Isolation and characterization of aDatura innoxia cell line resistant to ethanol

Abstract

A cell line ofDatura innoxia was selected in suspension culture to be resistant to 1% (vol/vol) ethanol (EtOHR). EtOHR cells were cross-resistant to 1% (vol/vol) methanol and 1% (vol/vol) 2-propanol but not 1% (vol/vol)n-propanol orn-butanol, whereas wild type (WT) cells were resistant only to methanol. Resistance in EtOHR cells is probably a result of a very low level of alcohol dehydrogenase (ADH) activity which was only 9 to 10% of that in WT cells and was undetectable during much of the EtOHR growth cycle. In the absence of ethanol, EtOHR cells have a I50 for the toxic ethanol analog allyl alcohol, which is nearly 3 times higher than that in WT cells. In the presence of ethanol, EtOHR cells have an I50 for allyl alcohol which is 12 times more than WT cells. This difference correlated well with the decrease in ADH activity found in EtOHR cells grown on ethanol. When ethanol was removed from the suspension medium, ADH activity in EtOHR cells gradually increased to WT levels. When re-exposed to ethanol after 200 cell generations, ADH activity quickly decreased and growth resumed after a 4- to 6-day lag period. Lipid analysis showed a 37% increase in total lipid in EtOHR cells, mostly in polar lipids, di- and triglycerides. The fatty acid composition of these lipid classes was shifted toward the more polyunsaturated. These lipid changes were probably a reflection of the increased plastid number in the EtOHR cells and may be a result of growth in ethanol rather than a reason for resistance. EtOHR cells seem to be regulatory mutants able to quickly lower ADH activity in the presence of ethanol.