采用污染土盆栽法，研究箭管豌豆（Viciasativa L．）在镉（Cd）添加量为2．5和10．0mg／kg土壤甲的生长状况、营养物质吸收和镉富集特征。结果表明，土壤镉添加对箭箸豌豆地上部和地下部生物量没有显著影响，但使种子的生物量降低。轻度和重度镉污染条件下，箭箸豌豆地下部的镉富集系数为21．12-26．39，地上部的镉富集系数为0．47～1．00，种子的镉富集系数为0．46～1．16，镉的转运系数为0．02～0．05。地下部镉含量达268．97mg／kg，地上部和种子的镉含量超国家食品限量标准，营养元素尤其是Fe、Zn、Mn和P的含量，受到镉添加的显著影响。因此，箭等豌豆可用于镉污染土壤的植物修复，但需防范地上部和种子食用和饲用的安全风险；镉对营养元素吸收的影响是其生长受抑制的原因之一。

Cadmium (Cd) is an inorganic mineral in the earth's crust. Cadmium entry into the environment occurs through geogenic and anthropogenic sources. Industrial activities including mining, electroplating, iron and steel plants, and battery production employ Cd during their processes and often release Cd into the environment. When disseminated into soil, Cd can be detrimental to agro-ecosystems because it is relatively mobile and phytotoxic even at low concentrations. Cadmium's phytotoxicity is due to reductions in the rate of transpiration and photosynthesis and chlorophyll concentration resulting in retardation of plant growth, and an alteration in the nutrient concentration in roots and leaves. In response to Cd toxicity, plants have developed protective cellular mechanisms such as synthesis of phytochelatins and metallothioneins, metal compartmentalization in vacuoles, and the increased activity of antioxidant enzymes to neutralize Cd-induced toxicity. While these direct protective mechanisms can help alleviate Cd toxicity, other indirect mechanisms such as microelements (zinc, iron, manganese, and selenium) interfering with Cd uptake may decrease Cd concentration in plants. This comprehensive review encompasses the significance of Cd, portals of contamination and toxicity to plants, and implications for crop production. Various mitigation strategies with the beneficial effects of zinc, iron, manganese, and selenium in activating defence mechanisms against Cd stress are discussed. Furthermore, this review systematically identifies and summarises suitable strategies for mitigating Cd-induced toxicity in plants.

<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">The concentrations of heavy metals in <em>Phytolacca americana</em> L. and corresponding soil samples from three contaminated sites and an uncontaminated site were studied. Hydroponic experiments were also conducted to investigate the Cd uptake ability and mechanism of <em>P. americana</em>. The field results showed that the average Cd concentration was 42&#xA0;mg&#xA0;kg^&minus;1 in <em>P. americana</em> leaves, with the highest concentration of 402&#xA0;mg&#xA0;kg^&minus;1 found at Datianwan. A significant relationship was observed between the concentrations of Cd in leaves and those of corresponding soils on a logarithmic scale. Under laboratory hydroponic conditions, the maximum Cd concentration in aerial tissues of <em>P. americana</em> was 637&#xA0;mg&#xA0;kg^&minus;1, under treatment with 100&#xA0;μM Cd. The population from the uncontaminated site (Zijinshan) also had a remarkable ability to accumulate Cd in shoots to concentrations well in excess of 100&#xA0;μM in the hydroponic experiment, similar to the population from contaminated site, suggesting that Cd accumulation is a constitutive trait of <em>P. americana</em>. In the presence of 100&#xA0;μM Cd, the addition of polyethylene glycol decreased leaf transpiration, the shoot Cd concentration, and the shoot/root Cd concentration ratio. There was a significantly positive relationship between the shoot Cd concentration and the leaf transpiration of <em>P. americana</em>. A similar significant positive correlation was also obtained between the shoot/root Cd concentration and leaf transpiration. Moreover, pretreatment with 5&#xA0;μM abscisic acid or 5&#xA0;μM HgCl₂ significantly decreased the Cd concentration in <em>P</em>. <em>americana</em> shoots. These results suggest that transpiration has an important role in Cd accumulation in shoots of <em>P</em>. <em>americana</em>.</p>

Bark tissue of Populus 3 canescens can hyperaccumulate cadmium, but microstructural, transcriptomic, and physiological response mechanisms are poorly understood. Histochemical assays, transmission electron microscopic observations, energy-dispersive x-ray microanalysis, and transcriptomic and physiological analyses have been performed to enhance our understanding of cadmium accumulation and detoxification in P. x 3 canescens. Cadmium was allocated to the phloem of the bark, and subcellular cadmium compartmentalization occurred mainly in vacuoles of phloem cells. Transcripts involved in microstructural alteration, changes in nutrition and primary metabolism, and stimulation of stress responses showed significantly differential expression in the bark of P. x 3 canescens exposed to cadmium. About 48% of the differentially regulated transcripts formed a coregulation network in which 43 hub genes played a central role both in cross talk among distinct biological processes and in coordinating the transcriptomic regulation in the bark of P. x 3 canescens in response to cadmium. The cadmium transcriptome in the bark of P. x 3 canescens was mirrored by physiological readouts. Cadmium accumulation led to decreased total nitrogen, phosphorus, and calcium and increased sulfur in the bark. Cadmium inhibited photosynthesis, resulting in decreased carbohydrate levels. Cadmium induced oxidative stress and antioxidants, including free proline, soluble phenolics, ascorbate, and thiol compounds. These results suggest that orchestrated microstructural, transcriptomic, and physiological regulation may sustain cadmium hyperaccumulation in P. x 3 canescens bark and provide new insights into engineering woody plants for phytoremediation.

High expression of zinc (Zn)-regulated, iron-regulated transporter-like protein (ZIP) genes increases root Zn uptake in dicots, leading to high accumulation of Zn in shoots. However, none of the ZIP genes tested previously in monocots could enhance shoot Zn accumulation. In this report, barley (Hordeum vulgare) HvZIP7 was investigated for its functions in Zn transport.The functions of HvZIP7 in planta were studied using in situ hybridization and transient analysis of subcellular localization with a green fluorescent protein (GFP) reporter. Transgenic barley lines overexpressing HvZIP7 were also generated to further understand the functions of HvZIP7 in metal transport.HvZIP7 is strongly induced by Zn deficiency, primarily in vascular tissues of roots and leaves, and its protein was localized in the plasma membrane. These properties are similar to its closely related homologs in dicots. Overexpression of HvZIP7 in barley plants increased Zn uptake when moderately high concentrations of Zn were supplied. Significantly, there was a specific enhancement of shoot Zn accumulation, with no measurable increase in iron (Fe), manganese (Mn), copper (Cu) or cadmium (Cd). HvZIP7 displays characteristics of low-affinity Zn transport.The unique function of HvZIP7 provides new insights into the role of ZIP genes in Zn homeostasis in monocots, and offers opportunities to develop Zn biofortification strategies in cereals.

