Overexpression in Arabidopsis of a plasma membrane-targeting glutamate receptor from small radish increases glutamate-mediated Ca2+ influx and delays fungal infection

Ionotropic glutamate receptors (iGluRs) are ligand-gated nonselective cation channels that mediate fast excitatory neurotransmission. Although homologues of the iGluRs have been identified in higher plants, their roles are largely unknown. In this work we isolated a full-length cDNA clone (RsGluR) encoding a putative glutamate receptor from small radish. An RsGluR: mGFP fusion protein was localized to the plasma membrane. In Arabidopsis thaliana overexpressing the full-length cDNA, glutamate treatment triggered greater Ca2+ influx in the root cells of transgenic seedlings than in those of the wild type. Transgenic plants exhibited multiple morphological changes such as necrosis at their tips and the margins of developing leaves, dwarf stature with multiple secondary inflorescences, and retarded growth, as previously observed in transgenic Arabidopsis overexpressing AtGluR3.2 [Kim et al. (2001)]. Microarray analysis showed that jasmonic acid (JA)-responsive genes including defensins and JA-biosynthetic genes were up-regulated. RsGluR overexpression also inhibited growth of a necrotic fungal pathogen Botrytis cinerea possibly due to up-regulation of the defensins. Based on these results, we suggest that RsGluR is a glutamate-gated Ca2+ channel located in the plasma membrane of higher plants and plays a direct or indirect role in defense against pathogen infection by triggering JA biosynthesis.     

Overexpression of RCI2A decreases Na+ uptake and mitigates salinity-induced damages in Arabidopsis thaliana plants

We have investigated whether the overexpression of RCI2A gene causes an enhanced salt-tolerant phenotype in Arabidopsis thaliana . Although the growth of RCI2A -overexpressing transgenic plants was comparable with that of wild type under normal conditions, high salinity treatment caused decreased accumulation of Na⁺ and ameliorated suppression of the shoot growth of transgenic plants than that of wild type. Under high salinity treatment, the chlorophyll content of the shoots of wild-type plants significantly decreased compared with transgenic plants. The increases of malondialdehyde (MDA) and of H₂O₂ production caused by high salinity were greater in the shoots of wild type than in that of transgenic plants. These results suggest that overexpression of RCI2A can alleviate salinity-induced growth suppression and photooxidative damages via reducing Na⁺ uptake into the shoots.     

水稻NHX1基因启动子和C末端的调控功能研究

为了解水稻Na+/H+逆向转运蛋白(OsNHX1)在植物应答非生物胁迫中的分子调控机制,采用RT-PCR方法克隆OsNHX1基因上游2 000bp的启动子序列,并通过基因枪轰击瞬时转化洋葱表皮细胞,检测不同非生物胁迫下启动子的活性和表达模式;同时,分别克隆全长和C末端缺失的OsNHX1基因,通过花序浸染法转化拟南芥,研究OsNHX1基因及其C末端的功能。结果显示:OsNHX1启动子受逆境胁迫诱导,在盐、干旱、脱落酸胁迫处理下GUS表达活性明显升高;过表达OsNHX1的转基因拟南芥中,种子萌发率、根长、丙二醛含量和相对含水量的测定结果均显示其胁迫耐受性得到改善,但过表达OsNHX1C末端缺失基因对转基因植株的胁迫耐受性无明显影响。研究表明,Na+/H+逆向转运蛋白有助于提高植物耐盐性,且其C末端区域对该转运蛋白活性的发挥具有关键作用。     

Identification of a putative voltage-gated Ca2+ -permeable channel (OsTPC1) involved in Ca2+ influx and regulation of growth and development in rice

