Ethylene Signaling Components Research Articles

Ethylene Signaling in Regulating Plant Growth, Development, and Stress Responses

Ethylene is a gaseous plant hormone that plays a crucial role in coordinating various physiological processes in plants. It acts as a key mediator, integrating both endogenous developmental cues and external environmental signals to regulate a wide range of functions, including growth, fruit ripening, leaf abscission, and responses to stress. The signaling pathway is initiated when ethylene binds to its receptor. After decades of research, the key components of ethylene signaling have been identified and characterized. Although the molecular mechanisms of the sensing of ethylene signal and its transduction have been studied extensively, a new area of research is how respiration and epigenetic modifications influence ethylene signaling and ethylene response. Here, we summarize the research progress in recent years and review the function and importance of ethylene signaling in plant growth and stress responses. In addition, we also describe the current understanding of how epigenetic modifications regulate ethylene signaling and the ethylene response. Together, our review sheds light on the new signaling mechanisms of ethylene.

PpNAP4 and ethylene act in a regulatory loop to modulate peach fruit ripening and softening.

Ripening significantly influences fruit quality and commercial value. Peaches (Prunus persica), a climacteric fruit, exhibit increased ethylene biosynthesis and decreased fruit firmness during ripening. NAC-like proteins activated by AP3/P1 (NAP) proteins are a subfamily of NAC transcription factors, and certain NAPs have been shown to intervene in fruit ripening. Here, we revealed that one NAP member PpNAP4, along with ethylene, positively regulated peach ripening and softening. Positive regulation of fruit ripening by PpNAP4 was demonstrated by overexpressing PpNAP4 in both peaches and tomatoes, resulting in enhanced fruit ripening through targeted modulation of specific ethylene biosynthesis and cell wall degradation-related genes. Further investigation revealed that PpNAP4 targets and upregulates key ethylene biosynthesis genes PpACS1, PpACO1 and PpEIN2, which is the core component of ethylene signaling. PpNAP4 positively modulates fruit softening by binding to and activating the promoters of cell wall degradation-related genes PpPL1 and PpPL15. Additionally, expression of PpPL1 and PpPL15 was directly affected by ethylene, with further investigation revealing that their promoters were clearly induced by ethylene. Our findings demonstrate a synergistic role played by the interaction between PpNAP4 and PpNAP6, enhancing the expression of PpACS1, PpACO1, PpPL1, PpPL15 and PpEIN2, thereby contributing to fruit ripening and softening. Overall, our study revealed the intricate mechanisms responsible for PpNAP4, PpNAP6, and ethylene roles during peach fruit ripening, highlighting a regulatory loop in which PpNAP4 and ethylene mutually enhance each other during the ripening process. These enhancements further contribute to peach fruit softening by upregulating specific cell wall degradation-related genes.

Ethylene in fruits: beyond ripening control.

Fruits are a rich source of nutrients, minerals, and dietary fibers for both humans and animals. While the gaseous phytohormone ethylene is well-known for its role in controlling fruit ripening, there is growing evidence that ethylene also plays crucial roles in regulating other developmental processes of fruits, such as sex determination, fruit set, and fruit growth. In this review, we aim to revisit these findings from various species like cucumber, melon, tomato, rice, maize, and more. These studies not only enhance our understanding of ethylene's function in fruits but also highlight the potential for manipulating ethylene to improve crops. Furthermore, we discuss recent studies that show the ethylene precursor ACC (1-AMINOCYCLOPROPANE-1-CARBOXYLATE), and the ethylene signaling components EIN2 (ETHYLENE INSENSITIVE2) and EIN3 (ETHYLENE INSENSITIVE3) have ethylene-independent function in specific conditions. This phenomenon, combined with findings of dosage-dependent ethylene functions in certain conditions, highlights the importance of analyzing mutants with completely blocked ethylene pathways in different species at specific developmental stages and tissue types. Overall, this review offers a timely and essential summary of ethylene's role in sex determination, fruit formation, and fruit growth, which could be beneficial for horticulture crop breeding.

