Publications - Stress and Develop Biology

Lipoxygenases (LOX; linoleate:oxygen oxidoreductase EC 1.13.11.12) consist of a class of enzymes that catalyze the regio‐ and stereo specific dioxygenation of polyunsaturated fatty acids. Here we characterize two proteins that belong to the less studied class of 9‐LOXs, Solanum tuberosum StLOX1 and Arabidopsis thaliana AtLOX1. The proteins were recombinantly expressed in E. coli and the product specificity of the enzymes was tested against different fatty acid substrates. Both enzymes showed high specificity against all tested C18 fatty acids and produced (9S )‐hydroperoxides. However, incubation of the C20 fatty acid arachidonic acid with AtLOX1 gave a mixture of racemic hydroperoxides. On the other hand, with StLOX1 we observed the formation of a mixture of products among which the (5S )‐hydroperoxy eicosatetraenoic acid (5S‐ H(P)ETE) was the most abundant. Esterified fatty acids were no substrates. We used site directed mutagenesis to modify a conserved valine residue in the active site of StLOX1 and examine the importance of space within the active site, which has been shown to play a role in determining the positional specificity. The Val576Phe mutant still catalyzed the formation of (9S )‐hydroperoxides with C18 fatty acids, while it exhibited altered specificity against arachidonic acid and produced mainly (11S )‐H(P)ETE. These data confirm the model that in case of linoleate 9‐LOX binding of the substrate takes place with the carboxyl‐group first.

The F2 progeny of a third backcross(BC3) line, BC line 240, derived from a Turkish accession of wild barley (Hordeum vulgare ssp. spontaneum),segregated for resistance to scald (Rhynchosporium secalis) in a manner indicating the presence of a single dominant resistance gene. Two SCAR marker slinked to this resistance were developed from AFLP markers. Screens of disomic and ditelosomic wheat-barley addition lines with the SCAR markers demonstrated that the scald resistance gene is located in the centromeric region of barley chromosome 3H,a region previously reported to contain a major scald resistance locus, Rrs1. Markers that flank the Rrs1 locus were used to screen the wild barley-derivedBC3F2 population. These markers also flank the wild barley-derived scald resistance, indicating that it maps to the same locus as Rrs1; it may beallelic, or a separate gene within a complex locus. However, BC line 240 does not respond to treatment with the Rhynchosporium secalis avirulence factorNIP1 in the same way as the Rrs1-carrying cultivar Atlas46. This suggests that the scald resistance gene derived from wild barley confers a different specificity of response to theRrs1 allele in Atlas46.In order to increase the durability of scald resistance in the field, we suggest that at least two scald resistances should be combined into barley cultivars before release. The scald resistance gene described here will be of value in the Australian environment, and the several markers linked to it will facilitate pyramiding.