Heavy Metal ATPase Research Articles

Molecular mechanisms of phosphate and zinc signalling crosstalk in plants: Phosphate and zinc loading into root xylem in Arabidopsis

Inorganic phosphate (Pi) and zinc (Zn) are an essential macro- and micronutrients for plant survival. Control of Pi and Zn content in tissues is of major importance for normal plant growth and development. Zn deficiency typically leads to Pi over-accumulation in shoots (and vice versa), signifying the presence of complex interactions that link the homeostatic regulation of these two nutrients. Despite their primary importance, the molecular bases of these interactions remains poorly understood. Recent research has placed the co-regulation of these two elements at a limiting step in Pi and Zn distribution within plants, e.g. the loading of Pi and Zn into root xylem. In Arabidopsis thaliana, this process mainly involves members of the Phosphate 1 (PHO1 and PHO1;H1) family (for Pi) and the heavy metal ATPases protein (HMA2 and HMA4) family (for Zn). This review examines recent progress in determining the molecular mechanisms that regulate the loading of Pi and Zn into root xylem, by individually describing these specific genes. The first molecular evidence for their signalling crosstalk at this particular step of their transport in plants is also presented, with an emerging role for PHO1;H3. This recent progress is important for biotechnological and agronomic strategies aimed at enhancing Pi and Zn transfer to the aerial part of plants.

Arabidopsis HY1 Confers Cadmium Tolerance by Decreasing Nitric Oxide Production and Improving Iron Homeostasis

Up-regulation of the gene that encodes intracellular heme oxygenase 1 (HO1) benefits plants under cadmium (Cd(2+)) stress; however, the molecular mechanisms remain unclear. Here, we elucidate the role of Arabidopsis HY1 (AtHO1) in Cd(2+) tolerance by using genetic and molecular approaches. Analysis of two HY1 null mutants, three HY1 overexpression lines, HO double or triple mutants, as well as phyA and phyB mutants revealed the specific hypersensitivity of hy1 to Cd(2+) stress. Supplementation with two enzymatic by-products of HY1, carbon monoxide (CO) and iron (Fe, especially), rescued the Cd(2+)-induced inhibition of primary root (PR) elongation in hy1-100. The mutation of HY1, which exhibited lower glutathione content than Col-0 in root tissues, was able to induce nitric oxide (NO) overproduction, Cd(2+) accumulation, and severe Fe deficiency in root tissues. However, the contrasting responses appeared in 35S:HY1-4. Additionally, reduced levels of Ferric Reduction Oxidase 2 (FRO2) and Iron-Regulated Transporter 1 (IRT1) transcripts, and increased levels of Heavy Metal ATPase 2/4 (HMA2/4) transcripts bolster the notion that HY1 up-regulation ameliorates Fe deficiency, and might increase Cd(2+) exclusion. Taken together, these results showed that HY1 plays a common link in Cd(2+) tolerance by decreasing NO production and improving Fe homeostasis in Arabidopsis root tissues.

How do heavy metal ATPases contribute to hyperaccumulation?

AbstractPlants contain metal chelators and transporters that regulate metal homeostasis. The HMA3 and HMA4 genes encode proteins belonging to the PIB‐type. Recently, some studies on the characterization of the HMA2 gene from several plants for potential application in phytoremediation have been reported. This ATPase family of integral membrane transporter proteins that transport transition metals are probably involved in mediating metal‐resistant and metal‐hyperaccumulating traits. While HMA3 may contribute to metal detoxification by sequestering Cd into the vacuole, HMA4 acts as a physiological master switch during the process of metal hyperaccumulation, and HMA2 and HMA4 play roles in root to shoot metal translocation. The focus of this review is the discussion on the different functions of HMA3, HMA4, and HMA2 in various plant species. It is hypothesized that a refined distinction and description of the roles of several types of these metal transporters in plants will be essential for the development of schemes to genetically modify plants that accumulate specific metals, with subsequent use in either phytoremediation or in improving human nutrition. Enhanced expression of HMA3 and HMA4 is a prerequisite for hyperaccumulation and hyperresistance in hyperaccumulators. Through genetic engineering, these genes are able to generate nontoxic, metal‐free crops to produce safe food.

