Cyanide is an adequate agonist of the plant hormone ethylene for studying signalling of sensor kinase ETR1 at the molecular level.

The plant hormone ethylene is involved in many developmental processes and responses to environmental stresses in plants. Although the elements of the signalling cascade and the receptors operating the ethylene pathway have been identified, a detailed understanding of the molecular processes related to signal perception and transfer is still lacking. Analysis of these processes using purified proteins in physical, structural and functional studies is complicated by the gaseous character of the plant hormone. In the present study, we show that cyanide, a π-acceptor compound and structural analogue of ethylene, is a suitable substitute for the plant hormone for in vitro studies with purified proteins. Recombinant ethylene receptor protein ETR1 (ethylene-resistant 1) showed high level and selective binding of [(14)C]cyanide in the presence of copper, a known cofactor in ethylene binding. Replacement of Cys(65) in the ethylene-binding domain by serine dramatically reduced binding of radiolabelled cyanide. In contrast with wild-type ETR1, autokinase activity of the receptor is not reduced in the ETR1-C65S mutant upon addition of cyanide. Additionally, protein-protein interaction with the ethylene signalling protein EIN2 (ethylene-insensitive 2) is considerably sustained by cyanide in wild-type ETR1, but is not affected in the mutant. Further evidence for the structural and functional equivalence of ethylene and cyanide is given by the fact that the ethylene-responsive antagonist silver, which is known to allow ligand binding but prevent intrinsic signal transduction, also allows specific binding of cyanide, but shows no effect on autokinase activity and ETR1-EIN2 interaction.

EIN2, the central regulator of ethylene signalling, is localized at the ER membrane where it interacts with the ethylene receptor ETR1.

Genetic studies have identified the membrane protein EIN2 (ethylene insensitive 2) as a central component of ethylene signalling in Arabidopsis. In addition, EIN2 might take part in multiple hormone signalling pathways and in response to pathogens as demonstrated by recent genetic and biochemical studies. Here we show, by an integrated approach using in vivo and in vitro fluorescence techniques, that EIN2 is localized at the ER (endoplasmic reticulum) membrane where it shows specific interaction with the ethylene receptor protein ETR1.

Peptides interfering with protein-protein interactions in the ethylene signaling pathway delay tomato fruit ripening.

The plant hormone ethylene is involved in the regulation of several processes with high importance for agricultural applications, e.g. ripening, aging and senescence. Previous work in our group has identified a small peptide (NOP-1) derived from the nuclear localization signal of the Arabidopsis ethylene regulator ETHYLENE INSENSITIVE-2 (EIN2) C-terminal part as efficient inhibitor of ethylene responses. Here, we show that NOP-1 is also able to efficiently disrupt EIN2-ETR1 complex formation in tomato, indicating that the NOP-1 inhibition mode is conserved across plant species. Surface application of NOP-1 on green tomato fruits delays ripening similar to known inhibitors of ethylene perception (MCP) and ethylene biosynthesis (AVG). Fruits treated with NOP-1 showed similar ethylene production as untreated controls underlining that NOP-1 blocks ethylene signaling by targeting an essential interaction in this pathway, while having no effect on ethylene biosynthesis.

Targeting Plant Ethylene Responses by Controlling Essential Protein-Protein Interactions in the Ethylene Pathway.

The gaseous plant hormone ethylene regulates many processes of high agronomic relevance throughout the life span of plants. A central element in ethylene signaling is the endoplasmic reticulum (ER)-localized membrane protein ethylene insensitive2 (EIN2). Recent studies indicate that in response to ethylene, the extra-membranous C-terminal end of EIN2 is proteolytically processed and translocated from the ER to the nucleus. Here, we report that the conserved nuclear localization signal (NLS) mediating nuclear import of the EIN2 C-terminus provides an important domain for complex formation with ethylene receptor ethylene response1 (ETR1). EIN2 lacking the NLS domain shows strongly reduced affinity for the receptor. Interaction of EIN2 and ETR1 is also blocked by a synthetic peptide of the NLS motif. The corresponding peptide substantially reduces ethylene responses in planta. Our results uncover a novel mechanism and type of inhibitor interfering with ethylene signal transduction and ethylene responses in plants. Disruption of essential protein-protein interactions in the ethylene signaling pathway as shown in our study for the EIN2-ETR1 complex has the potential to guide the development of innovative ethylene antagonists for modern agriculture and horticulture.

New paradigm in ethylene signaling: EIN2, the central regulator of the signaling pathway, interacts directly with the upstream receptors.

The membrane protein ETHYLENE INSENSITIVE2 (EIN2), which is supposed to act between the soluble serine/threonine kinase CTR1 and the EIN3/EIL family of transcription factors, is a central and most critical element of the ethylene signaling pathway in Arabidopsis. In a recent study, we have identified that EIN2 interacts tightly with all members of the Arabidopsis ethylene receptor family - proteins that mark the starting point of the signaling pathway. Our studies show consistently that the kinase domain of the receptors is essential for the formation of the EIN2-receptor complex. Furthermore, mutational analysis demonstrates that phosphorylation is a key mechanism in controlling the interaction of EIN2 and the ethylene receptors. Interaction studies in the presence of the ethylene agonist cyanide revealed a causal link between hormone binding and complex formation. In the presence of the plant hormone agonist the auto-kinase activity of the receptors is inhibited and the non-phosphorylated kinase domain of the receptors binds tightly to the carboxyl-terminal domain of EIN2. In the absence of cyanide inhibition of the auto-kinase activity is relieved and complex formation with the phosphorylated kinase domain of the receptors is reduced. Our data suggest a novel model on the integration of EIN2 in the ethylene signaling pathway.

New insight in ethylene signaling: autokinase activity of ETR1 modulates the interaction of receptors and EIN2.

Ethylene insensitive 2 (EIN2), an integral membrane protein of the ER network, has been identified as the central regulator of the ethylene signaling pathway. Still, the mechanism by which the ethylene signal is transferred from the receptors to EIN2 has not been solved yet. Here, we show that protein phosphorylation is a key mechanism to control the interaction of EIN2 and the receptors. In vivo and in vitro fluorescence studies reveal that the kinase domain of the receptors is essential for the interaction. Cyanide, an ethylene agonist, which is known to reduce auto-phosphorylation of the ethylene receptor ethylene resistant 1 (ETR1) or a mutation in the kinase domain of ETR1 that prevents auto-phosphorylation (H353A), increases the affinity of the receptors for EIN2. On the other hand, mimicking permanent auto-phosphorylation of ETR1 as in the mutant H353E releases the EIN2-ETR1 interaction from the control by the plant hormone. Based on our data, we propose a novel model on the integration of EIN2 in the ethylene signaling cascade.