Functional analyses of TaHMA2, a P1B‐type ATPase in wheat

Currently, there are few studies concerning the function of heavy metal ATPase 2 (HMA2), particularly in monocotyledons, and the potential application of this protein in biofortification and phytoremediation. Thus, we isolated and characterized the TaHMA2 gene from wheat (Triticum aestivum L.). Our results indicate that TaHMA2 is localized to the plasma membrane and stably expressed, except in the nodes, which showed relatively high expression. Zinc/cadmium (Zn/Cd) resistance was observed in TaHMA2‐transformed yeast. The over‐expression of TaHMA2 increased the elongation and decreased the seed‐setting rate in rice (Oryza sativa L.), but not Arabidopsis thaliana, tobacco (Nicotiana tabacum L.) or wheat. TaHMA2 over‐expression also improved root‐shoot Zn/Cd translocation, especially in rice. The seeds of transgenic rice and wheat, not tobacco, showed decreased Zn concentrations. The Zn concentration was decreased in all parts of the transgenic rice seeds, but was decreased only in the ventral endosperm of wheat, which showed an increased Zn concentration in the embryo and aleurone. The over‐expression of TaHMA2 improved plant tolerance under moderate Zn stress and Zn deficiency, but Zn and Cd resistance decreased under high levels of Zn and Cd stress, respectively. The Cd concentration in transgenic rice seedlings was dramatically increased under Zn deficiency. Thus, over‐expression of TaHMA2 showed a more obvious phenotype in monocotyledons than in dicotyledons. These findings provide important information for TaHMA2, and more efforts should be made in the future to characterize the reduced Zn concentration in TaHMA2 transgenic grains and the diversity of TaHMA2 substrate specificity.     

Development of Zn‐related necrosis in tobacco is enhanced by expressing AtHMA4 and depends on the apoplastic Zn levels

AtHMA4 was previously shown to contribute to the control of Zn root‐to‐shoot translocation and tolerance to high Zn. However, heterologous expression of 35S::AtHMA4 in tobacco (Nicotiana tabacum cv. Xanthi) results in enhanced Zn sensitivity. This study provides a better understanding of the development of this Zn‐sensitive phenotype and demonstrates that substantial modifications of Zn homeostasis occur due to AtHMA4 expression. We show that ectopically expressing AtHMA4 in tobacco results in overloading the root and leaf apoplast with Zn. The tissue and cellular distribution of Zn, monitored using Zinpyr‐1, was altered in the AtHMA4‐expressing plants compared with wild type. Increased loading of the leaf apoplast with Zn in AtHMA4 transformants induced necrosis; this appeared at lower levels of Zn supply in the transgenics compared with wild type. This study suggests that Zn concentration may be sensed in the apoplast of leaves, and if concentrations are above a certain threshold then particular groups of cells accumulate Zn and necrosis is initiated. Therefore, this could be considered as a mechanism for protecting the other parts of the photosynthetically active leaf from Zn toxicity.     

Plant Cd2+ and Zn2+ status effects on root and shoot heavy metal accumulation in Thlaspi caerulescens

* In this study we address the impact of changes in plant heavy metal, (i.e. zinc (Zn) and cadmium (Cd)) status on metal accumulation in the Zn/Cd hyperaccumulator, Thlaspi caerulescens. * Thlaspi caerulescens plants were grown hydroponically on both high and low Zn and Cd regimes and whole-shoot and -root metal accumulation, and root (109)Cd(2+) influx were determined. * High-Zn-grown (500 microm Zn) plants were found to be more Cd-tolerant than plants grown in standard Zn conditions (1 microm Zn). Furthermore, shoot Cd accumulation was significantly greater in the high-Zn-grown plants. A positive correlation was also found between shoot Zn accumulation and increased plant Cd status. Radiotracer (109)Cd root flux experiments demonstrated that high-Zn-grown plants maintained significantly higher root Cd(2+) influx than plants grown on 1 microm Zn. It was also found that both nickel (Ni) and copper (Cu) shoot accumulation were stimulated by high plant Zn status, while manganese (Mn) accumulation was not affected. * A speculative model is presented to explain these findings, suggesting that xylem loading may be one of the key sites responsible for the hyperaccumulation of Zn and Cd accumulation in Thlaspi caerulescens.     