Silicon mitigates heavy metal stress by regulating P-type heavy metal ATPases, Oryza sativalow silicon genes, and endogenous phytohormones

BackgroundSilicon (Si) application has been known to enhance the tolerance of plants against abiotic stresses. However, the protective mechanism of Si under heavy metals contamination is poorly understood. The aim of this study was to assess the role of Si in counteracting toxicity due to cadmium (Cd) and copper (Cu) in rice plants (Oryza sativa).ResultsSi significantly improved the growth and biomass of rice plants and reduced the toxic effects of Cd/Cu after different stress periods. Si treatment ameliorated root function and structure compared with non-treated rice plants, which suffered severe root damage. In the presence of Si, the Cd/Cu concentration was significantly lower in rice plants, and there was also a reduction in lipid peroxidation and fatty acid desaturation in plant tissues. The reduced uptake of metals in the roots modulated the signaling of phytohormones involved in responses to stress and host defense, such as abscisic acid, jasmonic acid, and salicylic acid. Furthermore, the low concentration of metals significantly down regulated the mRNA expression of enzymes encoding heavy metal transporters (OsHMA2 and OsHMA3) in Si-metal-treated rice plants. Genes responsible for Si transport (OsLSi1 and OsLSi2), showed a significant up-regulation of mRNA expression with Si treatment in rice plants.ConclusionThe present study supports the active role of Si in the regulation of stresses from heavy metal exposure through changes in root morphology.

Many rivers to cross: the journey of zinc from soil to seed

An important goal of micronutrient biofortification is to enhance the amount of bioavailable zinc in the edible seed of cereals and more specifically in the endosperm. The picture is starting to emerge for how zinc is translocated from the soil through the mother plant to the developing seed. On this journey, zinc is transported from symplast to symplast via multiple apoplastic spaces. During each step, zinc is imported into a symplast before it is exported again. Cellular import and export of zinc requires passage through biological membranes, which makes membrane-bound transporters of zinc especially interesting as potential transport bottlenecks. Inside the cell, zinc can be imported into or exported out of organelles by other transporters. The function of several membrane proteins involved in the transport of zinc across the tonoplast, chloroplast or plasma membranes are currently known. These include members of the ZIP (ZRT-IRT-like Protein), and MTP (Metal Tolerance Protein) and heavy metal ATPase (HMA) families. An important player in the transport process is the ligand nicotianamine that binds zinc to increase its solubility in living cells and in this way buffers the intracellular zinc concentration.

Inactivation of two newly identified tobacco heavy metal ATPases leads to reduced Zn and Cd accumulation in shoots and reduced pollen germination.

Cadmium (Cd) is a non-essential heavy metal, which is classified as a "known human carcinogen" by the International Agency for Research on Cancer (IARC). Understanding the mechanisms controlling Cd distribution in planta is essential to develop phytoremediation approaches as well as for food safety. Unlike most other plants, tobacco (Nicotiana tabacum) plants translocate most of the Cd taken up from the soil, out of the roots and into the shoots, leading to high Cd accumulation in tobacco shoots. Two orthologs of the Arabidopsis thaliana HMA2 and HMA4 Zn and Cd ATPases that are responsible for zinc (Zn) and Cd translocation from roots to shoots were identified in tobacco and sequenced. These genes, named NtHMAα and NtHMAβ, were more highly expressed in roots than in shoots. NtHMAα was expressed in the vascular tissues of both roots and leaves as well as in anthers. No visual difference was observed between wild-type plants and plants in which the NtHMAα and NtHMAβ genes were either mutated or silenced. These mutants showed reduced Zn and Cd accumulation in shoots as well as increased Cd tolerance. When both NtHMA genes were silenced, plant development was altered and pollen germination was severely impaired due to Zn deficiency. Interestingly, seeds from these lines also showed decreased Zn concentration but increased iron (Fe) concentration.

Hard selective sweep and ectopic gene conversion in a gene cluster affording environmental adaptation.