Cytosolic free Ca2+ serves as an important second messenger participating in signal transduction of various environmental stresses. However, molecular bases for the plasma membrane Ca2+ influx and its regulation remain largely unknown. We here identified a gene (OsTPC1) encoding a putative voltage-gated Ca2+ channel from rice, ubiquitously expressed in mature leaves, shoots and roots as well as in cultured cells. OsTPC1 rescued the Ca2+ uptake activity and growth rate of a yeast mutant cch1. To elucidate its physiological roles, we generated transgenic rice plants and cultured cells overexpressing OsTPC1 mRNA. Furthermore, a retrotransposon (Tos17) insertional knockout mutant of OsTPC1 was isolated. OsTPC1-overexpressing cells showed hypersensitivity to excess Ca2+ but higher growth rate under Ca2+ limitation, while growth of the OsTPC1-knockout cultured cells was less sensitive to extracellular free Ca2+ concentration, suggesting that OsTPC1 has Ca2+ transport activity across the plasma membrane. OsTPC1-overexpressing plants showed reduced growth and abnormal greening of roots. Growth of Ostpc1 seedlings was comparable to the control on agar plates, while significantly reduced in adult plants. These results suggest that OsTPC1 functions as a Ca2+ -permeable channel involved in the regulation of growth and development.     

Induction of enhanced disease resistance and oxidative stress tolerance by overexpression of pepper basic PR-1 gene in Arabidopsis

The pathogen- and ethylene-inducible pepper-basic pathogenesis-related (PR)-1 gene, CABPR1 , was strongly expressed in pepper leaves by osmotic and oxidative stresses. The pepper CABPR1 was introduced into the Arabidopsis plants under the control of the cauliflower mosaic virus 35S promoter. Polymerase chain reaction-amplification with the Arabidopsis genomic DNA and Northern blot analyses confirmed that the pepper CABPR1 gene was integrated into the Arabidopsis genome, where it was overexpressed in the transgenic Arabidopsis plants under normal growth conditions. The constitutive overexpression of CABPR1 induced the expression of the Arabidopsis PR-genes including PR-4 , PR-5 and PDF1.2 . Enhanced resistance to phytopathogenic bacteria, Pseudomonas syringae pv. tomato DC3000, was also observed in the transgenic Arabidopsis plants. CABPR1 overexpression in the transgenic Arabidopsis caused enhanced seed germination under NaCl (ionic) and mannitol (non-ionic) osmotic stresses. Enhanced tolerances to high salinity and dehydration stresses during seed germination of the transgenic plants were not found at the early seedling stage. The transgenic Arabidopsis plants exhibited a higher tolerance to oxidative stress by methyl viologen at the seed germination, seedling and adult plant stages. These results suggest that the CABPR1 gene may function in the enhanced disease resistance and oxidative stress tolerance of transgenic Arabidopsis plants.     

拟南芥AtUNE12基因的耐盐功能初探

bHLH转录因子家族成员在植物生长发育、生理代谢及非生物胁迫响应过程中起重要作用。本研究选取拟南芥抗逆相关bHLH转录因子家族中AtUNE12基因为研究对象,对其进行耐盐功能初探。首先构建AtUNE12基因的植物过表达载体（pROKⅡ-AtUNE12）,通过农杆菌介导的浸花法转化拟南芥,利用qRT-PCR技术检测获得T₃代AtUNE12过表达转基因植株。在盐胁迫下,分析过表达AtUNE12与野生型拟南芥长势、根长及鲜重;比较过表达AtUNE12与野生型植株的电解质渗透率、失水率、MDA含量、POD与SOD活性及H₂O₂含量,鉴定AtUNE12基因是否具有耐盐能力。结果表明：过表达AtUNE12基因降低了拟南芥植株的失水率、电解质渗透率及MDA含量,保护细胞膜结构的完整性;增强了POD与SOD活性,降低了拟南芥植株内的H₂O₂含量,进而增强拟南芥植株的ROS清除能力,从而提高拟南芥的耐盐能力。     

超表达AVP1基因提高转基因百脉根的耐盐性和抗旱性

本研究以超表达拟南芥液泡膜H＋-焦磷酸酶编码基因AVPI的转基因百脉根为材料,对其耐盐性和抗旱性进行了检测。结果显示：在200mmol·L^-1 NaCl下处理或自然干旱7d后,转基因植株的生长虽然受到抑制,但受抑程度明显低于野生型植株,前者叶片相对含水量比后者分别高18％和14％,净光合速率分别高20％和21％,而MDA含量则分别低35％和27％,相对质膜透性分别低28％和27％。此外,随着盐和干旱胁迫的加剧,与野生型植株相比,转基因植株体内积累了更多Na＋、K＋和Ca2＋。以上结果表明,AVPI基因的超表达可能提高了百脉根细胞Na＋区域化能力,既减轻了过量Na＋对细胞质的毒害作用,也提高了植株的渗透调节能力,从而增强了百脉根的耐盐性和抗旱性。     

Co-expression of the Arabidopsis SOS genes enhances salt tolerance in transgenic tall fescue (Festuca arundinacea Schreb.)

