Abstract
Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.
This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Bell, P. R. and Helmsley, A. R. (2000) Green Plants: Their Origin and Diversity. Cambridge University Press, Cambridge, p. 361.
Harkins, K. R. and Galbraith, D. W. (1984) Flow sorting and culture of plant protoplasts. Physiol Plant 60, 43–52.
Galbraith, D. W. (1990) Isolation and flow cytometric characterization of plant protoplasts. Methods Cell Biol 33, 527–547.
Galbraith, D. W., Bartos, J., and Dolezel, J. (2005) Flow cytometry and cell sorting in plant biotechnology. In Flow Cytometry in Biotechnology (Sklar, L.A., ed.), Oxford University Press, New York, pp. 291–322.
Galbraith, D. W., Grebenok, R. J., Lambert, G. M., and Sheen, J. (1995) Flow cytometric analysis of transgene expression in higher plants: green fluorescent protein. Methods Cell Biol 50, 3–12.
Sheen, J., Hwang, S., Niwa, Y., Kobayashi, H., and Galbraith, D. W. (1995) Green fluorescent protein as a new vital marker in plant cells. Plant J 8, 777–784.
Galbraith, D. W., Herzenberg, L. A., and Anderson, M. (1999) Flow cytometric analysis of transgene expression in higher plants: green fluorescent protein. Methods Enzymol 320, 296–315.
Birnbaum, K., Shasha, D. E., Wang, J. Y., Jung, J. W., Lambert, G. M., Galbraith, D. W., and Benfey, P. N. (2003) A gene expression map of the Arabidopsis root. Science 302, 1956–1960.
Birnbaum, K., Jung, J. W., Wang, J. Y., Lambert, G. M., Hirst, J. A., Galbraith, D. W., and Benfey, P. N. (2005) Cell-type specific expression profiling in plants using fluorescent reporter lines, protoplasting, and cell sorting. Nat Methods 2, 1–5.
Yadav, R. K., Girke, T., Pasala, S., Xie, M. T., and Reddy, V. (2009) Gene expression map of the Arabidopsis shoot apical meristem stem cell niche. Proc Natl Acad Sci U S A 106, 4941–4946.
Sheen, J. (2002) A transient expression assay using Arabidopsis mesophyll protoplasts. http://genetics.mgh.harvard.edu/sheenweb/
Petersson, S. V., Johansson, A. I., Kowalczyk, M., Makoveychuk, A., Wang, J. Y., Moritz, T., Grebe, M., Benfey, P. N., Sandberg, G., and Ljung, K (2009) 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.
Galbraith, D. W., Harkins, K. R., and Jefferson, R. A. (1988) Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts. Cytometry 9, 75–83.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.
Snapp, E. L. (2009) Fluorescent proteins: a cell biologist’s user guide. Trends Cell Biol 19, 649–655.
Berg, R. H. and Beachy, R. N. (2008) Fluorescent protein applications in plants. Methods Cell Biol 85, 153–177.
Galbraith, D. W. (2004) The rainbow of fluorescent proteins. Methods Cell Biol 75, 153–169.
Nelson, B. K., Cai, X., and Nebenfuehr, A. (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51, 1126–1136.
Millar, A. H., Carrie, C., Pogson, B., and Whelan, J. (2009) Exploring the function-location nexus: using multiple lines of evidence in defining the subcellular location of plant proteins. Plant Cell 21, 1625–1631.
Grebenok, R. J., Pierson, E. A., Lambert, G. M., Gong, F. -C., Afonso, C. L., Haldeman-Cahill, R., Carrington, J. C., and Galbraith, D. W. (1997) Green-fluorescent protein fusions for efficient characterization of nuclear localization signals. Plant J 11, 573–586.
Grebenok, R. J., Lambert, G. M., and Galbraith, D. W. (1997) Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants. Plant J 12, 685–696.
Chytilova, E., Macas, J., Sliwinska, E., Rafelski, S., Lambert, G. M., and Galbraith, D. W. (2000) Nuclear dynamics in Arabidopsis thaliana. Mol Biol Cell 11, 2733–2741.
