Brad M. Binder

Auxin and ethylene: collaborators or competitors?

The individual roles of auxin and ethylene in controlling the growth and development of young seedlings have been well studied. In recent years, these two hormones have been shown to act synergistically to control specific growth and developmental processes, such as root elongation and root hair formation, as well as antagonistically in other processes, such as lateral root formation and hypocotyl elongation. This review examines the growth and developmental processes that are regulated by crosstalk between these two hormones and explores the mechanistic basis for the regulation of these processes. The emerging trend from these experiments is that ethylene modulates auxin synthesis, transport, and signaling with unique targets and responses in a range of tissues to fine-tune seedling growth and development.

The Arabidopsis EIN3 Binding F-Box Proteins EBF1 and EBF2 Have Distinct but Overlapping Roles in Ethylene Signaling

Ethylene signaling in Arabidopsis thaliana converges on the ETHYLENE-INSENSITIVE3 (EIN3)/EIN3-Like (EIL) transcription factors to induce various responses. EIN3 BINDING F-BOX1 (EBF1) and EBF2 were recently shown to function in ethylene perception by regulating EIN3/EIL turnover. In the absence of ethylene, EIN3 and possibly other EIL proteins are targeted for ubiquitination and subsequent degradation by Cullin 1–based E3 complexes containing EBF1 and 2. Ethylene appears to block this ubiquitination, allowing EIN3/EIL levels to rise and mediate ethylene signaling. Through analysis of mutant combinations affecting accumulation of EBF1, EBF2, EIN3, and EIL1, we show that EIN3 and EIL1 are the main targets of EBF1/2. Kinetic analyses of hypocotyl growth inhibition in response to ethylene and growth recovery after removal of the hormone revealed that EBF1 and 2 have temporally distinct but overlapping roles in modulating ethylene perception. Whereas EBF1 plays the main role in air and during the initial phase of signaling, EBF2 plays a more prominent role during the latter stages of the response and the resumption of growth following ethylene removal. Through their coordinated control of EIN3/EIL1 levels, EBF1 and EBF2 fine-tune ethylene responses by repressing signaling in the absence of the hormone, dampening signaling at high hormone concentrations, and promoting a more rapid recovery after ethylene levels dissipate.

Arabidopsis Seedling Growth Response and Recovery to Ethylene. A Kinetic Analysis

Responses to the plant hormone ethylene are mediated by a family of five receptors in Arabidopsis that act in the absence of ethylene as negative regulators of response pathways. In this study, we examined the rapid kinetics of growth inhibition by ethylene and growth recovery after ethylene withdrawal in hypocotyls of etiolated seedlings of wild-type and ethylene receptor-deficient Arabidopsis lines. This analysis revealed that there are two phases to growth inhibition by ethylene in wild type: a rapid phase followed by a prolonged, slower phase. Full recovery of growth occurs approximately 90 min after ethylene removal. None of the receptor null mutations tested had a measurable effect on the two phases of growth inhibition. However, loss-of-function mutations in ETR1, ETR2, and EIN4 significantly prolonged the time for recovery of growth rate after ethylene was removed. Plants with an etr1-6;etr2-3;ein4-4 triple loss-of-function mutation took longer to recover than any of the single mutants, while the ers1;ers2 double mutant had no effect on recovery rate, suggesting that receiver domains play a role in recovery. Transformation of the ers1-2;etr1-7 double mutant with wild-type genomic ETR1 rescued the slow recovery phenotype, while a His kinase-inactivated ETR1 construct did not. To account for the rapid recovery from growth inhibition, a model in which clustered receptors act cooperatively is proposed.