Plant hormone ethylene: A leading edge in conferring drought stress tolerance

AbstractAgricultural sufficient productivity is of paramount importance for ensuring food security and conserving soil health to support the world's agronomy. Climatic abruptions have been emerging as one of the most nerve‐pressing issues for the sustainment of the planet Earth in the twenty‐first century. Among the various environmental constraints, drought stress stands out as a potent factor restricting crop growth and productivity. It triggers a myriad of intricate responses in plants to combat the underlying stress‐mediated adversities. Gaining a comprehensive understanding of the key physiological and molecular mechanisms that enable plants to withstand drought stress is crucial for developing effective strategies to enhance crop resilience. Ethylene, a gaseous plant hormone, influences the adaptive measures adopted by plants subjected to drought stress by regulating the drought stress‐mediated signal transduction‐associated responses. The present review article provides an in‐depth understanding of the critical roles of ethylene in enhancing plants' ability to restrain the severity of drought stress. It also highlights the significance of ethylene signaling components in regulating plant survival and drought stress tolerance. Additionally, we have illustrated the additive and antagonistic interactions of ethylene with other plant growth regulators, which instigate the tolerance responses. Conclusively, this review emphasizes the significance of complex networks involved in ethylene‐mediated drought tolerance, providing valuable insights for future research and uncovering novel studies in the field of ethylene biology.

Uncovering the proximal proteome of CTR1 through TurboID-mediated proximity labeling.

Protein-protein interactions play a crucial role in driving cellular processes and enabling appropriate physiological responses in organisms. The plant hormone ethylene signaling pathway is complex and regulated by the spatiotemporal regulation of its signaling molecules. Constitutive Triple Response 1 (CTR1), a key negative regulator of the pathway, regulates the function of Ethylene-Insensitive 2 (EIN2), a positive regulator of ethylene signaling, at the endoplasmic reticulum (ER) through phosphorylation. Our recent study revealed that CTR1 can also translocate from the ER to the nucleus in response to ethylene and positively regulate ethylene responses by stabilizing EIN3. To gain further insights into the role of CTR1 in plants, we used TurboID-based proximity labeling and mass spectrometry to identify the proximal proteomes of CTR1 in Nicotiana benthamiana. The identified proximal proteins include known ethylene signaling components, as well as proteins involved in diverse cellular processes such as mitochondrial respiration, mRNA metabolism, and organelle biogenesis. Our study demonstrates the feasibility of proximity labeling using the N. benthamiana transient expression system and identifies the potential interactors of CTR1 in vivo, uncovering the potential roles of CTR1 in a wide range of cellular processes.

Postharvest fruit quality of tomatoes influenced by an ethylene signaling component during long-term cold storage.

Ethylene production is essential for improving cold resistance of postharvest tomatoes. However, the role of ethylene signaling pathway in maintaining fruit quality during long-term cold storage remains poorly understood. Here, we demonstrated that a partial loss of function in ethylene signaling by mutation of Ethylene Response Factor 2 (SlERF2), worsened fruit quality during cold storage, as determined by visual characterization, and physiological analyses of membrane damage and reactive oxygen species metabolism. In addition, the transcriptions of genes related to abscisic acid (ABA) biosynthesis and signaling were also altered by SlERF2 gene in response to cold storage. Furthermore, mutation of SlERF2 gene compromised cold-induced expression of genes in the C-repeat/dehydration-responsive binding factor (CBF) signaling pathway. Therefore, it's concluded that an ethylene signaling component, SlERF2 contributed to the regulations of ABA biosynthesis and signaling, as well as CBF cold signaling pathway, ultimately affecting the fruit quality during long-term cold storage of tomatoes.

Humic Substances Isolated from Recycled Biomass Trigger Jasmonic Acid Biosynthesis and Signalling.

