LeCTR2, a CTR1-like protein kinase from tomato, plays a role in ethylene signalling, development and defence

Arabidopsis AtCTR1 is a Raf-like protein kinase that interacts with ETR1 and ERS and negatively regulates ethylene responses. In tomato, several CTR1-like proteins could perform this role. We have characterized LeCTR2, which has similarity to AtCTR1 and also to EDR1, a CTR1-like Arabidopsis protein involved in defence and stress responses. Protein–protein interactions between LeCTR2 and six tomato ethylene receptors indicated that LeCTR2 interacts preferentially with the subfamily I ETR1-type ethylene receptors LeETR1 and LeETR2, but not the NR receptor or the subfamily II receptors LeETR4, LeETR5 and LeETR6. The C-terminus of LeCTR2 possesses serine/threonine kinase activity and is capable of auto-phosphorylation and phosphorylation of myelin basic protein in vitro . Overexpression of the LeCTR2 N-terminus in tomato resulted in altered growth habit, including reduced stature, loss of apical dominance, highly branched inflorescences and fruit trusses, indeterminate shoots in place of determinate flowers, and prolific adventitious shoot development from the rachis or rachillae of the leaves. Expression of the ethylene-responsive genes E4 and chitinase B was upregulated in transgenic plants, but ethylene production and the level of mRNA for the ethylene biosynthetic gene ACO1 was unaffected. The leaves and fruit of transgenic plants also displayed enhanced susceptibility to infection by the fungal pathogen Botrytis cinerea , which was associated with much stronger induction of pathogenesis-related genes such as PR1b1 and chitinase B compared with the wild-type. The results suggest that LeCTR2 plays a role in ethylene signalling, development and defence, probably through its interactions with the ETR1-type ethylene receptors of subfamily I.     

Protein kinase domain of CTR1 from Arabidopsis thaliana promotes ethylene receptor cross talk

Ethylene controls many aspects of plant growth and development. Signaling by the gaseous phytohormone is initiated by disulfide-linked membrane-bound receptors, and the formation of heteromeric receptor clusters contributes to the broad range of ethylene responsiveness. In Arabidopsis thaliana, the TCS-like ethylene receptors interact with the cytosolic serine/threonine kinase constitutive triple response 1 (CTR1), a proposed mitogen-activated protein kinase kinase kinase. In the absence of the hormone, the receptor and therefore CTR1 are active. Hence, ethylene acts as an inverse agonist of its signaling pathway. The three-dimensional structures of the active, triphosphorylated and the unphosphorylated, inactive kinase domain of CTR1 in complex with staurosporine illustrate the conformational rearrangements that form the basis of activity regulation. Additionally, in analytical ultracentrifugation experiments, active kinase domains form back-to-back dimers, while inactive and activation loop variants are monomers. Together with a front-to-front activation interface, the active protein kinase dimers thereby engage in interactions that promote CTR1-mediated cross talk between ethylene receptor clusters. This model provides a structural foundation for the observed high sensitivity of plants to ethylene.     

Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis

In Arabidopsis (Arabidopsis thaliana), ethylene is perceived by a receptor family consisting of five members. Subfamily 1 members ETHYLENE RESPONSE1 (ETR1) and ETHYLENE RESPONSE SENSOR1 (ERS1) have histidine kinase activity, unlike the subfamily 2 members ETR2, ERS2, and ETHYLENE INSENSITIVE4 (EIN4), which lack amino acid residues critical for this enzymatic activity. To resolve the role of histidine kinase activity in signaling by the receptors, we transformed an etr1-9;ers1-3 double mutant with wild-type and kinase-inactive versions of the receptor ETR1. Both wild-type and kinase-inactive ETR1 rescue the constitutive ethylene-response phenotype of etr1-9;ers1-3, restoring normal growth to the mutant in air. However, the lines carrying kinase-inactive ETR1 exhibit reduced sensitivity to ethylene based on several growth response assays. Microarray and real-time polymerase chain reaction analyses of gene expression support a role for histidine kinase activity in eliciting the ethylene response. In addition, protein levels of the Raf-like kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), which physically associates with the ethylene receptor ETR1, are less responsive to ethylene in lines containing kinase-inactive ETR1. These data indicate that the histidine kinase activity of ETR1 is not required for but plays a modulating role in the regulation of ethylene responses. Models for how enzymatic and nonenzymatic regulation may facilitate signaling from the ethylene receptors are discussed.     