Crop productivity is greatly affected by soil salinity; therefore, improvement in salinity tolerance of crops is a major goal in salt-tolerant breeding. The Salt Overly Sensitive (SOS) signal-transduction pathway plays a key role in ion homeostasis and salt tolerance in plants. Here, we report that overexpression of Arabidopsis thaliana SOS1+SOS2+SOS3 genes enhanced salt tolerance in tall fescue. The transgenic plants displayed superior growth and accumulated less Na⁺ and more K⁺ in roots after 350 mM NaCl treatment. Moreover, Na⁺ enflux, K⁺ influx, and Ca²⁺ influx were higher in the transgenic plants than in the wild-type plants. The activities of the enzyme superoxide dismutase, peroxidase, catalase, and proline content in the transgenic plants were significantly increased; however, the malondialdehyde content decreased in transgenic plants compared to the controls. These results suggested that co-expression of A. thaliana SOS1+SOS2+SOS3 genes enhanced the salt tolerance in transgenic tall fescue.     

Development of transgenic rice plants overexpressing the Arabidopsis H+/Ca2+ antiporter CAX1 gene

The gene of the Arabidopsis thaliana H+/Ca2+ transporter, CAX1 (cation exchanger 1) was introduced into Japonica cultivars of rice (Ilpumbyeo) by Agrobacterium-mediated transformation, and a large number of transgenic plants were produced. The neomycin phosphotransferase II (NPTII) gene was used as a selectable marker. The activity of neomycin phosphotransferase could be successfully detected in transgenic rice callus. The introduction of the CAX1 gene was also proven by PCR using CAX1-specific oligonucleotide primers in regenerated plants. Stable integration and expression of the CAX1 gene in T0 plants and T1 progeny were confirmed by DNA hybridization, Northern blot analysis, and luminescent analysis.     

In planta regulation of the Arabidopsis Ca(2+)/H(+) antiporter CAX1

Vacuolar localized Ca(2+)/H(+) exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca(2+) homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. In yeast expression assays, a 36 amino acid N-terminal truncation of CAX1, termed sCAX1, and variants with specific mutations in this N-terminus, show CAX1-mediated Ca(2+)/H(+) antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca(2+) accumulation and heightened activity of vacuolar Ca(2+)/H(+) antiport. Here the properties of N-terminal CAX1 variants in plants and yeast expression systems are compared and contrasted to determine if autoinhibition of CAX1 is occurring in planta. Initially, using ionome analysis, it has been demonstrated that only yeast cells expressing activated CAX1 transporters have altered total calcium content and fluctuations in zinc and nickel. Tobacco plants expressing activated CAX1 variants displayed hypersensitivity to ion imbalances, increased calcium accumulation, heightened concentrations of other mineral nutrients such as potassium, magnesium and manganese, and increased activity of tonoplast-enriched Ca(2+)/H(+) transport. Despite high in planta gene expression, CAX1 and N-terminal variants of CAX1 which were not active in yeast, displayed none of the aforementioned phenotypes. Although several plant transporters appear to contain N-terminal autoinhibitory domains, this work is the first to document clearly N-terminal-dependent regulation of a Ca(2+) transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits.     