Among the rare colonizers of heavy-metal rich toxic soils, Arabidopsis halleri is a compelling model extremophile, physiologically distinct from its sister species A. lyrata, and A. thaliana. Naturally selected metal hypertolerance and extraordinarily high leaf metal accumulation in A. halleri both require Heavy Metal ATPase4 (HMA4) encoding a PIB-type ATPase that pumps Zn2+ and Cd2+ out of specific cell types. Strongly enhanced HMA4 expression results from a combination of gene copy number expansion and cis-regulatory modifications, when compared to A. thaliana. These findings were based on a single accession of A. halleri. Few studies have addressed nucleotide sequence polymorphism at loci known to govern adaptations. We thus sequenced 13 DNA segments across the HMA4 genomic region of multiple A. halleri individuals from diverse habitats. Compared to control loci flanking the three tandem HMA4 gene copies, a gradual depletion of nucleotide sequence diversity and an excess of low-frequency polymorphisms are hallmarks of positive selection in HMA4 promoter regions, culminating at HMA4-3. The accompanying hard selective sweep is segmentally eclipsed as a consequence of recurrent ectopic gene conversion among HMA4 protein-coding sequences, resulting in their concerted evolution. Thus, HMA4 coding sequences exhibit a network-like genealogy and locally enhanced nucleotide sequence diversity within each copy, accompanied by lowered sequence divergence between paralogs in any given individual. Quantitative PCR corroborated that, across A. halleri, three genomic HMA4 copies generate overall 20- to 130-fold higher transcript levels than in A. thaliana. Together, our observations constitute an unexpectedly complex profile of polymorphism resulting from natural selection for increased gene product dosage. We propose that these findings are paradigmatic of a category of multi-copy genes from a broad range of organisms. Our results emphasize that enhanced gene product dosage, in addition to neo- and sub-functionalization, can account for the genomic maintenance of gene duplicates underlying environmental adaptation.

Preferential Delivery of Zinc to Developing Tissues in Rice Is Mediated by P-Type Heavy Metal ATPase OsHMA2

Developing tissues such as meristems and reproductive organs require high zinc, but the molecular mechanisms of how zinc taken up by the roots is preferentially delivered to these tissues with low transpiration are unknown. Here, we report that rice (Oryza sativa) heavy metal ATPase2 (OsHMA2), a member of P-type ATPases, is involved in preferential delivery of zinc to the developing tissues in rice. OsHMA2 was mainly expressed in the mature zone of the roots at the vegetative stage, but higher expression was also found in the nodes at the reproductive stage. The expression was unaffected by either zinc deficiency or zinc excess. OsHMA2 was localized at the pericycle of the roots and at the phloem of enlarged and diffuse vascular bundles in the nodes. Heterologous expression of OsHMA2 in yeast (Saccharomyces cerevisiae) showed influx transport activity for zinc as well as cadmium. Two independent Tos17 insertion lines showed decreased zinc concentration in the crown root tips, decreased concentration of zinc and cadmium in the upper nodes and reproductive organs compared with wild-type rice. Furthermore, a short-term labeling experiment with (67)Zn showed that the distribution of zinc to the panicle and uppermost node I was decreased, but that, to the lower nodes, was increased in the two mutants. Taken together, OsHMA2 in the nodes plays an important role in preferential distribution of zinc as well as cadmium through the phloem to the developing tissues.

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.

Zinc uptake and radial transport in roots of Arabidopsis thaliana: a modelling approach to understand accumulation.

