A GFP–MAP4 Reporter Gene for Visualizing Cortical Microtubule Rearrangements in Living Epidermal Cells

Correspondence to: Richard J. Cyr, rjc8{at}psu.edu (E-mail), 814-865-9131 (fax).

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.

Related articles in Plant Cell:

To Shape a Plant—The Cytoskeleton in Plant Morphogenesis

Whitney E. Hable, Sherryl R. Bisgrove, and Darryl L. Kropf
Plant Cell 1998 10: 1772-1774. [Full Text]  

This article has been cited by other articles:

				M. J. Kim, S. Ciani, and D. P. Schachtman A Peroxidase Contributes to ROS Production during Arabidopsis Root Response to Potassium Deficiency Mol Plant, February 5, 2010; (2010) ssp121v1. [Abstract] [Full Text] [PDF]

				S. K. A. Natesan, J. A. Sullivan, and J. C. Gray Myosin XI Is Required for Actin-Associated Movement of Plastid Stromules Mol Plant, November 1, 2009; 2(6): 1262 - 1272. [Abstract] [Full Text] [PDF]

				M. J. Kim, R. Shin, and D. P. Schachtman A Nuclear Factor Regulates Abscisic Acid Responses in Arabidopsis Plant Physiology, November 1, 2009; 151(3): 1433 - 1445. [Abstract] [Full Text] [PDF]

				E. J. Wiltshire and D. A. Collings New Dynamics in an Old Friend: Dynamic Tubular Vacuoles Radiate Through the Cortical Cytoplasm of Red Onion Epidermal Cells Plant Cell Physiol., October 1, 2009; 50(10): 1826 - 1839. [Abstract] [Full Text] [PDF]

				J. M. Plett, J. Mathur, and S. Regan Ethylene receptor ETR2 controls trichome branching by regulating microtubule assembly in Arabidopsis thaliana J. Exp. Bot., September 1, 2009; 60(13): 3923 - 3933. [Abstract] [Full Text] [PDF]

				N. Frey, J. Klotz, and P. Nick Dynamic Bridges--A Calponin-Domain Kinesin From Rice Links Actin Filaments and Microtubules in Both Cycling and Non-Cycling Cells Plant Cell Physiol., August 1, 2009; 50(8): 1493 - 1506. [Abstract] [Full Text] [PDF]

				E. F. Crowell, V. Bischoff, T. Desprez, A. Rolland, Y.-D. Stierhof, K. Schumacher, M. Gonneau, H. Hofte, and S. Vernhettes Pausing of Golgi Bodies on Microtubules Regulates Secretion of Cellulose Synthase Complexes in Arabidopsis PLANT CELL, April 1, 2009; 21(4): 1141 - 1154. [Abstract] [Full Text] [PDF]

				P. Ruggenthaler, D. Fichtenbauer, J. Krasensky, C. Jonak, and E. Waigmann Microtubule-Associated Protein AtMPB2C Plays a Role in Organization of Cortical Microtubules, Stomata Patterning, and Tobamovirus Infectivity Plant Physiology, March 1, 2009; 149(3): 1354 - 1365. [Abstract] [Full Text] [PDF]

				L. Guo, C.-M. K. Ho, Z. Kong, Y.-R. J. Lee, Q. Qian, and B. Liu Evaluating the microtubule cytoskeleton and its interacting proteins in monocots by mining the rice genome Ann. Bot., February 1, 2009; 103(3): 387 - 402. [Abstract] [Full Text] [PDF]

				P. A. Harries, K. Palanichelvam, W. Yu, J. E. Schoelz, and R. S. Nelson The Cauliflower Mosaic Virus Protein P6 Forms Motile Inclusions That Traffic along Actin Microfilaments and Stabilize Microtubules Plant Physiology, February 1, 2009; 149(2): 1005 - 1016. [Abstract] [Full Text] [PDF]

				E. Kawamura and G. O. Wasteneys MOR1, the Arabidopsis thaliana homologue of Xenopus MAP215, promotes rapid growth and shrinkage, and suppresses the pausing of microtubules in vivo J. Cell Sci., December 15, 2008; 121(24): 4114 - 4123. [Abstract] [Full Text] [PDF]

				Y. Gu, Z. Deng, A. R. Paredez, S. DeBolt, Z.-Y. Wang, and C. Somerville Prefoldin 6 is required for normal microtubule dynamics and organization in Arabidopsis PNAS, November 18, 2008; 105(46): 18064 - 18069. [Abstract] [Full Text] [PDF]

				A. J. Bernal, C.-M. Yoo, M. Mutwil, J. K. Jensen, G. Hou, C. Blaukopf, I. Sorensen, E. B. Blancaflor, H. V. Scheller, and W. G.T. Willats Functional Analysis of the Cellulose Synthase-Like Genes CSLD1, CSLD2, and CSLD4 in Tip-Growing Arabidopsis Cells Plant Physiology, November 1, 2008; 148(3): 1238 - 1253. [Abstract] [Full Text] [PDF]

