Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2

Plant copper P_1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu⁺ (K_m ∼1 or 0.5 μm, respectively) and similar affinity to Ag⁺ (K_m ∼2.5 μm). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu⁺ transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess.     

The role of heavy-metal ATPases,HMAs, in zinc and cadmium transport in rice

The P_1B-type heavy metal ATPases (HMAs) are diverse in terms of tissue distribution, subcellular localization, and metal specificity. Functional studies of HMAs have shown that these transporters can be divided into two subgroups based on their metal-substrate specificity: a copper (Cu)/silver (Ag) group and a zinc (Zn)/cobalt (Co)/cadmium (Cd)/lead (Pb) group. Studies on Arabidopsis thaliana and metal hyperaccumulator plants indicate that HMAs play an important role in the translocation or detoxification of Zn and Cd in plants. Rice possesses nine HMA genes, of which OsHMA1–OsHMA3 belong to the Zn/Co/Cd/Pb subgroup. OsHMA2 plays an important role in root-to-shoot translocation of Zn and Cd, and participates in Zn and Cd transport to developing seeds in rice. OsHMA3 transports Cd and plays a role in the sequestration of Cd into vacuoles in root cells. Modification of the expression of these genes might be an effective approach for reducing the Cd concentration in rice grains.     

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.     

Comparative expression analysis of heavy metal ATPase subfamily genes between Cd-tolerant and Cd-sensitive turnip landraces

The heavy metal ATPase(HMA)subfamily is mainly involved in heavy metal(HM)tolerance and transport in plants,but an understanding of the definite roles and mechanisms of most HMA members are still limited.In the present study,we identified 14 candidate HMA genes named BrrHMAl—BrrHMA8 from the turnip genome and analyzed the phylogeny,gene structure,chromosome distribution,and conserved domains and motifs of HMAs in turnip(Brassica rapa var.rapa).According to our phylogenetic tree,the BrrHMAs are divided into a Zn/Cd/Co/Pb subclass and Cu/Ag subclass.The BrrHMA members show similar structural characteristics within subclasses.To explore the roles of BrrHMAs in turnip,we compared the gene sequences and expression patterns of the BrrHMA genes between a Cd-tolerant landrace and a Cd-sensitive landrace.Most BrrHMA genes showed similar spatial expression patterns in both Cd-tolerant and Cd-sensitive turnip landraces;some BrrHMA genes,however,were differentially expressed in specific tissue in Cd-tolerant and Cd-sensitive turnip.Specifically,BrrHMA genes in the Zn/Cd/Co/Pb subclass shared the same coding sequence but were differentially expressed in Cd-tolerant and Cd-sensitive turnip landraces under Cd stress.Our findings suggest that the stable expression and up-regulated expression of BrrHMA Zn/Cd/Co/Pb subclass genes under Cd stress may contribute to the higher Cd tolerance of turnip landraces.     

Leaching of Zinc,Cadmium, and Lead in a Sandy Soil Due to Application of Poultry Litter

Dissolved organic matter in poultry litter could contribute organic ligands to form complexes with heavy metals in soil. The soluble complexes with heavy metals can be transported downward and possibly deteriorate groundwater quality. To better understand metal mobilization by soluble organic ligands in poultry litter, soil columns were employed to investigate the movement of zinc (Zn), cadmium (Cd), and lead (Pb). Uncontaminated soil was amended with Zn, Cd, and Pb at rates of 400, 8, and 200 mg kg ^{? 1} soil, respectively. Glass tubes, 4.9-cm-diameter and 40-cm-long, were packed with either natural or metal-amended soil. The resulting 20-cm-long column of soils had bulk density of about 1.58 g cm ^{? 3} . Columns repacked with natural or amended soil were leached with distilled water, 0.01 M EDTA, 0.01 M CaCl ₂ , or poultry litter extract (PLE) solutions. Low amounts of Zn, Cd, and Pb were leached from natural soil with the solutions. Leaching of Zn, Cd, or Pb was negligible with distilled water. In the metal-amended soil, EDTA solubilized more Zn, Cd, and Pb than CaCl ₂ and PLE. The breakthrough curves of Zn and Pb in the PLE and CaCl ₂ were similar, indicating they have similar ability to displace Zn and Pb from soils. Compared with Zn and Cd the PLE had a small ability to solubilize Pb from metal-amended soil. Thus, the application of poultry litter on metal-contaminated soils might enhance the mobility of Zn and Cd.     

