TY - JOUR
AU1 - Talke, Ina N.
AU2 - Hanikenne, Marc
AU3 - Krämer, Ute
AB - Abstract The metal hyperaccumulator Arabidopsis halleri exhibits naturally selected zinc (Zn) and cadmium (Cd) hypertolerance and accumulates extraordinarily high Zn concentrations in its leaves. With these extreme physiological traits, A. halleri phylogenetically belongs to the sister clade of Arabidopsis thaliana. Using a combination of genome-wide cross species microarray analysis and real-time reverse transcription-PCR, a set of candidate genes is identified for Zn hyperaccumulation, Zn and Cd hypertolerance, and the adjustment of micronutrient homeostasis in A. halleri. Eighteen putative metal homeostasis genes are newly identified to be more highly expressed in A. halleri than in A. thaliana, and 11 previously identified candidate genes are confirmed. The encoded proteins include HMA4, known to contribute to root-shoot transport of Zn in A. thaliana. Expression of either AtHMA4 or AhHMA4 confers cellular Zn and Cd tolerance to yeast (Saccharomyces cerevisiae). Among further newly implicated proteins are IRT3 and ZIP10, which have been proposed to contribute to cytoplasmic Zn influx, and FRD3 required for iron partitioning in A. thaliana. In A. halleri, the presence of more than a single genomic copy is a hallmark of several highly expressed candidate genes with possible roles in metal hyperaccumulation and metal hypertolerance. Both A. halleri and A. thaliana exert tight regulatory control over Zn homeostasis at the transcript level. Zn hyperaccumulation in A. halleri involves enhanced partitioning of Zn from roots into shoots. The transcriptional regulation of marker genes suggests that in the steady state, A. halleri roots, but not the shoots, act as physiologically Zn deficient under conditions of moderate Zn supply. Metal hyperaccumulation is a rare trait found in approximately 440 plant taxa, 11 of which have been reported to hyperaccumulate zinc (Zn; Reeves and Baker, 2000). Individuals of these taxa accumulate very high Zn concentrations of more than 10,000 μg g−1 Zn in leaf dry biomass (Baker and Brooks, 1989). The accumulation of higher metal concentrations in shoots than in roots has been described as a characteristic of hyperaccumulation in both hydroponic and soil culture (Baker et al., 1994; Krämer et al., 1996; Weber et al., 2004). All known metal hyperaccumulator taxa occur primarily on geologically metal-rich or metal-contaminated soils and possess extraordinarily high metal tolerance. The number of Zn and nickel hyperaccumulator taxa is higher in the Brassicaceae family than in any other plant family, and includes a large number of nickel hyperaccumulator species in the section Odontarrhena of the genus Alyssum and the two most commonly studied Zn hyperaccumulator model species Thlaspi caerulescens and Arabidopsis halleri (Reeves and Baker, 2000). Both subspecies of A. halleri, subsp. gemmifera and subsp. halleri, are hyperaccumulators of Zn, and subsp. halleri is able to grow healthily and to accumulate very high leaf Zn concentrations of between 3,000 and 22,000 μg g−1 dry biomass within a wide dynamic range of external Zn concentrations in the field (Bert et al., 2000; Dahmani-Muller et al., 2000; Kubota and Takenaka, 2003). The leaves of individuals of some populations have additionally been reported to contain hyperaccumulator levels of cadmium (Cd) of more than 100 μg g−1 dry biomass (Dahmani-Muller et al., 2000). Together, A. halleri and its nonaccumulating close relative Arabidopsis lyrata form the sister clade of Arabidopsis thaliana (Koch et al., 2000; Yogeeswaran et al., 2005). A. halleri and A. thaliana share on average approximately 94% nucleotide sequence identity within coding regions (Becher et al., 2004). Leaves of both A. lyrata and A. thaliana commonly accumulate between 40 and 80 μg g−1 Zn, and gradually develop chlorosis when they contain significantly higher metal concentrations (Macnair et al., 1999; Desbrosses-Fonrouge et al., 2005). In hydroponic culture, the growth of A. halleri roots has been reported to tolerate at least 30-fold higher Zn and 10-fold higher Cd concentrations than root growth of A. lyrata (Macnair et al., 1999; Bert et al., 2003). These observations illustrate the dramatic alterations in metal homeostasis of A. halleri compared to both A. thaliana and A. lyrata, despite a very close phylogenetic relationship and high overall sequence conservation among these three species (Clemens et al., 2002; Krämer and Clemens, 2005). Researchers have begun to address the major differences between the metal homeostasis networks of closely related metal hyperaccumulator and nonaccumulator plants at the molecular level (Salt and Krämer, 2000; Clemens et al., 2002; Krämer and Clemens, 2005). Using single gene and transcriptomic approaches, the most notable differences observed in hyperaccumulator compared to closely related nonaccumulator plants were high relative transcript levels (RTLs) of several candidate genes (Pence et al., 2000; Assunção et al., 2001; Becher et al., 2004; Dräger et al., 2004; Papoyan and Kochian, 2004; Weber et al., 2004; Hammond et al., 2006; Mirouze et al., 2006). Differences in the functions of hyperaccumulator candidate gene products, when compared to their A. thaliana orthologs, have not extensively been documented so far (Bernard et al., 2004; Roosens et al., 2004). The observed high transcript levels of candidate genes in A. halleri and in other hyperaccumulator model plants are based on a modified regulation of transcript levels (Pence et al., 2000; Becher et al., 2004; Weber et al., 2004). This could involve at least four possible mechanisms. First, it is conceivable that for some A. halleri genes, the presence or activity of proximal cis-elements or trans-factors controlling transcript levels could be altered in comparison to A. thaliana. Second, it is possible that the affinity of a Zn sensing machinery that down-regulates transcript levels of genes encoding Zn acquisitory proteins may be reduced in A. halleri. Third, Zn requirement of A. halleri may merely be higher than in closely related nonaccumulator plants. Finally, the high expression of a specific gene of A. halleri could be an indirect consequence of another alteration in a different element of the metal homeostasis network, which is complex and tightly regulated in plants. Before dissecting the molecular basis underlying the modified regulation of expression of individual candidate genes in A. halleri, it is therefore important to understand globally how the regulation of the metal homeostasis network is altered. For A. halleri MTP1, which is involved in Zn hypertolerance, the amplification of gene copy number contributes to enhanced MTP1 transcript levels, when compared to A. thaliana or A. lyrata (Dräger et al., 2004). It has not been investigated whether genomic copy number is also increased for other genes involved in naturally selected metal hyperaccumulation or hypertolerance of A. halleri. Previously, root and shoot transcript profiles were established in separate studies using A. halleri and A. thaliana grown under different conditions with Affymetrix 8 K chips representing only a limited number of genes (Becher et al., 2004; Weber et al., 2004). To obtain a comprehensive account of directly comparable expression profiles for both roots and shoots of the two species, we employed the next generation of Affymetrix Arabidopsis GeneChips, the ATH1 microarray, covering more than 22,500 genes. Here we present genome-wide transcriptional profiles of hydroponically cultivated A. halleri and A. thaliana plants maintained under control conditions or upon a short-term exposure to high Zn concentrations. We report a set of candidate genes for metal hyperaccumulation and tolerance, for the adjustment of micronutrient homeostasis, and for the protection from secondary abiotic stress in A. halleri. For several of the most highly expressed candidate genes, genomic copy number is estimated to be higher in A. halleri than in A. thaliana by DNA gel blot. The detailed analysis of transcriptional regulation of candidate genes by real-time reverse transcription (RT)-PCR is combined with existing and newly generated knowledge on gene product function to develop a model of the alterations in the metal homeostasis network underlying metal hyperaccumulation in A. halleri. RESULTS Establishment of Experimental Conditions To compare the effect of external Zn supply on Zn accumulation and partitioning in A. thaliana and A. halleri, plants were cultivated hydroponically and supplied with different subtoxic Zn concentrations for 3 weeks (Fig. 1, A and B Figure 1. Open in new tabDownload slide Long-term Zn accumulation in A. halleri and A. thaliana roots and shoots. Hydroponically grown 4.5-week-old plants were supplied with different Zn concentrations in the culture medium for 3 weeks before harvest. The Zn concentrations in root (A) and shoot (B) tissues were determined by inductively coupled plasma optical emission spectroscopy. White circles, A. thaliana; black circles: A. halleri. C illustrates the ratio of Zn concentrations in shoot versus root tissues. Note that at 10 μ m ZnSO4, the Zn concentration in A. thaliana roots exceeds that of A. halleri roots by more than 30-fold. In preliminary experiments, exposure of hydroponically grown A. thaliana to 30 μ m ZnSO4 for a period of 3 weeks led to the onset of mild toxicity symptoms; therefore, this Zn condition was excluded. Data are from one experiment representative of two independent experiments. Values represent mean ± se, calculated from n = 3 culture vessels per Zn condition. For C, data were log2 transformed. Data were plotted with Origin-Pro 7 SR4 (v 7.0552; OriginLab Corporation) and logarithmic fits to the data were performed for A and B, a cubic B-spline connection was used for the smooth curves in C. Figure 1. Open in new tabDownload slide Long-term Zn accumulation in A. halleri and A. thaliana roots and shoots. Hydroponically grown 4.5-week-old plants were supplied with different Zn concentrations in the culture medium for 3 weeks before harvest. The Zn concentrations in root (A) and shoot (B) tissues were determined by inductively coupled plasma optical emission spectroscopy. White circles, A. thaliana; black circles: A. halleri. C illustrates the ratio of Zn concentrations in shoot versus root tissues. Note that at 10 μ m ZnSO4, the Zn concentration in A. thaliana roots exceeds that of A. halleri roots by more than 30-fold. In preliminary experiments, exposure of hydroponically grown A. thaliana to 30 μ m ZnSO4 for a period of 3 weeks led to the onset of mild toxicity symptoms; therefore, this Zn condition was excluded. Data are from one experiment representative of two independent experiments. Values represent mean ± se, calculated from n = 3 culture vessels per Zn condition. For C, data were log2 transformed. Data were plotted with Origin-Pro 7 SR4 (v 7.0552; OriginLab Corporation) and logarithmic fits to the data were performed for A and B, a cubic B-spline connection was used for the smooth curves in C. ). In A. halleri, the ratio of shoot Zn concentrations to root Zn concentrations was above unity in plants cultivated in a Zn-deficient hydroponic solution containing no added Zn for 3 weeks (Fig. 1C). Continuous supply of Zn concentrations of between 1 and 5 μ m led to an increase in the shoot-to-root ratio of Zn concentrations, with maximum ratios approximately tripling the ratio determined under Zn-deficient conditions. By contrast, A. thaliana partitioned into the shoots only a small proportion of the Zn concentration accumulated in the roots (Fig. 1, A and B). This was reflected in shoot-to-root Zn concentration ratios substantially below unity, which decreased further with increasing Zn supply (Fig. 1C). These results indicated that A. halleri accumulates Zn predominantly in the shoot even at very low Zn supply, whereas A. thaliana primarily immobilizes Zn in the roots irrespective of the Zn supply. When A. halleri was cultivated in media containing high Zn concentrations of 30 or 300 μ m Zn, shoot-to-root ratios of Zn concentrations were below unity and only about one-quarter of the ratios measured at 0.3 μ m Zn. In addition to the dramatic interspecies differences in Zn partitioning between roots and shoots, the data suggest a species-specific modulation of Zn partitioning dependent on the Zn supply. To select suitable conditions and time points for transcript profiling, transcript levels of three metal homeostasis genes, ZIP4, ZIP9, and NAS2, were determined in preliminary experiments (Fig. 2 Figure 2. Open in new tabDownload slide Expression analysis of known Zn-regulated marker genes. Real-time RT-PCR analysis of expression of ZIP4, ZIP9, and NAS2 genes in roots (top section) and shoots (bottom section) of hydroponically grown A. halleri (black bars) and A. thaliana (light gray bars). Two different basal Zn conditions upon which additional Zn was supplied were assessed: Short Zn oversupply denotes a short-term high Zn supply to plants precultivated at control Zn concentrations; for A. thaliana, 30 μ m Zn, and for A. halleri, 300 μ m Zn were added to the culture medium for 2 and 8 h before harvest. Zn deficiency/resupply denotes a supply of 5 μ m Zn to Zn-deficient plants for 2, 8, and 24 h before harvest. Note that substantial changes in root transcript levels occur as soon as 2 h after the change of Zn concentration in the culture medium whereas shoot transcript levels reflect the change in external Zn supply only at a later time point. Data shown are transcript levels relative to EF1α from one experiment representative of two independent biological experiments. Tissues from at least three culture vessels containing three plants each were pooled for each condition. Genes were concluded to be not expressed (n.e.) when C T values were above a threshold of 35 and reaction efficiencies (REs) with the respective cDNA were more than 5% below the mean RE for the respective primer pair (see also “Material and Methods”). Figure 2. Open in new tabDownload slide Expression analysis of known Zn-regulated marker genes. Real-time RT-PCR analysis of expression of ZIP4, ZIP9, and NAS2 genes in roots (top section) and shoots (bottom section) of hydroponically grown A. halleri (black bars) and A. thaliana (light gray bars). Two different basal Zn conditions upon which additional Zn was supplied were assessed: Short Zn oversupply denotes a short-term high Zn supply to plants precultivated at control Zn concentrations; for A. thaliana, 30 μ m Zn, and for A. halleri, 300 μ m Zn were added to the culture medium for 2 and 8 h before harvest. Zn deficiency/resupply denotes a supply of 5 μ m Zn to Zn-deficient plants for 2, 8, and 24 h before harvest. Note that substantial changes in root transcript levels occur as soon as 2 h after the change of Zn concentration in the culture medium whereas shoot transcript levels reflect the change in external Zn supply only at a later time point. Data shown are transcript levels relative to EF1α from one experiment representative of two independent biological experiments. Tissues from at least three culture vessels containing three plants each were pooled for each condition. Genes were concluded to be not expressed (n.e.) when C T values were above a threshold of 35 and reaction efficiencies (REs) with the respective cDNA were more than 5% below the mean RE for the respective primer pair (see also “Material and Methods”). ). These genes are known to be Zn responsive at the transcript level in A. thaliana (Grotz et al., 1998; Wintz et al., 2003) and they have previously been identified as candidate genes for Zn accumulation and tolerance in A. halleri (Becher et al., 2004; Weber et al., 2004). Plants of two different Zn steady states were subjected to short-term