转AtNHX1基因杨树Tr品系的耐盐性研究

以不同盐分强度处理欧美107杨(Populus × euramericana ‘Neva’) (Wt)和转拟南芥液泡膜Na⁺/H⁺逆向转运蛋白基因AtNHX1欧美107杨新品系(Tr)幼苗, 揭示Tr和Wt两品系幼苗耐盐性的差异, 探索拟南芥液泡膜Na⁺/H⁺逆向转运蛋白基因AtNHX1对提高杨树耐盐能力的效应。结果表明: 低盐处理下, Wt植株生长明显受到抑制, 其干重显著低于对照, 盐分强度加大后, 抑制作用更大, 其干重只有对照的50%; 而Tr植株在低盐处理下干重与对照差异不显著, 高盐处理时其干重为对照的74%。同时, 不同盐度处理下, Tr的干重均显著高于Wt, 且随着盐度升高, 两品系间植株干重差异增大。盐处理后, Tr植株叶片叶绿素和类胡萝卜素的含量均显著高于Wt, 并能维持较高的净光合速率(P_n)和PSII最大光化学效率(F_v/F_m); 在盐处理下虽然Tr叶片和根系均较Wt积累了更多的Na⁺, 但同时也维持了更高的K⁺和K⁺/Na⁺比率, 而且叶片对K⁺选择性的运输明显高于Wt; 同时, Tr叶片MDA含量和电解质渗漏率显著低于Wt。可见, 在盐处理下转AtNHX1植株较未转基因植株维持了更高的生长量、光合色素、光合能力和叶片质膜稳定性, 说明AtNHX1的转入能够显著提高欧美107杨的耐盐性。     

植物适应长期缺钾——积累叶片膜脂、维持根膜脂组成不变

环境对植物的胁迫可能是短期快速的、也可能是长期而缓慢的,而植物应对这两种胁迫的策略可能不同。膜脂组成变化是植物响应环境胁迫的主要手段之一,其响应长期胁迫和短期胁迫的样式也可能不同。植物膜脂组成对短期缺钾胁迫的响应已经有报道,但是对长期缺钾的响应如何尚且未知。我们设置了4种（51,051,0051和0mmol·L-1）不同的钾浓度,比较了拟南芥（Arabidopsis thaliana）及其生长于贫钾生境中的近缘种须弥芥（Crucihimalaya himalaica）长期缺钾后（18天）的生理和生化变化,发现须弥芥具有耐受贫钾的能力。我们进一步运用脂类组学的方法检测比较了拟南芥和须弥芥在长期缺钾胁迫下脂类组成的变化,发现：(1) 两种植物叶片中总脂以及几乎所有脂类的含量明显上升;(2) 两种植物都是地上部分膜脂的变化幅度大于根部膜脂的变化幅度;(3) 地上部分膜脂变化幅度,须弥芥的大于拟南芥的;地下部分的膜脂变化幅度,须弥芥的小于拟南芥的;(4) 拟南芥叶片和根中PA的含量显著上升,与PA相对应的是PE含量的显著下降,由此我们推测拟南芥中PA的积累主要来自于PE的水解。上述结果提示,在细胞水平上,植物主要通过积累叶片膜脂和维持根部膜脂组成基本不变来适应长期缺钾。     

Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis

The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.     

A history of stress alters drought calcium signalling pathways in Arabidopsis

Environmental stresses commonly encountered by plants lead to rapid transient elevations in cytosolic free calcium concentration ([Ca2+]cyt) (Bush, 1995; Knight et al., 1991). These cellular calcium (Ca2+) signals lead ultimately to the increased expression of stress-responsive genes, including those encoding proteins of protective function (Knight et al., 1996; Knight et al., 1997). The kinetics and magnitude of the Ca2+ signal, or 'calcium signature', differ between different stimuli and are thought to contribute to the specificity of the end response (Dolmetsch et al., 1997; McAinsh and Hetherington, 1998). We measured [Ca2+]cyt changes during treatment with mannitol (to mimic drought stress) in whole intact seedlings of Arabidopsis thaliana. The responses of plants which were previously exposed to osmotic and oxidative stresses were compared to those of control plants. We show here that osmotic stress-induced Ca2+ responses can be markedly altered by previous encounters with either osmotic or oxidative stress. The nature of the alterations in Ca2+ response depends on the identity and severity of the previous stress: oxidative stress pre-treatment reduced the mannitol-induced [Ca2+]cyt response whereas osmotic stress pretreatment increased the [Ca2+]cyt response. Therefore, our data show that different combinations of environmental stress can produce novel Ca2+ signal outputs. These alterations are accompanied by corresponding changes in the patterns of osmotic stress-induced gene expression and, in the case of osmotic stress pre-treatment, the acquisition of stress-tolerance. This suggests that altered Ca2+ responses encode a 'memory' of previous stress encounters and thus may perhaps be involved in acclimation to environmental stresses.     

Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol

Ascorbate (vitamin C) is a potent antioxidant protecting plants against oxidative damage imposed by environmental stresses such as ozone and drought. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is one of the two important enzymes functioning in the regeneration of ascorbate (AsA). To examine the protective role of DHAR against oxidative stress, we developed transgenic tobacco plants overexpressing cytosolic DHAR gene from Arabidopsis thaliana . Incorporation of the transgene in the genome of tobacco plants was confirmed by polymerase chain reaction and Southern blot analysis, and its expression was confirmed by Northern and Western blot analyses. These transgenic plants exhibited 2.3–3.1 folds higher DHAR activity and 1.9–2.1 folds higher level of reduced AsA compared with non-transformed control plants. The transgenic plants showed maintained redox status of AsA and exhibited an enhanced tolerance to ozone, drought, salt, and polyethylene glycol stresses in terms of higher net photosynthesis. In this study, we report for the first time that the elevation of AsA level by targeting DHAR overexpression in cytosol properly provides a significantly enhanced oxidative stress tolerance imposed by drought and salt.     

Vacuolar membrane localization of the Arabidopsis 'two-pore' K+ channel KCO1

Potassium (K+) channels play multiple roles in higher plants, and have been characterized electrophysiologically in various subcellular membranes. The K+ channel AtKCO1 from Arabidopsis thaliana is the prototype of a new family of plant K+ channels. In a previous study the protein has been functionally characterized after heterologous expression in Baculovirus-infected insect cells. In order to obtain further information on the physiological function of AtKCO1, the gene expression pattern and subcellular localization of the protein in plants were investigated. The regulatory function of the 5' region of the AtKCO1 gene was examined in transgenic A. thaliana plants carrying beta-glucuronidase (GUS) fusion constructs. Our analysis demonstrates that the AtKCO1 promoter is active in various tissues and cell types, and the highest GUS activity could be detected in mitotically active tissues of the plant. Promoter activity was strongly dependent on the presence of a 5' leader intron. The same overall structure was identified in two genes encoding AtKCO1-like K+ channels from Solanum tuberosum (StKCO1alpha and StKCO1beta). To investigate the subcellular localization of AtKCO1, the channel protein, as well as a fusion protein of AtKCO1 with green fluorescence protein (GFP), were expressed in transgenic tobacco BY2 cells. In sucrose density gradients, both proteins co-fractionate with tonoplast markers (Nt-TIPa, vATPase). In fluorescence images from transgenic AtKCO1-GFP BY2 cells fluorescence was exclusively detected in the tonoplast. Thus AtKCO1 is the first cloned K+ channel demonstrated to be a vacuolar K+ channel.     

Transformation of peanut using a modified bacterial mercuric ion reductase gene driven by an actin promoter from Arabidopsis thaliana

In order to test an alternative selectable marker system for the production of transgenic peanut plants (Arachis hypogaea), the bacterial mercuric ion reductase gene, merA, was introduced into embryogenic cultures via microprojectile bombardment. MerA reduces toxic Hg(II) to the volatile and less toxic metallic mercury molecule, Hg(0), and renders its source Gram-negative bacterium mercury resistant. A codon-modified version of the merA gene, MerApe9, was cloned into a plant expression cassette containing the ACT2 promoter from Arabidopsis thaliana and the NOS terminator. The expression cassette also was inserted into a second vector containing the hygromycin resistance gene driven by the UBI3 promoter from potato. Stable transgenic plants were recovered through hygromycin-based selection from somatic embryo tissues bombarded with the plasmid containing both genes. However, no transgenic somatic embryos were recovered from selection on 50-100 micromol/L HgCl2. Expression of merA as mRNA was detected by Northern blot analysis in leaf tissues of transgenic peanut, but not in somatic embryos. Western blot analysis showed the production of the mercuric ion reductase protein in leaf tissues. Differential responses to HgCl2 of embryo-derived explants from segregating R1 seeds of one transgenic line also were observed.