Wuyi Wang

Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission.

Ethylene signaling in plants is mediated by a family of receptors related to bacterial two-component histidine kinases. Of the five members of the Arabidopsis ethylene receptor family, members of subfamily I (ETR1 and ERS1) contain completely conserved histidine kinase domains, whereas members of subfamily II (ETR2, EIN4, and ERS2) lack conserved residues thought to be necessary for kinase activity. To examine the role of the conserved histidine kinase domain in receptor signaling, ers1;etr1 loss-of-function double mutants were generated. The double mutants exhibited a severe constitutive ethylene response phenotype consistent with the negative regulator model for receptor function. The adult ers1-2;etr1-6 and ers1-2;etr1-7 phenotypes included miniature rosette size, delayed flowering, and both male and female sterility, whereas etiolated-seedling responses were less affected. Chimeric transgene constructs in which the ETR1 promoter was used to drive expression of cDNAs for each of the five receptor isoforms were transferred into the ers1-2;etr1-7 double-mutant plants. Subfamily I constructs restored normal growth, whereas subfamily II constructs failed to rescue the double mutant, providing evidence for a unique role for subfamily I in receptor signaling. However, transformation of either the ers1-2;etr1-6 or ers1-2;etr1-7 mutant with a kinase-inactivated ETR1 genomic clone also resulted in complete restoration of normal growth and ethylene responsiveness in the double-mutant background, leading to the conclusion that canonical histidine kinase activity by receptors is not required for ethylene receptor signaling.

Cell Surface ABP1-TMK Auxin-Sensing Complex Activates ROP GTPase Signaling

A Different Route The plant hormone auxin regulates a variety of developmental processes and responses to environmental inputs, often via changes in gene transcription. Xu et al. (p. 1025) analyzed a signaling pathway involving ABP1 (auxin-binding protein 1) that affects the cytoskeleton and endocytosis in Arabidopsis without changing gene transcription. Instead, ABP1 functions at the cell surface to bind auxin and a family of membrane kinases, thereby activating intracellular guanosine triphosphatases to initiate important developmental changes in cell shape. An alternate signaling route for the plant hormone auxin goes directly inside from the cell surface. Auxin-binding protein 1 (ABP1) was discovered nearly 40 years ago and was shown to be essential for plant development and morphogenesis, but its mode of action remains unclear. Here, we report that the plasma membrane–localized transmembrane kinase (TMK) receptor–like kinases interact with ABP1 and transduce auxin signal to activate plasma membrane–associated ROPs [Rho-like guanosine triphosphatases (GTPase) from plants], leading to changes in the cytoskeleton and the shape of leaf pavement cells in Arabidopsis. The interaction between ABP1 and TMK at the cell surface is induced by auxin and requires ABP1 sensing of auxin. These findings show that TMK proteins and ABP1 form a cell surface auxin perception complex that activates ROP signaling pathways, regulating nontranscriptional cytoplasmic responses and associated fundamental processes.

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.

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.

Ethylene Stimulates Nutations That Are Dependent on the ETR1 Receptor

Ethylene influences a number of processes in Arabidopsis (Arabidopsis thaliana) through the action of five receptors. In this study, we used high-resolution, time-lapse imaging to examine the long-term effects of ethylene on growing, etiolated Arabidopsis seedlings. These measurements revealed that ethylene stimulates nutations of the hypocotyls with an average delay in onset of over 6 h. The nutation response was constitutive in ctr1-2 mutants maintained in air, whereas ein2-1 mutants failed to nutate when treated with ethylene. Ethylene-stimulated nutations were also eliminated in etr1-7 loss-of-function mutants. Transformation of the etr1-7 mutant with a wild-type genomic ETR1 transgene rescued the nutation phenotype, further supporting a requirement for ETR1. Loss-of-function mutations in the other receptor isoforms had no effect on ethylene-stimulated nutations. However, the double ers1-2 ers2-3 and triple etr2-3 ers2-3 ein4-4 loss-of-function mutants constitutively nutated in air. These results support a model where all the receptors are involved in ethylene-stimulated nutations, but the ETR1 receptor is required and has a contrasting role from the other receptor isoforms in this nutation phenotype. Naphthylphthalamic acid eliminated ethylene-stimulated nutations but had no effect on growth inhibition caused by ethylene, pointing to a role for auxin transport in the nutation phenotype.