Identification of Important Regions for Ethylene Binding and Signaling in the Transmembrane Domain of the ETR1 Ethylene Receptor of Arabidopsis

The ethylene binding domain (EBD) of the Arabidopsis thaliana ETR1 receptor is modeled as three membrane-spanning helices. We surveyed ethylene binding activity in different kingdoms and performed a bioinformatic analysis of the EBD. Ethylene binding is confined to land plants, Chara, and a group of cyanobacteria but is largely absent in other organisms, consistent with our finding that EBD-like sequences are overrepresented among plant and cyanobacterial species. We made amino acid substitutions in 37 partially or completely conserved residues of the EBD and assayed their effects on ethylene binding and signaling. Mutations primarily in residues in Helices I and II midregions eliminated ethylene binding and conferred constitutive signaling, consistent with the inverse-agonist model of ethylene receptor signaling and indicating that these residues define the ethylene binding pocket. The largest class of mutations, clustered near the cytoplasmic ends of Helices I and III, gave normal ethylene binding activity yet still conferred constitutive signaling. Therefore, these residues may play a role in turning off the signal transmitter domain of the receptor. By contrast, only two mutations were loss of function with respect to signaling. These findings yield insight into the structure and function of the EBD and suggest a conserved role of the EBD as a negative regulator of the signal transmitter domain.

Short-Term Growth Responses to Ethylene in Arabidopsis Seedlings Are EIN3/EIL1 Independent

Kinetic studies indicate there are two phases to growth inhibition by ethylene for the hypocotyls of etiolated Arabidopsis seedlings. Phase I is transient, while phase II results in sustained growth inhibition. The EIN2 membrane protein is required for both the first and second phases of growth inhibition by ethylene, while the transcription factors EIN3 and EIL1 are required for the second phase but not the first phase. The first phase lasts no more than 2 h. It is less sensitive to the ethylene response inhibitor 1-methylcyclopropene and more sensitive to ethylene than the second phase. The first phase shows adaptation at low concentrations of ethylene (≤0.01 μL L−1) with a relative refractory period of 5 h after ethylene is added. A modified signal transduction model is proposed that accounts for the two phases of growth inhibition.

The BTB ubiquitin ligases ETO1, EOL1 and EOL2 act collectively to regulate ethylene biosynthesis in Arabidopsis by controlling type-2 ACC synthase levels

Ethylene biosynthesis is directed by a family of 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS) that convert S-adenosyl-l-methionine to the immediate precursor ACC. Members of the type-2 ACS subfamily are strongly regulated by proteolysis with various signals stabilizing the proteins to increase ethylene production. In Arabidopsis, this turnover is mediated by the ubiquitin/26 S proteasome system, using a broad complex/tramtrack/bric-a-brac (BTB) E3 assembled with the ETHYLENE OVERPRODUCER 1 (ETO1) BTB protein for target recognition. Here, we show that two Arabidopsis BTB proteins closely related to ETO1, designated ETO1-like (EOL1) and EOL2, also negatively regulate ethylene synthesis via their ability to target ACSs for breakdown. Like ETO1, EOL1 interacts with type-2 ACSs (ACS4, ACS5 and ACS9), but not with type-1 or type-3 ACSs, or with type-2 ACS mutants that stabilize the corresponding proteins in planta. Whereas single and double mutants affecting EOL1 and EOL2 do not show an ethylene-related phenotype, they exaggerate the effects caused by inactivation of ETO1, and further increase ethylene production and the accumulation of ACS5 in eto1 plants. The triple eto1 eol1 eol2 mutant phenotype can be effectively rescued by the ACS inhibitor aminoethoxyvinylglycine, and by silver, which antagonizes ethylene perception. Together with hypocotyl growth assays showing that the sensitivity and response kinetics to ethylene are normal, it appears that ethylene synthesis, but not signaling, is compromised in the triple mutant. Collectively, the data indicate that the Arabidopsis BTB E3s assembled with ETO1, EOL1 and EOL2 work together to negatively regulate ethylene synthesis by directing the degradation of type-2 ACS proteins.