Zinc uptake in roots is believed to be mediated by ZIP (ZRT-, IRT-like proteins) transporters. Once inside the symplast, zinc is transported to the pericycle, where it exits by means of HMA (heavy metal ATPase) transporters. The combination of symplastic transport and spatial separation of influx and efflux produces a pattern in which zinc accumulates in the pericycle. Here, mathematical modelling was employed to study the importance of ZIP regulation, HMA abundance and symplastic transport in creation of the radial pattern of zinc in primary roots of Arabidopsis thaliana. A comprehensive one-dimensional dynamic model of radial zinc transport in roots was developed and used to conduct simulations. The model accounts for the structure of the root consisting of symplast and apoplast and includes effects of water flow, diffusion and cross-membrane transport via transporters. It also incorporates the radial geometry and varying porosity of root tissues, as well as regulation of ZIP transporters. Steady-state patterns were calculated for various zinc concentrations in the medium, water influx and HMA abundance. The experimentally observed zinc gradient was reproduced very well. An increase of HMA or decrease in water influx led to loss of the gradient. The dynamic behaviour for a change in medium concentration and water influx was also simulated showing short adaptation times in the range of seconds to minutes. Slowing down regulation led to oscillations in expression levels, suggesting the need for rapid regulation and existence of buffering agents. The model captures the experimental findings very well and confirms the hypothesis that low abundance of HMA4 produces a radial gradient in zinc concentration. Surprisingly, transpiration was found also to be a key parameter. The model suggests that ZIP regulation takes place on a comparable timescale as symplastic transport.

Differences in root-to-shoot Cd and Zn translocation and by HMA3 and 4 could influence chlorophyll and anthocyanin content in Arabidopsis Ws and Col-0 ecotypes under excess metals

Judging from the ecotypic variability in Arabidopsis thaliana L., Columbia-0 (Col-0) appears to be less tolerant to cadmium (Cd) than the Wassilewskija (Ws) ecotype that possesses the full-length Heavy Metal ATPase3 (HMA3) cDNA. In this study, the Ws and Col-0 were tested to determine toxic metal response between Ws and Col-0 due to AtHMA3 point mutation and/or other factors. The growth inhibition of Col-0 mediated by Cd and zinc (Zn) was more serious than the inhibition of Ws, while no significant difference was evident by lead (Pb) and cobalt (Co). In the presence of Cd stress, chlorosis in leaves of Col-0 was more serious than the Ws ecotype. When grown under hydroponic culture containing 500 µM Zn, leaves of Col-0 showed a remarkable increase in the anthocyanin content in a dose-dependent manner and the expression of genes encoding enzymes involved in anthocyanin synthesis in the leaves. The rate of root-to-shoot translocation of Cd and Zn in the Col-0 was 2 times higher when compared with the Ws, whereas roots of the Col-0 accumulated 2 times lower Cd and Zn concentrations than those of the Ws. Real-time polymerase chain reaction (PCR) analyses indicated that not only the alteration of the expression of HMA3 but also of the HMA4 was responsible for the root-to-shoot translocation of toxic metals. The results demonstrate that the Col-0 is readily translocating Cd and Zn to the aerial parts but not the Ws, thereby induce the alteration of phenotype in leaf color.

Variation in HMA4 gene copy number and expression among Noccaea caerulescens populations presenting different levels of Cd tolerance and accumulation

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-Félix-de-Pallières, 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 Félix-de-Pallières) 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.

Phytoextraction of Zinc: Physiological and Molecular Mechanism

Zinc is an essential trace element, necessary for plants, animals, and microorganisms. Zn is required for many enzymes as a catalytic cofactor, for photosynthetic CO2 fixation, and in maintaining the integrity of bio-membranes. However, Zn is potentially toxic when accumulated beyond cellular needs. Phytoextraction technique, which is a part of phytoremediation, has opened new avenues for remediation of Zn-contaminated places. Hyperaccumulators like Thlaspi caerulescens and Arabidopsis halleri have been identified, which can accumulate up to 40,000 mg kg−1 Zn in the aerial parts of the plant body. Carboxylic acids, primarily malate, citrate, and oxalate, and amino acids are found to play an important role in Zn hyperaccumulation. Transmembrane metal transporters are assumed to play a key role in Zn metal uptake, xylem loading, and vacuolar sequestration. Members of CDF (cation diffusion facilitator) and ZIP (zinc-regulated transporter, iron-regulated transporter like protein) family have been implicated in Zn-metal-tolerance mechanisms. A potential metal-binding motif, containing multiple histidine residues, is found in the variable regions of almost all of the ZIP family, including ZIP1, ZIP2, ZIP4, ZRT1, and ZRT2. Overexpression of some Zn metal transporter genes like TcZNT1 (Thlaspi caerulescens Zn transporter1), TcHMA4 (Thlaspi caerulescens heavy metal ATPase) in Thlaspi caerulescens, AhMTP1;3 (Arabidopsis halleri metal transporter1;3) in Arabidopsis halleri, and PtdMTP1(Poplar metal transporter1) from a hybrid poplar confer Zn hypertolerance in Thlaspi, Arabidopsis, and Poplar plant species.

