Ethylene is an important phytohormone that regulates growth, development and stress responses in land plants and charophycean green algae. In Arabidopsis thaliana, ethylene is perceived by a family of five receptors. One of these five receptors, ETR1, is dependent on REVERSION-TO-ETHYLENE1 (RTE1) and Cytochrome B5 (Cb5) while the other four receptors are not. We found that RTE1 and Cb5 interact in planta and used genetic analyses to place Cb5 upstream of RTE1 in the ethylene signaling pathway. After comparing different ethylene receptors we identified an N-terminally localized proline that is important in determining whether a receptor is RTE1-dependent. Our results suggest that Cb5 receives electrons from upstream redox molecules, passes these electrons to RTE1; RTE1 is then able to activate the ETR1 receptor possibly by acting a molecular chaperone that refolds the ETR1 receptor into an active conformation.

The ethylene signal transduction pathway is functionally conserved in the charophycean green algae such as Spirogyra pratensis, suggesting that this signaling pathway was present in the common ancestor of charophytes and land plants over 450 million years ago. However, it is unclear whether the central regulator of ethylene response, EIN2, was conserved in charophytes. Furthermore, the details of ethylene biosynthesis in charophytes were unresolved. After examining the genomes and transcriptomes of many green algae we are able to report that EIN2 is conserved in most charophytes and even some of the more distantly related chlorophycean green algae. Moreover, the Spirogyra EIN2 is functionally conserved and able to activate ethylene responses in Arabidopsis.

Ethylene is synthesized via a two-step reaction involving the conversion of S-adenosyl-L-methionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase (ACS), followed by oxidation of ACC to ethylene gas by the enzyme ACC oxidase (ACO). We identified S. pratensis ACS homologs and demonstrated that S. pratensis can synthesize ACC. S. pratensis lacks ACO homologs but we find it is still capable of producing low levels of ethylene. From our results we conclude that the ethylene biosynthesis and signaling pathways were established in early charophytes allowing these algae to establish ethylene as an important signalling molecule.