Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families

Plant Methods

Jari PT Valkonen¹  Vidmantas Stanys²  Danas Baniulis²  Minna-Liisa Rajamäki¹  Sidona Sikorskaite¹ 

[1] Department of Agricultural Sciences, University of Helsinki, PO Box 27, FIN-00014 Helsinki, Finland;Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Kaunas st 30, Babtai, LT-54333, Kaunas, Lithuania

关键词: Apple;    Potato;    Tobacco;    Rosaceae;    Solanaceae;    Nuclei isolation;   

Others  :  812656
DOI  :  10.1186/1746-4811-9-31

 received in 2013-05-03, accepted in 2013-07-24,  发布年份 2013

【 摘 要 】

In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.

【 授权许可】

   
2013 Sikorskaite et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Shaw PJ: Nuclear organization in plants. Essays in Biochem 1996, 31:77-89.
• [2]Shaw PJ, Brown JW: Plant nuclear bodies. Curr Opin Plant Biol 2004, 7(6):614-620.
• [3]Folta KM, Kaufman LS: Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Nat Protoc 2006, 1(6):3094-3100.
• [4]Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS: Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci U S A 2005, 102(10):3691-3696.
• [5]Hoffer P, Ivashuta S, Pontes O, Vitins A, Pikaard C, Mroczka A, Wagner N, Voelker T: Posttranscriptional gene silencing in nuclei. Proc Natl Acad Sci 2011, 108(1):409-414.
• [6]Lutz K, Wang W, Zdepski A, Michael T: Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing. BMC Biotechnology 2011, 11(1):54. BioMed Central Full Text
• [7]Galbraith D, Harkins K, Maddox J, Ayres N, Sharma D, Firoozabady E: Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 1983, 220(4601):1049-1051.
• [8]Arumuganathan K, Earle E: Estimation of nuclear DNA content of plants by flow cytometry. Plant Mol Biol Rep 1991, 9(3):229-241.
• [9]Doleźel J, Greilhuber J, Suda J: Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2007, 2:2233-2244.
• [10]Cires E, Cuesta C, Fernández Casado MÁ, Nava HS, Vázquez VM, Fernández Prieto JA: solation of plant nuclei suitable for flow cytometry from species with extremely mucilaginous compounds: an example in the genus Viola L. (Violaceae). An Jar Bot Madr 2011, 68(2):139-154.
• [11]Loureiro J, Rodriguez E, Doležel J, Santos C: Comparison of four nuclear isolation buffers for plant DNA flow cytometry. Ann Bot 2006, 98(3):679-689.
• [12]Bae MS, Cho EJ, Choi E, Park OK: Analysis of the Arabidopsis nuclear proteome and its response to cold stress. Plant J 2003, 36(5):652-663.
• [13]Jones AME, MacLean D, Studholme DJ, Serna-Sanz A, Andreasson E, Rathjen JP, Peck SC: Phosphoproteomic analysis of nuclei-enriched fractions from Arabidopsis thaliana. J Proteomics 2009, 72(3):439-451.
• [14]Khan MMK, Komatsu S: Rice proteomics: recent developments and analysis of nuclear proteins. Phytochemistry 2004, 65(12):1671-1681.
• [15]Repetto O, Rogniaux H, Firnhaber C, Zuber H, Küster H, Larré C, Thompson R, Gallardo K: Exploring the nuclear proteome of Medicago truncatula at the switch towards seed filling. Plant J 2008, 56(3):398-410.
• [16]Pandey A, Choudhary MK, Bhushan D, Chattopadhyay A, Chakraborty S, Datta A, Chakraborty N: The nuclear proteome of chickpea (Cicer arietinum L.) reveals predicted and unexpected proteins. J Proteome Res 2006, 5(12):3301-3311.
• [17]Pendle AF, Clark GP, Boon R, Lewandowska D, Lam YW, Andersen J, Mann M, Lamond AI, Brown JWS, Shaw PJ: Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions. Mol Biol Cell 2005, 16(1):260-269.
• [18]Calikowski TT, Meulia T, Meier I: A proteomic study of the Arabidopsis nuclear matrix. J Cell Biochem 2003, 90(2):361-378.
• [19]Tan F, Li G, Chitteti BR, Peng Z: Proteome and phosphoproteome analysis of chromatin associated proteins in rice (Oryza sativa). Proteomics 2007, 7(24):4511-4527.
• [20]Aki T, Yanagisawa S: Application of rice nuclear proteome analysis to the identification of evolutionarily conserved and glucose-responsive nuclear proteins. J Proteome Res 2009, 8(8):3912-3924.
• [21]Pandey A, Chakraborty S, Datta A, Chakraborty N: Proteomics approach to identify dehydration responsive nuclear proteins from chickpea (Cicer arietinum L.). Mol Cell Proteomics 2008, 7(1):88-107.
