Ideal Cereals With Lower Arsenic and Cadmium by Accurately Enhancing Vacuolar Sequestration Capacity.

Cereals are a staple food for many people around the world; however, they are also a major dietary source of toxic metal(loid)s. Many agricultural regions throughout the world are contaminated with toxic metal(loid)s, which can accumulate to high levels in the grains of cereals cultivated in these regions, posing serious health risks to consumers. Arsenic (As) and cadmium (Cd) are efficiently accumulated in cereals through metal transport pathways. Therefore, there is an urgent need to develop crops that contain greatly reduced levels of toxic metal(loid)s. Vacuolar sequestration of toxic metal(loid)s is a primary strategy for reducing toxic metal(loid)s in grains. However, until recently, detailed strategies and mechanisms for reducing toxic metal(loid)s in grain were limited by the lack of experimental data. New strategies to reduce As and Cd in grain by enhancing vacuolar sequestration in specific tissues are critical to develop crops that lower the daily intake of As and Cd, potentially improving human health. This review provides insights and strategies for developing crops with strongly reduced amounts of toxic metal(loid)s without jeopardizing agronomic traits.

Engineering rice with lower grain arsenic.

Arsenic (As) is a poisonous element that causes severe skin lesions and cancer in humans. Rice (Oryza sativa L.) is a major dietary source of As in humans who consume this cereal as a staple food. We hypothesized that increasing As vacuolar sequestration would inhibit its translocation into the grain and reduce the amount of As entering the food chain. We developed transgenic rice plants expressing two different vacuolar As sequestration genes, ScYCF1 and OsABCC1, under the control of the RCc3 promoter in the root cortical and internode phloem cells, along with a bacterial γ-glutamylcysteine synthetase driven by the maize UBI promoter. The transgenic rice plants exhibited reduced root-to-shoot and internode-to-grain As translocation, resulting in a 70% reduction in As accumulation in the brown rice without jeopardizing agronomic traits. This technology could be used to reduce As intake, particularly in populations of South East Asia suffering from As toxicity and thereby improve human health.

Root avoidance of toxic metals requires the GeBP-LIKE 4 transcription factor in Arabidopsis thaliana.

Plants reorganize their root architecture to avoid growth into unfavorable regions of the rhizosphere. In a screen based on chimeric repressor gene-silencing technology, we identified the Arabidopsis thaliana GeBP-LIKE 4 (GPL4) transcription factor as an inhibitor of root growth that is induced rapidly in root tips in response to cadmium (Cd). We tested the hypothesis that GPL4 functions in the root avoidance of Cd by analyzing root proliferation in split medium, in which only half of the medium contained toxic concentrations of Cd. The wild-type (WT) plants exhibited root avoidance by inhibiting root growth in the Cd side but increasing root biomass in the control side. By contrast, GPL4-suppression lines exhibited nearly comparable root growth in the Cd and control sides and accumulated more Cd in the shoots than did the WT. GPL4 suppression also altered the root avoidance of toxic concentrations of other essential metals, modulated the expression of many genes related to oxidative stress, and consistently decreased reactive oxygen species concentrations. We suggest that GPL4 inhibits the growth of roots exposed to toxic metals by modulating reactive oxygen species concentrations, thereby allowing roots to colonize noncontaminated regions of the rhizosphere.

Tilianin Inhibits MUC5AC Expression Mediated Via Down-Regulation of EGFR-MEK-ERK-Sp1 Signaling Pathway in NCI-H292 Human Airway Cells.

In the human airway, mucus exists to protect the respiratory system as a primary barrier of the innate immune system. However, hyperexpressed mucus limits airflow, resulting in a decrease of lung function. Among more than 20 mucin family members, MUC5AC and MUC5B are major glycoproteins in human airway mucus. The epidermal growth factor receptor (EGFR) signaling pathway is one of the mechanisms of these mucins expression and specificity protein-1 (Sp1) transcription factor is the downstream signal of this pathway, playing pivotal roles in mucin expression. Even though there are some drugs for treating mucus hypersecretion, no drug has proven effects on humans. We found that the flavonoid tilianin regulated MUC5AC expression and also inhibited Sp1 phosphorylation. In this study, we investigated how tilianin would modulate EGFR signaling and regulate mucin production. In conclusion, tilianin inhibited MUC5AC expression mediated via modulating the EGFRMEK-ERK-Sp1 signaling pathway in NCI-H292 human airway epithelial cells. This study may provide the basis for the novel treatment of mucus hypersecretion.

Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content.

Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid-targeted 2-lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn-2 position of a 2-lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0-CoA to 18:1-CoA as an acyl donor. Fluorescent protein-tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen-deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid-targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.

