Ethylene advances the transition from vegetative growth to flowering in Arabidopsis thaliana

The transition from vegetative growth to flowering is the most drastic change in plant development. In order to examine the involvement of ethylene in growth transition, we compared the development of ethylene-related mutants, eto1, etr1, ein2-1 and ein3-1, with the wild type (WT) in Arabidopsis thaliana. The ethylene sensitivity of two WT and the mutants is decreased in the following order: eto1 = WT < ein3-1 < ein2-1 = etr1-1. Bolting time was also delayed in nearly the same order: eto1 < WT < ein3-1 < ein2-1 < etr1. Leaf numbers increased according to the delay of bolting time, indicating that the delay of bolting time was caused by the delay of transition from vegetative to reproductive growth. Other growth parameters, including leaf area and number of flowers opening at the same time, increased in the same order, indicating that these changes were caused by a single factor, the amount of ethylene signal which was transferred though an ethylene signal transduction pathway. These results suggest that ethylene is involved in the transition from vegetative to reproductive growth in Arabidopsis thaliana.     

Ethylene and shoot regeneration: hookless1 modulates de novo shoot organogenesis in Arabidopsis thaliana

We have investigated the role of ethylene in shoot regeneration from cotyledon explants of Arabidopsis thaliana. We examined the ethylene sensitivity of five ecotypes representing both poor and prolific shoot regenerators and identified Dijon-G, a poor regenerator, as an ecotype with dramatically enhanced ethylene sensitivity. However, inhibiting ethylene action with silver nitrate generally reduced shoot organogenesis in ecotypes capable of regeneration. In ecotype Col-0, we found that ethylene-insensitive mutants (etr1-1, ein2-1, ein4, ein7) exhibited reduced shoot regeneration rates, whereas constitutive ethylene response mutants (ctr1-1, ctr1-12) increased the proportion of explants producing shoots. Our experiments with ethylene over-production mutants (eto1, eto2 and eto3) indicate that the ethylene biosynthesis inhibitor gene, ETO1, can act as an inhibitor of shoot regeneration. Pharmacological elevation of ethylene levels was also found to significantly increase the proportion of explants regenerating shoots. We determined that the hookless1 (hls1-1) mutant, a suppressor of the ethylene response phenotypes of ctr1 and eto1 mutants, is capable of dramatically enhancing shoot organogenesis. The effects of ACC and loss of HLS1 function on shoot organogenesis were found to be largely additive.     

Effect of octanoic acid on ethylene-mediated processes in Arabidopsis

We have examined whether octanoic acid (OA) one of the short chain saturated fatty acids (SCSFA), increases ethylene response in the following three ethylene-mediated processes: a) hypocotyl growth in darkness; b) formation of new flowers; c) flower abscission. These processes were examined in the presence or absence of exogenous ethylene in Arabidopsis wild type (WT) and in the ethylene-insensitive mutants, etr1-3 and ein2-1 and in the ethylene over-producer mutant eto1-1. Our results show that OA decreased hypocotyl length of WT in the absence or presence of exogenous ethylene, apparently showing that OA acts via augmentation of ethylene action. However, the hypocotyl growth inhibition could not be ascribed to increased ethylene sensitivity since application of inhibitors of ethylene synthesis (aminoethoxyvinylglycine; AVG) or action (1-methylcyclopropene;1-MCP) to WT seedlings did not prevent specifically the OA-induced growth inhibition. Also, OA inhibited hypocotyl growth in the mutants etr1-3 and ein2-1 in a similar pattern to that obtained in WT. On the other hand, OA had no effect on flower formation neither in WT, etr1-3 and eto1-1, in which ethylene reduced flower formation, nor in the ein2-1 mutant, in which ethylene had no effect. OA also did not increase flower abscission in WT or in the mutants etr1-3 and ein2-1 neither in the absence nor in the presence of ethylene. However, OA has augmented flower abscission in the mutant eto1-1 only in the absence of exogenous ethylene. This result might indicate that the effect of OA on eto1-1 is specific to this mutant and is not due to general deleterious effects inflicted by OA. Taken together, our results show that in general OA does not augment ethylene response in Arabidopsis, but it might affect ethylene action in flower abscission of the ethylene-overproducer mutant.     

