematic drawing of an Arabidopsis root. Middle, root hair staging and timeline of recruitment of polar proteins to the root hair initiation domain (RHID, in red). Top right, interplay between ROP, anionic lipids and lipid kinases during root hair growth. Bottom right, the recruitment of ROPs at the RHID requires the presence of GEF3 [3]. Note that ROPs are synthesized as soluble proteins in the cytosol. They are then prenylated at the surface of the endoplasmic reticulum (ER) and are subsequently targeted to the plasma membrane via YIP4-dependent secretion from the TGN [6]. Root drawing by Benjamin Peret.
genomes lack the Frizzled pathway, yet Rho-of-plants (ROPs) proteins are polarized early at the root hair initiation domain (RHID), which is positioned close to the distal (rootward) end of hair-forming cells (Figure 1) [2]. How ROP proteins are precisely positioned at the RHID in the absence of the core planar polarity pathway is still unclear. A new study in this issue of Current Biology tackled this question and uncovered ROP GEF3 as a key protein driving the initial ROP polarization for the correct positioning of root hairs [3]. ROPs are highly conserved small GTPases, which function as molecular switches due to conformational differences between an inactive GDPbound state and an active GTP-bound state. These activation–inactivation cycles are regulated in time and space by guanine nucleotide exchange factors (GEFs) that promote the GTP-bound state and GTPase-activating proteins (GAPs) that enhance GTP hydrolysis [4].
The Arabidopsis thaliana genome encodes 11 ROPs, which control fundamental cellular processes, including cytoskeleton dynamics and vesicular trafficking [5]. To determine the timing of ROP polarization at the RHID, Denninger, Reichelt et al. took advantage of the fact that cells along the root tip are ordered according to their developmental age, with younger cells located rootward and older cells shootward [3]. In this developmental timeline, the cell with the first visible root hair bulge is numbered as +1 (Figure 1). Using this staging system, the authors separated root hair formation into three phases: initiation (from cells -7 to -1, in blue), bulging (cells +1 and +2, in green), and polar growth/elongation (starting at cell +3 and onward, in pink) [3]. Using confocal imaging and fluorescently-tagged ROPs, Denninger, Reichelt et al. showed that three ROPs (ROP2, ROP4 and ROP6) polarize at the RHID much before root hair initiation (i.e., at cell -4
R406 Current Biology 29, R403–R424, June 3, 2019
before root hair bulging; Figure 1). Accordingly, genetic analyses with double and triple rop mutants suggest that these three ROPs redundantly regulate root hair development [3,6]. Strikingly, all the known downstream or upstream ROP regulators polarize much later, at the stage of root hair bulging or after (Figure 1) [3]. This suggests a two-phase assembly of the tip growth machinery — an initiation phase, during which the RHID is positioned and predefined, followed by the tip growth phase. To uncover whether GEF could participate in ROP membrane targeting, Denninger, Reichelt et al. localized, via translational fusion reporters, all the GEF that are expressed in the root epidermis (6 GEFs in total). This analysis uncovered two GEFs (GEF3, GEF4 and to a lesser extent also GEF12 and GEF14) with polar localization in root hair cells. Interestingly, GEF3 polarizes early to the RHID, while GEF4 localizes at the root hair initiation site during bulging. gef3 and gef4 mutant analyses confirmed that gef3 is involved in root hair initiation, while gef4 is involved in root hair polar growth, suggesting that distinct GEFs are responsible for polar initiation and polarized growth, respectively. Elegant live imaging analyses using microfluidics suggested that GEF3 localizes to the RHID prior to ROP proteins (i.e., at cell -5) and could therefore be an early landmark for root hair positioning acting upstream of ROPs [3]. Biochemical interactions, structure–function analyses and over-expression studies validated that GEF3 polarizes first to the RHID and in turn recruits ROP proteins to this polar domain. Strikingly, upon ectopic expression in cells that do not normally form hairs (in the root and the hypocotyl) GEF3 accumulates into plasma membrane domains that resembled those of the RHID. Ectopic accumulation of GEF3 in these RHID-like domains subsequently drove the ectopic ROP2 localization in the same membrane patches.
