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.     

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.     

Roles of ethylene receptor NTHK1 domains in plant growth, stress response and protein phosphorylation

Ethylene receptors sense ethylene and regulate downstream signaling events. Tobacco ethylene receptor NTHK1, possessing Ser/Thr kinase activity, has been found to function in plant growth and salt-stress responses. NTHK1 contains transmembrane domains, a GAF domain, a kinase domain and a receiver domain. We examined roles of these domains in regulation of plant leaf growth, salt-stress responses and salt-responsive gene expressions using an overexpression approach. We found that the transgenic Arabidopsis plants harboring the transmembrane domain plus kinase domain exhibited large rosettes, had reduction in ethylene sensitivity, and showed enhanced salt sensitivity. The transgenic plants harboring the transmembrane domain plus GAF domain also showed larger rosettes. Truncations of NTHK1 affected salt-induced gene expressions. Transmembrane domain plus kinase domain promoted RD21A and VSP2 expression but decreased salt-induction of AtNAC2. The kinase domain itself promoted AtERF4 gene expression. The GAF domain itself enhanced Cor6.6 induction. Moreover, the NTHK1 functional kinase domain phosphorylated the HIS and ATP subdomains, and five putative phosphorylation sites were identified in these two subdomains. In addition, the salt-responsive element of the NTHK1 gene was in the transmembrane-coding region but not in the promoter region. These results indicate that NTHK1 domains or combination of them have specific functions in plant leaf growth, salt-stress response, gene expression and protein phosphorylation.     

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。     

Regulation of Ethylene Biosynthesis Under Salt Stress in Red Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) by 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-producing Halotolerant Bacteria

The present study was carried out to understand the mechanism of salt stress amelioration in red pepper plants by inoculation of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing halotolerant bacteria. In general, ethylene production, ACC concentration, ACC synthase (ACS), and ACC oxidase (ACO) enzyme activities increased with increasing levels of salt stress. Treatment with halotolerant bacteria reduced ethylene production by 47–64%, ACC concentration by 47–55% and ACO activity by 18–19% in salt-stressed (150 mmol NaCl) red pepper seedlings compared to uninoculated controls. ACS activity was lower in red pepper seedlings treated with Bacillus aryabhattai RS341 but higher in seedlings treated with Brevibacterium epidermidis RS15 (44%) and Micrococcus yunnanensis RS222 (23%) under salt-stressed conditions as compared to uninoculated controls. A significant increase was recorded in red pepper plant growth under salt stress when treated with ACC deaminase-producing halotolerant bacteria as compared to uninoculated controls. The results of this study collectively suggest that salt stress enhanced ethylene production by increasing enzyme activities of the ethylene biosynthetic pathway. Inoculation with ACC deaminase-producing halotolerant bacteria plays an important role in ethylene metabolism, particularly by reducing the ACC concentration, although a direct effect on reducing ACO activity was also observed. It is suggested that growth promotion in inoculated red pepper plants under inhibitory levels of salt stress is due to ACC deaminase activity present in the halotolerant bacteria.     

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.     

Expression of Indica rice <Emphasis Type="Italic">OsBADH1</Emphasis> gene under salinity stress in transgenic tobacco

Glycine betaine has been reported as an osmoprotectant compound conferring tolerance to salinity and osmotic stresses in plants. We previously found that the expression of betaine aldehyde dehydrogenase 1 gene (OsBADH1), encoding a key enzyme for glycine betaine biosynthesis pathway, showed close correlation with salt tolerance of rice. In this study, the expression of the OsBADH1 gene in transgenic tobacco was investigated in response to salt stress using a transgenic approach. Transgenic tobacco plants expressing the OsBADH1 gene were generated under the control of a promoter from the maize ubiquitin gene. Three homozygous lines of T₂ progenies with single transgene insert were chosen for gene expression analysis. RT-PCR and western blot analysis results indicated that the OsBADH1 gene was effectively expressed in transgenic tobacco leading to the accumulation of glycine betaine. Transgenic lines demonstrated normal seed germination and morphology, and normal growth rates of seedlings under salt stress conditions. These results suggest that the OsBADH1 gene could be an excellent candidate for producing plants with osmotic stress tolerance.     

Involvement of NtERF3 in the cell death signalling pathway mediated by SIPK/WIPK and WRKY1 in tobacco plants

We previously reported that one of the ethylene response factors (ERFs), NtERF3, and other members of the subgroup VIII‐a ERFs of the AP2/ERF family exhibit cell death‐inducing ability in tobacco leaves. In this study, we focused on the involvement of NtERF3 in a cell death signalling pathway in tobacco plants, particularly downstream of NtSIPK/NtWIPK and NtWRKY1, which are mitogen‐activated protein kinases and a phosphorylation substrate of NtSIPK, respectively. An ERF‐associated amphiphilic repression (EAR) motif‐deficient NtERF3b mutant (NtERF3bΔEAR) that lacked cell death‐inducing ability suppressed the induction of cell death caused by NtERF3a. The transient co‐expression of NtERF3bΔEAR suppressed the hypersensitive reaction (HR)‐like cell death induced by NtSIPK and NtWRKY1. The induction of cell death by NtSIPK and NtWRKY1 was also inhibited in transgenic plants expressing NtERF3bΔEAR. Analysis of gene expression, ethylene production and cell death symptoms in salicylic acid‐deficient tobacco plants suggested the existence of some feedback regulation in the HR cell death signalling pathway mediated by SIPK/WIPK and WRKY1. Overall, these results suggest that NtERF3 functions downstream of NtSIPK/NtWIPK and NtWRKY1 in a cell death signalling pathway, with some feedback regulation.     

