Lipoxygenases during Brassica napus seed germination

Abstract

The peroxidation of polyunsaturated fatty acids is mostly catalyzed by members of the lipoxygenase enzyme family. Lipoxygenase products can be metabolized further in the oxylipin pathway and are known as signalling substances that play a role in plant development as well as in plant responses to wounding and pathogen attack. Apart from accumulating data in model plants like Arabidopsis, information on the relevance of lipid peroxide metabolism in the crop plant oilseed rape is scarce. Thus we aimed to analyze lipoxygenases and oxylipin patterns in seedlings of oilseed rape. RNA isolated from 3 day etiolated seedlings contains mRNAs for at least two different lipoxygenases. These have been cloned as cDNAs and named Bn-Lox-1fl and Bn-Lox-2fl. The protein encoded by Bn-Lox-2fl was identified as a 13-lipoxygenase by expression in Escherichia coli. The Bn-Lox-1fl yielded an inactive protein when expressed in E. coli. Based on Bn-Lox-1fl active site determinants and on sequence homology the Bn-Lox-1fl is most likely a 9-lipoxygenase. Both genes are expressed in light-grown and etiolated cotyledons as well as in leaves. Bn-Lox-2fl protein is more abundant in cotyledons of etiolated seedlings than in cotyledons of green seedlings. Both 13- and 9-lipoxygenase-derived hydroperoxides can be detected during germination. Etiolated seedlings contain more lipoxygenase-derived hydroperoxides in non esterified fatty acids than green seedlings. The 13-lipoxygenase derivatives are 6-8-fold more abundant than the 9-derivatives. Lipoxygenase-derived hydroperoxides in esterified lipids are almost not present during germination. These results suggest that 13-lipoxygenases acting on free fatty acids dominate during B. napus seed germination.