Characterisation of the xanthine oxidase protein family in Arabidopsis: biochemistry and physiological importance for the synthesis of phytohormones, ureides, and reactive oxygen species

• Applicant: Dr. Christian Gehl, since 10/2015
• Subject Area: Plant Biochemistry and Biophysics
• Term: from 2013 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 234281575

The xanthine oxidase family comprises aldehyde oxidase (AO) and xanthine dehydrogenase (XDH) proteins, with different plant species encoding a varying number of isoenzymes. In Arabidopsis four AO isoforms, AAO1-AAO4, are existing, which form homodimers as well as heterodimers. While AAO3 is well accepted as a key enzyme in abscisic acid synthesis, the physiological functions of the other isoforms are mostly uncharacterised and only poorly understood. Yet, based on substrate specificities it is assumed that AAO1 and/or AAO2 catalyse the oxidation of indole acetaldehyde to indole acetic acid, by which they could be involved in one out of several possible ways of indole acetic acid synthesis. Although AAO4 is assumed to play a role in glucosinolate synthesis, convincing evidence for this is still lacking. Thus, it is one aspect of the present project to study in detail and to identify the physiological functions of the aldehyde oxidase isoforms AAO1, 2, and 4.In Arabidopsis, two XDH genes are exisiting with AtXDH2 representing a pseudogene and AtXDH1 being a key enzyme in purine breakdown that catalyses the oxidation of hypoxanthine via xanthine to uric acid. XDH is the evolutionary ancestor of AO and thus, both enzymes share certain properties: Both enzymes are able to transfer substrate-derived electrons to molecular oxygen with simultaneous formation of superoxides in case of XDH and superoxides and hydrogen peroxide in case of AO. Moreover, we were able to show that both enzymes harbor an intrinsic NADH oxidase activity which is likewise associated with the formation of superoxides. Interestingly, all physiological conditions associated with increased activities of AAO3 and/or AtXDH1 are likewise characterised by a requirement for increased levels of reactive oxygen species (e.g. drought stress, senescence). Since a physiological significance of reactive oxygen species produced by AO and/or XDH has not even been investigated as yet, this will be another subject of this proposal.In our preliminary work, AAO3 and AtXDH1 have been demonstrated to be regulated by ubiquitination, with a loss of ubiquitination capacity resulting in accumulation of these enzymes and premature senescence. Besides corresponding ubiquitination motives, AAO3 and AtXDH1 also possess pronounced sumoylation motives, with the latter not having been considered in earlier studies. Since such motives are also found in other AO isoforms, we wish to elucidate whether all or some enzymes of the xanthine oxidase family underly these post-translational modifications, and if so, for what physiological reason.Finally, by using recombinant proteins we will analyse several biochemical properties of AO and XDH proteins with a special focus on the characterisation of substrate specificities and a possible production of nitric oxide, which is controversially discussed for a long time.

• DFG Programme: Research Grants
• International Connection: Japan
• Participating Persons: Professor Dr. Stefan Hoth; Professor Dr. Tomokazu Koshiba
• Ehemaliger Antragsteller: Dr. Florian Bittner, until 10/2015