The roles of abscisic acid and ethylene in the abscission and senescence of cocoa flowers

Cocoa flowers have a limited period of longevity; more than 90% of unpollinated flowers abscised within 32 h after anthesis. Abscisic acid (ABA) levels increased significantly prior to abscission. By 21 h after anthesis, ABA levels had increased almost 10-fold, and by 32 h flowers had 20-fold higher levels of ABA than at anthesis. Fluridone completely inhibited both the increase in ABA, the formation of an abscission zone, and the abscission and senescence of flowers. In contrast, ethylene production increased only slightly 21 h after anthesis and was only 2-fold higher after 32 h. Aminoethoxyvinylglycine (AVG) delayed but did not prevent abscission. In cocoa flowers, ABA is the primary regulator of abscission; ethylene accelerates abscission but only in the presence of ABA. Naphthalene acetic acid (NAA) treatment of flowers at anthesis prevented abscission zone formation and flower abscission, but did not induce fruit set. All parts of the NAA-treated flower except the pedicel senesced after 6 days. NAA+AVG treatment only delayed, whereas fluridone treatment completely prevented flower senescence.     

Ethylene- and dark-induced flower abscission in potted Plectranthus: Sensitivity,prevention by 1-MCP,and expression of ethylene biosynthetic genes

Prevention of ethylene- and shipping-induced flower abscission is necessary to maintain the quality of both cut flowers and potted plants during handling, transport and retail display. The aims of the present work were to determine the sensitivity of Plectranthus cultivars to applied ethylene, to alleviate ethylene- and shipping-induced flower abscission in intact potted plants using 1-methylcyclopropene (1-MCP), and to investigate the possible causes of dark-induced flower abscission. All cultivars were sensitive to ethylene in a concentration-dependent manner, and complete abscission occurred within 24 h with 1 and 2 μl l^− 1 ethylene. Unopened buds were more sensitive to applied ethylene, and exhibited greater abscission than open flowers. Ethylene synthesis remained below detection limits at all time points under control and continuous dark conditions. Dark treatment significantly increased flower abscission in Plectranthus cultivars, and like ethylene-induced flower abscission, this could be prevented by continuous 1-MCP treatment. Gene expression of ethylene biosynthetic enzymes ACS and ACO was examined as possible causes for the accelerated flower abscission observed in plants kept in continuous darkness. Expression patterns of ACS and ACO varied between different cultivars of Plectranthus. In some cases, increased expression of ACS and ACO led to increased flower abscission. Gene expression was higher in open flowers when compared to unopened flowers suggesting a cause for the observed preferential shedding of open flowers in some cultivars. Although the cause of dark-induced abscission in Plectranthus remains elusive, it can be effectively controlled by treatment with 1-MCP.     

Soluble carbohydrates in Delphinium and their influence on sepal abscission in cut flowers

A carbohydrate other than sucrose, glucose, fructose and myo -inositol was detected in sepal extracts of Delphinium . This compound was identified as mannitol by ¹H-NMR. Mannitol was the major carbohydrate in all examined organs: the sepal, the other parts of the flower, the stem and leaves. Mannitol as well as glucose (both at 0.55 M ), fed to cut Delphinium flowers, similarly delayed the abscission of sepals. 3- O -methyl glucose (3-OMG) and polyethylene glycol 200 at the same molar concentrations had no such effect. The treatment with glucose markedly increased the concentrations of glucose and fructose in the sepals without changing the concentrations of sucrose and mannitol. On the other hand, the treatment with mannitol increased the concentrations of glucose and fructose in addition to mannitol in the sepals, suggesting that mannitol is metabolized in Delphinium flowers. The treatment with 3-OMG increased the concentration of 3-OMG but not other carbohydrates. Mannitol and glucose similarly delayed the increase in ethylene production in flowers, but 3-OMG did not. The sensitivity to ethylene was similarly reduced by the treatment with glucose and mannitol, but not by 3-OMG. These results suggest that the treatment with mannitol, a major carbohydrate in Delphinium , delayed the abscission of sepals by reducing the sensitivity to ethylene. Mannitol further acted, not merely as an osmolyte, but as an apparent source for carbohydrate metabolism in the flower.     

