Ctr1-1 Mutant Research Articles

Ethylene signaling in salt-stressed Arabidopsis thaliana ein2-1 and ctr1-1 mutants – A dissection of molecular mechanisms involved in acclimation

To pinpoint ethylene-mediated molecular mechanisms involved in the adaptive response to salt stress we conducted a comparative study of Arabidopsis thaliana wild type (Col-0), ethylene insensitive (ein2-1), and constitutive signaling (ctr1-1) mutant plants. Reduced germination and survival rates were observed in ein2-1 plants at increasing NaCl concentrations. By contrast, ctr1-1 mutation conferred salt stress tolerance during early vegetative development, corroborating earlier studies. Аll genotypes experienced strong stress as evidenced by the accumulation of reactive oxygen species (ROS) and increased membrane lipid peroxidation. However, the isoenzyme profiles of ROS scavenging enzymes demonstrated a higher peroxidase (POX) activity in ctr1-1 individuals under control and salt stress conditions. A markedly elevated free L-Proline (L-Pro) content was detected in the ethylene constitutive mutant. This coincided with the increased levels of Delta-1-Pyrroline-5-Carboxylate Synthase (P5CS) which is the rate-limiting enzyme from the proline biosynthetic pathway. A stabilized upregulation of a stress-induced P5CS1 splice variant was observed in the ctr1-1 background, which was not documented in the ethylene insensitive mutant ein2-1. Transcript profiling of the major SALT OVERLY SENSITIVE (SOS) pathway players (SOS1, SOS2, and SOS3) revealed altered gene expression in the organs of the ethylene signaling mutants. Overall suppressed SOS expression was observed in the ein2-1 mutants while only the SOS transcript profiles in the ctr1-1 roots were similar to the wild type. Altogether, we provide experimental evidence for ethylene-mediated molecular mechanisms implicated in the acclimation response to salt stress in Arabidopsis, which operate mainly through the regulation of free proline accumulation and enhanced ROS scavenging.

The Diverse Salt-Stress Response of Arabidopsis ctr1-1 and ein2-1Ethylene Signaling Mutants Is Linked to Altered Root Auxin Homeostasis.

We explored the interplay between ethylene signals and the auxin pool in roots exposed to high salinity using Arabidopsis thaliana wild-type plants (Col-0), and the ethylene-signaling mutants ctr1-1 (constitutive) and ein2-1 (insensitive). The negative effect of salt stress was less pronounced in ctr1-1 individuals, which was concomitant with augmented auxin signaling both in the ctr1-1 controls and after 100 mM NaCl treatment. The R2D2 auxin sensorallowed mapping this active auxin increase to the root epidermal cells in the late Cell Division (CDZ) and Transition Zone (TZ). In contrast, the ethylene-insensitive ein2-1 plants appeared depleted in active auxins. The involvement of ethylene/auxin crosstalk in the salt stress response was evaluated by introducing auxin reporters for local biosynthesis (pTAR2::GUS) and polar transport (pLAX3::GUS, pAUX1::AUX1-YFP, pPIN1::PIN1-GFP, pPIN2::PIN2-GFP, pPIN3::GUS) in the mutants. The constantly operating ethylene-signaling pathway in ctr1-1 was linked to increased auxin biosynthesis. This was accompanied by a steady expression of the auxin transporters evaluated by qRT-PCR and crosses with the auxin transport reporters. The results imply that the ability of ctr1-1 mutant to tolerate high salinity could be related to the altered ethylene/auxin regulatory loop manifested by a stabilized local auxin biosynthesis and transport.

CONSTITUTIVE TRIPLE RESPONSE1 and PIN2 act in a coordinate manner to support the indeterminate root growth and meristem cell proliferating activity in Arabidopsis seedlings

The plant hormone ethylene induces auxin biosynthesis and transport and modulates root growth and branching. However, its function on root stem cells and the identity of interacting factors for the control of meristem activity remains unclear. Genetic analysis for primary root growth in wild-type (WT) Arabidopsis thaliana seedlings and ethylene-related mutants showed that the loss-of-function of CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) inhibits cell division and elongation. This phenotype is associated with an increase in the expression of the auxin transporter PIN2 and a drastic decrease in the expression of key factors for stem cell niche maintenance such as PLETHORA1, SHORT ROOT and SCARECROW. While the root stem cell niche is affected in ctr1 mutants, its maintenance is severely compromised in the ctr1-1eir1-1(pin2) double mutant, in which an evident loss of proliferative capacity of the meristematic cells leads to a fully differentiated root meristem shortly after germination. Root traits affected in ctr1-1 mutants could be restored in ctr1-1ein2-1 double mutants. These results reveal that ethylene perception via CTR1 and EIN2 in the root modulates the proliferative capacity of root stem cells via affecting the expression of genes involved in the two major pathways, AUX-PIN-PLT and SCR-SHR, which are key factors for proper root stem cell niche maintenance.