Current Biology
Dispatches Together, the GEF3–ROP interplay appears critical to position the RHID, which is later translated into root hair tip growth via the recruitment of GEF4. Meanwhile, Gendre and collaborators studied the mechanisms and factors underlying ROP localization at the root hair plasma membrane with a focus on membrane trafficking [6]. They demonstrated that the membrane trafficking machinery acts as a central component in ROP-dependent root hair formation. Indeed, the trans-Golgi Network (TGN)-localized YPTINTERACTING PROTEIN (YIP)4a and YIP4b are required for the activation and plasma membrane accumulation of ROPs at the RHID (Figure 1). Another important factor that regulates ROP localization and signaling activity is its interaction with lipids. ROP2, 4 and 6 carboxyl termini are prenylated (i.e., geranylgeranylation, a covalently linked lipid anchor), which provides hydrophobic anchoring and is required for membrane association [5]. ROP6 is also S-acylated (i.e., palmitoylation), which is a reversible lipid modification required for ROP6 function [7]. Furthermore, all ROPs possess in their carboxy-terminal tail a polybasic region, which interacts with anionic phospholipids, at least in the case of ROP6 [8]. The plasma membrane is highly enriched in anionic phospholipids and hence is strongly electronegative [9,10]. Consequently, ROP6 polybasic tail interacts with this plasma membrane electrostatic field and is required for the proper targeting of ROP to the cell surface [8]. At the bulging stage, the RHID is enriched in the anionic phospholipid PI(4,5)P2, and ROP2 colocalizes and interacts with PIP5K3, the kinase that produces PI(4,5)P2 [3,11,12] (Figure 1). Interestingly, this situation is not restricted to ROP2, but also ROP10, which localizes in a complementary domain at the shank of growing root hairs [11] (Figure 1). ROP10 colocalizes and interacts with both the anionic phospholipids PI(3,5)P2 and the lipid kinase that produces it (i.e., FAB1; Figure 1) [11]. This suggests that ROP activity may regulate lipid synthesis, while in turn lipids themselves control ROP activity and/or localization. Therefore, ROP and anionic lipids may
form a self-organizing system for the focal recruitment and activation of Rho GTPases in plants. However, given that PIP5K3 and PI(4,5)P2 polarize relatively late at the RHID (Figure 1) [3], it is likely that they act as an amplifying system rather than the initial polarizing cue. Taken together, this evidence suggests that ROP localization and focal activation in root hairs may require multiple factors, including enzymatic activation, lipid modification, direct interaction with lipids and vesicular trafficking. An open question is to understand what is/are the primary signal(s) that defines the position of the RHID; that is, what are the factors that initially drive GEF3 polarity? In other contexts, ROP GEFs act downstream of receptorlike kinases (RLK), which are membrane receptors able to translate signal(s) across the plasma membrane [5]. One may speculate that there are root hair-expressed receptors that localize at the RHID. Alternatively, such receptors would not necessarily have to be polarized themselves but could rather perceive a polarized signal. Such a signal could include the concentration gradient of the plant hormone auxin at the root tip, which is critical to determine ROP localization at the RHID [13]. In addition, auxin promotes ROP2 and ROP6 activation [14], but the machinery that perceives and transduces the auxin signal across the plasma membrane is still elusive [15]. One of the future challenges in understanding planar cell polarity in plants will be to elucidate how ROP polarization is linked with extracellular signals, such as auxin.
