Ethylene insensitivity conferred by a mutated Arabidopsis ethylene receptor gene alters nodulation in transgenic Lotus japonicus

Background and Aims

Transgenics are used to demonstrate a causal relationship between ethylene insensitivity of a seedling legume plant, the level of ethylene receptor gene expression, lateral root growth and Mesorhizobium loti-induced nodule initiation.

Methods

Lotus japonicus plants expressing the dominant etr1-1 allele of the Arabidopsis thaliana gene encoding a well-characterized mutated ethylene receptor were created by stable Agrobacterium tumefaciens transformation. Single insertion, homozygous lines were characterized for symbiotic properties.

Key Results

Transgenic plants were ethylene insensitive as judged by the lack of the ‘Triple Response’, and their continued ability to grow and nodulate in the presence of inhibitory concentrations of ACC (1-aminocyclopropane-1-carboxylic acid; an ethylene precursor). Transgenic plants with high insensitivity to ACC had significantly fewer lateral roots and exhibited increased nodulation while showing no altered nitrate sensitivity or lack of systemic autoregulation. Whereas ACC-insensitive shoot growth and nodulation were observed in transformants, root growth was inhibited similarly to the wild type. Increased nodulation was caused by increased infection and a seven-fold increase in nodules developing between xylem poles. Bacteroid numbers per symbiosome increased about 1·7-fold in ethylene-insensitive plants.

Conclusions

The study further demonstrates multiple roles for ethylene in nodule initiation by influencing root cell infections and radial positioning, independent of autoregulation and nitrate inhibition of nodulation.Key words: Ethylene insensitivity, Lotus japonicus, symbiosis, phytohormone, nodulation, signal transduction     

Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis

Background  

Ethylene receptor single mutants of Arabidopsis do not display a visibly prominent phenotype, but mutants defective in multiple ethylene receptors exhibit a constitutive ethylene response phenotype. It is inferred that ethylene responses in Arabidopsis are negatively regulated by five functionally redundant ethylene receptors. However, genetic redundancy limits further study of individual receptors and possible receptor interactions. Here, we examined the ethylene response phenotype in two quadruple receptor knockout mutants, (ETR1) ers1 etr2 ein4 ers2 and (ERS1) etr1 etr2 ein4 ers2, to unravel the functions of ETR1 and ERS1. Their functions were also reciprocally inferred from phenotypes of mutants lacking ETR1 or ERS1. Receptor protein levels are correlated with receptor gene expression. Expression levels of the remaining wild-type receptor genes were examined to estimate the receptor amount in each receptor mutant, and to evaluate if effects of ers1 mutations on the ethylene response phenotype were due to receptor functional compensation. As ers1 and ers2 are in the Wassilewskija (Ws) ecotype and etr1, etr2, and ein4 are in the Columbia (Col-0) ecotype, possible effects of ecotype mixture on ethylene responses were also investigated.     

Ethylene perception by the ERS1 protein in Arabidopsis

Ethylene perception in Arabidopsis is controlled by a family of five genes, including ETR1, ERS1 (ethylene response sensor 1), ERS2, ETR2, and EIN4. ERS1, the most highly conserved gene with ETR1, encodes a protein with 67% identity to ETR1. To clarify the role of ERS1 in ethylene sensing, we biochemically characterized the ERS1 protein by heterologous expression in yeast. ERS1, like ETR1, forms a membrane-associated, disulfide-linked dimer. In addition, yeast expressing the ERS1 protein contains ethylene-binding sites, indicating ERS1 is also an ethylene-binding protein. This finding supports previous genetic evidence that isoforms of ETR1 also function in plants as ethylene receptors. Further, we used the ethylene antagonist 1-methylcyclopropene (1-MCP) to characterize the ethylene-binding sites of ERS1 and ETR1. We found 1-MCP to be both a potent inhibitor of the ethylene-induced seedling triple response, as well as ethylene binding by yeast expressing ETR1 and ERS1. Yeast expressing ETR1 and ERS1 showed nearly identical sensitivity to 1-MCP, suggesting that the ethylene-binding sites of ETR1 and ERS1 have similar affinities for ethylene.     

Arabidopsis RTE1 is essential to ethylene receptor ETR1 amino-terminal signaling independent of CTR1

