Etr1-1 Mutants Research Articles

Visible light regulates anthocyanin synthesis via malate dehydrogenases and the ethylene signaling pathway in plum (Prunus salicina L.)

Light regulates anthocyanins synthesis in plants. Upon exposure to visible light, the inhibition of photosynthetic electron transfer significantly lowered the contents of anthocyanins and the expression levels of key genes involved in anthocyanins synthesis in plum fruit peel. Meanwhile, the expression levels of PsmMDH2 (encoding the malate dehydrogenase in mitochondria) and PschMDH (encoding the malate dehydrogenase in chloroplasts) decreased significantly. The contents of anthocyanins and the levels of the key genes involved in anthocyanin synthesis decreased significantly with the treatment of 1-MCP (an inhibitor of ethylene perception) but were enhanced by the exogenous application of ethylene. The ethylene treatment could also recover the anthocyanin synthesis capacity lowered by the photosynthetic electron transfer inhibition. Silencing PsmMDH2 and PschMDH significantly lowered the contents of anthocyanins in plum fruit. At low temperature, visible light irradiation induced anthocyanin accumulation in Arabidopsis leaves. However, the mmdh, chmdh, and etr1-1 mutants had significantly lower anthocyanins content and expressions of the key genes involved in anthocyanins synthesis compared to wild type. Overall, the present study demonstrates that both photosynthesis and respiration were involved in the regulation of anthocyanin synthesis in visible light. The visible light regulates anthocyanin synthesis by controlling the malate metabolism via MDHs and the ethylene signaling pathway.

A Salt-Signaling Network Involving Ethylene, Extracellular ATP, Hydrogen Peroxide, and Calcium Mediates K+/Na+ Homeostasis in Arabidopsis.

This work aimed at investigating the interactive effects of salt-signaling molecules, i.e., ethylene, extracellular ATP (eATP), H2O2, and cytosolic Ca2+ ([Ca2+]cyt), on the regulation of K+/Na+ homeostasis in Arabidopsis thaliana. The presence of eATP shortened Col-0 hypocotyl length under no-salt conditions. Moreover, eATP decreased relative electrolyte leakage and lengthened root length significantly in salt-treated Col-0 plants but had no obvious effects on the ethylene-insensitive mutants etr1-1 and ein3-1eil1-1. Steady-state ionic flux kinetics showed that exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) and eATP-Na2 (an eATP donor) significantly increased Na+ extrusion and suppressed K+ loss during short-term NaCl treatment. Moreover, ACC remarkably raised the fluorescence intensity of salt-elicited H2O2 and cytosolic Ca2+. Our qPCR data revealed that during 12 h of NaCl stress, application of ACC increased the expression of AtSOS1 and AtAHA1, which encode the plasma membrane (PM) Na+/H+ antiporters (SOS1) and H+-ATPase (H+ pumps), respectively. In addition, eATP markedly increased the transcription of AtEIN3, AtEIL1, and AtETR1, and ACC treatment of Col-0 roots under NaCl stress conditions caused upregulation of AtRbohF and AtSOS2/3, which directly contribute to the H2O2 and Ca2+ signaling pathways, respectively. Briefly, ethylene was triggered by eATP, a novel upstream signaling component, which then activated and strengthened the H2O2 and Ca2+ signaling pathways to maintain K+/Na+ homeostasis under salinity.

Coupling of Cell Division and Differentiation in Arabidopsis thaliana Cultured Cells with Interaction of Ethylene and ABA Signaling Pathways.