When plants are subjected to high metal exposure, different plant species take different strategies in response to metal-induced stress. Largely, plants can be distinguished in four groups: metal-sensitive species, metal-resistant excluder species, metal-tolerant non-hyperaccumulator species, and metal-hypertolerant hyperaccumulator species, each having different molecular mechanisms to accomplish their resistance/tolerance to metal stress or reduce the negative consequences of metal toxicity. Plant responses to heavy metals are molecularly regulated in a process called metal homeostasis, which also includes regulation of the metal-induced reactive oxygen species (ROS) signaling pathway. ROS generation and signaling plays an important duel role in heavy metal detoxification and tolerance. In this review, we will compare the different molecular mechanisms of nutritional (Zn) and non-nutritional (Cd) metal homeostasis between metal-sensitive and metal-adapted species. We will also include the role of metal-induced ROS signal transduction in this comparison, with the aim to provide a comprehensive overview on how plants cope with Zn/Cd stress at the molecular level.

<a name="Abs1"></a>The ZRT-and IRT-like proteins (ZIP) comprise a large family of transition metal transporters in plants that have diverse functions to transport zinc, iron, copper, etc. Here, we provided a complete overview of this gene family in rice (<i>Oryza sativa</i> L.). Based on the hidden Markov model and BLAST analysis, a total of 17 ZIP-coding genes were identified and further studied by semi-quantitative RT-PCR analysis. Sequence analysis revealed 17 putative genes distributed randomly on eight chromosomes. Although most of the predicted proteins had typical characteristics of the ZIP protein family, the extent of their sequence similarity varied considerably. The expression patterns of <i>OsZIP1, OsZIP3</i>, and <i>OsZIP4</i>, which encode Zn²⁺ transporters in rice, were studied in the Zn-efficient and Zn-inefficient rice genotypes (IR8192 and Erjiufeng) by semi-quantitative RT-PCR analysis of roots, shoots, and panicle from the plants grown under Zn deficiency and normal conditions. <i>OsZIP1</i> was expressed only in the roots and very weakly if at all in the panicles, while the other two genes were expressed in all parts of plants under study. The Zn-deficient conditions up-regulated the expression of <i>OsZIP1, OsZIP3</i>, and <i>OsZIP4</i> in the roots and that of <i>OsZIP4</i> in the shoots of both genotypes, indicating that all these genes may participate in rice zinc nutrition. Furthermore, the expression of <i>OsZIP3</i> and <i>OsZIP4</i> was found to be much stronger in the roots of IR8192 than those of Erjiufeng, which suggests that these genes may contribute to high Zn efficiency in rice. The expression patterns and the roles of other <i>OsZIPs</i> are also discussed on the basis of the phylogenetic tree of ZIP proteins and RT-PCR analysis of the two rice genotypes with different zinc efficiency.

Metal hyperaccumulator plants accumulate and detoxify extraordinarily high concentrations of metal ions in their shoots. Metal hyperaccumulation is a fascinating phenomenon, which has interested scientists for over a century. Hyperaccumulators constitute an exceptional biological material for understanding mechanisms regulating plant metal homeostasis as well as plant adaptation to extreme metallic environments. Our understanding of metal hyperaccumulation physiology has recently increased as a result of the development of molecular tools. This review presents key aspects of our current understanding of plant metal - in particular cadmium (Cd), nickel (Ni) and zinc (Zn) - hyperaccumulation.

In this paper, we conducted a detailed analysis of the ZIP family transporter, NcZNT1, in the zinc (Zn)/cadmium (Cd) hyperaccumulating plant species, Noccaea caerulescens, formerly known as Thlaspi caerulescens. NcZNT1 was previously suggested to be the primary root Zn/Cd uptake transporter. Both a characterization of NcZNT1 transport function in planta and in heterologous systems, and an analysis of NcZNT1 gene expression and NcZNT1 protein localization were carried out. We show that NcZNT1 is not only expressed in the root epidermis, but also is highly expressed in the root and shoot vasculature, suggesting a role in long-distance metal transport. Also, NcZNT1 was found to be a plasma membrane transporter that mediates Zn but not Cd, iron (Fe), manganese (Mn) or copper (Cu) uptake into plant cells. Two novel regions of the NcZNT1 promoter were identified which may be involved in both the hyperexpression of NcZNT1 and its ability to be regulated by plant Zn status. In conclusion, we demonstrate here that NcZNT1 plays a role in Zn and not Cd uptake from the soil, and based on its strong expression in the root and shoot vasculature, could be involved in long-distance transport of Zn from the root to the shoot via the xylem.