Ethylene Receptor ETHYLENE RECEPTOR1 Domain Requirements for Ethylene Responses in Arabidopsis Seedlings

Ethylene influences many processes in Arabidopsis (Arabidopsis thaliana) through the action of five receptor isoforms. We used high-resolution, time-lapse imaging of dark-grown Arabidopsis seedlings to better understand the roles of each isoform in the regulation of growth in air, ethylene-stimulated nutations, and growth recovery after ethylene removal. We found that ETHYLENE RECEPTOR1 (ETR1) is both necessary and sufficient for nutations. Transgene constructs in which the ETR1 promoter was used to drive expression of cDNAs for each of the five receptor isoforms were transferred into etr1-6;etr2-3;ein4-4 triple loss-of-function mutants that have constitutive growth inhibition in air, fail to nutate in ethylene, and take longer to recover a normal growth rate when ethylene is removed. The patterns of rescue show that ETR1, ETR2, and ETHYLENE INSENSITIVE4 (EIN4) have the prominent roles in rapid growth recovery after removal of ethylene whereas ETR1 was the sole isoform that rescued nutations. ETR1 histidine kinase activity and phosphotransfer through the receiver domain are not required to rescue nutations. However, REVERSION TO SENSITIVITY1 modulates ethylene-stimulated nutations but does not modulate the rate of growth recovery after ethylene removal. Several chimeric receptor transgene constructs where domains of EIN4 were swapped into ETR1 were also introduced into the triple mutant. The pattern of phenotype rescue by the chimeric receptors used in this study supports a model where a receptor with a receiver domain is required for normal growth recovery and that nutations specifically require the full-length ETR1 receptor.

The TMK subfamily of receptor-like kinases in Arabidopsis display an essential role in growth and a reduced sensitivity to auxin.

Mechanisms that govern the size of plant organs are not well understood but believed to involve both sensing and signaling at the cellular level. We have isolated loss-of-function mutations in the four genes comprising the transmembrane kinase TMK subfamily of receptor-like kinases (RLKs) in Arabidopsis. These TMKs have an extracellular leucine-rich-repeat motif, a single transmembrane region, and a cytoplasmic kinase domain. While single mutants do not display discernable phenotypes, unique double and triple mutant combinations result in a severe reduction in organ size and a substantial retardation in growth. The quadruple mutant displays even greater severity of all phenotypes and is infertile. The kinematic studies of root, hypocotyl, and stamen filament growth reveal that the TMKs specifically control cell expansion. In leaves, TMKs control both cell expansion and cell proliferation. In addition, in the tmk double mutants, roots and hypocotyls show reduced sensitivity to applied auxin, lateral root induction and activation of the auxin response reporter DR5: GUS. Thus, taken together with the structural and biochemical evidence, TMKs appear to orchestrate plant growth by regulation of both cell expansion and cell proliferation, and as a component of auxin signaling.

Botryomycosis presenting as nasal cutaneous fistulas caused by Prevotella melaninogenica

Summary Botryomycosis is an uncommon chronic suppurative granulomatous bacterial infection that can affect the skin and viscera. Clinically, lesions typically consist of small tender nodules from which draining sinuses may develop to expel a purulent discharge. Histopathological features include characteristic aggregation of microorganisms (grain) within the inflammatory infiltrate. The commonest causative organisms are Staphylococcus aureus and Pseudomonas aeruginosa, of others. Botryomycosis resulting from Prevotella melaninogenica has not been reported previously. We report the case of a middle‐aged patient with botryomycosis presenting as nasal cutaneous fistulas caused by P. melaninogenica, which was successfully treated with surgical intervention combined with systemic antibiotic treatment.

Ethylene stimulates nutations of etiolated Arabidopsis hypocotyls that are dependent on the ETR1 receptor

1 University of Wisconsin in Madison, Department of Botany, 430 Lincoln Drive, Madison, WI, 53706, USA; 2 Current address: U.W. Madison, Department of Horticulture, 1575 Linden Drive, Madison, WI 53706; (608) 262-1779; 3 Current address: Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037; 4 Current address: Ceres Inc., Ceres, Inc.1535 Rancho Conejo Blvd., Thousand Oaks, CA 91320, USA; 5 Deceased. (*Corresponding author: bmbinder@wisc.edu)