increases in external Zn supply. In one experiment, plants grown under control (1 μ m) Zn conditions were exposed to excess Zn concentrations of 30 μ m Zn for A. thaliana and 300 μ m Zn for A. halleri, for 2 and 8 h. The choice of concentrations reflects naturally selected Zn tolerance in A. halleri, whereas A. thaliana possesses only basic Zn tolerance (Clemens, 2001; Becher et al., 2004). (In preliminary experiments, the chosen concentrations were the maximum Zn concentrations that did not cause a reduction in biomass gain in A. thaliana and A. halleri, respectively, during 4 d of exposure [M. Becher, I.N. Talke, A.N. Chardonnens, and U. Krämer, unpublished data].) In an additional experiment, Zn-deficient plants cultivated in a medium lacking added Zn for 3 weeks were resupplied with a Zn concentration of 5 μ m (Fig. 2). In roots, Zn-induced changes in transcript levels were observed as early as 2 h after increasing the Zn concentrations in the hydroponic medium (Fig. 2, top row: ZIP4, ZIP9). In shoots, transcriptional responses were observed 2 or 8 h after the increase in Zn concentrations in the hydroponic medium (Fig. 2, bottom row: ZIP4, ZIP9, and NAS2). Transcripts of ZIP9 encoding a transporter of the ZIP family and NAS2 encoding a nicotianamine synthase were not detectable in shoots of either Arabidopsis species grown in the presence of sufficient (control) Zn, but were strongly induced upon Zn deficiency (Fig. 2, ZIP9, NAS2). This suggested that neither of the two species experienced Zn deficiency under the chosen control conditions. Comparison of A. halleri and A. thaliana Transcript Profiles with ATH1 GeneChips According to the results from the preliminary experiments, microarrays were hybridized with labeled cRNAs prepared from roots and shoots of A. halleri and A. thaliana grown at 1 μ m Zn and after 2 and 8 h of exposure to excess Zn for roots and shoots, respectively (see “Material and Methods”). Under control conditions, higher signal intensities were detected for a total of 628 probe sets in roots, and for 1,739 probe sets in shoots of A. halleri compared to A. thaliana (as outlined in “Materials and Methods;” for visualization see all colored, nongray datapoints in Fig. 3, A and B Figure 3. Open in new tabDownload slide The relation between ATH1 microarray signals of GeneChips hybridized with cRNA from A. halleri and from A. thaliana grown under control Zn conditions. For each of the 22,810 probe sets on the ATH1 array, the mean signal derived from the two replicate arrays of roots (A) and shoots (B) of A. halleri was plotted on the y axis and the respective mean signal of A. thaliana was plotted on the x axis. Signal values represent mean signal intensities calculated from MAS 5.0-scaled raw signals of replicate arrays, without further normalization. Symbols and colors reflect the data filtering which was applied to identify candidate genes more highly expressed in A. halleri compared to A. thaliana and involved in metal ion homeostasis or related processes. Gray diamonds represent all probe sets that did not pass any filter. Yellow squares represent probe sets that exhibited a 4-fold higher signal intensity in A. halleri than in A. thaliana and passed the value filter (for details see “Materials and Methods”). Blue triangles represent probe sets that, in addition, passed a first annotation filter (I) of genes annotated as metal ion (Zn, Fe, Cu, and Mn) binding, membrane and transport associated, oxidative stress protection associated, pathogen response related, and RNA metabolism associated (Supplemental Table XI). Red circles represent probe sets that passed a further annotation filter (II) that included only genes with a predicted function in metal ion homeostasis. Candidate genes that passed the value filter and either annotation filter and that were chosen for further analysis by real-time RT-PCR are represented by green triangles (passed annotation filter I) or circles (passed annotation filter II) with a black outline (for details see Table I). Figure 3. Open in new tabDownload slide The relation between ATH1 microarray signals of GeneChips hybridized with cRNA from A. halleri and from A. thaliana grown under control Zn conditions. For each of the 22,810 probe sets on the ATH1 array, the mean signal derived from the two replicate arrays of roots (A) and shoots (B) of A. halleri was plotted on the y axis and the respective mean signal of A. thaliana was plotted on the x axis. Signal values represent mean signal intensities calculated from MAS 5.0-scaled raw signals of replicate arrays, without further normalization. Symbols and colors reflect the data filtering which was applied to identify candidate genes more highly expressed in A. halleri compared to A. thaliana and involved in metal ion homeostasis or related processes. Gray diamonds represent all probe sets that did not pass any filter. Yellow squares represent probe sets that exhibited a 4-fold higher signal intensity in A. halleri than in A. thaliana and passed the value filter (for details see “Materials and Methods”). Blue triangles represent probe sets that, in addition, passed a first annotation filter (I) of genes annotated as metal ion (Zn, Fe, Cu, and Mn) binding, membrane and transport associated, oxidative stress protection associated, pathogen response related, and RNA metabolism associated (Supplemental Table XI). Red circles represent probe sets that passed a further annotation filter (II) that included only genes with a predicted function in metal ion homeostasis. Candidate genes that passed the value filter and either annotation filter and that were chosen for further analysis by real-time RT-PCR are represented by green triangles (passed annotation filter I) or circles (passed annotation filter II) with a black outline (for details see Table I). ; complete gene lists with annotations in Supplemental Tables I and II). We also identified genes that responded at the transcript level to excess Zn in A. halleri (see “Materials and Methods;” Supplemental Tables III and IV). To group the identified genes according to functional classes, a genome-wide annotation list was assembled; this list includes the class of metal homeostasis-related genes, which contained all functionally characterized A. thaliana genes encoding proteins involved in metal transport, chelation, binding, and trafficking and all other members of the respective protein families, and genes encoding members of protein families known to participate in metal homeostasis in other organisms (Supplemental Table XI: class “Metal ion homeostasis;” Fig. 3, see “Material and Methods”). The intersection of the gene list of metal homeostasis-related genes and of the genes that are either more highly expressed (Supplemental Tables I and II) or Zn regulated in A. halleri (Supplemental Tables III and IV) contained a total of 17 genes in the roots and 19 genes in the shoots, with seven genes in common between both roots and shoots (Fig. 3; Table I Table I. Summary of microarray data for A. halleri root and shoot candidate genes ATH1 probe set identifiers and the corresponding A. thaliana locus identifiers are given for candidate genes more highly expressed in A. halleri than in A. thaliana under control conditions (genes that passed the value filter as described in “Materials and Methods”). In addition, genes were included that showed an at least 2-fold up-regulation or down-regulation upon short-term Zn supply in A. halleri. Raw signal denotes mean signal value calculated from signal values exported from MAS 5.0 using GeneSpring. Microarray data for short-term Zn supply and Zn deficiency are given where the regulation compared to controls was at least 2-fold. One sample Student's t tests were performed in GeneSpring on ratios from cross species and on fold changes from within-species comparisons, P values are indicated as follows: * = P < 0.05; ** = P < 0.01; *** = P < 0.001. ATH1 Probe Set Identifier . AGI Locus Identifier . Gene Name . A. halleri Raw Signal . A. halleri/A. thaliana Signal Ratio . A. halleri Fold Change −Zn(II) . A. halleri Fold Change +Zn(II), Root: 2 h, Shoot: 8 h . A. thaliana Fold Change +Zn(II), Root: 2 h, Shoot: 8 h . Annotation . Root Candidates Cytoplasmic metal influx     262546_at At1g31260 ZIP10 a 141 111** 3.74 ZIP family of metal transporters     253413_at At4g33020 ZIP9 617 36.2*** ZIP family of metal transporters     260462_at At1g10970 ZIP4 722 24.6*** 2.29 0.31 ZIP family of metal transporters     259723_at At1g60960 IRT3 a 738 4.47* 2.79 0.28 0.37* ZIP family of metal transporters     257715_at At3g12750 ZIP1 194 4.08* 2.05 ZIP family of metal transporters     266336_at At2g32270 ZIP3 a 849 1.71* 0.37 0.30* ZIP family of metal transporters Root-to-shoot metal transport     258646_at At3g08040 FRD3 a 2,890 44.6*** Multidrug and toxin efflux family transporter Other membrane transport     266184_s_at At2g38940 PHT1;4 a 822 19.1** Phosphate:H+ symporter family, ScPho84 like     253658_at At4g30120 HMA3 457 9.14* P1B CPX metal ATPase family     267266_at At2g23150 NRAMP3 685 6.61*** Natural resistance-associated macrophage protein family     249255_at At5g41610 CHX18 a 34.1 6.52* Cation/H+ exchanger of unknown specificity, CPA2 family     266718_at At2g46800 MTP1 1,561 6.40*** Cation diffusion facilitator family     267029_at At2g38460 IREG2 a 248 4.49*** Similar to human Fe-regulated Fe transporter Nicotianamine/thiol biosynthesis pathways     248048_at At5g56080 NAS2 2,765 48.4*** Nicotianamine synthase     259632_at At1g56430 NAS4 b 70.1 13.7 2.42 Nicotianamine synthase     264261_at At1g09240 NAS3 106 5.22** 3.81* Nicotianamine synthase     263838_at At2g36880 SAMS3 a 5,353 4.31* S-adenosyl-Met synthetase     250832_at At5g04950 NAS1 b 90.1 0.81 Nicotianamine synthase Other metal handling     257365_x_at At2g26020 PDF1.2b 57.15 37.2* Plant defensin, antifungal protein AFP5d Shoot Candidates Cytoplasmic metal influx     267304_at At2g30080 ZIP6 288 7.34*** ZIP family of metal transporters     260462_at At1g10970 ZIP4 256 4.04* 12.70** ZIP family of metal transporters Root-to-shoot metal transport     267488_at At2g19110 HMA4 a 113 52.5** P1B CPX metal ATPase family Other membrane transport     253658_at At4g30120 HMA3 796 358** P1B CPX metal ATPase family     251620_at At3g58060 MTP8 a 108 65.0** Cation diffusion facilitator family     266718_at At2g46800 MTP1 4,862 27.9*** Cation diffusion facilitator family     246276_at At4g37270 HMA1 416 12.6** P1B CPX metal ATPase family     267266_at At2g23150 NRAMP3 309 7.30*** Natural resistance-associated macrophage protein family     266963_at At2g39450 MTP11 a 465 4.74*** Cation diffusion facilitator family     253339_at At4g33520 HMA6 a 148 4.58** P1B CPX metal ATPase family; PAA1     257789_at At3g27020 YSL6 a 256 4.42* 2.54 Yellow-stripe-like transporter family Nicotianamine/thiol biosynthesis pathways     256930_at At3g22460 OASA2 c 188 10.6*** 2.43 O-acetyl-Ser thiol lyase     260913_at At1g02500 SAMS1 a 988 10.4** 3.68 S-adenosyl-Met synthetase     255552_at At4g01850 SAMS2 a 1,765 8.54*** 2.10 S-adenosyl-Met synthetase     264261_at At1g09240 NAS3 316 7.89** 5.01* Nicotianamine synthase     263838_at At2g36880 SAMS3 a 2,392 5.31** 2.04 S-adenosyl-Met synthetase Other metal handling     256416_at At3g11050 FER2 a 83.1 11.0* Ferritin, Fe(III) binding     257365_x_at At2g26020 PDF1.2b 5,023 7.95* Plant defensin, antifungal protein AFP5d     251109_at At5g01600 FER1 a 6,509 5.13** Ferritin, Fe(III) binding Stress protection     262504_at At1g21750 PDI1 a 2,538 39.5** 3.76 Protein disulfide isomerase     259757_at At1g77510 PDI2 c 742 17.5** 2.65 Protein disulfide isomerase ATH1 Probe Set Identifier . AGI Locus Identifier . Gene Name . A. halleri Raw Signal . A. halleri/A. thaliana Signal Ratio . A. halleri Fold Change −Zn(II) . A. halleri Fold Change +Zn(II), Root: 2 h, Shoot: 8 h . A. thaliana Fold Change +Zn(II), Root: 2 h, Shoot: 8 h . Annotation . Root Candidates Cytoplasmic metal influx     262546_at At1g31260 ZIP10 a 141 111** 3.74 ZIP family of metal transporters     253413_at At4g33020 ZIP9 617 36.2*** ZIP family of metal transporters     260462_at At1g10970 ZIP4 722 24.6*** 2.29 0.31 ZIP family of metal transporters     259723_at At1g60960 IRT3 a 738 4.47* 2.79 0.28 0.37* ZIP family of metal transporters     257715_at At3g12750 ZIP1 194 4.08* 2.05 ZIP family of metal transporters     266336_at At2g32270 ZIP3 a 849 1.71* 0.37 0.30* ZIP family of metal transporters Root-to-shoot metal transport     258646_at At3g08040 FRD3 a 2,890 44.6*** Multidrug and toxin efflux family transporter Other membrane transport     266184_s_at At2g38940 PHT1;4 a 822 19.1** Phosphate:H+ symporter family, ScPho84 like     253658_at At4g30120 HMA3 457 9.14* P1B CPX metal ATPase family     267266_at At2g23150 NRAMP3 685 6.61*** Natural resistance-associated macrophage protein family     249255_at At5g41610 CHX18 a 34.1 6.52* Cation/H+ exchanger of unknown specificity, CPA2 family     266718_at At2g46800 MTP1 1,561 6.40*** Cation diffusion facilitator family     267029_at At2g38460 IREG2 a 248 4.49*** Similar to human Fe-regulated Fe transporter Nicotianamine/thiol biosynthesis pathways     248048_at At5g56080 NAS2 2,765 48.4*** Nicotianamine synthase     259632_at At1g56430 NAS4 b 70.1 13.7 2.42 Nicotianamine synthase     264261_at At1g09240 NAS3 106 5.22** 3.81* Nicotianamine synthase     263838_at At2g36880 SAMS3 a 5,353 4.31* S-adenosyl-Met synthetase     250832_at At5g04950 NAS1 b 90.1 0.81 Nicotianamine synthase Other metal handling     257365_x_at At2g26020 PDF1.2b 57.15 37.2* Plant defensin, antifungal protein AFP5d Shoot Candidates Cytoplasmic metal influx     267304_at At2g30080 ZIP6 288 7.34*** ZIP family of metal transporters     260462_at At1g10970 ZIP4 256 4.04* 12.70** ZIP family of metal transporters Root-to-shoot metal transport     267488_at At2g19110 HMA4 a 113 52.5** P1B CPX metal ATPase family Other membrane transport     253658_at At4g30120 HMA3 796 358** P1B CPX metal ATPase family     251620_at At3g58060 MTP8 a 108 65.0** Cation diffusion facilitator family     266718_at At2g46800 MTP1 4,862 27.9*** Cation diffusion facilitator family     246276_at At4g37270 HMA1 416 12.6** P1B CPX metal ATPase family     267266_at At2g23150 NRAMP3 309 7.30*** Natural resistance-associated macrophage protein family     266963_at At2g39450 MTP11 a 465 4.74*** Cation diffusion facilitator family     253339_at At4g33520 HMA6 a 148 4.58** P1B CPX metal ATPase family; PAA1     257789_at At3g27020 YSL6 a 256 4.42* 2.54 Yellow-stripe-like transporter family Nicotianamine/thiol biosynthesis pathways     256930_at At3g22460 OASA2 c 188 10.6*** 2.43 O-acetyl-Ser thiol lyase     260913_at At1g02500 SAMS1 a 988 10.4** 3.68 S-adenosyl-Met synthetase     255552_at At4g01850 SAMS2 a 1,765 8.54*** 2.10 S-adenosyl-Met synthetase     264261_at At1g09240 NAS3 316 7.89** 5.01* Nicotianamine synthase     263838_at At2g36880 SAMS3 a 2,392 5.31** 2.04 S-adenosyl-Met synthetase Other metal handling     256416_at At3g11050 FER2 a 83.1 11.0* Ferritin, Fe(III) binding     257365_x_at At2g26020 PDF1.2b 5,023 7.95* Plant defensin, antifungal protein AFP5d     251109_at At5g01600 FER1 a 6,509 5.13** Ferritin, Fe(III) binding Stress protection     262504_at At1g21750 PDI1 a 2,538 39.5** 3.76 Protein disulfide isomerase     259757_at At1g77510 PDI2 c 742 17.5** 2.65 Protein disulfide isomerase a Newly identified candidate gene. b Data for 259632_at, NAS4, and 250832_at, NAS1, in roots are given for comparison. c Identified as false positive by real-time RT-PCR (Table II). d Mirouze et al. (2006). Open in new tab Table I. Summary of microarray data for A. halleri root and shoot candidate genes ATH1 probe set identifiers and the corresponding A. thaliana locus identifiers are given for candidate genes more highly expressed in A. halleri than in A. thaliana under control conditions (genes that passed the value filter as described in “Materials and Methods”). In addition, genes were included that showed an at least 2-fold up-regulation or down-regulation upon short-term Zn supply in A. halleri. Raw signal denotes mean signal value calculated from signal values exported from MAS 5.0 using GeneSpring. Microarray data for short-term Zn supply and Zn deficiency are given where the regulation compared to controls was at least 2-fold. One sample Student's t tests were performed in GeneSpring on ratios from cross species and on fold changes from within-species comparisons, P values are indicated as follows: * = P < 0.05; ** = P < 0.01; *** = P < 0.001. ATH1 Probe Set Identifier . AGI Locus Identifier . Gene Name . A. halleri Raw Signal . A. halleri/A. thaliana Signal Ratio . A. halleri Fold Change −Zn(II) . A. halleri Fold Change +Zn(II), Root: 2 h, Shoot: 8 h . A. thaliana Fold Change +Zn(II), Root: 2 h, Shoot: 8 h . Annotation . Root Candidates Cytoplasmic metal influx     262546_at At1g31260 ZIP10 a 141 111** 3.74 ZIP family of metal transporters     253413_at At4g33020 ZIP9 617 36.2*** ZIP family of metal transporters     260462_at At1g10970 ZIP4 722 24.6*** 2.29 0.31 ZIP family of metal transporters     259723_at At1g60960 IRT3 a 738 4.47* 2.79 0.28 0.37* ZIP family of metal transporters     257715_at At3g12750 ZIP1 194 4.08* 2.05 ZIP family of metal transporters     266336_at At2g32270 ZIP3 a 849 1.71* 0.37 0.30* ZIP family of metal transporters Root-to-shoot metal transport     258646_at At3g08040 FRD3 a 2,890 44.6*** Multidrug and toxin efflux family transporter Other membrane transport     266184_s_at At2g38940 PHT1;4 a 822 19.1** Phosphate:H+ symporter family, ScPho84 like     253658_at At4g30120 HMA3 457 9.14* P1B CPX metal ATPase family     267266_at At2g23150 NRAMP3 685 6.61*** Natural resistance-associated macrophage protein family     249255_at At5g41610 CHX18 a 34.1 6.52* Cation/H+ exchanger of unknown specificity, CPA2 family     266718_at At2g46800 MTP1 1,561 6.40*** Cation diffusion facilitator family     267029_at At2g38460 IREG2 a 248 4.49*** Similar to human Fe-regulated Fe transporter Nicotianamine/thiol biosynthesis pathways     248048_at At5g56080 NAS2 2,765 48.4*** Nicotianamine synthase     259632_at At1g56430 NAS4 b 70.1 13.7 2.42 Nicotianamine synthase     264261_at At1g09240 NAS3 106 5.22** 3.81* Nicotianamine synthase     263838_at At2g36880 SAMS3 a 5,353 4.31* S-adenosyl-Met synthetase     250832_at At5g04950 NAS1 b 90.1 0.81 Nicotianamine synthase Other metal handling     257365_x_at At2g26020 PDF1.2b 57.15 37.2* Plant defensin, antifungal protein AFP5d Shoot Candidates Cytoplasmic metal influx     267304_at At2g30080 ZIP6 288 7.34*** ZIP family of metal transporters     260462_at At1g10970 ZIP4 256 4.04* 12.70** ZIP family of metal transporters Root-to-shoot metal transport     267488_at At2g19110 HMA4 a 113 52.5** P1B CPX metal ATPase family Other membrane transport     253658_at At4g30120 HMA3 796 358** P1B CPX metal ATPase family     251620_at At3g58060 MTP8 a 108 65.0** Cation diffusion facilitator family     266718_at At2g46800 MTP1 4,862 27.9*** Cation diffusion facilitator family     246276_at At4g37270 HMA1 416 12.6** P1B CPX metal ATPase family     267266_at At2g23150 NRAMP3 309 7.30*** Natural resistance-associated macrophage protein family     266963_at At2g39450 MTP11 a 465 4.74*** Cation diffusion facilitator family     253339_at At4g33520 HMA6 a 148 4.58** P1B CPX metal ATPase family; PAA1     257789_at At3g27020 YSL6 a 256 4.42* 2.54 Yellow-stripe-like transporter family Nicotianamine/thiol biosynthesis pathways     256930_at At3g22460 OASA2 c 188 10.6*** 2.43 O-acetyl-Ser thiol lyase     260913_at At1g02500 SAMS1 a 988 10.4** 3.68 S-adenosyl-Met synthetase     255552_at At4g01850 SAMS2 a 1,765 8.54*** 2.10 S-adenosyl-Met synthetase     264261_at At1g09240 NAS3 316 7.89** 5.01* Nicotianamine synthase     263838_at At2g36880 SAMS3 a 2,392 5.31** 2.04 S-adenosyl-Met synthetase Other metal handling     256416_at At3g11050 FER2 a 83.1 11.0* Ferritin, Fe(III) binding     257365_x_at At2g26020 PDF1.2b 5,023 7.95* Plant defensin, antifungal protein AFP5d     251109_at At5g01600 FER1 a 6,509 5.13** Ferritin, Fe(III) binding Stress protection     262504_at At1g21750 PDI1 a 2,538 39.5** 3.76 Protein disulfide isomerase     259757_at At1g77510 PDI2 c 742 17.5** 2.65 Protein disulfide isomerase ATH1 Probe Set Identifier . AGI Locus Identifier . Gene Name . A. halleri Raw Signal . A. halleri/A. thaliana Signal Ratio . A. halleri Fold Change −Zn(II) . A. halleri Fold Change +Zn(II), Root: 2 h, Shoot: 8 h . A. thaliana Fold Change +Zn(II), Root: 2 h, Shoot: 8 h . Annotation . Root Candidates Cytoplasmic metal influx     262546_at At1g31260 ZIP10 a 141 111** 3.74 ZIP family of metal transporters     253413_at At4g33020 ZIP9 617 36.2*** ZIP family of metal transporters     260462_at At1g10970 ZIP4 722 24.6*** 2.29 0.31 ZIP family of metal transporters     259723_at At1g60960 IRT3 a 738 4.47* 2.79 0.28 0.37* ZIP family of metal transporters     257715_at At3g12750 ZIP1 194 4.08* 2.05 ZIP family of metal transporters     266336_at At2g32270 ZIP3 a 849 1.71* 0.37 0.30* ZIP family of metal transporters Root-to-shoot metal transport     258646_at At3g08040 FRD3 a 2,890 44.6*** Multidrug and toxin efflux family transporter Other membrane transport     266184_s_at At2g38940 PHT1;4 a 822 19.1** Phosphate:H+ symporter family, ScPho84 like     253658_at At4g30120 HMA3 457 9.14* P1B CPX metal ATPase family     267266_at At2g23150 NRAMP3 685 6.61*** Natural resistance-associated macrophage protein family     249255_at At5g41610 CHX18 a 34.1 6.52* Cation/H+ exchanger of unknown specificity, CPA2 family     266718_at At2g46800 MTP1 1,561 6.40*** Cation diffusion facilitator family     267029_at At2g38460 IREG2 a 248 4.49*** Similar to human Fe-regulated Fe transporter Nicotianamine/thiol biosynthesis pathways     248048_at At5g56080 NAS2 2,765 48.4*** Nicotianamine synthase     259632_at At1g56430 NAS4 b 70.1 13.7 2.42 Nicotianamine synthase     264261_at At1g09240 NAS3 106 5.22** 3.81* Nicotianamine synthase     263838_at At2g36880 SAMS3 a 5,353 4.31* S-adenosyl-Met synthetase     250832_at At5g04950 NAS1 b 90.1 0.81 Nicotianamine synthase Other metal handling     257365_x_at At2g26020 PDF1.2b 57.15 37.2* Plant defensin, antifungal protein AFP5d Shoot Candidates Cytoplasmic metal influx     267304_at At2g30080 ZIP6 288 7.34*** ZIP family of metal transporters     260462_at At1g10970 ZIP4 256 4.04* 12.70** ZIP family of metal transporters Root-to-shoot metal transport     267488_at At2g19110 HMA4 a 113 52.5** P1B CPX metal ATPase family Other membrane transport     253658_at At4g30120 HMA3 796 358** P1B CPX metal ATPase family     251620_at At3g58060 MTP8 a 108 65.0** Cation diffusion facilitator family     266718_at At2g46800 MTP1 4,862 27.9*** Cation diffusion facilitator family     246276_at At4g37270 HMA1 416 12.6** P1B CPX metal ATPase family     267266_at At2g23150 NRAMP3 309 7.30*** Natural resistance-associated macrophage protein family     266963_at At2g39450 MTP11 a 465 4.74*** Cation diffusion facilitator family     253339_at At4g33520 HMA6 a 148 4.58** P1B CPX metal ATPase family; PAA1     257789_at At3g27020 YSL6 a 256 4.42* 2.54 Yellow-stripe-like transporter family Nicotianamine/thiol biosynthesis pathways     256930_at At3g22460 OASA2 c 188 10.6*** 2.43 O-acetyl-Ser thiol lyase     260913_at At1g02500 SAMS1 a 988 10.4** 3.68 S-adenosyl-Met synthetase     255552_at At4g01850 SAMS2 a 1,765 8.54*** 2.10 S-adenosyl-Met synthetase     264261_at At1g09240 NAS3 316 7.89** 5.01* Nicotianamine synthase     263838_at At2g36880 SAMS3 a 2,392 5.31** 2.04 S-adenosyl-Met synthetase Other metal handling     256416_at At3g11050 FER2 a 83.1 11.0* Ferritin, Fe(III) binding     257365_x_at At2g26020 PDF1.2b 5,023 7.95* Plant defensin, antifungal protein AFP5d     251109_at At5g01600 FER1 a 6,509 5.13** Ferritin, Fe(III) binding Stress protection     262504_at At1g21750 PDI1 a 2,538 39.5** 3.76 Protein disulfide isomerase     259757_at At1g77510 PDI2 c 742 17.5** 2.65 Protein disulfide isomerase a Newly identified candidate gene. b Data for 259632_at, NAS4, and 250832_at, NAS1, in roots are given for comparison. c Identified as false positive by real-time RT-PCR (Table II). d Mirouze et al. (2006). Open in new tab ). We considered these genes as an initial set of major candidate genes for an involvement in naturally selected metal tolerance and hyperaccumulation in A. halleri. The metal homeostasis genes newly identified here as candidate genes encode proteins encompassing both cellular functions known to be of primary importance in metal homeostasis: membrane transport of metals and metal binding/chelation. The 12 new candidate genes encoding proteins that are likely to be involved in the transport of Zn or other metals across membranes were Zn-regulated transporter/iron-regulated transporter-like protein 10 (ZIP10; roots, 111-fold), metal tolerance protein 8 (MTP8; shoots, 65-fold), heavy metal-associated domain-containing protein 4 or heavy metal ATPase 4 (HMA4; shoots, 53-fold), ferric reductase defective 3 (FRD3; roots, 45-fold), phosphate transporter 1;4 (PHT1;4; roots, 19-fold), cation/H+ exchanger 18 (CHX18; roots, 6.5-fold), MTP11 (shoots, 4.7-fold), P-type ATPase from Arabidopsis 1 (PAA1/HMA6; shoots, 4.6-fold), iron-regulated transporter 2 (IREG2; roots, 4.5-fold), iron-regulated transporter 3 (IRT3; roots, 4.5-fold), yellow-stripe 1-like protein 6 (YSL6; shoots, 4.4-fold), and ZIP3 (down-regulated 2.7-fold in roots following exposure to excess Zn; Fristedt et al., 1999; Curie et al., 2001; Mäser et al., 2001; Rogers and Guerinot, 2002; Blaudez et al., 2003; Jensen et al., 2003; Lahner et al., 2003; Shikanai et al., 2003; Green and Rogers, 2004; Hussain et al., 2004; Shin et al., 2004). A total of five new candidate genes encoding metal-binding proteins or proteins involved in the biosynthesis of metal chelators included ferritin 2 (FER2; shoots, 11-fold), S-adenosyl-Met synthetase 1 (SAMS1; shoots, 10-fold), SAMS2 (shoots, 8.5-fold), FER1 (shoots, 5.1-fold), and SAMS3 (roots and shoots, around 5-fold; Peleman et al., 1989; Petit et al., 2001). The compound S-adenosyl-Met is the substrate for nicotianamine synthase, and the A. halleri nicotianamine synthase genes NAS2 and NAS3 have previously been implicated in Zn tolerance of A. halleri (Becher et al., 2004; Weber et al., 2004; see also Table I). Two additional, closely related genes encoding the protein disulfide isomerases 1 and 2 were included among the set of candidate genes analyzed here because of their interesting regulation and potential function in metal detoxification (PDI1: shoots, 39-fold higher in A. halleri; PDI2: shoots, 17-fold; Table I; Rensing et al., 1997; Narindrasorasak et al., 2003). These proteins are the closest sequence relatives of a previously characterized endoplasmic reticulum-localized protein from castor bean (Ricinus communis), which catalyzes the isomerization of disulfide bridges within proteins. Enhanced PDI activity is thought to protect plants from stress that results in the modification of thiol groups of endoplasmic reticulum proteins (Houston et al., 2005). Microarray Analysis of Zn-Dependent Transcriptional Regulation Among candidate genes, the microarray data suggested a trend toward decreasing transcript levels in response to excess Zn in roots of both A. thaliana and A. halleri (Table I). Conversely, in A. halleri roots transcript levels of ZIP and NAS genes were increased under Zn deficiency, when compared to control conditions (Table I). In shoots of A. thaliana, transcript levels of several candidate genes were increased in response to excess Zn, namely for PDI and SAMS genes, and for YSL6 (Table I). In A. halleri, transcript levels of these genes were constitutively high and unchanged in response to excess Zn (Table I). Our data suggested that, especially in shoots, A. halleri responds differently to excess Zn than A. thaliana at the transcript level. We investigated whether the species-specific patterns observed in metal-dependent transcriptional regulation of candidate genes are also reflected globally across the entire panel of genes represented on the ATH1 microarray. In roots of A. halleri and A. thaliana, exposure to excess Zn resulted in increased transcript levels for 0.5% and 0.9%, respectively, of all expressed genes, and in decreased transcript levels for only 0.09% and 0.07%, respectively, of all expressed genes (Supplemental Tables III and V, P < 0.1). Thus, the predominant directions of transcriptional responses to excess Zn were identical in roots of A. halleri and A. thaliana, with a global trend toward up-regulation opposing a trend toward down-regulation among our set of candidate genes in both species. In shoots of A. thaliana, among all expressed genes, transcriptional up-regulation was observed in response to excess Zn (0% down, 3.2% up, P < 0.1; Supplemental Table VI). By comparison, in shoots of A. halleri a smaller proportion of expressed genes responded, and transcriptional down-regulation was predominant (0.8% down, 0.2% up, P < 0.1; Supplemental Table IV). This indicated that primarily in the shoots, the two species respond differently to excess Zn also at the whole transcriptome level. Confirmation of Candidate Genes by Real-Time RT-PCR To assess the reliability of the cross species expression differences between A. halleri and A. thaliana detected using the microarrays, we determined transcript levels by an alternative method, quantitative real-time RT-PCR, for a subset of 18 genes (Table II Table II. Summary of real-time RT-PCR data for selected candidate genes at control Zn concentrations Transcript levels relative to EF1α of selected candidate genes as determined by real-time RT-PCR are given for shoot and root tissues from plants grown under control conditions. Values are mean and se of between three and five independent experiments for A. halleri and A. thaliana. The statistical significances of the differences between A. halleri and A. thaliana expression levels was assessed with a Student's t test (two sample assuming unequal variances), P values are indicated as follows: * = P < 0.05; ** = P < 0.01; *** = P < 0.001. In real-time RT-PCR experiments, genes were determined as not expressed (n.e.) when C T values were above a threshold of 35 and reaction efficiencies with the respective cDNA were more than 5% below the mean RE for the respective primer pair; n.d. = not determined.The normalized -fold difference between A. halleri and A. thaliana from ATH1 microarray data is given for comparison (see Table I for further details). Flags denote calls as given by MAS5: P = present; M = marginal; A = absent. NAS1 and NAS4 were included for comparison with the other members of the NAS family. Gene Name . Root . . . . . . . Shoot . . . . . . . Tissue with Higher Expression in A. halleri Compared to A. thaliana . . . Real-Time RT-PCR . . . . Microarray . . . Real-Time RT-PCR . . . . Microarray . . . . . . A. halleri Transcript Level . . A. thaliana Transcript Level . A. halleri/A. thaliana Transcript Ratio . A. halleri/A. thaliana Signal Ratio . A. halleri Flag . A. thaliana Flag . A. halleri Transcript Level . . A. thaliana Transcript Level . A. halleri/A. thaliana Transcript Ratio . A. halleri/A. thaliana Signal Ratio . A. halleri Flag . A. thaliana Flag . Real-Time RT-PCR . Microarray . Cytoplasmic metal influx ZIP3 498 ± 46.0 ** 108 ± 46.1 4.62 1.71* P P 10.2 ± 2.62 * 1.29 ± 0.43 7.89 2.37 A A Root, shoot ZIP4 155 ± 19.6 *** 13.1 ± 1.74 11.9 24.6*** P A 52.6 ± 7.96 92.1 ± 44.3 0.57 4.04* P P Root Root, shoot ZIP9 654 ± 189 * 50.0 ± 4.61 13.1 36.2*** P P, A n.e. n.e. n.d. 6.32 A A Root Root ZIP10 17.9 ± 4.98 * 2.89 ± 0.71 6.18 111** P, M A n.e. n.e. n.d. 3.24 A A Root Root IRT3 191 ± 