Ethylene-insensitive Arabidopsis Research Articles

EIN3/EIL (Ethylene Insensitive3 / Ethylene Insensitive3 Like) Protein Family in Phaseolus vulgaris: Identification, Evolution and Expression Analysis within the Genome

Ethylene insensitive-3 (EIN3) / Ethylene insensitive-3-like (EIL) protein family is a small family of transcription factors specific to plants that play role in plant growth and development under various environmental conditions. In this study, various bioinformatics approaches were used to make an in-depth identification of the EIN3/EIL family at both the gene and protein levels. So, 11 Pvul-EIL genes were identified and their approximate locations were determined. Various biochemical and physicochemical properties of EIL proteins in Phaseolus vulgaris have been described. It was determined that Pvul-EIL proteins had a length of 447-651 amino acids and a molecular weight of 51.08-70.68 kDa. All duplications occurring in the Pvul-EIL genome were segmental type. It was observed that conserved motif, gene structure and phylogeny analyses all yielded similar results. For instance, it has been understood that genes with same motif type and number have similar gene structures and were located under the same branch in the phylogenetic tree. Pvul-EIL protein homology modeling showed that DNA binding properties and protein structure were similar to Arabidopsis EIN3. According to cis-element analysis, Pvul-EIL genes are engaged in a wide range of functions, including tissue-specific, stress, and hormone-sensitive expression. Additionally, RNAseq data was used to perform a comparative expression analysis of EIL genes. Various Pvul-EIL gene expression levels were detected under salt and drought stress. This is the first study to check the gene expression levels in P. vulgaris using in-silico detection and characterization of EIL genes. Therefore, obtained results can form the basis for future studies.

Light Modulates Ethylene Synthesis, Signaling, and Downstream Transcriptional Networks to Control Plant Development.

The inhibition of hypocotyl elongation by ethylene in dark-grown seedlings was the basis of elegant screens that identified ethylene-insensitive Arabidopsis mutants, which remained tall even when treated with high concentrations of ethylene. This simple approach proved invaluable for identification and molecular characterization of major players in the ethylene signaling and response pathway, including receptors and downstream signaling proteins, as well as transcription factors that mediate the extensive transcriptional remodeling observed in response to elevated ethylene. However, the dark-adapted early developmental stage used in these experiments represents only a small segment of a plant’s life cycle. After a seedling’s emergence from the soil, light signaling pathways elicit a switch in developmental programming and the hormonal circuitry that controls it. Accordingly, ethylene levels and responses diverge under these different environmental conditions. In this review, we compare and contrast ethylene synthesis, perception, and response in light and dark contexts, including the molecular mechanisms linking light responses to ethylene biology. One powerful method to identify similarities and differences in these important regulatory processes is through comparison of transcriptomic datasets resulting from manipulation of ethylene levels or signaling under varying light conditions. We performed a meta-analysis of multiple transcriptomic datasets to uncover transcriptional responses to ethylene that are both light-dependent and light-independent. We identified a core set of 139 transcripts with robust and consistent responses to elevated ethylene across three root-specific datasets. This “gold standard” group of ethylene-regulated transcripts includes mRNAs encoding numerous proteins that function in ethylene signaling and synthesis, but also reveals a number of previously uncharacterized gene products that may contribute to ethylene response phenotypes. Understanding these light-dependent differences in ethylene signaling and synthesis will provide greater insight into the roles of ethylene in growth and development across the entire plant life cycle.

Ethylene-insensitive Arabidopsis mutants etr1-1 AND ein2-1 have decreased freezing tolerance

The freezing tolerance of Arabidopsis thaliana (L.) Heynh. was studied in relation to functioning of the ethylene signaling pathway. Constitutive freezing tolerance was compared in wild-type plants (ecotype Col-0) and ethylene-insensitive mutants etr1-1 and ein2-1. For the first time it was established that ethylene-insensitive mutants had by 25-30% lower net photosynthesis rate, the decreased content of soluble sugars and, as a result, lower freezing tolerance. Our work provides evidence that perception and transduction of ethylene signal are necessary for constitutive tolerance of Arabidopsis to low temperature.

Molecular Characterization and Functional Analysis of Two Petunia PhEILs.

