The squash gain-of-function mutant etr2b disrupts the ethylene-binding domain of ethylene receptor CpETR2B, conferring partial ethylene insensitivity, changes in flower and fruit development, and enhanced salt tolerance. In this paper, we found that etr2b also confers a growth advantage as well as a physiological and metabolomic response that make the mutant better adapted to drought. Mutant plants had a higher root and leaf biomass than WT under both well-watered and drought conditions, but the reduction in growth parameters in response to drought was similar in WT and etr2b. Water deficit reduced all gas-exchange parameters in both WT and etr2b, but under moderate drought the mutant increased photosynthesis rate in comparison with control conditions, and showed a higher leaf CO2 concentration, transpiration rate, and stomata conductance than WT. The response of etr2b to drought indicates that ethylene is a negative regulator of plant growth under both control and drought. Since etr2b increased ABA content in well-watered plant, but prevented the induction of ABA production in response to drought, it is likely that the etr2b response under drought is not mediated by ABA. A 1H NMR metabolomic analysis revealed that etr2b enhances the accumulation of osmolytes (soluble sugars and trigonelline), unsaturated and polyunsaturated fatty acids, and phenolic compounds under drought, concomitantly with a reduction of malic- and fumaric-acid. The role of CpETR2B and ethylene in the regulation of these drought-protective metabolites is discussed.

CONCLUSIONS: Taetr1-1 can promote enhanced seed dormancy and ethylene insensitivity in wheat, indicating a conserved function of ETR1 in regulating seed dormancy. Lots of wheat cultivars have weak dormant seed. Weak seed dormancy can cause pre-harvest sprouting (PHS) in grain which significantly reduces grain yield and quality. The mining of causal genes of PHS resistance will serve to enhance breeding selection and cultivar development. In a previous study in Arabidopsis, we identified reduced dormancy 3 as a loss-of-function mutant of the ethylene receptor 1 (ETR1), which can control seed dormancy through the ERF12-TPL-DOG1 pathway. However, it is unknown whether ETR1 also functions in the regulation of wheat seed dormancy. To identify the regulatory role of ETR1 in wheat, we cloned TaETR1 and overexpressed the gain-of-function mutant Taetr1-1. The result indicated that overexpression of Taetr1-1 can promote enhanced seed dormancy and ethylene insensitivity in wheat. This study contributed to our understanding of the molecular basis for the regulation of wheat PHS resistance.

The gaseous hormone ethylene is perceived in plants by membrane-bound receptors, the best studied of these being ETR1 from Arabidopsis. Ethylene receptors can mediate a response to ethylene concentrations at less than one part per billion; however, the mechanistic basis for such high-affinity ligand binding has remained elusive. Here we identify an Asp residue within the ETR1 transmembrane domain that plays a critical role in ethylene binding. Site-directed mutation of the Asp to Asn results in a functional receptor that has a reduced affinity for ethylene, but still mediates ethylene responses in planta. The Asp residue is highly conserved among ethylene receptor-like proteins in plants and bacteria, but Asn variants exist, pointing to the physiological relevance of modulating ethylene-binding kinetics. Our results also support a bifunctional role for the Asp residue in forming a polar bridge to a conserved Lys residue in the receptor to mediate changes in signaling output. We propose a new structural model for the mechanism of ethylene binding and signal transduction, one with similarities to that found in a mammalian olfactory receptor.

The sex-control system involves several mechanisms in melon. The present study identified a novel bisexual flower control gene from the hermaphroditic melon germplasm, different from the previously recognized one. Genetic analysis showed that a single recessive gene in the newly identified locus b controlled the bisexual flower phenotype in melons. We generated 1431 F2 segregating individuals for genetic mapping of locus b, which was delimited to a 47.94 kb region. Six candidate genes were identified in the delimited interval, and candidate No. 4 encoding melon CPR5 protein was selected as the suitable one for locus b and was denoted CmCPR5. CPR5 reportedly interacted with ethylene receptor ETR1 to regulate ethylene signal transduction. Moreover, the ethephon assays showed that the parental lines (unisexual line and bisexual line) had contrasting expression patterns of CmCPR5. The BiFC and LCI assays also confirmed that CmCPR5 interacted with CmETR1 in 0426 but not in Y101. However, crossover tests showed that CmETR1 functioned normally in both parental lines, suggesting CPR5 malfunction in Y101. This study proposed a corollary mechanism of bisexual flower regulation during stamen primordium development in which the inhibition of stamen primordia development was prevented by the malfunctioning CmCPR5, resulting in bisexual flowers.