Interactions between ethylene,abscisic acid and cytokinin during germination and seedling establishment in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In order to investigate the interaction of the plant hormones ethylene, abscisic acid (ABA) and cytokinin in seed germination and early seedling development, we studied germination in ethylene-related mutants of Arabidopsis. Mutations in the genes etr1 and ein2, which reduce ethylene responses, showed increased dormancy and a delay in germination in comparison with wild type. Mutations in etr1, ein2 and ein6 also resulted in increased sensitivity to ABA with respect to inhibition of germination. Conversely, mutations in ctr1 and eto3, which lead to an increased ethylene response and overproduction of ethylene, respectively, decreased sensitivity to ABA during germination. Increased ABA sensitivity was also effected in wild type seeds by the presence during germination of AgNO₃, an inhibitor of ethylene action. The addition of the cytokinin N-6 benzyl adenine (BA) reversed the increased sensitivity of ethylene-resistant mutants to ABA. The action of cytokinin in reversing increased ABA sensitivity of ethylene-resistant mutants also suggests that at least part of the action of cytokinin in promoting germination is independent of its role in stimulating ethylene production. These observations further extend the evidence in support of interaction between ethylene, ABA and cytokinin signalling in controlling seed germination and early seedling development in Arabidopsis.     

Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis

To examine the cross talk between the abscisic acid (ABA) and ethylene signal transduction pathways, signaling events during ABA-induced stomatal closure were examined in Arabidopsis (Arabidopsis thaliana) wild-type plants, in an ethylene-overproducing mutant (eto1-1), and in two ethylene-insensitive mutants (etr1-1 and ein3-1). Using isolated epidermal peels, stomata of wild-type plants were found to close within a few minutes in response to ABA, whereas stomata of the eto1-1 mutant showed a similar but less sensitive ABA response. In addition, ABA-induced stomatal closure could be inhibited by application of ethylene or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). In contrast, stomata of the etr1-1 and ein3-1 mutants were able to close in response to concomitant ABA and ACC application, although to a lesser extent than in wild-type plants. Moreover, expression of the ABA-induced gene RAB18 was reduced following ACC application. These results indicate that ethylene delays stomatal closure by inhibiting the ABA signaling pathway. The same inhibitive effects of ethylene on stomatal closure were observed in ABA-irrigated plants and the plants in drought condition. Furthermore, upon drought stress, the rate of transpiration was greater in eto1-1 and wild-type plants exposed to ethylene than in untreated wild-type control plants, indicating that the inhibitive effects of ethylene on ABA-induced stomatal closure were also observed in planta.     

Ethylene is one of the key elements for cell death and defense response control in the Arabidopsis lesion mimic mutant vad1

Although ethylene is involved in the complex cross talk of signaling pathways regulating plant defense responses to microbial attack, its functions remain to be elucidated. The lesion mimic mutant vad1-1 (for vascular associated death), which exhibits the light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, is a good model for studying the role of ethylene in programmed cell death and defense. Here, we demonstrate that expression of genes associated with ethylene synthesis and signaling is enhanced in vad1-1 under lesion-promoting conditions and after plant-pathogen interaction. Analyses of the progeny from crosses between vad1-1 plants and either 35SERF1 transgenic plants or ein2-1, ein3-1, ein4-1, ctr1-1, or eto2-1 mutants revealed that the vad1-1 cell death and defense phenotypes are dependent on ethylene biosynthesis and signaling. In contrast, whereas vad1-1-dependent increased resistance was abolished by ein2, ein3, and ein4 mutations, positive regulation of ethylene biosynthesis (eto2-1) or ethylene responses (35SERF1) did not exacerbate this phenotype. In addition, VAD1 expression in response to a hypersensitive response-inducing bacterial pathogen is dependent on ethylene perception and signaling. These results, together with previous data, suggest that VAD1 could act as an integrative node in hormonal signaling, with ethylene acting in concert with salicylic acid as a positive regulator of cell death propagation.     

Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana

Lateral root branching is a genetically defined and environmentally regulated process. Auxin is required for lateral root formation, and mutants that are altered in auxin synthesis, transport or signaling often have lateral root defects. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in the regulation of Arabidopsis lateral root formation are not well characterized. This study utilized Arabidopsis mutants altered in ethylene signaling and synthesis to explore the role of ethylene in lateral root formation. We find that enhanced ethylene synthesis or signaling, through the eto1-1 and ctr1-1 mutations, or through the application of 1-aminocyclopropane-1-carboxylic acid (ACC), negatively impacts lateral root formation, and is reversible by treatment with the ethylene antagonist, silver nitrate. In contrast, mutations that block ethylene responses, etr1-3 and ein2-5 , enhance root formation and render it insensitive to the effect of ACC, even though these mutants have reduced root elongation at high ACC doses. ACC treatments or the eto1-1 mutation significantly enhance radiolabeled indole-3-acetic acid (IAA) transport in both the acropetal and the basipetal directions. ein2-5 and etr1-3 have less acropetal IAA transport, and transport is no longer regulated by ACC. DR5-GUS reporter expression is also altered by ACC treatment, which is consistent with transport differences. The aux1-7 mutant, which has a defect in an IAA influx protein, is insensitive to the ethylene inhibition of root formation. aux1-7 also has ACC-insensitive acropetal and basipetal IAA transport, as well as altered DR5-GUS expression, which is consistent with ethylene altering AUX1-mediated IAA uptake, and thereby blocking lateral root formation.     

Antagonism between abscisic acid and ethylene in Arabidopsis acts in parallel with the reciprocal regulation of their metabolism and signaling pathways

Although abscisic acid (ABA) and ethylene have antagonistic functions in the control of plant growth and development, including seed germination and early seedling development, it remains unknown whether a convergent point exists between these two signaling pathways or whether they operate in parallel in Arabidopsis thaliana. To elucidate this issue, four ethylene mutants, ctr1, ein2, ein3, and ein6, were crossed with aba2 (also known as gin1-3) to generate double mutants. Genetic epistasis analysis revealed that all of the resulting double mutants displayed aba2 mutant phenotypes with a small plant size and wiltiness when grown in soil or on agar plates. Further ethylene sensitivity or triple response analyses demonstrated that these double mutants also retained the ctr1 or ein mutant phenotypes, showing ethylene constitutive triple and insensitive responses, respectively. Our current data therefore demonstrate that ABA and ethylene act in parallel, at least in primary signal transduction pathways. Moreover, by microarray analysis we found that an ACC oxidase (ACO) was significantly upregulated in the aba2 mutant, whereas the 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) gene in ein2 was upregulated, and both the ABSCISIC ACID INSENSITIVE1 (ABI1) and cytochrome P450, family 707, subfamily A, polypeptide 2 (CYP707A2) genes in etr1-1 were downregulated. These data further suggest that ABA and ethylene may control the hormonal biosynthesis, catabolism, or signaling of each other to enhance their antagonistic effects upon seed germination and early seedling growth.     

Morphological analysis of seed shape in Arabidopsis thaliana reveals altered polarity in mutants of the ethylene signaling pathway

The shape of Arabidopsis thaliana dry seed is described here as a prolate spheroid. The accuracy of this approximation is discussed. Considering its limitations, it allows a geometric approximation to the analysis of changes occurring in seed shape during imbibition prior to seed germination as well as the differences in shape between genotypes and their changes during imbibition. The triple mutant ein2-1, ers1-2, etr1-7 presents notable alterations in seed shape. In addition, seeds of this and other mutants in the ethylene signaling pathway (ctr1-1, eto1-1, etr1-1, ein2-1) show different response to imbibition than the wild type. Imbibed seeds of the wild type increase their asymmetry compared with the dry seeds. This is detected by the relative changes in the curvature values in both poles. Thus, during imbibition of the wild-type seeds, the reduction in curvature values observed in the basal pole gives them an ovoid shape. In contrast, in the seeds of the ethylene mutants, reduction in curvature values occurs in both basal and apical poles, and its shape remains as a prolate spheroid. Our data indicate that the ethylene signaling pathway is involved, in general, in the complex regulation of seed shape and, in particular, in the establishment of polarity in seeds, controlling curvature values in the seed poles.     