Intensive agriculture maintains high crop yields through chemical inputs, which are well known for their adverse effects on environmental quality and human health. Innovative technologies are required to reduce the risk generated by the extensive and harmful use of pesticides. The plant biostimulants made from humic substances isolated from recyclable biomass offer an alternative approach to address the need for replacing conventional agrochemicals without compromising the crop yield. The stimulatory effects of humic substances are commonly associated with plant hormones, particularly auxins. However, jasmonic acid (JA) is crucial metabolite in mediating the defence responses and governing plant growth and development. This work aimed to evaluate the changes in the biosynthesis and signalling pathway of JA in tomato seedlings treated with humic acids (HA) isolated from vermicompost. We use the tomato model system cultivar Micro-Tom (MT) harbouring a reporter gene fused to a synthetic promoter that responds to jasmonic acid (JERE::GUS). The transcript levels of genes involved in JA generation and activity were also determined using qRT-PCR. The application of HA promoted plant growth and altered the JA status, as revealed by both GUS and qRT-PCR assays. Both JA enzymatic synthesis (LOX, OPR3) and JA signalling genes (JAZ and JAR) were found in higher transcription levels in plants treated with HA. In addition, ethylene (ETR4) and auxin (ARF6) signalling components were positively modulated by HA, revealing a hormonal cross-talk. Our results prove that the plant defence system linked to JA can be emulated by HA application without growth inhibition.

A role for ethylene signaling and biosynthesis in regulating and accelerating CO2 - and abscisic acid-mediated stomatal movements in Arabidopsis.

Little is known about long-distance mesophyll-driven signals that regulate stomatal conductance. Soluble and/or vapor-phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance in Arabidopsis thaliana by CO2 /abscisic acid (ABA) was examined. We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophyll-dependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene-signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO2 ]-shifts. According to our research, higher [CO2 ] causes Arabidopsis rosettes to produce more ethylene. An ACC-synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO2 -induced stomatal movements. Ethylene-insensitive receptor (gain-of-function), etr1-1 and etr2-1, and signaling, ein2-5 and ein2-1, mutants showed intact stomatal responses to [CO2 ]-shifts, whereas loss-of-function ethylene receptor mutants, including etr2-3;ein4-4;ers2-3, etr1-6;etr2-3 and etr1-6, showed markedly accelerated stomatal responses to [CO2 ]-shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC-synthase octuple mutant and accelerated stomatal responses in the etr1-6;etr2-3, and etr1-6, but not in the etr2-3;ein4-4;ers2-3 mutants. These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO2 and ABA.

High temperature elevates carotenoid accumulation of banana fruit via upregulation of MaEIL9 module

Banana is a good source of carotenoids, which are bioactive metabolites with health beneficial properties for human. However, the molecular mechanism of carotenoid accumulation in banana fruit is largely unclear. In this study, we found that high temperature elevated carotenoid production in banana pulp, which is presumably due to upregulation of a subset of carotenogenic genes as well as a carotenoid biosynthesis regulator MaSPL16. Moreover, an ethylene signaling component MaEIL9 was identified, whose transcript and protein contents were also induced by high temperature. In addition, MaEIL9 positively regulates transcription of MaDXR1, MaPDS1, MaZDS1 and MaSPL16 through directly targeting their promoters. Overexpression of MaEIL9 in tomato fruit substantially increased the expression of carotenoid formation genes and elevated carotenoid content. Importantly, transiently silencing MaEIL9 in banana fruit weakened carotenoid production caused by high temperature. Taken together, these results indicate that high temperature induces carotenoid production in banana fruit, at least in part, through MaEIL9-mediated activation of MaDXR1, MaPDS1, MaZDS1 and MaSPL16 expression.

Methionine oxidation and reduction of the ethylene signaling component MaEIL9 are involved in banana fruit ripening.