REFERENCES 1. Goodrich, L.V., and Strutt, D. (2011). Principles of planar polarity in animal development. Development 138, 1877–1892. 2. Nakamura, M., Kiefer, C.S., and Grebe, M. (2012). Planar polarity, tissue polarity and planar morphogenesis in plants. Curr. Opin. Plant Biol. 15, 593–600. 3. Denninger, P., Reichelt, A., Schmidt, V.A.F., Mehlhorn, D.G., Asseck, L.Y., Stanley, C.E., Keinath, N.F., Evers, J.-F., Grefen, C., and Grossmann, G. (2019). Distinct RopGEFs successively drive polarization and
outgrowth of root hairs. Curr. Biol. 29, 1854– 1865. 4. Berken, A., and Wittinghofer, A. (2008). Structure and function of Rho-type molecular switches in plants. Plant Physiol. Biochem. 46, 380–393. 5. Feiguelman, G., Fu, Y., and Yalovsky, S. (2018). ROP GTPases structure-function and signaling pathways. Plant Physiol. 176, 57–79. 6. Gendre, D., Baral, A., Dang, X., Esnay, N., Boutte, Y., Stanislas, T., Vain, T., Claverol, S., Gustavsson, A., Lin, D., et al. (2019). Rho-ofplant-activated root hair formation requires Arabidopsis YIP4a/b gene function. Development 146, dev168559. 7. Sorek, N., Segev, O., Gutman, O., Bar, E., Richter, S., Poraty, L., Hirsch, J.A., Henis, Y.I., Lewinsohn, E., Jurgens, G., et al. (2010). An S-acylation switch of conserved G domain cysteines is required for polarity signaling by ROP GTPases. Curr. Biol. 20, 914–920. 8. Platre, M.P., Bayle, V., Armengot, L., Bareille, J., Marques-Bueno, M.M., Creff, A., ManetaPeyret, L., Fiche, J.B., Nolmann, M., Mie`ge, C., et al. (2019). Developmental control of plant Rho GTPase nano-organization by the lipid phosphatidylserine. Science 364, 57–62. 9. Platre, M.P., Noack, L.C., Doumane, M., Bayle, V., Simon, M.L.A., Maneta-Peyret, L., Fouillen, L., Stanislas, T., Armengot, L., Pejchar, P., et al. (2018). A combinatorial lipid code shapes the electrostatic landscape of plant endomembranes. Dev. Cell 45, 465–480, e411. 10. Simon, M.L., Platre, M.P., Marques-Bueno, M.M., Armengot, L., Stanislas, T., Bayle, V., Caillaud, M.C., and Jaillais, Y. (2016). A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants. Nat. Plants 2, 16089. 11. Hirano, T., Konno, H., Takeda, S., Dolan, L., Kato, M., Aoyama, T., Higaki, T., TakigawaImamura, H., and Sato, M.H. (2018). PtdIns(3,5)P2 mediates root hair shank hardening in Arabidopsis. Nat. Plants 4, 888–897. 12. Stanislas, T., Hu¨ser, A., Barbosa, I.C., Kiefer, P., Brackmann, K., Pitra, S., Gustavsson, A., Zourelidou, M., Schwechheimer, C., and Grebe, M. (2015). Arabidopsis D6PK is a lipid domain-dependent mediator of root epidermal planar polarity. Nat. Plants 1, 15162. 13. Ikeda, Y., Men, S., Fischer, U., Stepanova, A.N., Alonso, J.M., Ljung, K., and Grebe, M. (2009). Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis. Nat. Cell Biol. 11, 731–738. 14. Xu, T., Wen, M., Nagawa, S., Fu, Y., Chen, J.G., Wu, M.J., Perrot-Rechenmann, C., Friml, J., Jones, A.M., and Yang, Z. (2010). Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis. Cell 143, 99–110. 15. Armengot, L., Marques-Bueno, M.M., and Jaillais, Y. (2016). Regulation of polar auxin transport by protein and lipid kinases. J. Exp. Bot. 67, 4015–4037.
Current Biology 29, R403–R424, June 3, 2019 R407
Dispatches another colour to the visual image? That is highly unlikely. Snakes express three opsin genes in their retina, UV-sensitive and long wavelength-sensitive cone opsins and rod opsin [14]. Interactions between cone and rod signals occur as early as the retina in vertebrates, and a nocturnal snake uses colour-blind rod vision at night, when the pit organ is of most use. Therefore, it seems much more likely that the images generated by the two sensory modalities — the thermal image and the visual image — are overlaid as two separate spatial representations of the snake’s prey. Which of the two is more important seems to differ between different species: snakes with larger eyes have smaller pit organs and vice versa [15], but the combination of both sensory modalities improves the hunting success [16]. While the visual and infrared signals are likely analyzed separately, the neural processing of the thermal image seems to have evolved to be surprisingly similar to retinal mechanisms. The new findings by Bothe et al. [3] have made a big step towards understanding the neural mechanisms underlying the amazing ability of the pit vipers to strike accurately even in total darkness.