The phylogenetic tree gathering the plant Zn/Cd/Pb/Co P1B-ATPases appears to be structured according to the botanical families

Plant Zn/Cd/Pb/Co P_1B-ATPases (HMAs) play different roles, among which are the control of metal transport from the roots to the shoot and/or from the cytoplasm into the cell vacuole. Transferring the knowledge acquired on HMAs from model species to HMAs from other species requires one to identify orthologues in these other species. Through an extensive screening of the public sequence databases, 96 plant P_1B-ATPases showing orthology to any of the AtHMA1, AtHMA2, AtHMA3 or AtHMA4 isoforms were identified from 32 plant species belonging to 15 botanical families. The number of paralogues within a species varied greatly from species to species, even within a specific botanical family, suggesting that gene duplication events occurred after speciation. The phylogenetic tree gathering the Zn/Cd/Pb/Co P_1B-ATPases was strongly structured according to the botanical family to which the sequences could be related to. In particular, no strict orthology relationship links the Brassicaceae HMAs to the non-Brassicaceae or the Poaceae ones. Recent data showed that the sole rice HMA characterised to date displays different functional properties from the Arabidopsis HMAs. Altogether, data suggest that it might be risky to directly transfer the knowledge acquired through the study of HMAs in model plant species to HMAs from other species.     

P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis

Arabidopsis thaliana has eight genes encoding members of the type 1_B heavy metal–transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter- and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.     

Risk Element (As,Cd, Cu,Pb, and Zn) Contamination of Soils and Edible Vegetables in the Vicinity of Guixi Smelter,South China

Risk element (As, Cd, Cu, Pb, and Zn) contamination in soils and in two edible vegetables (Solanum melongena L. and Capsicum annum L.) was investigated in the vicinity of Guixi Smelter, South China. Soil As concentrations averaged 23.9 mg/kg. Sites near the smelter tailings recorded the highest levels of As and heavy metals in soils. The concentration order of heavy metals in soils was Cd < Pb < Zn < Cu. Cu and Cd in soils were abundant in the exchangeable and bound to carbonate fraction, while Pb and Zn were in the residual fraction, limiting their potential toxicity as pollutants. The proportions of the metals in the mobile fraction followed the order Pb < Zn < Cu < Cd. In Solanum melongena L. and Capsicum annum L., Zn concentration was the highest, followed by Cu, Cd, and Pb, different from that in soils and in the mobile fraction. Concentrations of heavy metals in the labile fractions in soils and in vegetables presented significant correlation (p < 0.05). Both of the two vegetables are not the Cu and Zn accumulators. As for Cd and As, Capsicum annum L. poses a higher risk to animal and human health than Solanum melongena L., with soil-plant transfer coefficients more than three. Root-stem is the main barrier for most of the heavy metals and As in the two vegetables, resulting in higher metal concentrations in roots relative to other plant tissues. The low stem-fruit transfer coefficients for Zn in Solanum melongena L. and for Pb in Capsicum annum L. suggested that very few of them could reach the fruits.     

Mycorrhizal symbiosis and phosphorus fertilization effects on Zea mays growth and heavy metals uptake

Arbuscular mycorrhizal fungi (AMF) can promote plant growth and reduce plant uptake of heavy metals. Phosphorus (P) fertilization can affect this relationship. We investigated maize (Zea mays L.) uptake of heavy metals after soil AMF inoculation and P fertilization. Maize biomass, glomaline and chlorophyll contents and uptake of Fe, Mn, Zn, Cu, Cd and Pb have been determined in a soil inoculated with AMF (Glomus aggregatum, or Glomus intraradices) and treated with 30 or 60 µg P-K₂HPO₄ g^?1 soil. Consistent variations were found between the two mycorrhizal species with respect to the colonization and glomalin content. Shoot dry weight and chlorophyll content were higher with G. intraradices than with G. aggregatum inoculation. The biomass was highest with 30 µg P g^?1 soil. Shoot concentrations of Cd, Pb and Zn decreased with G. aggregatum inoculation, but that of Cd and Pb increased with G. intraradices inoculation. Addition of P fertilizers decreased Cd and Zn concentrations in the shoot. AMF with P fertilization greatly reduced maize content of heavy metals. The results provide that native AMF with a moderate application rate of P fertilizers can be exploited in polluted soils to minimize the heavy metals uptake and to increase maize growth.     

