Ethylene signaling in Arabidopsis involves feedback regulation via the elaborate control of EBF2 expression by EIN3

EIN3 is a key transcription factor in the signaling pathway of the plant hormone ethylene in Arabidopsis. Ethylene signaling suppresses the 26S proteasome-mediated proteolysis of EIN3, the accumulation of which induces ethylene-responsive gene expression. The proteolysis of EIN3 has been suggested to be mediated by the E3 ubiquitin ligase SCF(EBF1/2) that contains either of the two closely related F-box proteins, EBF1 or EBF2. Here, we demonstrate feedback regulation of ethylene signaling that results from the direct upregulation of the EBF2 gene by EIN3. Although EBF1 and EBF2 show comparable activities as repressors of EIN3-dependent transcription, our analysis of transgenic Arabidopsis plants has revealed that the EBF2 promoter, but not the EBF1 promoter, is activated by ethylene. Furthermore, the results of protoplast transient assays in vivo and electrophoretic mobility shift assays in vitro have revealed that EIN3 can bind and activate the EBF2 promoter, indicating that EIN3 modulates EBF2 gene expression in planta. An ebf2 mutant transformed with the EBF2 gene under the control of a mutant promoter in which the EIN3-binding site was disrupted still showed an ethylene hyper-responsive phenotype, indicating the physiological relevance of EIN3-mediated activation of the EBF2 gene in the downregulation of ethylene signaling. We show an additional finding that a sequence downstream of the EBF2 coding region is also involved in modulations of both the EBF2 expression level and sensitivity to ethylene. These results thus indicate that the fine-tuning of ethylene signaling involves the intricate regulation of EBF2 expression in Arabidopsis.