探明超积累植物对重金属超积累作用的生理生化过程和分子机理,不仅有利于植物修复技术的发展和推广应用,而且有助于丰富植物营养学理论,是当前国内外研究热点。超积累生态型东南景天(Sedum alfredii Hance)是我国原生的锌/镉超积累植物,对锌、镉具有很强的耐性和很高的积累量,对其超积累作用的分子机理研究,有望克隆具有自主知识产权的超积累作用相关基因。转运蛋白是一类位于膜上的运输蛋白,广泛参与金属元素的吸收、转运和区隔过程。其中锌铁调节转运蛋白(ZRT, IRT-like Protein, ZIP)广泛存在于细菌、真菌、动物、植物真核生物中,被认为在锌、铁、锰、镉、铜等金属元素的根系吸收、木质部长距离运输以及地上部稳态维持中发挥重要功能。基于转录组测序,本实验室已克隆得到多个超积累生态型东南景天的ZIP家族基因,但其功能尚不明晰。在上述基础上,本论文利用酵母功能互补、洋葱表皮定位、q-RT-PCR、转基因技术等分子生物学方法,系统探究了超积累东南景天中SaZIP2和SaZIP3基因的功能,取得的主要研究结果如下：1、使用醋酸锂酵母转化法,选用野生型DY1457和DY4743、锌镉敏感型突变体△zrc1、锌吸收缺陷型突变体ZHY3、锰吸收缺陷型突变体△smfl、铁吸收缺陷型突变体DDY4分别研究SaZIP2和SaZIP3基因对镉、锌、锰、铁的转运能力。转化SaZIP2和SaZIP3均使锌镉敏感型突变体△zrc1无法在含镉培养基上生长,而对酵母镉积累量没有影响,说明SaZIP2和SaZIP3在质膜而非液泡膜上转运镉；转化SaZIP2和SaZIP3均能恢复锌吸收缺陷型突变体ZHY3吸收锌的能力,且转化SaZIP2和SaZIP3后,突变体锌含量分别提高了63.7%和251.4%,说明SaZIP2和SaZIP3均能转运锌,且SaZIP3转运锌的能力大于SaZIP2;转化SaZIP3的锰吸收缺陷型突变体△smfl锰含量较对照提高了13.4%,但并末达到野生型酵母锰含量的水平,说明表达SaZIP3只在一定程度上恢复了突变体吸收锰的功能,而SaZIP2在酵母中不具备转运锰的能力；转化SaZIP2和SaZIP3更加减弱了铁吸收缺陷型突变体DDY4吸收铁的能力,但不影响突变体铁含量。上述酵母功能互补试验说明,SaZIP2和SaZIP3均能介导镉、锌和铁的转运,且SaZIP3能介导锰的转运。2、利用Gateway技术构建GFP融合载体,采用基因枪技术将其导入洋葱表皮细胞,暗培养16h后用激光共聚焦显微镜检测绿色荧光,SaZIP2-GFP融合载体表达产物在细胞质液泡以外的地方呈现点状分布,SaZIP3-GFP融合载体表达产物在细胞膜和细胞核膜上分布,暗示SaZIP2是分布在除液泡膜以外细胞器质膜上的转运蛋白,SaZIP3是分布在细胞质膜和细胞核膜上的转运蛋白。但SaZIP2究竟分布在何种细胞器质膜上,以及SaZIP3分布在核膜上的作用还有待进一步研究。3、采用营养液培养试验,利用q-RT-PCR比较分析了锌/镉处理下ZIP2和ZIP3在两种生态型东南景天(超积累生态型HE,非超积累生态型NHE)中的表达水平,及其与东南景天的锌、镉含量的关系。长时间缺锌处理会增加NHE和HE地上部、根部ZIP2和ZIP3的表达量,加锌处理则有抑制效果,说明ZIP2和ZIP3是高亲和的锌转运蛋白。5μM镉处理NHE24h和8d, SnZIP2基因在其地上部表达分别上调2倍和7倍,在根部表达分别下调25倍和4倍,说明SnZIP2在地上部能特异性转运锌,在根部还转运镉。HE只在100 μM镉处理8d的情况下,地上部SaZIP2表达量显著上调,说明SaZIP2在HE中非特异性吸收锌,还参与了镉从根部到地上部的转运。5μM镉处理NHE24h和8d, SnZIP3基因在其地上部表达分别上调近5倍和下调近4倍,在根部表达分别上调近6倍和近8倍,说明SnZIP3在NHE中是高亲和且特异性强的锌转运蛋白。镉处理对HE地上部SaZIP3表达有抑制作用,且随着镉浓度的增加,抑制程度加强,低浓度的镉处理对HE根部SaZIP3表达有促进作用,而高浓度的镉长时间处理有抑制作用,说明在HE中SaZIP3非特异性转运锌,还参与了根部镉吸收。4、通过农杆菌介导的方法将SaZIP2和SaZIP3基因过表达载体分别转入模式植物拟南芥中,采用平板和水培的方法观察转基因拟南芥的锌、镉耐性和积累量变化,以验证SaZIP2和SaZIP3基因的功能。过表达SaZIP2或SaZIP3的拟南芥同野生型(WT)在锌、镉处理下根系生长受抑制程度一致,根系长度明显变短,说明它们并不参与锌镉的区隔解毒过程,与它们并不定位在液泡膜上的研究结果相符。50μM ZnSO4处理拟南芥一周,转基因株系间地上部锌含量没有明显差异,均显著高于WT,是WT的90倍多,根部锌含量有同样的趋势,转基因株系根部锌含量相对WT增加25%以上,转基因株系锌转运系数是WT的7倍,说明SaZIP2和SaZIP3参与锌的吸收及向地上部的转运过程。过表达SaZIP2株系的镉转运系数最高,地上部和根部镉含量均高于WT,但差异不显著,过表达SaZIP3的株系地上部和根部镉含量分别是WT的1.5倍和3倍,显著高于WT,但镉转运系数最低,说明SaZIP2参与镉向地上部的转运过程,SaZIP3参与镉的吸收过程,这与前文酵母功能互补实验和镉处理时基因表达水平研究所得结论相符,即SaZIP2和SaZIP3特异性不强,除转运锌外,在镉的吸收及转运中也有重要作用。

Abstract Methyl jasmonate (MeJA), a vital cellular regulator, mediates diverse developmental processes and defense responses against environmental stresse. Recently, a novel gasotransmitter, hydrogen sulfide (H2S), was found to have similar functions, but the interactions between H2S and MeJA in the acquisition of cadmium (Cd) tolerance have not been reported. Treating foxtail millet with 1 microM MeJA not only enhanced Cd tolerance and alleviated growth inhibitions but also decreased the contents of hydrogen peroxide, malondialdehyde and Cd in seedlings under 200 microM of Cd stress. Exogenous application of MeJA inhibited the transcript levels of the Natural Resistance-Associated Macrophage Protein (NRAMP1 and NRAMP6) and intensified Cd-induced expression of the homeostasis-related genes (MTP1, MTP12, CAX2 and ZIP4, besides HMA3). In addition, treatment with MeJA induced the production of endogenous H2S. Fumigation with sodium hydrosulfide (H2S donor) significantly enhanced MeJA-induced Cd tolerance, but this ability was weakened when H2S biosynthesis was inhibited with hydroxylamine. These results suggest that pretreatment with MeJA alleviated Cd stress and that this improvement was mediated by H2S in foxtail millet.

Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically....

Metals like manganese (Mn) and iron (Fe) are essential for metabolism, while cadmium (Cd) is toxic for virtually all living organisms. Understanding the transport of these metals is important for breeding better crops. We have identified that OsNRAMP5 contributes to Mn, Fe and Cd transport in rice. OsNRAMP5 expression was restricted to roots epidermis, exodermis, and outer layers of the cortex as well as in tissues around the xylem. OsNRAMP5 localized to the plasma membrane, and complemented the growth of yeast strains defective in Mn, Fe, and Cd transport. OsNRAMP5 RNAi (OsNRAMP5i) plants accumulated less Mn in the roots, and less Mn and Fe in shoots, and xylem sap. The suppression of OsNRAMP5 promoted Cd translocation to shoots, highlighting the importance of this gene for Cd phytoremediation. These data reveal that OsNRAMP5 contributes to Mn, Cd, and Fe transport in rice and is important for plant growth and development.

Abstract The Natural Resistance Associated Macrophage Protein (Nramp) represents a transporter family for metal ions in all organisms. Here, we functionally characterized a member of Nramp family in barley (Hordeum vulgare), HvNramp5. This member showed different expression patterns, transport substrate specificity, and cellular localization from its close homolog in rice (Oryza sativa), OsNramp5, although HvNramp5 was also localized to the plasma membrane. HvNramp5 was mainly expressed in the roots and its expression was not affected by Cd and deficiency of Zn, Cu, and Mn, but slightly up-regulated by Fe deficiency. Spatial expression analysis showed that the expression of HvNramp5 was higher in the root tips than that in the basal root regions. Furthermore, analysis with laser microdissection revealed higher expression of HvNramp5 in the outer root cell layers. HvNramp5 showed transport activity for both Mn 2+ and Cd 2+ , but not for Fe 2+ when expressed in yeast. Immunostaining with a HvNramp5 antibody showed that this protein was localized in the root epidermal cells without polarity. Knockdown of HvNramp5 in barley resulted in a significant reduction in the seedling growth at low Mn supply, but this reduction was rescued at high Mn supply. The concentration of Mn and Cd, but not other metals including Cu, Zn, and Fe, was decreased in both the roots and shoots of knockdown lines compared with the wild-type barley. These results indicate that HvNramp5 is a transporter required for uptake of Mn and Cd, but not for Fe, and that barley has a distinct uptake system from rice. 2016 American Society of Plant Biologists. All Rights Reserved.

