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Relationship between programmed cell death and the cell cycle in the tobacco BY-2 cell line

Orchard, Craig Brailsford 2004. Relationship between programmed cell death and the cell cycle in the tobacco BY-2 cell line. PhD Thesis, Cardiff University.

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Ethylene is an established plant growth regulator linked with programmed cell death (PCD). To investigate the relationship between the cell cycle and PCD, ethylene was used to see if it induced mortality in a cell cycle specific manner. Tobacco BY-2 cultures synchronized with aphidicolin were treated with ethylene. Cell cycle progression and mortality, measured at hourly intervals, showed distinct peaks of mortality at the G2/M boundary and S-phase. In conjunction with this, DNA fragmentation increased at G2/M. Furthermore, ethylene caused a significant reduction in cell size of the cycling population. Simultaneous addition of silver nitrate with ethylene ameliorated ethylene-induced G2/M mortality, although a toxic effect of silver alone was evident. Due to the toxicity of silver, 1-MCP, an alternative chemical for blocking ethylene receptors was used. 1-MCP neither affected the BY-2 cell cycle nor mortality levels. In addition, 1-MCP ameliorated ethylene-induced G2/M mortality. To balance the chemical approaches to blocking ethylene receptors, tobacco BY-2 cells were transformed with Atetrl that encodes a dominant insensitive form of the Arabidopsis ETR1 ethylene receptor. Atetrl expression caused a massive perturbation to the tobacco BY-2 cell cycle, especially in S-phase, and resulted in high levels of mortality throughout the cell cycle. Ethylene treatment caused a doubling of G2 duration but did not affect temporal distribution of mortality. However, ethylene treatment generated a peak of mortality in S-phase. These results suggest that ethylene induces PCD at G2/M through the known ethylene signaling pathway. Furthermore, it confirms that 1-MCP and Atetrl result in ethylene insensitivity. To examine the G2/M transition, Spcdc25, a positive regulator of G2/M in fission yeast was transformed into the tobacco BY-2 cell line. This resulted in premature entry into mitosis, a shortened cell cycle, and reduced cell size. This was similar to Spcdc25 over-expression in fission yeast and suggests the presence of a CDC25-like phosphatase in plants.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Biosciences
ISBN: 9781303200304
Funders: University of Wales, University College Worcester
Date of First Compliant Deposit: 30 March 2016
Last Modified: 12 Feb 2016 23:15

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