Serotonin modulates Arabidopsis root growth via changes in reactive oxygen species and jasmonic acid–ethylene signaling

Serotonin (5‐hydroxytryptamine) is a bioactive indoleamine with neurotransmitter function in vertebrates, which represents an emerging signaling molecule in plants, playing key roles in the development and defense. In this study, the role of reactive oxygen species (ROS) and jasmonic acid (JA)–ethylene (Et) signaling in root developmental alterations induced by serotonin was investigated. An Arabidopsis thaliana mutant defective at the RADICAL‐INDUCED CELL DEATH1 (RCD1) locus was resistant to paraquat‐induced ROS accumulation in primary roots and showed decreased inhibition or root growth in response to serotonin. A suite of JA‐ and Et‐related mutants including coronatine insensitive1, jasmonic acid resistant1 (jar1), etr1, ein2 and ein3 showed tolerance to serotonin in the inhibition of primary root growth and ROS redistribution within the root tip when compared with wild‐type (WT) seedlings. Competence assays between serotonin and AgNO₃, a well‐known blocker of Et action, showed that primary root growth in medium supplemented with serotonin was normalized by AgNO_3, whereas roots of eto3, an Et overproducer mutant, were oversensitive to serotonin. Comparison of ROS levels in WT, etr1, jar1 and rcd1 primary root tips using the ROS‐specific probe 2′,7′‐dichlorofluorescein diacetate and confocal imaging showed that serotonin inhibition of primary root growth likely occurs independently of its conversion into melatonin. Our results provide compelling evidence that serotonin affects ROS distribution in roots, involving RCD1 and components of the JA–Et signaling pathways.     

Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis

Background  

Ethylene receptor single mutants of Arabidopsis do not display a visibly prominent phenotype, but mutants defective in multiple ethylene receptors exhibit a constitutive ethylene response phenotype. It is inferred that ethylene responses in Arabidopsis are negatively regulated by five functionally redundant ethylene receptors. However, genetic redundancy limits further study of individual receptors and possible receptor interactions. Here, we examined the ethylene response phenotype in two quadruple receptor knockout mutants, (ETR1) ers1 etr2 ein4 ers2 and (ERS1) etr1 etr2 ein4 ers2, to unravel the functions of ETR1 and ERS1. Their functions were also reciprocally inferred from phenotypes of mutants lacking ETR1 or ERS1. Receptor protein levels are correlated with receptor gene expression. Expression levels of the remaining wild-type receptor genes were examined to estimate the receptor amount in each receptor mutant, and to evaluate if effects of ers1 mutations on the ethylene response phenotype were due to receptor functional compensation. As ers1 and ers2 are in the Wassilewskija (Ws) ecotype and etr1, etr2, and ein4 are in the Columbia (Col-0) ecotype, possible effects of ecotype mixture on ethylene responses were also investigated.     

Use of Mutants to Dissect the Role of Ethylene Signalling in Organ Senescence and the Regulation of Yield in Arabidopsis thaliana

The role of ethylene in regulating organ senescence in Arabidopsis has been investigated by studying the development of mutants that have an attenuated capacity to perceive the gas. The onset of leaf senescence and floral organ abscission was delayed in the ethylene-insensitive mutant etr1. The photosynthetic life span of rosette leaves was similarly extended in the gain-of-function mutant ers2, and this mutant also exhibited a delay in the timing of pod dehiscence primarily as a consequence of an extension in the final stages of senescence. A detailed analysis of yield revealed that whilst thousand grain weight was increased, by as much as 20 %, in etr1, ein4, and the loss-of-function mutant etr2, only the latter showed a significant increase in total weight of seeds produced per plant. The other studied mutants exhibited a reduction in total seed yield of almost 40 %. These observations are discussed in the context of the possible role of ethylene in regulating organ senescence and their significance in the breeding of crop plants with enhanced phenotypic characteristics.     

Involvement of ethylene in UV-B-induced changes in polyamine content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Components of the ethylene signal perception and transduction pathway (ethylene signaling pathway, ESP) were studied in respect to their involvement in regulation of UV-B-induced changes in levels of polyamines in plants Arabidopsis thaliana (L.) Heynh. Experiments were performed on 15-day old wild type (WT) plants, the mutant etr1-1 with impaired ethylene reception, and the ethylene-insensitive mutant ctr1-1 with constitutively activated ESP. The plants were cultivated aseptically. It was found that exogenous ethylene or an inhibitor of its action 1-methylcyclopropen (1-MCP), which blocks ethylene receptors did not affect the polyamine content in leaf rosettes of plants, which had not been subjected to UV-B stress. A day after UV-B irradiation at intermediate (9 kJ/m²) or high doses (18 kJ/m²), the putrescine levels increased, respectively, 6.4 and 3.0 times in WT, 4.5 and 3.2 times in etr1-1, and 5.5 and 4.7 in ctr1-1. Pretreatment with ethylene (1 μL/L) for 24 h reduced the putrescine accumulation along with the loss in spermidine and spermine pools in WT plants and, to a lesser extent, in etr1-1 mutant. Treatment with 1-MCP (50 nL/L, 3 h before and 24 h after the irradiation) enhanced plant sensitivity to UV-B, putrescine accumulation, as well as spermidine and spermine consumption in WT and, to a lesser degree, in etr1-1. The mutant ctr1-1 was insensitive to both ethylene and 1-MCP. The results show that the activation of ESP by ethylene increases plant resistance to UV-B because the irradiation stimulates accumulation of putrescine, which converts to spermidine and spermine functioning as ROS traps.     