Recent studies indicate direct links between molecular cell cycle and cell differentiation machineries. Ethylene and abscisic acid (ABA) are known to affect cell division and differentiation, but the mechanisms of such effects are poorly understood. As ethylene and ABA signaling routes may interact, we examined their involvement in cell division and differentiation in cell tissue cultures derived from several Arabidopsis thaliana plants: wild type (Col-0), and ethylene-insensitive mutants etr1-1, ctr1-1, and ein2-1. We designed an experimental setup to analyze the growth-related parameters and molecular mechanisms in proliferating cells upon short exposure to ABA. Here, we provide evidence for the ethylene–ABA signaling pathways’ interaction in the regulation of cell division and differentiation as follows: (1) when the ethylene signal transduction pathway is functionally active (Col-0), the cells actively proliferate, and exogenous ABA performs its function as an inhibitor of DNA synthesis and division; (2) if the ethylene signal is not perceived (etr1-1), then, in addition to cell differentiation (tracheary elements formation), cell death can occur. The addition of exogenous ABA can rescue the cells via increasing proliferation; (3) if the ethylene signal is perceived, but not transduced (ein2-1), then cell differentiation takes place—the latter is enhanced by exogenous ABA while cell proliferation is reduced; (4) when the signal transduction pathway is constitutively active, the cells begin to exit the cell cycle and proceed to endo-reduplication (ctr1-1). In this case, the addition of exogenous ABA promotes reactivation of cell division.

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.

Ethylene-Insensitive Arabidopsis Mutants etr1-1 and ein2-1 Have a 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 the ethylene-insensitive mutants had a 25-30% lower net photosynthesis rate, a decreased content of soluble sugars, and, as a result, a lower freezing tolerance. Our work provides evidence that the perception and transduction of ethylene signal are necessary for constitutive tolerance of Arabidopsis to low temperature.

Application of 1-MCP as a liquid formulation prevents ethylene-induced senescence in Phalaenopsis orchid flowers and Kalanchoë blossfeldiana inflorescences

ABSTRACT This study provides a very first report on the use of a sprayable formulation of 1-MCP, HarvistaTM, on ornamental plant species. Individual florets of Phalaenopsis and inflorescences of Kalanchoë blossfeldiana were sprayed with 1, 2, 4, or 6% HarvistaTM and this was compared to the commonly used 1-MCP formulation, SmartFreshTM, which is administered as a powder releasing gas. HarvistaTM treatments for Kalanchoë were in addition also compared to plants treated with an alternative ethylene receptor blocker, STS, as well as with transgenic plants expressing the etr1-1 mutant gene of Arabidopsis thaliana. In the presence of ethylene, HarvistaTM at all concentrations (except 1% for Kalanchoë), as well as the conventional SmartFreshTM treatment, prevented wilting of florets and significantly extended their display life compared to that of control treatments: up to twofold for Kalanchoë and up to fourfold for Phalaenopsis. For Kalanchoë the best display life was, however, presented by etr1-1 transgenic plants. The efficacy and suitability of using HarvistaTM, a sprayable formulation of 1-MCP, was compared to using the gaseous application of SmartFreshTM, or the environmental hazardous silver treatment, against having to consider expensive and lengthy genetic modification with etr1-1 as commercial alternatives to achieve protection against the detrimental effects of ethylene. Abbreviations: 1-MCP, 1-methylcyclopropene; STS, silver thiosulfate

Improved leaf and flower longevity by expressing the etr1-1 allele in Pelargonium zonale under control of FBP1 and SAG12 promoters

The ethylene receptor etr1-1 (ETHYLENE RESPONSE 1) mutant gene from Arabidopsis thaliana, with its expression under the control of either the Petunia floral binding protein FBP1 promoter or the Arabidopsis senescence-specific SAG12 gene promoter, was introduced into Pelargonium zonale cv. ‘Katinka’. The heterologous expression of these transgenes and the response of flowers and leaves to exogenous ethylene were then studied. Transgenic etr1-1 lines grown in the greenhouse did not differ morphologically or physiologically from nontransformed plants nor was the rooting ability of cuttings affected. Reverse transcription (RT)-PCR revealed that transcripts from an FBP1 promoter were present from green tissue to the roots, whereas the SAG12 promoter was only active in processes associated with aging or senescence of flower petals and leaves. The sensitivity to ethylene exposure was markedly reduced by using both promoter constructs. Flower petals from nontransformed controls were fully senescent 4 days after ethylene exposure, while more than 90% of the flower petals from FBP1-promoter lines and over 50% of the flower petals from SAG12-promoter lines remained unchanged, without showing any signs of wilting or any response to ethylene. However, ethylene sensitivity of leaves was only delayed in the best lines but not suppressed through etr1-1 expression. Foliage displayed less intense leaf yellowing and wilting, which were similar for both promoter variants compared to wild type controls.