The ethylene insensitive 3/ethylene insensitive 3-like (EIN3/EIL) plays an indispensable role in fruit ripening. However, the regulatory mechanism that links post-translational modification of EIN3/EIL to fruit ripening is largely unknown. Here, we studied the expression of 13 MaEIL genes during banana fruit ripening, among which MaEIL9 displayed higher enhancement particularly in the ripening stage. Consistent with its transcript pattern, abundance of MaEIL9 protein gradually increased during the ripening process, with maximal enhancement in the ripening. DNA affinity purification (DAP)-seq analysis revealed that MaEIL9 directly targets a subset of genes related to fruit ripening, such as the starch hydrolytic genes MaAMY3D and MaBAM1. Stably overexpressing MaEIL9 in tomato fruit hastened fruit ripening, whereas transiently silencing this gene in banana fruit retarded the ripening process, supporting a positive role of MaEIL9 in fruit ripening. Moreover, oxidation of methionines (Met-129, Met-130, and Met-282) in MaEIL9 resulted in the loss of its DNA-binding capacity and transcriptional activation activity. Importantly, we identified MaEIL9 as a potential substrate protein of methionine sulfoxide reductase A MaMsrA4, and oxidation of Met-129, Met-130, and Met-282 in MaEIL9 could be restored by MaMsrA4. Collectively, our findings reveal a novel regulatory network controlling banana fruit ripening, which involves MaMsrA4-mediated redox regulation of the ethylene signaling component MaEIL9.

APETALA2/ethylene responsive factor in fruit ripening: Roles, interactions and expression regulation

Insects and animals are attracted to, and feed on ripe fruit, thereby promoting seed dispersal. As a vital vitamin and nutrient source, fruit make up an indispensable and enjoyable component of the human diet. Fruit ripening involves a series of physiological and biochemical changes in, among others, pigmentation, chlorophyll (Chl) degradation, texture, sugar accumulation, and flavor. Growing evidence indicates that the coordinated and ordered trait changes during fruit ripening depend on a complex regulatory network consisting of transcription factors, co-regulators, hormonal signals, and epigenetic modifications. As one of the predominant transcription factor families in plants and a downstream component of ethylene signaling, more and more studies are showing that APETALA2/ethylene responsive factor (AP2/ERF) family transcription factors act as critical regulators in fruit ripening. In this review, we focus on the regulatory mechanisms of AP2/ERFs in fruit ripening, and in particular the recent results on their target genes and co-regulators. We summarize and discuss the role of AP2/ERFs in the formation of key fruit-ripening attributes, the enactment of their regulatory mechanisms by interaction with other proteins, their role in the orchestration of phytohormone-signaling networks, and the epigenetic modifications associated with their gene expression. Our aim is to provide a multidimensional perspective on the regulatory mechanisms of AP2/ERFs in fruit ripening, and a reference for understanding and furthering research on the roles of AP2/ERF in fruit ripening.

Jasmonate Hypersensitive 3 negatively regulates both jasmonate and ethylene-mediated responses in Arabidopsis.

Jasmonate (JA) is an important hormone involved in regulating diverse responses to environmental factors as well as growth and development, and its signalling is influenced by other hormones such as ethylene (ET). However, our understanding of the regulatory relationship between the JA and ET signalling pathways is limited. In this study, we isolated an Arabidopsis JA-hypersensitive mutant, jah3 (jasmonate hypersensitive3)-1. Map-based cloning revealed that the JAH3 gene corresponds to At4g16535. JAH3 encodes a protein of unknown function whose amino acid sequence has similarity to leukocyte receptor cluster-like protein. The mutation in jah3-1 is caused by a single nucleotide change from A to T at position 220 of 759 bp. Using CRISPR-Cas9, we generated a second allele, jah3-2, that encodes a truncated protein. Both of these loss-of-function alleles resulted in hypersensitivity to JA, ET-induced root growth inhibition, and accelerated dark-induced senescence. Double mutant analyses employing coronatine insensitive 1 (coi1) and ethylene insensitive 3 (ein3) mutants (jah3 coi1 and jah3 ein3) demonstrated that the hypersensitive phenotypes of the jah3 mutants are mediated by JA and ET signalling components COI1 and EIN3. Therefore, we propose that JAH3 is a negative regulator of both JA and ET signalling.

Relatively Low Light Intensity Promotes Phosphorus Absorption and Enhances the Ethylene Signaling Component EIN3 in Maize, Wheat, and Oilseed Rape

Previous studies have found that once seedlings break the soil, light can induce the degradation of the key ethylene signaling element ethylene insensitive 3 (EIN3), so as to indirectly inhibit the synthesis of ethylene. Ethylene is the most important hormone in phosphorus absorption by plants, which induces the expression of acid phosphatase (APase) and phosphorus starvation response genes. Therefore, it might be speculated that changes in light intensity could regulate phosphorus absorption to some degree. However, there are few reports on the mechanism by which light intensity regulates phosphorus metabolism. In this study, the effects of different light intensities on phosphorus assimilation and metabolism in plants were studied. The results showed that relatively low light intensity could promote the secretion of APase, so as to increase the concentration of plant total phosphorus and cellular Pi. However, the low light intensity may also inhibit plant growth. Among the three species, oilseed rape was the most sensitive to the low light intensity. The steady-state level of the EIN3 protein decreased significantly under a relatively high light intensity; while the ethylene level also decreased under the high light intensity. Therefore, appropriate reductions in light intensity may simultaneously promote phosphorus assimilation and maintain plant growth.

Lack of ethylene does not affect reproductive success and synergid cell death in Arabidopsis

The signaling pathway of the gaseous hormone ethylene is involved in plant reproduction, growth, development, and stress responses. During reproduction, the two synergid cells of the angiosperm female gametophyte both undergo programmed cell death (PCD)/degeneration but in a different manner: PCD/degeneration of one synergid facilitates pollen tube rupture and thereby the release of sperm cells, while PCD/degeneration of the other synergid blocks supernumerary pollen tubes. Ethylene signaling was postulated to participate in some of the synergid cell functions, such as pollen tube attraction and the induction of PCD/degeneration. However, ethylene-mediated induction of synergid PCD/degeneration and the role of ethylene itself have not been firmly established. Here, we employed the CRISPR/Cas9 technology to knock out the five ethylene-biosynthesis 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes and created Arabidopsis mutants free of ethylene production. The ethylene-free mutant plants showed normal triple responses when treated with ethylene rather than 1-aminocyclopropane-1-carboxylic acid, but had increased lateral root density and enlarged petal sizes, which are typical phenotypes of mutants defective in ethylene signaling. Using these ethylene-free plants, we further demonstrated that production of ethylene is not necessarily required to trigger PCD/degeneration of the two synergid cells, but certain components of ethylene signaling including transcription factors ETHYLENE-INSENSITIVE 3 (EIN3) and EIN3-LIKE 1 (EIL1) are necessary for the death of the persistent synergid cell.

Tomato and cotton G protein beta subunit mutants display constitutive autoimmune responses.

Heterotrimeric G protein Gβ‐deficient mutants in rice and maize display constitutive immune responses, whereas Arabidopsis Gβ mutants show impaired defense, suggesting the existence of functional differences between monocots and dicots. Using CRISPR/Cas9, we produced one hemizygous tomato line with a mutated SlGB1 Gβ gene. Homozygous slgb1 knockout mutants exhibit all the hallmarks of autoimmune mutants, including development of necrotic lesions, constitutive expression of defense‐related genes, and high endogenous levels of salicylic acid (SA) and reactive oxygen species, resulting in early seedling lethality. Virus‐induced silencing of Gβ in cotton reproduced the symptoms observed in tomato mutants, confirming that the autoimmune phenotype is not limited to monocot species but is also shared by dicots. Even though multiple genes involved in SA and ethylene signaling are highly induced by Gβ silencing in tomato and cotton, co‐silencing of SA or ethylene signaling components in cotton failed to suppress the lethal phenotype, whereas co‐silencing of the oxidative burst oxidase RbohD can repress lethality. Despite the autoimmune response observed in slgb1 mutants, we show that SlGB1 is a positive regulator of the pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) response in tomato. We speculate that the phenotypic differences observed between Arabidopsis and tomato/cotton/rice/maize Gβ knockouts do not necessarily reflect divergences in G protein‐mediated defense mechanisms.