REFERENCES 1. Newman, E.A., and Hartline, P.H. (1982). The infrared ‘‘vision’’ of snakes. Sci. Am. 246, 116–127. 2. Hartline, P.H., Kass, L., and Loop, M.S. (1978). Merging of modalities in the optic tectum: infrared and visual integration in rattlesnakes. Science 199, 1225–1229. 3. Bothe, M.S., Luksch, H., Straka, H., and Kohl, T. (2019). Neuronal substrates for infrared contrast enhancement and motion detection in rattlesnakes. Curr. Biol. 29, 1827– 1832. 4. Ros, M. (1935). Die Lippengruben der Pythonen als Temperaturorgane. Jenaische Zeitschrift fuer Naturwissenschaft 70, 1–32. 5. Noble, G.K., and Schmidt, A. (1937). The structure and function of the facial and labial pits of snakes. Proc. Am. Philos. Soc. 77, 263–288. 6. Krochmal, A.R., and Bakken, G.S. (2003). Thermoregulation is the pits- use of thermal radiation for retreat site selection by rattlesnakes. J. Exp. Biol. 206, 2539–2545. 7. Chen, Q., Liu, Y., Brauth, S.E., Fang, G., and Tang, Y. (2017). The thermal background determines how the infrared and visual systems interact in pit vipers. J. Exp. Biol. 220, 3103–3109. 8. Bothe, M.S., Luksch, H., Straka, H., and Kohl, T. (2018). Synaptic convergence of afferent inputs in primary infrared-sensitive nucleus (LTTD) neurons of rattlesnakes (Crotalinae) as the origin for sensory contrast enhancement. J. Exp. Biol. 221, jeb185611.
9. Hartline, H.K., Wagner, H.G., and Ratliff, F. (1956). Inhibition in the eye of Limulus. J. Gen. Psychol. 39, 651–671. 10. Barlow, H.B., Fitzhugh, R., and Kuffler, S.W. (1957). Change in organisation in the receptive fields of the cat’s retina during dark adaptation. J. Physiol. 137, 338–354. ke sy, G. (1960). Neural inhibitory units of the 11. Be eye and skin. Quantitative description of contrast phenomena. J. Opt. Soc. Am. 50, 1060–1070. 12. Hassenstein, B., and Reichardt, W. (1956). Systemtheoretische Analyse der Zeit, Reihenfolgen, und Vorzeichenauswertung bei €fers. der Bewegungsperzepion des Ru¨sselka Chlorophanus. Naturforsch. 11b, 513–524. 13. Borst, A., and Helmstaedter, M. (2015). Common circuit design in fly and mammalian motion vision. Nat. Neurosci. 18, 1067–1076. 14. Simo˜es, B.F., Sampaio, F.L., Douglas, R.H., Kodandaramaiah, U., Casewell, N.R., Harrison, R.A., Hart, N.S., Partridge, J.C., Hunt, D.M., and Gower, D.J. (2016). Visual pigments, ocular filters and the evolution of snake vision. Mol. Biol. Evol. 33, 2483–2495. 15. Liu, Y., Chen, Q., Papenfuss, T.J., Lu, F., and Tang, Y. (2016). Eye and pit size are inversely correlated in Crotalinae: implications for selection pressure relaxation. J. Morphol. 277, 107–117. 16. Chen, Q., Liu, Y., Brauth, S.E., Fang, G., and Tang, Y. (2017). The thermal background determines how the infrared and visual systems interact in pit vipers. J. Exp. Biol. 220, 3103–3109.
Plant Cell Biology: How to Give Root Hairs Enough ROPs? Thomas Stanislas1 and Yvon Jaillais2,* 1Center
for Plant Molecular Biology (ZMBP), University of Tu¨bingen, Auf der Morgenstelle 32, 72076, Tu¨bingen, Germany veloppement des Plantes, Universite de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, Lyon 69342, France Reproduction et De *Correspondence: [email protected] https://doi.org/10.1016/j.cub.2019.04.023
2Laboratoire
Root hairs are precisely positioned close to the rootward end of epidermal cells. A new study shows that the successful production of root hairs is a two-step process with different molecular players driving the initial cell polarization and subsequent hair outgrowth.
