Spatial expression and characterization of a putative ethylene receptor protein NTHK1 in tobacco

A putative ethylene receptor gene NTHK1 encodes a protein with a putative signal peptide, three transmembrane segments, a putative histidine kinase domain and a putative receiver domain. The receiver domain was expressed in an Escherichia coli expression system, purified and used to generate polyclonal antibodies for immunohistochemistry analysis. The spatial expression of the NTHK1 protein was then investigated. We found that NTHK1 was abundant during flower and ovule development. It was also expressed in glandular hairs, stem, and in leaves that had been wounded. The NTHK1 gene was further introduced into the tobacco plant and we found that, in different transgenic lines, the NTHK1 gene was transcribed to various degrees. Upon ACC treatment, the etiolated transgenic seedlings showed reduced ethylene sensitivity when compared with the control, indicating that NTHK1 is a functional ethylene receptor in plants.     

烟草乙烯受体类似物NTHK2全长基因的克隆及其激酶结构域生化特性

应用5＇-RACE方法克隆到烟草NTHK2的全长cDNA.其全长cDNA共有3 216bp,其中5'非编码区为509bp,3＇非编码区为427bp,编码区为2 280bp,编码产物为760个氨基酸.NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性,具有推测的组氨酸激酶结构域和接受域;但是,在激酶结构域中没有保守的组氨酸,而是被一个天冬氨酸残基所替代.为了研究其生化特性,在酵母中以融合蛋白的形式表达了激酶结构域.体外激酶分析表明,当有Mg2+存在的情况下NTHK2能够自我磷酸化.进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体,参与乙烯的信号传导过程.     

烟草乙烯受体类似物NTHK2全长基因的克隆及其激酶结构域生化特性

应用5′-ARCE方法克隆到烟草NTHK2的全长cDNA。其全长cDNA共有3216bp,其中5′非编码区为509bp,3′非编码区为427bp,编码区为2280bp,编码产物为760个氨基酸。NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性,具有推测的组氨酸激酶结构域和接受域。但是,在激酶结构域中没有保守的组氨酸,而是被一个天冬氨酸残基所替代。为了研究其生化特性,在酵母中以融合蛋白的形式表达了激酶结构域,体外激酶分析表明,当有Mg^2 存在的情况下NTHK2能够自我磷酸化。进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体。参与乙烯的信号传导过程。     

Expression of tobacco ethylene receptor NTHK1 alters plant responses to salt stress

Ethylene has been regarded as a stress hormone involved in many stress responses. However, ethylene receptors have not been studied for the roles they played under salt stress condition. Previously, we characterized an ethylene receptor gene NTHK1 from tobacco, and found that NTHK1 is salt-inducible. Here, we report a further investigation towards the function of NTHK1 in response to salt stress by using a transgenic approach. We found that NTHK1 promotes leaf growth in the transgenic tobacco seedlings but affects salt sensitivity in these transgenic seedlings under salt stress condition. Differential Na+/K+ ratio was observed in the control Xanthi and NTHK1-transgenic plants after salt stress treatment. We further found that the NTHK1 transgene is also salt-inducible in the transgenic plants, and the higher NTHK1 expression results in early inductions of the ACC (1-aminocyclopropane-1-carboxylic acid) oxidase gene NtACO3 and ethylene responsive factor (ERF) genes NtERF1 and NtERF4 under salt stress. However, NTHK1 suppresses the salt-inducible expression of the ACC synthase gene NtACS1. These results indicate that NTHK1 regulates salt stress responses by affecting ion accumulation and related gene expressions, and hence have significance in elucidation of ethylene receptor functions during stress signal transduction.     

Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco

A histidine kinase-based signaling system has been proposed to function in ethylene signal transduction pathway of plants and one ethylene receptor has been found to possess His kinase activity. Here we demonstrate that a His kinase-like ethylene receptor homologue NTHK1 from tobacco has serine/threonine (Ser/Thr) kinase activity, but no His kinase activity. Evidence obtained by analyzing acid/base stability, phosphoamino acid and substrate specificity of the phosphorylated kinase domain, supports this conclusion. In addition, mutation of the presumptive phosphorylation site His (H378) to Gln did not affect the kinase activity whereas deletion of the ATP-binding domain eliminated it, indicating that the conserved His (H378) is not required for the kinase activity and this activity is intrinsic to the NTHK1-KD. Moreover, confocal analysis of NTHK1 expression in insect cells and plant cells suggested the plasma membrane localization of the NTHK1 protein. Thus, NTHK1 may represent a distinct Ser/Thr kinase-type ethylene receptor and function in an alternative mechanism for ethylene signal transduction.     

Evidence for serine/threonine and histidine kinase activity in the tobacco ethylene receptor protein NTHK2

Ethylene plays important roles in plant growth, development, and stress responses. Two ethylene receptors, ETR1 from Arabidopsis and NTHK1 from tobacco (Nicotiana tabacum), have been found to have His kinase (HK) activity and Ser/Thr kinase activity, respectively, although both show similarity to bacterial two-component HK. Here, we report the characterization of another ethylene receptor homolog gene, NTHK2, from tobacco. This gene also encodes a HK-like protein and is induced by dehydration and CaCl(2) but not significantly affected by NaCl and abscisic acid treatments. The biochemical properties of the yeast (Schizosaccharomyces pombe)-expressed NTHK2 domains were further characterized. We found that NTHK2 possessed Ser/Thr kinase activity in the presence of Mn(2+) and had HK activity in the presence of Ca(2+). Several lines of evidence supported this conclusion, including hydrolytic stability, phosphoamino acid analysis, mutation, deletion, and substrate analysis. These properties have implications in elucidation of the complexity of the ethylene signal transduction pathway and understanding of ethylene functions in plants.     

Characterization of an ethylene receptor homolog gene from rice

Ethylene is a gaseous hormone and plays important roles in plant growth and development, including seed germination, root hair development, flowering, pollination, abscission, and fruit ripening[1]. It is also involved in plant responses to biotic stress such as pathogen attack, and abiotic stresses such as wounding, drought and freezing[1]. Mutational and genetic analysis of Arabidopsis has led to the identification of five ethylene receptor genes, i.e. ETR1, ERS1, ETR2, EIN4 and ERS2. …     

Characterization of an ethylene receptor homolog gene from rice

Ethylene plays important roles in plant growth, development and stress responses. Its receptor genes have been studied in dicots such asArabidopsis, tobacco and tomato. However, no research has been reported for the ethylene receptors from monocots currently. In the present study, we cloned an ethylene receptor geneOSPK2 from rice and found that its encoded protein was divergent from the ethylene receptors from dicots. OSPK2 had a long extension in its N-terminal, followed by three transmembrane segments, a GAF domain, a putative kinase domain and a putative receiver domain. Although most of the domains were conserved, the expected phosphorylation site His and the phosphate receiver Asp have been replaced by Gln and Asn, respectively. This fact indicates that OSPK2 may not function as a histidine kinase in a phosphorelay manner, but rather play roles by other mechanism, probably through Ser/Thr kinase activity. The expression of theOSPK2 gene was investigated by RT-PCR method under different conditions. We found that this gene was apparently induced by wounding and PEG treatment, but not significantly affected by salt and ABA treatments. The differential expression of theOSPK2 gene may reflect its roles in mediating different abiotic stress responses, consistent with our previous studies on tobacco ethylene receptors.     