The Arabidopsis (Arabidopsis thaliana) ethylene receptor Ethylene Response1 (ETR1) can mediate the receptor signal output via its carboxyl terminus interacting with the amino (N) terminus of Constitutive Triple Response1 (CTR1) or via its N terminus (etr1^1-349 or the dominant ethylene-insensitive etr1-1^1-349) by an unknown mechanism. Given that CTR1 is essential to ethylene receptor signaling and that overexpression of Reversion To Ethylene Sensitivity1 (RTE1) promotes ETR1 N-terminal signaling, we evaluated the roles of CTR1 and RTE1 in ETR1 N-terminal signaling. The mutant phenotype of ctr1-1 and ctr1-2 was suppressed in part by the transgenes etr1^1-349 and etr1-1^1-349, with etr1-1^1-349 conferring ethylene insensitivity. Coexpression of 35S:RTE1 and etr1^1-349 conferred ethylene insensitivity in ctr1-1, whereas suppression of the ctr1-1 phenotype by etr1^1-349 was prevented by rte1-2. Thus, RTE1 was essential to ETR1 N-terminal signaling independent of the CTR1 pathway. An excess amount of the CTR1 N terminus CTR1^7-560 prevented ethylene receptor signaling, and the CTR1^7-560 overexpressor CTR1-Nox showed a constitutive ethylene response phenotype. Expression of the ETR1 N terminus suppressed the CTR1-Nox phenotype. etr1^1-349 restored the ethylene insensitivity conferred by dominant receptor mutant alleles in the ctr1-1 background. Therefore, ETR1 N-terminal signaling was not mediated by full-length ethylene receptors; rather, full-length ethylene receptors acted cooperatively with the ETR1 N terminus to mediate the receptor signal independent of CTR1. ETR1 N-terminal signaling may involve RTE1, receptor cooperation, and negative regulation by the ETR1 carboxyl terminus.The gaseous plant hormone ethylene is perceived by a small family of ethylene receptors. Arabidopsis (Arabidopsis thaliana) has five ethylene receptors that are structurally similar to prokaryotic two-component histidine kinase (HK) proteins. Mutants defective in multiple ethylene receptor genes show a constitutive ethylene response phenotype, which indicates a negative regulation of ethylene responses by the receptor genes (Hua and Meyerowitz, 1998).The receptor N terminus has three or four transmembrane domains that bind ethylene. The GAF (for cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain, which follows the transmembrane helices, mediates noncovalent receptor heterodimerization and may have a role in receptor cooperation (Gamble et al., 2002; O’Malley et al., 2005; Xie et al., 2006; Gao et al., 2008). The subfamily I receptors Ethylene Response1 (ETR1) and Ethylene Response Sensor1 (ERS1) have a conserved HK domain following the GAF domain. For subfamily II members ETR2, Ethylene Insensitive4 (EIN4), and ERS2, the HK domain is less conserved, and they lack most signature motifs essential for HK activity (Chang et al., 1993; Gamble et al., 1998; Hua et al., 1998; Qu and Schaller, 2004; Xie et al., 2006). Among the five receptors, ETR1, ETR2, and EIN4 have a receiver domain following the HK domain. The ETR1 HK domain may have a role in mediating the receptor signal to downstream components, and the HK activity facilitates the ethylene signaling (Clark et al., 1998; Huang et al., 2003; Hall et al., 2012). The receiver domain can dimerize and could involve receptor cooperation (Müller-Dieckmann et al., 1999). However, differential receptor cooperation occurs between the receiver domain-lacking ERS1 and the other ethylene receptors, which does not support the hypothesis that the domains involve receptor cooperation (Liu and Wen, 2012).Acting downstream of the ethylene receptors is Constitutive Triple Response1 (CTR1), a MEK kinase (mitogen-activated protein kinase kinase kinase) with Ser/Thr kinase activity, and the kinase domain locates at the C terminus. The CTR1 N terminus does not share sequence similarity to known domains and can physically interact with the ethylene-receptor HK domain (Clark et al., 1998; Huang et al., 2003). ctr1 mutants showing attenuated CTR1 kinase activity or the ETR1-CTR1 association exhibit various degrees of the constitutive ethylene-response phenotype. For example, the ctr1-1 and ctr1^btk mutations result from the D694E and E626K substitutions, respectively, in the CTR1 kinase domain, and ctr1-1 shows a stronger ethylene-response phenotype than ctr1^btk, with ctr1-1 having much weaker kinase activity than ctr1^btk (Kieber et al., 1993; Huang et al., 2003; Ikeda et al., 2009). The ctr1-8 mutation results in the G354E substitution that prevents the ETR1-CTR1 association, and the mutant exhibits a constitutive ethylene-response phenotype. Overexpression of the CTR1 N terminus CTR1^7-560, which is responsible for interaction with ethylene receptors, leads to constitutive ethylene responses, possibly by titrating out available ethylene receptors (Kieber et al., 1993; Huang et al., 2003). These studies suggest that CTR1 kinase activity and the interaction of CTR1 with the receptor HK domain may be important to the ethylene receptor signal output in suppressing constitutive ethylene responses.Although the ETR1-CTR1 interaction via the HK domain is essential to the ethylene receptor signal output, evidence suggests that the ETR1 receptor signal output can also be independent of the HK activity or domain. The etr1 ers1 loss-of-function mutant displays extreme growth defects. The etr1[HGG] mutation inactivates ETR1 HK activity, and expression of the getr1[HGG] transgene rescues the etr1 ers1 growth defects, which indicates a lack of association of ETR1 receptor signaling and its kinase activity (Wang et al., 2003). The dominant etr1-1 mutation results in the C65Y substitution and confers ethylene insensitivity (Chang et al., 1993), and the expression of the HK domain-lacking etr1^1-349 and ethylene-insensitive etr1-1^1-349 isoforms partially suppresses the growth defects of etr1 ers1-2. Loss-of-function mutations of subfamily II members do not affect etr1-1^1-349 functions. Therefore, etr1-1^1-349 predominantly cooperates with subfamily I receptors to mediate the ethylene receptor signal output (Xie et al., 2006). Biochemical and transformation studies showing that ethylene receptors can form heterodimers and that each receptor is a component of high-molecular-mass complexes explain how ethylene receptors may act cooperatively (Gao et al., 2008; Gao and Schaller, 2009; Chen et al., 2010).Reversion To Ethylene Sensitivity1 (RTE1), a Golgi/endoplasmic reticulum protein, was isolated from a suppressor screen of the dominant ethylene-insensitive etr1-2 mutation. The cross-species complementation of the rte1-2 loss-of-function mutation by the rice (Oryza sativa) RTE Homolog1 (OsRTH1) suggests a conserved mechanism that modulates the ethylene receptor signaling across higher plant species (Zhang et al., 2012). RTE1 and OsRTH1 overexpression led to ethylene insensitivity in wild-type Arabidopsis but not the etr1-7 loss-of-function mutant, and expression of etr1^1-349 restored ethylene insensitivity with RTE1 overexpression in etr1-7 (Resnick et al., 2006; Zhou et al., 2007; Zhang et al., 2010). Coimmunoprecipitation of epitope-tagged ETR1 and RTE1 and Trp fluorescence spectroscopy revealed the physical interaction of RTE1 and ETR1 (Zhou et al., 2007; Dong et al., 2008, 2010). Therefore, RTE1 may directly promote ETR1 receptor signal output through the ETR1 N terminus, but whether RTE1 has an essential role in ETR1 N-terminal signaling remains to be addressed.Currently, the biochemical nature of the ethylene receptor signal is unknown, and the underlying mechanisms of mediation of the ethylene receptor signal output remain uninvestigated. Genetic and biochemical studies suggest that activation of CTR1 by ethylene receptors may suppress constitutive ethylene responses; upon ethylene binding, the receptors are converted to an inactive state and fail to activate CTR1, and the suppression of ethylene responses by CTR1 is alleviated (Hua and Meyerowitz, 1998; Klee, 2004; Wang et al., 2006; Hall et al., 2007). However, this model does not address how the ETR1 N terminus, which does not have the CTR1-interacting site, mediates the receptor signal to suppress constitutive ethylene responses. The receptor signal of the truncated etr1 isoforms may be mediated by other full-length ethylene receptors and then activate CTR1; alternatively, the ETR1 N-terminal signal may be mediated by a pathway independent of CTR1 (Gamble et al., 2002; Qu and Schaller, 2004; Xie et al., 2006). Results showing that mutants defective in multiple ethylene receptor genes exhibit a more severe ethylene-response phenotype than ctr1 and that ctr1 mutants are responsive to ethylene support the presence of a CTR1-independent pathway (Hua and Meyerowitz, 1998; Cancel and Larsen, 2002; Huang et al., 2003; Liu et al., 2010).In this study, we investigated whether mediation of ETR1 N-terminal signaling is independent of CTR1 and whether RTE1 is essential to the CTR1-independent ETR1 N-terminal signaling. The ETR1 N-terminal signaling was not mediated via other full-length ethylene receptors, but the signal of full-length ethylene receptors could be mediated by the ETR1 N terminus independent of CTR1. The ETR1 C terminus may inhibit ETR1 N-terminal signaling, whereby deletion of the C terminus facilitates N-terminal signaling. We propose a model for the possible modulation of ETR1 receptor signaling.     

