Superoxide-responsive gene expression in Arabidopsis thaliana and Zea mays

- Of 12 genes previously reported to be induced by O2− in Arabidopsis, only 6 were selective for O2−.
- Of the genes with the highest selectivity for O2−, AtZAT12 had the highest absolute expression.
- Expression of a AtZAT12::Luc reporter was reduced by an inhibitor of superoxide dismutase.
- Of Arabidopsis genes with highest selectivity for O2−, 3 (AtUCP1, AtUCP2, AtZAT12) had maize homologs.
- One AtUCP1 homolog in maize (GRMZM5G852877) was significantly upregulated by methyl viologen.

Superoxide (O2−) and other reactive oxygen species (ROS) are generated in response to numerous biotic and abiotic stresses. Different ROS have been reported to elicit different transcriptional responses in plants, and so ROS-responsive marker genes and promoter::reporter gene fusions have been proposed as indirect means of detecting ROS and discriminating among different species. However, further information about the specificity of transcriptional responses to O2− is needed in order to assess potential markers for this critical stress-responsive signaling molecule. Using qRT-PCR, the expression of 12 genes previously reported to be upregulated by O2− was measured in Arabidopsis thaliana plants exposed to elicitors of common stress-responsive ROS: methyl viologen (an inducer of O2−), rose bengal (an inducer of singlet oxygen, 1ΔO2), and exogenous hydrogen peroxide (H2O2). Surprisingly, Zinc-Finger Protein 12 (AtZAT12), which had previously been used as a reporter for H2O2, responded more strongly to O2− than to H2O2; moreover, the expression of an AtZAT12 promoter-reporter fusion (AtZAT12::Luc) was enhanced by diethyldithiocarbamate, which inhibits dismutation of O2− to H2O2. These results suggest that AtZAT12 is transcriptionally upregulated in response to O2−, and that AtZAT12::Luc may be a useful biosensor for detecting O2− generation in vivo. In addition, transcripts encoding uncoupling proteins (AtUCPs) showed selectivity for O2− in Arabidopsis, and an AtUCP homolog upregulated by methyl viologen was also identified in maize (Zea mays L.), indicating that there are O2−-responsive members of this family in monocots. These results expand our limited knowledge of ROS-responsive gene expression in monocots, as well as O2−-selective responses in dicots.