Zhang, C. Q., Gong, F. C., Lambert, G. M., and Galbraith, D. W. (2005) Cell type-specific characterization of nuclear DNA contents within complex tissues and organs. Plant Methods 1, 7, doi:10.1186/1746-4811-1-7.
Zhang, C. Q., Barthelson, R. A., Lambert, G. M., and Galbraith, D. W. (2008) Characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiol 147, 30–40.
Harkins, K. R., Jefferson, R. A., Kavanagh, T. A., Bevan, M. W., and Galbraith, D. W. (1990) Expression of photosynthesis-related gene fusions is restricted by cell-type in transgenic plants and in transfected protoplasts. Proc Natl Acad Sci USA 87, 816–820.
Nawy, T., Lee, J. -Y., Colinas, J., Wang, J. Y., Thongrod, S. C., Malamy, J. E., Birnbaum, K., and Benfey, P. N. (2005) Transcriptional profile of the Arabidopsis root quiescent center. Plant Cell 17, 1908–1925.
Brady, S. M., Orlando, D. A., Lee, J. Y., Wang, J. Y., Koch, J., Dinneny, J. R., Mace, D., Ohler, U., and Benfey, P. N. (2007) A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 318, 801–806.
Dinneny, J. R., Long, T. A., Wang, J. Y., Jung, J. W., Mace, D., Pointer, S., Barron, C., Brady, S. M., Schiefelbein, J., and Benfey, P. N. (2008) Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science 320, 942–945.
Bargmann, B. O. R. and Birnbaum, K. D. (2009) Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts. Plant Physiol 149, 1231–1239.
Galbraith, D. W. and Lucretti, S. (2000) Large particle sorting. In Flow Cytometry and Cell Sorting, 2nd edition (Radbruch, A., ed.), Springer-Verlag, Berlin, pp. 293–317.
Harkins, K. R. and Galbraith, D. W. (1987) Factors governing the flow cytometric analysis and sorting of large biological particles. Cytometry 8, 60–71.
Galbraith, D. W., Harkins, K. R., Maddox, J. R., Ayres, N. M., Sharma, D. P., and Firoozabady, E. (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051.
Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470.
Deyholos, M. K. and Galbraith, D. W. (2001) High-density DNA microarrays for gene expression analysis. Cytometry 43, 229–238.
Galbraith, D. W. (2006) Microarray analyses in higher plants. OMICS 10, 455–473.
Wilhelm, B. T. and Landry, J. R. (2009) RNA-Seq-quantitative measurement of expression through massively parallel RNA-sequencing. Methods 48, 249–257.
Larson, D. R., Singer, R. H., and Zenklusen, D. (2009) A single molecule view of gene expression. Trends Cell Biol 19, 630–637.
Applied Biosystems Technical Application Note (2008) SOLiD™ System 2.0 Library preparation protocol for the whole transcriptome analysis of a single cell.
Kurimoto, K., Yabuta, Y., Ohinata, Y., and Saitou, M. (2007) Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis. Nat Protoc 2, 739–752.
Fare, T. L., Coffey, E. M., Dai, H. Y., He, Y. D. D., Kessler, D. A., Kilian, K. A., Koch, J. E., LeProust, E., Marton, M. J., Meyer, M. R., Stoughton, R. B., Tokiwa, G. Y., and Wang, Y. Q. (2003) Effects of atmospheric ozone on microarray data quality. Anal Chem 75, 4672–4675.
Skibbe, D. S., Wang, X. J., Zhao, X. F., Borsuk, L. A., Nettleton, D., and Schnable, P. S. (2006) Scanning microarrays at multiple intensities enhances discovery of differentially expressed genes. Bioinformatics 22, 1863–1870.
Acknowledgments
Part of the development of the methods described in this chapter was done with support from the NSF Plant Genome program.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Galbraith, D.W., Janda, J., Lambert, G.M. (2011). Multiparametric Analysis, Sorting, and Transcriptional Profiling of Plant Protoplasts and Nuclei According to Cell Type. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 699. Humana Press. https://doi.org/10.1007/978-1-61737-950-5_20
Download citation
DOI: https://doi.org/10.1007/978-1-61737-950-5_20
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61737-949-9
Online ISBN: 978-1-61737-950-5
eBook Packages: Springer Protocols