				C.-M. Yoo, J. Wen, C. M. Motes, J. A. Sparks, and E. B. Blancaflor A Class I ADP-Ribosylation Factor GTPase-Activating Protein Is Critical for Maintaining Directional Root Hair Growth in Arabidopsis Plant Physiology, August 1, 2008; 147(4): 1659 - 1674. [Abstract] [Full Text] [PDF]

				A. R. Paredez, S. Persson, D. W. Ehrhardt, and C. R. Somerville Genetic Evidence That Cellulose Synthase Activity Influences Microtubule Cortical Array Organization Plant Physiology, August 1, 2008; 147(4): 1723 - 1734. [Abstract] [Full Text] [PDF]

				M. M. Castellano and R. Sablowski Phosducin-Like Protein 3 Is Required for Microtubule-Dependent Steps of Cell Division but Not for Meristem Growth in Arabidopsis PLANT CELL, April 1, 2008; 20(4): 969 - 981. [Abstract] [Full Text] [PDF]

				S. DeBolt, R. Gutierrez, D. W. Ehrhardt, C. V. Melo, L. Ross, S. R. Cutler, C. Somerville, and D. Bonetta Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement PNAS, April 3, 2007; 104(14): 5854 - 5859. [Abstract] [Full Text] [PDF]

				Z. Chu, H. Chen, Y. Zhang, Z. Zhang, N. Zheng, B. Yin, H. Yan, L. Zhu, X. Zhao, M. Yuan, et al. Knockout of the AtCESA2 Gene Affects Microtubule Orientation and Causes Abnormal Cell Expansion in Arabidopsis Plant Physiology, January 1, 2007; 143(1): 213 - 224. [Abstract] [Full Text] [PDF]

				H. S. Sardar, J. Yang, and A. M. Showalter Molecular Interactions of Arabinogalactan Proteins with Cortical Microtubules and F-Actin in Bright Yellow-2 Tobacco Cultured Cells Plant Physiology, December 1, 2006; 142(4): 1469 - 1479. [Abstract] [Full Text] [PDF]

				S. D.X. Chuong, V. R. Franceschi, and G. E. Edwards The Cytoskeleton Maintains Organelle Partitioning Required for Single-Cell C4 Photosynthesis in Chenopodiaceae Species PLANT CELL, September 1, 2006; 18(9): 2207 - 2223. [Abstract] [Full Text] [PDF]

				J. G. DUBROVSKY, M. GUTTENBERGER, A. SARALEGUI, S. NAPSUCIALY-MENDIVIL, B. VOIGT, F. BALUSKA, and D. MENZEL Neutral Red as a Probe for Confocal Laser Scanning Microscopy Studies of Plant Roots Ann. Bot., June 1, 2006; 97(6): 1127 - 1138. [Abstract] [Full Text] [PDF]

				P. E. Dowd, S. Coursol, A. L. Skirpan, T.-h. Kao, and S. Gilroy Petunia Phospholipase C1 Is Involved in Pollen Tube Growth PLANT CELL, June 1, 2006; 18(6): 1438 - 1453. [Abstract] [Full Text] [PDF]

				M. Pastuglia, J. Azimzadeh, M. Goussot, C. Camilleri, K. Belcram, J.-L. Evrard, A.-C. Schmit, P. Guerche, and D. Bouchez {gamma}-Tubulin Is Essential for Microtubule Organization and Development in Arabidopsis PLANT CELL, June 1, 2006; 18(6): 1412 - 1425. [Abstract] [Full Text] [PDF]

				A. Genre, M. Chabaud, T. Timmers, P. Bonfante, and D. G. Barker Arbuscular Mycorrhizal Fungi Elicit a Novel Intracellular Apparatus in Medicago truncatula Root Epidermal Cells before Infection PLANT CELL, December 1, 2005; 17(12): 3489 - 3499. [Abstract] [Full Text] [PDF]

				J.-Z. Liu, E. B. Blancaflor, and R. S. Nelson The Tobacco Mosaic Virus 126-Kilodalton Protein, a Constituent of the Virus Replication Complex, Alone or within the Complex Aligns with and Traffics along Microfilaments Plant Physiology, August 1, 2005; 138(4): 1853 - 1865. [Abstract] [Full Text] [PDF]

				P. Vincent, M. Chua, F. Nogue, A. Fairbrother, H. Mekeel, Y. Xu, N. Allen, T. N. Bibikova, S. Gilroy, and V. A. Bankaitis A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs J. Cell Biol., February 28, 2005; 168(5): 801 - 812. [Abstract] [Full Text] [PDF]

