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http://dx.doi.org/10.14348/molcells.2016.0209

Optimized Methods for the Isolation of Arabidopsis Female Central Cells and Their Nuclei  

Park, Kyunghyuk (Department of Biological Sciences, Seoul National University)
Frost, Jennifer M. (Department of Plant and Microbial Biology, University of California)
Adair, Adam James (Department of Plant and Microbial Biology, University of California)
Kim, Dong Min (Department of Biological Sciences, Seoul National University)
Yun, Hyein (Department of Biological Sciences, Seoul National University)
Brooks, Janie S. (Department of Science, Seoul Foreign School)
Fischer, Robert L. (Department of Plant and Microbial Biology, University of California)
Choi, Yeonhee (Department of Biological Sciences, Seoul National University)
Publication Information
Molecules and Cells / v.39, no.10, 2016 , pp. 768-775 More about this Journal
Abstract
The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75-90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction.
Keywords
Arabidopsis central cell; embryo sac; nuclei isolation; protoplast;
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1 Bechtold, N., and Pelletier, G. (1998). In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol. Biol. 82, 259-266.
2 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 via cell sorting of protoplasts from fluorescent reporter lines. Nat. Methods 2, 615-619.   DOI
3 Boyes, D.C., Zayed, A.M., Ascenzi, R., McCaskill, A.J., Hoffman, N.E., Davis, K.R.,, and Gorlach, J. (2001). Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13, 1499-1510.   DOI
4 Chen, J., Yi, Q., Song, Q., Gu, Y., Zhang, J., Hu, Y., Liu, H., Liu, Y., Yu, G., and Huang, Y. (2015). A highly efficient maize nucellus protoplast system for transient gene expression and studying programmed cell death-related processes. Plant Cell Rep. 34, 1239-1251.   DOI
5 Choi, Y., Gehring, M., Johnson, L., Hannon, M., Harada, J.J., Goldberg, R.B., Jacobsen, S.E., and Fischer, R.L. (2002). DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Cell 110, 33-42.   DOI
6 Cocking, E.C. (1960). A method for the isolation of plant protoplasts and vacuoles. Nature 187, 962-963.   DOI
7 Deal, R.B., and Henikoff, S. (2010). A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev. Cell 18, 1030-1040.   DOI
8 Deal, R.B., and Henikoff, S. (2011). The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat. Protoc. 6, 56-68.   DOI
9 Faraco, M., Di Sansebastiano, G.P., Spelt, K., Koes, R.E., and Quattrocchio, F.M. (2011). One protoplast is not the other! Plant Physiol. 156, 474-478.   DOI
10 Fischer, R., and Hain, R. (1995). Tobacco protoplast transformation and use for functional analysis of newly isolated genes and gene constructs. Methods Cell. Biol. 50, 401-410.   DOI
11 Gehring, M., Bubb, K.L., and Henikoff, S. (2009). Extensive demethylation of repetitive elements during seed development underlies gene imprinting. Science 324, 1447-1451.   DOI
12 Gronlund, J.T., Eyres, A., Kumar, S., Buchanan-Wollaston, V. and Gifford, M.L. (2012). Cell specific analysis of Arabidopsis leaves using fluorescence activated cell sorting. J. Vis. Exp. 4, pii: 4214.
13 Harholt, J., Suttangkakul, A., and Vibe Scheller, H. (2010). Biosynthesis of pectin. Plant Physiol. 153, 384-395.   DOI
14 Henry, G.L., Davis, F.P., Picard, S., and Eddy, S.R. (2012). Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704.   DOI
15 Hsieh, T.F., Shin, J., Uzawa, R., Silva, P., Cohen, S., Bauer, M.J., Hashimoto, M., Kirkbride, R.C., Harada, J.J., Zilberman, D., et al. (2011). Regulation of imprinted gene expression in Arabidopsis endosperm. Proc. Natl. Acad. Sci. USA 108, 1755-1762.   DOI
16 Hong, S.-Y., Seo, P.J., Cho, S.-H., and Park, C.-M. (2012). Preparation of leaf mesophyll protoplasts for transient gene expression in Brachypodium distachyon. J. Plant Biol. 55, 390-397.   DOI
17 Hoshino, Y., Murata, N., and Shinoda, K. (2006). Isolation of individual egg cells and zygotes in Alstroemeria followed by manual selection with a microcapillary-connected micropump. Ann Bot 97, 1139-1144.   DOI
18 Hsieh, T.F., Ibarra, C.A., Silva, P., Zemach, A., Eshed-Williams, L., Fischer, R.L., and Zilberman, D. (2009). Genome-wide demethylation of Arabidopsis endosperm. Science 324, 1451-1454.   DOI
19 Im, J.H., and Yoo, S.D. (2014). Transient expression in Arabidopsis leaf mesophyll protoplast system for cell-based functional analysis of MAPK cascades signaling. Methods Mol. Biol. 1171, 3-12.   DOI
20 Ibarra, C.A., Feng, X., Schoft, V.K., Hsieh, T.F., Uzawa, R., Rodrigues, J.A., Zemach, A., Chumak, N., Machlicova, A., Nishimura, T., et al. (2012). Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, 1360-1364.   DOI
21 Kelley, D.R., and Gasser, C.S. (2009). Ovule development: genetic trends and evolutionary considerations. Sex Plant Reprod. 22, 229-234.   DOI
22 Kim, M., Ohr, H., Lee, J.W., Hyun, Y., Fischer, R.L., and Choi, Y. (2008). Temporal and spatial downregulation of Arabidopsis MET1 activity results in global DNA hypomethylation and developmental defects. Mol. Cells 26, 611-615.