Planar polarity is the coordination of cell polarity across a two-dimensional tissue layer [1]. Two classical examples of planar polarity are the polar emergence of hairs on epidermal cells of the
Drosophila wing and the Arabidopsis root [1,2]. However, the establishment of hair planar polarity is regulated by distinct mechanisms in animals and plants. In animals, the core planar
polarity pathway (also known as the Frizzled pathway) interprets positional cues and then positions and regulates Rho GTPases, which execute downstream cellular effects [1]. Plant
Current Biology 29, R403–R424, June 3, 2019 ª 2019 Elsevier Ltd. R405
Current Biology
Dispatches Polar growth
Epidermis
+3
Bulging
+3 +2 RLK (FER) Exocyst (SEC3A)
FAB1
Exocytosis (SYP123) +1 Actin (ARPC2) GEF4 Calcium
Initiation
-1
PIP5K3 / PI(4,5)P2 ROS (RBOHC)
PI(3,5)P2
ROP2 PI(4,5)P2
PIP5K3
Root hair
ROP
Synthesis
ER
-2 ROP
Prenylation (lipid anchor) GDP (inactive) ROP ROP
Prenylation TGN
-3 -4
Golgi
-4
Root tip
GEF4
ROP10
ROP2 ROP4 ROP6 -5 GEF3 -6 -7 First recruitment at the RHID
ROP
YIP4
ROP
ROPGTP (active)
GEF3 Root hair initiation ROP domain (RHID)
Current Biology
Figure 1. Successive phases in root hair initiation and outgrowth. Left, schematic drawing of an Arabidopsis root. Middle, root hair staging and timeline of recruitment of polar proteins to the root hair initiation domain (RHID, in red). Top right, interplay between ROP, anionic lipids and lipid kinases during root hair growth. Bottom right, the recruitment of ROPs at the RHID requires the presence of GEF3 [3]. Note that ROPs are synthesized as soluble proteins in the cytosol. They are then prenylated at the surface of the endoplasmic reticulum (ER) and are subsequently targeted to the plasma membrane via YIP4-dependent secretion from the TGN [6]. Root drawing by Benjamin Peret.
genomes lack the Frizzled pathway, yet Rho-of-plants (ROPs) proteins are polarized early at the root hair initiation domain (RHID), which is positioned close to the distal (rootward) end of hair-forming cells (Figure 1) [2]. How ROP proteins are precisely positioned at the RHID in the absence of the core planar polarity pathway is still unclear. A new study in this issue of Current Biology tackled this question and uncovered ROP GEF3 as a key protein driving the initial ROP polarization for the correct positioning of root hairs [3]. ROPs are highly conserved small GTPases, which function as molecular switches due to conformational differences between an inactive GDPbound state and an active GTP-bound state. These activation–inactivation cycles are regulated in time and space by guanine nucleotide exchange factors (GEFs) that promote the GTP-bound state and GTPase-activating proteins (GAPs) that enhance GTP hydrolysis [4].
The Arabidopsis thaliana genome encodes 11 ROPs, which control fundamental cellular processes, including cytoskeleton dynamics and vesicular trafficking [5]. To determine the timing of ROP polarization at the RHID, Denninger, Reichelt et al. took advantage of the fact that cells along the root tip are ordered according to their developmental age, with younger cells located rootward and older cells shootward [3]. In this developmental timeline, the cell with the first visible root hair bulge is numbered as +1 (Figure 1). Using this staging system, the authors separated root hair formation into three phases: initiation (from cells -7 to -1, in blue), bulging (cells +1 and +2, in green), and polar growth/elongation (starting at cell +3 and onward, in pink) [3]. Using confocal imaging and fluorescently-tagged ROPs, Denninger, Reichelt et al. showed that three ROPs (ROP2, ROP4 and ROP6) polarize at the RHID much before root hair initiation (i.e., at cell -4
R406 Current Biology 29, R403–R424, June 3, 2019
before root hair bulging; Figure 1). Accordingly, genetic analyses with double and triple rop mutants suggest that these three ROPs redundantly regulate root hair development [3,6]. Strikingly, all the known downstream or upstream ROP regulators polarize much later, at the stage of root hair bulging or after (Figure 1) [3]. This suggests a two-phase assembly of the tip growth machinery — an initiation phase, during which the RHID is positioned and predefined, followed by the tip growth phase. To uncover whether GEF could participate in ROP membrane targeting, Denninger, Reichelt et al. localized, via translational fusion reporters, all the GEF that are expressed in the root epidermis (6 GEFs in total). This analysis uncovered two GEFs (GEF3, GEF4 and to a lesser extent also GEF12 and GEF14) with polar localization in root hair cells. Interestingly, GEF3 polarizes early to the RHID, while GEF4 localizes at the root hair initiation site during bulging. gef3 and gef4 mutant analyses confirmed that gef3 is involved in root hair initiation, while gef4 is involved in root hair polar growth, suggesting that distinct GEFs are responsible for polar initiation and polarized growth, respectively. Elegant live imaging analyses using microfluidics suggested that GEF3 localizes to the RHID prior to ROP proteins (i.e., at cell -5) and could therefore be an early landmark for root hair positioning acting upstream of ROPs [3]. Biochemical interactions, structure–function analyses and over-expression studies validated that GEF3 polarizes first to the RHID and in turn recruits ROP proteins to this polar domain. Strikingly, upon ectopic expression in cells that do not normally form hairs (in the root and the hypocotyl) GEF3 accumulates into plasma membrane domains that resembled those of the RHID. Ectopic accumulation of GEF3 in these RHID-like domains subsequently drove the ectopic ROP2 localization in the same membrane patches.