Mutations in Rice (Oryza sativa) Heavy Metal ATPase 2 (OsHMA2) Restrict the Translocation of Zinc and Cadmium

Widespread soil contamination with heavy metals has fostered the need for plant breeders to develop new crops that do not accumulate heavy metals. Metal-transporting transmembrane proteins that transport heavy metals across the plant plasma membrane are key targets for developing these new crops. Oryza sativa heavy metal ATPase 3 (OsHMA3) is known to be a useful gene for limiting cadmium (Cd) accumulation in rice. OsHMA2 is a close homolog of OsHMA3, but the function of OsHMA2 is unknown. To gain insight into the function of OsHMA2, we analyzed three Tos17 insertion mutants. The translocation ratios of zinc (Zn) and Cd were clearly lower in all mutants than in the wild type, suggesting that OsHMA2 is a major transporter of Zn and Cd from roots to shoots. By comparing each allele in the OsHMA2 protein structure and measuring the Cd translocation ratio, we identified the C-terminal region as essential for Cd translocation into shoots. Two alleles were identified as good material for breeding rice that does not contain Cd in the grain but does contain some Zn, and that grows normally.

P-type ATPases at a glance

P-type ATPases are a large family of integral membrane transporters that are of vital importance in all kingdoms of life. In eponymous distinction from the other main classes of transport ATPase – the FF1 (F-), the vacuolar (V-) and the ATP-binding cassette (ABC-) type – the P-type ATPases form

Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance in a Cd‐hyperaccumulating ecotype of Thlaspi caerulescens

Cadmium (Cd) is a highly toxic heavy metal for plants, but several unique Cd-hyperaccumulating plant species are able to accumulate this metal to extraordinary concentrations in the aboveground tissues without showing any toxic symptoms. However, the molecular mechanisms underlying this hypertolerance to Cd are poorly understood. Here we have isolated and functionally characterized an allelic gene, TcHMA3 (heavy metal ATPase 3) from two ecotypes (Ganges and Prayon) of Thlaspi caerulescens contrasting in Cd accumulation and tolerance. The TcHMA3 alleles from the higher (Ganges) and lower Cd-accumulating ecotype (Prayon) share 97.8% identity, and encode a P(1B)-type ATPase. There were no differences in the expression pattern, cell-specificity of protein localization and transport substrate-specificity of TcHMA3 between the two ecotypes. Both alleles were characterized by constitutive expression in the shoot and root, a tonoplast localization of the protein in all leaf cells and specific transport activity for Cd. The only difference between the two ecotypes was the expression level of TcHMA3: Ganges showed a sevenfold higher expression than Prayon, partly caused by a higher copy number. Furthermore, the expression level and localization of TcHMA3 were different from AtHMA3 expression in Arabidopsis. Overexpression of TcHMA3 in Arabidopsis significantly enhanced tolerance to Cd and slightly increased tolerance to Zn, but did not change Co or Pb tolerance. These results indicate that TcHMA3 is a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens.

