The NADPH oxidase Rboh D is involved in primary hypoxia signalling and modulates expression of hypoxia-inducible genes under hypoxic stress

• The transcription profiles of 10 Rbohs under hypoxic stress were characterized.
• Rboh D functions at an early stage to modulate the expression of down-stream genes.
• Ethylene modulates H2O2 signalling via regulation of expression of Rboh genes in hypoxia pathway.
• The feedback mechanism helps the plant to preserve ethylene and H2O2 homeostasis.

In plants, hydrogen peroxide (H2O2) acts as a signalling molecule in biotic or abiotic stress responses. Under oxygen deprivation (hypoxia), H2O2 accumulates and regulates the expression of a set of genes encoding heat-shock proteins and other groups of reactive-oxygen-species-mediated proteins. The plasma membrane NADPH oxidase, or respiratory burst oxidase, is the main source of H2O2 in cells. In this study, the transcription profiles of 10 Rbohs (respiratory burst oxidase homologs) in Arabidopsis under hypoxic stress were characterized. The transcript levels of five Rboh genes (Rboh A, B, D, G and I) were increased under hypoxia. In particular, the transcript levels of Rboh D were significantly increased at an early stage in the hypoxia response. Ethylene signalling, which is involved in the hypoxia pathway, affected the transcript levels of the five hypoxia-inducible Rbohs. H2O2 accumulated to lower levels in a rbohd-knockout line than in wild type under hypoxic stress. Under hypoxic conditions, compared with wild-type, the rbohd-knockout line showed lower transcript levels of ACS7 and ACS8, which are involved in ethylene synthesis; lower transcript levels of downstream hypoxia-inducible genes ERF73/HRE1 and ADH1; lower transcript levels of Rboh A, B, and G (but not Rboh I); and higher transcript levels of genes encoding peroxidases and cytochrome P450s. Together, these results show that Rboh D plays a major role at an early stage to modulate the expression of down-stream hypoxia-inducible genes under hypoxic stress, and that ethylene signalling modulates H2O2 signalling via regulation of expression of Rboh genes in the hypoxia response pathway.