Natural Resistance Associated Macrophage Protein (NRAMP) homologues are evolutionarily conserved divalent metal transporters. In Arabidopsis, AtNRAMP3 and AtNRAMP4 play a key role in iron nutrition of the germinating plantlet by remobilizing vacuolar Fe stores. Here we describe the molecular and physiological characterization of AtNRAMP6. AtNRAMP6 is predominantly expressed in the dry seed embryo and to a lesser extent in aerial parts. Its promoter activity is found diffusely distributed in cotyledons and hypocotyls as well as in the vascular tissue region of leaf and flower. We show that AtNRAMP6 transcript coexists with a partially spliced isoform in all tested shoot cell types. When expressed in yeast, AtNRAMP6, but not its misspliced derivative, increased sensitivity to cadmium without affecting Cd content in the cell. Likewise, Arabidopsis transgenic plants overexpressing AtNRAMP6 were hypersensitive to Cd although plant Cd content remained unchanged. Consistently, a null allele of AtNRAMP6, named nramp6-1, was more tolerant to Cd toxicity, a phenotype that was reverted by expressing AtNRAMP6 in the mutant background. We used an AtNRAMP6::HA fusion, shown to be functional in yeast, to demonstrate through immunoblot analysis of membrane fractions and immunofluorescence localization that, in yeast cells, AtNRAMP6 is targeted to a vesicular-shaped endomembrane compartment distinct from the vacuole or mitochondria. We therefore propose that AtNRAMP6 functions as an intracellular metal transporter, whose presence, when modified, is likely to affect distribution/availability of Cd within the cell.

The P 1B -ATPases are integral membrane proteins that couple ATP hydrolysis to metal cation transport. Widely distributed across all domains of life, these enzymes have been previously shown to transport copper, zinc, cobalt, and other thiophilic heavy metals. Recent data suggest that these enzymes may also be involved in nickel and/or iron transport. Here we have exploited large amounts of genomic data to examine and classify the various P 1B -ATPase subfamilies. Specifically, we have combined new methods of data partitioning and network visualization known as Transitivity Clustering and Protein Similarity Networks with existing biochemical data to examine properties such as length, speciation, and metal-binding motifs of the P 1B -ATPase subfamily sequences. These data reveal interesting relationships among the enzyme sequences of previously established subfamilies, indicate the presence of two new subfamilies, and suggest the existence of new regulatory elements in certain subfamilies. Taken together, these findings underscore the importance of P 1B -ATPases in homeostasis of nearly every biologically relevant transition metal and provide an updated framework for future studies.

The specific transport of metal ions, mediated by membrane-localized metal transporters, is of fundamental importance in all eukaryotes. Genome-wide analysis of metal transporters was undertaken, making use of whole genome sequences of the green alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii, the monocots rice and sorghum, and the dicots Arabidopsis thaliana, poplar, grapevine, as well as of the yeast Saccharomyces cerevisiae. A repertoire of 430 metal transporters was found in total across eight photosynthetic plants, as well as in S. cerevisiae. Seventy-two full-length metal transporter genes were identified in the Populus genome alone, which is the largest number of metal transporters genes identified in any single species to date. Diversification of some transporter family gene clusters appears to have occurred in a lineage-specific manner. Expression analysis of Populus metal transporters indicates that some family members show tissue-specific transcript abundance. Taken together, the data provide a picture into the diversification of these important gene families.

The Arabidopsis thaliana AtHMA1 protein is a member of the P(IB)-ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase)-type pumps that is involved in inhibition by thapsigargin. To analyze the ion specificity of AtHMA1, we performed functional complementation assays using mutant yeast strains defective in Ca(2+) homeostasis or heavy metal transport. The heterologous expression of AtHMA1 complemented the phenotype of both types of mutants and, interestingly, increased heavy metal tolerance of wild-type yeast. Biochemical analyses were performed to describe the activity of AtHMA1 in microsomal fractions isolated from complemented yeast. Zinc, copper, cadmium, and cobalt activate the ATPase activity of AtHMA1, which corroborates the results of metal tolerance assays. The outcome establishes the role of AtHMA1 in Cd(2+) detoxification in yeast and suggests that this pump is able to transport other heavy metals ions. Further analyses were performed to typify the active Ca(2+) transport mediated by AtHMA1. Ca(2+) transport displayed high affinity with an apparent K(m) of 370 nm and a V(max) of 1.53 nmol mg(-1) min(-1). This activity was strongly inhibited by thapsigargin (IC(50) = 16.74 nm), demonstrating the functionality of its SERCA-like stalk segment. In summary, these results demonstrate that AtHMA1 functions as a Ca(2+)/heavy metal pump. This protein is the first described plant P-type pump specifically inhibited by thapsigargin.

The Arabidopsis (Arabidopsis thaliana) Heavy Metal Associated3 (AtHMA3) protein belongs to the $P_{1B - 2}$ subgroup of the P-type ATPase family, which is involved in heavy metal transport. In a previous study, we have shown, using heterologous expression in the yeast Saccharomyces cerevisiae, that in the presence of toxic metals, AtHMA3 was able to phenotypically complement the cadmium/lead (Cd/Pb)-hypersensitive strain ycf1 but not the zinc (Zn)-hypersensitive strain zrc1. In this study, we demonstrate that AtHMA3 in planta is located in the vacuolar membrane, with a high expression level in guard cells, hydathodes, vascular tissues, and the root apex. Confocal imaging in the presence of the Zn/Cd fluorescent probe BTC-5N revealed that AtHMA3 participates in the vacuolar storage of Cd. A T-DNA insertional mutant was found more sensitive to Zn and Cd. Conversely, ectopic overexpression of AtHMA3 improved plant tolerance to Cd, cobalt, Pb, and Zn; Cd accumulation increased by about 2-to 3-fold in plants overexpressing AtHMA3 compared with wild-type plants. Thus, AtHMA3 likely plays a role in the detoxification of biological (Zn) and nonbiological (Cd, cobalt, and Pb) heavy metals by participating in their vacuolar sequestration, an original function for a $P_{1B - 2}$ ATPase in a multicellular eukaryote.

There is huge variability among populations of the hyperaccumulator Noccaea caerulescens (formerly Thlaspi caerulescens) in their capacity to tolerate and accumulate cadmium. To gain new insights into the mechanisms underlying this variability, we estimated cadmium fluxes and further characterized the N. caerulescens heavy metal ATPase 4 (NcHMA4) gene in three populations (two calamine, Saint-Felix-de-Pallieres, France and Prayon, Belgium; one serpentine, Puente Basadre, Spain) presenting contrasting levels of tolerance and accumulation. Cadmium uptake and translocation varied among populations in the same way as accumulation; the population with the highest cadmium concentration in shoots (Saint Felix-de-Pallieres) presented the highest capacity for uptake and translocation. We demonstrated that the four NcHMA4 copies identified in a previous study are not fixed at the species level, and that the copy truncated in the C-terminal part encodes a functional protein. NcHMA4 expression and gene copy number was lower in the serpentine population, which was the least efficient in cadmium translocation compared to the calamine populations. NcHMA4 expression was associated with the vascular tissue in all organs, with a maximum at the crown. Overall, our results indicate that differences in cadmium translocation ability of the studied populations appear to be controlled, at least partially, by NcHMA4, while the overexpression of NcHMA4 in the two calamine populations may result from convergent evolution.