烟草乙烯受体研究进展(综述)

对近几年有关烟草乙烯受体基因研究的最新进展作简要介绍,并就今后该领域的研究方向进行探讨。已知烟草乙烯受体家族至少包括NtETR1、NtERS1、NTHK1和NTHK2等4种基因,其中NTHK1和NTHK2同源且有相似结构,两者的激酶活性与细菌双组分调节系统非常相似,激酶活性需要一些二价阳离子的参与。烟草乙烯受体在细胞内的作用位点还缺少研究。     

A two-component gene (NTHK1) encoding a putative ethylene-receptor homolog is both developmentally and stress regulated in tobacco

The full-length of a two-component gene NTHK1 (Nicotiana tabacum histidine kinase-l) was isolated from tobacco (N. tabacum var. Xanthi) using a previously obtained NTHK1 cDNA fragment as a probe. Sequence analysis revealed that NTHK1 shared high homology with LeETR4 from tomato and encoded an ethylene- receptor homolog. The predicted NTHK1 protein had a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. The putative autophosphorylation site at His378 and the phosphate receiver site at Asp689 were also identified. By using the in situ hybridization technique, NTHK1 mRNA was detected during flower organ development. It is also highly expressed in the processes of pollen formation and embryo development. The expression of NTHK1 in response to wounding and other stresses was investigated using competitive RT-PCR. The results demonstrated that NTHK1 was inducible upon wounding (cutting). Floating of the cut leaf pieces in 0.5× MS, with shaking, led to a relatively rapid and strong expression. This phenomenon was confirmed by the in situ hybridization results. In addition to the up-regulation by wounding, NTHK1 expression was also induced following NaCl and PEG treatment, indicating a possible role for NTHK1 in multiple stress responses. Received: 28 June 2000 / Accepted: 1 August 2000     

Structural Model of the Cytosolic Domain of the Plant Ethylene Receptor 1 (ETR1)

Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.     

A kinase subdomain of transforming growth factor-beta (TGF-beta) type I receptor determines the TGF-beta intracellular signaling specificity.

Transforming growth factor-beta (TGF-beta) signals through a heteromeric complex of related type I and type II serine/threonine kinase receptors. In Mv1Lu cells the type I receptor TbetaRI mediates TGF-beta-induced gene expression and growth inhibition, while the closely related type I receptors Tsk7L and TSR1 are inactive in these responses. Using chimeras between TbetaRI and Tsk7L or TSR1, we have defined the structural requirements for TGF-beta signaling by TbetaRI. The extracellular/transmembrane or cytoplasmic domains of TbetaRI and Tsk7L were functionally not equivalent. The juxtamembrane domain, including the GS motif, and most regions in the kinase domain can functionally substitute for each other, but the alphaC-beta4-beta5 region from kinase subdomains III to V conferred a distinct signaling ability. Replacement of this sequence in TbetaRI by the corresponding domain of Tsk7L inactivated TGF-beta signaling, whereas its introduction into Tsk7L conferred TGF-beta signaling. The differential signaling associated with this region was narrowed down to a sequence of eight amino acids, the L45 loop, which is exposed in the three-dimensional kinase structure and diverges highly between TbetaRI and Tsk7L or TSR1. Replacement of the L45 sequence in Tsk7L with that of TbetaRI conferred TGF-beta responsiveness to the Tsk7L cytoplasmic domain in Mv1Lu cells. Thus, the L45 sequence between kinase subdomains IV and V specifies TGF-beta responsiveness of the type I receptor.     

The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus

In Myxococcus xanthus the extracellular matrix is essential for type IV pili-dependent motility and starvation-induced fruiting body formation. Proteins of two-component systems including the orphan DNA binding response regulator DigR are essential in regulating the composition of the extracellular matrix. We identify the orphan hybrid histidine kinase SgmT as the partner kinase of DigR. In addition to kinase and receiver domains, SgmT consists of an N-terminal GAF domain and a C-terminal GGDEF domain. The GAF domain is the primary sensor domain. The GGDEF domain binds the second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP) and functions as a c-di-GMP receptor to spatially sequester SgmT. We identify the DigR binding site in the promoter of the fibA gene, which encodes an abundant extracellular matrix metalloprotease. Whole-genome expression profiling experiments in combination with the identified DigR binding site allowed the identification of the DigR regulon and suggests that SgmT/DigR regulates the expression of genes for secreted proteins and enzymes involved in secondary metabolite synthesis. We suggest that SgmT/DigR regulates extracellular matrix composition and that SgmT activity is regulated by two sensor domains with ligand binding to the GAF domain resulting in SgmT activation and c-di-GMP binding to the GGDEF domain resulting in spatial sequestration of SgmT.     

