Root hairs elongate by tip growth and simultaneously harden the shank by constructing the inner secondary cell wall layer. While much is known about the process of tip growth1, almost nothing is known about the mechanism by which root hairs harden the shank. Here we show that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), the enzymatic product of FORMATION OF APLOID AND BINUCLEATE CELLS 1 (FAB1), is involved in the hardening of the shank in root hairs in Arabidopsis. FAB1 and PtdIns(3,5)P2 localize to the plasma membrane along the shank of growing root hairs. By contrast, phosphatidylinositol 4-phosphate 5-kinase 3 (PIP5K3) and PtdIns(4,5)P2 localize to the apex of the root hair where they are required for tip growth. Reduction of FAB1 function results in the formation of wavy root hairs while those of the wild type are straight. The localization of FAB1 in the plasma membrane of the root hair shank requires the activity of Rho-related GTPases from plants 10 (ROP10) and localization of ROP10 requires FAB1 activity. Computational modelling of root hair morphogenesis successfully reproduces the wavy root hair phenotype. Taken together, these data demonstrate that root hair shank hardening requires PtdIns(3,5)P2/ROP10 signalling.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $5.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
All data appearing in this study are available from the authors upon reasonable request.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Grierson, C., Nielsen, E., Ketelaarc, T. & Schiefelbein, J. Root hairs. The Arabidopsis Book 12, e0172 (2014).
Xu, T. et al. Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis. Cell 143, 99–110 (2010).
Akkerman, M. et al. Texture of cellulose microfibrils of root hair cell walls of Arabidopsis thaliana, Medicago truncatula and Vicia sativa. J. Microsc. 247, 60–67 (2012).
Campanoni, P. & Blatt, M. R. Membrane trafficking and polar growth in root hairs and pollen tubes. J. Exp. Bot. 58, 65–74 (2007).
Feiguelman, G., Fu, Y. & Yalovsky, S. ROP GTPases structure–function and signaling pathways. Plant Physiol. 176, 57–79 (2018).
Gerth, K. et al. Guilt by association: a phenotype-based view of the plant phosphoinositide network. Annu. Rev. Plant Biol. 68, 349–374 (2017).
Yalovsky, S., Bloch, D., Sorek, N. & Kost, B. Regulation of membrane trafficking, cytoskeleton dynamics, and cell polarity by ROP/RAC GTPases. Plant Physiol. 147, 1527–1543 (2008).
Jones, M. A. et al. The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth. Plant Cell 14, 763–776 (2002).
Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006).
Kusano, H. et al. The Arabidopsis phosphatidylinositol phosphate 5-kinase PIP5K3 is a key regulator of root hair tip growth. Plant Cell 20, 367–380 (2008).
Braun, M., Baluska, F., von Witsch, M. & Menzel, D. Redistribution of actin, profilin and phosphatidylinositol-4,5-bisphosphate in growing and maturing root hairs. Planta 209, 435–443 (1999).
Serrazina, S., Dias, F. V. & Malhó, R. Characterization of FAB1 phosphatidylinositol kinases in Arabidopsis pollen tube growth and fertilization. New Phytologist 203, 784–793 (2014).
Hirano, T., Stecker, K., Munnik, T., Xu, H. & Sato, M. H. Visualization of phosphatidylinositol 3,5-bisphosphate dynamics by a tandem ML1N-based fluorescent protein probe in Arabidopsis. Plant Cell Physiol. 58, 1185–1195 (2017).
Stenzel, I., Ischebeck, T. & Ko, S. The type B phosphatidylinositol-4-phosphate 5-kinase 3 is essential for root hair formation in Arabidopsis thaliana. Plant Cell 20, 124–141 (2008).
Simon, M. L. A. et al. A multi-colour/multi-affinity marker set to visualize phosphoinositide dynamics in Arabidopsis. Plant J. 77, 322–337 (2014).
Park, S., Szumlanski, A. L., Gu, F., Guo, F. & Nielsen, E. A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells. Nat. Cell Biol. 13, 973–980 (2011).
Blake, A. W. et al. Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes. J. Biol. Chem. 281, 29321–29329 (2006).
Larson, E. R., Tierney, M. L., Tinaz, B. & Domozych, D. S. Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in Arabidopsis. Plant Methods 10, 30 (2014).
Oda, Y. & Fukuda, H. Initiation of cell wall pattern by a Rho- and microtubule-driven symmetry breaking. Science 337, 1333–1336 (2012).
Hogetsu, T. Detection of hemicelluloses specific to the cell wall of tracheary elements and phloem cells by fluorescein-conjugated lectins. Protoplasma 156, 67–73 (1990).
Bibikova, T. N., Blancaflor, E. B. & Gilroy, S. Microtubules regulate tip growth and orientation in root hairs of Arabidopsis thaliana. Plant J. 17, 657–665 (1999).
Hirano, T., Munnik, T. & Sato, M. H. Phosphatidylinositol 3-phosphate 5-kinase, FAB1/PIKfyve kinase mediates endosome maturation to establish endosome–cortical microtubule interaction in Arabidopsis. Plant Physiol. 169, 1961–1974 (2015).
Komaki, S. et al. Nuclear-localized subtype of end-binding 1 protein regulates spindle organization in Arabidopsis. J. Cell. Sci. 123, 451–459 (2010).
Jones, M. A. The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth. Plant Cell 14, 763–776 (2002).
Fu, Y., Gu, Y., Zheng, Z., Wasteneys, G. & Yang, Z. Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis. Cell 120, 687–700 (2005).