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn2+/H+ antiporter of the cation diffusion facilitator family. Here we present a structurefunction analysis of AtMTP1-mediated transport and its remarkable Zn2+ selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn2+-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn2+-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn2+ into the intra-membrane Zn2+-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn2+ entry into the pore. This would allow dynamic coupling of Zn2+ transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn2+ levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP.

The maintenance of ion homeostasis in plant cells is a fundamental physiological requirement for sustainable plant growth, development and production. Plants exposed to high concentrations of heavy metals must respond in order to avoid the deleterious effects of heavy metal toxicity at the structural, physiological and molecular levels. Plant strategies for coping with heavy metal toxicity are genotype-specific and, at least to some extent, modulated by environmental conditions. There is considerable interest in the mechanisms underpinning plant metal tolerance, a complex process that enables plants to survive metal ion stress and adapt to maintain growth and development without exhibiting symptoms of toxicity. This review briefly summarizes some recent cell biological, molecular and proteomic findings concerning the responses of plant roots to heavy metal ions in the rhizosphere, metal ion-induced reactions at the cell wall-plasma membrane interface, and various aspects of heavy metal ion uptake and transport in plants via membrane transporters. The molecular and genetic approaches that are discussed are analyzed in the context of their potential practical applications in biotechnological approaches for engineering increased heavy metal tolerance in crops and other useful plants.

<h3>Background&nbsp;&nbsp;</h3><div class="normal">The homologues of human disease genes are expected to contribute to better understanding of physiological and pathogenic processes. We made use of the present availability of vertebrate genomic sequences, and we have conducted the most comprehensive comparative genomic analysis of the prion protein gene <i>PRNP</i> and its homologues, shadow of prion protein gene <i>SPRN</i> and doppel gene <i>PRND</i>, and prion testis-specific gene <i>PRNT</i> so far.

Abstract<br/>Rice (<em class="a-plus-plus">Oryza sativa</em> L. ‘Nipponbare’) cDNA subtractive suppression hybridization (SSH) libraries constructed using cadmium (Cd)-treated seedling roots were screened to isolate Cd-responsive genes. A cDNA clone, encoding the rice homolog of <em class="a-plus-plus">Metal Tolerance Protein</em> (<em class="a-plus-plus">OsMTP1</em>), was induced by Cd treatment. Plant MTPs belong to cation diffusion facilitator (CDF) protein family, which are widespread in bacteria, fungi, plants, and animals. <em class="a-plus-plus">OsMTP1</em> heterologous expression in yeast mutants showed that <em class="a-plus-plus">OsMTP1</em> was able to complement the mutant strains’ hypersensitivity to Ni, Cd, and Zn, but not other metals including Co and Mn. <em class="a-plus-plus">OsMTP1</em> expression increased tolerance to Zn, Cd, and Ni in wild-type yeast BY4741 during the exponential growth phase. OsMTP1 fused to green fluorescent protein was localized in onion epidermal cell plasma membranes, consistent with an <em class="a-plus-plus">OsMTP1</em> function in heavy metal transporting. <em class="a-plus-plus">OsMTP1</em> dsRNAi mediated by transgenic assay in rice seedlings resulted in heavy metal sensitivity and changed the heavy metal accumulation in different organs of mature rice under low-concentration heavy metal stress. Taken together, our results show that OsMTP1 is a bivalent cation transporter localized in the cell membrane, which is necessary for efficient translocation of Zn, Cd and other heavy metals, and maintain ion homeostasis in plant.<br/>

Brassica juncea is promising for metal phytoremediation, but little is known about the functional role of most metal transporters in this plant. The functional characterization of two B. juncea cation-efflux family proteins BjCET3 and BjCET4 is reported here. The two proteins are closely related to each other in amino acid sequence, and are members of Group III of the cation-efflux transporters. Heterologous expression of BjCET3 and BjCET4 in yeast confirmed their functions in exporting Zn, and possibly Cd, Co, and Ni. Yeast transformed with BjCET4 showed higher metal resistance than did BjCET3 transformed. The two BjCET-GFP fusion proteins were localized to the plasma membrane in the roots when expressed in tobacco, and significantly enhanced the plants' Cd tolerance ability. Under Cd stress, tobacco plants transformed with BjCET3 accumulated significant amounts of Cd in shoots, while maintaining similar shoot biomass production with vector-control subjects. Transformed BjCET4 tobacco plants showed significantly enhanced shoot biomass production with markedly decreased shoot Cd content. The two transporter genes have a lower basal transcript expression in B. juncea seedling tissues when grown in normal conditions than under metal-stress, however, their transcripts levels could be substantially increased by Zn, Cd, NaCl or PEG, suggesting that BjCET3 and BjCET4 may play roles in several stress conditions, roles which appear to be different from those of previous characterized cation-efflux transporters, for example, AtMTP1, BjCET2, and BjMTP1.

<a name="Abs1"></a> <i>Brassica juncea</i> L. is a Zn/Cd accumulator. To determine the physiological basis of its metal accumulation phenotype, the functional properties and role of the metal efflux transporter <i>BjCET2</i> were investigated using transgenic technology. Heterologous expression of <i>BjCET2</i> in the double mutant yeast strain <i>&#916;zrc1&#916;cot1</i> enhanced the metal tolerance of the yeast strain and led to decrease in Zn or Cd accumulation. Detection of green fluorescence from green fluorescent protein (GFP) in the root tip of transgenic tobacco further revealed that BjCET2::GFP is localized at the plasma membrane. Semi-quantitative RT-PCR analysis showed that <i>BjCET2</i> was most abundant in the root and was weakly expressed in the stem and leaves. The expression of <i>BjCET2</i> was up-regulated by heavy metals. However, exposure to low temperature, salt and drought did not affect the expression of <i>BjCET2</i>. Overexpression of <i>BjCET2</i> in transgenic <i>B. juncea</i> plants conferred heavy metal tolerance and increased Cd/Zn accumulation in the leaves. <i>BjCET2</i>-deficient <i>B. juncea</i> mediated by antisense RNA resulted in hypersensitivity to heavy metals and decreased Zn/Cd accumulation in the plants. These results suggest that the heavy metal efflux of <i>BjCET2</i> plays important roles in the metal tolerance of <i>B. juncea</i> and in Zn/Cd accumulation in <i>B. juncea</i>.