Ethylene plays an important role in flower senescence of many plants. Arabidopsis ETHYLENE INSENSITIVE3 (EIN3) and its homolog EIL1 are the downstream component of ethylene signaling transduction. However, the function of EILs during flower senescence remains unknown. Here, a petunia EIL gene, PhEIL2, was isolated. Phylogenetic tree showed that PhEIL1, whose coding gene is previously isolated, and PhEIL2 are the homologs of Arabidopsis AtEIL3 and AtEIL1, respectively. The expression of both PhEIL1 and PhEIL2 is the highest in corollas and increased during corolla senescence. Ethylene treatment increased the mRNA level of PhEIL1 but reduced that of PhEIL2. VIGS-mediated both PhEIL1 and PhEIL2 silencing delayed flower senescence, and significantly reduced ethylene production and the expression of PhERF3 and PhCP2, two senescence-associated genes in petunia flowers. The PhEIL2 protein activating transcription domain is identified in the 353-612-amino acids at C-terminal of PhEIL2 and yeast two-hybrid and bimolecular fluorescence complementation assays show that PhEIL2 interacts with PhEIL1, suggesting that PhEIL1 and PhEIL2 might form heterodimers to recognize their targets. These molecular characterizations of PhEIL1 and PhEIL2 in petunia are different with those of in Vigna radiata and Arabidopsis.

Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis

Ethylene participates in the regulation of numerous cellular events and biological processes, including water loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a 15N stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eil1-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene-regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-0 and ein3eil1 genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up-regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs.

Biochemical and Structural Insights into the Mechanism of DNA Recognition by Arabidopsis ETHYLENE INSENSITIVE3.

Gaseous hormone ethylene regulates numerous stress responses and developmental adaptations in plants by controlling gene expression via transcription factors ETHYLENE INSENSITIVE3 (EIN3) and EIN3-Like1 (EIL1). However, our knowledge regarding to the accurate definition of DNA-binding domains (DBDs) within EIN3 and also the mechanism of specific DNA recognition by EIN3 is limited. Here, we identify EIN3 82–352 and 174–306 as the optimal and core DBDs, respectively. Results from systematic biochemical analyses reveal that both the number of EIN3-binding sites (EBSs) and the spacing length between two EBSs affect the binding affinity of EIN3; accordingly, a new DNA probe which has higher affinity with EIN3 than ERF1 is also designed. Furthermore, we show that palindromic repeat sequences in ERF1 promoter are not necessary for EIN3 binding. Finally, we provide, to our knowledge, the first crystal structure of EIN3 core DBD, which contains amino acid residues essential for DNA binding and signaling. Collectively, these data suggest the detailed mechanism of DNA recognition by EIN3 and provide an in-depth view at molecular level for the transcriptional regulation mediated by EIN3.

A role for Ethylene-Insensitive3 in the regulation of hydrogen peroxide production during seed germination under high salinity in Arabidopsis

The Arabidopsis Ethylene-Insensitive3 (EIN3) has received attention recently and has been shown to be involved in the regulation of multitude of responses ranging from biotic stress defense and development to hormone interaction. To better understand the roles of EIN3 in plants response to salinity stress during germination and post-germination development, seeds of two EIN3 deficient mutant and a EIN3 overexpression mutant of Arabidopsis were analyzed under salinity and compared with Col-0 as control. The results showed that the ein3-1eil1-1 double mutant (lacking EIN3 and EIN3-Like1) and ein3-1 (lacking EIN3) were hypersensitive to high salinity (>150 mM NaCl), while EIN3 overexpression mutant (EIN3ox) displayed enhanced tolerance, indicating that EIN3 plays important roles during seed germination under salinity. In addition, we also found that the two EIN3 deficient mutant seedlings accumulate high levels of hydrogen peroxide (H2O2), which was thought to be an inhibitor of germination under salinity before, suggesting that EIN3 may function as a negative regulator of reactive oxygen species metabolism in germinating seeds under salinity. Taken together, our studies provide insights that EIN3 promotes seed germination under salinity, at least in part, through modulating concentration of H2O2 in germinating seeds.

Isolation and expression analysis of three EIN3-like genes in tree peony (Paeonia suffruticosa)

