Fallon, Kevin Michael; (1990) Polyamines in cell culture. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Described is an investigation into the role of polyamines in plant cells. The study was performed throughout with the use of a liquid suspension culture of carrot (Daucus carota cv Chantenay) cells as the model system. Polyamine synthesis and accumulation were studied during the culture growth period, with biosynthetic and catabolic enzyme assays performed to determine rates of polyamine synthesis and free-polyamine analysis to determine cellular polyamine levels. Methods of manipulating polyamine levels within the cell were studied. These included varying the constituents of the growth medium of the suspension culture, such as the plant hormone levels and the balance and level of the available nitrogen sources. The possibility that polyamine supplements might replace plant hormones in the growth medium was investigated and polyamine precursors were added to the medium in an attempt to boost cellular polyamine levels. A substantial comparative study was also made into the effects of inhibitors of polyamine biosynthesis on polyamine biosynthesis and accumulation. Included were determinations of the effects of a range of inhibitors on polyamine biosynthetic and catabolic enzyme activities, on the accumulation of polyamines within the cell and on growth over the culture period. Finally, the relationship between polyamines and other solutes, and cellular responses to short and long-term stress conditions was studied - both in cells previously adapted to stress conditions, and unadapted cell lines. Principal findings were: a) Difluoromethylarginine strongly inhibits arginine decarboxylase and radically reduces putrescine and total amine content, while having no effect on cell number and stimulating fresh weight through increased cell expansion. b) Difluoromethylornithine strongly inhibits ornithine decarboxylase activity but has little effect on either polyamine levels or culture growth. c) Ethylmethylglyoxalbisguanylhydrazone strongly inhibits S- adenosylmethionine decarboxylase activity with little effect on fresh weight. d) Proline and other organic solutes increase in concentration in response to polyethylene glycol induced water stress in adapted and unadapted cell lines while polyamines and glycinebetaine do not increase significantly.

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