Analysis of the functional conservation of ethylene receptors between maize and Arabidopsis

Ethylene, a regulator of plant growth and development, is perceived by specific receptors that act as negative regulators of the ethylene response. Five ethylene receptors, i.e., ETR1, ERS1, EIN4, ETR2, and ERS2, are present in Arabidopsis and dominant negative mutants of each that confer ethylene insensitivity have been reported. In contrast, maize contains just two types of ethylene receptors: ZmERS1, encoded by ZmERS1a and ZmERS1b, and ZmETR2, encoded by ZmETR2a and ZmETR2b. In this study, we introduced a Cys to Tyr mutation in the transmembrane domain of ZmERS1b and ZmETR2b that is present in the etr1-1 dominant negative mutant and expressed each protein in Arabidopsis. Mutant Zmers1b and Zmetr2b receptors conferred ethylene insensitivity and Arabidopsis expressing Zmers1b or Zmetr2b were larger and exhibited a delay in leaf senescence characteristic of ethylene insensitive Arabidopsis mutants. Zmers1b and Zmetr2b were dominant and functioned equally well in a hemizygous or homozygous state. Expression of the Zmers1b N-terminal transmembrane domain was sufficient to exert dominance over endogenous Arabidopsis ethylene receptors whereas the Zmetr2b N-terminal domain failed to do so. Neither Zmers1b nor Zmetr2b functioned in the absence of subfamily 1 ethylene receptors, i.e., ETR1 and ERS1. These results suggest that Cys65 in maize ZmERS1b and ZmETR2b plays the same role that it does in Arabidopsis receptors. Moreover, the results demonstrate that the mutant maize ethylene receptors are functionally dependent on subfamily 1 ethylene receptors in Arabidopsis, indicating substantial functional conservation between maize and Arabidopsis ethylene receptors despite their sequence divergence.     

A strong constitutive ethylene-response phenotype conferred on Arabidopsis plants containing null mutations in the ethylene receptors <Emphasis Type="Italic">ETR1</Emphasis> and <Emphasis Type="Italic">ERS1</Emphasis>

Background  

The ethylene receptor family of Arabidopsis consists of five members, falling into two subfamilies. Subfamily 1 is composed of ETR1 and ERS1, and subfamily 2 is composed of ETR2, ERS2, and EIN4. Although mutations have been isolated in the genes encoding all five family members, the only previous insertion allele of ERS1 (ers1-2) is a partial loss-of-function mutation based on our analysis. The purpose of this study was to determine the extent of signaling mediated by subfamily-1 ethylene receptors through isolation and characterization of null mutations.     