20.5 *** 40.9 ± 14.6 4.68 4.47* P P 85.0 ± 4.70 63.8 ± 38.9 1.33 1.70* P P Root Root ZIP6 122 ± 27.9 * 26.3 ± 4.78 4.64 2.16* P P 183 ± 33.5 ** 18.2 ± 5.35 10.1 7.34*** P M, A Root, shoot Shoot Root-to-shoot metal transport HMA4 2,092 ± 401 ** 327 ± 29.2 6.39 0.71 P P 1,141 ± 332 * 37.9 ± 31.4 30.1 52.5** P A Root, shoot Shoot FRD3 292 ± 87.8 * 18.8 ± 4.95 15.5 44.6*** P P 46.5 ± 13.3 * 7.45 ± 3.05 6.23 37.1* P A Root, shoot Root Other membrane transport MTP8 10.6 ± 3.53 * 1.39 ± 0.53 7.65 15.9* A A 32.4 ± 7.84 * 2.86 ± 1.45 11.3 65.0** P, A A Root, shoot Shoot PHT1;4 77.6 ± 15.9 * 18.7 ± 1.43 4.15 19.1** P P 108 ± 10.2 *** 20.2 ± 10.3 5.33 446* P A Root, shoot Root Nicotianamine/thiol biosynthesis pathways NAS1 52.0 ± 10.4 49.5 ± 24.9 1.05 0.81 P P 37.4 ± 9.27 79.3 ± 26.7 0.47 0.54 P, M P NAS2 437 ± 113 * 27.5 ± 14.9 15.9 48.4*** P P n.e. n.e. n.d. 3.14 P, A A Root Root NAS3 13.9 ± 2.55 ** 0.20 ± 0.05 71.4 5.22** P A 44.7 ± 5.06 * 15.8 ± 6.43 2.82 7.89** P P Root, shoot Root, shoot NAS4 12.3 ± 1.54 6.93 ± 2.12 1.78 13.7 P, M A 34.5 ± 10.4 * 125 ± 34.6 0.28 0.36* P P OASA2 22.7 ± 5.28 83.5 ± 27.1 0.27 3.25* P P 13.3 ± 2.08 64.7 ± 30.8 0.21 10.6*** P P Shoot SAMS2 881 ± 140 ** 158 ± 36.4 5.59 2.60* P P 422 ± 65.4 ** 70.6 ± 24.6 5.97 8.54*** P P Root, shoot Shoot Stress protection PDI1 362 ± 56.7 * 173 ± 42.9 2.10 3.50** P P 355 ± 63.3 * 91.3 ± 47.1 3.89 39.5** P P Root, shoot Shoot PDI2 118 ± 20.4 125 ± 48.8 0.94 3.08* P P 129 ± 16.9 127 ± 49.3 1.01 17.5** P P Shoot Gene Name . Root . . . . . . . Shoot . . . . . . . Tissue with Higher Expression in A. halleri Compared to A. thaliana . . . Real-Time RT-PCR . . . . Microarray . . . Real-Time RT-PCR . . . . Microarray . . . . . . A. halleri Transcript Level . . A. thaliana Transcript Level . A. halleri/A. thaliana Transcript Ratio . A. halleri/A. thaliana Signal Ratio . A. halleri Flag . A. thaliana Flag . A. halleri Transcript Level . . A. thaliana Transcript Level . A. halleri/A. thaliana Transcript Ratio . A. halleri/A. thaliana Signal Ratio . A. halleri Flag . A. thaliana Flag . Real-Time RT-PCR . Microarray . Cytoplasmic metal influx ZIP3 498 ± 46.0 ** 108 ± 46.1 4.62 1.71* P P 10.2 ± 2.62 * 1.29 ± 0.43 7.89 2.37 A A Root, shoot ZIP4 155 ± 19.6 *** 13.1 ± 1.74 11.9 24.6*** P A 52.6 ± 7.96 92.1 ± 44.3 0.57 4.04* P P Root Root, shoot ZIP9 654 ± 189 * 50.0 ± 4.61 13.1 36.2*** P P, A n.e. n.e. n.d. 6.32 A A Root Root ZIP10 17.9 ± 4.98 * 2.89 ± 0.71 6.18 111** P, M A n.e. n.e. n.d. 3.24 A A Root Root IRT3 191 ± 20.5 *** 40.9 ± 14.6 4.68 4.47* P P 85.0 ± 4.70 63.8 ± 38.9 1.33 1.70* P P Root Root ZIP6 122 ± 27.9 * 26.3 ± 4.78 4.64 2.16* P P 183 ± 33.5 ** 18.2 ± 5.35 10.1 7.34*** P M, A Root, shoot Shoot Root-to-shoot metal transport HMA4 2,092 ± 401 ** 327 ± 29.2 6.39 0.71 P P 1,141 ± 332 * 37.9 ± 31.4 30.1 52.5** P A Root, shoot Shoot FRD3 292 ± 87.8 * 18.8 ± 4.95 15.5 44.6*** P P 46.5 ± 13.3 * 7.45 ± 3.05 6.23 37.1* P A Root, shoot Root Other membrane transport MTP8 10.6 ± 3.53 * 1.39 ± 0.53 7.65 15.9* A A 32.4 ± 7.84 * 2.86 ± 1.45 11.3 65.0** P, A A Root, shoot Shoot PHT1;4 77.6 ± 15.9 * 18.7 ± 1.43 4.15 19.1** P P 108 ± 10.2 *** 20.2 ± 10.3 5.33 446* P A Root, shoot Root Nicotianamine/thiol biosynthesis pathways NAS1 52.0 ± 10.4 49.5 ± 24.9 1.05 0.81 P P 37.4 ± 9.27 79.3 ± 26.7 0.47 0.54 P, M P NAS2 437 ± 113 * 27.5 ± 14.9 15.9 48.4*** P P n.e. n.e. n.d. 3.14 P, A A Root Root NAS3 13.9 ± 2.55 ** 0.20 ± 0.05 71.4 5.22** P A 44.7 ± 5.06 * 15.8 ± 6.43 2.82 7.89** P P Root, shoot Root, shoot NAS4 12.3 ± 1.54 6.93 ± 2.12 1.78 13.7 P, M A 34.5 ± 10.4 * 125 ± 34.6 0.28 0.36* P P OASA2 22.7 ± 5.28 83.5 ± 27.1 0.27 3.25* P P 13.3 ± 2.08 64.7 ± 30.8 0.21 10.6*** P P Shoot SAMS2 881 ± 140 ** 158 ± 36.4 5.59 2.60* P P 422 ± 65.4 ** 70.6 ± 24.6 5.97 8.54*** P P Root, shoot Shoot Stress protection PDI1 362 ± 56.7 * 173 ± 42.9 2.10 3.50** P P 355 ± 63.3 * 91.3 ± 47.1 3.89 39.5** P P Root, shoot Shoot PDI2 118 ± 20.4 125 ± 48.8 0.94 3.08* P P 129 ± 16.9 127 ± 49.3 1.01 17.5** P P Shoot Open in new tab Table II. Summary of real-time RT-PCR data for selected candidate genes at control Zn concentrations Transcript levels relative to EF1α of selected candidate genes as determined by real-time RT-PCR are given for shoot and root tissues from plants grown under control conditions. Values are mean and se of between three and five independent experiments for A. halleri and A. thaliana. The statistical significances of the differences between A. halleri and A. thaliana expression levels was assessed with a Student's t test (two sample assuming unequal variances), P values are indicated as follows: * = P < 0.05; ** = P < 0.01; *** = P < 0.001. In real-time RT-PCR experiments, genes were determined as not expressed (n.e.) when C T values were above a threshold of 35 and reaction efficiencies with the respective cDNA were more than 5% below the mean RE for the respective primer pair; n.d. = not determined.The normalized -fold difference between A. halleri and A. thaliana from ATH1 microarray data is given for comparison (see Table I for further details). Flags denote calls as given by MAS5: P = present; M = marginal; A = absent. NAS1 and NAS4 were included for comparison with the other members of the NAS family. Gene Name . Root . . . . . . . Shoot . . . . . . . Tissue with Higher Expression in A. halleri Compared to A. thaliana . . . Real-Time RT-PCR . . . . Microarray . . . Real-Time RT-PCR . . . . Microarray . . . . . . A. halleri Transcript Level . . A. thaliana Transcript Level . A. halleri/A. thaliana Transcript Ratio . A. halleri/A. thaliana Signal Ratio . A. halleri Flag . A. thaliana Flag . A. halleri Transcript Level . . A. thaliana Transcript Level . A. halleri/A. thaliana Transcript Ratio . A. halleri/A. thaliana Signal Ratio . A. halleri Flag . A. thaliana Flag . Real-Time RT-PCR . Microarray . Cytoplasmic metal influx ZIP3 498 ± 46.0 ** 108 ± 46.1 4.62 1.71* P P 10.2 ± 2.62 * 1.29 ± 0.43 7.89 2.37 A A Root, shoot ZIP4 155 ± 19.6 *** 13.1 ± 1.74 11.9 24.6*** P A 52.6 ± 7.96 92.1 ± 44.3 0.57 4.04* P P Root Root, shoot ZIP9 654 ± 189 * 50.0 ± 4.61 13.1 36.2*** P P, A n.e. n.e. n.d. 6.32 A A Root Root ZIP10 17.9 ± 4.98 * 2.89 ± 0.71 6.18 111** P, M A n.e. n.e. n.d. 3.24 A A Root Root IRT3 191 ± 20.5 *** 40.9 ± 14.6 4.68 4.47* P P 85.0 ± 4.70 63.8 ± 38.9 1.33 1.70* P P Root Root ZIP6 122 ± 27.9 * 26.3 ± 4.78 4.64 2.16* P P 183 ± 33.5 ** 18.2 ± 5.35 10.1 7.34*** P M, A Root, shoot Shoot Root-to-shoot metal transport HMA4 2,092 ± 401 ** 327 ± 29.2 6.39 0.71 P P 1,141 ± 332 * 37.9 ± 31.4 30.1 52.5** P A Root, shoot Shoot FRD3 292 ± 87.8 * 18.8 ± 4.95 15.5 44.6*** P P 46.5 ± 13.3 * 7.45 ± 3.05 6.23 37.1* P A Root, shoot Root Other membrane transport MTP8 10.6 ± 3.53 * 1.39 ± 0.53 7.65 15.9* A A 32.4 ± 7.84 * 2.86 ± 1.45 11.3 65.0** P, A A Root, shoot Shoot PHT1;4 77.6 ± 15.9 * 18.7 ± 1.43 4.15 19.1** P P 108 ± 10.2 *** 20.2 ± 10.3 5.33 446* P A Root, shoot Root Nicotianamine/thiol biosynthesis pathways NAS1 52.0 ± 10.4 49.5 ± 24.9 1.05 0.81 P P 37.4 ± 9.27 79.3 ± 26.7 0.47 0.54 P, M P NAS2 437 ± 113 * 27.5 ± 14.9 15.9 48.4*** P P n.e. n.e. n.d. 3.14 P, A A Root Root NAS3 13.9 ± 2.55 ** 0.20 ± 0.05 71.4 5.22** P A 44.7 ± 5.06 * 15.8 ± 6.43 2.82 7.89** P P Root, shoot Root, shoot NAS4 12.3 ± 1.54 6.93 ± 2.12 1.78 13.7 P, M A 34.5 ± 10.4 * 125 ± 34.6 0.28 0.36* P P OASA2 22.7 ± 5.28 83.5 ± 27.1 0.27 3.25* P P 13.3 ± 2.08 64.7 ± 30.8 0.21 10.6*** P P Shoot SAMS2 881 ± 140 ** 158 ± 36.4 5.59 2.60* P P 422 ± 65.4 ** 70.6 ± 24.6 5.97 8.54*** P P Root, shoot Shoot Stress protection PDI1 362 ± 56.7 * 173 ± 42.9 2.10 3.50** P P 355 ± 63.3 * 91.3 ± 47.1 3.89 39.5** P P Root, shoot Shoot PDI2 118 ± 20.4 125 ± 48.8 0.94 3.08* P P 129 ± 16.9 127 ± 49.3 1.01 17.5** P P Shoot Gene Name . Root . . . . . . . Shoot . . . . . . . Tissue with Higher Expression in A. halleri Compared to A. thaliana . . . Real-Time RT-PCR . . . . Microarray . . . Real-Time RT-PCR . . . . Microarray . . . . . . A. halleri Transcript Level . . A. thaliana Transcript Level . A. halleri/A. thaliana Transcript Ratio . A. halleri/A. thaliana Signal Ratio . A. halleri Flag . A. thaliana Flag . A. halleri Transcript Level . . A. thaliana Transcript Level . A. halleri/A. thaliana Transcript Ratio . A. halleri/A. thaliana Signal Ratio . A. halleri Flag . A. thaliana Flag . Real-Time RT-PCR . Microarray . Cytoplasmic metal influx ZIP3 498 ± 46.0 ** 108 ± 46.1 4.62 1.71* P P 10.2 ± 2.62 * 1.29 ± 0.43 7.89 2.37 A A Root, shoot ZIP4 155 ± 19.6 *** 13.1 ± 1.74 11.9 24.6*** P A 52.6 ± 7.96 92.1 ± 44.3 0.57 4.04* P P Root Root, shoot ZIP9 654 ± 189 * 50.0 ± 4.61 13.1 36.2*** P P, A n.e. n.e. n.d. 6.32 A A Root Root ZIP10 17.9 ± 4.98 * 2.89 ± 0.71 6.18 111** P, M A n.e. n.e. n.d. 3.24 A A Root Root IRT3 191 ± 20.5 *** 40.9 ± 14.6 4.68 4.47* P P 85.0 ± 4.70 63.8 ± 38.9 1.33 1.70* P P Root Root ZIP6 122 ± 27.9 * 26.3 ± 4.78 4.64 2.16* P P 183 ± 33.5 ** 18.2 ± 5.35 10.1 7.34*** P M, A Root, shoot Shoot Root-to-shoot metal transport HMA4 2,092 ± 401 ** 327 ± 29.2 6.39 0.71 P P 1,141 ± 332 * 37.9 ± 31.4 30.1 52.5** P A Root, shoot Shoot FRD3 292 ± 87.8 * 18.8 ± 4.95 15.5 44.6*** P P 46.5 ± 13.3 * 7.45 ± 3.05 6.23 37.1* P A Root, shoot Root Other membrane transport MTP8 10.6 ± 3.53 * 1.39 ± 0.53 7.65 15.9* A A 32.4 ± 7.84 * 2.86 ± 1.45 11.3 65.0** P, A A Root, shoot Shoot PHT1;4 77.6 ± 15.9 * 18.7 ± 1.43 4.15 19.1** P P 108 ± 10.2 *** 20.2 ± 10.3 5.33 446* P A Root, shoot Root Nicotianamine/thiol biosynthesis pathways NAS1 52.0 ± 10.4 49.5 ± 24.9 1.05 0.81 P P 37.4 ± 9.27 79.3 ± 26.7 0.47 0.54 P, M P NAS2 437 ± 113 * 27.5 ± 14.9 15.9 48.4*** P P n.e. n.e. n.d. 3.14 P, A A Root Root NAS3 13.9 ± 2.55 ** 0.20 ± 0.05 71.4 5.22** P A 44.7 ± 5.06 * 15.8 ± 6.43 2.82 7.89** P P Root, shoot Root, shoot NAS4 12.3 ± 1.54 6.93 ± 2.12 1.78 13.7 P, M A 34.5 ± 10.4 * 125 ± 34.6 0.28 0.36* P P OASA2 22.7 ± 5.28 83.5 ± 27.1 0.27 3.25* P P 13.3 ± 2.08 64.7 ± 30.8 0.21 10.6*** P P Shoot SAMS2 881 ± 140 ** 158 ± 36.4 5.59 2.60* P P 422 ± 65.4 ** 70.6 ± 24.6 5.97 8.54*** P P Root, shoot Shoot Stress protection PDI1 362 ± 56.7 * 173 ± 42.9 2.10 3.50** P P 355 ± 63.3 * 91.3 ± 47.1 3.89 39.5** P P Root, shoot Shoot PDI2 118 ± 20.4 125 ± 48.8 0.94 3.08* P P 129 ± 16.9 127 ± 49.3 1.01 17.5** P P Shoot Open in new tab ). Overall, there was good qualitative agreement between the ratios of microarray signals in A. halleri relative to A. thaliana and the ratios of transcript levels as determined by real-time RT-PCR (Table II). The false negatives according to microarray hybridization, ZIP3, and HMA4 (in roots), could be attributed to a high divergence of the A. halleri sequence from the A. thaliana sequence in the region covered by the oligonucleotide probes on the microarray, in combination with a modest magnitude of the A. halleri to A. thaliana ratio of transcript levels. For several other genes, quantitative discrepancies between microarray and real-time RT-PCR data are likely to be attributable to cross hybridization between high transcript levels of a gene in A. halleri and the probes for a closely related member of the same gene family (NAS2 on NAS4 in roots, NAS3 on NAS2 in shoots, NAS2 on NAS3 in shoots under Zn deficiency, and PDI1 on PDI2). It was reported earlier that very low signals (for example, for ZIP10, MTP8, and ZIP9 in A. thaliana) are often quantified imprecisely on the ATH1 chip (Czechowski et al., 2004), and for these genes in particular, the real-time RT-PCR results are likely to be more accurate. Functional Analysis of the Novel Candidate Protein AhHMA4 Out of all candidate genes identified here, absolute transcript levels were highest for A. halleri HMA4 (see Table II), which encodes a membrane transport protein of the P1B transition metal pump family of the P-type ATPases (Axelsen and Palmgren, 2001). To determine the cellular function of this candidate gene, which had not previously been implicated in metal hyperaccumulation or metal tolerance in A. halleri, we expressed AhHMA4 and AtHMA4 in metal hypersensitive mutants of budding yeast (Saccharomyces cerevisiae; Fig. 4 Figure 4. Open in new tabDownload slide Functional analysis of AtHMA4 and AhHMA4 cDNAs in yeast mutant strains. Expression of Arabidopsis HMA4 cDNAs in the Zn-hypersensitive zrc1 cot1 double mutant (A) and in the Cd-hypersensitive ycf1 mutant (B) is shown. Wild-type cells were transformed with pFL38H-GW (ev), and mutant cells were transformed with pFL38H-GW (ev) and with wild-type and mutated (D401A) cDNAs of both AtHMA4 and AhHMA4 in pFL38H-GW. The phosphorylation of the Asp residue (here D401) of the conserved phosphorylation motif is an indispensable step of the reaction cycle in all P-type ATPases. Serial dilutions of transformants were spotted on LSP plates containing the indicated concentrations of ZnSO4 (A) or CdSO4 (B). The 100 dilution corresponds to an OD600 of 0.5. Plates were incubated at 30°C for 4 d. Figure 4. Open in new tabDownload slide Functional analysis of AtHMA4 and AhHMA4 cDNAs in yeast mutant strains. Expression of Arabidopsis HMA4 cDNAs in the Zn-hypersensitive zrc1 cot1 double mutant (A) and in the Cd-hypersensitive ycf1 mutant (B) is shown. Wild-type cells were transformed with pFL38H-GW (ev), and mutant cells were transformed with pFL38H-GW (ev) and with wild-type and mutated (D401A) cDNAs of both AtHMA4 and AhHMA4 in pFL38H-GW. The phosphorylation of the Asp residue (here D401) of the conserved phosphorylation motif is an indispensable step of the reaction cycle in all P-type ATPases. Serial dilutions of transformants were spotted on LSP plates containing the indicated concentrations of ZnSO4 (A) or CdSO4 (B). The 100 dilution corresponds to an OD600 of 0.5. Plates were incubated at 30°C for 4 d. ). Both the A. thaliana and the A. halleri HMA4 proteins were similarly able to complement Zn hypersensitivity of the zrc1 cot1 double mutant and Cd hypersensitivity of the ycf1 mutant of yeast (Fig. 4, A and B). When the transport functions of AtHMA4 and AhHMA4 were disrupted by converting the conserved Asp residues (D401) of the phosphorylation motifs (Axelsen and Palmgren, 2001) into Alas, respectively, complementation was no longer observed. Consequently, Zn and Cd are substrates of both Arabidopsis HMA4 proteins in yeast, and the observed complementation is attributable to the transport functions of the proteins and not to the known ability of the cytoplasmic C termini of AtHMA4 and related proteins to bind metal ions (Bernard et al., 2004; Papoyan and Kochian, 2004). Gene Copy Number of Highly Expressed Candidate Genes in A. halleri Previously, high transcript levels of the Zn tolerance gene MTP1 were partly attributed to the presence of several MTP1 gene copies in the genome of A. halleri, whereas the genome of the closely related nontolerant species A. thaliana contains only a single MTP1 gene copy (Dräger et al., 2004). To estimate the genomic copy number for other candidate genes in A. halleri, we generated genomic DNA blots for 12 of these genes (Kim et al., 1998; Marquardt et al., 2000; Small and Wendel, 2000; Ishizaki et al., 2002). Complex band patterns suggested that more than one gene copy is present in the A. halleri genome for the genes ZIP3, ZIP6, ZIP9, and HMA4 (Fig. 5, A to D Figure 5. Open in new tabDownload slide Genomic DNA gel blot for ZIP9 (A), HMA4 (B), FRD3 (C), and MTP8 (D) in A. thaliana and in A. halleri. Blots were performed using genomic DNA extracted from A. thaliana (Col accession) and from two A. halleri individuals (Lan 3-1 and Lan 5 of the accession Langelsheim). A to D, Five micrograms of genomic DNA were digested with EcoRI, HindIII, or NcoI, resolved on a 0.9% (w/v) agarose gel, blotted, and hybridized with radiolabeled A. halleri-derived cDNA probes. A, An EcoRI restriction site is present in the region spanned by the ZIP9 probe in A. thaliana only, a HindIII restriction site is present in the intron spanned by the probe in A. halleri only, and an NcoI restriction