Identifying signal transduction components acting downstream of reactive oxygen species (ROS) in Arabidopsis thaliana

Abstract

Traditionally, reactive oxygen species (ROS) have been regarded as toxic by-products of aerobic metabolism. However, in recent years it has become apparent that plants actively produce ROS as signalling molecules. ROS are able to mediate adaptive responses to various environmental stresses as well as processes such as stomatal closure and development. Downstream signalling events that are modulated by ROS include calcium mobilisation, protein phosphorylation and gene expression. This study investigated signalling proteins acting downstream of ROS, in order to understand how ROS are perceived and transduced to elicit such a wide range of responses. To establish a molecular profile provoked by ROS, a microarray experiment of Arabidopsis plants exposed to exogenous H(_2)O(_2) was analysed. Of the 895 differentially expressed transcripts, a substantial proportion had predicted functions in cell rescue and defence, including heat shock, disease resistance and antioxidant genes. Genes encoding candidate H(_2)O(_2) signalling components were identified from this microarray experiment and their H(_2)O(_2) - induced expression was verified by northern RNA-blot analysis. Two transcription factors of the ethylene response factor (ERF) family (AtERFS [At5g47230]) and AtERF6 [At4g17490])and an ankyrin protein kinase (APK [At4g18950]) were selected for further study. Northern blot analysis and comparison with publicly available transcriptome data sets demonstrated that the expression of these three genes was induced by various stress treatments, such as UV-B irradiation, cold and elicitor challenge. To unravel the potential in vivo function of these proteins, loss- and gain-of-function lines were generated and analysed. No abnormal plant phenotypes were observed during development or in response to the stress and hormone treatments tested. A high level of functional redundancy may exist between AtERFS and AtERF6. Microarray analyses were performed on the over-expression lines. Genes that were differentially regulated in APK over-expressor lines gave no indication of its function. However, the microarray analyses revealed that AtERFS and AtERF6 have roles in the plant pathogen defence response, since their over-expression induced defence gene expression. Analysis of cis elements in the promoters of the ERF-differentially regulated genes revealed that both transcription factors displayed GCC box binding activity. However, the GCC box was not over-represented in the promoters of H202-differentially regulated genes, which suggests that this element has a ROS independent regulation.