Gershater, Markus Christian (2006) Plant carboxylesterases involved in pesticide hydrolysis. Doctoral thesis, Durham University.
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Abstract
Many herbicides are applied in the form of carboxylic esters to increase their hydrophobicity and hence aid their passage through the waxy cuticle. Hydrolysis in planta of these pro-herbicide esters releases the active acid or alcohol and the rate of this cleavage can be a factor in determining herbicide selectivity. Protein extracts from 13 crop and weed species were assayed for carboxylesterase activity toward multiple xenobiotic and pesticide ester substrates, including 2,4-D-methyl, aryloxyphenoxypropionate esters and p-nitrophenyl esters. A diversity of activities was exhibited by the different species, with Arabidopsis thaliana extracts showing high hydrolytic activity toward several xenobiotic esters, particularly 2,4-D- methyl.The major 2,4-D-methyl hydrolysing enzyme in arabidopsis cell cultures was purified through three rounds of chromatography, then selectively labelled with a biotinylated fluorophosphonate probe (FP-biotin). Following streptavidin affinity purification, the labelled protein was identified by proteomics as the previously uncharacterised serine hydrolase AtCXE12. Recombinant AtCXE12 was subsequently confirmed to effectively hydrolyse 2,4-D-methyl.A T-DNA insertion knockout line that did not express AtCXE12 was identified and characterised. Protein from the knockout plants did not contain AtCXE12 and was found to have a reduced rate of 2,4-D-methyl hydrolysis compared to wild-type plant extracts. This translated into a higher tolerance of 2,4-D-methyl in young atcxel2 plants, due to a lower rate of bioactivation of the pro-herbicide.The fluorophosphonate-based chemical probe was subsequently used to identify other major serine hydrolases in arabidopsis. AtCXE12 and three previously uncharacterised hydrolases were identified, each belonging to a distinct enzyme family.
Item Type: | Thesis (Doctoral) |
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Award: | Doctor of Philosophy |
Thesis Date: | 2006 |
Copyright: | Copyright of this thesis is held by the author |
Deposited On: | 09 Sep 2011 09:57 |