植物抗病基因同源序列及其在抗病基因克隆与定位中的应用

近10年来已有20多个植物抗病基因被克隆,测序,这些抗病基因所编码的蛋白中大多含有核苷酸结合位点,富含亮氨酸重复序列,蛋白激酶,亮氨酸拉链结构,跨膜结构域,Toll白介素－1区域等保守结构域。利用这些保守结构域合成PCR引物,已扩增出大量的植物抗病基因同源序列（RGA）。对RGA与抗病基因的关系进行了分析,讨论了RGA在研究抗病基因进化中的作用,指出RGA在抗病基因定位和转基因中具有重要意义。     

Characterization of the tandem GAF domain of human phosphodiesterase 5 using a cyanobacterial adenylyl cyclase as a reporter enzyme

We analyzed cGMP signaling by the human phosphodiesterase 5 (hPDE5) tandem GAF domain based on a functional activation assay. The C-terminal catalytic domain of the cyanobacterial adenylyl cyclase (AC) cyaB1 was used as a reporter enzyme. We demonstrate functional coupling between the hPDE5 GAF ensemble and the AC resulting in a chimera stimulated 10-fold by cGMP. The hPDE5 GAF domain has an inhibitory effect on AC activity, which is released upon cGMP activation. Removal of 109 amino acids from the N terminus resulted in partial disengagement of the GAF domain and AC, i.e. in a 10-fold increase in basal activity, and affected cGMP affinity. The Ser-102 phosphorylation site of hPDE5 increased cGMP affinity, as shown by a 5-fold lower K(D) for cGMP in a S102D mutant, which mimicked complete modification. The function of the NKFDE motif, which is a signature of all GAF domains with known cyclic nucleotide binding capacity, was elucidated by targeted mutations. Data with either single and double mutants in either GAF A or GAF B or a quadruple mutant affecting both subdomains simultaneously indicated that it is impossible to functionally assign cGMP binding and intramolecular signaling to either GAF A or B of hPDE5. Both subdomains are structurally and functionally interdependent and act in concert in regulating cycaB1 AC and, most likely, also hPDE5.     

Changes in purine specificity in tandem GAF chimeras from cyanobacterial cyaB1 adenylate cyclase and rat phosphodiesterase 2

The C-terminal catalytic domains of the 11 mammalian phosphodiesterase families (PDEs) are important drug targets. Five of the 11 PDE families contain less well-characterized N-terminal GAF domains. cGMP is the ligand for the GAF domains in PDEs 2, 5, 6 and 11, and cAMP is the ligand for PDE10. Structurally related tandem GAF domains signalling via cAMP are present in the cyanobacterial adenylate cyclases cyaB1 and cyaB2. Because current high-resolution crystal structures of the tandem GAF domains of PDE2 and cyaB2 do not reveal how cNMP specificity is encoded, we generated chimeras between the tandem GAF domains of cyaB1 and PDE2. Both bind the ligand in the GAF B subdomains. Segmental replacements in the highly divergent beta1-beta3 region of the GAF B subdomain of cyaB1 by the corresponding PDE2 regions switched signalling from cAMP to cGMP. Using 10 chimeric constructs, we demonstrated that, for this switch in purine specificity, only 11% of the sequence of the cyanobacterial GAF B needs to be replaced by PDE2 sequences. We were unable, however, to switch the purine specificity of the PDE2 tandem GAF domain from cGMP to cAMP in reverse constructs, i.e. by replacement of PDE2 segments with those from the cyaB1 GAF tandem domain. The data provide a novel view on the structure-function relationships underlying the purine specificity of cNMP-binding GAF domains and indicate that, as potential drug targets, they must be characterized structurally and biochemically one by one.