Cross-regulation of Arabidopsis root growth by plant hormones auxins and ethylene

We investigated the cross-talk between auxin (IAA) and ethylene in the control of root growth of Arabidopsis plants (Arabidopsis thaliana). The root growth of ethylene insensitive etr1-1 and auxin insensitive tir1 mutants under the effect of IAA and 1-methylcyclopropene (1-MCP, inhibitor of ethylene perception) was compared, respectively, with that of the wild type ecotype Columbia (Col-0). Roots of Col-0 were shorter than those of the etr1-1 mutant. The addition of IAA (5.7 × 10–6 М) to the growth medium led to 20 % root shortening in Col-0 plants, but not in etr1-1 mutants. Thus, the capacity of plants for ethylene perception contributes to the control of root length and its sensitivity to IAA. Roots of etr1-1 were less heavy than in Col-0, indicating that ethylene maintains root mass accumulation. Treatment with IAA caused a decrease in root mass of both genotypes (resulting in a 25 and 10 % decline in the root mass of Col-0 and etr1-1 as compared to the corresponding control), suggesting that IAA may influence root biomass accumulation independently of ethylene. However, sensitivity to ethylene increases plant responsiveness to IAA. Mutation in the auxin receptor decreased the sensitivity of roots to inactivation of ethylene receptors: treatment with 1-MCP resulted in a 40 % decline in the root mass of Col-0 and only a 10 % decrease in tir1. The decrement in sensitivity to auxins in the tir1 mutant decreased the responsiveness of root biomass to 1-MCP-treatment. These data suggest an additive action of ethylene and auxins on accumulation of root biomass.

Ethylene promotes pollen tube growth by affecting actin filament organization via the cGMP-dependent pathway in Arabidopsis thaliana

Ethylene and cGMP are key regulators of plant developmental processes. In this study, we demonstrate that ethylene or cGMP promote pollen tube growth in a dose-dependent manner. The etr1-1 mutant was found to be insensitive to ethylene with regard to pollen tube growth, while the growth-promoting effect of ethylene in etr2-2, ein4-4, or ein4-7 did not change, suggesting that ethylene signaling was mainly perceived by ETR1. However, the function of cGMP was not inhibited in etr1-1 and pollen tubes became insensitive to ethylene when the endogenous cGMP level was artificially decreased. This shows that cGMP is necessary for the control of pollen tube growth and that it might be a downstream component of ETR1 in the ethylene signaling pathway. Our study also found that ethylene or cGMP increase the actin bundles and elevated the percentage of relative amount of F-actin, while removal of cGMP decreased actin bundles abundance and altered the ratio of F-actin in the tip and base regions of pollen tubes. In conclusion, our data suggests that ethylene functions as the upstream signal of cGMP, and that both signals promote pollen germination and tube growth by regulating F-actin, which is essential for vesicular transport and cytoplasmic streaming.

The Biphasic Root Growth Response to Abscisic Acid in Arabidopsis Involves Interaction with Ethylene and Auxin Signalling Pathways.

Exogenous abscisic acid (ABA) is known to either stimulate or inhibit root growth, depending on its concentration. In this study, the roles of ethylene and auxin in this biphasic effect of ABA on root elongation were investigated using chemical inhibitors and mutants. Inhibitors of ethylene perception and biosynthesis and an auxin influx inhibitor were all found to block the inhibitory effect of high ABA concentrations, but not the stimulatory effect of low ABA concentrations. In addition, three ethylene-insensitive mutants (etr1-1, ein2-1, and ein3-1), two auxin influx mutants (aux1-7, aux1-T) and an auxin-insensitive mutant (iaa7/axr2-1) were all insensitive to the inhibitory effect of high ABA concentrations. In the case of the stimulatory effect of low ABA concentrations, it was blocked by two different auxin efflux inhibitors and was less pronounced in an auxin efflux mutant (pin2/eir1-1) and in the iaa7/axr2-1 auxin-insensitive mutant. Thus it appears that the stimulatory effect seen at low ABA concentrations is via an ethylene-independent pathway requiring auxin signalling and auxin efflux through PIN2/EIR1, while the inhibitory effect at high ABA concentrations is via an ethylene-dependent pathway requiring auxin signalling and auxin influx through AUX1.

