SummaryCadmium (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-AtPDR8 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.

The posttranslational addition of ubiquitin (Ub) profoundly controls the half-life, interactions, and/or trafficking of numerous intracellular proteins. Using stringent two-step affinity methods to purify Ub-protein conjugates followed by high-sensitivity mass spectrometry, we identified almost 950 ubiquitylation substrates in whole Arabidopsis thaliana seedlings. The list includes key factors regulating a wide range of biological processes, including metabolism, cellular transport, signal transduction, transcription, RNA biology, translation, and proteolysis. The ubiquitylation state of more than half of the targets increased after treating seedlings with the proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-Leu-al), strongly suggesting that Ub addition commits many to degradation by the 26S proteasome. Ub-attachment sites were resolved for a number of targets, including six of the seven Lys residues on Ub itself with a Lys-48>Lys-63>Lys-11>>>Lys-33/Lys-29/Lys-6 preference. However, little sequence consensus was detected among conjugation sites, indicating that the local environment has little influence on global ubiquitylation. Intriguingly, the level of Lys-11-linked Ub polymers increased substantially upon MG132 treatment, revealing that they might be important signals for proteasomal breakdown. Taken together, this proteomic analysis illustrates the breadth of plant processes affected by ubiquitylation and provides a deep data set of individual targets from which to explore the roles of Ub in various physiological and developmental pathways.

Transcription activator-like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.

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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 synthetaseconjugated Cd(II) across the mitochondrial membrane.

Supercapacitors with porous carbon structures have high energy storage capacity. However, the porous nature of the carbon electrode, composed mainly of carbon nanotubes (CNTs) and graphene oxide (GO) derivatives, negatively impacts the volumetric electrochemical characteristics of the supercapacitors because of poor packing density (<0.5 g cm(-3)). Herein, we report a simple method to fabricate highly dense and vertically aligned reduced graphene oxide (VArGO) electrodes involving simple hand-rolling and cutting processes. Because of their vertically aligned and opened-edge graphene structure, VArGO electrodes displayed high packing density and highly efficient volumetric and areal electrochemical characteristics, very fast electrolyte ion diffusion with rectangular CV curves even at a high scan rate (20 V/s), and the highest volumetric capacitance among known rGO electrodes. Surprisingly, even when the film thickness of the VArGO electrode was increased, its volumetric and areal capacitances were maintained.

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.

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