Heteromeric Interactions among Ethylene Receptors Mediate Signaling in Arabidopsis

The gaseous hormone ethylene is perceived in Arabidopsis by a five member receptor family that consists of the subfamily 1 receptors ETR1 and ERS1 and the subfamily 2 receptors ETR2, ERS2, and EIN4. Previous work has demonstrated that the basic functional unit for the ethylene receptor, ETR1, is a disulfide-linked homodimer. We demonstrate here that ethylene receptors isolated from Arabidopsis also interact with each other through noncovalent interactions. Evidence that ETR1 associates with other ethylene receptors was obtained by co-purification of ETR1 with tagged versions of ERS1, ETR2, ERS2, and EIN4 from Arabidopsis membrane extracts. ETR1 preferentially associated with the subfamily 2 receptors compared with the subfamily 1 receptor ERS1, but ethylene treatment affected the interactions and relative composition of the receptor complexes. When transgenically expressed in yeast, ETR1 and ERS2 can form disulfide-linked heterodimers. In plant extracts, however, the association of ETR1 and ERS2 can be largely disrupted by treatment with SDS, supporting a higher order noncovalent interaction between the receptors. Yeast two-hybrid analysis demonstrated that the receptor GAF domains are capable of mediating heteromeric receptor interactions. Kinetic analysis of ethylene-insensitive mutants of ETR1 is consistent with their dominance being due in part to an ability to associate with other ethylene receptors. These data suggest that the ethylene receptors exist in plants as clusters in a manner potentially analogous to that found with the histidine kinase-linked chemoreceptors of bacteria and that interactions among receptors contribute to ethylene signal output.

Mechanisms of signal transduction by ethylene: overlapping and non-overlapping signalling roles in a receptor family

The plant hormone ethylene regulates growth and development as well as stress responses. This review focuses on recent discoveries that support a model for ethylene signal transduction that involves overlapping and non-overlapping roles for members of the ethylene receptor family. The roles of ethylene receptors in regulating plant growth, pathogen responses, and development are discussed. Mechanisms are proposed by which receptors can modulate downstream responses together and independently.

Ethylene Receptors Function as Components of High-Molecular-Mass Protein Complexes in Arabidopsis

Background The gaseous plant hormone ethylene is perceived in Arabidopsis thaliana by a five-member receptor family composed of ETR1, ERS1, ETR2, ERS2, and EIN4. Methodology/Principal Findings Gel-filtration analysis of ethylene receptors solubilized from Arabidopsis membranes demonstrates that the receptors exist as components of high-molecular-mass protein complexes. The ERS1 protein complex exhibits an ethylene-induced change in size consistent with ligand-mediated nucleation of protein-protein interactions. Deletion analysis supports the participation of multiple domains from ETR1 in formation of the protein complex, and also demonstrates that targeting to and retention of ETR1 at the endoplasmic reticulum only requires the first 147 amino acids of the receptor. A role for disulfide bonds in stabilizing the ETR1 protein complex was demonstrated by use of reducing agents and mutation of Cys4 and Cys6 of ETR1. Expression and analysis of ETR1 in a transgenic yeast system demonstrates the importance of Cys4 and Cys6 of ETR1 in stabilizing the receptor for ethylene binding. Conclusions/Significance These data support the participation of ethylene receptors in obligate as well as ligand-dependent non-obligate protein interactions. These data also suggest that different protein complexes may allow for tailoring of the ethylene signal to specific cellular environments and responses.

The Ethylene Signal Transduction Pathway

The molecular details of early steps in ethylene signal transduction are becoming firmly established. Many key components have been identified through the study of mutations that affect a broad range of ethylene responses in the plant. Members of the ETR gene family can cause dominant ethylene insensitivity when mutated [1]. Loss of function mutations in EIN2 and EIN3 also result in ethylene insensitivity in the plant; while loss of function mutations in the CTR1 gene leads to constitutive activation of ethylene response pathways [2]. Genetic epistasis analysis indicated that the CTR1 protein acts between the ETR receptors and EIN2 and EIN3 in the signal transduction chain. The genes responsible for these all of these mutant phenotypes have been cloned by a variety of techniques and the derived amino acid sequences have provided important clues as to the biochemical functions of the gene products [3]. The ETR family shows homology to the two-component histidine-kinase receptors common in bacteria. CTR1 shows homology to eukaryotic serine/threonine protein kinases that initiate MAP kinase cascades in eukaryotes. EIN2 is related to a family of metal transporters found in eukaryotes while EIN3 represents a family of transcription factors found only in plants. These components of ethylene signaling and their evolutionary relationships are depicted in Figure 1.