Adenosine receptor expression in rheumatoid synovium: a basis for methotrexate action

Introduction

Methotrexate (MTX) exerts at least part of its anti-inflammatory effects through adenosine receptors (ADOR). The aims of this study were to determine the expression of all four adenosine receptor genes (ADORA₁, ADORA_2A, ADORA_2B, ADORA₃ and ADORA_3variant) in rheumatoid synovial tissue and any influence of MTX exposure on this expression. Furthermore, we investigated whether polymorphisms within ADORA₃ were associated with response and/or adverse effects associated with MTX.

Methods

Adenosine receptor gene expression was undertaken using PCR in 20 rheumatoid arthritis (RA) synovial samples. A separate cohort of 225 RA patients receiving MTX was genotyped for SNPs in the ADORA₃ receptor gene. Double immunofluorescence was used to identify cells expressing ADOR protein.

Results

All ADOR genes were expressed in all synovial samples. ADORA₃ and A_3variant were the dominant subtypes expressed irrespective of MTX therapy. Expression of ADORA_2A and ADORA_2B was increased in patients receiving MTX compared to those not receiving MTX. There was no association between the ADORA₃ rs1544224 SNP and high and low disease activity or MTX-associated adverse effects. ADORA_2B protein expression was most obvious in vascular endothelial cells whereas ADORA₃ protein was more abundant and expressed by synovial fibroblasts.

Conclusions

We have shown that adenosine receptors are expressed in RA synovium. There is differential expression of receptors such that ADORA₃ is expressed at significantly higher levels. This evidence demonstrates the potential for MTX to exert its anti-inflammatory effects at the primary site of pathology within the joints of patients with RA.     

Ethylene-Stimulated Nutations Do Not Require ETR1 Receptor Histidine Kinase Activity

Ethylene influences the growth and development of plants through the action of receptors that have homology to bacterial two-component receptors. In bacteria these receptors function via autophosphorylation of a His residue in the kinase domain followed by phosphotransfer to a conserved Asp residue in a response regulator protein. In Arabidopsis, two of the five receptor isoforms are capable of His kinase activity. However, the role of His kinase activity and phosphotransfer is unclear in ethylene signaling. A previous study showed that ethylene stimulates nutations of the hypocotyl in etiolated Arabidopsis seedlings that are dependent on the ETR1 receptor isoform. The ETR1 receptor is the only isoform in Arabidopsis that contains both a functional His kinase domain and a receiver domain for phosphotransfer. Therefore, we examined the role that ETR1 His kinase activity and phosphotransfer plays in ethylene-stimulated nutations.Key Words: ethylene, nutations, signal transduction, receptors, histidine kinase, phosphotransfer, two component signallingThe gaseous plant hormone ethylene has a role in a variety of physiological events in higher plants such as seed germination, abscission, senescence, fruit ripening, and growth regulation.¹ In etiolated Arabidopsis seedlings, ethylene causes reduced growth of the hypocotyl and root, increased radial expansion of the hypocotyl, and increased tightening of the apical hook.^2,3Previous studies have identified components in the ethylene signaling pathway and led to an inverse-agonist model for signal transduction.^4,5 According to this model, responses to ethylene are mediated by a family of five receptors (ETR1, ERS1, ETR2, EIN4, ERS2) in Arabidopsis that have homology to bacterial two-component receptors.^6–9 In bacterial systems, two-component receptors transduce signal via the autophosphorylation of a His residue in the kinase domain, followed by the transfer of phosphate to a conserved Asp residue in the receiver domain of a response regulator protein.¹⁰ The ethylene receptors of plants can be divided into two subfamilies based on sequence homology in the ethylene-binding domains.¹¹ ETR1 and ERS1 belong to subfamily I, contain all amino acid residues needed for His kinase activity,^6,12 and show His kinase activity in vitro.^13,14 ETR2, EIN4, and ERS2 belong to subfamily II, contain degenerate His kinase domains^7,9 and have Ser/Thr kinase activity in vitro.¹⁴ ERS1 shows both His and Ser/Thr kinase activities in vitro depending on the assay conditions used.¹⁴ While the kinase domain of ETR1 appears to be required for signaling,¹⁵ kinase activity is not.^15–17 It is unclear whether or not histidine kinase activity is involved in ethylene signaling, although, this activity might be involved in growth recovery after ethylene removal.¹⁷Recently, high-resolution, time-lapse imaging revealed that prolonged treatment with ethylene stimulates nutational bending of etiolated Arabidopsis hypocotyls.¹⁸ Nutations are oscillatory bending movements caused by localized differential growth¹⁹ that were originally termed “circumnutations”.²⁰ Nutations have been posited to be important for seedlings to penetrate through the soil²⁰ and thus could be critical for seedling survival. In support of this hypothesis, nutations of rice roots have been reported to increase soil penetration.²¹Mutational analysis revealed that many of the known ethylene signaling components including CTR1, EIN2, EIN3 and EIL1 are involved in signaling leading to ethylene-stimulated nutations.¹⁸ Surprisingly, loss-of-function mutations in ETR1 eliminated ethylene-stimulated nutations while combinatorial loss-of-function mutations in the other four receptor isoforms led to constitutive nutations in air.¹⁸ These results support a model where all the receptors are involved in ethylene-stimulated nutations but the ETR1 receptor is required for and has a contrasting role from the other receptor isoforms in this nutation phenotype. Since the ETR1 receptor is the only receptor isoform that contains both a functional His-kinase domain and a receiver domain,^6,13,14 the roles of His kinase activity and phosphorelay in the nutation phenotype were examined in the current study.Previous work showed that the nutation phenotype in etr1-7 loss-of-function mutants could be rescued with a wild-type, genomic ETR1 transgene.¹⁸ Etr1-7 mutants transformed with a kinase-inactivated genomic ETR1 transgene (gETR1 (G₂)) where the two conserved glycines in the G₂ box of the histidine kinase domain (G545, G547) were changed to alanines were examined to determine if ETR1 His kinase activity is required for ethylene-stimulated nutations. This construct lacks histidine autophosphorylation in vitro.²² Figure 1 shows that ethylene stimulates nutations in etr1-7 gETR1(G₂) seedlings. The period of these nutations was 4.7 ± 1.5 h which is similar to values obtained previously for wild-type seedlings (4.7 ± 1h) and somewhat longer than etr1-7 seedlings transformed with wild-type, genomic ETR1 (3.2 ± 0.6 h). However, the amplitude of these nutations (3.7 ± 1.0°) was approximately half that of nutations previously observed in wild-type seedlings (9.1 ± 6.0°) as well as etr1-7 seedlings transformed with wild-type, genomic ETR1 (8.2 ± 3.6°). This suggests that ETR1 histidine kinase activity is not required for ethylene-stimulated nutations but might have a role in modulating nutation amplitudes.Open in a separate windowFigure 1Ethylene stimulates nutations of etr1-7 seedlings transformed with a kinase-inactivated ETR1 transgene. The hypocotyl angles for four etr1-7 mutants transformed with a kinase-inactivated genomic ETR1 transgene (gETR1(G₂)) are shown. Transformants were obtained from Eric Schaller and have been described previously.²² In this and the following figure, etiolated Arabidopsis seedlings were imaged from the side at 15 min intervals while growing along a vertically orientated agar plate and the hypocotyl angle measured as described previously.¹⁸ Black and gray lines are used to help distinguish the movements of individual seedlings. All seedlings were grown in the presence of 5 µM AVG to block biosynthesis of ethylene by the seedlings. Seedlings were grown in air for 2 h prior to treatment with 10 µL L⁻¹ ethylene (Open in a separate window).To determine whether phosphotransfer through the receiver domain of ETR1 is required for the nutation phenotype, seedlings deficient in ethylene receptor isoforms containing a receiver domain (ETR1, ETR2, EIN4) were transformed with a mutant ETR1 transgene lacking the conserved Asp₆₅₉ required for phosphotransfer (getr1-[D]). Previous work showed that etr1-6 etr2-3 ein4-4 triple loss-of-function mutant seedlings failed to nutate and this nutation phenotype could be rescued when these mutants were transformed with wild-type, genomic ETR1 transgene.¹⁸ Similarly, transformation of the etr1-6 etr2-3 ein4-4 triple mutants with getr1-[D] rescued the nutation phenotype in most seedlings observed (Fig. 2). However, some seedlings (four of the eleven observed) failed to nutate. The reason for this variable rescue is unclear but could reflect differences in expression levels of the mutant transgene in individual plants. Alternatively, this variable rescue could reflect functional differences between the mutant and wild-type transgene suggesting a modulating role for phosphotransfer through the receiver domain of ETR1. Two independent lines were observed with similar results. Of those that did nutate, the period of nutations was 5.0 ± 1.2 h and the amplitude 7.6 ± 3.8° which is similar to values obtained previously for wild-type plants as well as plants transformed with a wild-type, genomic ETR1 transgene.¹⁸Open in a separate windowFigure 2Ethylene stimulates nutations of etr1-6 etr2-3 ein4-4 seedlings transformed with an ETR1 transgene mutated at Asp₆₅₉. The hypocotyl angles from seven etr1-6 etr2-3 ein4-4 triple mutants transformed with an ETR1 transgene mutated at Asp₆₅₉ (getr1[D]) are shown in two panels. One seedling in (A) (black) had no measurable nutations while one in (B) (black) had very small nutations.Conclusions from this and the previous study are that the ETR1 receptor has a unique role in ethylene-stimulated nutations. However, this role does not require either histidine kinase activity or phosphotransfer through the receiver domain of ETR1.     