Combined expression of the Arabidopsis metallothionein MT2b and the heavy metal transporting ATPase HMA4 enhances cadmium tolerance and the root to shoot translocation of cadmium and zinc in tobacco

We expressed the AtMt2b and AtHMA4 genes under the 35S cauliflower mosaic virus promoter simultaneously in Nicotiana tabacum (SR1), using leaf disc transformation. A single AtMT2b tobacco T2 line was used for re-transformation with AtHMA4 to obtain the double transformant. Cadmium (Cd) and zinc (Zn) tolerance, uptake and translocation were measured in the double transformant, and compared to untransformed (‘wild type’) tobacco and single gene transformants. The double transformant exhibited enhanced Cd-tolerance, enhanced Cd and Zn root to shoot transport, but unaltered Zn tolerance and Cd and Zn uptake, compared with wild type.The single transformant lines did not show significant phenotypes. Our results suggest that the phenotypes of the double transformant are due to synergistic interaction between the transgenes. Except for Cd tolerance, the phenotypes were moderate for Cd and Zn root to shoot transport, which may be due to use of the 35S promotor, resulting in incorrect tissue-specificity.     

Functional expression of AtHMA4, a P1B-type ATPase of the Zn/Co/Cd/Pb subclass

Mechanisms are required by all organisms to maintain the concentration of essential heavy metals (e.g. Zn and Cu) within physiological limits and to minimise the detrimental effects of non-essential heavy metals (e.g. Cd). Heavy-metal P-type ATPases (HMAs) are a subgroup of the P-type ATPase superfamily that may contribute to metal homeostasis in plants. We cloned and characterised a member of this family, AtHMA4, from Arabidopsis thaliana that clusters with the Zn/Co/Cd/Pb subclass of HMAs on phylogenetic analysis. Sequencing of the AtHMA4 cDNA showed that it contained the conserved motifs found in all P-type ATPases and also motifs that are characteristic of heavy-metal ATPases. Escherichia coli mutants defective in the HMAs, CopA and ZntA, were used in functional complementation studies. AtHMA4 was able to restore growth at high [Zn] in the zntA mutant but not at high [Cu] in the copA mutant, suggesting a role in zinc transport. Heterologous expression of AtHMA4 in Saccharomyces cerevisiae made the yeast more resistant to Cd but did not affect sensitivity to other metals compared with vector-transformed controls. The organ specificity of AtHMA4 was analysed in Arabidopsis and showed that AtHMA4 was expressed in a range of tissues with highest expression in roots. AtHMA4 was upregulated in roots exposed to elevated levels of Zn and Mn but downregulated by Cd. Possible physiological roles of this transporter in Arabidopsis are discussed.     

AtHMA1 contributes to the detoxification of excess Zn(II) in Arabidopsis

AtHMA1 is a member of the heavy metal-transporting ATPase family. It exhibits amino acid sequence similarity to two other Zn(II) transporters, AtHMA2 and AtHMA4, and contains poly-His motifs that are commonly found in Zn(II)-binding proteins, but lacks some amino acids that are typical for this class of transporters. AtHMA1 localizes to the chloroplast envelope. In comparison with wild-type plants, we observed a more pronounced sensitivity in the presence of high Zn(II) concentrations, and increased accumulation of Zn in the chloroplast of T-DNA insertional mutants in AtHMA1 . The Zn(II)-sensitive phenotype of AtHMA1 knock-out plants was complemented by the expression of AtHMA1 under the control of its own promoter. The Zn(II)-transporting activity of AtHMA1 was confirmed in a heterologous expression system, Saccharomyces cerevisiae . The sensitivity of yeast to high concentrations of Zn(II) was altered by the expression of AtHMA1 lacking its N-terminal chloroplast-targeting signal. Taken together, these results suggest that under conditions of excess Zn(II), AtHMA1 contributes to Zn(II) detoxification by reducing the Zn content of Arabidopsis thaliana plastids.     

The plant P1B-type ATPase AtHMA4 transports Zn and Cd and plays a role in detoxification of transition metals supplied at elevated levels

The transition metal Zn is essential for many physiological processes in plants, yet at elevated concentrations this, and the related non-essential metal Cd, can be toxic. Arabidopsis thaliana HMA4, belonging to the Type P1B subfamily of P-type ATPases, has recently been implicated in Zn nutrition, having a role in root to shoot Zn translocation. Using Arabidopsis insertional mutants, it is shown here that disruption of AtHMA4 function also results in increased sensitivity to elevated levels of Cd and Zn, suggesting that AtHMA4 serves an important role in metal detoxification at higher metal concentrations. AtHMA4 and a truncated form lacking the last 457 amino acids both confer Cd and Zn resistance to yeast but a mutant version of the full-length AtHMA4 (AtHMA4-C357G) does not; this demonstrates that the C-terminal region is not essential for this function. Evidence is presented that AtHMA4 functions as an efflux pump.     

