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 O-1(2) from an unknown non-photosynthetic source. Reduced root growth can be alleviated by application of the O-1(2) scavenger histidine (HIS). Here, we examined the possibility that O-1(2) production involves Russell reactions occurring among the enzymatic products of lipoxygenases (LOXs), the fatty acid hydroperoxides. 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 LOX inhibitor, but not by HIS, whereas O-1(2) production was inhibited by both. D2O, which specifically extends the half-life of O-1(2), augmented the LOX-dependent generation of O-1(2), as expected from a Russell-type reaction. The addition of linoleic acid to roots stimulated O-1(2) production and inhibited growth, suggesting that the availability of LOX 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 LOXs showed downregulation of LOX protein and activity. The lines with downregulated LOX displayed significantly less O-1(2) formation, improved root growth in osmotic stress, and an altered transcriptome response compared with wild type. The results show that LOXs can serve as an enzymatic source of "dark" O-1(2) during osmotic stress and demonstrate a role for O-1(2) in defining the physiological response.