Ethylene is a critical signal that influences cut flower opening and senescence in tree peony (Paeonia suffruticosa), which is a traditional ornamental plant in China. Arabidopsis ETHYLENE-INSENSITIVE3 (EIN3) acts as a key transcription factor of the ethylene signaling pathway, suggesting a possible role for its homologues in regulation of cut flower postharvest development. In this study, three EIN3 homologous genes including PsEIL1, PsEIL2, and PsEIL3 have been isolated from petals of tree peony. Deduced amino acid sequences of conserved domains of PsEILs share high similarities with the Arabidopsis EIN3 protein. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that three PsEILs were differentially expressed in various tissues of tree peony, and none was flower specific. During cut flower postharvest development, PsEIL1 transcript in petals accumulated at a relatively low level at the opening stage and reached the highest level of mRNA accumulation at senescence; PsEIL2 and PsEIL3 transcripts were gradually increased and peaked at full opening stage followed by a decline when petals wilted. However, these PsEIL genes exhibited differential responses to ethylene and 1-methycyclopropene (1-MCP) treatments. Compared with the control, the mRNA level of PsEIL1 was not influenced by either ethylene or 1-MCP treatment, whereas both PsEIL2 and PsEIL3 transcripts were significantly increased by exogenous ethylene, and only PsEIL3 was strongly inhibited by 1-MCP. These results suggest that PsEIL transcripts are spatiotemporally regulated, and the transcriptional regulation of PsEIL3 may play an important role in ethylene-mediated cut flower opening and senescence in tree peony.

Cloning, characterization, and expression of LeEIL-1, an Arabidopsis EIN3 homolog, in Lithospermum erythrorhizon

Ethylene is a crucial signal in regulating the biosynthesis of shikonin in Lithospermum erythrorhizon. Arabidopsis ethylene-insensitive 3 (EIN3) is the key transcriptional factor of the ethylene signal transduction pathway; thus, EIN3 homologs might play an important role in shikonin formation. Here, LeEIL-1, a gene with high similarity to EIN3, was cloned from L. erythrorhizon using a combination method of touch-down PCR and rapid amplification of cDNA ends. The full-length cDNA of LeEIL-1 is 2,359 bp, encoding a polypeptide of 635 amino acids. By inserting the entire coding region of LeEIL-1 into the pET32a (+) expression vector, the prokaryotic expression of LeEIL-1 was successfully induced by isopropyl β-d-1-thiogalactopyranoside in BL21(DE3)pLysS cells. Moreover, the recombinant LeEIL-1 protein was verified by western blotting with the Arabidopsis anti-EIN3 antibody. Real-time PCR results show that the mRNA level of LeEIL-1 was first dramatically induced within 3 h when the L. erythrorhizon cells were transferred from B5 to M9 medium for shikonin formation; subsequently, the transcripts of LeEIL-1 decreased to a relatively stable level. Tissue-specific expression analysis indicates that less LeEIL-1 mRNA accumulated in the stem and leaf than in the root, where shikonin was biosynthesized. These results imply that LeEIL-1 could be a possible reverse genetic target for revealing the relationship between ethylene and shikonin formation.

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.

Arabidopsis enhanced ethylene response 4 encodes an EIN3-interacting TFIID transcription factor required for proper ethylene response, including ERF1 induction

eer4 was isolated as an Arabidopsis mutant with an extreme response to ethylene in dark-grown seedlings that was also found to have partial ethylene insensitivity at the level of ethylene-dependent gene expression, including ERF1. Subsequent cloning of eer4 revealed an inappropriate stop codon in a previously uncharacterized TFIID-interacting transcription factor homologous to human TAF12 and yeast TAF61. Genetic and pharmacological analysis demonstrated that the eer4 phenotype is strictly ethylene dependent in seedlings, yet a double mutant with the partially ethylene-insensitive Arabidopsis mutant, ein3-1, had restored ethylene responsiveness, indicating that eer4 also regulates a previously unknown resetting or dampening mechanism for the ethylene signalling pathway. Consistent with the absolute requirement of EER4 for ERF1 expression, biochemical analysis showed that EER4 is localized to the nucleus where it probably recruits EIN3 and probably other transcription factors along with components of the TFIID complex for expression of a subset of genes required for either manifestation or subsequent dampening of the response to ethylene.

OsEIL1, a Rice Homolog of the Arabidopsis EIN3 Regulates the Ethylene Response as a Positive Component

The plant gaseous hormone ethylene regulates many aspects of plant growth, development and responses to the environment. ETHYLENE INSENSITIVE3 (EIN3) is a transcription factor involved in the ethylene signal transduction pathway in Arabidopsis. To gain a better understanding of the ethylene signal transduction pathway in rice, six EIN3-like genes (designated OsEIL1-6) were identified. OsEIL1, which showed highest similarity with EIN3, was isolated and functionally characterized. Ectopic expression of OsEIL1 in Arabidopsis can partially complement the ein3-1 mutant. The transgenic rice plants with overexpression of OsEIL1 exhibit short root, coiled primary root and slightly short shoot phenotype and elevated response to exogenous ethylene. OsEBP89, an ethylene responsive element binding protein (EREBP) and OsACO1, an ACC (1-aminocyclopropane-1-carboxylic acid) oxidase gene were enhanced in the OsEIL1 overexpressing transgenic plants. These results indicate that OsEIL1 is involved in ethylene signal transduction pathway and acts as a positive regulator of ethylene response in rice.

