Investigating the molecular mechanism of RTE1 activation of the ethylene receptor ETR1 in Arabidopsis

Abstract

The plant hormone ethylene plays a vital role in regulating plant growth and development as well as plant defense to biotic and abiotic stresses during the entire life of the plant. In Arabidopsis, ethylene is perceived by a family of five receptors, one of which is ETR1. The Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) gene is a positive regulator of ETR1. RTE1 encodes a novel integral membrane protein that interacts with ETR1 at the Golgi apparatus and the endoplasmic reticulum (ER). Genetic evidence indicates that RTE1 is required for the formation of a functional ETR1 receptor, whereas the other ethylene receptors in Arabidopsis do not require RTE1. But the molecular mechanism by which RTE1 specifically activates ETR1 remains unknown. I took different approaches to gain insights into the molecular function of RTE1 and the basis for the specificity for activating ETR1. In a library screen for RTE1–interacting proteins using the yeast split–ubiquitin assay, an ER–localized cytochrome b5 isoform (AtCb5–D) was identified. Cb5 is a small hemoprotein that functions in oxidation/reduction reactions. Mutants of three AtCb5 isoforms show phenotypes in ethylene responses that are similar to those of the rte1 mutant, suggesting the functional parallel between AtCb5 and RTE1 in ethylene signaling. Additional genetic analyses suggest that AtCb5 might act in the same pathway as RTE1 and that AtCb5 is specific to ETR1 like RTE1. Moreover, using a hemin–agarose affinity chromatography assay, I found that RTE1 homologs are able to bind heme in vitro, raising the possibility that RTE1 carries out redox with cytochrome b5s. I also found that the specificity for regulating ETR1 by RTE1 is largely due to a unique proline (P9) conserved only in ETR1 orthologs; introduction of P9 into the Arabidopsis ERS1 ethylene receptor was sufficient to convert ERS1 into an RTE1–dependent receptor. I propose that P9 may interfere with the proper folding of ETR1 EBD and formation of the ETR1 homodimer by affecting the conserved disulfide bond–forming cysteines (C4, C6) in the ETR1 homodimer. Taken together, our results suggest a model in which RTE1, together with cytochrome b5, promotes the active conformation of ETR1 through oxidative folding.