Amino Acid Features of PIB-ATPase Heavy Metal Transporters Enabling small Numbers of Organisms to Cope with Heavy Metal Pollution

Phytoremediation refers to the use of plants for extraction and detoxification of pollutants, providing a new and powerful weapon against a polluted environment. In some plants, such as Thlaspi spp, heavy metal ATPases are involved in overall metal ion homeostasis and hyperaccumulation. P1B-ATPases pump a wide range of cations, especially heavy metals, across membranes against their electrochemical gradients. Determination of the protein characteristics of P1B-ATPases in hyperaccumulator plants provides a new opportuntity for engineering of phytoremediating plants. In this study, using diverse weighting and modeling approaches, 2644 protein characteristics of primary, secondary, and tertiary structures of P1B-ATPases in hyperaccumulator and nonhyperaccumulator plants were extracted and compared to identify differences between proteins in hyperaccumulator and nonhyperaccumulator pumps. Although the protein characteristics were variable in their weighting, tree and rule induction models; glycine count, frequency of glutamine-valine, and valine-phenylalanine count were the most important attributes highlighted by 10, five, and four models, respectively. In addition, a precise model was built to discriminate P1B-ATPases in different organisms based on their structural protein features. Moreover, reliable models for prediction of the hyperaccumulating activity of unknown P1B-ATPase pumps were developed. Uncovering important structural features of hyperaccumulator pumps in this study has provided the knowledge required for future modification and engineering of these pumps by techniques such as site-directed mutagenesis.

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn(2+) nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn(2+) and Cd(2+) chelator with capacity to bind 10 Zn(2+) ions per C terminus. When AtHMA4 is expressed in a Zn(2+)-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn(2+) and Cd(2+) tolerance and lowered Zn(2+) and Cd(2+) content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn(2+) and Cd(2+) chelator (sensor) and as a regulator of the efficiency of Zn(2+) and Cd(2+) export. The identification of a post-translational handle on Zn(2+) and Cd(2+) transport efficiency opens new perspectives for regulation of Zn(2+) nutrition and tolerance in eukaryotes.

OsHMA3, a P1B‐type of ATPase affects root‐to‐shoot cadmium translocation in rice by mediating efflux into vacuoles

• The cadmium (Cd) over-accumulating rice (Oryza sativa) cv Cho-Ko-Koku was previously shown to have an enhanced rate of root-to-shoot Cd translocation. This trait is controlled by a single recessive allele located at qCdT7. • In this study, using positional cloning and transgenic strategies, heavy metal ATPase 3 (OsHMA3) was identified as the gene that controls root-to-shoot Cd translocation rates. The subcellular localization and Cd-transporting activity of the gene products were also investigated. • The allele of OsHMA3 that confers high root-to-shoot Cd translocation rates (OsHMA3mc) encodes a defective P(1B) -ATPase transporter. OsHMA3 fused to green fluorescent protein was localized to vacuolar membranes in plants and yeast. An OsHMA3 transgene complemented Cd sensitivity in a yeast mutant that lacks the ability to transport Cd into vacuoles. By contrast, OsHMA3mc did not complement the Cd sensitivity of this yeast mutant, indicating that the OsHMA3mc transport function was lost. • We propose that the root cell cytoplasm of Cd-overaccumulating rice plants has more Cd available for loading into the xylem as a result of the lack of OsHMA3-mediated transportation of Cd to the vacuoles. This defect results in Cd translocation to the shoots in higher concentrations. These data demonstrate the importance of vacuolar sequestration for Cd accumulation in rice.

Phylogenetic analysis of heavy-metal ATPases in fungi and characterization of the copper-transporting ATPase of Cochliobolus heterostrophus

We performed a phylogenetic analysis of heavy-metal ATPases (HMAs) in fungi and found that HMAs can be divided into three groups, A, B, and C. Group A is predicted to deliver copper ions to copper-containing proteins, while Groups B and C are thought to function as cell-membrane copper-efflux pumps. Furthermore, Groups B and C consist of fungal-specific HMAs, while Group A consists of fungal orthologues that have been well conserved in eukaryotes. We also cloned and characterized a Group A-type HMA gene ( i.e., ChCcc2) of the filamentous plant pathogen, Cochliobolus heterostrophus. Mutation of ChCcc2 severely affected growth, pigmentation, conidiation, and colonial morphology. Activity of the copper-containing protein, laccase, was also lost in ChCcc2 mutants, suggesting that ChCCC2 plays an important role in growth and morphology by activating various copper-containing proteins in C. heterostrophus.