Use of differential display for the identification of touch-induced genes from an ethylene-insensitive Arabidopsis mutant and partial characterization of these genes

Touch has been shown to affect plant growth and development and ethylene has been shown to have similar effects. However, the mechanisms responsible for touch-induced responses remain unclear. Differential display PCR was used to identify touch-regulated genes from 3-week-light-grown ethylene-insensitive etr1-3 Arabidopsis (Columbia ecotype) mutant plants. The differential display PCR screening process yielded 32 cDNA fragments. Subsequent screening of the 32 fragments using northern analysis yielded three touch-inducible clones (A8A, G5A and G7F). These three cDNA were then used to screen a cDNA library. A 1.2kb fragment for OPR3 was obtained from A8A screenings. This cDNA fragment encodes 12-oxophytodienoate-10, 11-reductase (OPR), an enzyme in the jasmonic acid biosynthetic pathway. OPR3 was found to be induced by touch, wounding, methyl jasmonate (MeJA), NaCl and CaCl(2) while ethylene and darkness had no effect. A 2kb cDNA encoding a calcium-dependent protein kinase (CDPK32) was obtained with G5A screenings. CDPK32 was shown to be induced by touch, wounding, NaCl and darkness while ethylene and MeJA had little or no effect. A 1.4kb cDNA encoding a novel protein was recovered from the cDNA library screenings with a G7F fragment. This cDNA had some sequence similarity to GDA1 and was designated GDL for GDA1-like cDNA. GDL was activated by touch, wounding, MeJA, NaCl and CaCl(2) while there was no induction with ethylene and darkness. Using differential display PCR we have successfully been able to identify three clones that are inducible by touch and not by ethylene.

Salicylate Activity. 1. Protection of Plants from Paraquat Injury

Paraquat (1,1'-dimethyl-4,4'-bipyridinium; methylviologen) is a widely used, nonselective contact herbicide that rapidly stimulates free radical generation. It has been found that the addition of sodium salicylate (sodium 2-hydroxybenzoate; NaSA) to paraquat spray solutions significantly decreased herbicidal activity. This protection was observed in tobacco (Nicotiana tabacum) regardless of whether NaSA was foliar-applied along with or prior to paraquat application or NaSA was soil-applied prior to paraquat application. Because salicylic acid (SA) is an inducer of systemic acquired resistance (SAR) to plant disease, paraquat protection by three SAR inducers (acibenzolar-S-methyl, harpin, and probenazole) and selected salicylate derivatives was assessed. Twenty-two of 24 compounds tested decreased herbicidal activity when foliar-applied with paraquat. Protection from paraquat was greatest with 5-chlorosalicylate, and no protection was observed with benzoic acid. NaSA decreased paraquat activity on npr1-2, an Arabidopsis mutant that is compromised in NaSA-induced SAR, and on ein2-1, an ethylene-insensitive Arabidopsis mutant. Thus, salicylate protection from paraquat is independent of disease resistance and ethylene perception. This suggests the existence of an NaSA-mediated pathway capable of protecting plants from reactive oxygen stress.

Constitutive expression of EIL-like transcription factor partially restores ripening in the ethylene-insensitive Nr tomato mutant.

Climacteric fruit ripening is regulated by the phytohormone ethylene. ETHYLENE-INSENSITIVE3 (EIN3) is a transcription factor that functions downstream from the ethylene receptors in the Arabidopsis ethylene signal transduction pathway. Three homologues of the Arabidopsis EIN3 gene have been identified in tomato, Lycopersicon esculentum, EIN3-like or LeEIL, LeEIL1, LeEIL2, and LeEIL3. These transcription factors have been proposed to be functionally redundant positive regulators of multiple ethylene responses. In order to test the role of such factors in the ethylene signal transduction pathway during ripening, EIL1 fused to green fluorescent protein (GFP) has been over-expressed in the ethylene-insensitive non-ripening Nr mutant of tomato. Increased levels of LeEIL1 compensated for the normally reduced levels of LeEIL1 in the Nr mutant, and transgenic Nr plants that exhibited high-level constitutive expression of LeEIL1GFP phenotypically resembled wild-type plants, the fruit ripened and the leaves exhibited epinasty, unlike Nr plants. The EIL1GFP fusion protein was located in the cell nuclei of ripe tomato fruit. The mRNA profile of these plants showed that the expression of certain ethylene-dependent ripening genes was up-regulated, including polygalacturonase and TOMLOX B. However, not all ripening genes and ethylene responses, such as seedling triple response, were restored. These results demonstrate that expressing candidate genes in the Nr ethylene-insensitive background is a valuable general approach for testing the role of putative downstream components in the ethylene-signalling pathway.