Abstract Ca(2+)/cation antiporter (CaCA) proteins are integral membrane proteins that transport Ca(2+) or other cations using the H(+) or Na(+) gradient generated by primary transporters. The CAX (for CAtion eXchanger) family is one of the five families that make up the CaCA superfamily. CAX genes have been found in bacteria, Dictyostelium, fungi, plants, and lower vertebrates, but only a small number of CAXs have been functionally characterized. In this study, we explored the diversity of CAXs and their phylogenetic relationships. The results demonstrate that there are three major types of CAXs: type I (CAXs similar to Arabidopsis thaliana CAX1, found in plants, fungi, and bacteria), type II (CAXs with a long N-terminus hydrophilic region, found in fungi, Dictyostelium, and lower vertebrates), and type III (CAXs similar to Escherichia coli ChaA, found in bacteria). Some CAXs were found to have secondary structures that are different from the canonical six transmembrane (TM) domains-acidic motif-five TM domain structure. Our phylogenetic tree indicated no evidence to support the cyanobacterial origin of plant CAXs or the classification of Arabidopsis exchangers CAX7 to CAX11. For the first time, these results clearly define the CAX exchanger family and its subtypes in phylogenetic terms. The surprising diversity of CAXs demonstrates their potential range of biochemical properties and physiologic relevance.

<a name="Abs1"></a>Sequestration mechanisms that prevent high concentrations of free metal ions from persisting in metabolically active compartments of cells are thought to be central in tolerance of plants to high levels of divalent cation metals. Expression of <i>AtCAX2</i> or <i>AtCAX4</i>, which encode divalent cation/proton antiporters, in <i>Nicotiana tabacum</i> cv. KY14 results in enhanced Cd- and Zn-selective transport into root tonoplast vesicles, and enhanced Cd accumulation in roots of plants exposed to moderate, 0.02&nbsp;&#956;M Cd in solution culture (Korenkov et al. in Planta 225:403&#8211;411, <cite>2007</cite>). Here we investigated effects of expressing <i>AtCAX2</i> and <i>AtCAX4</i> in the same lines on tolerance to growth with near-incipient toxicity levels of Cd, Zn and Mn. Less growth inhibition (higher tolerance) to all three metals was observed in <i>35S::AtCAX2</i> and <i>FS3::AtCAX4</i> expressing plants. Consistent with the tolerance observed for Cd was the finding that while root tonoplast vesicle proton pump activities of control and FS3AtCAX4 expressing plants grown in 3&nbsp;&#956;M Cd were similarly reduced, and vesicle proton leak was enhanced, root tonoplast vesicle antiporter activity of these plants remained elevated above that in controls. We suggest that CAX antiporters, unlike tonoplast proton pump and membrane integrity, are not negatively impacted by high Cd, and that supplementation of tonoplast with AtCAX compensates somewhat for reduced tonoplast proton pump and proton leak, and thereby results in sufficient vacuolar Cd sequestration to provide higher tolerance. Results are consistent with the view that CAX2 and CAX4 antiporters of tonoplast play a role in tolerance to high, toxic levels of Cd, Zn, and Mn in tobacco.

Inorganic cations play decisive roles in many cellular and physiological processes and are essential components of plant nutrition. Therefore, the uptake of cations and their redistribution must be precisely controlled. Vacuolar antiporters are important elements in mediating the intracellular sequestration of these cations. These antiporters are energized by the proton gradient across the vacuolar membrane and allow the rapid transport of cations into the vacuole. CAXs (for CAtion eXchanger) are members of a multigene family and appear to predominately reside on vacuoles. Defining CAX regulation and substrate specificity have been aided by utilising yeast as an experimental tool. Studies in plants suggest CAXs regulate apoplastic Ca2+ levels in order to optimise cell wall expansion, photosynthesis, transpiration and plant productivity. CAX studies provide the basis for making designer transporters that have been used to develop nutrient enhanced crops and plants for remediating toxic soils.

Phytoremediation is a cost-effective and minimally invasive technology to cleanse soils contaminated with heavy metals. However, few plant species are suitable for phytoremediation of metals such as cadmium (Cd). Genetic engineering offers a powerful tool to generate plants that can hyperaccumulate Cd. An Arabidopsis CAX1 mutant (CAXcd), which confers enhanced Cd transport in yeast, was ectopically expressed in petunia to evaluate whether the CAXcd expression would enhance Cd tolerance and accumulation in planta. The CAXcd-expressing petunia plants showed significantly greater Cd tolerance and accumulation than the controls. After being treated with either 50 or 100 mu M CdCl2 for 6 weeks, the CAXcd-expressing plants showed more vigorous growth compared with controls, and the transgenic plants accumulated significantly more Cd (up to 2.5-fold) than controls. Moreover, the accumulation of Cd did not affect the development and morphology of the CAXcd-expressing petunia plants until the flowering and ultimately the maturing of seeds. Therefore, petunia has the potential to serve as a model species for developing herbaceous, ornamental plants for phytoremediation. (C) 2010 Elsevier GmbH. All rights reserved.

Research into plant nutrition focuses on how plants maintain elemental differences from the surrounding environment. Classic genetic analysis has been hindered because it is not possible to accurately screen for perturbations in this disequilibrium. Recent work by Lahner and colleagues has enabled efficient screening and identification of plants that have altered elemental profiles.

<a name="Abs1"></a><i>Thlaspi caerulescens</i> (Ganges ecotype) is a known Cd hyperaccumulator, however, the ligands which coordinate to Cd ions in the leaves have not been identified. In the present study, the chemical form of Cd was investigated by using ¹¹³Cd-nuclear magnetic resonance (NMR) spectroscopy. Plants were grown hydroponically with a highly enriched ¹¹³Cd stable isotope. Measurements of ¹¹³Cd-NMR with intact leaves showed a signal at the chemical shift of around &#x2013;16&nbsp;ppm. Crude leaf sap also gave a similar chemical shift. Purification by gel filtration (Sephadex G-10), followed by cationic and anionic exchange chromatography, showed that Cd occurred only in the anionic fraction, which gave the same chemical shift as intact leaves. Further purification of the anionic fraction, combined with ¹¹³Cd- and ¹H-NMR studies, revealed that only the fraction containing malate showed a chemical shift similar to the intact leaves. These results indicate that Cd was coordinated mainly with malate in the leaves of <i>T. caerulescens</i>. The malate concentration in the leaves was not affected by increasing Cd concentration in the solution, suggesting that malate synthesis is not induced by Cd. Because the Cd-malate complex is relatively weak, we suggest that the complex forms inside the vacuoles as a result of an efficient tonoplast transport of Cd and a constitutively high concentration of malate in the vacuoles, and that the formation of the Cd-malate complex may lead to a decrease of subsequent Cd efflux to the cytoplasm.

<i>O</i>-Acetylserine (thiol) lyase (OASTL, EC 2.5.1.47) catalyzes the final step in cysteine biosynthesis. The soybean <i>GmOASTL4</i> gene was overexpressed in tobacco. Transgenic plants showed markedly increased accumulation of transcripts and higher cysteine content compared with the wild-type. Upon exposure to cadmium stress, OASTL activity and cysteine levels increased significantly in transgenic plants. Cadmium accumulation and the activity of both superoxide dismutase and catalase enzymes were enhanced in transformants. These results demonstrate that overexpression of <i>GmOASTL4</i> in tobacco can enhance cysteine levels and increase tolerance to cadmium stress.