Involvement of ethylene in UV-B-induced changes in polyamine content in Arabidopsis thaliana 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.

Dependence of root biomass accumulation on the content and metabolism of cytokinins in ethylene-insensitive plants

Diverse functions of ethylene in plants may depend on its ability to interact with other hormones. We studied the participation of ethylene in the regulation of accumulation and metabolism of cytokinins comparing ethylene-insensitive mutant plants of arabidopsis (Arabidopsis thaliana [L.] Heynh., etr1-1) with the plants of original ecotype Columbia (Col-0). Because cytokinins can regulate growth of both leaves and roots, we determined the weights of these organs and the ratio between them. The content of zeatin and its riboside in the roots of etr1-1 plants was two times greater than in Col-0 plants, which could be accounted for by inhibition of conversion of these forms of cytokinins into 9-N-glucosides. In the leaves of mutant plants, expression of IPT3 gene responsible for the synthesis of cytokinins was more intense than in Col-0 plants, which could also contribute to a rise in the content of cytokinins. In this case, the weight of roots in etr1-1 mutants was lower than in the plants of original ecotype. Because high concentrations of cytokinins can inhibit root growth, suppression of accumulation of their biomass in mutant plants may be related to a greater content of cytokinins therein. The obtained results suggest that ethylene can suppress accumulation of cytokinins and, thereby, maintain redistribution of biomass in favor of the roots, which is important for plant adaptation to a shortage of water and ions.

Ethylene Modulates Sphingolipid Synthesis in Arabidopsis.

Sphingolipids have essential structural and bioactive functions in membranes and in signaling. However, how plants regulate sphingolipid biosynthesis in the response to stress remains unclear. Here, we reveal that the plant hormone ethylene can modulate sphingolipid synthesis. The fungal toxin Fumonisin B1 (FB1) inhibits the activity of ceramide synthases, perturbing sphingolipid homeostasis, and thus inducing cell death. We used FB1 to test the role of ethylene signaling in sphingolipid synthesis in Arabidopsis thaliana. The etr1-1 and ein2 mutants, which have disrupted ethylene signaling, exhibited hypersensitivity to FB1; by contrast, the eto1-1 and ctr1-1 mutants, which have enhanced ethylene signaling, exhibited increased tolerance to FB1. Gene expression analysis showed that during FB1 treatment, transcripts of genes involved in de novo sphingolipid biosynthesis were down-regulated in ctr1-1 mutants but up-regulated in ein2 mutants. Strikingly, under normal conditions, ctr1-1 mutants contained less ceramides and hydroxyceramides, compared with wild type. After FB1 treatment, ctr1-1 and ein2 mutants showed a significant improvement in sphingolipid contents, except the ctr1-1 mutants showed little change in hydroxyceramide levels. Treatment of wild-type seedlings with the ethylene precursor 1-aminocyclopropane carboxylic acid down-regulated genes involved in the sphingolipid de novo biosynthesis pathway, thus reducing sphingolipid contents and partially rescuing FB1-induced cell death. Taking these results together, we propose that ethylene modulates sphingolipids by regulating the expression of genes related to the de novo biosynthesis of sphingolipids.

Ethylene signalling is mediating the early cadmium-induced oxidative challenge in Arabidopsis thaliana

Cadmium (Cd) induces the generation of reactive oxygen species (ROS) and stimulates ethylene biosynthesis. The phytohormone ethylene is a regulator of many developmental and physiological plant processes as well as stress responses. Previous research indicated various links between ethylene signalling and oxidative stress. Our results support a correlation between the Cd-induced oxidative challenge and ethylene signalling in Arabidopsis thaliana leaves. The effects of 24 or 72h exposure to 5μM Cd on plant growth and several oxidative stress-related parameters were compared between wild-type (WT) and ethylene insensitive mutants (etr1-1, ein2-1, ein3-1). Cadmium-induced responses observed in WT plants were mainly affected in etr1-1 and ein2-1 mutants, of which the growth was less inhibited by Cd exposure as compared to WT and ein3-1 mutants. Both etr1-1 and ein2-1 showed a delayed response in the glutathione (GSH) metabolism, including GSH levels and transcript levels of GSH synthesising and recycling enzymes. Furthermore, the expression of different oxidative stress marker genes was significantly lower in Cd-exposed ein2-1 mutants, evidencing that ethylene signalling is involved in early responses to Cd stress. A model for the cross-talk between ethylene signalling and oxidative stress is proposed.

