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Flow cytometric analysis of protein content in Taxus protoplasts and single cells as compared to aggregated suspension cultures

  • Cell Biology and Morphogenesis
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Abstract

Plant suspension cultures are highly aggregated, preventing the direct application of flow cytometry for the study of population dynamics. The utility of single cells to accurately represent aggregated suspension cultures was tested through the analysis of total protein content. Specifically, protein content of two Taxus cuspidata suspension culture lines was studied using the Bradford assay for aggregated suspension cultures, and flow cytometry with fluorescein isothiocyanate staining for protoplasts and single cells. Taxus protein levels were measured at 75–160 mg per gram dry weight via the Bradford assay. Aggregated suspension cultures, protoplasts, and single cells predicted the same trend of protein content over the culture period (21 days). Normalized protein content of isolated single cells was statistically equivalent to aggregated suspensions for both cell lines. However, normalized protein content of isolated protoplasts showed significant differences from aggregated suspensions for one of the two cell lines. Elicitation with methyl jasmonate (MJ) is commonly utilized to increase paclitaxel accumulation in suspension cultures, and therefore the effect of MJ elicitation on protein content in aggregated suspensions, isolated single cells and protoplasts was assessed. Aggregated suspension cultures, protoplasts, and single cells did not show any change in total protein content following elicitation with MJ at 200 μM on day 7. This study illustrates the usefulness of flow cytometry for obtaining culture population information and the value of using intact single cells for the study of plant metabolism.

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References

  • Aoyagi H, DiCosmo F, Tanaka H (2002) Efficient paclitaxel production using protoplasts isolated from cultured cells of Taxus cuspidata. Planta Med 68:420–424

    Article  CAS  PubMed  Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of dye-binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brincat M, Gibson DM, Shuler ML (2002) Alterations in Taxol production in plant cell culture via manipulation of the phenylalanine ammonia lyase pathway. Biotechnol Prog 18:1149–1156

    Article  CAS  PubMed  Google Scholar 

  • Choi H, Kim S, Song J, Son J, Hong S, Durzan DJ, Lee HJ (2001) Localization of paclitaxel in suspension cultures of Taxus chinensis. J Microbiol Biotechnol 11:458–462

    CAS  Google Scholar 

  • Crissman HA, Darzynkiewicz Z, Tobey RA, Steinkamp JA (1985) Correlated measurements of DNA, RNA, and protein in individual cells by flow cytometry. Science 228:1321–1324

    Google Scholar 

  • Do C, Cormier F (1991) Effect of low nitrate and high sugar concentration on the accumulation of anthocyanins in grape (Vitis vinifera L.) cell suspensions. Plant Cell Rep 9:500–504

    CAS  Google Scholar 

  • Dong H, Zhong J (2001) Significant improvement of taxane production in suspension cultures of Taxus chinensis by combining elicitation with sucrose feed. Biochem Eng J 8:145–150

    Article  CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    CAS  PubMed  Google Scholar 

  • Gray JW, Dolbeare F, Pallavicini MG (1990) Quantitative cell-cycle analysis. In: Melamed MR, Lindmo T, Mendelsohn ML (eds) Flow cytometry and sorting. Wiley-Liss, New York, pp 445–468

    Google Scholar 

  • Hermsmeier D, Schittko U, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. I. Large-scale changes in accumulation of growth- and defense-related plant mRNAs. Plant Physiol 125:683–700

    Article  CAS  PubMed  Google Scholar 

  • Ketchum R, Gibson DM, Croteau RB, Shuler ML (1999) The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate. Biotechnol Bioeng 62:97–105

    Article  CAS  PubMed  Google Scholar 

  • Kim J, Yun JH, Hwang YS, Byun SY, Kim DI (1995) Production of taxol and related taxanes in Taxus brevifolia cell cultures: effect of sugar. Biotechnol Lett 17:101–106

    CAS  Google Scholar 

  • Kim S, Choi H, Kim J, Lee H, Hong S (2001) Effect of osmotic pressure on paclitaxel production in suspension cell cultures of Taxus chinensis. Enzyme Microb Technol 28:202–209

    Article  CAS  PubMed  Google Scholar 

  • Laskaris G, Bounkhay M, Theodoris G, van der Heijden R, Verpoorte R, Jaziri M (1999) Induction of geranylgeranyl diphosphate synthase activity and taxane accumulation in Taxus baccata cell cultures after elicitation by methyl jasmonate. Plant Sci 147:1–8

    Article  CAS  Google Scholar 

  • Mirjalili N, Linden JC (1996) Methyl jasmonate induced production of Taxol in suspension cultures of Taxus cuspidata. Biotechnol Bioeng 12:110–118

    Article  CAS  Google Scholar 

  • Naill M, Roberts SC (2004) Preparation of isolated cells from Taxus supension cultures for population analysis. Biotechnol Bioeng 86:817–826

    Article  CAS  PubMed  Google Scholar 

  • Roberts SC, Naill M, Gibson DM, Shuler ML (2003) Localization of paclitaxel storage and a simple method for enhancing paclitaxel release from Taxus cell suspension cultures. Plant Cell Rep 21:1217–1220

    Article  CAS  PubMed  Google Scholar 

  • Rudge K, Morris P (1983) The effect of osmotic stress on growth and alkaloid accumulation in Cathanranthus roseus. In: Morris P, Scragg AH, Stafford A, Fowler MW (eds) Secondary metabolism in plant cell cultures. Cambridge University Press, Cambridge, pp 75–82

    Google Scholar 

  • Swiatek A, Lenjou M, Van Bockstaele D, Inze D, Van Onckelen H (2002) Differential effect of jasmonic acid and abscisic acid on cell cycle progression in Tobacco BY-2 cells. Plant Physiol 128:201–211

    Article  CAS  PubMed  Google Scholar 

  • Wink M (1999) Biochemistry, role, and biotechnology of secondary metabolites. In: Wink M (ed) Functions of plant secondary metabolites and their exploitation in biotechnology. Sheffield Academic, Sheffield, pp 1–17

    Google Scholar 

  • Yukimune Y, Tabata H, Higashi Y, Hara Y (1996) Methyl jasmonate-induced overproduction of paclitaxel and baccatin III in Taxus cell suspension cultures. Nat Biotechnol 14:1129–1132

    PubMed  Google Scholar 

  • Zhang Y, Zhong JJ, Yu JT (1995) Effect of osmotic pressure on cell growth and production of ginseng saponin and polysaccharide in suspension cultures of Panax notoginseng. Biotechnol Lett 17:101–106

    CAS  Google Scholar 

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Acknowledgements

This work was supported by the National Science Foundation (BES 9984463). The authors would like to thank Dr. Donna Gibson of the US Plant Soil and Nutrition Laboratory of the USDA for the Taxus cell cultures and Dr. Barbara Osborne and coworkers for assistance with flow cytometry. Michael Naill additionally acknowledges support from a National Research Service Award T32 GM08515 from the National Institutes of Health and from a University of Massachusetts Faculty Research Grant.

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  1. Department of Chemical Engineering, 159 Goessmann Laboratory, University of Massachusetts-Amherst, Amherst, MA, 01003, USA

    Michael C. Naill & Susan C. Roberts

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  1. Michael C. Naill
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  2. Susan C. Roberts
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Correspondence to Susan C. Roberts.

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Communicated by K.K. Kamo

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Naill, M.C., Roberts, S.C. Flow cytometric analysis of protein content in Taxus protoplasts and single cells as compared to aggregated suspension cultures. Plant Cell Rep 23, 528–533 (2005). https://doi.org/10.1007/s00299-004-0875-y

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  • DOI: https://doi.org/10.1007/s00299-004-0875-y

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