We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter)Several heavy metals are essential during plant growth and development, but their excess can easily lead to toxic effects. Contamination of soils with heavy metals, either by natural causes or due to pollution, often has pronounced effects on the vegetation, resulting in the appearance of metallophytes, heavy-metal-tolerant plants. The precise mechanisms of uptake, transport, and accumulation of heavy metals in plants are poorly understood, but several genes likely to be involved in these processes have been described. Recently, a family of ZIP genes that are expressed in roots upon Zn deficiency was isolated from Arabidopsis (Grotz et al., 1998). The proteins encoded by the ZIP genes have eight predicted TM regions and a high degree of similarity to the ZRT genes from yeast that are involved in Zn uptake. Expression of the ZIP genes in yeast conferred Zn-uptake activities to these cells, demonstrating that they are probably functional homologs of the yeast ZRT genes (Grotz et al., 1998). The only other metaltransporting protein recently identified in plants belongs to the large family of cation-transporting P-type ATPases (Tabata et al., 1997), but these proteins are structurally very different from the metal-transporting proteins mentioned above.Recent data have provided more insight into the mechanisms of heavy-metal tolerance. Metallophytes often exhibit tolerance to several different heavy metals, but all of these metals need not be present at toxic levels in their habitat (Schat and ten Bookum, 1992a; Schat and Vooijs, 1997, and refs. therein;Schat and Verkleij, 1998). Although such a feature is suggestive of a general mechanism of heavy-metal tolerance, recent genetic evidence has shown that a number of different mechanisms must exist, each with its own metal specificity (Schat and Vooijs, 1997). In Arabidopsis, a plant species with a typical level of tolerance to heavy metals, it has been demonstrated that the Cd-sensitive mutants cad1 and cad2 are defective in the synthesis of the metal-binding compound phytochelatin (Howden et al., 1995). cad1 plants were only slightly more sensitive to Cu and Zn, indicating that phytochelatinmediated detoxification is not sufficient for Cu and Zn detoxification (Howden et al., 1995b). Metallothioneins appear to be of major importance for constitutive Cu tolerance in Arabidopsis (Zhou and Goldsbrough, 1994).Aside from complexation of heavy metals by heavymetal-binding proteins, there is evidence that transportmediated sequestration can contribute to heavy-metal tolerance. In the Zn-tolerant plant Silene vulgaris it was shown that Zn transport across the tonoplast was about 2.5 times higher than in Zn-sensitive plants of the same species (Verkleij et al

SummaryZinc-finger nucleases (ZFNs) are artificial restriction enzymes, custom designed for induction of double-strand breaks (DSBs) at a specific locus. These DSBs may result in site-specific mutagenesis or homologous recombination at the repair site, depending on the DNA repair pathway that is used. These promising techniques for genome

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Summary A library of genes for zinc finger artificial transcription factors (ZF‐ATF) was generated by fusion of DNA sequences encoding three‐finger Cys2His2 ZF domains to the VP16 activation domain under the control of the promoter of the ribosomal protein gene RPS5A from Arabidopsis thaliana. After introduction of this library into an Arabidopsis homologous recombination (HR) indicator line, we selected primary transformants exhibiting multiple somatic recombination events. After PCR‐mediated rescue of ZF sequences, reconstituted ZF‐ATFs were re‐introduced in the target line. In this manner, a ZF‐ATF was identified that led to a 200–1000‐fold increase in somatic HR (replicated in an independent second target line). A mutant plant line expressing the HR‐inducing ZF‐ATF exhibited increased resistance to the DNA‐damaging agent bleomycin and was more sensitive to methyl methanesulfonate (MMS), a combination of traits not described previously. Our results demonstrate that the use of ZF‐ATF pools is highly rewarding when screening for novel dominant phenotypes in Arabidopsis.

Previously, we showed that ZFN-mediated induction of double-strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium-mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild-type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T-DNA with an incomplete PPO gene, missing the 5′ coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10−3 per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10−3 per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so-called true GT events, repaired via homologous recombination (HR) at the 5′ and the 3′ end of the gene. One plant line contained a PPO gene repaired only at the 5′ end via HR. Most plant lines contained extra randomly integrated T-DNA copies. Two plant lines did not contain extra T-DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.