Interactions between abscisic acid and ethylene signaling cascades

We screened for mutations that either enhanced or suppressed the abscisic acid (ABA)-resistant seed germination phenotype of the Arabidopsis abi1-1 mutant. Alleles of the constitutive ethylene response mutant ctr1 and ethylene-insensitive mutant ein2 were recovered as enhancer and suppressor mutations, respectively. Using these and other ethylene response mutants, we showed that the ethylene signaling cascade defined by the ETR1, CTR1, and EIN2 genes inhibits ABA signaling in seeds. Furthermore, epistasis analysis between ethylene- and ABA-insensitive mutations indicated that endogenous ethylene promotes seed germination by decreasing sensitivity to endogenous ABA. In marked contrast to the situation in seeds, ein2 and etr1-1 roots were resistant to both ABA and ethylene. Our data indicate that ABA inhibition of root growth requires a functional ethylene signaling cascade, although this inhibition is apparently not mediated by an increase in ethylene biosynthesis. These results are discussed in the context of the other hormonal regulations controlling seed germination and root growth.     

The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination

In Arabidopsis thaliana, the etr1-2 mutation confers dominant ethylene insensitivity and results in a greater proportion of mature seeds that exhibit dormancy compared with mature seeds of the wild-type. We investigated the impact of the etr1-2 mutation on other plant hormones by analyzing the profiles of four classes of plant hormones and their metabolites by HPLC-ESI/MS/MS in mature seeds of wild-type and etr1-2 plants. Hormone metabolites were analyzed in seeds imbibed immediately under germination conditions, in seeds subjected to a 7-day moist-chilling (stratification) period, and during germination/early post-germinative growth. Higher than wild-type levels of abscisic acid (ABA) appeared to contribute, at least in part, to the greater incidence of dormancy in mature seeds of etr1-2. The lower levels of abscisic acid glucose ester (ABA-GE) in etr1-2 seeds compared with wild-type seeds under germination conditions (with and without moist-chilling treatments) suggest that reduced metabolism of ABA to ABA-GE likely contributed to the accumulation of ABA during germination in the mutant. The mutant seeds exhibited generally higher auxin levels and a large build-up of indole-3-aspartate when placed in germination conditions following moist-chilling. The mutant manifested increased levels of cytokinin glucosides through zeatin-O-glucosylation (Z-O-Glu). The resulting increase in Z-O-Glu was the largest and most consistent change associated with the ETR1 gene mutation. There were more gibberellins (GA) and at higher concentrations in the mutant than in wild-type. Our results suggest that ethylene signaling modulates the metabolism of all the other plant hormone pathways in seeds. Additionally, the hormone profiles of etr1-2 seed during germination suggest a requirement for higher than wild-type levels of GA to promote germination in the absence of a functional ethylene signaling pathway.     

Ethylene modulates flavonoid accumulation and gravitropic responses in roots of Arabidopsis

Plant organs change their growth direction in response to reorientation relative to the gravity vector. We explored the role of ethylene in Arabidopsis (Arabidopsis thaliana) root gravitropism. Treatment of wild-type Columbia seedlings with the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC) reduced root elongation and gravitropic curvature. The ethylene-insensitive mutants ein2-5 and etr1-3 had wild-type root gravity responses, but lacked the growth and gravity inhibition by ACC found in the wild type. We examined the effect of ACC on tt4(2YY6) seedlings, which have a null mutation in the gene encoding chalcone synthase, the first enzyme in flavonoid synthesis. The tt4(2YY6) mutant makes no flavonoids, has elevated indole-3-acetic acid transport, and exhibits a delayed gravity response. Roots of tt4(2YY6), the backcrossed line tt4-2, and two other tt4 alleles had wild-type sensitivity to growth inhibition by ACC, whereas the root gravitropic curvature of these tt4 alleles was much less inhibited by ACC than wild-type roots, suggesting that ACC may reduce gravitropic curvature by altering flavonoid synthesis. ACC treatment induced flavonoid accumulation in root tips, as judged by a dye that becomes fluorescent upon binding flavonoids in wild type, but not in ein2-5 and etr1-3. ACC also prevented a transient peak in flavonoid synthesis in response to gravity. Together, these experiments suggest that elevated ethylene levels negatively regulate root gravitropism, using EIN2- and ETR1-dependent pathways, and that ACC inhibition of gravity response occurs through altering flavonoid synthesis.     

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 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.     

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.     