Ethylene regulates mycelial growth, primordium formation, and postharvest mushroom maturation and senescence in the white button mushroom, Agaricus bisporus. However, it remains unknown how ethylene is detected by the mushroom. In this study, we found that two hybrid histidine kinases in the mushroom, designated AbETR1 and AbETR2, showed domain structures similar to those of plant ethylene receptors. The transmembrane helices of AbETR1 and AbETR2 were expressed in yeast cells and showed ethylene-binding activities. Mushroom strains with downregulated expressions of AbETR1 and AbETR2 showed reduced sensitivity to the ethylene inhibition of mycelial growth, ethylene regulation of their own synthesis, postharvest mushroom maturation, and senescence and expression of maturation- and senescence-related genes. Therefore, AbETR1 and AbETR2 are expected to be biologically functional ethylene receptors and exhibit a different mode of action from that of the receptors of plants. Here, we fill gaps in the knowledge pertaining to higher fungus ethylene receptors, discover a novel mode of action of ethylene receptors, confirm ethylene as a novel fungal hormone, and provide a facilitated approach for preventing the maturation and senescence of postharvest button mushrooms. IMPORTANCE Ethylene regulates diverse physiological activities in bacteria, cyanobacteria, fungi, and plants, but how to perceive ethylene by fungi only remains unknown. In this study, we identify two biologically functional ethylene receptors in the basidiomycete fungus Agaricus bisporus, which fills the gaps of deficient fungal ethylene receptors. Furthermore, we found that decreased expression of the ethylene receptors facilitates preventing the maturation and senescence of postharvest button mushrooms, indicating that the two fungal ethylene receptors positively regulate the ethylene response, in contrast to that in plants.

Following successful pollination, Dendrobium orchid flowers rapidly undergo senescence. In Dendrobium cv. Khao Chaimongkol, compatible pollination resulted in faster ethylene production and more rapid development of senescence symptoms, such as drooping, epinasty, venation and yellowing, compared with non-pollinated controls or pollination with incompatible pollinia. The DenACS1 and DenACO1 genes in the perianth of florets that had been pollinated with compatible pollinia were expressed more highly than those in non-pollinated open florets. Incompatible pollinia reduced the expression of DenACS1 and DenACO1 genes in the perianth. Transcript levels of the ethylene receptor gene DenERS1 and signaling genes DenEIL1 and DenERF1 showed differential spatial regulation with greater expression in the perianth than in the column plus ovary following compatible pollination. Compatible pollinia increased ethylene production concomitant with premature senescence and the increased expression of the DenACS1 and DenACO1 genes, and suppressed the ethylene receptor gene DenERS1, whereas incompatible pollinia did not stimulate ethylene production nor induce premature senescence but induced higher expression of DenERS1 both in the perianth and in the column plus ovary. These results suggest that the increased ethylene production in open florets pollinated with compatible pollen was partially due to an increase in the expression of DenACS1 and DenACO1 genes. The compatible pollinia induced a negative regulation of DenERS1 which may play an important role in ethylene perception and in modulating ethylene signaling transduction during pollinia-induced flower senescence.

Ripening of climacteric fruits is initiated when the gaseous plant hormone ethylene is perceived by the cell. Ethylene binding to membrane-associated receptors (ETRs) triggers a series of biochemical events through multiple components, resulting in the induction of numerous ripening-related genes. In tomato (Solanum lycopersicum L.), there are seven members of the ETR family, which each contributes to the regulation of fruit ripening. However, the relative contribution of each individual receptor to ethylene signaling remains unknown. Here, we demonstrated the formation of heteromeric receptor complexes across the two ETR subfamilies in tomato fruit. Immunoprecipitation of subfamily II SlETR4 resulted in co-purification of subfamily I (SlETR1, SlETR2, and SlETR3), but not subfamily II members (SlETR5, SlETR6, and SlETR7). Such biased interactions were verified in yeast two-hybrid assays, and in transgenic Arabidopsis plants, in which heterologous SlETR4 interacts with subfamily I ETRs. Our analysis also revealed that the receptor complexes engage the Raf-like protein kinases SlCTR1 and SlCTR3, which are potential regulators of signaling. Here, we suggest that tomato receptor members form heteromeric complexes to fine-tune signal output to the downstream pathway, which is similar to that of the Arabidopsis system but appears to be partially diverged.