Differential expression of antisense in regenerated tobacco plants transformed with an antisense version of a tomato ACC oxidase gene

Ethylene production was measured during vegetative and reproductive development in normal tobacco plants and in transgenic tobacco plants carrying antisense genes for tomato ACC oxidase driven by the 35S CaMV promoter (Hamilton et al., 1990). When expressed in three independently derived transgenic plants, the antisense ethylene gene failed to affect ethylene production in young/mature leaves or in stems but it did inhibit ethylene production in roots by 37–58%. Ethylene production in developing flowers (i.e. from small unopened flower buds up until open flowers at anthesis) was not affected in transgenic plants but ethylene production in fruits was inhibited by 35%. The most dramatic effect on ethylene production in transgenic plants was seen immediately after wounding leaf tissue, in which case the antisense gene inhibited wound ethylene production by 72%. Thus, the antisense gene composed of a 35S CaMV promoter driving a heterologous ACC oxidase sequence had differential effects on ethylene production in tobacco plants.     

水稻受盐抑制基因OsZFP1的转基因分析

OsZFP1(水稻锌指蛋白1)基因编码的蛋白含有3个推测的Cys2/Cys2-型锌指结构域,它的表达受盐胁迫负调控。构建了以35S为启动子的OsZFP1基因的植物表达载体,并将其转入拟南芥(ArabidopsisthalianaL.)植物和水稻(OryzasativaL.)愈伤组织中以过量表达OsZFP1基因。转基因的拟南芥植株和水稻愈伤组织对盐处理的敏感性都比野生型要高。这一结果表明OsZFP1基因可能编码一种负调控蛋白,它可能抑制某些盐诱导基因的表达。在ABA处理下,转基因拟南芥植株比野生型植株抽苔晚,说明OsZFP1基因的作用可能受ABA调节。     

Inhibition of Ethylene Biosynthesis Enhances Vegetative Bud Formation without Affecting Growth and Development of Transgenic Tobacco Plants

The role of ethylene in vegetative bud formation was investigated using transgenic tobacco plants expressing an antisense tomato 1-aminocyclopropane-carboxylic acid synthase (ACS) gene. Northern blot hybridization showed that the accumulation of ACS mRNA was strongly reduced in the bud-forming leaf explants of the transgenic plants. Consequently, these transgenic tissues exhibited low ACS enzyme activity, 1-aminocyclopropane-carboxylic acid (ACC) content and ethylene production, and at the same time the tissue capacity to generate buds was greatly enhanced. However, it was also noted that the antisense ACS gene did not inhibit the endogenous ACS gene expression in intact transgenic tobacco plants. The growth and development of the transgenic tobacco was almost identical to control plants with respect to height, internode number, leaf morphology, and flowering time. Furthermore, mature leaves of transgenic tobacco had similar chlorophyll content, stomatal conductance, photosynthetic ability, and transpiration rates compared to control plants. These results demonstrated that ethylene plays an important role in bud formation in tobacco tissue culture.     

Molecular cloning and characterization of a cDNA for a novel ethylene receptor, NT-ERS1, of tobacco (Nicotiana tabacum L.)

The cDNA encoding a novel member (NT-ERS1) of ethylene receptor family of tobacco (Nicotiana tabacum L.) was obtained by a combination of RT-PCR and 5'-/3'-RACE cloning. The cDNA was 2,092 nucleotides long and had an open reading frame of 1,911 bp encoding 637 amino acids. The deduced polypeptide lacked a response regulator domain, indicating that the ethylene receptor belongs to an ERS-group. The amino acid sequence was similar to respective members of the tobacco ethylene receptor family: 67.8% to NT-ETR1, 39.1% to NTHK1 and 31.1% to NTHK2. Comparison of amino acid sequence suggested that NT-ERS1 is the counterpart of Nr in the ethylene receptor family of tomato, which belongs to Solanaceae as does tobacco. Northern blot analysis showed that mRNA of NT-ERS1 was present in leaf, shoot and root tissues, and accumulated in leaves treated with exogenous ethylene. A mutated NT-ERS1 cDNA transgene, obtained by introducing one nucleotide substitution into NT-ETR1 cDNA, conferred ethylene insensitivity in tobacco plants, indicating that the translation product of the cDNA actually functioned in the plants.     

水稻受盐抑制基因OsZFP1的转基因分析

OsZFP1(水稻锌指蛋白1)基因编码的蛋白含有3个推测的Cys2/Cys2-型锌指结构域,它的表达受盐胁迫负调控.构建了以35S为启动子的OsZFP1基因的植物表达载体,并将其转入拟南芥(Arabidopsis thaliana L.)植物和水稻(Oryza sativa L.)愈伤组织中以过量表达OsZFP1基因.转基因的拟南芥植株和水稻愈伤组织对盐处理的敏感性都比野生型要高.这一结果表明OsZFP1基因可能编码一种负调控蛋白,它可能抑制某些盐诱导基因的表达.在ABA处理下,转基因拟南芥植株比野生型植株抽苔晚,说明OsZFP1基因的作用可能受ABA调节.