Dynamic analysis of Arabidopsis seed shape reveals differences in cellulose mutants

In a previous work, the shape of Arabidopsis seed was described as a cardioid modified by a factor of Phi. In addition, J index was defined as the similarity of the seed (in an orthogonal, bi-dimensional image) to a cardioid, thus allowing the quantitative comparison of seed shape in seeds of varieties and mutants at different stages of development. Here, J index is used for modeling changes in seed morphology during the dynamic process of seed imbibition before germination. The analysis was carried out by means of a general linear model with two fixed factors (genotype and time) applied to two Arabidopsis varieties: Columbia and Wassilewskija and two mutants in cellulose synthesis: prc1-1 (CESA6 in Columbia) and kor1-1 (in Wassilewskija). Equations representing the changes in seed form during imbibition are given. The analysis of changes in seed shape by this procedure provides (1) a quantitative method to record changes in seed shape and to compare between genotypes or treatments showing the time points with maximum differences, and (2) the observation of remarkable differences between wild-type seeds and mutants in cellulose biosynthesis, indicating new phenotypic characteristics previously unknown in the latter. While wild-type seeds increase their J index values during imbibition, in the cellulose mutants J index values decrease. In addition, shape comparisons were done with other mutants. Seeds of ga1-1 mutants behave like cellulose mutants, whereas different ethylene mutants present varied responses. Quantitative analysis of seed morphology is a new basis for the record of differences between wild-type and mutants as well as for phenotypic characterization.     

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.     

EIN2 regulates salt stress response and interacts with a MA3 domain-containing protein ECIP1 in Arabidopsis

Ethylene signalling regulates plant growth and development. However, its roles in salt stress response are less known. Here we studied functions of EIN2, a central membrane protein of ethylene signalling, and its interacting protein ECIP1 in salt stress responses. Mutation of EIN2 led to extreme salt sensitivity as revealed by phenotypic and physiological changes, and overexpression of C-terminus of EIN2 suppressed salt sensitivity in ein2-5, indicating that EIN2 is required for salt tolerance. Downstream components EIN3 and EIL1 are also essential for salt tolerance because ein3-1eil1-1 double mutant showed extreme salt-sensitive phenotype. A MA3 domain-containing protein ECIP1 was further identified to interact with EIN2 in yeast two-hybrid assay and GST pull-down assay. Loss-of-function of ECIP1 resulted in enhanced ethylene response but altered salt response during seed germination and plant growth. Double mutant analysis revealed that ein2-1 was epistatic to ecip1, and ecip1 mutation partially suppressed ethylene-insensitivity of etr2-1 and ein4-1. These studies strengthen that interactions between ECIP1 and EIN2 or ethylene receptors regulate ethylene response and stress response.     

Hydrogen peroxide treatment results in reduced curvature values in the Arabidopsis root apex

Curvature of a plane curve is a measurement related to its shape. A Mathematica code was developed [Cervantes E, Tocino A. J Plant Physiol 2005;162:1038-1045] to obtain parametric equations from microscopic images of the Arabidopsis thaliana root apex. In addition, curvature values for these curves were given. It was shown that ethylene-insensitive mutants (etr1-1 and ein2-1) have reduced curvature values in the root apex. It has also been shown that blocking ethylene action by norbornadiene, an ethylene inhibitor, results in reduced curvature values in the two outer cell layers of the root apex [Noriega A, Cervantes E, Tocino A. J Plant Physiol 2008, in press]. Because ethylene action has been related with hydrogen peroxide [Desikan R, Hancock JT, Bright J, Harrison J, Weir I, Hooley R, Neill SJ. Plant Physiol 2005;137:831-834], the effect of a treatment with hydrogen peroxide in the curvature values of three successive layers of the root apex in Arabidopsis thaliana was investigated by confocal microscopy. Treatment with 10mM hydrogen peroxide resulted in reduced curvature values in the three layers. The effect was associated with smaller cells having higher circularity indices. The results are discussed in the context of the role of ethylene in development.     

Arabidopsis seedling growth response and recovery to ethylene. A kinetic analysis

Responses to the plant hormone ethylene are mediated by a family of five receptors in Arabidopsis that act in the absence of ethylene as negative regulators of response pathways. In this study, we examined the rapid kinetics of growth inhibition by ethylene and growth recovery after ethylene withdrawal in hypocotyls of etiolated seedlings of wild-type and ethylene receptor-deficient Arabidopsis lines. This analysis revealed that there are two phases to growth inhibition by ethylene in wild type: a rapid phase followed by a prolonged, slower phase. Full recovery of growth occurs approximately 90 min after ethylene removal. None of the receptor null mutations tested had a measurable effect on the two phases of growth inhibition. However, loss-of-function mutations in ETR1, ETR2, and EIN4 significantly prolonged the time for recovery of growth rate after ethylene was removed. Plants with an etr1-6;etr2-3;ein4-4 triple loss-of-function mutation took longer to recover than any of the single mutants, while the ers1;ers2 double mutant had no effect on recovery rate, suggesting that receiver domains play a role in recovery. Transformation of the ers1-2;etr1-7 double mutant with wild-type genomic ETR1 rescued the slow recovery phenotype, while a His kinase-inactivated ETR1 construct did not. To account for the rapid recovery from growth inhibition, a model in which clustered receptors act cooperatively is proposed.     