site is present in an exon spanned by the probe in both species. B, A HindIII restriction site is present in one of the introns spanned by the HMA4 probe in A. halleri. C, A HindIII restriction site is present in the region spanned by the FRD3 probe in both species. D, After restriction with NcoI, a predicted 20.9 kb fragment in A. thaliana was not detected by the MTP8 probe, probably because of the large size of this fragment (see also Table III). Note that for clarity, lanes were rearranged in the ZIP9 blot. Figure 5. Open in new tabDownload slide Genomic DNA gel blot for ZIP9 (A), HMA4 (B), FRD3 (C), and MTP8 (D) in A. thaliana and in A. halleri. Blots were performed using genomic DNA extracted from A. thaliana (Col accession) and from two A. halleri individuals (Lan 3-1 and Lan 5 of the accession Langelsheim). A to D, Five micrograms of genomic DNA were digested with EcoRI, HindIII, or NcoI, resolved on a 0.9% (w/v) agarose gel, blotted, and hybridized with radiolabeled A. halleri-derived cDNA probes. A, An EcoRI restriction site is present in the region spanned by the ZIP9 probe in A. thaliana only, a HindIII restriction site is present in the intron spanned by the probe in A. halleri only, and an NcoI restriction site is present in an exon spanned by the probe in both species. B, A HindIII restriction site is present in one of the introns spanned by the HMA4 probe in A. halleri. C, A HindIII restriction site is present in the region spanned by the FRD3 probe in both species. D, After restriction with NcoI, a predicted 20.9 kb fragment in A. thaliana was not detected by the MTP8 probe, probably because of the large size of this fragment (see also Table III). Note that for clarity, lanes were rearranged in the ZIP9 blot. ; Table III Table III. Gene copy number estimated by genomic DNA gel blot for selected candidate genes in A. halleri Five micrograms of genomic DNA isolated from A. thaliana (Col-0 accession) and from two A. halleri individuals (Lan 3-1 and Lan 5 of the Langelsheim accession) were digested with EcoRI, HindIII, or NcoI, resolved on a 0.9% (w/v) agarose gel, blotted, and hybridized with radiolabelled A. halleri-derived cDNA probes. The size of the detected DNA fragments is given in kilobase pair (detectable fragments were in the range of 0.2 and 14 kb). Note that in A. halleri, there are a maximum of two alleles per gene copy. Blots for the four underlined genes are presented in Figure 5, and other blots are shown in Supplemental Figure 1. Gene . Copy No. in A. halleri . A. halleri Lan 3.1 . . . A. halleri Lan 5 . . . A. thaliana Col-0 . . . . . EcoRI . HindIII . NcoI . EcoRI . HindIII . NcoI . EcoRI . HindIII . NcoI . ZIP3 >1 3.5 6.5 >10 3.5 6.5 >10 3.6 7.4 15.2 1.2 5.8 1.2 5.8 (3.8)a (5.4)a (7.2)a 1.5 1.5 (1.7)a ZIP4 ∼1 4.2 2.2 0.6 4.2 2.2 0.6 0.8 7.0 2.0 0.8 1.5 0.8 1.5 (6.3)b (5.5)b (4.5)b (2.0)bc ZIP9 >1 5.5 2.5d 4e 5.5 2.5d 4e 4.2d 4.4 4.0e 1.7 2 3.5 1.7 2.2 0.5 0.2 0.7 0.2 0.7 2 0.2 ZIP10 ∼1 3.2 2.8 –c 3.23 3 4 14.4c 2.9 28.6c 3 2.8 IRT3 ∼1 1.5 2.2e 9.5 1.5 2.2e 9.5 6.3 (7.0)b 4.5 0.9 2 0.8 0.9 2 0.8 (0.8)b 5.5f (2.0)b 1.5 1.5 2.0 1 0.06b ZIP6 >1 10 5.8 >10 5 6 >10 2.7c 8.4 11.7 5 4 9 2.5 3.8 9 2.5 2.2 2 HMA4 >1 10 6d 9 10 6d 9 15.7 2.8d 7.3 8 4.5 7.5 8 4.5 7.5 0.6 (4.1)g 1.7 3.8 4 1.2 1.2 0.6 0.6 FRD3 ∼1 >10 3.5ce 10 >10 3.5ce 10 1.5 3.5e 14.9 8 1.0 MTP8 ∼1 5 1.1 –c 5 1.1 –c 5.7 1.1 20.9c PHT1;4 ∼1 1 –c 4 1 –c 4 10.0 6.0 4.1 (2.0)h (2.5)h (5.9)h PDI1 ∼1 1.5 3.3 2.2 2.4 3.3 2.2 (3.4)i 3.0d,e 2.2 1 1 2.4 (2.4)i (2.0)i 0.7 0.7 (1.5)j (1.0)i 0.6 0.6 0.65 0.56 PDI2 ∼1 1.5 3.3 2.2 2.4 3.3 2.2 3.4 (3.0)i 2.8e 1 1 (2.4)i 2.4d 2.0 0.7 0.7 (1.5)j 1.0 (2.2)i 0.6 0.6 (0.65)i (0.56)i Gene . Copy No. in A. halleri . A. halleri Lan 3.1 . . . A. halleri Lan 5 . . . A. thaliana Col-0 . . . . . EcoRI . HindIII . NcoI . EcoRI . HindIII . NcoI . EcoRI . HindIII . NcoI . ZIP3 >1 3.5 6.5 >10 3.5 6.5 >10 3.6 7.4 15.2 1.2 5.8 1.2 5.8 (3.8)a (5.4)a (7.2)a 1.5 1.5 (1.7)a ZIP4 ∼1 4.2 2.2 0.6 4.2 2.2 0.6 0.8 7.0 2.0 0.8 1.5 0.8 1.5 (6.3)b (5.5)b (4.5)b (2.0)bc ZIP9 >1 5.5 2.5d 4e 5.5 2.5d 4e 4.2d 4.4 4.0e 1.7 2 3.5 1.7 2.2 0.5 0.2 0.7 0.2 0.7 2 0.2 ZIP10 ∼1 3.2 2.8 –c 3.23 3 4 14.4c 2.9 28.6c 3 2.8 IRT3 ∼1 1.5 2.2e 9.5 1.5 2.2e 9.5 6.3 (7.0)b 4.5 0.9 2 0.8 0.9 2 0.8 (0.8)b 5.5f (2.0)b 1.5 1.5 2.0 1 0.06b ZIP6 >1 10 5.8 >10 5 6 >10 2.7c 8.4 11.7 5 4 9 2.5 3.8 9 2.5 2.2 2 HMA4 >1 10 6d 9 10 6d 9 15.7 2.8d 7.3 8 4.5 7.5 8 4.5 7.5 0.6 (4.1)g 1.7 3.8 4 1.2 1.2 0.6 0.6 FRD3 ∼1 >10 3.5ce 10 >10 3.5ce 10 1.5 3.5e 14.9 8 1.0 MTP8 ∼1 5 1.1 –c 5 1.1 –c 5.7 1.1 20.9c PHT1;4 ∼1 1 –c 4 1 –c 4 10.0 6.0 4.1 (2.0)h (2.5)h (5.9)h PDI1 ∼1 1.5 3.3 2.2 2.4 3.3 2.2 (3.4)i 3.0d,e 2.2 1 1 2.4 (2.4)i (2.0)i 0.7 0.7 (1.5)j (1.0)i 0.6 0.6 0.65 0.56 PDI2 ∼1 1.5 3.3 2.2 2.4 3.3 2.2 3.4 (3.0)i 2.8e 1 1 (2.4)i 2.4d 2.0 0.7 0.7 (1.5)j 1.0 (2.2)i 0.6 0.6 (0.65)i (0.56)i a Cross hybridization of AhZIP3 probe with AtZIP5 genomic sequence. b Cross hybridization of AhIRT3 and AhZIP4 probes with AtZIP4 and AtIRT3 genomic sequences, respectively. c Not detected, predicted fragment given for A. thaliana. d Enzyme cuts once in one of the introns spanned by the cDNA probe. e Enzyme cuts once in the probe. f Enzyme cuts twice in the probe. g Cross hybridization of AhHMA4 probe with AtHMA2 genomic sequence. h Cross hybridization of AhPht1;4 probe with At3g54700 genomic sequence. i Cross hybridization of AhPDI1 and AhPDI2 probes with AtPDI2 and AtPDI1 genomic sequences, respectively. Note that it is likely that cross hybridization also occurs of AhPDI1 and AhPDI2 probes with AhPDI2 and AhPDI1 genomic sequences. j Additional band, identity not known. Open in new tab Table III. Gene copy number estimated by genomic DNA gel blot for selected candidate genes in A. halleri Five micrograms of genomic DNA isolated from A. thaliana (Col-0 accession) and from two A. halleri individuals (Lan 3-1 and Lan 5 of the Langelsheim accession) were digested with EcoRI, HindIII, or NcoI, resolved on a 0.9% (w/v) agarose gel, blotted, and hybridized with radiolabelled A. halleri-derived cDNA probes. The size of the detected DNA fragments is given in kilobase pair (detectable fragments were in the range of 0.2 and 14 kb). Note that in A. halleri, there are a maximum of two alleles per gene copy. Blots for the four underlined genes are presented in Figure 5, and other blots are shown in Supplemental Figure 1. Gene . Copy No. in A. halleri . A. halleri Lan 3.1 . . . A. halleri Lan 5 . . . A. thaliana Col-0 . . . . . EcoRI . HindIII . NcoI . EcoRI . HindIII . NcoI . EcoRI . HindIII . NcoI . ZIP3 >1 3.5 6.5 >10 3.5 6.5 >10 3.6 7.4 15.2 1.2 5.8 1.2 5.8 (3.8)a (5.4)a (7.2)a 1.5 1.5 (1.7)a ZIP4 ∼1 4.2 2.2 0.6 4.2 2.2 0.6 0.8 7.0 2.0 0.8 1.5 0.8 1.5 (6.3)b (5.5)b (4.5)b (2.0)bc ZIP9 >1 5.5 2.5d 4e 5.5 2.5d 4e 4.2d 4.4 4.0e 1.7 2 3.5 1.7 2.2 0.5 0.2 0.7 0.2 0.7 2 0.2 ZIP10 ∼1 3.2 2.8 –c 3.23 3 4 14.4c 2.9 28.6c 3 2.8 IRT3 ∼1 1.5 2.2e 9.5 1.5 2.2e 9.5 6.3 (7.0)b 4.5 0.9 2 0.8 0.9 2 0.8 (0.8)b 5.5f (2.0)b 1.5 1.5 2.0 1 0.06b ZIP6 >1 10 5.8 >10 5 6 >10 2.7c 8.4 11.7 5 4 9 2.5 3.8 9 2.5 2.2 2 HMA4 >1 10 6d 9 10 6d 9 15.7 2.8d 7.3 8 4.5 7.5 8 4.5 7.5 0.6 (4.1)g 1.7 3.8 4 1.2 1.2 0.6 0.6 FRD3 ∼1 >10 3.5ce 10 >10 3.5ce 10 1.5 3.5e 14.9 8 1.0 MTP8 ∼1 5 1.1 –c 5 1.1 –c 5.7 1.1 20.9c PHT1;4 ∼1 1 –c 4 1 –c 4 10.0 6.0 4.1 (2.0)h (2.5)h (5.9)h PDI1 ∼1 1.5 3.3 2.2 2.4 3.3 2.2 (3.4)i 3.0d,e 2.2 1 1 2.4 (2.4)i (2.0)i 0.7 0.7 (1.5)j (1.0)i 0.6 0.6 0.65 0.56 PDI2 ∼1 1.5 3.3 2.2 2.4 3.3 2.2 3.4 (3.0)i 2.8e 1 1 (2.4)i 2.4d 2.0 0.7 0.7 (1.5)j 1.0 (2.2)i 0.6 0.6 (0.65)i (0.56)i Gene . Copy No. in A. halleri . A. halleri Lan 3.1 . . . A. halleri Lan 5 . . . A. thaliana Col-0 . . . . . EcoRI . HindIII . NcoI . EcoRI . HindIII . NcoI . EcoRI . HindIII . NcoI . ZIP3 >1 3.5 6.5 >10 3.5 6.5 >10 3.6 7.4 15.2 1.2 5.8 1.2 5.8 (3.8)a (5.4)a (7.2)a 1.5 1.5 (1.7)a ZIP4 ∼1 4.2 2.2 0.6 4.2 2.2 0.6 0.8 7.0 2.0 0.8 1.5 0.8 1.5 (6.3)b (5.5)b (4.5)b (2.0)bc ZIP9 >1 5.5 2.5d 4e 5.5 2.5d 4e 4.2d 4.4 4.0e 1.7 2 3.5 1.7 2.2 0.5 0.2 0.7 0.2 0.7 2 0.2 ZIP10 ∼1 3.2 2.8 –c 3.23 3 4 14.4c 2.9 28.6c 3 2.8 IRT3 ∼1 1.5 2.2e 9.5 1.5 2.2e 9.5 6.3 (7.0)b 4.5 0.9 2 0.8 0.9 2 0.8 (0.8)b 5.5f (2.0)b 1.5 1.5 2.0 1 0.06b ZIP6 >1 10 5.8 >10 5 6 >10 2.7c 8.4 11.7 5 4 9 2.5 3.8 9 2.5 2.2 2 HMA4 >1 10 6d 9 10 6d 9 15.7 2.8d 7.3 8 4.5 7.5 8 4.5 7.5 0.6 (4.1)g 1.7 3.8 4 1.2 1.2 0.6 0.6 FRD3 ∼1 >10 3.5ce 10 >10 3.5ce 10 1.5 3.5e 14.9 8 1.0 MTP8 ∼1 5 1.1 –c 5 1.1 –c 5.7 1.1 20.9c PHT1;4 ∼1 1 –c 4 1 –c 4 10.0 6.0 4.1 (2.0)h (2.5)h (5.9)h PDI1 ∼1 1.5 3.3 2.2 2.4 3.3 2.2 (3.4)i 3.0d,e 2.2 1 1 2.4 (2.4)i (2.0)i 0.7 0.7 (1.5)j (1.0)i 0.6 0.6 0.65 0.56 PDI2 ∼1 1.5 3.3 2.2 2.4 3.3 2.2 3.4 (3.0)i 2.8e 1 1 (2.4)i 2.4d 2.0 0.7 0.7 (1.5)j 1.0 (2.2)i 0.6 0.6 (0.65)i (0.56)i a Cross hybridization of AhZIP3 probe with AtZIP5 genomic sequence. b Cross hybridization of AhIRT3 and AhZIP4 probes with AtZIP4 and AtIRT3 genomic sequences, respectively. c Not detected, predicted fragment given for A. thaliana. d Enzyme cuts once in one of the introns spanned by the cDNA probe. e Enzyme cuts once in the probe. f Enzyme cuts twice in the probe. g Cross hybridization of AhHMA4 probe with AtHMA2 genomic sequence. h Cross hybridization of AhPht1;4 probe with At3g54700 genomic sequence. i Cross hybridization of AhPDI1 and AhPDI2 probes with AtPDI2 and AtPDI1 genomic sequences, respectively. Note that it is likely that cross hybridization also occurs of AhPDI1 and AhPDI2 probes with AhPDI2 and AhPDI1 genomic sequences. j Additional band, identity not known. Open in new tab ; Supplemental Fig. 1; for data evaluation see also “Materials and Methods”). For comparison, the maximum expression of these genes was 8-, 10-, 13-, and 30-fold higher in A. halleri than in A. thaliana, respectively, as determined by real-time RT-PCR (Table II). By contrast, the simple band patterns of the genomic DNA blots indicated that a number of other candidate genes are most likely to be present as single copies in the A. halleri genome. These include FRD3, ZIP4, MTP8, ZIP10, IRT3, PHT1;4, and PDI1, the maximum expression of which was 16-, 12-, 11-, 6-, 5-, 5-, and 4-fold in A. halleri compared to A. thaliana, respectively (Tables II and III; Fig. 5). Among the group of candidate genes for which DNA gel-blot analysis was performed, more than single gene copies were detected for each of the three candidate genes exhibiting the highest transcript levels in A. halleri (normalized to EF1α transcript levels), i.e. HMA4 and ZIP9 and ZIP3 (compare Table II, left section). Based on sequence data and restriction analysis of genomic PCR products, it is unlikely that additional bands on the DNA gel blots originated from uncharacterized restriction sites rather than additional gene copies in the A. halleri genome (see Table III; M. Hanikenne and U. Krämer, unpublished data). Regulation of Candidate Gene Expression Transcriptional Zn responses were further investigated for a subset of 18 candidate genes using real-time RT-PCR (Fig. 6 Figure 6. Open in new tabDownload slide Transcriptional responses of selected candidate genes to Zn and other ions. Transcript levels relative to EF1α were assessed in roots (A) and shoots (B) of hydroponically grown plants subjected to short-term treatment with high Zn, long-term Zn deficiency, or short-term treatment with Cd, Cu, or Na (see “Materials and Methods” for details). RTLs within a treatment were normalized to the RTL of the control treatment in the respective experiment (“RTL normalized to control”). To determine transcriptional responses for each treatment and each gene, the mean RTL normalized to control was calculated from the respective values from two independent biological experiments. Bold letters indicate genes that exhibit higher transcript levels in A. halleri compared to A. thaliana after cultivation in the presence of control Zn concentrations. Note that some genes were not expressed in some conditions, see Figures 2 and 7 for details. Figure 6. Open in new tabDownload slide Transcriptional responses of selected candidate genes to Zn and other ions. Transcript levels relative to EF1α were assessed in roots (A) and shoots (B) of hydroponically grown plants subjected to short-term treatment with high Zn, long-term Zn deficiency, or short-term treatment with Cd, Cu, or Na (see “Materials and Methods” for details). RTLs within a treatment were normalized to the RTL of the control treatment in the respective experiment (“RTL normalized to control”). To determine transcriptional responses for each treatment and each gene, the mean RTL normalized to control was calculated from the respective values from two independent biological experiments. Bold letters indicate genes that exhibit higher transcript levels in A. halleri compared to A. thaliana after cultivation in the presence of control Zn concentrations. Note that some genes were not expressed in some conditions, see Figures 2 and 7 for details. ). In the roots of A. halleri, short-term exposure to 300 μ m Zn for 2 and 8 h resulted in the down-regulation of transcript levels of a group of genes, which encode several ZIP family Zn transporters with likely functions in Zn influx into the cytoplasm (Fig. 6A). Down-regulation of transcript levels of this group of genes was clearly less pronounced in the roots of A. thaliana after short-term exposure to 30 μ m Zn. It has to be kept in mind that in roots of A. halleri the Zn-induced decrease in transcript levels occurred from very high transcript levels under control conditions. Consequently, despite a more pronounced down-regulation in A. halleri, final root transcript levels 8 h after supply of excess Zn were generally higher in A. halleri than in A. thaliana (compare Fig. 2). Different from roots, shoot transcript levels of most ZIP genes were generally as low in A. halleri as in A. thaliana under control conditions (see Fig. 2; Table II). In shoots of A. halleri, there was virtually no transcriptional response to a short-term oversupply of Zn, whereas A. thaliana shoots responded by a marked down-regulation of several genes including primarily ZIP genes and PHT1;4 (Fig. 6B). To test whether the shoots of A. halleri were able to respond transcriptionally to changes in Zn supply, we investigated gene expression under Zn deficiency (Fig. 6). Zn deficiency resulted in a very pronounced increase in shoot transcript levels of genes encoding ZIP family transporters and nicotianamine synthase proteins (Fig. 6B; see Table I). In roots of A. halleri, Zn deficiency resulted in a moderate increase in transcript levels of these genes, when compared to the high transcript levels observed under control conditions (Fig. 6A; for comparison with A. thaliana, see Figs. 2 and 7 Figure 7. Open in new tabDownload slide Zn-dependent transcriptional regulation of selected candidate genes. Real-time RT-PCR analysis of expression of HMA4, ZIP3, and IRT3 genes in roots (top section) and shoots (bottom section) of hydroponically grown A. halleri (black bars) and A. thaliana (light gray bars). Two different basal Zn conditions upon which additional Zn was supplied were assessed: Short Zn oversupply and Zn deficiency/resupply. For a detailed description, see Figure 2 and “Materials and Methods.” Data shown are transcript levels relative to EF1α from one experiment representative of two independent biological experiments. Tissues from at least three culture vessels with three plants each were pooled for each condition. Genes were concluded to be not expressed (n.e.) when C T values were above a threshold of 35 and reaction efficiencies with the respective cDNA were more than 5% below the mean RE for the respective primer pair. Note that ZIP3 was not expressed in shoots only for the condition A. thaliana, 30 μ m Zn supply, 8 h. At all other conditions, ZIP3 transcripts were detectable but RTLs were very low (A. halleri: 4.3 [control], 3.5 [2 h], and 2.6 [8 h]; A. thaliana: 1.7 [control], 1.7 [2 h], and 3.9 [control Zn deficiency]), and therefore