A gene encoding a protein similar to ethylene receptor was isolated from maize (Zea mays L.), which was named as ZmERS4.The gene was 1,905 bp in length with an open reading frame that encoded a protein consisting of 634 amino acids. The homologous analysis showed that ZmERS4 shared high similarity with the ethylene receptor protein, OsERS1, from rice (Oryza sativa L.). ZmERS4 grouped into the ETR1 subfamily of ethylene receptors based on its conserved domain and phylogenetic status. Tissue-specific and induced expression analyses indicated that ZmERS4 was differentially expressed in maize tissues, predominantly in the stems and leaves, and was induced by salicylic acid (SA). Overexpression of ZmERS4 in Arabidopsis improved resistance against the bacterial pathogen, PstDC3000, by inducing the expression of SA signaling-related genes. Moreover, treatment with flg22 induced the expression of the defense-related gene, PR1, in maize protoplasts that transiently expressed ZmERS4. Furthermore, the ultra-high-performance liquid chromatography (UPLC) analysis showed that the SA contents in ZmERS4-overexpressing Arabidopsis lines were significantly higher than the control lines. Additionally, the improved resistance of ZmERS4-overexpressing Arabidopsis against PstDC3000 was blocked after pretreatment with the SA biosynthetic inhibitor, ABT. Based on the collective findings, we hypothesize that ZmERS4 plays an important role in disease resistance through SA-mediated signaling pathways.

Ethylene is the key regulator of sex determination in monoecious species of the family Cucurbitaceae. This hormone determines which individual floral meristems develop as female or male flowers and the female flowering transition. The sex determination genes discovered so far code for ethylene biosynthesis enzymes, but little is known about the importance of ethylene signaling components. In this paper we characterize two novel ethylene-insensitive mutations (etr1a-1 and etr1b) which block the female flowering transition of Cucurbita pepo; this makes plants produce male flowers indefinitely (androecy). Two missense mutations in the ethylene-binding domain of the ethylene receptors CpETR1A or CpETR1B were identified as the causal mutations of these phenotypes by using whole-genome resequencing. The distinctive phenotypes of single and double mutants for four etr mutations have demonstrated that the final level of ethylene insensitivity depends upon the strength and dosage of mutant alleles for at least three cooperating ETR genes, and that the level of ethylene insensitivity determines the final sex phenotype of the plant. The sex phenotype ranges from monoecy in ethylene-sensitive wild-type plants to androecy in the strongest ethylene-insensitive ones, via andromonoecy in partially ethylene-insensitive plants. The induction of female flowering transition was found to be associated with upregulation of CpACS11, CpACO2 and CpACS27, three ethylene biosynthesis genes required for female flower development. A model is proposed herein, integrating both ethylene biosynthesis and receptor genes into the genetic network which regulates sex determination in C. pepo.

Ethylene is a gaseous phytohormone that is perceived by two-component histidine kinase-type receptors. Recent studies identified choline transporter-like 1 (CTL1) essential for Arabidopsis growth and development, including apical hook development in the etiolated seedlings. Here, we report that CTL1 contributes to apical hook development by enhancing ethylene response. The expression of CTL1 was highly correlated with the intensity of ethylene response and was enriched in the apical hook, cotyledon tip and hypocotyl. Genetic analysis showed that the dark-grown ctl1 mutant displayed a defect in ethylene-induced apical hook development as compared with the wild type. Accordingly, the expression of ethylene signaling reporter EBS::GUS in ctl1 mutant was greatly reduced in leaves, apical hook, hypocotyl and root, suggesting that the disruption of CTL1 impairs the ethylene signaling. Furthermore, protein-protein interaction assays demonstrated that CTL1 may interact with ethylene receptors, including ETR1, ETR2, ERS1, ERS2. Importantly, the abundance of CTL1 was diminished when ETR1 was disrupted upon ethylene response. Taken together, our results suggest that CTL1 functions as a positive regulator in ethylene signaling which in turn contributes to apical hook development of etiolated plant seedlings.