hOGG1 Ser326Cys Polymorphism and Risk of Hepatocellular Carcinoma among East Asians: A Meta-Analysis

Background

The hOGG1 gene encodes a DNA glycosylase enzyme responsible for DNA repair. The Ser326Cys polymorphism in this gene may influence its repair ability and thus plays a role in carcinogenesis. Several case-control studies have been conducted on this polymorphism and its relationship with the risk of hepatocellular carcinoma (HCC) among East Asians. However, their results are inconsistent.

Methods

We performed a meta-analysis of published case-control studies assessing the association of the hOGG1 Ser326Cys polymorphism with HCC risk among East Asians. PubMed, EMBASE, SCI, BIOSIS, CNKI and WanFang databases were searched. A random-effect model was used to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs). Analyses were conducted for additive, dominant and recessive genetic models.

Results

Eight studies were identified involving 2369 cases and 2442 controls assessing the association of the hOGG1 Ser326Cys polymorphism with HCC risk among East Asians. Applying a dominant genetic model, only in the Chinese population, the Cys allele was significantly associated with increased risk of HCC (OR 1.56, 95% CI 1.12–2.17). However, two studies influenced this finding according to sensitivity analysis. Furthermore, considerable heterogeneity and bias existed among Chinese studies.

Conclusion

There is limited evidence to support that the hOGG1 Ser326Cys polymorphism is associated with HCC risk among East Asians. Well-designed and large-sized studies are required to determine this relationship.     