Arabidopsis ROOT HAIR DEFECTIVE3 is involved in nitrogen starvation-induced anthocyanin accumulation

Anthocyanin accumulation is a common phenom-enon seen in plants under environmental stress. In this study, we identified a new allele of ROOT HAIR DEFECTIVE3 (RHD3) showing an anthocyanin overaccumulat...     

Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.

Alterations in the response of dark-grown seedlings to ethylene (the "triple response") were used to isolate a collection of ethylene-related mutants in Arabidopsis thaliana. Mutants displaying a constitutive response (eto1) were found to produce at least 40 times more ethylene than the wild type. The morphological defects in etiolated eto1-1 seedlings reverted to wild type under conditions in which ethylene biosynthesis or ethylene action were inhibited. Mutants that failed to display the apical hook in the absence of ethylene (his1) exhibited reduced ethylene production. In the presence of exogenous ethylene, hypocotyl and root of etiolated his1-1 seedlings were inhibited in elongation but no apical hook was observed. Mutants that were insensitive to ethylene (ein1 and ein2) produced increased amounts of ethylene, displayed hormone insensitivity in both hypocotyl and root responses, and showed an apical hook. Each of the "triple response" mutants has an effect on the shape of the seedling and on the production of the hormone. These mutants should prove to be useful tools for dissecting the mode of ethylene action in plants.     

Reducing jasmonic acid levels causes ein2 mutants to become ethylene responsive

It has previously been shown that jasmonic acid affects the ethylene signaling pathway. EIN2 is a central component of ethylene signaling that is downstream of the receptors. EIN2 has previously been shown to be required for ethylene responses. We found that reducing jasmonic acid levels, either mutationally or chemically, caused ein2 ethylene-insensitive mutants to become ethylene responsive. This effect was not seen with the ethylene-insensitive etr1-1 mutants that affect receptor function. Based upon these results, we propose a model where jasmonic acid is inhibiting ethylene signal transduction down-stream of the ethylene receptors. This may involve an EIN2-independent pathway.     

ETR1-, JAR1- and PAD2-dependent signaling pathways are involved in C6-aldehyde-induced defense responses of Arabidopsis

C6-aldehydes [(E)-2-hexenal and (Z)-3-hexenal] induce expression of defense genes, accumulation of antifungal substances, and resistance against Botrytis cinerea with Arabidopsis. In order to elucidate the signaling pathway mediating the defense responses induced by C6-aldehydes in Arabidopsis, we compared the responses of Arabidopsis mutants deficient in the signaling pathways; i.e., etr1-1 (ethylene resistant), jar1-1 (jasmonate resistant), npr1-1 (salicylic acid insensitive), or pad2-1 (phytoalexin-deficient) with those of wild type (WT) plants. Induction of some, but not all of the defense genes in response to C6-aldehydes was significantly repressed in jar1-1, etr1-1, and pad2-1, but not at all in npr1-1. C6-aldehyde-treatment enhanced accumulation of camalexin with WT and npr1-1, but only partially with etr1-1 and jar1-1. pad2-1 showed little accumulation of camalexin. npr1-1 accumulated the antifungal substances as WT did, however, etr1-1, jar1-1 and pad2-1 exhibited only partial accumulation. The treatment enhanced resistance of etr1-1, jar1-1 and npr1-1 against B. cinerea, but failed to enhance the resistance of pad2-1. Taken together, it was suggested that ETR1-, JAR1-, and PAD2-dependent signaling pathways were simultaneously activated by C6-aldehyde-treatment. Among these, PAD2-dependent signaling appeared to be most important. In contrast, involvement of NPR1-dependent signaling was minimal.