Current Biology
Dispatches Together, the GEF3–ROP interplay appears critical to position the RHID, which is later translated into root hair tip growth via the recruitment of GEF4. Meanwhile, Gendre and collaborators studied the mechanisms and factors underlying ROP localization at the root hair plasma membrane with a focus on membrane trafficking [6]. They demonstrated that the membrane trafficking machinery acts as a central component in ROP-dependent root hair formation. Indeed, the trans-Golgi Network (TGN)-localized YPTINTERACTING PROTEIN (YIP)4a and YIP4b are required for the activation and plasma membrane accumulation of ROPs at the RHID (Figure 1). Another important factor that regulates ROP localization and signaling activity is its interaction with lipids. ROP2, 4 and 6 carboxyl termini are prenylated (i.e., geranylgeranylation, a covalently linked lipid anchor), which provides hydrophobic anchoring and is required for membrane association [5]. ROP6 is also S-acylated (i.e., palmitoylation), which is a reversible lipid modification required for ROP6 function [7]. Furthermore, all ROPs possess in their carboxy-terminal tail a polybasic region, which interacts with anionic phospholipids, at least in the case of ROP6 [8]. The plasma membrane is highly enriched in anionic phospholipids and hence is strongly electronegative [9,10]. Consequently, ROP6 polybasic tail interacts with this plasma membrane electrostatic field and is required for the proper targeting of ROP to the cell surface [8]. At the bulging stage, the RHID is enriched in the anionic phospholipid PI(4,5)P2, and ROP2 colocalizes and interacts with PIP5K3, the kinase that produces PI(4,5)P2 [3,11,12] (Figure 1). Interestingly, this situation is not restricted to ROP2, but also ROP10, which localizes in a complementary domain at the shank of growing root hairs [11] (Figure 1). ROP10 colocalizes and interacts with both the anionic phospholipids PI(3,5)P2 and the lipid kinase that produces it (i.e., FAB1; Figure 1) [11]. This suggests that ROP activity may regulate lipid synthesis, while in turn lipids themselves control ROP activity and/or localization. Therefore, ROP and anionic lipids may
form a self-organizing system for the focal recruitment and activation of Rho GTPases in plants. However, given that PIP5K3 and PI(4,5)P2 polarize relatively late at the RHID (Figure 1) [3], it is likely that they act as an amplifying system rather than the initial polarizing cue. Taken together, this evidence suggests that ROP localization and focal activation in root hairs may require multiple factors, including enzymatic activation, lipid modification, direct interaction with lipids and vesicular trafficking. An open question is to understand what is/are the primary signal(s) that defines the position of the RHID; that is, what are the factors that initially drive GEF3 polarity? In other contexts, ROP GEFs act downstream of receptorlike kinases (RLK), which are membrane receptors able to translate signal(s) across the plasma membrane [5]. One may speculate that there are root hair-expressed receptors that localize at the RHID. Alternatively, such receptors would not necessarily have to be polarized themselves but could rather perceive a polarized signal. Such a signal could include the concentration gradient of the plant hormone auxin at the root tip, which is critical to determine ROP localization at the RHID [13]. In addition, auxin promotes ROP2 and ROP6 activation [14], but the machinery that perceives and transduces the auxin signal across the plasma membrane is still elusive [15]. One of the future challenges in understanding planar cell polarity in plants will be to elucidate how ROP polarization is linked with extracellular signals, such as auxin.
