Lipoxygenase functions in 1O2 production during root responses to osmotic stress

Drought induces osmotic stress in roots, a condition simulated by the application of high-molecular-weight polyethylene glycol. Osmotic stress results in the reduction of Arabidopsis thaliana root growth and production of 1O2 from an unknown non-photosynthetic source. Reduced root growth can be alleviated by application of the 1O2 scavenger histidine. Here we examined the possibility that 1O2 production involves Russell reactions occurring among the enzymatic products of lipoxygenases, the fatty acid hydroperoxides. Lipoxygenase (LOX) activity was measured for purified soybean (Glycine max) LOX1 and in crude Arabidopsis root extracts using linoleic acid as substrate. Formation of the 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid product was inhibited by salicylhdroxamic acid, which is a lipoxygenase inhibitor, but not by histidine, whereas 1O2 production was inhibited by both. D2O, which specifically extends the half-life of 1O2, augmented the lipoxygenase-dependent generation of 1O2, as expected from a Russell-type reaction. The addition of linoleic acid to roots stimulated 1O2 production and inhibited growth, suggesting that the availability of lipoxygenase substrate is a rate-limiting step. Indeed, water stress rapidly increased linoleic and linolenic acids by 2.5-fold in roots. Mutants with root-specific microRNA repression of lipoxygenases showed downregulation of lipoxygenase protein and activity. The lines with downregulated lipoxygenase displayed significantly less 1O2 formation, improved root growth in osmotic stress, and an altered transcriptome response compared to wild type. The results show that lipoxygenases can serve as an enzymatic source of ‘dark’ 1O2 during osmotic stress and demonstrate a role for 1O2 in defining the physiological response.