do not appear as visible bars in the diagram. Figure 7. Open in new tabDownload slide Zn-dependent transcriptional regulation of selected candidate genes. Real-time RT-PCR analysis of expression of HMA4, ZIP3, and IRT3 genes in roots (top section) and shoots (bottom section) of hydroponically grown A. halleri (black bars) and A. thaliana (light gray bars). Two different basal Zn conditions upon which additional Zn was supplied were assessed: Short Zn oversupply and Zn deficiency/resupply. For a detailed description, see Figure 2 and “Materials and Methods.” Data shown are transcript levels relative to EF1α from one experiment representative of two independent biological experiments. Tissues from at least three culture vessels with three plants each were pooled for each condition. Genes were concluded to be not expressed (n.e.) when C T values were above a threshold of 35 and reaction efficiencies with the respective cDNA were more than 5% below the mean RE for the respective primer pair. Note that ZIP3 was not expressed in shoots only for the condition A. thaliana, 30 μ m Zn supply, 8 h. At all other conditions, ZIP3 transcripts were detectable but RTLs were very low (A. halleri: 4.3 [control], 3.5 [2 h], and 2.6 [8 h]; A. thaliana: 1.7 [control], 1.7 [2 h], and 3.9 [control Zn deficiency]), and therefore do not appear as visible bars in the diagram. ). To analyze the specificity of transcriptional Zn responses, candidate gene regulation in A. halleri was also investigated following exposure to moderate excesses of Cd (30 μ m) or copper (Cu; 5 μ m) and NaCl (100 mm) for 2 and 8 h (Fig. 6). The results suggested that overall, Zn responses were rather specific. Some of the Zn-responsive genes, primarily NAS and ZIP genes, were also down-regulated in response to excess Cu, as for excess Zn. The transcriptional responses to excess Zn and excess Cd, however, were distinct. Furthermore, there was a second group of genes exhibiting a pattern of regulation that was clearly different from the Zn- and Cu-repressed genes. Expression of these genes was induced in response to exposure to 100 mm NaCl and included MTP8, PHT1;4, FRD3, ZIP6, and OASA2. Transcript levels of subsets of these genes were also increased following exposure to excess Cu or Cd, supporting their responsiveness to abiotic stress. PDI transcript levels showed few changes over the entire range of conditions, with an increase in root PDI2 transcript levels in response to excess Cu in A. halleri and an increase in PDI1 transcript levels in response to Zn in shoots of A. thaliana. Transcript levels of HMA4 appeared to be constitutively very high in the roots of A. halleri across all treatments, and only slightly induced under Zn deficiency in A. halleri shoots (Fig. 6, A and B). A detailed analysis confirmed the extremely high expression of HMA4 in roots and shoots of A. halleri (Fig. 7). Under Zn deficiency, shoot HMA4 transcript levels were slightly increased, and they were reduced to control levels shortly after readdition of Zn to the medium (Fig. 7). We investigated transcript levels of the additional newly identified candidate genes ZIP3 and IRT3, under different conditions of Zn deficiency, resupply, and oversupply (Fig. 7). There were overall similarities in short-term Zn responses of plants maintained under control (1 μ m added Zn) and Zn-deficient (0 μ m added Zn) conditions (Fig. 7; see also Fig. 2). In response to resupply of 5 μ m Zn to Zn-deficient plants, there was a decrease in transcript levels for these Zn-deficiency-induced genes in both A. halleri and A. thaliana. This demonstrated clearly that there is no general defect in the magnitude or time scale of transcriptional Zn responses in A. halleri (Fig. 7; see also Fig. 2: ZIP4 and ZIP9 in controls and at time points 8 and 24 h). In roots of A. halleri, after the initial down-regulation, which was strongest 2 h after resupply of Zn, a general trend of a gradual increase in transcript levels was observed at later time points (Fig. 7). DISCUSSION In a microarray-based cross species comparative transcript analysis in A. thaliana and A. halleri, we identified a set of candidate genes expressed at higher levels in A. halleri than in A. thaliana or regulated by Zn in A. halleri (Fig. 3; Table I; Supplemental Tables I–IV). Microarray data were confirmed by an alternative technique of transcript quantification, real-time RT-PCR, for a representative subset of genes. Among the genes identified using microarrays, combined in silico and real-time RT-PCR analyses revealed a total of 29 genes encoding putative metal homeostasis proteins, which we consider here as an initial set of major candidates for a role in naturally selected metal hyperaccumulation and associated metal hypertolerance in A. halleri. Of these, 18 genes have not previously been implicated in these traits, and 11 confirm earlier findings (Tables I and II; Becher et al., 2004; Weber et al., 2004; Mirouze et al., 2006). We performed DNA gel blots to estimate genomic copy numbers for a subset of 12 candidate genes (Fig. 5; Table III; Supplemental Fig. 1). The obtained data are important for future studies of segregation of candidate genes and for the cloning of genomic sequences from A. halleri. Moreover, the combination of DNA gel blot with steady-state transcript level data suggested the presence of more than a single genomic copy in A. halleri of the genes, for which the highest transcript levels were detected, HMA4, ZIP9, and ZIP3 (Tables II and III). As reported previously for MTP1 (Dräger et al., 2004), increased gene copy numbers of these genes may contribute to the ability of A. halleri to express these genes at very high levels, and possibly allow for regulatory diversification among the copies of these genes. However, high expression of these candidate genes cannot be explained without additionally invoking an altered regulation of transcript levels. Moreover, substantially higher transcript levels in A. halleri than in A. thaliana, as also found for FRD3, ZIP4, and MTP8, were not in all cases associated with indications for more than a single genomic copy of the respective candidate gene. Ratios of shoot-to-root Zn concentrations are generally below unity in A. thaliana and other nonaccumulator plants, and typically above unity in A. halleri and other Zn hyperaccumulating plants (Baker et al., 1994; Dahmani-Muller et al., 2000; Becher et al., 2004; Weber et al., 2004; Fig. 1). Zn concentrations partitioned into shoots relative to the roots were dependent on the external Zn supply (Fig. 1). In addition, the adjustment of Zn partitioning as a function of Zn supply differed between A. thaliana and A. halleri, with maximum relative Zn concentrations partitioned into the shoot at approximately 5 μ m in A. halleri, and under Zn deficiency in A. thaliana. In A. halleri the partitioning of Zn into the shoot was restricted at high external Zn supply of 30 μ m Zn and above. This may contribute to Zn hypertolerance in the Langelsheim accession of A. halleri. It has been reported that individuals from the most Zn-hypertolerant populations of A. halleri, which include the Langelsheim accession, accumulate lower leaf Zn concentrations than individuals from less hypertolerant populations (Bert et al., 2000, 2002). As expected based on Zn partitioning, we observed between-species differences in gene expression under control conditions, as well as species-specific short-term transcriptional Zn responses of candidate genes in A. halleri and A. thaliana (Tables I and II; Figs. 1, 2, 6, and 7). Transcript levels of genes known to be transcriptionally regulated by Zn, such as ZIP1, ZIP3, ZIP4, ZIP9, NAS2, and NAS3 can be used as indicators of the Zn status of A. thaliana plants (Grotz et al., 1998; Wintz et al., 2003). The expression of a group of Zn deficiency marker genes suggested that both roots and shoots of A. thaliana are largely Zn sufficient at 1 or 5 μ m Zn, and concertedly experience Zn deficiency after prolonged growth in a medium lacking added Zn (Figs. 2 and 7). Similarly, the expression of the same set of genes in A. halleri plants grown at 1 or 5 μ m Zn suggests that the shoots are Zn sufficient under these conditions (see NAS2, ZIP9, and ZIP4 transcript levels in shoots in Figs. 2, 6, and 7). By contrast, the roots of A. halleri act transcriptionally as Zn deficient after prolonged growth at 1 or 5 μ m Zn. Thus, transcript levels of a common set of Zn status marker genes can be interpreted to indicate that roots and shoots of A. halleri experience different Zn stati in the steady state. There was a rapid and strong down-regulation of transcript levels of ZIP3, ZIP4, and IRT3 in roots of A. halleri following exposure to excess Zn or resupply of Zn to roots of Zn-deficient plants grown in a medium lacking added Zn (Figs. 2 and 7). Overall, this suggests that short-term Zn-dependent down-regulation of Zn deficiency-induced transcripts in roots of A. halleri is generally as functional and efficient as in A. thaliana. Only NAS2 and ZIP9 transcript levels were not or only partly Zn responsive in roots of A. halleri, respectively. The Zn-dependent control of transcript abundance of these two genes in A. thaliana appears to be partially or fully inactive in roots, but not in shoots, of A. halleri. The simplest explanation for the high expression of Zn deficiency responsive genes in the roots of A. halleri at normal Zn supplies could be a loss of Zn from the roots via high root-to-shoot Zn transport rates (see Fig. 1). The proteins encoded by two of the candidate genes have been implicated in root-to-shoot metal translocation in A. thaliana: HMA4 and FRD3. The A. thaliana HMA4 protein is a P1B-type metal pump that localizes to the plasma membrane and mediates cellular metal efflux. Together with the related AtHMA2, AtHMA4 has a role in the transport of Zn from the root to the shoot and in metal detoxification (Hussain et al., 2004; Verret et al., 2004; Mills et al., 2005). According to real-time RT-PCR analysis (Fig. 7; Table II), HMA4 transcript levels are between 4- and 10-fold higher in roots and at least 30-fold higher in shoots of A. halleri than in A. thaliana. In A. halleri, HMA2 transcript levels are much lower than HMA4 transcript levels (less than 0.25% in shoots and less than 0.2% in roots), and HMA2 transcript levels are lower in A. halleri than in A. thaliana (approximately 25% in shoots and 8% in roots; I.N. Talke, L. Krall, and U. Krämer, unpublished data). Heterologous expression in yeast indicated that AhHMA4 and AtHMA4 are similarly able to complement Zn and Cd hypersensitivity of yeast mutants (Fig. 4), and that complementation is dependent on the transport functions of the proteins. Together, our data suggest that AhHMA4 is functionally highly similar to AtHMA4, and that HMA4 expression is very high in A. halleri and predominant over the expression of HMA2. Thus, in the context of the data published on AtHMA4, the data presented here implicate A. halleri HMA4 in Zn and Cd hypertolerance and in Zn hyperaccumulation, i.e. the partitioning of Zn predominantly to the shoot. The A. thaliana and A. halleri FRD3 proteins are members of the multidrug and toxin efflux family of membrane transport proteins. Although the transport function of AtFRD3 has not yet been directly established, the available data suggest that AtFRD3 has a role in maintaining iron (Fe) mobility between the root and the shoot. In the frd3 mutant, root Fe deficiency responses are constitutively active, leading to secondary accumulation of a number of metals including manganese (Mn), cobalt, Cu, Zn, and Fe in both roots and shoots (Delhaize, 1996; Rogers and Guerinot, 2002; Lahner et al., 2003; Green and Rogers, 2004). Based on published data, one may thus expect FRD3 to be expressed at low levels in hyperaccumulator plants. Unexpectedly however, FRD3 is expressed at very high levels in A. halleri (Tables I and II; Fig. 6). In agreement with this, Zn hyperaccumulation in A. halleri is specific both in terms of the accumulated metal and in terms of the accumulating tissue, which is the shoot (Becher et al., 2004). In addition, Zn accumulation is highest in the vacuoles of leaf mesophyll cells in A. halleri (Küpper et al., 2000; Sarret et al., 2002), whereas in the A. thaliana frd3 mutant Fe and Mn appear to accumulate in the leaf apoplast (Green and Rogers, 2004). In conclusion, based on the available evidence, it can be hypothesized that the high expression of FRD3 in A. halleri contributes to metal homeostasis, but not specifically to the high accumulation of Zn in shoots of A. halleri. In addition to FRD3, several other genes found to be highly expressed in A. halleri (Table I) were previously associated not with Zn or Cd homeostasis, but with the homeostasis of other transition metals, among them FER1 (Fe), FER2 (Fe), NRAMP3 (Fe and Mn), IREG2 (Fe), HMA6/PAA1 (Cu), MTP8, and MTP11 (Mn; Thomine et al., 2000; Petit et al., 2001; Delhaize et al., 2003; Shikanai et al., 2003; Thomine et al., 2003; McKie and Barlow, 2004; Lanquar et al., 2005). The encoded proteins may contribute to the tolerance and hyperaccumulation phenotypes by adjusting the homeostasis of Fe, Mn, and Cu to the alterations in Zn and Cd homeostasis of A. halleri. Alternatively, these proteins may have direct roles in Zn or Cd homeostasis. It will be important to establish the functions of these proteins in the future. To address the specificity of Zn-dependent regulation of candidate gene transcript levels, we exposed plants to an excess of Cu, Cd, or NaCl in the hydroponic medium. Root ZIP3 and ZIP4 transcript levels and transcript levels of all four NAS genes were suppressed following exposure of A. halleri to excess Cu (Fig. 6). Wintz et al. (2003) demonstrated that expression of A. thaliana ZIP4 can complement the Cu uptake defect of the ctr1 yeast mutant. AtZIP3-dependent uptake of 65Zn2+ into yeast cells was decreased by more than 55% in the presence of a 10-fold excess of CuCl2 (Grotz et al., 1998). Root-to-shoot transport of Cu in the xylem is likely to occur as a Cu-nicotianamine complex (Pich et al., 1994; Pich and Scholz, 1996; von Wiren et al., 1999). The Cu-induced down-regulation of ZIP and NAS transcript levels observed here in A. halleri (Fig. 6) may constitute a regulatory response counteracting excessive Cu uptake and transport to the shoot. Based on the chemical similarity between Cd2+ and Zn2+ ions, Cd toxicity may involve the disruption of plant Zn homeostasis. However, none of the ZIP or NAS genes known to have a role in Zn acquisition and distribution were regulated in response to an excess of Cd (Fig. 6). This may reflect the Cd hypertolerance of A. halleri (Bert et al., 2003). There appear to be similarities between major candidate genes expressed at high levels in A. halleri and in the metal-tolerant Zn and Cd hyperaccumulator model species T. caerulescens. The A. halleri/A. lyrata clade is phylogenetically closest to A. thaliana, with an estimated divergence time of between 3.5 and 5.8 million years ago (Koch et al., 2001; Yogeeswaran et al., 2005). For comparison, T. caerulescens belongs to the clade containing Sinapis alba, which is thought to have diverged from the A. thaliana lineage more than 20 million years ago (Gao et al., 2005). In contrast to A. halleri, multiple genomic copies have not been reported for any T. caerulescens candidate genes so far. High transcript levels have been observed for T. caerulescens ZNT1 (Lasat et al., 2000; Pence et al., 2000; Assunção et al., 2001) as well as for its sequence ortholog AhZIP4 (Becher et al., 2004), and for TcZNT2 (Assunção et al., 2001) and its sequence ortholog AhIRT3 (Fig. 7), which are likely to encode cellular Zn uptake systems. The sequence-orthologous TcZTP1 (Assunção et al., 2001), Thlaspi goesingense MTP1 (Persans et al., 2001), and AhMTP1 