REFERENCES 1. Goodrich, L.V., and Strutt, D. (2011). Principles of planar polarity in animal development. Development 138, 1877–1892. 2. Nakamura, M., Kiefer, C.S., and Grebe, M. (2012). Planar polarity, tissue polarity and planar morphogenesis in plants. Curr. Opin. Plant Biol. 15, 593–600. 3. Denninger, P., Reichelt, A., Schmidt, V.A.F., Mehlhorn, D.G., Asseck, L.Y., Stanley, C.E., Keinath, N.F., Evers, J.-F., Grefen, C., and Grossmann, G. (2019). Distinct RopGEFs successively drive polarization and
outgrowth of root hairs. Curr. Biol. 29, 1854– 1865. 4. Berken, A., and Wittinghofer, A. (2008). Structure and function of Rho-type molecular switches in plants. Plant Physiol. Biochem. 46, 380–393. 5. Feiguelman, G., Fu, Y., and Yalovsky, S. (2018). ROP GTPases structure-function and signaling pathways. Plant Physiol. 176, 57–79. 6. Gendre, D., Baral, A., Dang, X., Esnay, N., Boutte, Y., Stanislas, T., Vain, T., Claverol, S., Gustavsson, A., Lin, D., et al. (2019). Rho-ofplant-activated root hair formation requires Arabidopsis YIP4a/b gene function. Development 146, dev168559. 7. Sorek, N., Segev, O., Gutman, O., Bar, E., Richter, S., Poraty, L., Hirsch, J.A., Henis, Y.I., Lewinsohn, E., Jurgens, G., et al. (2010). An S-acylation switch of conserved G domain cysteines is required for polarity signaling by ROP GTPases. Curr. Biol. 20, 914–920. 8. Platre, M.P., Bayle, V., Armengot, L., Bareille, J., Marques-Bueno, M.M., Creff, A., ManetaPeyret, L., Fiche, J.B., Nolmann, M., Mie`ge, C., et al. (2019). Developmental control of plant Rho GTPase nano-organization by the lipid phosphatidylserine. Science 364, 57–62. 9. Platre, M.P., Noack, L.C., Doumane, M., Bayle, V., Simon, M.L.A., Maneta-Peyret, L., Fouillen, L., Stanislas, T., Armengot, L., Pejchar, P., et al. (2018). A combinatorial lipid code shapes the electrostatic landscape of plant endomembranes. Dev. Cell 45, 465–480, e411. 10. Simon, M.L., Platre, M.P., Marques-Bueno, M.M., Armengot, L., Stanislas, T., Bayle, V., Caillaud, M.C., and Jaillais, Y. (2016). A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants. Nat. Plants 2, 16089. 11. Hirano, T., Konno, H., Takeda, S., Dolan, L., Kato, M., Aoyama, T., Higaki, T., TakigawaImamura, H., and Sato, M.H. (2018). PtdIns(3,5)P2 mediates root hair shank hardening in Arabidopsis. Nat. Plants 4, 888–897. 12. Stanislas, T., Hu¨ser, A., Barbosa, I.C., Kiefer, P., Brackmann, K., Pitra, S., Gustavsson, A., Zourelidou, M., Schwechheimer, C., and Grebe, M. (2015). Arabidopsis D6PK is a lipid domain-dependent mediator of root epidermal planar polarity. Nat. Plants 1, 15162. 13. Ikeda, Y., Men, S., Fischer, U., Stepanova, A.N., Alonso, J.M., Ljung, K., and Grebe, M. (2009). Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis. Nat. Cell Biol. 11, 731–738. 14. Xu, T., Wen, M., Nagawa, S., Fu, Y., Chen, J.G., Wu, M.J., Perrot-Rechenmann, C., Friml, J., Jones, A.M., and Yang, Z. (2010). Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis. Cell 143, 99–110. 15. Armengot, L., Marques-Bueno, M.M., and Jaillais, Y. (2016). Regulation of polar auxin transport by protein and lipid kinases. J. Exp. Bot. 67, 4015–4037.
Current Biology 29, R403–R424, June 3, 2019 R407