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.

Arabidopsis acyl-CoA-binding protein ACBP3 participates in plant response to hypoxia by modulating very-long-chain fatty acid metabolism.

In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by a family of six genes (ACBP1 to ACBP6), and are essential for diverse cellular activities. Recent investigations suggest that the membrane-anchored ACBPs are involved in oxygen sensing by sequestration of group VII ethylene-responsive factors under normoxia. Here, we demonstrate the involvement of Arabidopsis ACBP3 in hypoxic tolerance. ACBP3 transcription was remarkably induced following submergence under both dark (DS) and light (LS) conditions. ACBP3-overexpressors (ACBP3-OEs) showed hypersensitivity to DS, LS and ethanolic stresses, with reduced transcription of hypoxia-responsive genes as well as accumulation of hydrogen peroxide in the rosettes. In contrast, suppression of ACBP3 in ACBP3-KOs enhanced plant tolerance to DS, LS and ethanol treatments. By analyses of double combinations of OE-1 with npr1-5, coi1-2, ein3-1 as well as ctr1-1 mutants, we observed that the attenuated hypoxic tolerance in ACBP3-OEs was dependent on NPR1- and CTR1-mediated signaling pathways. Lipid profiling revealed that both the total amounts and very-long-chain species of phosphatidylserine (C42:2- and C42:3-PS) and glucosylinositolphosphorylceramides (C22:0-, C22:1-, C24:0-, C24:1-, and C26:1-GIPC) were significantly lower in ACBP3-OEs but increased in ACBP3-KOs upon LS exposure. By microscale thermophoresis analysis, the recombinant ACBP3 protein bound VLC acyl-CoA esters with high affinities in vitro. Further, a knockout mutant of MYB30, a master regulator of very-long-chain fatty acid (VLCFA) biosynthesis, exhibited enhanced sensitivities to LS and ethanolic stresses, phenotypes that were ameliorated by ACBP3-RNAi. Taken together, these findings suggest that Arabidopsis ACBP3 participates in plant response to hypoxia by modulating VLCFA metabolism.

Ectopic expression of CsCTR1, a cucumber CTR-like gene, attenuates constitutive ethylene signaling in an Arabidopsis ctr1-1 mutant and expression pattern analysis of CsCTR1 in cucumber (Cucumis sativus).

The gaseous plant hormone ethylene regulates many aspects of plant growth, development and responses to the environment. Constitutive triple response 1 (CTR1) is a central regulator involved in the ethylene signal transduction pathway. To obtain a better understanding of this particular pathway in cucumber, the cDNA-encoding CTR1 (designated CsCTR1) was isolated from cucumber. A sequence alignment and phylogenetic analyses revealed that CsCTR1 has a high degree of homology with other plant CTR1 proteins. The ectopic expression of CsCTR1 in the Arabidopsis ctr1-1 mutant attenuates constitutive ethylene signaling of this mutant, suggesting that CsCTR1 indeed performs its function as negative regulator of the ethylene signaling pathway. CsCTR1 is constitutively expressed in all of the examined cucumber organs, including roots, stems, leaves, shoot apices, mature male and female flowers, as well as young fruits. CsCTR1 expression gradually declined during male flower development and increased during female flower development. Additionally, our results indicate that CsCTR1 can be induced in the roots, leaves and shoot apices by external ethylene. In conclusion, this study provides a basis for further studies on the role of CTR1 in the biological processes of cucumber and on the molecular mechanism of the cucumber ethylene signaling pathway.

Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase

Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on (15)N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein-protein interaction filter revealed a total of 14 kinase-substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light harvesting chlorophyll A/B binding protein 1.1, and flowering bHLH 3 proteins in a dual-and-opposing fashion.

Genetic identification of a second site modifier of ctr1-1 that controls ethylene-responsive and gravitropic root growth in Arabidopsis thaliana.