Ethylene responsiveness changes in Pelargonium×domesticum florets

Florets of Pelargonium x domesticum L. H. Bailey respond to ethylene with rapid petal abscission. Florets become increasingly responsive to ethylene with age, with the greatest change in responsiveness occurring between the day of anthesis and 2 days later. The ethylene responsiveness coefficient (the ratio of the concentrations of ethylene giving 90% and 10% abscission) declined from 21 in 1-day-old florets to slightly greater than 1 in older florets; therefore abscission was ultraresponsive (responsiveness coefficient <81) even in 1-day old florets. Younger florets required a higher concentration of ethylene and longer duration of ethylene exposure to induce the abscission response. Even florets aged 2–3 days after anthesis required at least 40 min of exposure to ethylene for the abscission response to occur, irrespective of the concentration.     

乙烯的生物合成与信号传递

乙烯是气体植物激素, 它在植物的生长发育过程中有很多作用。所以了解乙烯的生物合成及其信号转导是非常重要的。二十年来, 通过筛选有异于正常三重反应的突变体, 人们发现了乙烯信号转导的粗略轮廓。在拟南芥中, 有5个受体蛋白感受乙烯, ETR1、ERS1、ETR2、ERS2、EIN4。它们表现出功能冗余, 是乙烯信号的负调控因子, 在植物体内以二聚体的形式存在。ETR1的N端与乙烯结合时需要 铜离子(Ⅰ)的参与。尽管已经发现ETR1有组氨酸激酶活性, 而其它受体有丝氨酸/苏氨酸激酶活性, 但受体参与乙烯信号转导的机制还不是很清楚。受体与Raf类蛋白激酶CTR1相互作用, CTR1是乙烯反应的负调控因子。CTR1蛋白失活使EIN2蛋白活化。EIN2的N端是跨膜结构域, 与Nramp家族金属离子转运蛋白的跨膜结构域类似。EIN2的C端是一个新的未知结构域, 与乙烯信号途径的下游组分相互作用。EIN3位于EIN2的下游, EIN3和EILs诱导ERF1和其它转录因子的表达, 这些转录因子依次激活乙烯反应目的基因的表达, 表现出乙烯的反应。EIN3受到蛋白酶体介导的蛋白降解途径的调节。由于乙烯是一种多功能的植物激素, 其信号途径与其它信号途径有多重的交叉。     

Enhanced ethylene responsiveness in the Arabidopsis eer1 mutant results from a loss-of-function mutation in the protein phosphatase 2A A regulatory subunit,RCN1

Ethylene signaling in Arabidopsis begins with a family of five ethylene receptors that regulate the activity of the Raf-like kinase, CTR1. Recent work to identify novel factors required for modulating ethylene signaling resulted in the isolation of enhanced ethylene response 1 (eer1), a mutant that displays both increased sensitivity and increased amplitude of response to ethylene. Molecular cloning of eer1 reveals that its mutant phenotype results from a loss-of-function mutation in the previously characterized RCN1, one of three PP2A A regulatory subunits in Arabidopsis. Our analysis shows that neither RCN1 expression nor PP2A activity is regulated by ethylene. Instead, we found that Arabidopsis PP2A-1C, a PP2A catalytic subunit previously characterized as interacting with RCN1, associates strongly with the kinase domain of CTR1 in vitro. This likely represents a role for PP2A in modulation of CTR1 activity because an in vitro kinase assay did not reveal phosphorylation of either RCN1 or PP2A-1C by CTR1, indicating that neither of them is a substrate for CTR1. PP2A activity is required for Ras-dependent activation of mammalian Raf, with reductions in PP2A activity significantly compromising the effectiveness of this mechanism. Our genetic and biochemical results suggest that a similar requirement for PP2A activity exists for ethylene signaling, with loss-of-function mutations affecting PP2A activity possibly reducing the effectiveness of CTR1 activation, thus lowering the threshold required for manifestation of ethylene response.     

EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis.

The Arabidopsis ethylene receptor gene ETR1 and two related genes, ERS1 and ETR2, were identified previously. These three genes encode proteins homologous to the two-component regulators that are widely used for environment sensing in bacteria. Mutations in these genes confer ethylene insensitivity to wild-type plants. Here, we identified two Arabidopsis genes, EIN4 and ERS2, by cross-hybridizing them with ETR2. Sequence analysis showed that they are more closely related to ETR2 than they are to ETR1 or ERS1. EIN4 previously was isolated as a dominant ethylene-insensitive mutant. ERS2 also conferred dominant ethylene insensitivity when certain mutations were introduced into it. Double mutant analysis indicated that ERS2, similar to ETR1, ETR2, ERS1, and EIN4, acts upstream of CTR1. Therefore, EIN4 and ERS2, along with ETR1, ETR2, and ERS1, are members of the ethylene receptor-related gene family of Arabidopsis. RNA expression patterns of members of this gene family suggest that they might have distinct as well as redundant functions in ethylene perception.     

Lack of ethylene involvement in tulip tepal abscission

The tepals of cut flowers of Tulipa hybrida cv. Golden Apeldoorn and Tulipa kaufmanniana cv. Shakespeare abscise 3–4 days after harvest. The weakening of the abscission zones is accompanied by cell wall breakdown and the separation of 3–4 rows of intact cells at the base of the tepal. During senescence, there is no ethylene climacteric and ethylene production rates remain low, between 0.07 and 0.4 nl g⁻¹ fresh weight h⁻¹. Adding 3–5 μl l⁻¹ ethylene slightly accelerated the weakening of the abscission zones but had no effect on the time of first abscission. Neither 0.5 m M silver thiosulphate nor 5 m M aminoethoxyvinylglycine delayed the time to abscission. It is concluded that tulip tepal fall does not involve primary regulation by ethylene, unlike the majority of other abscission systems that have been studied.     

Nm23-H1 metastasis suppressor phosphorylation of kinase suppressor of Ras via a histidine protein kinase pathway

The metastasis-suppressive activity of Nm23-H1 was previously correlated with its in vitro histidine protein kinase activity, but physiological substrates have not been identified. We hypothesized that proteins that interact with histidine kinases throughout evolution may represent partners for Nm23-H1 and focused on the interaction of Arabidopsis "two-component" histidine kinase ERS with CTR1. A mammalian homolog of CTR1 was previously reported to be c-Raf; we now report that CTR1 also exhibits homology to the kinase suppressor of Ras (KSR), a scaffold protein for the mitogen-activated protein kinase (MAPK) cascade. Nm23-H1 co-immunoprecipitated KSR from lysates of transiently transfected 293T cells and at endogenous protein expression levels in MDA-MB-435 breast carcinoma cells. Autophosphorylated recombinant Nm23-H1 phosphorylated KSR in vitro. Phosphoamino acid analysis identified serine as the major target, and two peaks of Nm23-H1 phosphorylation were identified upon high performance liquid chromatography analysis of KSR tryptic peptides. Using site-directed mutagenesis, we found that Nm23-H1 phosphorylated KSR serine 392, a 14-3-3-binding site, as well as serine 434 when serine 392 was mutated. Phosphorylated MAPK but not total MAPK levels were reduced in an nm23-H1 transfectant of MDA-MB-435 cells. The data identify a complex in vitro histidine-to-serine protein kinase pathway, which may contribute to signal transduction and metastasis.     

Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.     