Midostaurin preferentially attenuates proliferation of triple-negative breast cancer cell lines through inhibition of Aurora kinase family

Background

Breast cancer is classified into three subtypes by the expression of biomarker receptors such as hormone receptors and human epidermal growth factor receptor 2. Triple-negative breast cancer (TNBC) expresses none of these receptors and has an aggressive phenotype with a poor prognosis, which is insensitive to the drugs that target the hormone receptors and human epidermal growth factor receptor 2. It is, thus, required to develop an effective therapeutic reagent to treat TNBC.

Results

The study using a panel of 19 breast cancer cell lines revealed that midostaurin, a multi-target protein kinase inhibitor, suppresses preferentially the growth of TNBC cells comparing with non-TNBC cells. Clustering analysis of the drug activity data for the panel of cancer cell lines predicted that midostaurin shares the target with Aurora kinase inhibitors. Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells.

Conclusion

Midostaurin suppresses the proliferation of TNBC cells among the breast cancer cell lines presumably through the inhibition of the Aurora kinase family. The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0150-2) contains supplementary material, which is available to authorized users.     

The role of receptor interactions in regulating ethylene signal transduction

The phytohormone ethylene is perceived in Arabidopsis by a five-member receptor family. Earlier work has demonstrated that the basic functional unit for an ethylene receptor is a disulfide-linked homodimer. We recently reported in The Journal of Biological Chemistry that the ethylene-receptor ETR1 physically associates with other ethylene receptors through higher order interactions, suggesting the existence of receptor clusters. Here we consider the implications of such clusters upon the mechanism of ethylene signal transduction. In particular, we consider how such clustering provides a cooperative mechanism, akin to what has been found for the prokaryotic chemoreceptors, by which plant sensitivity to ethylene may be increased. In addition, we consider how the dominant ethylene insensitivity conferred by some receptor mutations, such as etr1-1, may also be propagated by interactions among members of the ethylene receptor family.Key words: ethylene, receptor, ETR1, cooperativity, ArabidopsisThe plant hormone ethylene regulates growth and development, and is perceived by a five-member family of receptors (ETR1, ERS1, ETR2, ERS2 and EIN4) in Arabidopsis.¹ Genetic analysis indicates that ethylene receptors are functionally redundant and negatively regulate ethylene responses through interactions with the Raf-like kinase CTR1.^2–5 The functional unit of an ethylene receptor in a disulfide-linked homodimer, with each homodimer capable of binding one ethylene molecule.^6,7 However, several observations suggest that propagation of the ethylene signal through the receptors is likely to involve more than just ethylene-induced changes within individual receptor homodimers. First, Arabidopsis is amazingly sensitive to ethylene and can respond to ethylene concentrations as low as 0.2 nl/L,⁸ 300-fold lower than the K_d of the receptors for ethylene, which suggests that some mechanism exists for amplifying the input signal.^7,9 Second, ethylene-insensitive mutations in the binding sites of the receptors exhibit greater dominance than would be predicted solely from a lesion within one member of the receptor family.¹⁰In our paper published in The Journal of Biological Chemistry,¹¹ we demonstrate that the Arabidopsis ethylene receptor ETR1 physically associates with other ethylene receptors through higher order interactions. Such physical interactions suggest that the receptors exist in plants as clusters, and that models for cooperative signaling previously applied to the histidine-kinaselinked chemoreceptors of bacteria may also be applicable to the evolutionarily related ethylene receptors of plants. In bacteria, the highly packed chemoreceptors are found in clusters at one or both poles of the cell.^12,13 Structural studies indicate that chemoreceptors can associate to form a ‘trimer of dimers’^14,15 and also support the possibility that domain swapping may occur to produce a large interconnected array of receptors. 16 Our studies indicate that ethylene receptors can interact through their cytosolic GAF domains, identifying one possible interface through which conformational changes could be propagated in an ethylene receptor cluster.A higher-order cooperative mechanism among the ethylene receptors may explain the high sensitivity of plants to ethylene. In this model, the ethylene receptors amplify ethylene signaling by lateral signal output. Binding of ethylene to one receptor induces the conformation change of the receptor from a tense state (T) to a relaxed state (R). This conformational change is then propagated to other empty receptors in the cluster due to their physical associations with the receptor in the R state. As a result empty receptors also adopt the relaxed state (R′), resulting in amplification of the initial signal. It should be noted here that mutational evidence supports the unbound state of the receptors (T state) as being the lower energy conformation of the receptors.¹⁷ Thus, according to this model, part of the energy from ligand binding would be used to transmit conformational changes to the neighboring receptors.An alternative model that may also explain the high sensitivity of ethylene responsiveness in plants, and one that is not necessarily incompatible with the previous model, is a conjugation model.¹⁸ Here it is hypothesized that, due to the physical proximity of the ethylene receptors, that ethylene released from one receptor then binds to another receptor rather than diffusing away. Through this conjugation mechanism, one ethylene molecule could amplify its signal by converting the conformations of multiple ethylene receptors from the ethylene-unbound state (T) to the ethylene-bound state (R). This model is based on several assumptions. One assumption is that a single ethylene molecule can bind ethylene receptors in the same cluster multiple times due to the dynamic binding of ethylene and ethylene receptor. A second assumption is that, after ethylene is released from one ethylene receptor, the recovery time for that receptor to resume the T state is longer than the time required for the released ethylene to bind to and convert another receptor from the T to the R state.Models for cooperativity need to also explain the dominant ethylene insensitivity of various mutant receptors such as etr1-1, in which a missense mutation results in a receptor incapable of binding ethylene. Several studies indicate that the etr1-1 mutant receptor acts cooperatively to affect the signal output from other wild-type receptors (i.e., the presence of the etr1-1 receptor in its T state increases the likelihood of other receptors adopting the T state).^10,11 This observation can be most readily explained if the dominant ethylene-insensitive mutations result in a receptor that requires more energy to undergo the T to R transition than do the wild-type receptors. For example, the etr1-1 mutation may increase the stability of the T form (a T′ state). There is evidence to support this possibility. The etr1-1 missense mutation results in a receptor unable to chelate a copper cofactor necessary for ethylene binding,¹⁹ but the effects of this mutation on signaling are different from wild-type receptors that lack their copper cofactor. The etr1-1 mutant receptor appears locked in its T state, whereas wild-type receptors lacking the copper cofactor appear to be in the R state.²⁰ Thus etr1-1 is truly a gain-of-function mutation that alters the conformation of the receptor in ways not necessarily predicted from just the loss of the copper cofactor.In conclusion, we have attempted here to provide models that can resolve an apparent contradiction in the cooperative signaling behavior exhibited by ethylene receptors. The high sensitivity of plants to ethylene suggest cooperative changes in which an R state can be propagated within a receptor cluster, but the dominance of the ethylene ethylene-insensitive mutant etr1-1 suggests that the T state can also be propagated within a receptor cluster. It should be born in mind, however, that ethylene signaling is mediated by multiple signaling components. The ethylene receptors regulate ethylene responses through interaction with and modulation of CTR1 kinase activity. Thus, the total kinase activity of CTR1 represents the signal output from the receptors. This situation is very similar to that of the bacterial chemoreceptors, which regulate the activity of an associated histidine kinase, and, as with the chemoreceptors, the stoichiometry of CTR1 interactions with the ethylene receptors and the means by which its kinase activity is regulated are important for the elucidation of the mechanism of ethylene signal transduction.     