The influence of pH and chloride on the retention of cadmium,lead, mercury,and zinc by soils

The extent of contamination of soils by toxic heavy metals not only depends on the rate of loading of the metal but also on the nature of the adsorbing surfaces, the degree of alkalinity or acidity of the soil and the presence of aqueous complexant ligands. This work reports on the role of pH on the retention of Cd, Hg, Pb and Zn by two soils and on the influence of the chloride, Cl‐, ion on the chemical speciation and retention of the four metals. Batch adsorption experiments were conducted from pH 3 to 7 in the presence of either 0.1 M LiCl or LiClO₄. The results of the study showed that high concentrations of Cl^‐ ions can greatly decrease the retention of Hg and have an increasingly lesser effect on Cd, Pb and Zn retention. The effect of the Cl^‐ons was directly related to the metal‐Cl formation constants. The results of computer modeling of Cd and Hg retention by goethite and humic acid fractions indicated the relative importance of aqueous vs. surface complexation on metal retention. For organic surfaces, which do not form ternary surface complexes, the presence of aqueous complexant ligands should always decrease the adsorption of the metal. For mineral surfaces, which do form ternary surface complexes, there may be increased or decreased metal retention depending on the formation constant of the aqueous metal‐ligand species, the intrinsic complexation constants for the various binary and ternary complexes of the metal and the concentration of the complexant ligand. Thus for Hg, which forms very strong aqueous species with Cl^‐ ions, reduced adsorption on goethite was predicted in the presence of 0.1 M LiCl, while enhanced adsorption was predicted for Cd and Pb. The results suggest caution in the disposal of Cl‐containing wastes onto metal‐contaminated soils. The deleterious effects of Cl^‐ ion addition would be greatest for soils with relatively high organic matter contents and low contents of hydrous ferric oxides.     

Impairing both HMA4 homeologs is required for cadmium reduction in tobacco

In tobacco, the heavy metal P1B‐ATPases HMA4.1 and HMA4.2 function in root‐to‐shoot zinc and cadmium transport. We present greenhouse and field data that dissect the possibilities to impact the two homeologous genes in order to define the best strategy for leaf cadmium reduction. In a first step, both genes were silenced using an RNAi approach leading to >90% reduction of leaf cadmium content. To modulate HMA4 function more precisely, mutant HMA4.1 and HMA4.2 alleles of a Targeting Induced Local Lesions IN Genomes (TILLING) population were combined. As observed with RNAi plants, knockout of both homeologs decreased cadmium root‐to‐shoot transfer by >90%. Analysis of plants with segregating null and wild‐type alleles of both homeologs showed that one functional HMA4 allele is sufficient to maintain wild‐type cadmium levels. Plant development was affected in HMA4 RNAi and double knockout plants that included retarded growth, necrotic lesions, altered leaf morphology and increased water content. The combination of complete functional loss (nonsense mutation) in one homeologous HMA4 gene and the functional reduction in the other HMA4 gene (missense mutation) is proposed as strategy to limit cadmium leaf accumulation without developmental effects.     

新疆焉耆盆地辣椒地土壤重金属污染及生态风险预警

从新疆加工辣椒主产地(焉耆盆地)采集105个辣椒地典型土壤样品,测定其中As、Cd、Cr、Cu、Mn、Ni、Pb和Zn等8种重金属元素的含量。采用污染负荷指数(Pollution load index,PLI)、潜在生态风险指数(Potential ecological risk index,RI)和生态风险预警指数(Ecological risk warning index,I_(ER))对辣椒地土壤重金属污染及生态风险进行评价。结果表明:(1)焉耆盆地辣椒地土壤Cd、Cr、Ni、Pb和Zn含量的平均值分别超出新疆灌耕土背景值的1.65、1.40、1.32、3.21、6.42倍。辣椒地土壤Pb和Zn呈现重度污染,Cd、Cr和Ni轻度污染,As、Mn和Cu无污染。(2)土壤PLI平均值为1.40,呈现轻度污染。各重金属元素单项生态风险指数从大到小依次为:Cd、Ni、As、Cu、Pb、Cr、Zn。土壤RI平均值为18.40,属于轻微生态风险态势,IER平均值为-4.78,属于无警态势;博湖县辣椒地污染水平、潜在生态风险程度与生态风险预警等级最高,焉耆县污染水平、潜在生态风险程度与生态风险预警等级最低。(3)辣椒地土壤As、Cd、Pb与Zn主要受到人类活动的影响,Cr、Cu、Mn和Ni主要受到土壤地球化学作用的控制。Cd是焉耆盆地辣椒地生态风险等级最高的重金属元素,研究区农业生产过程中要防范Cd的污染风险。     