• [22]Choudhary MK, Basu D, Datta A, Chakraborty N, Chakraborty S: Dehydration-responsive nuclear proteome of rice (Oryza sativa L.) illustrates protein network, novel regulators of cellular adaptation, and evolutionary perspective. Mol Cell Proteomics 2009, 8(7):1579-1598.
• [23]Ohyama K, Lawrence EP, Horn D: A rapid, simple method for nuclei isolation from plant protoplasts. Plant Physiol 1977, 60(2):179-181.
• [24]Saxena PK, Fowke LC, King J: An efficient procedure for isolation of nuclei from plant protoplasts. Protoplasma 1985, 128(2–3):184-189.
• [25]Masuda K, Takahashi S, Nomura K, Inoue M: A simple procedure for the isolation of pure nuclei from carrot embryos in synchronized cultures. Plant Cell Rep 1991, 10(6–7):329-333.
• [26]Yamaguchi J, Lim P, Akazawa T: Isolation and characterization of nuclei from rice embryo. Cell Struct Funct 1992, 17:87-92.
• [27]Renouard S, Cyrielle C, Lopez T, Lamblin F, Laine E, Hano C: Isolation of nuclear proteins from flax (Linum usitatissimum L.) seed coats for gene expression regulation studies. BMC Res Notes 2012, 5(1):15. BioMed Central Full Text
• [28]Willmitzer L, Wagner KG: The isolation of nuclei from tissue-cultured plant cells. Exp Cell Res 1981, 135(1):69-77.
• [29]Ribeiro Silva TC, Santiago Abreu I, Carvalho CR: Improved and reproducible flow cytometry methodology for nuclei isolation from single root meristem. J Bot 2010, 2010:1-7.
• [30]Zhang H, Zhao X, Ding X, Paterson AH, Wing RA: Preparation of megabase-size DNA from plant nuclei. Plant J 1995, 7(1):175-184.
• [31]Cushman JC: Isolation of nuclei suitable for in vitro transcriptional studies. In Methods in Cell Biology. Volume 50. Edited by Galbraith DW, Bourque DP, Bohnert HJ. San Diego: Academic Press; 1995:113-128.
• [32]Loureiro J, Rodriguez E, Doležel J, Santos C: Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann Bot 2007, 100(4):875-888.
• [33]Guilfoyle TJ: Isolation and characterization of plant nuclei. In Methods in Cell Biology. Volume 50. Edited by Galbraith DW, Bourque DP, Bohnert HJ. San Diego: Academic Press; 1995:101-112.
• [34]McKeown P, Pendle AF, Shaw PJ: Preparation of Arabidopsis nuclei and nucleoli. In The Nucleus. Volume 1. Edited by Hancock R. New York: Humana Press; 2008:67-75.
• [35]Carrier G, Santoni S, Rodier-Goud M, Canaguier A: Kochko Ad, Dubreuil-Tranchant C, This P, Boursiquot J, Cunff LL: an efficient and rapid protocol for plant nuclear DNA preparation suitable for next generation sequencing methods. Am J Bot 2011, 98(1):e13-e15.
• [36]Deal RB, Henikoff S: The INTACT method for cell type–specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat Protoc 2011, 6:56-68.
• [37]Abdalla KO, Thomson JA, Rafudeen MS: Protocols for nuclei isolation and nuclear protein extraction from the resurrection plant Xerophyta viscosa for proteomic studies. Anal Biochem 2009, 384(2):365-367.
• [38]Valkonen J, Slack S, Plaisted R, Watanabe K: Extreme resistance is epistatic to hypersensitive resistance to potato virus Y in a Solanum tuberosum ssp. andigena -derived potato genotype. Plant Dis 1994, 78(12):1177-1180.
• [39]Meier U (Ed): Growth stages of mono- and dicotyledonous plants In BBCH Monograph. 2nd edition. : Federal Biological Research Centre for Agriculture and Forestry; 2001.
• [40]Busk PK, Pagés M: Microextraction of nuclear proteins from single maize embryos. Plant Mol Biol Rep 1997, 15(4):371-376.40.
• [41]Vlasák J: Effect of different disintegration techniques and media on yield and appearance of isolated nuclei. Biol Plant 1981, 23(6):406-413.
• [42]Watson JC, Thompson WF: Purification and restriction endonuclease analysis of plant nuclear DNA. In Methods in Enzymology. Volume 118. Edited by Weissbach A, Weissbach H. San Diego: Academic Press; 1986:57-75.
• [43]Graham J, Rickwood D: Subcellular fractionation: a practical approach. Oxford: IRL Press; 1997.
• [44]Harford JB, Bonifacino JS: Subcellular fractionation and isolation of organelles. Curr Protoc Cell Biol 2010, 48:3.0.1-3.0.8.
• [45]Millar AH, Liddell A, Leaver CJ: Isolation and subfractionation of mitochondria from plant. In Methods in Cell Biology. Volume 80. Edited by Pon LA, Schon EA. San Diego: Academic Press; 2001:65-90.