Arsenic Uptake and Translocation in Plants.

Arsenic (As) is a highly toxic metalloid that is classified as a non-threshold class-1 carcinogen. Millions of people worldwide suffer from As toxicity due to the intake of As-contaminated drinking water and food. Reducing the As concentration in drinking water and food is thus of critical importance. Phytoremediation of soil contaminated with As and the reduction of As contamination in food depend on a detailed understanding of As uptake and transport in plants. As transporters play essential roles in As uptake, translocation and accumulation in plant cells. In this review, we summarize the current understanding of As transport in plants, with an emphasis on As uptake, mechanisms of As resistance and the long-distance translocation of As, especially the accumulation of As in grains through phloem-mediated transport.

A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain.

Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the phloem companion cells of the nodes in rice.

Transgenic poplar trees expressing yeast cadmium factor 1 exhibit the characteristics necessary for the phytoremediation of mine tailing soil.

Genetic engineering of plants for phytoremediation is thought to be possible based on results using model plants expressing genes involved in heavy metal resistance, which improve the plant's tolerance of heavy metals and accumulation capacity. The next step of progress in this technology requires the genetic engineering of plants that produce large amounts of biomass and the testing of these transgenic plants in contaminated soils. Thus, we transformed a sterile line of poplar Populus alba X P. tremula var. glandulosa with a heavy metal resistance gene, ScYCF1 (yeast cadmium factor 1), which encodes a transporter that sequesters toxic metal(loid)s into the vacuoles of budding yeast, and tested these transgenic plants in soil taken from a closed mine site contaminated with multiple toxic metal(loid)s under greenhouse and field conditions. The YCF1-expressing transgenic poplar plants exhibited enhanced growth, reduced toxicity symptoms, and increased Cd content in the aerial tissue compared to the non-transgenic plants. Furthermore, the plants accumulated increased amounts of Cd, Zn, and Pb in the root, because they could establish an extensive root system in mine tailing soil. These results suggest that the generation of YCF1-expressing transgenic poplar represents the first step towards producing plants for phytoremediation. The YCF1-expressing poplar may be useful for phytostabilization and phytoattenuation, especially in highly contaminated regions, where wild-type plants cannot survive.

The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury.

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-phytochelatin 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.

Brassica juncea plant cadmium resistance 1 protein (BjPCR1) facilitates the radial transport of calcium in the root.

Calcium (Ca) is an important structural component of plant cell walls and an intracellular messenger in plants and animals. Therefore, plants tightly control the balance of Ca by regulating Ca uptake and its transfer from cell to cell and organ to organ. Here, we propose that Brassica juncea PCR1 (PCR1), a member of the plant cadmium resistance (PCR) protein family in Indian mustard, is a Ca(2+) efflux transporter that is required for the efficient radial transfer of Ca(2+) in the root and is implicated in the translocation of Ca to the shoot. Knock-down lines of BjPCR1 were greatly stunted and translocated less Ca to the shoot than did the corresponding WT. The localization of BjPCR1 to the plasma membrane and the preferential expression of BjPCR1 in the root epidermal cells of WT plants suggest that BjPCR1 antisense plants could not efficiently transfer Ca(2+) from the root epidermis to the cells located inside the root. Protoplasts isolated from BjPCR1 antisense lines had lower Ca(2+) efflux activity than did those of the WT, and membrane vesicles isolated from BjPCR1-expressing yeast exhibited increased Ca(2+) transport activity. Inhibitor studies, together with theoretical considerations, indicate that BjPCR1 exports one Ca(2+) in exchange for three protons. Root hair-specific expression of BjPCR1 in Arabidopsis results in plants that exhibit increased Ca(2+) resistance and translocation. In conclusion, our data support the hypothesis that BjPCR1 is an exporter required for the translocation of Ca(2+) from the root epidermis to the inner cells, and ultimately to the shoot.

Tonoplast-localized Abc2 transporter mediates phytochelatin accumulation in vacuoles and confers cadmium tolerance.