Over the past 200 years emissions of toxic heavy metals have risen tremendously and significantly exceed those from natural sources for practically all metals. Uptake and accumulation by crop plants represents the main entry pathway for potentially health-threatening toxic metals into human and animal food. Of major concern are the metalloids arsenic (As) and selenium (Se), and the metals cadmium (Cd), mercury (Hg), and lead (Pb). This review discusses the molecular mechanisms of toxic metal accumulation in plants and algae, the responses to metal exposure, as well as our understanding of metal tolerance and its evolution. The main emphasis will be on cadmium, which is by far the most widely studied of the non-essential toxic metals/metalloids. Entry via Zn, Fe, and Ca transporters is the molecular basis of Cd uptake into plant cells. Much less is known about the partitioning of non-essential metals and about the genes underlying the enormous diversity among plants with respect to Cd accumulation in different tissues. Numerous studies have described symptoms and responses of plants upon toxic metal exposure. Mysterious are primary targets of toxicity, the degree of specificity of responses, the sensing and the signaling events that lead to transcriptional activation. All plants apparently possess a basal tolerance of toxic non-essential metals. For Cd and As, this is largely dependent on the phytochelatin pathway. Not understood is the molecular biology of Cd hypertolerance in certain plant species such as the metallophytes Thlaspi caerulescens.

During their life, plants have to cope with a variety of abiotic stresses. Cadmium is highly toxic to plants, water soluble and therefore promptly adsorbed in tissues and its presence greatly influences the entire plant metabolism. In this review, we focus on the signal pathways responsible for the sensing and transduction of the “metal signal” inside the cell, ultimately driving the activation of transcription factors and consequent expression of genes that enable plants to counteract the heavy metal stress.

Zn deficiency is among the leading health risk factors in developing countries. Breeding of Zn-enriched crops is expected to be facilitated by molecular dissection of plant Zn hyperaccumulation (i.e., the ability of certain plants to accumulate Zn to levels > 100-fold higher than normal plants). The model hyperaccumulators Arabidopsis halleri and Noccaea caerulescens share elevated nicotianamine synthase (NAS) expression relative to nonaccumulators among a core of alterations in metal homeostasis. Suppression of Ah-NAS2 by RNA interference (RNAi) resulted in strongly reduced root nicotianamine (NA) accumulation and a concomitant decrease in root-to-shoot translocation of Zn. Speciation analysis by size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry showed that the dominating Zn ligands in roots were NA and thiols. In NAS2-RNAi plants, a marked increase in Zn-thiol species was observed. Wild-type A. halleri plants cultivated on their native soil showed elemental profiles very similar to those found in field samples. Leaf Zn concentrations in NAS2-RNAi lines, however, did not reach the Zn hyperaccumulation threshold. Leaf Cd accumulation was also significantly reduced. These results demonstrate a role for NAS2 in Zn hyperaccumulation also under near-natural conditions. We propose that NA forms complexes with Zn(II) in root cells and facilitates symplastic passage of Zn(II) toward the xylem.

Abstract We describe the first complete inventory of ATP-binding cassette (ABC) proteins from a multicellular organism, the model plant Arabidopsis thaliana. By the application of several search criteria, Arabidopsis was found to contain a total of 129 open reading frames (ORFs) capable of encoding ABC proteins, of which 103 possessed contiguous transmembrane spans and were identified as putative intrinsic membrane proteins. Fifty-two of the putative intrinsic membrane proteins contained at least two transmembrane domains (TMDs) and two nucleotide-binding folds (NBFs) and could be classified as belonging to one of five subfamilies of full-molecule transporters. The other 51 putative membrane proteins, all of which were half-molecule transporters, fell into five subfamilies. Of the remaining ORFs identified, all of which encoded proteins lacking TMDs, 11 could be classified into three subfamilies. There were no obvious homologs in other organisms for 15 of the ORFs which encoded a heterogeneous group of non-intrinsic ABC proteins (NAPs). Unrooted phylogenetic analyses substantiated the subfamily designations. Notable features of the Arabidopsis ABC superfamily was the presence of a large yeast-like PDR subfamily, and the absence of genes encoding bona fide cystic fibrosis transmembrane conductance regulator (CFTR), sulfonylurea receptor (SUR), and heavy metal tolerance factor 1 (HMT1) homologs. Arabidopsis was unusual in its large allocation of ORFs (a minimum of 0.5%) to members of the ABC protein superfamily.

The ATP‐binding cassette (ABC) superfamily consists of membrane proteins that transport a wide variety of substrates across membranes. Mutations in ABC transporters cause or contribute to a number of different Mendelian disorders, including adrenoleukodystrophy, cystic fibrosis, retinal degeneration, cholesterol, and bile transport defects. In addition, the genes are involved in an increasing number of complex disorders. The proteins play essential roles in the protection of organisms from toxic metabolites and compounds in the diet and are involved in the transport of compounds across the intestine, blood–brain barrier, and the placenta. There are 48 ABC genes in the human genome divided into seven subfamilies based in gene structure, amino acid alignment, and phylogenetic analysis. These seven subfamilies are found in all other sequenced eukaryotic genomes and are of ancient origin. Further characterization of all ABC genes from humans and model organisms will lead to additional insights into normal physiology and human disease.

The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products. A consolidated nomenclature will provide much-needed clarity and a framework for future research.

We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination. We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast. DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium. Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals. These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium.

Heavy metals such as cadmium (Cd) and mercury (Hg) are toxic pollutants that are detrimental to living organisms. Plants employ a two-step mechanism to detoxify toxic ions. First, phytochelatins bind to the toxic ion, and then the metal hytochelatin complex is sequestered in the vacuole. Two ABCC-type transporters, AtABCC1 and AtABCC2, that play a key role in arsenic detoxification, have recently been identified in Arabidopsis thaliana. However, it is unclear whether these transporters are also implicated in phytochelatin-dependent detoxification of other heavy metals such as Cd(II) and Hg(II). Here, we show that atabcc1 single or atabcc1 atabcc2 double knockout mutants exhibit a hypersensitive phenotype in the presence of Cd(II) and Hg(II). Microscopic analysis using a Cd-sensitive probe revealed that Cd is mostly located in the cytosol of protoplasts of the double mutant, whereas it occurs mainly in the vacuole of wild-type cells. This suggests that the two ABCC transporters are important for vacuolar sequestration of Cd. Heterologous expression of the transporters in Saccharomyces cerevisiae confirmed their role in heavy metal tolerance. Over-expression of AtABCC1 in Arabidopsis resulted in enhanced Cd(II) tolerance and accumulation. Together, these results demonstrate that AtABCC1 and AtABCC2 are important vacuolar transporters that confer tolerance to cadmium and mercury, in addition to their role in arsenic detoxification. These transporters provide useful tools for genetic engineering of plants with enhanced metal tolerance and accumulation, which are desirable characteristics for phytoremediation.