Structural basis for metal binding specificity: the N-terminal cadmium binding domain of the P1-type ATPase CadA

In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a betaalphabetabetaalphabeta fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sphere was investigated by X-ray absorption spectroscopy. A novel metal binding environment was found, comprising the two conserved Cys residues of the metal binding motif and a Glu in loop 5. A bioinformatic search identifies an ensemble of highly homologous sequences presumably with the same function. Another group of highly homologous sequences is found which can be referred to as zinc-detoxifying P1-type ATPases with the metal binding pattern DCXXC in the N terminus. Because no carboxylate groups participate in Cu(I) or Ag(I) binding sites, we suggest that the acidic residue plays a key role in the coordination properties of divalent cations, hence conferring a function to the N terminus in the metal specificity of the ATPase.     

Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein

The use of microalgal biomass for metal pollutant bioremediation might be improved by genetic engineering to modify the selectivity or capacity of metal biosorption. A plant cadmium (Cd) and zinc (Zn) transporter (AtHMA4) was used as a transgene to increase the ability of Chlamydomonas reinhardtii to tolerate 0.2 mM Cd and 0.3 mM Zn exposure. The transgenic cells showed increased accumulation and internalization of both metals compared to wild-type. AtHMA4 was expressed either as the full-length (FL) protein or just the C-terminal (CT) tail, which is known to have metal-binding sites. Similar Cd and Zn tolerance and accumulation was observed with expression of either the FL protein or CT domain, suggesting that enhanced metal tolerance was mainly due to increased metal binding rather than metal transport. The effectiveness of the transgenic cells was further examined by immobilization in calcium alginate to generate microalgal beads that could be added to a metal contaminated solution. Immobilization maintained metal tolerance, while AtHMA4-expressing cells in alginate showed a concentration-dependent increase in metal biosorption that was significantly greater than alginate beads composed of wild-type cells. This demonstrates that expressing AtHMA4 FL or CT has great potential as a strategy for bioremediation using microalgal biomass.     

Plastid transformation of high‐biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV‐1) p24 antigen

Chloroplast transformation of the high‐biomass tobacco variety Maryland Mammoth has been assessed as a production platform for the human immunodeficiency virus type 1 (HIV‐1) p24 antigen. Maryland Mammoth offers the prospect of higher yields of intact functional protein per unit floor area of contained glasshouse per unit time prior to flowering. Two different transformation constructs, pZSJH1p24 (for the insertion of a native p24 cDNA between the rbcL and accD genes) and pZF5 (for the insertion of a chloroplast‐codon‐optimized p24 gene between trnfM and trnG) were examined for the production of p24. Plants generated with construct pZSJH1p24 exhibited a normal green phenotype, but p24 protein accumulated only in the youngest leaves (up to approximately 350 µg/g fresh weight or ~2.5% total soluble protein) and was undetectable in mature leaves. In contrast, some of the plants generated with pZF5 exhibited a yellow phenotype (pZF5‐yellow) with detectable p24 accumulation (up to approximately 450 µg/g fresh weight or ~4.5% total soluble protein) in all leaves, regardless of age. Total protein in pZF5‐yellow leaves was reduced by ~40%. The pZF5‐yellow phenotype was associated with recombination between native and introduced direct repeat sequences of the rbcL 3′ untransformed region in the plastid genome. Chloroplast‐expressed p24 was recognized by a conformation‐dependent monoclonal antibody to p24, and p24 protein could be purified from pZF5‐yellow leaves using a simple procedure, involving ammonium sulphate precipitation and ion‐exchange chromatography, without the use of an affinity tag. The purified p24 was shown to be full length with no modifications, such as glycosylation or phosphorylation, using N‐terminal sequencing and mass spectrometry.     

CyclinD1、CDK4、P16和结核菌L型感染在前列腺癌中的表达及临床意义

目的探讨CyclinD1、CDK4和P16在前列腺癌中的表达以及结核菌L型感染率及临床意义。方法应用免疫组化和抗酸染色等方法检测了65例前列腺癌（carcinoma of prostate,PCa）和30例良性前列腺增生（benignprostatic hyperplasia,BPH）中的CyclinD1、CDK4和P16的表达,以及结核菌L型的检出率。并对前列腺肿瘤主要临床资料和病理分级参数进行比较,用χ^2检验进行统计学处理。结果 CyclinD1、CDK4阳性表达前列腺癌明显高于前列腺增生（P〈0.01）;并与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.01-0.05）呈正相关。P16阳性表达前列腺增生明显高于前列腺癌（P〈0.01）;与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.01-0.05）呈负正相关。结核菌L型检出率前列腺癌明显高于前列腺增生;与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.05）。结论 CyclinD1、CDK4和P16在前列腺肿瘤中不同程度异常表达以及结核菌L型检出率与肿瘤的临床分期、病理分级和转移相关,因此研究CyclinD1、CDK4和P16的阳性表达和结核菌L型感染与前列腺癌发生发展中可能有协同作用,具有重要的临床应用价值。     

