Ethylene insensitivity impedes a subset of responses to phosphorus deficiency in tomato and petunia

The role of ethylene in growth and developmental responses to low phosphorus stress was evaluated using ethylene-insensitive 'Never-ripe' (Nr) tomato and etr1 petunia plants. Low phosphorus increased adventitious root formation in 'Pearson' (wild-type) tomato plants, but not in Nr, supporting a role for ethylene in adventitious root development and showing that ethylene is important for this aspect of phosphorus response. Low phosphorus reduced ethylene production by adventitious roots of both genotypes, suggesting that ethylene perception--not production--regulates carbon allocation to adventitious roots at the expense of other roots under low phosphorus stress. With the exception of its effect on adventitious rooting, Nr had positive effects on growth and biomass accumulation in tomato whereas etr1 tended to have negative effects on petunia. This was particularly evident during the recovery from transplanting, when the effective quantum yield of photosystem II of etr1 petunia grown with low phosphorus was significantly lower than 'Mitchell Diploid', suggesting that etr1 petunia plants may undergo more severe post-transplant stress at low phosphorus availability. Our results demonstrate that ethylene mediates adventitious root formation in response to phosphorus stress and plays an important role for quick recovery of plants exposed to multiple environmental stresses, i.e. transplanting and low phosphorus.     

Analysis of combinatorial loss-of-function mutants in the Arabidopsis ethylene receptors reveals that the ers1 etr1 double mutant has severe developmental defects that are EIN2 dependent

Ethylene responses in Arabidopsis are controlled by the ETR receptor family. The receptors function as negative regulators of downstream signal transduction components and fall into two distinct subfamilies based on sequence similarity. To clarify the levels of functional redundancy between receptor isoforms, combinatorial mutant lines were generated that included the newly isolated ers1-2 allele. Based on the etiolated seedling growth response, all mutant combinations tested exhibited some constitutive ethylene responsiveness but also remained responsive to exogenous ethylene, indicating that all five receptor isoforms can contribute to signaling and no one receptor subtype is essential. On the other hand, light-grown seedlings and adult ers1 etr1 double mutants exhibited severe phenotypes such as miniature rosette size, delayed flowering, and sterility, revealing a distinct role for subfamily I receptors in light-grown plants. Introduction of an ein2 loss-of-function mutation into the ers1 etr1 double mutant line resulted in plants that phenocopy ein2 single mutants, indicating that all phenotypes observed in the ers1 etr1 double mutant are EIN2 dependent.     

Genetic Analysis of Ethylene Signal Transduction in Arabidopsis Thaliana: Five Novel Mutant Loci Integrated into a Stress Response Pathway

The response of Arabidopsis thaliana etiolated seedlings to the plant hormone ethylene is a conspicuous phenotype known as the triple response. We have identified genes that are required for ethylene perception and response by isolating mutants that fail to display a triple response in the presence of exogenous ethylene. Five new complementation groups have been identified. Four of these loci, designated ein4, ein5, ein6 and ein7, are insensitive to ethylene. The fifth complementation group, eir1, is defined by a novel class of mutants that have agravitropic and ethylene-insensitive roots. Double-mutant phenotypes have allowed the positioning of these loci in a genetic pathway for ethylene signal transduction. The ethylene-response pathway is defined by the following loci: ETR1, EIN4, CTR1, EIN2, EIN3, EIN5, EIN6, EIN7, EIR1, AUX1 and HLS1. ctr1-1 is epistatic to etr1-3 and ein4, indicating that CTR1 acts after both ETR1 and EIN4 in the ethylene-response pathway. Mutations at the EIN2, EIN3, EIN5, EIN6 and EIN7 loci are all epistatic to the ctr1 seedling phenotype. The EIR1 and AUX1 loci define a root-specific ethylene response that does not require EIN3 or EIN5 gene activity. HLS1 appears to be required for differential cell growth in the apical hook. The EIR1, AUX1 and HLS1 genes may function in the interactions between ethylene and other plant hormones that occur late in the signaling pathway of this simple gas.     

Heterologous expression of the Arabidopsis etr1-1 allele inhibits the senescence of carnation flowers

The Arabidopsis thaliana etr1-1 allele, capable of conferring ethylene insensitivity in a heterologous host, was introduced into transgenic carnation plants. This gene was expressed under control of either its own promoter, the constitutive CaMV 35S promoter or the flower-specific petunia FBP1 promoter. In about half of the transgenic plants obtained flower senescence was delayed by at least 6 days relative to control flowers, with a maximum delay of 16 days, a 3-fold increase in vase life. These flowers did not show the petal inrolling phenotype typical of ethylene-dependent carnation flower senescence. Instead, petals remained firm and finally started to rot and decolorize.In transgenic plants with delayed flower senescence, expression of the Arabidopsis etr1-1 gene was detectable and the expression pattern followed the activity of the upstream promoter. In these flowers expression of the ACO1 gene, encoding the final enzyme in the ethylene biosynthesis pathway, ACC oxidase, was down-regulated. This indicates that the autocatalytic induction of ethylene biosynthesis, required to initiate and regulate the flower senescence process, is absent in etr1-1 transgenic plants due to dominant ethylene insensitivity.The delay in senescence observed in transgenic etr1-1 flowers was longer than in flowers pretreated with chemicals that inhibit either ethylene biosynthesis (amino-oxyacetic acid) or the ethylene response (silver thiosulfate). This may have important implications for post-harvest management of carnation flowers.     