				D. Basu, J. Le, S. E.-D. El-Essal, S. Huang, C. Zhang, E. L. Mallery, G. Koliantz, C. J. Staiger, and D. B. Szymanski DISTORTED3/SCAR2 Is a Putative Arabidopsis WAVE Complex Subunit That Activates the Arp2/3 Complex and Is Required for Epidermal Morphogenesis PLANT CELL, February 1, 2005; 17(2): 502 - 524. [Abstract] [Full Text] [PDF]

				R. Dixit and R. Cyr Encounters between Dynamic Cortical Microtubules Promote Ordering of the Cortical Array through Angle-Dependent Modifications of Microtubule Behavior PLANT CELL, December 1, 2004; 16(12): 3274 - 3284. [Abstract] [Full Text] [PDF]

				N. Van Bruaene, G. Joss, and P. Van Oostveldt Reorganization and in Vivo Dynamics of Microtubules during Arabidopsis Root Hair Development Plant Physiology, December 1, 2004; 136(4): 3905 - 3919. [Abstract] [Full Text] [PDF]

				W. Li, C. Chen, U. Markmann-Mulisch, L. Timofejeva, E. Schmelzer, H. Ma, and B. Reiss The Arabidopsis AtRAD51 gene is dispensable for vegetative development but required for meiosis PNAS, July 20, 2004; 101(29): 10596 - 10601. [Abstract] [Full Text] [PDF]

				R. Saedler, N. Mathur, B. P. Srinivas, B. Kernebeck, M. Hulskamp, and J. Mathur Actin Control Over Microtubules Suggested by DISTORTED2 Encoding the Arabidopsis ARPC2 Subunit Homolog Plant Cell Physiol., July 15, 2004; 45(7): 813 - 822. [Abstract] [Full Text] [PDF]

				P. Dhonukshe, A. M. Laxalt, J. Goedhart, T. W. J. Gadella, and T. Munnik Phospholipase D Activation Correlates with Microtubule Reorganization in Living Plant Cells PLANT CELL, November 1, 2003; 15(11): 2666 - 2679. [Abstract] [Full Text] [PDF]

				F. Kragler, M. Curin, K. Trutnyeva, A. Gansch, and E. Waigmann MPB2C, a Microtubule-Associated Plant Protein Binds to and Interferes with Cell-to-Cell Transport of Tobacco Mosaic Virus Movement Protein Plant Physiology, August 1, 2003; 132(4): 1870 - 1883. [Abstract] [Full Text] [PDF]

				M. Sivaguru, S. Pike, W. Gassmann, and T. I. Baskin Aluminum Rapidly Depolymerizes Cortical Microtubules and Depolarizes the Plasma Membrane: Evidence that these Responses are Mediated by a Glutamate Receptor Plant Cell Physiol., July 15, 2003; 44(7): 667 - 675. [Abstract] [Full Text] [PDF]

				T. Bouquin, O. Mattsson, H. Naested, R. Foster, and J. Mundy The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth J. Cell Sci., March 1, 2003; 116(5): 791 - 801. [Abstract] [Full Text] [PDF]

				P. Dhonukshe and T. W. J. Gadella Jr. Alteration of Microtubule Dynamic Instability during Preprophase Band Formation Revealed by Yellow Fluorescent Protein-CLIP170 Microtubule Plus-End Labeling PLANT CELL, March 1, 2003; 15(3): 597 - 611. [Abstract] [Full Text] [PDF]

				J.-Y. Jung, Y.-W. Kim, J. M. Kwak, J.-U. Hwang, J. Young, J. I. Schroeder, I. Hwang, and Y. Lee Phosphatidylinositol 3- and 4-Phosphate Are Required for Normal Stomatal Movements PLANT CELL, October 1, 2002; 14(10): 2399 - 2412. [Abstract] [Full Text] [PDF]

				B. J. Sieberer, A. C.J. Timmers, F. G.P. Lhuissier, and A. M. C. Emons Endoplasmic Microtubules Configure the Subapical Cytoplasm and Are Required for Fast Growth of Medicago truncatula Root Hairs Plant Physiology, October 1, 2002; 130(2): 977 - 988. [Abstract] [Full Text] [PDF]

				V. Kirik, P. E. Grini, J. Mathur, I. Klinkhammer, K. Adler, N. Bechtold, M. Herzog, J.-M. Bonneville, and M. Hulskamp The Arabidopsis TUBULIN-FOLDING COFACTOR A Gene Is Involved in the Control of the {alpha}/{beta}-Tubulin Monomer Balance PLANT CELL, September 1, 2002; 14(9): 2265 - 2276. [Abstract] [Full Text] [PDF]

