Uncertainty of EIN2Ser645/Ser924 Inactivation by CTR1-Mediated Phosphorylation Reveals the Complexity of Ethylene Signaling.

ETHYLENE INSENSITIVE2 (EIN2) is a key component of ethylene signaling whose activity is inhibited upon phosphorylation of Ser645 and Ser924 by the Raf-like CONSTITUTIVE TRIPLE-RESPONSE 1 (CTR1) in the absence of ethylene. Ethylene prevents CTR1 activity and thus EIN2Ser645/Ser924 phosphorylation, and subcellular trafficking of a proteolytically cleaved EIN2 C terminus (EIN2-C) from the endoplasmic reticulum to the nucleus and processing bodies triggers ethylene signaling. Here, we report an unexpected complexity of EIN2-activated ethylene signaling. EIN2 activation in part requires ethylene in the absence of CTR1-mediated negative regulation. The ein2 mutant was complemented by the transgenes encoding EIN2, EIN2 variants with mutations that either prevent or mimic Ser645/Ser924 phosphorylation, or EIN2-C; and all the transgenic lines carrying these EIN2-derived transgenes responded to ethylene. Furthermore, we found that the fluorescence protein-tagged EIN2 and its variants were affected little by ethylene and exhibited similar subcellular distribution patterns: in the cytosolic particles and nuclear speckles. Of note, the subcellular localization patterns of EIN2 proteins fused with a fluorescence protein either at the N or C terminus were similar, whereas EIN2-C-YFP was primarily observed in the cytosol but not in the nucleus. Western blots and mass spectrum analyses suggested a high complexity of EIN2, which is likely proteolytically processed into multiple fragments. Our results suggested a nuclear localization of the full-length EIN2, weak association of the EIN2Ser645/Ser924 phosphorylation status and ethylene signaling, and the complexity of ethylene signaling caused by EIN2 and its proteolytic products in different subcellular compartments. We propose an alternative model to explain EIN2-activated ethylene signaling.

The Predicted RNA-Binding Protein ETR-1/CELF1 Acts in Muscles To Regulate Neuroblast Migration in Caenorhabditis elegans.

Neuroblast migration is a critical aspect of nervous system development (e.g, neural crest migration). In an unbiased forward genetic screen, we identified a novel player in neuroblast migration, the ETR-1/CELF1 RNA binding protein. CELF1 RNA binding proteins are involved in multiple aspects of RNA processing including alternative splicing, stability, and translation. We find that a specific mutation in alternatively-spliced exon 8 results in migration defects of the AQR and PQR neurons, and not the embryonic lethality and body wall muscle defects of complete knockdown of the locus. Surprisingly, ETR-1 was required in body wall muscle cells for AQR/PQR migration (i.e., it acts cell non-autonomously). Genetic interactions indicate that ETR-1 acts with Wnt signaling, either in the Wnt pathway or in a parallel pathway. Possibly, ETR-1 is involved in the production of a Wnt signal or a parallel signal by the body wall muscles that controls AQR and PQR neuronal migration. In humans, CELF1 is involved in a number of neuromuscular disorders. If the role of ETR-1/CELF1 is conserved, these disorders might also involve cell or neuronal migration. Finally, we describe a technique of amplicon sequencing to detect rare, cell-specific genome edits by CRISPR/Cas9 in vivo (CRISPR-seq) as an alternative to the T7E1 assay.

Canonical and noncanonical ethylene signaling pathways that regulate Arabidopsis susceptibility to the cyst nematode Heterodera schachtii.

Plant-parasitic cyst nematodes successfully exploit various phytohormone signaling pathways to establish a new hormonal equilibrium that facilitates nematode parasitism. Although it is largely accepted that ethylene regulates plant responses to nematode infection, a mechanistic understanding of how ethylene shapes plant-nematode interactions remains largely unknown. In this study, we examined the involvement of various components regulating ethylene perception and signaling in establishing Arabidopsis susceptibility to the cyst nematode Heterodera schachtii using a large set of well-characterized single and higher order mutants. Our analyses revealed the existence of two pathways that separately engage ethylene with salicylic acid (SA) and cytokinin signaling during plant response to nematode infection. One pathway involves the canonical ethylene signaling pathway in which activation of ethylene signaling results in suppression of SA-based immunity. The second pathway involves the ethylene receptor ETR1, which signals independently of SA acid to affect immunity, instead altering cytokinin-mediated regulation of downstream components. Our results reveal important mechanisms through which cyst nematodes exploit components of ethylene perception and signaling to affect the balance of hormonal signaling through ethylene interaction with SA and cytokinin networks. This hormonal interaction overcomes plant defense and provokes a susceptible response.