Genetic analysis of involvement of ETR1 in plant response to salt and osmotic stress

The involvement of ethylene and ethylene receptor Ethylene Response 1 (ETR1) in plant stress responses has been highlighted. However, the physiological processes involved remain unclear. In this study, we have investigated the physiological response of two alleles etr1-1 and etr1-7 mutants during germination and post-germination seedling development in response to salt and osmotic stress. The etr1-1 mutants showed increased sensitivity to osmotic (200 mM or higher mannitol) and salt stress (50 mM NaCl or higher) during germination and seedling development, whereas the etr1-7 mutants displayed enhanced tolerance to the severe stresses (500 mM mannitol or 200 mM NaCl). These results provide physiological and genetic evidence that ethylene receptor ETR1 modulates plant response to abiotic stress. Furthermore, the etr1-1 and etr1-7 mutants showed different responses to exogenous abscisic acid (ABA) inhibition. The etr1-1 mutants were more sensitive to ABA than the wild type during germination, and young seedling development. In sharp contrast, the etr1-7 mutants showed enhanced insensitivity to ABA treatment (>1 μM ABA) in post-germination development including root elongation and greening of cotyledons of the treated seedlings, although the germination was not greatly altered at the tested doses of ABA. The results suggest that ETR1-modulated stress response may mediate ABA. Youning Wang and Tao Wang contributed equally to this report.     

Defence responses regulated by jasmonate and delayed senescence caused by ethylene receptor mutation contribute to the tolerance of petunia to Botrytis cinerea

Ethylene and jasmonate (JA) have powerful effects when plants are challenged by pathogens. The inducible promoter‐regulated expression of the Arabidopsis ethylene receptor mutant ethylene‐insensitive1‐1 (etr1‐1) causes ethylene insensitivity in petunia. To investigate the molecular mechanisms involved in transgenic petunia responses to Botrytis cinerea related to the ethylene and JA pathways, etr1‐1‐expressing petunia plants were inoculated with Botrytis cinerea. The induced expression of etr1‐1 by a chemical inducer dexamethasone resulted in retarded senescence and reduced disease symptoms on detached leaves and flowers or intact plants. The extent of decreased disease symptoms correlated positively with etr1‐1 expression. The JA pathway, independent of the ethylene pathway, activated petunia ethylene response factor (PhERF) expression and consequent defence‐related gene expression. These results demonstrate that ethylene induced by biotic stress influences senescence, and that JA in combination with delayed senescence by etr1‐1 expression alters tolerance to pathogens.     

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.     

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.     

Arabidopsis EIN2 modulates stress response through abscisic acid response pathway

The nuclear protein ETHYLENE INSENSITIVE2 (EIN2) is a central component of the ethylene signal transduction pathway in plants, and plays an important role in mediating cross-links between several hormone response pathways, including abscisic acid (ABA). ABA mediates stress responses in plants, but there is no report on the role of EIN2 on plant response to salt and osmotic stresses. Here, we show that EIN2 gene regulates plant response to osmotic and salt stress through an ABA-dependent pathway in Arabidopsis. The expression of the EIN2 gene is down-regulated by salt and osmotic stress. An Arabidopsis EIN2 null mutant was supersensitive to both salt and osmotic stress conditions. Disruption of EIN2 specifically altered the expression pattern of stress marker gene RD29B in response to the stresses, but not the stress- or ABA-responsive genes RD29A and RD22, suggesting EIN2 modulates plant stress responses through the RD29B branch of the ABA response. Furthermore, disruption of EIN2 caused substantial increase in ABA. Lastly, our data showed that mutations of other key genes in ethylene pathway also had altered sensitivity to abiotic stresses, indicating that the intact ethylene may involve in the stress response. Taken together, the results identified EIN2 as a cross-link node in ethylene, ABA and stress signaling pathways, and EIN2 is necessary to induce developmental arrest during seed germination, and seedling establishment, as well as subsequent vegetative growth, thereby allowing the survival and growth of plants under the adverse environmental conditions. Youning Wang and Chuang Liu contributed equally to this work.     

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.