Summary
During the past decade the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis root cells, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. Parallel to our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use nonembedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development.
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Blancaflor, E. B. and Gilroy, S. (2000) Plant cell biology in the new millennium: new tools and new insights. Am. J. Bot. 87, 1547–1560.
Dixit, R., Cyr, R. and Gilroy, S. (2006) Using intrinsically fluorescent proteins for plant cell imaging. Plant J. 45, 599–615.
Marc, J., Granger, C. L., Brincat, J., Fisher, D. D., Kao, T.-h., McCubbin, A. G. and Cyr, R. J. (1998) A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10, 1927–1940.
Kost, B., Spielhofer, P. and Chua N. H. (1998) A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J. 16, 393–401.
Wang, Y.-S., Motes, C. M., Mohamalawari, D. R. and Blancaflor, E. B. (2004) Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots. Cell Motil. Cytoskeleton 59, 79–93.
Sheahan, M. B., Staiger, C. J., Rose, R. J. and McCurdy, D. W. (2004) A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells. Plant Physiol. 136, 3968–3978.
Voigt, B., Timmers, A. C. J., Samaj, J., Muller, J., Baluška, F. and Menzel, D. (2005) GFP-ABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings. Eur. J. Cell Biol. 84, 595–608.
Wang, Y.-S., Yoo, C.-M. and Blancaflor, E. B. (2008) Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C and N termini of the fimbrin actin binding domain 2. New Phytol. 177, 525–536.
Lovy-Wheeler, A., Wilsen, K. L., Baskin, T. I. and Hepler, P. K. (2005) Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube. Planta 221, 95–104.
Collings, D. A. and Wasteneys, G. O. (2005) Actin microfilament and microtubule distribution patterns in the expanding root of Arabidopsis thaliana. Can. J. Bot. 83, 579–590.
Wilsen, K. L., Lovy-Wheeler, A., Voigt, B., Menzel, D., Kunkel, J. G. and Hepler, P. K. (2006) Imaging the actin cytoskeleton in growing pollen tubes. Sex. Plant Reprod. 19, 51–62.
Brown, R. C. and Lemmon, B. E. (1995) Methods in plant immunolight microscopy. In: Methods in Cell Biology (Galbraith, D. W., Bohnert, H. J., Bourque, D. P., eds). Academic, San Diego, CA, pp 85–107.
Blancaflor, E. B. and Hasenstein, K. H. (2000) Methods for detection and identification of F-actin organization in plant tissues. In: Actin: A Dynamic Framework for Multiple Plant Cell Functions (Staiger, C., Baluška, F., Volkmann, D., Barlow P. W., eds). Kluwer, Dordrecht, the Netherlands, pp 601–618.
Blancaflor, E. B., Wang, Y.-S. and Motes, C. M. (2006) Organization and function of the actin cytoskeleton in developing root cells. Int. Rev. Cytol. 252, 153–198.
Blancaflor, E. B. (2000) Cortical actin filaments potentially interact with cortical microtubules in regulating polarity of cell expansion in primary roots of maize (Zea mays L.). J. Plant Growth Regul. 19, 406–414.
Blancaflor, E. B., Zhao, L. and Harrison, M. J. (2001) Microtubule organization in root cells of Medicago truncatula during development of an arbuscular mycorrhizal symbiosis with Glomus versiforme. Protoplasma 217, 154–165.
Blancaflor, E. B., Hou, G. and Chapman, K. D. (2003) Elevated levels of N-Lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings. Planta 217, 206–217.
Blancaflor, E. B. and Hasenstein, K. H. (1997) The organization of the actin cytoskeleton in vertical and graviresponding primary roots of maize. Plant Physiol. 113, 1447–1455.
Hou, G., Mohamalawari, D. R. and Blancaflor, E. B. (2003) Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton. Plant Physiol. 131, 1360–1373.
Hou, G., Kramer, V. L., Wang, Y.-S., Chen, R., Perbal, G., Gilroy, S. and Blancaflor, E. B. (2004) The promotion of gravitropism in Arabidopsis roots upon actin disruption is coupled with the extended alkalinization of the columella cytoplasm and a persistent lateral auxin gradient. Plant J. 31, 113–125.
Acknowledgements
Work on the plant cytoskeleton in the authors’ laboratory is supported by the Noble Foundation and grants from the National Science Foundation (DBI-0722635) and the Oklahoma Center for the Advancement of Science and Technology (OCAST PSB-003).
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Dyachok, J., Yoo, CM., Palanichelvam, K., Blancaflor, E.B. (2009). Sample Preparation for Fluorescence Imaging of the Cytoskeleton in Fixed and Living Plant Roots. In: Gavin, R. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology, vol 586. Humana Press. https://doi.org/10.1007/978-1-60761-376-3_8
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DOI: https://doi.org/10.1007/978-1-60761-376-3_8
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