Ethylene controls myriad aspects of plant growth throughout developmental stages in higher plants. It has been well established that ethylene-responsive growth entails extensive crosstalk with other plant hormones, particularly auxin. Here, we report a genetic mutation, named 1-aminocyclopropane carboxylic acid (ACC) resistant root1-1 (are1-1) in Arabidopsis thaliana (L.) Heynh. The CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) encodes a Raf-related protein, functioning as an upstream negative regulator of ethylene signaling in Arabidopsis thaliana. We found that the ctr1-1, a kinase-inactive allele exhibited slightly, but significantly, longer root length, compared to ACC-treated wild-type or ctr1-3, a null allele. Our genetic studies unveiled the existence of are1-1 mutation in the ctr1-1 mutant, as a second-site modifier which confers root-specific ethylene-resistance. Based on well-characterized crosstalk between ethylene and auxin during ethylene-responsive root growth, we performed various physiological analyses. Whereas are1-1 displayed normal sensitivity to synthetic auxins, it showed modest resistance to an auxin transport inhibitor, 1-Nnaphthylphthalamic acid. In addition, are1-1 mutant exhibited ectopically altered DR5:GUS activity upon ethylenetreatment. The results implicated the involvement of are1-1 in auxin-distribution, but not in auxin-biosynthesis, -uptake, or -sensitivity. In agreement, are1-1 mutant exhibited reduced gravitropic root growth and defective redistribution of DR5:GUS activity upon gravi-stimulation. Taken together with genetic and molecular analysis, our results suggest that ARE1 defines a novel locus to control ethylene-responsive root growth as well as gravitropic root growth presumably through auxin distribution in Arabidopsis thaliana.

UV-B stress-induced ABA production in Arabidopsis thaliana mutants defective in ethylene signal transduction pathway

In this study, we examined the influence of UV-B radiation (280–320 nm) on ABA accumulation in 14-day-old Arabidopsis thaliana (L.) Heynh plants of wild type (WT), ethylene receptor mutant (etr1-1), and mutant with a constitutively active ethylene signal transduction pathway (ctr1-1). ABA content in nonirradiated WT plants was twice higher than in each mutant. UV-B irradiation caused dose-dependent ABA accumulation in WT plants. In the etr1-1 mutant, the amount of accumulated ABA was significantly less. In the ctr1-1 mutant, ABA content didn’t increase after UV-B irradiation. These data suggest that start of stress-induced ABA formation requires the adjustable ethylene signal pathway. In the ctr1-1 mutant, a constitutively active (nonadjustable) ethylene signal pathway blocks stress-induced ABA accumulation.

Effect of ABA on the UV-B-Induced Ethylene Evolution by the etr and ctr Mutants of Arabidopsis thaliana

The responses of 14-day-old Arabidopsis thaliana (L.) Heynh. plants to UV-B irradiation (280–320 nm) and ABA treatment were investigated. Wild-type plants as well as ethylene-insensitive etr1-1 and ctr1-1 mutants were used. Theetr1-1 mutant considerably differed from the ctr1-1 one in the fresh weight production after UV-B treatment (29.5 kJ/m2). The irradiated etr1-1 plants fell well behind the nonirradiated ones during the first two days after stress, but by the 8th day, their weight attained 70% of control plant weight. In contrast, Ctr1-1 mutant weight comprised 70% of control level after two days of stress but, by the 8th day, it was only 56% of the weight of control plants. In wild-type and ctr1-1 plants, ABA, in the 8 × 10–6 to 2 × 10–4 M concentration range, increased the difference between the weights of nonirradiated and irradiated plants, but in etr1-1 plants, ABA decreased this difference. The etr1-1, ctr1-1, and wild-type plants were very similar in the dynamics of ethylene evolution after UV-B treatment (7.4 kJ/m2). In wild-type, etr1-1, and ctr1-1 plants, ABA, in a concentration-dependent manner, inhibited UV-B-induced ethylene evolution to the same extent. The results obtained show that ABA exerted an opposite effect on UV-B-dependent growth in the plants with active (wild type and ctr1-1) and blocked (etr1-1) ethylene signal pathway, whereas the inhibition of ethylene synthesis by ABA was not related to ethylene signal transmission.

Ethylene regulates monomeric GTP-binding protein gene expression and activity in Arabidopsis.

Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction.