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Establishment of synchronization in carrot cell suspension culture and studies on stage specific activation of glyoxalase I

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Methods in Cell Science

Abstract

The present report summarizes and compares the effects of three cell cycle inhibitors, viz. aphidicolin, hydroxyurea and mimosine, in inducing synchronization of a rapidly proliferating suspension culture of carrot. These treatments efficiently synchronized the cell cycle as the doubling time of the cell population was roughly equal to the total length of one cell cycle. Protoplasts derived from mimosine treated cell suspension culture were resolved via flow cytometry to get an idea of the temporal organization of the cell cycle events. The biochemical analysis showed a rise in stage specific activity of glyoxalase I, an auxin inducible marker enzyme activated at G2-M. This activity peak could be shifted to an early phase of interphase in response to auxin treatment.

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References

  1. Amino S, Fujimura T, Komamine A (1983). Synchrony induced by double phosphate starvation in a suspension culture of Catharanthus roseus. Physiol Plant 59: 393–396.

    Google Scholar 

  2. Bradford MM (1976). A rapid and sensitive method for quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72: 213–220.

    Google Scholar 

  3. Constabel F, Kurz EGW, Chatson KS, Gamborg OL (1977). Partial synchrony in soybean cell suspension cultures induced by ethylene. Exp Cell Res 105: 263–268.

    Google Scholar 

  4. Darzynkiewics Z (1994). Cell cycle analysis by flow cytometry. In: Julio EC (ed), Cell biology: A laboratory handbook, pp 261–271. San Diego: Academic Press.

    Google Scholar 

  5. Droog FNJ, Hookyaas PJJ, Libbenga KR, Van der Zaal EJ (1993). Proteins encoded by an auxin regulated gene family of tobacco share limited but significant homology with GSTs and one member indeed shows in vitro GST activity. Plant Mol Biol 21: 965–972.

    Google Scholar 

  6. Eriksson T (1966). Partial synchronization of cell division in suspension culture of Haplopappus gracilis. Physiol Plant 19: 900–910.

    Google Scholar 

  7. Galbraith DW (1990). Isolation and flow characterization of plant protoplasts. Methods Cell Biology 33: 527–547.

    Google Scholar 

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

    Google Scholar 

  9. Geuskens M, Hardt N, Pedsrali-Noy G, Spadari S (1981). An autographic demonstration of nuclear DNA replication by DNA-polymerase α and of mitochondrial DNA synthesis by DNA polymerase α. Nucleic Acid Res 9: 1599–1613.

    Google Scholar 

  10. Hoffman BD, Hanauske-Abel HM, Flint A, Lalande M (1991). A new class of reversible cell cycle inhibitors. Cytometry 12: 26–32.

    Google Scholar 

  11. Ikegami S, Taguchi T, Ohashi M, Nagano H, Mano Y (1978). Aphidicolin prevents mitotic cell division by interfering the activity of DNA polymerase α. Nature 275: 458–460.

    Google Scholar 

  12. Ito M, Iwase M, Kodama H, Lavisse P, Komamine A, Nishihama R, Machida Y, Watanabe A (1998). A novel cis-acting element in promoters of plant B-type cyclin genes activates M phase-specific transcription. The Plant Cell 10: 331–341.

    Google Scholar 

  13. Jouanneau JP (1971). Contrôle par les cytokines de la synchronisation des mitosis dans les cellules de tabac. Exp Cell Res 67: 329–337.

    Google Scholar 

  14. Kalia S, Pal S, Guha-Mukherjee S (1998). Activation of glyoxalase I during the cell division cycle and its homology with auxin regulated genes. Plant Sci 132: 55–62.

    Google Scholar 

  15. King PJ (1980). Plant tissue culture and cell cycle. Adv Biol Eng 18: 1–38.

    Google Scholar 

  16. Mosca PJ, Dukwel PA, Hamlin JL (1992). The plant amino acid mimosine may inhibit initiation at origins of replication in chinese hamster cells. Mol Cell Bio 12: 4375–4383.

    Google Scholar 

  17. Nagata T, Nemoto Y, Hasezawa S (1992). Tobacco BY-2 cell line as the ‘Hela’ cell in the cell biology of higher plants. Int Rev Cytol 132: 1–30.

    Google Scholar 

  18. Nishinari N, Yamaki Y (1976). Relationship between cell division and endogenous auxin in synchronously cultured tobacco cells. Bot Mag Tokyo 89: 73–81.

    Google Scholar 

  19. Nishinari N, Syono K (1986). Induction of cell division synchrony and variation in cytokinin contents through the cell cycle in tobacco cells. Plant Cell Physiol 27: 147–153.

    Google Scholar 

  20. Nishinari N, Ogawa M, Syono K (1982). Synchronous culture and changes in cytokinin levels during the cell cycle in tobacco cells. In: Fujiwara A (ed), Plant tissue culture, pp 205–206. Tokyo: Japanese Association for Plant Tissue Culture.

    Google Scholar 

  21. Okumara S, Miyasaka K, Nishi A (1973). Synchronisation of carrot cell culture by starvation and cold treatment. Exp Cell Res 78: 467–470.

    Google Scholar 

  22. Paulus C, Kollner B, Jacobsen H-J (1993). Physiological and biochemical characterization of glyoxalase I-a general marker for cell proliferation from a soybean cell suspension. Planta 189: 561–566.

    Google Scholar 

  23. Perennes C, Qin LX, Nathalie G, Bergounioux C (1993). Petunia p34cdc2 protein kinase activity in G2/M cells obtained with a reversible cell cycle inhibitor, mimosine. FEBS 333: 141–145.

    Google Scholar 

  24. Ramaswamy O, Pal S, Guha-Mukherjee S, Sopory SK (1984). Correlation of glyoxalase I activity with cell proliferation in Daucus carota cultures. Plant Cell Rep 3: 121–124.

    Google Scholar 

  25. Reichheld J-P, Sonobe S, Clement B, Chaubet N, Gigot C (1995). Cell cycle-regulated histone gene expression in synchronized plant cells. The Plant J 7: 245–252.

    Google Scholar 

  26. Sala F, Parisi B, Burroni D, Amileni AR, Pedrali-Noy G, Spadari S (1980). Specific and reversible inhibition by aphidicolin of the α like DNA polymerase of plant cells. FEBS Lett 117: 93–98.

    Google Scholar 

  27. Takahashi Y, Kusuba M, Tanaka J, Machida Y, Tebebi I, Nagata T (1986). Isolation of an auxin regulated gene cDNA expressed during transition from G0-S phase in tobacco mesophyll protoplasts. Proc Natl Acad Sci USA 86: 9279–9283.

    Google Scholar 

  28. Wang I, Zhao J, Clapper J, Martin LD, Du C, DeVore ER, Harkins K, Dobbs DL, Benbow RM (1995). Mimosine differentially inhibits DNA replication and cell cycle progression in somatic cells compared to embryonic cells Xenopus laevis. Exp Cell Res 217: 84–91.

    Google Scholar 

  29. Watson PA, Hanauske-Abel HH, Flint A, Lalande M (1991). Mimosine reversibly arrests cell cycle progression at the G1-S phase border. Cytometry 12: 242–246.

    Google Scholar 

  30. Yeomen MM, Evans PK (1967). Growth and differentiation of plant tissue cultures. II. Synchronous cell divisions in developing callus cultures. Ann Bot 31: 323–332.

    Google Scholar 

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Ghosh, S., Sen, J., Kalia, S. et al. Establishment of synchronization in carrot cell suspension culture and studies on stage specific activation of glyoxalase I. Methods Cell Sci 21, 141–148 (1999). https://doi.org/10.1023/A:1009836923004

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