Ethylene-induced banana starch degradation mediated by an ethylene signaling component MaEIL2

Starch is the major reserve carbohydrate in nature, which possesses nutritional property and industrial applications. Starch degradation is an important contributor to softening and sweetening of banana fruit, but more details in this process are not fully understood. In this study, the contents of total starch, amylose and amylopectin were gradually decreased during banana fruit ripening, which is in parallel with the increased levels of total soluble sugars. Particularly, reduced sizes and elongated shapes of starch granules, as well as decreased crystallinity were observed as ripening proceeds, which is largely due to the digestion of starch degradation enzymes such as amylase and isoamylase. Importantly, an ethylene signaling component MaEIL2 bound to the promoters of amylase and isoamylase encoding genes MaAMY3, MaISA2 and MaISA3, and stimulated their transcription. Overall, these findings reveal that starch-sugar transformation during banana ripening is mediated by enzymatic hydrolysis, and that ethylene signaling component MaEIL2 positively modulates starch breakdown via trans-activation of MaAMY3, MaISA2 and MaISA3.

Modulation of Organogenesis and Somatic Embryogenesis by Ethylene: An Overview

Ethylene is a plant hormone controlling physiological and developmental processes such as fruit maturation, hairy root formation, and leaf abscission. Its effect on regeneration systems, such as organogenesis and somatic embryogenesis (SE), has been studied, and progress in molecular biology techniques have contributed to unveiling the mechanisms behind its effects. The influence of ethylene on regeneration should not be overlooked. This compound affects regeneration differently, depending on the species, genotype, and explant. In some species, ethylene seems to revert recalcitrance in genotypes with low regeneration capacity. However, its effect is not additive, since in genotypes with high regeneration capacity this ability decreases in the presence of ethylene precursors, suggesting that regeneration is modulated by ethylene. Several lines of evidence have shown that the role of ethylene in regeneration is markedly connected to biotic and abiotic stresses as well as to hormonal-crosstalk, in particular with key regeneration hormones and growth regulators of the auxin and cytokinin families. Transcriptional factors of the ethylene response factor (ERF) family are regulated by ethylene and strongly connected to SE induction. Thus, an evident connection between ethylene, stress responses, and regeneration capacity is markedly established. In this review the effect of ethylene and the way it interacts with other players during organogenesis and somatic embryogenesis is discussed. Further studies on the regulation of ERF gene expression induced by ethylene during regeneration can contribute to new insights on the exact role of ethylene in these processes. A possible role in epigenetic modifications should be considered, since some ethylene signaling components are directly related to histone acetylation.

Expression and protein levels of ethylene receptors, CTRs and EIN2 during tomato fruit ripening as affected by 1- MCP

Many studies have focused on the plant hormone ethylene because of its key role in controlling, among others, climacteric fruit ripening and fruit senescence. These processes can be controlled by applying 1-MCP, which tightly binds to the ethylene receptors thereby blocking the ethylene signaling pathway. 1-MCP is known to inhibit the action of ethylene and to delay the climacteric ripening of tomato fruit. Less is known about its long term effect when the inhibitory effect 1-MCP inhibition is eventually released. Our objective was to study this transient 1-MCP inhibition during tomato fruit ripening in terms of fruit quality, ethylene production, respiration rate and the expression and protein abundance of receptors, CTRs and EIN2. For the identification and quantification of proteins, we used an LC–MS based targeted method of Parallel Reaction Monitoring (PRM), while gene expression was done using real time qPCR. Different color stages of tomatoes were harvested and treated with 1-MCP and subsequently stored to follow up postharvest fruit ripening. The difference with previous 1-MCP studies is that we sampled 1-MCP treated tomatoes at different physiological stages during ripening (and not time), matching the color stages of the untreated control fruit. This allows to properly compare the underlying regulation of the ethylene signaling pathway during a 1-MCP-mediated suppression of ripening. We hypothesized that the levels of the ethylene signaling components would be different for 1-MCP treated fruit due to a reduced ethylene-mediated autocatalytic feed-back. Our results showed that fruit treated with 1-MCP at mature green stage showed a lower respiration rate during subsequent ripening as compared to the untreated fruit, suggesting that climacteric ripening was effectively inhibited by 1-MCP. However, these 1-MCP treated fruit showed a higher ethylene production as compared to untreated fruit. The 1-MCP treated fruit also showed lower to equal levels of gene expression and protein abundance of the ethylene receptors, CTRs and EIN2. As receptors and CTRs are negative regulators of ethylene signaling, decreasing the production of new signaling proteins could subsequently activate downstream ethylene signaling and with that expression of downstream genes. This could lead to higher ethylene production levels, which in turn can compensate 1-MCP mediated inhibition of fruit ripening.