Cadmium (Cd) and lead (Pb) are widespread pollutants that are toxic to plant growth. The expression of AtPDR8 was upregulated in cadmium- or lead-treated Arabidopsis thaliana. To test whether AtPDR8 is involved in heavy metal resistance, we examined transgenic Arabidopsis that over-expressed AtPDR8 and RNAi plants that exhibited a severely reduced AtPDR8 transcript level, as well as T-DNA insertion mutants of this ABC transporter. AtPDR8-over-expressing plants were more resistant to Cd 2+ or Pb 2+ than the wild-type and had lower Cd contents. In contrast, AtPDR8 RNAi transgenic plants and T-DNA insertion lines were more sensitive to Cd 2+ or Pb 2+ compared to wild-type plants and had higher Cd contents. The GFP tPDR8 protein was targeted to the plasma membrane, and GUS activity was present in most cells but strongest in the root hair and epidermal cells. Cd extrusion was higher in the AtPDR8-over-expressing plants in a flux assay using isolated protoplasts and radioactive 109 Cd, and was lower in the RNAi transgenic plants than in the wild-type. Together, these data strongly support a role for AtPDR8 as an efflux pump of Cd 2+ or Cd conjugates at the plasma membrane of Arabidopsis cells. As AtPDR8 has been suggested to be involved in the pathogen response and in the transport of chemicals that mediate pathogen resistance, this ABC protein is likely to transport a very broad range of substrates.

AtATM3, an ATP-binding cassette transporter of Arabidopsis (Arabidopsis thaliana), is a mitochondrial protein involved in the biogenesis of iron-sulfur clusters and iron homeostasis in plants. Our gene expression analysis showed that AtATM3 is upregulated in roots of plants treated with cadmium [Cd(II)] or lead (II); hence, we investigated whether this gene is involved in heavy metal tolerance. We found that AtATM3-overexpressing plants were enhanced in resistance to Cd, whereas atatm3 mutant plants were more sensitive to Cd than their wild-type controls. Moreover, atatm3 mutant plants expressing 35S promoter-driven AtATM3 were more resistant to Cd than wild-type plants. Since previous reports often showed that the cytosolic glutathione level is positively correlated with heavy metal resistance, we measured nonprotein thiols (NPSH) in these mutant plants. Surprisingly, we found that atatm3 contained more NPSH than the wild type under normal conditions. AtATM3-overexpressing plants did not differ under normal conditions, but contained less NPSH than wild-type plants when exposed to Cd(II). These results suggest a role for AtATM3 in regulating cellular NPSH level, a hypothesis that was further supported by our gene expression study. Genetic or pharmacological inhibition of glutathione biosynthesis led to the elevated expression of AtATM3, whereas expression of the glutathione synthase gene GSH1 was increased under Cd(II) stress and in the atatm3 mutant. Because the closest homolog of AtATM3 in fission yeast (Schizosaccharomyces pombe), HMT1, is a vacuolar membrane-localized phytochelatin-Cd transporter, it is tempting to speculate that glutathione-Cd(II) complexes formed in the mitochondria are exported by AtATM3. In conclusion, our data show that AtATM3 contributes to Cd resistance and suggest that it may mediate transport of glutamine synthetase-conjugated Cd(II) across the mitochondrial membrane.

Frequently, crop plants do not take up adequate amounts of iron from the soil, leading to chlorosis, poor yield and decreased nutritional quality. Extremely limited soil bioavailability of iron has led plants to evolve two distinct uptake strategies: chelation, which is used by the world's principal grain crops; and reduction, which is used by other plant groups. The chelation strategy involves extrusion of low-molecular-mass secondary amino acids (mugineic acids) known as 'phytosiderophores' which chelate sparingly soluble iron. The Fe(III)-phytosiderophore complex is then taken up by an unknown transporter at the root surface. The maize yellow stripe1 (ys1) mutant is deficient in Fe(III)-phytosiderophore uptake, therefore YS1 has been suggested to be the Fe(III)-phytosiderophore transporter. Here we show that ys1 is a membrane protein that mediates iron uptake. Expression of YS1 in a yeast iron uptake mutant restores growth specifically on Fe(III)-phytosiderophore media. Under iron-deficient conditions, ys1 messenger RNA levels increase in both roots and shoots. Cloning of ys1 is an important step in understanding iron uptake in grasses, and has implications for mechanisms controlling iron homeostasis in all plants.

Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. These are important candidate genes to study in metal tolerant and accumulator plants for their potential use in environmental clean up. We coupled a degenerate primer-based RT-PCR approach with a molecular fingerprinting technique based on amplified rDNA restriction analysis (ARDRA) to identify novel ESTs corresponding to heavy metal transporters from metal accumulator Brassica juncea. We utilized this technique to clone several family members of natural resistance-associated macrophage proteins (NRAMP) and yellow stripe-like proteins (YSL) in a high throughput manner to distinguish between closely related isoforms and/or allelic variants from the allopolyploid B. juncea. Partial clones of 23 Brassica juncea NRAMPs and 27 YSLs were obtained with similarity to known Arabidopsis thaliana and Noccaea (Thlaspi) caerulescens NRAMP and YSL genes. The cloned transporters showed Brassica-specific changes in domains, which can have important functional consequences. Semi-quantitative RT-PCR-based expression analysis of chosen members indicated that even closely related isoforms/allelic variants of BjNRAMP and BjYSL have distinct tissue-specific and metal-dependent expressions which might be essential for adaptive fitness and heavy metal tolerance. Consistent to this, BjYSL6.1 and BjYSL5.8 were found to show elevated expressions specifically in cadmium-treated shoots and lead-treated roots of B. juncea, respectively.

BjYSL7 encodes a plasma-localized metal-NA transporter and has transport Fe(II)-NA complexes activity. BjYSL7 is involved in the transport of Cd and Ni from roots to shoots.<br/>Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. In this study, we isolated a novel member of the yellow stripe-like (YSL) gene family BjYSL7 from the hyperaccumulator Brassica juncea. BjYSL7 is composed of 688 amino acids with 12 putative transmembrane domains and is over 90 % identical to TcYSL7 and AtYSL7. Real-time PCR analysis revealed that BjYSL7 mRNA was mainly expressed in the stem under normal condition. The expression of BjYSL7 was found to be up-regulated by 127.1-, 12.7-, and 3.4-fold in roots and 6.5-, 4.3-, and 2.8-fold in shoots under FeSO4, NiCl2, and CdCl2 stresses, respectively. We have demonstrated that BjYSL7 is a Fe(II)-NA influx transporter by yeast functional complementation. Moreover, a BjYSL7::enhanced green fluorescent protein (EGFP) fusion localized to the plasma membrane of onion epidermal cells. The BjYSL7-overexpressing transgenic tobacco plants exhibited longer root lengths, lower relative inhibition rate of lengths and superior root hair development compared to that of wild-type (WT) plants in the presence of CdCl2 and NiCl2. Furthermore, the concentrations of Cd and Ni in shoots of BjYSL7-overexpressing plants are significantly higher than that of WT plants. Compared with WT plants, BjYSL7-overexpressing plants exhibited Fe concentrations that were higher in the shoots and seeds and lower in the roots. Taken together, these results suggest that BjYSL7 might be involved in the transport of Fe, Cd and Ni to the shoot and improving heavy metal resistance in plants.