Novel functions for the RNA-binding protein ETR-1 in Caenorhabditis elegans reproduction and engulfment of germline apoptotic cell corpses.

RNA-binding proteins (RBPs) are essential regulators of gene expression that act through a variety of mechanisms to ensure the proper post-transcriptional regulation of their target RNAs. RBPs in multiple species have been identified as playing crucial roles during development and as having important functions in various adult organ systems, including the heart, nervous, muscle, and reproductive systems. ETR-1, a highly conserved ELAV-Type RNA-binding protein belonging to the CELF/Bruno protein family, has been previously reported to be involved in C. elegans muscle development. Animals depleted of ETR-1 have been previously characterized as arresting at the two-fold stage of embryogenesis. In this study, we show that ETR-1 is expressed in the hermaphrodite somatic gonad and germ line, and that reduction of ETR-1 via RNA interference (RNAi) results in reduced hermaphrodite fecundity. Detailed characterization of this fertility defect indicates that ETR-1 is required in both the somatic tissue and the germ line to ensure wild-type reproductive levels. Additionally, the ability of ETR-1 depletion to suppress the published WEE-1.3-depletion infertility phenotype is dependent on ETR-1 being reduced in the soma. Within the germline of etr-1(RNAi) hermaphrodite animals, we observe a decrease in average oocyte size and an increase in the number of germline apoptotic cell corpses as evident by an increased number of CED-1::GFP and acridine orange positive apoptotic germ cells. Transmission Electron Microscopy (TEM) studies confirm the significant increase in apoptotic cells in ETR-1-depleted animals, and reveal a failure of the somatic gonadal sheath cells to properly engulf dying germ cells in etr-1(RNAi) animals. Through investigation of an established engulfment pathway in C. elegans, we demonstrate that co-depletion of CED-1 and ETR-1 suppresses both the reduced fecundity and the increase in the number of apoptotic cell corpses observed in etr-1(RNAi) animals. Combined, this data identifies a novel role for ETR-1 in hermaphrodite gametogenesis and in the process of engulfment of germline apoptotic cell corpses.

Localization of the Ethylene-Receptor Signaling Complex to the Endoplasmic Reticulum: Analysis by Two-Phase Partitioning and Density-Gradient Centrifugation.

Ethylene receptors and other elements of the ethylene-signal transduction pathway localize to membranes of the endoplasmic reticulum (ER). New players in the ethylene signaling pathway continue to be discovered and so it is important to have methods by which to diagnose their cellular localization. Two methods for microsome isolation and fractionation are described here that can assist in determining if a protein localizes to the ER: aqueous two-phase partitioning and equilibrium density-gradient centrifugation. Two-phase partitioning serves to purify plasma membrane away from other cellular membranes and can thus discriminate whether a protein is localized to the plasma membrane or not. Equilibrium density-gradient centrifugation is particularly useful for resolving if a protein is localized to the ER. Ribosomes are associated with the rough ER in the presence of Mg2+ but are stripped away when Mg2+is removed from the medium, resulting in a reduction in the ER membrane density and a diagnostic shift in migration when analyzed by equilibrium density-gradient centrifugation. A method for growing plants in liquid culture is also provided because these microsomal membranes exhibit consistent fractionation by both two-phase partitioning and density-gradient centrifugation.

Single nucleotide polymorphism analysis reveals heterogeneity within a seedling tree population of a polyembryonic mango cultivar.