Petal Abscission in Rose Flowers: Effects of Water Potential, Light Intensity and Light Quality

Petal abscission was studied in roses (Rosa hybrida L.), cvs.Korflapei (trade name Frisco), Sweet Promise (Sonia) and CaraMia (trade name as officially registered cultivar name). Unlikeflowers on plants in greenhouses, cut flowers placed in waterin the greenhouse produced visible symptoms of water stress,depending on the weather during the experiment and on the cultivar.Cut Frisco roses showed no visible signs of water stress andthe time to petal abscission was as in uncut flowers. In Soniaroses the symptoms of water stress varied from mild to severe,and the number of flowers in which the petals abscised variedfrom 100% (mild stress) to 0% (severe stress). An antimicrobialcompound in the vase water of Sonia roses, or removal of theleaves, alleviated the symptoms of water stress and increasedthe number of stems in which the petals abscised. Cut Cara Miaroses showed severe symptoms of water stress in all experimentsand petal abscission was found in only a few flowers, even whenthe stems were placed at 20 °C and low photon flux (15 µmolm^-2s^-1). Abscission in Sonia and Cara Mia roses was low or absentwhen the water potential of the leaves reached values below-2.0 MPa within the first 5 d of the experiment; such low valueswere not reached in Frisco roses. Addition of sucrose to the vase solution, together with an effectiveantimicrobial compound, had no effect on the time to petal abscission,at any light intensity. Placing flowers in far-red light alsohad no effect on abscission, compared with flowers placed inred light or white light of the same photon fluence. It is concluded that petal abscission in the rose cultivarsstudied is not affected by their water status unless the plantsreach a low water potential (about -2 MPa) early on during vaselife. Petal abscission is not inhibited by low light intensitynor affected by the Pr/Pfr ratio. Abscission; light intensity; petals; phytochrome; Rosa hybrida L.; rose; sugars; water potential     

The MADS box gene, FOREVER YOUNG FLOWER, acts as a repressor controlling floral organ senescence and abscission in Arabidopsis

The ectopic expression of a MADS box gene FOREVER YOUNG FLOWER (FYF) caused a significant delay of senescence and a deficiency of abscission in flowers of transgenic Arabidopsis. The defect in floral abscission was found to be due to a deficiency in the timing of cell separation of the abscission zone cells. Down-regulation of INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) may contribute to the delay of the floral abscission in 35S:FYF flowers. FYF was found to be highly expressed in young flowers prior to pollination and was significantly decreased after pollination, a pattern that correlated with its function. Ethylene insensitivity in senescence/abscission and the down-regulation of ETHYLENE RESPONSE DNA-BINDING FACTOR 1 (EDF1) and EDF2, downstream genes in the ethylene response, in 35S:FYF Arabidopsis suggested a role for FYF in regulating senescence/abscission by suppressing the ethylene response. This role was further supported by the fact that 35S:FYF enhanced the delay of flower senescence/abscission in ethylene response 1 (etr1), ethylene-insensitive 2 (ein2) and constitutive triple response 1 (ctr1) mutants, which have defects in upstream genes of the ethylene signaling pathway. The presence of a repressor domain in the C-terminus of FYF and the enhancement of the delay of senescence/abscission in FYF+SRDX (containing a suppression motif) transgenic plants suggested that FYF acts as a repressor. Indeed, in FYF-DR+VP16 transgenic dominant-negative mutant plants, in which FYF was converted to a potent activator by fusion to a VP16-AD motif, the senescence/abscission of the flower organs was significantly promoted, and the expression of BOP2, IDA and EDF1/2 was up-regulated. Our data suggest a role for FYF in controlling floral senescence/abscission by repressing ethylene responses and regulating the expression of BOP2 and IDA in Arabidopsis.     

Ethylene, a regulator of young fruit abscission

In an earlier study we reported that detached cotton flowers produced sufficient ethylene before the period of natural abscission to suggest that ethylene might be a natural regulator of young fruit abscission. The present report explores this probability further. Intact cotton (Gossypium hirsutum L.) fruits produced ethylene at rates as high as 36 μl ethylene/kg fresh wt·hr during the 2 days before they abscised. Direct measurements of ethylene in gas samples withdrawn from fruits indicated that production of 1 μl ethylene/kg fresh wt·hr is equivalent to an internal concentration of approximately 0.1 μl/l. Fumigation of fruiting cotton plants with only 0.5 μl/l caused 100% abscission of young fruits and floral buds within 2 days. This correlated with the estimated endogenous levels of ethylene. Reduced pressure, which reduced the internal levels of ethylene, delayed abscission of young fruits and leaves, a result which supports our conclusion from this study— that ethylene is one of the regulators of young fruit abscission in cotton.