				J. E. Carette, K. Guhl, J. Wellink, and A. Van Kammen Coalescence of the Sites of Cowpea Mosaic Virus RNA Replication into a Cytopathic Structure J. Virol., June 15, 2002; 76(12): 6235 - 6243. [Abstract] [Full Text] [PDF]

				E. Lalanne and D. Twell Genetic Control of Male Germ Unit Organization in Arabidopsis Plant Physiology, June 1, 2002; 129(2): 865 - 875. [Abstract] [Full Text] [PDF]

				G. Jedd and N.-H. Chua Visualization of Peroxisomes in Living Plant Cells Reveals Acto-Myosin-Dependent Cytoplasmic Streaming and Peroxisome Budding Plant Cell Physiol., April 15, 2002; 43(4): 384 - 392. [Abstract] [Full Text] [PDF]

				K. J. Czymmek, T. M. Bourett, J. A. Sweigard, A. Carroll, and R. J. Howard Utility of cytoplasmic fluorescent proteins for live-cell imaging of Magnaporthe grisea in planta Mycologia, March 1, 2002; 94(2): 280 - 289. [Abstract] [Full Text] [PDF]

				J. Mathur, N. Mathur, and M. Hulskamp Simultaneous Visualization of Peroxisomes and Cytoskeletal Elements Reveals Actin and Not Microtubule-Based Peroxisome Motility in Plants Plant Physiology, March 1, 2002; 128(3): 1031 - 1045. [Abstract] [Full Text] [PDF]

				G. O. Wasteneys Microtubule organization in the green kingdom: chaos or self-order? J. Cell Sci., January 4, 2002; 115(7): 1345 - 1354. [Abstract] [Full Text] [PDF]

				F. Kumagai, A. Yoneda, T. Tomida, T. Sano, T. Nagata, and S. Hasezawa Fate of Nascent Microtubules Organized at the M/G1 Interface, as Visualized by Synchronized Tobacco BY-2 Cells Stably Expressing GFP-Tubulin: Time-Sequence Observations of the Reorganization of Cortical Microtubules in Living Plant Cells Plant Cell Physiol., July 1, 2001; 42(7): 723 - 732. [Abstract] [Full Text] [PDF]

				D. H. Burk, B. Liu, R. Zhong, W. H. Morrison, and Z.-H. Ye A Katanin-like Protein Regulates Normal Cell Wall Biosynthesis and Cell Elongation PLANT CELL, April 1, 2001; 13(4): 807 - 828. [Abstract] [Full Text]

				C. Boisnard-Lorig, A. Colon-Carmona, M. Bauch, S. Hodge, P. Doerner, E. Bancharel, C. Dumas, J. Haseloff, and F. Berger Dynamic Analyses of the Expression of the HISTONE::YFP Fusion Protein in Arabidopsis Show That Syncytial Endosperm Is Divided in Mitotic Domains PLANT CELL, March 1, 2001; 13(3): 495 - 509. [Abstract] [Full Text]

				A. I. Marcus, R. C. Moore, and R. J. Cyr The Role of Microtubules in Guard Cell Function Plant Physiology, January 1, 2001; 125(1): 387 - 395. [Abstract] [Full Text]

				E. B. Blancaflor and S. Gilroy Plant cell biology in the new millennium: new tools and new insights Am. J. Botany, November 1, 2000; 87(11): 1547 - 1560. [Abstract] [Full Text]

				J. Mathur and N.-H. Chua Microtubule Stabilization Leads to Growth Reorientation in Arabidopsis Trichomes PLANT CELL, April 1, 2000; 12(4): 465 - 478. [Abstract] [Full Text]

				J. Li, X. Wang, M. B. Watson, and S. M. Assmann Regulation of Abscisic Acid-Induced Stomatal Closure and Anion Channels by Guard Cell AAPK Kinase Science, January 14, 2000; 287(5451): 300 - 303. [Abstract] [Full Text]

				S. G. Lazarowitz and R. N. Beachy Viral Movement Proteins as Probes for Intracellular and Intercellular Trafficking in Plants PLANT CELL, April 1, 1999; 11(4): 535 - 548. [Full Text]

				W. E. Hable, S. R. Bisgrove, and D. L. Kropf To Shape a Plant—The Cytoskeleton in Plant Morphogenesis PLANT CELL, November 1, 1998; 10(11): 1772 - 1774. [Full Text] [PDF]

				C. Camilleri, J. Azimzadeh, M. Pastuglia, C. Bellini, O. Grandjean, and D. Bouchez The Arabidopsis TONNEAU2 Gene Encodes a Putative Novel Protein Phosphatase 2A Regulatory Subunit Essential for the Control of the Cortical Cytoskeleton PLANT CELL, April 1, 2002; 14(4): 833 - 845. [Abstract] [Full Text] [PDF]