Within a polyembryonic mango seedling tree population, the genetic background of individuals should be identical because vigorous plants for cultivation are expected to develop from nucellar embryos representing maternal clones. Due to the fact that the mango cultivar 'Hôi' is assigned to the polyembryonic ecotype, an intra-cultivar variability of ethylene receptor genes was unexpected. Ethylene receptors in plants are conserved, but the number of receptors or receptor isoforms is variable regarding different plant species. However, it is shown here that the ethylene receptor MiETR1 is present in various isoforms within the mango cultivar 'Hôi'. The investigation of single nucleotide polymorphisms revealed that different MiETR1 isoforms can not be discriminated simply by individual single nucleotide exchanges but by the specific arrangement of single nucleotide polymorphisms at certain positions in the exons of MiETR1. Furthermore, an MiETR1 isoform devoid of introns in the genomic sequence was identified. The investigation demonstrates some limitations of high resolution melting and ScreenClust analysis and points out the necessity of sequencing to identify individual isoforms and to determine the variability within the tree population.

Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling.

Ethylene as a gaseous plant hormone is directly involved in various processes during plant growth and development. Much is known regarding the ethylene receptors and regulatory factors in the ethylene signal transduction pathway. In Arabidopsis thaliana, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) can interact with and positively regulates the ethylene receptor ETHYLENE RESPONSE1 (ETR1). In this study we report the identification and characterization of an RTE1-interacting protein, a putative Arabidopsis lipid transfer protein 1 (LTP1) of unknown function. Through bimolecular fluorescence complementation, a direct molecular interaction between LTP1 and RTE1 was verified in planta. Analysis of an LTP1-GFP fusion in transgenic plants and plasmolysis experiments revealed that LTP1 is localized to the cytoplasm. Analysis of ethylene responses showed that the ltp1 knockout is hypersensitive to 1-aminocyclopropanecarboxylic acid (ACC), while LTP1 overexpression confers insensitivity. Analysis of double mutants etr1-2 ltp1 and rte1-3 ltp1 demonstrates a regulatory function of LTP1 in ethylene receptor signaling through the molecular association with RTE1. This study uncovers a novel function of Arabidopsis LTP1 in the regulation of ethylene response and signaling.

Dominant gain-of-function mutations in transmembrane domain III of ERS1 and ETR1 suggest a novel role for this domain in regulating the magnitude of ethylene response in Arabidopsis.

Prior work resulted in identification of an Arabidopsis mutant, eer5-1, with extreme ethylene response in conjunction with failure to induce a subset of ethylene-responsive genes, including AtEBP. EER5, which is a TREX-2 homolog that is part of a nucleoporin complex, functions as part of a cryptic aspect of the ethylene signaling pathway that is required for regulating the magnitude of ethylene response. A suppressor mutagenesis screen was carried out to identify second site mutations that could restore the growth of ethylene-treated eer5-1 to wild-type levels. A dominant gain-of-function mutation in the ethylene receptor ETHYLENE RESPONSE SENSOR 1 (ERS1) was identified, with the ers1-4 mutation being located in transmembrane domain III at a point nearly equivalent to the previously described etr1-2 mutation in the other Arabidopsis subfamily I ethylene receptor, ETHYLENE RESPONSE 1 (ETR1). Although both ers1-4 and etr1-2 partially suppress the ethylene hypersensitivity of eer5-1 and are at least in part REVERSION TO ETHYLENE SENSITIVITY 1 (RTE1)-dependent, ers1-4 was additionally found to restore the expression of AtEBP in ers1-4;eer5-1 etiolated seedlings after ethylene treatment in an EIN3-dependent manner. Our work indicates that ERS1-regulated expression of a subset of ethylene-responsive genes is related to controlling the magnitude of ethylene response, with hyperinduction of these genes correlated with reduced ethylene-dependent growth inhibition.

Four shades of detachment: regulation of floral organ abscission.

Abscission of floral organs from the main body of a plant is a dynamic process that is developmentally and environmentally regulated. In the past decade, genetic studies in Arabidopsis have identified key signaling components and revealed their interactions in the regulation of floral organ abscission. The phytohormones jasmonic acid (JA) and ethylene play critical roles in flower development and floral organ abscission. These hormones regulate the timing of floral organ abscission both independently and inter-dependently. Although significant progress has been made in understanding abscission signaling, there are still many unanswered questions. These include considering abscission in the context of reproductive development and interplay between hormones embedded in the developmental processes. This review summarizes recent advances in the identification of molecular components in Arabidopsis and discusses their relationship with reproductive development. The emerging roles of hormones in the regulation of floral organ abscission, particularly by JA and ethylene, are examined.

An alternate route of ethylene receptor signaling.