The AAA‐type ATPase AtSKD1 contributes to vacuolar maintenance of Arabidopsis thaliana

The vacuole is the most prominent organelle of plant cells. Despite its importance for many physiological and developmental aspects of plant life, little is known about its biogenesis and maintenance. Here we show that Arabidopsis plants expressing a dominant‐negative version of the AAA (ATPase associated with various cellular activities) ATPase AtSKD1 (SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1) under the control of the trichome‐specific GLABRA2 (GL2) promoter exhibit normal vacuolar development in early stages of trichome development. Shortly after its formation, however, the large central vacuole is fragmented and finally disappears completely. Secretion assays with amylase fused to the vacuolar sorting signal of Sporamin show that dominant‐negative AtSKD1 inhibits vacuolar trafficking of the reporter that is instead secreted. In addition, trichomes expressing dominant‐negative AtSKD1 frequently contain multiple nuclei. Our results suggest that AtSKD1 contributes to vacuolar protein trafficking and thereby to the maintenance of the large central vacuole of plant cells, and might play a role in cell‐cycle regulation.     

Expression of RPS4 in tobacco induces an AvrRps4-independent HR that requires EDS1, SGT1 and HSP90

The Arabidopsis RPS4 gene belongs to the Toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) class of plant resistance (R) genes. It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4. Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4-independent hypersensitive response (HR). The same phenotype is seen after expression of a full-length RPS4 cDNA. This indicates that alternative splicing of RPS4 is not involved in this HR. The extent of HR is correlated with RPS4 protein levels. Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype. Mutations in the P-loop motif of the NB domain abolish the HR. Using virus-induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90. All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR.     

Distinct functions of serial metal‐binding domains in the Escherichia coli P1B‐ATPase CopA

P_1B‐ATPases are among the most common resistance factors to metal‐induced stress. Belonging to the superfamily of P‐type ATPases, they are capable of exporting transition metal ions at the expense of adenosine triphosphate (ATP) hydrolysis. P_1B‐ATPases share a conserved structure of three cytoplasmic domains linked by a transmembrane domain. In addition, they possess a unique class of domains located at the N‐terminus. In bacteria, these domains are primarily associated with metal binding and either occur individually or as serial copies of each other. Within this study, the roles of the two adjacent metal‐binding domains (MBDs) of CopA, the copper export ATPase of Escherichia coli were investigated. From biochemical and physiological data, we deciphered the protein‐internal pathway of copper and demonstrate the distal N‐terminal MBD to possess a function analogous to the metallochaperones of related prokaryotic copper resistance systems, that is its involvement in the copper transfer to the membrane‐integral ion‐binding sites of CopA. In contrast, the proximal domain MBD2 has a regulatory role by suppressing the catalytic activity of CopA in absence of copper. Furthermore, we propose a general functional divergence of tandem MBDs in P_1B‐ATPases, which is governed by the length of the inter‐domain linker.     

骨肉瘤中P53及P21WAF1表达的生物学意义

应用免疫组化SP法观察34例骨肉瘤中P53及P21WAF1的表达情况,探讨P53及P21WAF1表达与骨肉瘸临床病理学特征之间的关系,分析P53和P21WAF1在骨肉瘤中的作用及其相关性。结果可见,在34例骨肉瘤中,18例表达P53,阳性表达率52.94%;12例表达P21WAF1,阳性表达率35.29%。P53阳性表达与性别、肿瘤分化及是否转移复发有关(P<0.05),P21WAF1与肿瘤分化有关(P<0.01)。P53在中、低分化骨肉瘤中阳性表达率较高,而在高分化骨肉瘤中阳性表达率较低(70%、28.57%);P21WAF1在高分化骨肉瘤中阳性表达率较高,而在中、低分化骨肉瘤阳性表达率较低(85.71%、0%)。P53及P21WAF1表达呈负相关性(r=-0.537,P=0.001)。在骨肉瘤组织中P53表达上调及P21WAF1表达下调与骨肉瘤的恶性进展有关。     

Synthesis and luminescent properties of a novel bluish‐white afterglow phosphor,b‐Zn3(PO4)2:Hf4+

A new bluish‐white long‐lasting phosphorescent material, Hf⁴⁺‐doped b‐Zn₃(PO₄)₂, was prepared by the conventional high‐temperature solid‐state method. The photoluminescence (PL) spectrum reveals that it exhibits a strong blue emission band centred at 470 nm, with asymmetry on the long wavelength side; this material emits bluish‐white light and shows strong afterglow phosphorescence after it is excited with a 254 nm UV lamp. The phosphorescence lasts nearly 40 min in the light perception of the dark‐adapted human eye (0.32 mcd/m²). The possible phosphorescence mechanism is also analysed. Copyright © 2008 John Wiley & Sons, Ltd.