Ethylene signaling may be involved in the regulation of tocopherol biosynthesis in Arabidopsis thaliana

Tocopherol biosynthesis was investigated in ein3-1, etr1-1 and eto1-1 mutants of Arabidopsis thaliana, which show a defect in ethylene signaling, perception and over-produce ethylene, respectively. A mutation in the EIN3 gene delayed the water-stress related increase in α-tocopherol and caused a reduction in the levels of this antioxidant by ca. 30% compared to the wild type. In contrast to the wild type and ein3-1 mutants, both etr1-1 and eto1-1 mutants showed a sharp (up to 5-fold) increase in α-tocopherol levels during leaf aging. It is concluded that ethylene perception and signaling may be involved in the regulation of tocopherol biosynthesis during water stress and leaf aging.     

The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene

In order to investigate the effects of a permanent increase in cellular H(2)O(2) on cation homeostasis we have studied a T-DNA insertion mutant of the Arabidopsis CATALASE 2 gene. This mutant (cat2-1) exhibits 20% of wild-type leaf catalase activity and accumulates more H(2)O(2) than the wild type under normal growth conditions. In addition to reduced size, a pale green color and great reduction in secondary roots, the cat2-1 mutant exhibited increased sensitivity to H(2)O(2), NaCl, norspermidine, high light and cold stress. On the other hand, the germination of the cat2-1 mutant is more tolerant to lithium than the wild type. This novel phenotype cannot be explained by changes in lithium transport. Actually, the uptake of lithium (and of other toxic cations such as sodium and norspermidine) is increased in the cat2-1 mutant while K(+) levels were decreased. The lithium tolerance of this mutant seems to result both from insensitivity to the inhibitory ethylene induced by this cation and a reduced capability for ethylene production. Accordingly, induction by ethylene of responsive genes such as PR4 and EBP/ERF72 is decreased in cat2-1. Mutants insensitive to ethylene such as etr1-1 and ein3-3 are lithium tolerant, and inhibition of ethylene biosynthesis with 2-aminoisobutyrate protects against lithium toxicity. Microarray analysis of gene expression indicates that the expression of genes related to cation transport and ethylene synthesis and perception was not altered in the cat2-1 mutant, suggesting that H(2)O(2) modulates these processes at the protein level. These results uncover a cross-talk between oxidative stress, cation homeostasis and ethylene.     

Maintenance of shoot growth by endogenous ABA: genetic assessment of the involvement of ethylene suppression

Previous work demonstrated that normal levels of endogenous abscisic acid (ABA) are required to maintain shoot growth in well-watered tomato plants independently of effects of hormone status on plant water balance. The results suggested that the impairment of shoot growth in ABA-deficient mutants is at least partly attributable to increased ethylene production. To assess the extent to which ABA maintains shoot growth by ethylene suppression, the growth of ABA-deficient (aba2-1) and ethylene-insensitive (etr1-1) single- and double-mutants of Arabidopsis was examined. To ensure that the results were independent of effects of hormone status on plant water balance, differential relative humidity regimes were used to achieve similar leaf water potentials in all genotypes and treatments. In aba2-1, shoot growth was substantially inhibited and ethylene evolution was doubled compared with the wild type, consistent with the results for tomato. In the aba2-1 etr1-1 double mutant, in which ABA was equally as deficient as in aba2-1 and shoot growth was shown to be insensitive to ethylene, shoot growth was substantially, although incompletely, restored relative to etr1-1. Treatment with ABA resulted in the complete recovery of shoot growth in aba2-1 relative to the wild type, and also significantly increased the growth of aba2-1 etr1-1 such that total leaf area and shoot fresh weight were not significantly lower than in etr1-1. In addition, ABA treatment of aba2-1 etr1-1 restored the wider leaf morphology phenotype exhibited by etr1-1. The results demonstrate that normal levels of endogenous ABA maintain shoot development, particularly leaf expansion, in well-watered Arabidopsis plants, partly by suppressing ethylene synthesis and partly by another mechanism that is independent of ethylene.     

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.