Ethylene insensitivity does not increase leaf area or relative growth rate in Arabidopsis, Nicotiana tabacum, and Petunia x hybrida.

The plant hormone ethylene plays a role in various growth related processes. In this detailed study of the vegetative growth of Arabidopsis, Nicotiana tabacum, and Petunia x hybrida plants, we show that ethylene insensitivity does not result in an increased total leaf area or relative growth rate (RGR) under optimal growth conditions. When grown in semiclosed containers, leaf area of ethylene-insensitive plants was larger compared to the wild type. This effect was caused by a buildup of ethylene inside these containers, which inhibited the growth of wild-type plants. Ethylene-insensitive Arabidopsis and N. tabacum plants had a lower biomass, which was mainly the result of a smaller seed mass. RGR of vegetative plants was not affected by ethylene insensitivity, but the underlying components of RGR differed; specific leaf area (leaf area per unit leaf mass) was higher, and unit leaf rate (growth rate per unit leaf area) was lower. The latter was a result of a slower rate of photosynthesis per unit leaf area in the ethylene-insensitive plants.

Members of the tomato LeEIL (EIN3‐like) gene family are functionally redundant and regulate ethylene responses throughout plant development

The plant hormone ethylene regulates many aspects of growth, development and responses to the environment. The Arabidopsis ETHYLENE INSENSITIVE3 (EIN3) protein is a nuclear-localized component of the ethylene signal-transduction pathway with DNA-binding activity. Loss-of-function mutations in this protein result in ethylene insensitivity in Arabidopsis. To gain a better understanding of the ethylene signal-transduction pathway in tomato, we have identified three homologs of the Arabidopsis EIN3 gene (LeEILs). Each of these genes complemented the ein3-1 mutation in transgenic Arabidopsis, indicating that all are involved in ethylene signal transduction. Transgenic tomato plants with reduced expression of a single LeEIL gene did not exhibit significant changes in ethylene response; reduced expression of multiple tomato LeEIL genes was necessary to reduce ethylene sensitivity significantly. Reduced LeEIL expression affected all ethylene responses examined, including leaf epinasty, flower abscission, flower senescence and fruit ripening. Our results indicate that the LeEILs are functionally redundant and positive regulators of multiple ethylene responses throughout plant development.

Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea.

Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola results in systemic induction of genes encoding a plant defensin (PDF1.2), a basic chitinase (PR-3), and an acidic hevein-like protein (PR-4). Pathogen-induced induction of these three genes is almost completely abolished in the ethylene-insensitive Arabidopsis mutant ein2-1. This indicates that a functional ethylene signal transduction component (EIN2) is required in this response. The ein2-1 mutants were found to be markedly more susceptible than wild-type plants to infection by two different strains of the gray mold fungus Botrytis cinerea. In contrast, no increased fungal colonization of ein2-1 mutants was observed after challenge with avirulent strains of either Peronospora parasitica or A. brassicicola. Our data support the conclusion that ethylene-controlled responses play a role in resistance of Arabidopsis to some but not all types of pathogens.

Activation of the Ethylene Gas Response Pathway in Arabidopsis by the Nuclear Protein ETHYLENE-INSENSITIVE3 and Related Proteins

Mutations in the Arabidopsis ETHYLENE-INSENSITIVE3 (EIN3) gene severely limit a plant's response to the gaseous hormone ethylene. ein3 mutants show a loss of ethylene-mediated effects including gene expression, the triple response, cell growth inhibition, and accelerated senescence. EIN3 acts downstream of the histidine kinase ethylene receptor, ETR1, and the Raf-like kinase, CTR1. The EIN3 gene encodes a novel nuclear-localized protein that shares sequence similarity, structural features, and genetic function with three EIN3-LIKE (EIL) proteins. In addition to EIN3, EIL1 or EIL2 were able to complement ein3, suggesting their participation in the ethylene signaling pathway. Overexpression of EIN3 or EIL1 in wild-type or ethylene-insensitive2 plants conferred constitutive ethylene phenotypes, indicating their sufficiency for activation of the pathway in the absence of ethylene.