transcripts (Becher et al., 2004; Dräger et al., 2004) have all been reported to occur at very high levels in the respective species and encode proteins mediating metal efflux from the cytoplasm. Finally, high transcript levels have been reported for TcHMA4 (Bernard et al., 2004; Papoyan and Kochian, 2004) and AhHMA4 (Fig. 7), and the encoded proteins are capable of mediating cellular Zn and Cd detoxification (see Fig. 4; Papoyan and Kochian, 2004). These observations raise the question of whether metal hyperaccumulation and metal hypertolerance are ancestral traits that have subsequently been lost in the majority of Brassicaceae species. We favor the alternative hypothesis that Zn hyperaccumulation evolved several times independently in the Brassicaceae through recent mutations leading to the convergent overexpression of an overlapping set of genes responsible for the Zn hyperaccumulation and hypertolerance traits. The comprehensive identification of candidate genes in several hyperaccumulator model plants is a prerequisite for their functional, regulatory, and sequence analysis. This will be an important step toward a better understanding of the evolution of naturally selected metal hyperaccumulation and associated metal hypertolerance, and of evolutionary genome dynamics in plants. Furthermore, this will allow a comparison with extreme traits in other Arabidopsis relative model systems, such as sodium (Na) tolerance in Thellungiella halophila (Inan et al., 2004; Taji et al., 2004; Gong et al., 2005). In summary, we have used cross species transcript profiling to successfully identify on a genome-wide scale a number of candidate genes for naturally selected metal tolerance and hyperaccumulation in A. halleri. By exploring candidate genes at the genome, functional, and regulatory level we have obtained insights into the complex molecular basis of an extreme physiological trait. MATERIALS AND METHODS Plant Material, Growth Conditions, and Experimental Treatments Plants of Arabidopsis thaliana (L. Heynhold, accession Columbia [Col]) and Arabidopsis halleri (L. O'Kane and Al-Shehbaz subsp. halleri, accession Langelsheim) were grown from seeds, which were the progeny of a single mother plant in a pool of six individuals grown in an open greenhouse from seeds collected at Langelsheim. Plants were cultivated in hydroponic culture as described in Becher et al. (2004), with a photoperiod of 11 h light, 13 h dark, at a photon flux density 145 μmol m−2 s−1 during the day, and day and night temperatures of 20°C and 18°C, respectively. In all experiments, each culture vessel of 400 mL hydroponic solution contained three individual plants, and all treatments were harvested at the same time. Experiments for subsequent RNA extraction were harvested by carefully separating roots and shoots of plants, pooling tissues according to treatment, and freezing in liquid nitrogen. Harvested tissues were stored at −80°C until further processing. Short-term high Zn, Cd, Cu, and Na treatments were initiated when plants were 6.5 weeks old. For short-term high Zn supply, the medium was replaced with fresh control medium (1 μ m ZnSO4) 8 h before harvest. Then nothing (controls) or 30 μ m ZnSO4 and 300 μ m ZnSO4 for A. thaliana and A. halleri, respectively, was added to the hydroponic solution 8 or 2 h before harvest. Three biologically independent experiments, each comprising five culture vessels per treatment (15 plants per treatment) were conducted. In experiments with Cd, Cu, and Na treatments of A. halleri, media were exchanged (1 μ m ZnSO4; Cd: none; Cu: 0.5 μ m CuSO4; Na: 0.1 μ m Na2MO4) 3 d before harvest; 30 μ m CdCl2, 5 μ m CuSO4, or 100 mm NaCl were added as treatments 8 or 2 h before harvest. Two biologically independent experiments were conducted, with at least two culture vessels (at least six plants in total) for each treatment. For Zn deficiency experiments with Zn resupply, the treatment was initiated when plants were 4.5 weeks old and lasted for 3 weeks. For deficiency, ZnSO4 was omitted from the hydroponic solution; the sufficient control solution contained 5 μ m ZnSO4. The last change of medium was done 3 d before harvest. For Zn resupply, 5 μ m ZnSO4 was added to the hydroponic solution of Zn-deficient plants 24, 8, and 2 h before harvest. Two biologically independent experiments were conducted, each comprising at least three culture vessels (at least nine plants) per treatment. In the experiments conducted to determine root and shoot Zn concentrations after long-term treatment with a range of different Zn supplies, plants were 4.5 weeks old when treatments were initiated. For A. halleri, the hydroponic solution contained 0, 0.3, 1, 5, 10, 30, or 300 μ m ZnSO4, for A. thaliana 0, 1, 5, or 10 μ m ZnSO4. Upon harvest after 3 weeks of treatment, shoots were briefly rinsed in ultrapure water and blotted dry, and roots from three individuals of the same species and hydroponic culture vessel were desorbed together in 150 mL of an ice-cold solution of 5 mm CaCl2 and 1 mm MES-KOH (pH 5.7) for 20 min, with a replacement of the solution after 5 min, followed by two washes in 150 mL ice-cold water, each for 3 min. Tissues were pooled by culture vessel and dried at 60°C for 3 d. Two biologically independent experiments were conducted, each comprising three culture vessels (nine plants) per treatment. Determination of Zn Concentrations Dried root and shoot material was homogenized, and for each sample, 35 to 70 mg were processed and elemental contents analyzed by inductively coupled plasma optical emission spectroscopy using an IRIS Advantage Duo ER/S (Thermo Jarrell Ash) as described previously (Becher et al., 2004). RNA Extraction For microarray expression profiling, total RNA was extracted with Trizol (Invitrogen Life Technologies) according to the manufacturer's instructions. RNeasy spin columns and the RNase-free DNase set for on-column DNase digest (Qiagen) were used according to the manufacturer's instructions for subsequent RNA purification and digestion of genomic DNA, and as an alternative method of total RNA isolation when transcript levels were determined by real-time RT-PCR only (RNeasy plant mini kit and RNase-free DNase set; Qiagen). Quality and quantity of RNA was checked visually by denaturing gel electrophoresis and by photometric analysis (A 260 and A 280). Microarray Expression Profiling and Data Analysis Synthesis of cDNA, cRNA labeling, and the hybridization on the GeneChip Arabidopsis ATH1 genome array were done as recommended by the manufacturer (Affymetrix; manual 701025 rev 1, https://www.affymetrix.com/support/downloads/manuals/expression_s2_manual.pdf). In short, 20 μg of total RNA were used for first-strand cDNA synthesis with 100 pmol T7(dT)24 primers and SuperScript II RT (Invitrogen), second-strand cDNA synthesis was carried out with Escherichia coli DNA ligase, DNA polymerase I, and RNase H (all Invitrogen). Biotin-labeled cRNAs were synthesized by in vitro transcription using the Enzo BioArray HighYield RNA transcript labeling kit (Enzo Diagnostics). Labeled cRNAs were quantified according to Affymetrix instructions, and a corrected amount of 25 μg cRNA were fragmented in fragmentation buffer (200 mm Tris-acetate, pH 8.1, 500 mm KOAc, 150 mm MgOAc). Before hybridization on ATH1 arrays, sample quality was assessed by hybridization on the test3-array (Affymetrix) for examination of 3′ to 5′ intensity ratios of houskeeping genes. ATH1 GeneChips were hybridized with cRNA from the following experiments: of the short-term Zn supply experiments, two for each treatment, tissue, and species; of the Zn deficiency experiments, one for each tissue from the 5 μ m Zn (sufficient) and 0 μ m Zn (deficient) treatment, respectively (Dr. F. Wagner, Resource Center and Primary Database, Berlin). The microarray suite software package (MAS 5.0, Affymetrix) was used to generate probe set signals of the scanned ATH1 arrays. At a target intensity of 100, scaling factors for arrays hybridized with cRNA from A. halleri were on average 2-fold higher than scaling factors for A. thaliana arrays (A. halleri: 0.943 ± 0.196; A. thaliana: 0.424 ± 0.093). The arithmetic means of signal intensities over all probe sets were 146.9 ± 8.3 for A. halleri and 135.4 ± 9.6 for A. thaliana. For A. thaliana an average of 13,907 (61.0%) and 11,646 (51.1%) of the total number of 22,810 probe sets were assigned a “present call” by the MAS 5.0 software in roots and shoots, respectively. On average 9,750 (42.8%) and 9,389 (41.2%) probe sets were assigned a “present call” in roots and shoots, respectively, for A. halleri. Pivot data of each hybridized ATH1 GeneChip containing raw signal values and present, marginal, and absent calls (flags) were imported from MAS 5.0 into GeneSpring GX (v 7.2; Agilent Technologies, http://www.chem.agilent.com/). Within GeneSpring, the following normalization steps were carried out for each chip: first, signals below 0.01 were set to 0.01; second, all signals within a dataset were normalized to the 50th percentile of the measurements of the chip, using only measurements with a raw signal of at least 12.5; third, in a per gene normalization, the signal for each probe set was normalized to the respective signal of the control chip (from the same experiment; chips from A. thaliana for interspecies comparisons, control condition for within-species comparisons). Calculation of mean values for raw and normalized signals, statistical analysis, and data filtering were performed in GeneSpring. For A. halleri versus A. thaliana comparisons, the following filters were applied sequentially to identify genes more highly expressed in A. halleri than in A. thaliana (value filter). First, to avoid the identification of false positives in a cross species evaluation, probe sets were selected that exhibited an at least 4-fold higher mean normalized signal in A. halleri than in A. thaliana. Second, among these probe sets, we selected probe sets for which the mean raw signal value was above 25 in A. halleri. Third, we selected only those probe sets for which in a one sample Student's t test the P value was below 0.05, with false discovery rate control adjusted at 5% (Benjamini and Hochberg, 1995). Fourth, only those probe sets were retained that were assigned at least one “present call” for A. halleri among the chips under comparison. For within-species comparisons, to identify genes up-regulated and/or down-regulated upon exposure to Zn, the following selection criteria were employed sequentially: For up-regulation/down-regulation in the experimental Zn condition (2 or 8 h high Zn supply; Zn deficiency), probe sets with (1) a mean normalized signal at least 2-fold higher/lower than in the control condition, (2) a mean raw signal above a threshold value (25 for A. halleri, 12.5 for A. thaliana), and (3) present calls in all biological replicates of the experimental condition (up-regulation)/control condition (down-regulation). To obtain an estimate of the percentage of genes regulated in within-species comparisons (as reported in the “Results”), a P-value cutoff of 0.1 was applied. The same cutoff was used for the gene lists shown in Supplemental Tables III, IV, and V. The P-value cutoffs were 0.1 for up-regulation and 0.2 for down-regulation for the gene lists shown in Supplemental Table VI, and 0.05 for up-regulation and 0.4 for down-regulation for Supplemental Tables VII and VIII. Lists of candidate probe sets and their respective signal data were exported from GeneSpring. Tables for assigning Affymetrix ATH1 probe set identifiers to Arabidopsis Genome Initiative locus identifiers were obtained from The Arabidopsis Information Resource (TAIR; http://www.arabidopsis.org) and Genomanalyse im biologischen System Pflanze (http://gabi.rzpd.de/services/Affymetrix.shtml). In cases where an ATH1 probe set could not be assigned to one single locus identifier, all locus identifiers represented by this probe set and its associated signal data are given. Annotation and classification of genes as metal ion homeostasis related, metal ion (Zn, Fe, Cu, and Mn) binding, membrane and transport associated, oxidative stress protection associated, pathogen response related, and RNA metabolism associated was done through literature review, keyword searches in TAIR, use of the PlantsT database (http://plantst.genomics.purdue.edu/), and using MapMan annotation based on TAIR5 (Usadel et al., 2005; http://gabi.rzpd.de/projects/MapMan/). Annotation of genes was obtained from TAIR. Table associations were done with Microsoft Access. In all Tables and Supplemental Tables, P values are given as exported from the program GeneSpring GX, i.e. from a one sample Student's t test and not adjusted for false discovery rate. Real-Time RT-PCR Synthesis of cDNA, primer design, and quality control were done as described in Becher et al. (2004). Primer sequences are given in Supplemental Table IX. Real-time RT-PCR reactions were performed in 384-well plates with an Applied Biosystems ABI Prism 7900HT sequence detection system (Applied Biosystems; http://www.appliedbiosystems.com/) using SYBR Green to monitor cDNA amplification. Equal amounts of cDNA, corresponding to approximately 0.06 ng of mRNA were used in each reaction. In addition, a reaction contained 5 μL of SYBR Green PCR Master Mix (Applied Biosystems) and 2.5 pmol of forward and reverse primers (Eurogentec) in a total volume of 10 μL. The following standard thermal profile was used: 2 min at 50°C, 10 min at 95°C, 40 repeats of 15 s at 95°C and 60 s at 60°C, and a final stage of 15 s at 95°C, 15 s at 60°C, and 15 s at 95°C to determine dissociation curves of the amplified products. Data were analyzed using 7900 HT sequence detection system software (v 2.2.1, Applied Biosystems). Threshold cycle (C T) values were determined for each reaction at a threshold value of the normalized reporter R n of 0.2. Furthermore, the reaction efficiency (RE) was determined for each PCR reaction with LinRegPCR v7.5 (Ramakers et al., 2003). Real-time RT-PCR was performed on material from at least two independent biological experiments. For confirmation of microarray data we used material from one experiment used for microarray hybdridization and from at least one additional independent experiment not used for microarray hybridization. At least two technical repeats were done for each combination of cDNA and primer pair, and the quality of the PCR reactions was checked through analysis of the dissociation and amplification curves. Only those reactions were used for data evaluation for which transcripts were reliably detectable (C T values below 35), and RE was above 1.5. Mean reaction efficiencies (REm) were determined for each primer pair and for each Arabidopsis species from all reactions passing the quality control (at least 40 reactions; Supplemental Table IX). Mean C T values (C T,m) were calculated from all quality-checked technical repeats of a cDNA-primer pair combination. Over all cDNAs used, the average C T value for the constitutively expressed control gene, elongation factor EF1α (At5g60390, Becher et al., 2004), was 18.68 with a sd of 1.03 (n = 76). Transcript levels of genes of interest (GI) within a cDNA were normalized to the respective transcript level of EF1α using the following formula: \[\mathrm{RTL}{\,}(\mathrm{GI}){=}[\mathrm{RE}_{\mathrm{m}}{\,}(EF1{\alpha})]{\,}^{\mathrm{C}_{\mathrm{T,m}}(EF1{\alpha})}{\times}[\mathrm{RE}_{\mathrm{m}}{\,}(\mathrm{GI})]^{{-}\mathrm{C}_{T,m}{\,}(\mathrm{GI})}.