Linking ethylene to nitrogen-dependent leaf longevity of grass species in a temperate steppe

Background and Aims

Leaf longevity is an important plant functional trait that often varies with soil nitrogen supply. Ethylene is a classical plant hormone involved in the control of senescence and abscission, but its role in nitrogen-dependent leaf longevity is largely unknown.

Methods

Pot and field experiments were performed to examine the effects of nitrogen addition on leaf longevity and ethylene production in two dominant plant species, Agropyron cristatum and Stipa krylovii, in a temperate steppe in northern China.

Key Results

Nitrogen addition increased leaf ethylene production and nitrogen concentration but shortened leaf longevity; the addition of cobalt chloride, an ethylene biosynthesis inhibitor, reduced leaf nitrogen concentration and increased leaf longevity. Path analysis indicated that nitrogen addition reduced leaf longevity mainly through altering leaf ethylene production.

Conclusions

These findings provide the first experimental evidence in support of the involvement of ethylene in nitrogen-induced decrease in leaf longevity.     

Decreased GABA receptor in the cerebral cortex of epileptic rats: effect of Bacopa monnieri and Bacoside-A

Abstact

Background

Gamma amino butyric acid (GABA), the principal inhibitory neurotransmitter in the cerebral cortex, maintains the inhibitory tones that counter balances neuronal excitation. When this balance is perturbed, seizures may ensue.

Methods

In the present study, alterations of the general GABA, GABA_A and GABA_B receptors in the cerebral cortex of the epileptic rat and the therapeutic application of Bacopa monnieri were investigated.

Results

Scatchard analysis of [³H]GABA, [³H]bicuculline and [³H]baclofen in the cerebral cortex of the epileptic rat showed significant decrease in B_max (P < 0.001) compared to control. Real Time PCR amplification of GABA receptor subunits such as GABA_Aά1, GABA_Aγ, GABA_Aδ, GABA_B and GAD where down regulated (P < 0.001) in epileptic rats. GABA_Aά5 subunit and Cyclic AMP responsible element binding protein were up regulated. Confocal imaging study confirmed the decreased GABA receptors in epileptic rats. Epileptic rats have deficit in radial arm and Y maze performance.

Conclusions

Bacopa monnieri and Bacoside-A treatment reverses epilepsy associated changes to near control suggesting that decreased GABA receptors in the cerebral cortex have an important role in epileptic occurrence; Bacopa monnieri and Bacoside-A have therapeutic application in epilepsy management.     

Filamin A-Hinge Region 1-EGFP: A Novel Tool for Tracking the Cellular Functions of Filamin A in Real-Time

Background

Filamin A (FLNa) is an actin-crosslinking protein necessary for stabilizing the cell surface, organizing protrusive activity and for promoting efficient cellular translocation. Recently, our group demonstrated the requirement of FLNa for the internalization of the chemokine receptor CCR2B.

Methodology and Principal Findings

In order to study the role of FLNa in vitro and in real-time, we have developed a fluorescent FLNa-EGFP construct. In this novel imaging tool, we introduced the EGFP-tag inside the flexible hinge 1 region of FLNa between two calpain cleavage sites. Our findings indicate that the FLNa-EGFP construct was correctly expressed, cleaved by calpain and colocalized with actin filaments as shown by immunostaining experiments in the human melanoma cell lines A7 (FLNa-repleted) and M2 (FLNa-deficient). In addition, scanning-electron microscopy (SEM) and micropatterning studies also provided clear evidence that the cell rigidity was restored. FLNa-EGFP allowed us to demonstrate the interaction of FLNa with the chemokine receptor CCR2B in endocytic vesicles after CCL2 ligand stimulation. Through live-cell imaging studies we show that the CCR2B receptor in Rab5-positive vesicles moves along filamin A-positive fibers.

