Association of cytochrome b5 with ETR1 ethylene receptor signaling through RTE1 in Arabidopsis

Ethylene plays important roles in plant growth, development and stress responses, and is perceived by a family of receptors that repress ethylene responses when ethylene is absent. Repression by the ethylene receptor ETR1 depends on an integral membrane protein, REVERSION TO ETHYLENE SENSITIVITY1 (RTE1), which acts upstream of ETR1 in the endoplasmic reticulum (ER) membrane and Golgi apparatus. To investigate RTE1 function, we screened for RTE1-interacting proteins using the yeast split-ubiquitin assay, which yielded the ER-localized cytochromeb(5) (Cb5) isoformD. Cb5s are small hemoproteins that perform electron transfer reactions in all eukaryotes, but their roles in plants are relatively uncharacterized. Using bimolecular fluorescence complementation (BiFC), we found that all four ER-localized Arabidopsis Cb5 isoforms (AtCb5-B, -C, -D and -E) interact with RTE1 in plant cells. In support of this interaction, atcb5 mutants exhibited phenotypic parallels with rte1 mutants in Arabidopsis. Phenotypes included partial suppression of etr1-2 ethylene insensitivity, and no suppression of RTE1-independent ethylene receptor isoforms. The single loss-of-function mutants atcb5-b, -c and -d appeared similar to the wild-type, but double mutant combinations displayed slight ethylene hypersensitivity. Over-expression of AtCb5-D conferred reduced ethylene sensitivity similar to that conferred by RTE1 over-expression, and genetic analyses suggested that AtCb5-D acts upstream of RTE1 in the ethylene response. These findings suggest an unexpected role for Cb5, in which Cb5 and RTE1 are functional partners in promoting ETR1-mediated repression of ethylene signaling.