Higaki, T. et al. Exogenous cellulase switches cell interdigitation to cell elongation in an RIC1-dependent manner in Arabidopsis thaliana cotyledon pavement cells. Plant Cell Physiol. 58, 106–119 (2017).
Takigawa-Imamura, H., Morita, R., Iwaki, T., Tsuji, T. & Yoshikawa, K. Tooth germ invagination from cell-cell interaction: Working hypothesis on mechanical instability. J. Theor. Biol. 382, 284–291 (2015).
Nakagawa, T. et al. Improved gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci. Biotechnol. Biochem. 71, 2095–2100 (2007).
Schwab, R., Ossowski, S., Riester, M., Warthmann, N. & Weigel, D. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18, 1121–1133 (2006).
Zuo, J., Niu, Q. W. & Chua, N. H. Technical advance: an estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265–273 (2000).
Hirano, T., Matsuzawa, T., Takegawa, K. & Sato, M. H. Loss-of-function and gain-of-function mutations in FAB1A/B impair endomembrane homeostasis, conferring pleiotropic developmental abnormalities in Arabidopsis. Plant Physiol. 155, 797–807 (2011).
Shimada, T. L., Shimada, T. & Hara-Nishimura, I. A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. Plant J. 61, 519–528 (2010).
Ichikawa, M. et al. Syntaxin of plant proteins SYP123 and SYP132 mediate root hair tip growth in Arabidopsis thaliana. Plant Cell Physiol. 55, 790–800 (2014).
Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).
Marc, J. et al. A GFP – MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10, 1927–1940 (2006).
Becker, J. D., Takeda, S., Borges, F., Dolan, L. & Feijó, J. A. Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signature. BMC Plant Biol. 14, 197 (2014).
Ueda, H. et al. Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells. Proc. Natl Acad. Sci. USA 107, 6894–6899 (2010).
Higaki, T., Kutsuna, N., Sano, T., Kondo, N. & Hasezawa, S. Quantification and cluster analysis of actin cytoskeletal structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsis guard cells. Plant J. 61, 156–165 (2010).
Dou, L., He, K., Higaki, T., Wang, X. A. & Mao, T. Ethylene signaling modulates cortical microtubule reassembly in response to salt stress. Plant Physiol. 176, 2071–2081 (2018).
Ando, T. et al. A high-speed atomic force microscope for studying biological macromolecules in action. ChemPhysChem 4, 1196–1202 (2003).
Ando, T., Uchihashi, T. & Fukuma, T. High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes. Prog. Surf. Sci. 83, 337–437 (2008).
Butt, H.-J. & Jaschke, M. Calculation of thermal noise in atomic force microscopy. Nanotechnology. 6, 1–7 (1995).
Cozier, G. E. et al. The phox homology (PX) domain-dependent, 3-phosphoinositide-mediated association of sorting nexin-1 with an early sorting endosomal compartment is required for its ability to regulate epidermal growth factor receptor degradation. J. Biol. Chem. 277, 48730–48736 (2002).
Morag, E. et al. Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum. Appl. Environ. Microbiol. 61, 1980–1986 (1995).
de Lucas, M. et al. A molecular framework for light and gibberellin control of cell elongation. Nature 451, 480–484 (2008).
We thank T. Miura (Kyushu University, Japan) for his helpful comments on the computational model, T. Nakagawa (Shimane University, Japan) for providing pGWB vectors, C. Ambrose (University of Saskatchewan, Canada) for providing the GFP–MBD-expressing line, T. Hashimoto (NAIST, Japan) for providing the EB1b–GFP-expression line, Y. Jailais (Université de Lyon, France) for providing the CITRINE–2:PHPLC-expressing line and fruitful discussion, and Y. Oda (National Institute for Genetics, Japan) and S. Sakamoto and N. Mitsuda (National Institute of Advanced Industrial Science and Technology, Japan) for fruitful discussion about cell wall components. We thank T. Ando and N. Kodera (Kanazawa University) for providing us with experimental instruments, and T. Nakayama-Watanabe for critical suggestions for data analysis. We also thank K. Tamura and I. Hara-Nishimura (Kyoto University) for fruitful discussion. This work was supported by JSPS KAKENHIJP16H05068 to M.H.S., JP17K08200 to T. Hirano, JP18K06260 to H.T.-I, JP16K06260 to T.A., 16KT0170 to T.A., 17K15238 to M.K., JP16H06280, 17K19380, 18H05492, a Grant for Basic Science Research Projects from The Sumitomo Foundation (160146), and a Grant from The Canon Foundation to T. Higaki, Marie Curie Actions; Incoming Interaction Fellowship (ID: 022275) to S.T.
Supplementary Figures 1–19 and Supplementary Video legends.
List of materials used for this study.
Time lapse images PI(3,5)P2 and PI(4,5)P2 fluorescence of initiation step of root hair elongation.
Time lapse images PtdIns(3,5)P2 and PtdIns(4,5)P2 fluorescence of elongating step of root hair.
Time lapse images PtdIns(3,5)P2 and PtdIns(4,5)P2 fluorescence of termination step of root hair elongation.
Computer simulation of the growing process of the wild type root hair in the air.
Computer simulation of the growing process of the wild type root hair in the gel.
Computer simulation of the wavy root hair in the air.
Computer simulation of the wavy root hair in the gel.
Computer simulation of the swollen root hair in the air.
Computer simulation of the swollen root hair in the gel.
About this article
Nature Communications (2019)
Nature Plants (2018)