23 Ohyama, K., Gamborg, O.L., and Miller, R.A. (1972). Isolation and properties of deoxyribonucleic acid from protoplasts of cell suspension cultures of Ammi visnaga and Carrot (Daucus carota). Plant Physiol. 50, 319-321.   DOI
24 Lenhard, M., Bohnert, A., Jurgens, G., and Laux, T. (2001). Termination of stem cell maintenance in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS. Cell 105, 805-814.   DOI
25 Long, J.A., Moan, E.I., Medford, J.I., and Barton, M.K. (1996). A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379, 66-69.   DOI
26 Moreno-Romero, J., Jiang, H., Santos-Gonzalez, J., and Kohler, C. (2016). Parental epigenetic asymmetry of PRC2-mediated histone modifications in the Arabidopsis endosperm. EMBO J. 35, 1298-1311.   DOI
27 Pillitteri, L.J., Bemis, S.M., Shpak, E.D., and Torii, K.U. (2007). Haploinsufficiency after successive loss of signaling reveals a role for ERECTA-family genes in Arabidopsis ovule development. Development 134, 3099-3109.   DOI
28 Rose, A., and Meier, I. (2001). A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim. Proc. Natl. Acad. Sci. USA 98, 15377-15382.   DOI
29 Schapire, A.L., and Lois, L.M. (2016). A simplified and rapid method for the isolation and transfection of Arabidopsis leaf mesophyll protoplasts for large-scale applications. Methods Mol. Biol. 1363, 79-88.   DOI
30 Schmidt, A., Schmid, M.W., and Grossniklaus, U. (2012). Analysis of plant germline development by high-throughput RNA profiling: technical advances and new insights. Plant J. 70, 18-29.   DOI
31 Sheen, J. (1993). Protein phosphatase activity is required for light-inducible gene expression in maize. EMBO J. 12, 3497-3505.
32 Steffen, J.G., Kang, I.H., Macfarlane, J., and Drews, G.N. (2007). Identification of genes expressed in the Arabidopsis female gametophyte. Plant J. 51, 281-292.   DOI
33 Sheen, J. (2001). Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol. 127, 1466-1475.   DOI
34 Shepard, J.F., and Totten, R.E. (1977). Mesophyll cell protoplasts of potato: isolation, proliferation, and plant regeneration. Plant Physiol. 60, 313-316.   DOI
35 Slotkin, R.K., Vaughn, M., Borges, F., Tanurdzic, M., Becker, J.D., Feijo, J.A., and Martienssen, R.A. (2009). Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136, 461-472.   DOI
36 Truernit, E., and Haseloff, J. (2007). A role for KNAT class II genes in root development. Plant Signal. Behav. 2, 10-12.   DOI
37 Weinhofer, I., Hehenberger, E., Roszak, P., Hennig, L., and Kohler, C. (2010). H3K27me3 profiling of the endosperm implies exclusion of polycomb group protein targeting by DNA methylation. PLoS Genet. 6.
38 Wu, F.H., Shen, S.C., Lee, L.Y., Lee, S.H., Chan, M.T., and Lin, C.S. (2009). Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method. Plant Methods 5, 16.   DOI
39 Wuest, S.E., Vijverberg, K., Schmidt, A., Weiss, M., Gheyselinck, J., Lohr, M., Wellmer, F., Rahnenfuhrer, J., von Mering, C., and Grossniklaus, U. (2010). Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes. Curr. Biol. 20, 506-512.   DOI
40 Yadegari, R., and Drews, G.N. (2004). Female gametophyte development. Plant Cell 16 Suppl, S133-141.   DOI
41 Yoo, S.D., Cho, Y.H., and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565-1572.   DOI
42 Zhang, Y., Su, J., Duan, S., Ao, Y., Dai, J., Liu, J., Wang, P., Li, Y., Liu, B., Feng, D., et al. (2011). A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7, 30.   DOI
43 Zhai, Z., Jung, H.I., and Vatamaniuk, O.K. (2009). Isolation of protoplasts from tissues of 14-day-old seedlings of Arabidopsis thaliana. J. Vis. Exp. 17, pii: 1149.