A novel heavy metal ATPase peptide from <Emphasis Type="Italic">Prosopis juliflora</Emphasis> is involved in metal uptake in yeast and tobacco

Heavy metal pollution of agricultural soils is one of the most severe ecological problems in the world. Prosopis juliflora, a phreatophytic tree species, grows well in heavy metal laden industrial sites and is known to accumulate heavy metals. Heavy Metal ATPases (HMAs) are ATP driven heavy metal pumps that translocate heavy metals across biological membranes thus helping the plant in heavy metal tolerance and phytoremediation. In the present study we have isolated and characterized a novel 28.9 kDa heavy metal ATPase peptide (PjHMT) from P. juliflora which shows high similarity to the C-terminal region of P_1B ATPase HMA1. It also shows the absence of the invariant signature sequence DKTGT, and the metal binding CPX motif but the presence of conserved regions like MVGEGINDAPAL (ATP binding consensus sequence), HEGGTLLVCLNS (metal binding domain) and MLTGD, GEGIND and HEGG motifs which play important roles in metal transport or ATP binding. PjHMT, was found to be upregulated under cadmium and zinc stress. Heterologous expression of PjHMT in yeast showed a higher accumulation and tolerance of heavy metals in yeast. Further, transgenic tobacco plants constitutively expressing PjHMT also showed increased accumulation and tolerance to cadmium. Thus, this study suggests that the transport peptide from P. juliflora may have an important role in Cd uptake and thus in phytoremediation.     

EFFECTS OF INOCULATION WITH ARBUSCULAR MYCORRHIZAL FUNGI ON MAIZE GROWN IN MULTI-METAL CONTAMINATED SOILS

Pot culture experiments were established to determine the effects of colonization by arbuscular mycorrhizal fungi (AMF) (Glomus mosseae and G. sp) on maize (Zea mays L.) grown in Pb, Zn, and Cd complex contaminated soils. AMF and non-AMF inoculated maize were grown in sterilized substrates and subjected to different soil heavy metal (Pb, Zn, Cd) concentrations. The root and shoot biomasses of inoculated maize were significantly higher than those of non-inoculated maize. Pb, Zn, and Cd concentrations in roots were significantly higher than those in shoots in both the inoculated and non-inoculated maize, indicating the heavy metals mostly accumulated in the roots of maize. The translocation rates of Pb, Zn, and Cd from roots to shoots were not significantly difference between inoculated and non-inoculated maize. However, at high soil heavy metal concentrations, Pb, Zn, and Cd in the shoots and Pb in the roots of inoculated maize were significantly reduced by about 50% compared to the non-inoculated maize. These results indicated that AMF could promote maize growth and decrease the uptake of these heavy metals at higher soil concentrations, thus protecting their hosts from the toxicity of heavy metals in Pb, Zn, and Cd complex contaminated soils.     

毛果杨HMA基因家族的生物信息学分析

重金属转运ATP酶（heavy metal transporting ATPase,HMA）是一种通过水解ATP跨膜运送重金属阳离子的转运蛋白,属于P-ATPase家族中一个亚类。不同HMA蛋白对重金属离子的转运具有选择性,在植物修复重金属污染土壤方面起着重要作用。依据毛果杨全基因组测序的结果,以及HMA基因蛋白的序列和功能特征,从毛果杨基因组中鉴定了13个HMA基因家族成员,分属于Zn亚类（Zn2＋/Co2＋/Cd2＋/Pb2＋P1B-ATPase）和Cu亚类（Cu＋/Ag＋P1B-ATPase）两个亚家族,主要分布于1、3号染色体上。生物信息学分析表明,毛果杨HMA基因的氨基酸序列一致性介于21.3%~89.3%,且具有保守的基序CPC、HP、DKTGT、TGEx、GDG、PxD和CxxC等。蛋白理化特征分析显示,多数毛果杨HMA蛋白稍偏酸性,结构稳定性较好,蛋白脂溶指数高,稍具疏水性。密码子偏好性分析显示,毛果杨HMA蛋白14个氨基酸中存在16个高频密码子,另有1个终止密码子为高频密码子,显示出毛果杨的种属特征。研究结果展示了毛果杨HMA基因家族的基本信息和特点,为深入研究毛果杨HMA基因的功能搭建了基础平台。     

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.