The gaseous plant hormone ethylene is perceived by a family of ethylene receptors and mediates an array of ethylene responses. In the absence of ethylene, receptor signaling is conveyed via the C-terminal histidine kinase domain to the N-terminus of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) protein kinase, which represses ethylene signaling mediated by ETHYLENE INSENSITIVE2 (EIN2) followed by EIN3. In the presence of ethylene, the receptors are inactivated when ethylene binds to their N-terminal domain, and consequently CTR1 is inactive, allowing EIN2 and EIN3 to activate ethylene signaling. Recent findings have shown that the ethylene receptor N-terminal portion can conditionally mediate the receptor signal output in mutants lacking CTR1, thus providing evidence of an alternative pathway from the ethylene receptors not involving CTR1. Here we highlight the evidence for receptor signaling to an alternative pathway and suggest that receptor signaling is coordinated via the N- and C-termini, as we address the biological significance of the negative regulation of ethylene signaling by the two pathways.

Cloning, overexpression, purification and preliminary X-ray analysis of the catalytic domain of the ethylene receptor ETR1 from Arabidopsis thaliana.

Ethylene is a gaseous plant hormone which controls many aspects of plant growth and development. It is perceived by membrane-bound receptors with a similarity to bacterial two-component systems. The catalytic and ATP-binding domain of the histidine kinase domain of ETR1 from Arabidopsis thaliana has been cloned, overexpressed and crystallized. The protein was crystallized together with various nucleotides. Crystals obtained in the presence of ADP belonged to space group I222 or I2(1)2(1)2(1) with one molecule per asymmetric unit. They diffracted X-ray radiation to beyond 1.85 Šresolution.

Current understanding on ethylene signaling in plants: the influence of nutrient availability.

The plant hormone ethylene is involved in many physiological processes, including plant growth, development and senescence. Ethylene also plays a pivotal role in plant response or adaptation under biotic and abiotic stress conditions. In plants, ethylene production often enhances the tolerance to sub-optimal environmental conditions. This role is particularly important from both ecological and agricultural point of views. Among the abiotic stresses, the role of ethylene in plants under nutrient stress conditions has not been completely investigated. In literature few reports are available on the interaction among ethylene and macro- or micro-nutrients. However, the published works clearly demonstrated that several mineral nutrients largely affect ethylene biosynthesis and perception with a strong influence on plant physiology. The aim of this review is to revisit the old findings and recent advances of knowledge regarding the sub-optimal nutrient conditions on the effect of ethylene biosynthesis and perception in plants. The effect of deficiency or excess of the single macronutrient or micronutrient on the ethylene pathway and plant responses are reviewed and discussed. The synergistic and antagonist effect of the different mineral nutrients on ethylene plant responses is critically analyzed. Moreover, this review highlights the status of information between nutritional stresses and plant response, emphasizing the topics that should be further investigated.

The role of receptor interactions in regulating ethylene signal transduction.

The phytohormone ethylene is perceived in Arabidopsis by a five-member receptor family. Earlier work has demonstrated that the basic functional unit for an ethylene receptor is a disulfide-linked homodimer. We recently reported in The Journal of Biological Chemistry that the ethylene-receptor ETR1 physically associates with other ethylene receptors through higher order interactions, suggesting the existence of receptor clusters. Here we consider the implications of such clusters upon the mechanism of ethylene signal transduction. In particular, we consider how such clustering provides a cooperative mechanism, akin to what has been found for the prokaryotic chemoreceptors, by which plant sensitivity to ethylene may be increased. In addition, we consider how the dominant ethylene insensitivity conferred by some receptor mutations, such as etr1-1, may also be propagated by interactions among members of the ethylene receptor family.

The role of ethylene perception in the control of photosynthesis.

The process of photosynthesis is under the control by several internal factors. Apart from the effect of abscisic acid on stomatal conductance, little is known about the interaction between hormonal signals and photosynthesis in fully-developed, nonsenescing leaves. Recently, we found that the ethylene transduction pathway is involved in the regulation of photosynthesis. Using an ethylene-insensitive tobacco genotype we showed that the absence of a functional ethylene receptor leads to a reduction in Rubisco content and photosynthetic capacity. In this addendum, we present some additional data indicating that photosynthetic capacity is also reduced in ethylene-insensitive Arabidopsis mutants.