RESUMO
The plant molybdenum-cofactor (Moco) and flavin-containing enzymes, xanthine dehydrogenase (XDH; EC 1.2.1.37) and aldehyde oxidase (AO; EC 1.2.3.1) are thought to play important metabolic roles in purine metabolism and hormone biosynthesis, respectively. Their animal counterparts contribute to reactive oxygen species (ROS) production in numerous pathologies and here we examined these enzymes as potential sources of ROS in plants. Novel in-gel assay techniques and Moco sulfurase mutants, lacking a sulfur ligand in their Moco active center, were employed to demonstrate that the native tomato and Arabidopsis XDHs are capable of producing O, but not H2O2, while the animal counterpart was shown to produce both, O and H2O2. Superoxide production was dependent on Moco sulfuration when using hypoxanthine/xanthine but not NADH as substrates. The activity was inhibited by diphenylene iodonium (DPI), a suicide inhibitor of FAD containing enzymes. Analysis of XDH in an Arabidopsis Atxdh1 T-DNA insertion mutant and RNA interference lines revealed loss of O activity, providing direct molecular evidence that plant XDH generates superoxides. Contrary to XDH, AO activity produced only H2O2 dissimilar to native animal AO, that can produce O as well. Surprisingly, H2O2 accumulation was not sensitive to DPI. Plant ROS production and transcript levels of AO and XDH were rapidly upregulated by application of abscisic acid and in water-stressed leaves and roots. These results, supported by in vivo measurement of ROS accumulation, indicate that plant AO and XDH are possible novel sources for ROS increase during water stress.