Pyocyanin, a Virulence Factor Produced by Pseudomonas aeruginosa, Alters Root Development Through Reactive Oxygen Species and Ethylene Signaling in Arabidopsis

Pyocyanin acts as a virulence factor in Pseudomonas aeruginosa, a plant and animal pathogen. In this study, we evaluated the effect of pyocyanin on growth and development of Arabidopsis seedlings. Root inoculation with P. aeruginosa PAO1 strain inhibited primary root growth in wild-type (WT) Arabidopsis seedlings. In contrast, single lasI- and double rhlI-/lasI- mutants of P. aeruginosa defective in pyocyanin production showed decreased root growth inhibition concomitant with an increased phytostimulation. Treatment with pyocyanin modulates root system architecture, inhibiting primary root growth and promoting lateral root and root hair formation without affecting meristem viability or causing cell death. These effects correlated with altered proportions of hydrogen peroxide and superoxide in root tips and with an inhibition of cell division and elongation. Mutant analyses showed that pyocyanin modulation of root growth was likely independent of auxin, cytokinin, and abscisic acid but required ethylene signaling because the Arabidopsis etr1-1, ein2-1, and ein3-1 ethylene-related mutants were less sensitive to pyocyanin-induced root stoppage and reactive oxygen species (ROS) distribution. Our findings suggest that pyocyanin is an important factor modulating the interplay between ROS production and root system architecture by an ethylene-dependent signaling.

Ethylene is involved in strawberry fruit ripening in an organ-specific manner.

The fruit of the strawberry Fragaria×ananassa has traditionally been classified as non-climacteric because its ripening process is not governed by ethylene. However, previous studies have reported the timely endogenous production of minor amounts of ethylene by the fruit as well as the differential expression of genes of the ethylene synthesis, reception, and signalling pathways during fruit development. Mining of the Fragaria vesca genome allowed for the identification of the two main ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Their expression pattern during fruit ripening was found to be stage and organ (achene or receptacle) specific. Strawberry plants with altered sensitivity to ethylene could be employed to unravel the role of ethylene in the ripening process of the strawberry fruit. To this end, independent lines of transgenic strawberry plants were generated that overexpress the Arabidopsis etr1-1 mutant ethylene receptor, which is a dominant negative allele, causing diminished sensitivity to ethylene. Genes involved in ethylene perception as well as in its related downstream processes, such as flavonoid biosynthesis, pectin metabolism, and volatile biosynthesis, were differently expressed in two transgenic tissues, the achene and the receptacle. The different transcriptional responsiveness of the achene and the receptacle to ethylene was also revealed by the metabolic profiling of the primary metabolites in these two organs. The free amino acid content was higher in the transgenic lines compared with the control in the mature achene, while glucose and fructose, and citric and malic acids were at lower levels. In the receptacle, the most conspicuous change in the transgenic lines was the depletion of the tricarboxylic acid cycle intermediates at the white stage of development, most probably as a consequence of diminished respiration. The results are discussed in the context of the importance of ethylene during strawberry fruit ripening.

