Grønlund, Jesper T., Eyres, Alison, Kumar, Sanjeev, Buchanan-Wollaston, Vicky and Gifford, M. L.. (2012) Cell specific analysis of Arabidopsis leaves using fluorescence activated cell sorting. Journal of Visualized Experiments (No.68). e4214. ISSN 1940-087X

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Abstract

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated. To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6. FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Item Type:	Journal Article
Subjects:	Q Science > QK Botany
Divisions:	Faculty of Science > Life Sciences (2010- ) Faculty of Science > Centre for Systems Biology
Library of Congress Subject Headings (LCSH):	Arabidopsis -- Analysis -- Methodology, Plant cells and tissues -- Analysis -- Methodology, Plant gene expression
Journal or Publication Title:	Journal of Visualized Experiments
Publisher:	JoVE
ISSN:	1940-087X
Date:	October 2012
Number:	No.68
Page Range:	e4214
Identification Number:	10.3791/4214
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Sixth Framework Programme (European Commission) (FP6), Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC)
Grant number:	LSHG-CT-2006-037704 (FP6), BB/H019502/1 (BBSRC)
References:	1. Efroni, I., Eshed, Y., & Lifschitz, E. Morphogenesis of Simple and Compound Leaves: A Critical Review. Plant Cell. 22, 1019-1032, Review., (2010). 2. Kleber, R. & Kehr, J. Preparation and Quality Assessment of RNA From Cell-Specific Samples Obtained by Laser Microdissection. Methods Mol. Biol. 323, 367-377 (2006). 3. Birnbaum, K., Shasha, D.E., Wang, J.Y., Jung, J.W., Lamberts, G.M., Galbraith, D.W., & Benfey, P.N. A Gene Expression Map of the Arabidopsis root. Science. 302, 1956-1960 (2003). 4. Warnasooriya, S.N. & Montgomery, B.L. Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana. J. Vis. Exp. (39), e1925, DOI: 10.3791/1925 (2010). 5. Beal, R.B. & Henikoff, S. A Simple Method For Gene Expression and Chromatin Profiling of Individual Cell Types Within a Tissue. Dev. Cell. 18, 1030-1040 (2010). 6. Mustroph, A., Zanetti, M.E., Jang, C.J.H., Holtan, H.E., Repetti, P.P., Galbraith, D.W., Girke, T., & Bailey-Serres, J. Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. PNAS. 106, 18843-18848 (2009). 7. Brady S.M., Orlando D.A., Lee J.Y., Wang, J.Y., Koch J., Dinneny J.R., Mace D., Ohler U., & Benfey P.N. Dominant Root Expression Patterns Revealed in a High-Resolution Spatiotemporal Expression Map. Science. 318, 801-806 (2007). 8. Gifford. M.L., Dean, A., Gutierrez, R.A., Coruzzi, G.M., & Birnbaum, K.D. Cell-specific nitrogen responses mediate developmental plasticity. PNAS. 105, 803-808 (2008). 9. Dinneny J.R., Long T.A., Wang J.Y., Jung J.W., Mace D. Pointer S., Barron C., Brady S.M., Schiefelbein J., & Benfey P.N. Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science. 320, 942-945 (2008). 10. Peterson, S.V., Johansson, A.I., Kowalczyk, M., Makoveychuk, A., Wang, J.Y., Moritz, T., Grebe, M., Benfey, P.N. 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. Plant Cell. 21, 1659-1668 (2009). 11. Yadav, R.K., Girke, T., Pasala, S., Xie, M., & Reddy, G.V. Gene expression map of the Arabidopsis shoot apical meristem stem cell niche. PNAS. 106, 4941-4946 (2009). 12. Harkins, K.R., Jefferson, R.A., Kavanagh, T.A., Bevan, M.W., & Galbraith, D.W. Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts. PNAS. 87, 816-820 (1990). 13. Yang, Y., Costa, A., Leonhardt, N., Siegel, R.S., & Schroeder, J.I. Isolation of a strong Arabidopsis guard cell promoter and it's potential as a research tool. Plant Methods. 4, 6 (2008). 14. Gardner, M.J., Baker, A.J., Assie, J.-M., Poethig, R.S., Haseloff, J.P., & Webb, A.A.R. GAL4 GFP enhancer trap lines for analysis of stomatal guard cell development and gene expression. J. Exp. Bot. 60, 213-226 (2009).
URI:	http://wrap.warwick.ac.uk/id/eprint/51017

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