Involvement of ethylene receptors in the salt tolerance response of Cucurbita pepo

Abiotic stresses have a negative effect on crop production, affecting both vegetative and reproductive development. Ethylene plays a relevant role in plant response to environmental stresses, but the specific contribution of ethylene biosynthesis and signalling components in the salt stress response differs between Arabidopsis and rice, the two most studied model plants. In this paper, we study the effect of three gain-of-function mutations affecting the ethylene receptors CpETR1B, CpETR1A, and CpETR2B of Cucurbita pepo on salt stress response during germination, seedling establishment, and subsequent vegetative growth of plants. The mutations all reduced ethylene sensitivity, but enhanced salt tolerance, during both germination and vegetative growth, demonstrating that the three ethylene receptors play a positive role in salt tolerance. Under salt stress, etr1b, etr1a, and etr2b germinate earlier than WT, and the root and shoot growth rates of both seedlings and plants were less affected in mutant than in WT. The enhanced salt tolerance response of the etr2b plants was associated with a reduced accumulation of Na+ in shoots and leaves, as well as with a higher accumulation of compatible solutes, including proline and total carbohydrates, and antioxidant compounds, such as anthocyanin. Many membrane monovalent cation transporters, including Na+/H+ and K+/H+ exchangers (NHXs), K+ efflux antiporters (KEAs), high-affinity K+ transporters (HKTs), and K+ uptake transporters (KUPs) were also highly upregulated by salt in etr2b in comparison with WT. In aggregate, these data indicate that the enhanced salt tolerance of the mutant is led by the induction of genes that exclude Na+ in photosynthetic organs, while maintaining K+/Na+ homoeostasis and osmotic adjustment. If the salt response of etr mutants occurs via the ethylene signalling pathway, our data show that ethylene is a negative regulator of salt tolerance during germination and vegetative growth. Nevertheless, the higher upregulation of genes involved in Ca2+ signalling (CpCRCK2A and CpCRCK2B) and ABA biosynthesis (CpNCED3A and CpNCED3B) in etr2b leaves under salt stress likely indicates that the function of ethylene receptors in salt stress response in C. pepo can be mediated by Ca2+ and ABA signalling pathways.

RPW8.1 enhances the ethylene-signaling pathway to feedback-attenuate its mediated cell death and disease resistance in Arabidopsis.

Summary The Arabidopsis RESISTANCE TO POWDERY MILDEW 8.1 (RPW8.1) activates confined cell death and defense against different pathogens. However, the underlying regulatory mechanisms still remain elusive.Here, we show that RPW8.1 activates ethylene signaling that, in turn, negatively regulates RPW8.1 expression. RPW8.1 binds and stabilizes 1‐aminocyclopropane‐1‐carboxylate oxidase 4 (ACO4), which may in part explain increased ethylene production and signaling in RPW8.1‐expressing plants. In return, ACO4 and other key components of ethylene signaling negatively regulate RPW8.1‐mediated cell death and disease resistance via suppressing RPW8.1 expression.Loss of function in ACO4, EIN2, EIN3 EIL1, ERF6, ERF016 or ORA59 increases RPW8.1‐mediated cell death and defense response. By contrast, overexpression of EIN3 abolishes or significantly compromises RPW8.1‐mediated cell death and disease resistance. Furthermore, ERF6, ERF016 and ORA59 appear to act as trans‐repressors of RPW8.1, with OAR59 being able to directly bind to the RPW8.1 promoter.Taken together, our results have revealed a feedback regulatory circuit connecting RPW8.1 and the ethylene‐signaling pathway, in which RPW8.1 enhances ethylene signaling, and the latter, in return, negatively regulates RPW8.1‐mediated cell death and defense response via suppressing RPW8.1 expression to attenuate its defense activity.