The rhizobacterium Variovorax paradoxus 5C-2, containing ACC deaminase, promotes growth and development of Arabidopsis thaliana via an ethylene-dependent pathway

Many plant-growth-promoting rhizobacteria (PGPR) associated with plant roots contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase and can metabolize ACC, the immediate precursor of the plant hormone ethylene, thereby decreasing plant ethylene production and increasing plant growth. However, relatively few studies have explicitly linked ethylene emission and/or action to growth promotion in these plant–microbe interactions. This study examined effects of the PGPR Variovorax paradoxus 5C-2 containing ACC deaminase on the growth and development of Arabidopsis thaliana using wild-type (WT) plants and several ethylene-related mutants (etr1-1, ein2-1, and eto1-1). Soil inoculation with V. paradoxus 5C-2 promoted growth (leaf area and shoot biomass) of WT plants and the ethylene-overproducing mutant eto1-1, and also enhanced floral initiation of WT plants by 2.5 days. However, these effects were not seen in ethylene-insensitive mutants (etr1-1 and ein2-1) even though bacterial colonization of the root system was similar. Furthermore, V. paradoxus 5C-2 decreased ACC concentrations of rosette leaves of WT plants by 59% and foliar ethylene emission of both WT plants and eto1-1 mutants by 42 and 37%, respectively. Taken together, these results demonstrate that a fully functional ethylene signal transduction pathway is required for V. paradoxus 5C-2 to stimulate leaf growth and flowering of A. thaliana.

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.

Metabolomic approaches reveal that cell wall modifications play a major role in ethylene‐mediated resistance against Botrytis cinerea

In Arabidopsis, resistance to the necrotrophic fungus Botrytis cinerea is conferred by ethylene via poorly understood mechanisms. Metabolomic approaches compared the responses of the wild-type, the ethylene-insensitive mutant etr1-1, which showed increased susceptibility, and the constitutively active ethylene mutants ctr1-1 and eto2 both exhibited decreased susceptibility to B.cinerea. Fourier transform-infrared (FT-IR) spectroscopy demonstrated reproducible biochemical differences between treatments and genotypes. To identify discriminatory mass-to-charge ratios (m/z) associated with resistance, discriminant function analysis was employed on spectra derived from direct injection electrospray ionisation-mass spectrometry on the derived principal components of these data. Ethylene-modulated m/z were mapped onto Arabidopsis biochemical pathways and many were associated with hydroxycinnamate and monolignol biosynthesis, both linked to cell wall modification. A high-resolution linear triple quadrupole-Orbitrap hybrid system confirmed the identity of key metabolites in these pathways. The contribution of these pathways to defence against B.cinerea was validated through the use of multiple Arabidopsis mutants. The FT-IR microspectroscopy indicated that spatial accumulation of hydroxycinnamates and monolignols at the cell wall to confine disease was linked ot ethylene. These data demonstrate the power of metabolomic approaches in elucidating novel biological phenomena, especially when coupled to validation steps exploiting relevant mutant genotypes.

Transcription Factor AtMYB44 Regulates Induced Expression of the ETHYLENE INSENSITIVE2 Gene in Arabidopsis Responding to a Harpin Protein

AtMYB44 is a transcription factor that functions in association with the ethylene-signaling pathway in Arabidopsis thaliana. The pathway depends on ETHYLENE INSENSITIVE2 (EIN2), an essential component of ethylene signaling, to regulate defense responses in the plant following treatment with HrpN(Ea), a harpin protein from a bacterial plant pathogen. Here, we show that AtMYB44 regulates induced expression of the EIN2 gene in HrpN(Ea)-treated Arabidopsis plants. A HrpN(Ea) and ethylene-responsive fragment of the AtMYB44 promoter is sufficient to support coordinate expression of AtMYB44 and EIN2 in specific transgenic Arabidopsis. In the plant, the AtMYB44 protein localizes to nuclei and binds the EIN2 promoter; the HrpN(Ea) treatment promotes AtMYB44 production, binding activity, and transcription of AtMYB44 and EIN2. AtMYB44 overexpression results in increased production of the AtMYB44 protein and the occurrence of AtMYB44-EIN2 interaction under all genetic backgrounds of wild-type Arabidopsis and the etr1-1, ein2-1, ein3-1, and ein5-1 mutants, which have defects in the ethylene receptor ETR1 and the signal regulators EIN2, EIN3, and EIN5. However, AtMYB44 overexpression leads to enhanced EIN2 expression only under backgrounds of wild type, ein3-1, and ein5-1 but not etr1-1 and ein2-1, suggesting that ethylene perception is necessary to the regulation of EIN2 transcription by AtMYB44.