Significance

Taken together, these results outline the functionality of the FLNa-EGFP and the importance of filamin A for receptor internalization and movement into endocytic vesicles.     

The HIV Matrix Protein p17 Promotes the Activation of Human Hepatic Stellate Cells through Interactions with CXCR2 and Syndecan-2

Background

The human immunodeficiency virus type 1 (HIV-1) p17 is a matrix protein involved in virus life''s cycle. CXCR2 and Syndecan-2, the two major coreceptors for the p17 protein, are expressed in hepatic stellate cells (HSCs), a key cell type involved in matrix deposition in liver fibrotic disorders.

Aim

In this report we have investigated the in vitro impact of p17 on HSCs transdifferentiation and function and underlying signaling pathways involved in these processes.

Methods

LX-2 cells, a human HSC line, and primary HSC were challenged with p17 and expressions of fibrogenic markers and of p17 receptors were assessed by qRT-PCR and Western blot. Downstream intracellular signaling pathways were evaluated with qRT-PCR and Western blot as well as after pre-treatment with specific pathway inhibitors.

Results

Exposure of LX2 cells to p17 increases their contractile force, reshapes the cytoskeleton fibers and upregulates the expression of transdifferentiation markers including αSMA, COL1α1 and endothelin-1 through the activation of Jak/STAT and Rho signaling pathways. These effects are lost in HSCs pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide. Confocal laser microscopy studies demonstrates that CXCR2 and syndecan-2 co-associate at the plasma membrane after exposure to p17. Immunostaining of HIV/HCV liver biopsies from co-infected patients reveals that the progression of liver fibrosis correlates with a reduced expression of CXCR2.

Conclusions

The HIV matrix protein p17 is pro-fibrogenic through its interactions both with CXCR2 and syndecan-2 on activated HSCs.     

Exogenous ethylene inhibits sprout growth in onion bulbs

Background and Aims

Exogenous ethylene has recently gained commercial interest as a sprouting inhibitor of onion bulbs. The role of ethylene in dormancy and sprouting of onions, however, is not known.

Methods

A cultivar (Allium cepa ‘Copra’) with a true period of dormancy was used. Dormant and sprouting states of onion bulbs were treated with supposedly saturating doses of ethylene or with the ethylene-action inhibitor 1-methylcyclopropene (1-MCP). Initial sprouting was determined during storage at 18 °C by monitoring leaf blade elongation in a specific size class of leaf sheaths. Changes in ATP content and sucrose synthase activity in the sprout leaves, indicators of the sprouting state, were determined. CO₂ and ethylene production of onion bulbs during storage were recorded.

Key results

Exogenous ethylene suppressed sprout growth of both dormant and already sprouting onion bulbs by inhibiting leaf blade elongation. In contrast to this growth-inhibiting effect, ethylene stimulated CO₂ production by the bulbs about 2-fold. The duration of dormancy was not significantly affected by exogenous ethylene. However, treatment of dormant bulbs with 1-MCP caused premature sprouting.

Conclusions

Exogenous ethylene proved to be a powerful inhibitor of sprout growth in onion bulbs. The dormancy breaking effect of 1-MCP indicates a regulatory role of endogenous ethylene in onion bulb dormancy.Key words: Bulb dormancy, Allium cepa, onion, sprout growth, ethylene, CO₂ production, respiration, 1-methylcyclopropene     

The Role of Siglec-1 and SR-BI Interaction in the Phagocytosis of Oxidized Low Density Lipoprotein by Macrophages

Background

Macrophages play a proatherosclerotic role in atherosclerosis via oxLDL uptake. As an adhesion molecular of I-type lectins, Siglec-1 is highly expressed on circulating monocytes and plaque macrophages of atherosclerotic patients, but the exact role of Siglec-1 has not been elucidated.

Methods

In this study, oxLDL was used to stimulate Siglec-1 and some oxLDL receptors (SR-BI, CD64, CD32B, LOX-1 and TLR-4) expression on bone marrow-derived macrophages, whereas small interfering RNA was used to down-regulate Siglec-1. Meanwhile, an ELISA-based assay for Siglec-1-oxLDL interaction was performed, and co-immunoprecipitation (co-IP) and laser scanning confocal microscopy (LSCM) were used to determine the role of Siglec-1 in oxLDL uptake by macrophages.

Results

We found that oxLDL could up-regulate the expression of various potential oxLDL receptors, including Siglec-1, in a dose-dependent manner. Moreover, down-regulation of Siglec-1 could attenuate oxLDL uptake by Oil red O staining. LSCM revealed that Siglec-1 and CD64/SR-BI may colocalize on oxLDL-stimulated macrophage surface, whereas co-IP showed that Siglec-1 and SR-BI can be immunoprecipitated by each other. However, no direct interaction between Siglec-1 and oxLDL was found in the in vitro protein interaction system.

Conclusions

Thus, Siglec-1 can interact with SR-BI in the phagocytosis of oxLDL by macrophages, rather than act as an independent receptor for oxLDL.     

The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis

Background

GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation.

Aims

To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis.

Methods

Colitis was induced in wild type and GP-BAR1^−/− mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies.

Results

GP-BAR1^−/− mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn''s disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1.

Conclusions

GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn''s disease. Ciprofloxacin is a GP-BAR1 ligand.