Phytochelatins mediate tolerance to heavy metals in plants and some fungi by sequestering phytochelatin-metal complexes into vacuoles. To date, only Schizosaccharomyces pombe Hmt1 has been described as a phytochelatin transporter and attempts to identify orthologous phytochelatin transporters in plants and other organisms have failed. Furthermore, recent data indicate that the hmt1 mutant accumulates significant phytochelatin levels in vacuoles, suggesting that unidentified phytochelatin transporters exist in fungi. Here, we show that deletion of all vacuolar ABC transporters abolishes phytochelatin accumulation in S. pombe vacuoles and abrogates (35)S-PC(2) uptake into S. pombe microsomal vesicles. Systematic analysis of the entire S. pombe ABC transporter family identified Abc2 as a full-size ABC transporter (ABCC-type) that mediates phytochelatin transport into vacuoles. The S. pombe abc1 abc2 abc3 abc4 hmt1 quintuple and abc2 hmt1 double mutant show no detectable phytochelatins in vacuoles. Abc2 expression restores phytochelatin accumulation into vacuoles and suppresses the cadmium sensitivity of the abc quintuple mutant. A novel, unexpected, function of Hmt1 in GS-conjugate transport is also shown. In contrast to Hmt1, Abc2 orthologs are widely distributed among kingdoms and are proposed as the long-sought vacuolar phytochelatin transporters in plants and other organisms.

Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers' yeast.

Cadmium causes the generation of reactive oxygen species, which in turn causes cell damage. We isolated a novel gene from a wheat root cDNA library, which conferred Cd(II)-specific tolerance when expressed in yeast (Saccharomyces cerevisiae). The gene, which we called TaTM20, for Triticum aestivum transmembrane 20, encodes a putative hydrophobic polypeptide of 889 amino acids, containing 20 transmembrane domains arranged as a 5-fold internal repeating unit of 4 transmembrane domains each. Expression of TaTM20 in yeast cells stimulated Cd(II) efflux resulting in a decrease in the content of yeast intracellular cadmium. TaTM20-induced Cd(II) tolerance was maintained in yeast even under conditions of reduced GSH. These results demonstrate that TaTM20 enhances Cd(II) tolerance in yeast through the stimulation of Cd(II) efflux from the cell, partially independent of GSH. Treatment of wheat seedlings with Cd(II) induced their expression of TaTM20, decreasing subsequent root Cd(II) accumulation and suggesting a possible role for TaTM20 in Cd(II) tolerance in wheat.

Arabidopsis metallothioneins 2a and 3 enhance resistance to cadmium when expressed in Vicia faba guard cells.

The Arabidopsis metallothionein genes AtMT1 and AtMT2 confer Cd(II) resistance to Cd(II)-sensitive yeast, but it has not been directly shown whether they or other metallothioneins provide the same protection to plants. We tested whether AtMT2a and AtMT3 can confer Cd(II) resistance to plant cells by introducing GFP- or RFP-fused forms into guard cells of Vicia faba by biolistic bombardment. AtMT2a and AtMT3 protected guard cell chloroplasts from degradation upon exposure to Cd(II), an effect that was confirmed using an FDA assay to test the viability of the exposed guard cells. AtMT2a- and AtMT3-GFP were localized in the cytoplasm both before and after treatment of V. faba guard cells or Arabidopsis protoplasts with Cd(II), and the levels of reactive oxygen species were lower in transformed guard cells than in non-transformed cells after Cd(II)-treatment. These results suggest that the Cd(II)-detoxification mechanism of AtMT2a and AtMT3 may not include sequestration into vacuoles or other organelles, but does involve reduction of the level of reactive oxygen species in Cd(II)-treated cells. Increased expression of AtMT2a and AtMT3 was observed in Arabidopsis seedlings exposed to Cd(II). Together, these data support a role for the metallothioneins AtMT2a and AtMT3 in Cd(II) resistance in intact plant cells.

A novel family of cys-rich membrane proteins mediates cadmium resistance in Arabidopsis.

Cadmium (Cd) is a widespread pollutant that is toxic to plant growth. However, only a few genes that contribute to Cd resistance in plants have been identified. To identify additional Cd(II) resistance genes, we screened an Arabidopsis cDNA library using a yeast (Saccharomyces cerevisiae) expression system employing the Cd(II)-sensitive yeast mutant ycf1. This screening process yielded a small Cys-rich membrane protein (Arabidopsis plant cadmium resistance, AtPcrs). Database searches revealed that there are nine close homologs in Arabidopsis. Homologs were also found in other plants. Four of the five homologs that were tested also increased resistance to Cd(II) when expressed in ycf1. AtPcr1 localizes at the plasma membrane in both yeast and Arabidopsis. Arabidopsis plants overexpressing AtPcr1 exhibited increased Cd(II) resistance, whereas antisense plants that showed reduced AtPcr1 expression were more sensitive to Cd(II). AtPcr1 overexpression reduced Cd uptake by yeast cells and also reduced the Cd contents of both yeast and Arabidopsis protoplasts treated with Cd. Thus, it appears that the Pcr family members may play an important role in the Cd resistance of plants.

Engineering tolerance and accumulation of lead and cadmium in transgenic plants.

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.