The gaseous phytohormone ethylene C ₂ H ₄ mediates numerous aspects of growth and development. Genetic analysis has identified a number of critical elements in ethylene signaling, but how these elements interact biochemically to transduce the signal from the ethylene receptor complex at the endoplasmic reticulum (ER) membrane to transcription factors in the nucleus is unknown. To close this gap in our understanding of the ethylene signaling pathway, the challenge has been to identify the target of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) Raf-like protein kinase, as well as the molecular events surrounding ETHYLENE-INSENSITIVE2 (EIN2), an ER membrane-localized Nramp homolog that positively regulates ethylene responses. Here we demonstrate that CTR1 interacts with and directly phosphorylates the cytosolic C-terminal domain of EIN2. Mutations that block the EIN2 phosphorylation sites result in constitutive nuclear localization of the EIN2 C terminus, concomitant with constitutive activation of ethylene responses in Arabidopsis . Our results suggest that phosphorylation of EIN2 by CTR1 prevents EIN2 from signaling in the absence of ethylene, whereas inhibition of CTR1 upon ethylene perception is a signal for cleavage and nuclear localization of the EIN2 C terminus, allowing the ethylene signal to reach the downstream transcription factors. These findings significantly advance our understanding of the mechanisms underlying ethylene signal transduction.

New insights into the ethylene signaling pathway, including dynamic regulatory mechanisms, provide a more comprehensive view of ethylene perception and signal t

Arabidopsis thaliana has five ethylene hormone receptors, which bind ethylene and elicit responses critical for plant growth and development. Here we describe a negative regulator of ethylene responses, REVERSION-TO-ETHYLENE SENSITIVITY1 ( RTE1 ), which regulates the function of at least one of the receptors, ETR1 , in Arabidopsis . RTE1 was identified based on the ability of rte1 mutations to suppress ethylene insensitivity of the dominant gain-of-function allele etr1-2 . rte1 loss-of-function mutants have an enhanced ethylene response that closely resembles the etr1 null phenotype. The etr1 rte1 double null mutant is identical to the etr1 and rte1 single null mutants, suggesting that the two genes act in the same pathway. rte1 is unable to suppress the etr1-1 gain-of-function allele, placing RTE1 at or upstream of ETR1 . rte1 also fails to suppress gain-of-function mutations in each of the four other ethylene receptor genes. RTE1 encodes a previously undescribed predicted membrane protein, which is highly conserved in plants and protists but absent in fungi and prokaryotes. Ethylene treatment induces RTE1 expression, and overexpression of RTE1 confers reduced ethylene sensitivity that partially depends on ETR1 . These findings demonstrate that RTE1 is a negative regulator of ethylene signaling and suggest that RTE1 plays an important role in ETR1 function.

Summary: Ethylene is an important plant growth regulator perceived by membrane‐bound ethylene receptors. The ETR1 ethylene receptor is positively regulated by a predicted membrane protein, RTE1, based on genetic studies in Arabidopsis. RTE1 homologs exist in plants, animals and protists, but the molecular function of RTE1 is unknown. Here, we examine RTE1 expression and subcellular protein localization in order to gain a better understanding of RTE1 and its function in relation to ETR1. Arabidopsis plants transformed with the RTE1 promoter fused to the β‐glucuronidase (GUS) reporter gene revealed that RTE1 expression partly correlates with previously described sites of ETR1 expression or sites of ethylene response, such as the seedling root, root hairs and apical hook. RTE1 transcript levels are also enhanced by ethylene treatment, and reduced by the inhibition of ethylene signaling. For subcellular localization of RTE1, a functional RTE1 fusion to red fluorescent protein (RFP) was expressed under the control of the native RTE1 promoter. Using fluorescence microscopy, RTE1 was observed primarily at the Golgi apparatus and partially at the endoplasmic reticulum (ER) in stably transformed Arabidopsis protoplasts, roots and root hairs. Next, a functional ETR1 fusion to a 5xMyc epitope tag was expressed under the control of the native ETR1 promoter. Immunohistochemistry of root hairs not only showed ETR1 residing at the ER as previously reported, but revealed substantial localization of ETR1 at the Golgi apparatus. Lastly, we demonstrated the subcellular co‐localization of RTE1 and ETR1. These findings support and enhance the genetic model that RTE1 plays a role in regulating ETR1.