\] For clarity, values shown in Tables and Figures are RTL × 103. Cloning All cloning and molecular biology procedures were carried out according to standard protocols unless indicated otherwise (Sambrook and Russel, 2001). For the candidate genes, A. halleri cDNA fragments were amplified with Red-Taq DNA polymerase (Sigma) using primers designed on the basis of the corresponding A. thaliana sequences (Supplemental Table X). The standard PCR program was as follows: 3 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 55°C, 1 min per kb at 72°C, and a final extension step of 7 min at 72°C. The PCR products were cloned into the pCR2.1 TOPO vector (Invitrogen) and the inserts of at least three independent plasmids were sequenced. Unless stated otherwise, A. halleri-cDNA-derived probes for DNA gel-blot hybridizations were prepared from pCR2.1 TOPO clones by excision of fragments using EcoRI. For ZIP3, ZIP6, PHT1;4, and MTP8, a 472 bp HindIII-SalI fragment, a 380 bp EcoRI-NdeI fragment, a 550 bp EcoRI fragment, and a 594 bp HindIII fragment were used, respectively. Full-length cDNAs for AtHMA4 and AhHMA4 were obtained as follows. cDNA libraries were prepared from mRNA of A. thaliana (Col-0 accession) inflorescence tissues and from root tissues of several individuals of A. halleri (Langelsheim accession), respectively, using the SMART RACE cDNA amplification kit according to the manufacturer's instructions (BD Biosciences). The open reading frames of AtHMA4 and AhHMA4 were subsequently amplified using a proofreading polymerase (Pfu Turbo; Stratagene) and the following PCR program: 3 min at 95°C, followed by 10 cycles of 30 s at 95°C, 30 s at 58°C, 8 min at 68°C, 20 cycles of 30 s at 95°C, 30 s at 55°C, 8 min at 68°C, and a final extension step of 7 min at 68°C. The products were cloned directionally into the GATEWAY entry vector pENTR TOPO (Invitrogen), and later subcloned into the yeast (Saccharomyces cerevisiae) centromeric expression vector pFL38H-GW by site-directed recombination according to the manufacturer's instructions (Invitrogen). pFL38H-GW was constructed by inserting a SmaI-BglII fragment from the vector pFL61-AKT1 (Sentenac et al., 1992) into the SmaI-BglII-digested vector pFL38 (Bonneaud et al., 1991), followed by replacement of the URA3 marker with the HIS3 marker, which was obtained from YDpH using BamHI, and inserted into the pFL38-derived vector following digestion with BglII. Subsequently, AKT1 was excised from the resulting vector using NotI, the linearized vector blunt ended using the Klenow fragment of DNA polymerase I, and a blunt Gateway cassette inserted according to the manufacturer's instructions (Invitrogen). To construct the AtHMA4 D401A and AhHMA4 D401A mutant cDNAs, linear amplification was performed of the AtHMA4 and AhHMA4 in the yeast expression vector pFL38H-GW in a PCR reaction using mutagenic primers (Supplemental Table X; 12 cycles of 95°C for 30 s, 55°C for 60 s, and 68°C for 16 min), followed by digestion of the parent DNA with DpnI, and subsequent transformation of E. coli. Constructs were verified by sequencing. Yeast Complementation Assays The following yeast strains were used in the assay: zrc1 cot1 (Mat a, zrc1∷natMX3, cot1∷kan-MX4, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0; Becher et al., 2004) and its parental strain BY4741 (Mat a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0), ycf1 (Mat α, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0, YDR135c∷kanMX4), and its parental strain BY4742 (Mat α, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0; http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html). Preparation of competent cells and transformations were carried out following the polyethylene glycol method (Dohmen et al., 1991). Both yeast mutant strains were transformed with pFL38H-GW empty vector (ev) or pFL38H containing AtHMA4 and AhHMA4, AtHMA4 D401A, and AhHMA4 D401A cDNAs. As control, the corresponding wild-type strains were transformed with pFL38H-GW ev. Transformants were selected and maintained on synthetic complete (SC) medium lacking His and with d-Glc as a carbon source. For complementation assays, transformants were grown overnight in 5 mL SC −His media to early stationary phase (OD600 approximately 1.0). Yeast cells were then washed twice with modified low sulfate/phosphate (LSP) medium (Conklin et al., 1992), which contained 80 mm NH4Cl, 0.5 mm KH2PO4, 2 mm MgSO4, 0.1 mm CaCl2, 2 mm NaCl, 10 mm KCl, trace elements, vitamins, and supplements −His as in SC, 2% [w/v] d-Glc, and serially diluted to identical OD600. Five microliters of each serial dilution were spotted onto LSP −His plates (1.5% agarose, Seakem, BMA) containing various concentrations of ZnSO4 (2 μ m for controls and between 100 and 400 μ m for experimental treatments) or CdSO4 (15–60 μ m). Results are from one transformant representative of at least three independent transformants for each experiment and two independent experiments. DNA Gel-Blot Analysis Genomic DNA of A. thaliana (Col-0 accession) and clones of two A. halleri individuals (Lan 3-1 and Lan 5) of the Langelsheim accession (Ernst, 1974) were isolated as described previously (Dräger et al., 2004). Five micrograms of genomic DNA were digested with 50 units EcoRI, HindIII, or NcoI in the appropriate buffer (Roche), supplemented with 100 μg mL−1 bovine casein (Sigma) overnight at 37°C, and separated on a 0.9% (w/v) TRIS-acetate-EDTA agarose gel. The DNA was transferred onto an uncharged nylon membrane (Hybond N; Amersham Biosciences) and cross-linked with UV light at 120 mJ (UV Stratalinker 1800; Stratagene). The probes were radiolabeled by random priming using [α 32P]dCTP (Hartmann Analytics) according to the manufacturer's instructions (ReadyPrime labeling kit; Amersham Biosciences). Stringent hybridization and washing conditions were used to minimize cross hybridization within gene families. The membranes were hybridized overnight at 60°C in the following buffer: 0.25 m Na phosphate (pH 7.2), 1 mm Na2EDTA, 6.7% (w/v) SDS, and 1% (w/v) bovine serum albumin. Blots were washed twice in 2× SSC containing 0.1% (w/v) SDS for 10 min at room temperature and once in 2× SSC containing 0.1% (w/v) SDS for 10 min at 60°C. X-ray films were exposed for between 5 and 30 d. As all blots were done using clones of the two A. halleri individuals named 3-1 and 5, there can be a minimum of one and a maximum of two alleles per gene copy per lane. When no restriction site was present in the region spanned by the probe, the presence of more than two bands in any one lane was interpreted as an indication for a gene copy number above one. When one restriction site was present in the region spanned by the probe, the presence of more than four bands (two per allele) in a lane was interpreted as an indication for a gene copy number above one. For one 200-bp NcoI fragment of ZIP9 located entirely within a single exon (see Table III), we expected no length polymorphisms between alleles of one gene copy, based on the sequences of multiple cloned A. halleri cDNAs (I.N. Talke, M. Hanikenne, U. Krämer, unpublished data). ACKNOWLEDGMENTS We thank Anne-Garlonn Desbrosses-Fonrouge for construction of the vector pFL38-GW and Astrid Schröder for technical support (Max Planck Institute of Molecular Plant Physiology). We thank Susanne Freund, Tomasz Czechowski, Björn Usadel, Toralf Senger, Leonard Krall (Max Planck Institute of Molecular Plant Physiology), and Florian Wagner (Resource Center and Primary Database, Berlin, Germany) for helpful discussions. LITERATURE CITED Assunção AGL, Da Costa Martins P, De Folter S, Vooijs R, Schat H, Aarts MGM ( 2001 ) Elevated expression of metal transporter genes in three accessions of the metal hyperaccumulator Thlaspi caerulescens. Plant Cell Environ 24 : 217 – 226 Crossref Search ADS Axelsen KB, Palmgren MG ( 2001 ) Inventory of the superfamily of P-type ion pumps in Arabidopsis. Plant Physiol 126 : 696 – 706 Crossref Search ADS PubMed Baker AJM, Brooks RR ( 1989 ) Terrestrial higher plants which hyperaccumulate metallic elements—a review of their distribution, ecology and phytochemistry. Biorecovery 1 : 81 – 126 Baker AJM, Reeves RD, Hajar ASM ( 1994 ) Heavy metal accumulation and tolerance in British populations of the metallophyte Thlaspi caerulescens J. & C. Presl (Brasicaceae). New Phytol 127 : 61 – 68 Crossref Search ADS PubMed Becher M, Talke IN, Krall L, Krämer U ( 2004 ) Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri. Plant J 37 : 251 – 268 Crossref Search ADS PubMed Benjamini Y, Hochberg Y ( 1995 ) Controlling false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B 57 : 289 – 300 Bernard C, Roosens N, Czernic P, Lebrun M, Verbruggen N ( 2004 ) A novel CPx-ATPase from the cadmium hyperaccumulator Thlaspi caerulescens. FEBS Lett 569 : 140 – 148 Crossref Search ADS PubMed Bert V, Bonnin I, Saumitou-Laprade P, de Laguerie P, Petit D ( 2002 ) Do Arabidopsis halleri from nonmetallicolous populations accumulate zinc and cadmium more effectively than those from metallicolous populations? New Phytol 155 : 47 – 57 Crossref Search ADS PubMed Bert V, Macnair MR, de Laguerie P, Saumitou-Laprade P, Petit D ( 2000 ) Zinc tolerance and accumulation in metallicolous and nonmetallicolous populations of Arabidopsis halleri (Brassicaceae). New Phytol 146 : 225 – 233 Crossref Search ADS PubMed Bert V, Meerts P, Saumitou-Laprade P, Salis P, Gruber W, Verbruggen N ( 2003 ) Genetic basis of Cd tolerance and hyperaccumulation in Arabidopsis halleri. Plant Soil 249 : 9 – 18 Crossref Search ADS Blaudez D, Kohler A, Martin F, Sanders D, Chalot M ( 2003 ) Poplar metal tolerance protein 1 (MTP1) confers zinc tolerance and is an oligomeric vacuolar zinc transporter with an essential s zipper motif. Plant Cell 15 : 2911 – 2928 Crossref Search ADS PubMed Bonneaud N, Ozier-Kalogeropoulos O, Li GY, Labouesse M, Minvielle-Sebastia L, Lacroute F ( 1991 ) A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7 : 609 – 615 Crossref Search ADS PubMed Clemens S ( 2001 ) Molecular mechanisms of plant metal tolerance and homeostasis. Planta 212 : 475 – 486 Crossref Search ADS PubMed Clemens S, Palmgren MG, Krämer U ( 2002 ) A long way ahead: understanding and engineering plant metal accumulation. Trends Plant Sci 7 : 309 – 315 Crossref Search ADS PubMed Conklin DS, McMaster JA, Culbertson MR, Kung C ( 1992 ) COT1, a gene involved in cobalt accumulation in Saccharomyces cerevisiae. Mol Cell Biol 12 : 3678 – 3688 PubMed Curie C, Panaviene Z, Loulergue C, Dellaporta SL, Briat JF, Walker EL ( 2001 ) Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 409 : 346 – 349 Crossref Search ADS PubMed Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK ( 2004 ) Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J 38 : 366 – 379 Crossref Search ADS PubMed Dahmani-Muller H, van Oort F, Gelie B, Balabane M ( 2000 ) Strategies of heavy metal uptake by three plant species growing near a metal smelter. Environ Pollut 109 : 231 – 238 Crossref Search ADS PubMed Delhaize E ( 1996 ) A metal-accumulator mutant of Arabidopsis thaliana. Plant Physiol 111 : 849 – 855 Crossref Search ADS PubMed Delhaize E, Kataoka T, Hebb DM, White RG, Ryan PR ( 2003 ) Genes encoding proteins of the cation diffusion facilitator family that confer manganese tolerance. Plant Cell 15 : 1131 – 1142 Crossref Search ADS PubMed Desbrosses-Fonrouge AG, Voigt K, Schröder A, Arrivault S, Thomine S, Krämer U ( 2005 ) Arabidopsis thaliana MTP1 is a Zn transporter in the vacuolar membrane which mediates Zn detoxification and drives leaf Zn accumulation. FEBS Lett 579 : 4165 – 4174 Crossref Search ADS PubMed Dohmen RJ, Strasser AW, Honer CB, Hollenberg CP ( 1991 ) An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7 : 691 – 692 Crossref Search ADS PubMed Dräger DB, Desbrosses-Fonrouge AG, Krach C, Chardonnens AN, Meyer RC, Saumitou-Laprade P, Krämer U ( 2004 ) Two genes encoding Arabidopsis halleri MTP1 metal transport proteins co-segregate with zinc tolerance and account for high MTP1 transcript levels. Plant J 39 : 425 – 439 Crossref Search ADS PubMed Ernst WHO ( 1974 ) Schwermetallvegetationen der Erde. Gustav Fischer Verlag, Stuttgart, Germany Fristedt U, van Der Rest M, Poolman B, Konings WN, Persson BL ( 1999 ) Studies of cytochrome c oxidase-driven H(+)-coupled phosphate transport catalyzed by the Saccharomyces cerevisiae Pho84 permease in co-reconstituted vesicles. Biochemistry 38 : 16010 – 16015 Crossref Search ADS PubMed Gao M, Li G, McCombie WR, Quiros CF ( 2005 ) Comparative analysis of a transposon-rich Brassica oleracea BAC clone with its corresponding sequence in A. thaliana. Theor Appl Genet 111 : 949 – 955 Crossref Search ADS PubMed Gong Q, Li P, Ma S, Indu Rupassara S, Bohnert HJ ( 2005 ) Salinity stress adaptation competence in the extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana. Plant J 44 : 826 – 839 Crossref Search ADS PubMed Green LS, Rogers EE ( 2004 ) FRD3 controls iron localization in Arabidopsis. Plant Physiol 136 : 2523 – 2531 Crossref Search ADS PubMed Grotz N, Fox T, Connolly E, Park W, Guerinot ML, Eide D ( 1998 ) Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency. Proc Natl Acad Sci USA 95 : 7220 – 7224 Crossref Search ADS PubMed Hammond JP, Bowen HC, White PJ, Mills V, Pyke KA, Baker AJ, Whiting SN, May ST, Broadley MR ( 2006 ) A comparison of the Thlaspi caerulescens and Thlaspi arvense shoot transcriptomes. New Phytol 170 : 239 – 260 Crossref Search ADS PubMed Houston NL, Fan C, Xiang JQ, Schulze JM, Jung R, Boston RS ( 2005 ) Phylogenetic analyses identify 10 classes of the protein disulfide isomerase family in plants, including single-domain protein disulfide isomerase-related proteins. Plant Physiol 137 : 762 – 778 Crossref Search ADS PubMed Hussain D, Haydon MJ, Wang Y, Wong E, Sherson SM, Young J, Camakaris J, Harper JF, Cobbett CS ( 2004 ) P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis. Plant Cell 16 : 1327 – 1339 Crossref Search ADS PubMed Inan G, Zhang Q, Li P, Wang Z, Cao Z, Zhang H, Zhang C, Quist TM, Goodwin SM, Zhu J, et al ( 2004 ) Salt cress: a halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles. Plant Physiol 135 : 1718 – 1737 Crossref Search ADS PubMed Ishizaki K, Shimizu-Ueda Y, Okada S, Yamamoto M, Fujisawa M, Yamato KT, Fukuzawa H, Ohyama K ( 2002 ) Multicopy genes uniquely amplified in the Y chromosome-specific repeats of the liverwort Marchantia polymorpha. Nucleic Acids Res 30 : 4675 – 4681 Crossref Search ADS PubMed Jensen LT, Ajua-Alemanji M, Culotta VC ( 2003 ) The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis. J Biol Chem 278 : 42036 – 42040 Crossref Search ADS PubMed Kim KN, Fisher DK, Gao M, Guiltinan MJ ( 1998 ) Genomic organization and promoter activity of the maize starch branching enzyme I gene. Gene 216 : 233 – 243 Crossref Search ADS PubMed Koch M, Haubold B, Mitchell-Olds T ( 2001 ) Molecular systematics of the Brassicaceae: evidence from coding plastidic matK and nuclear Chs sequences. Am J Bot 88 : 534 – 544 Crossref Search ADS PubMed Koch MA, Haubold B, Mitchell-Olds T ( 2000 ) Comparative evolutionary analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis, Arabis, and related genera (Brassicaceae). Mol Biol Evol 17 : 1483 – 1498 Crossref Search ADS PubMed Krämer U, Clemens S ( 2005 ) Functions and homeostasis of zinc, copper, and nickel in plants. In M Tamás, E Martinoia, eds, Molecular Biology of Metal Homeostasis and Detoxification, Top Curr Genet, Vol 14. Springer, Berlin, pp 216–271 Krämer U, Cotter-Howells JD, Charnock JM, Baker AJM, Smith JAC ( 1996 ) Free histidine as a metal chelator in plants that accumulate nickel. Nature 379 : 635 – 638 Crossref Search ADS Kubota H, Takenaka C ( 2003 ) Arabis gemmifera is a hyperaccumulator of Cd and Zn. Int J Phytoremediation 5 : 197 – 201 Crossref Search ADS PubMed Küpper H, Lombi E, Zhao FJ, McGrath SP ( 2000 ) Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta 212 : 75 – 84 Crossref Search ADS PubMed Lahner B, Gong J, Mahmoudian M, Smith EL, Abid KB, Rogers EE, Guerinot ML, Harper JF, Ward JM, McIntyre L, et al ( 2003 ) Genomic scale profiling of nutrient and trace elements in Arabidopsis thaliana. Nat Biotechnol 21 : 1215 – 1221 Crossref Search ADS PubMed Lanquar V, Lelievre F, Bolte S, Hames C, Alcon C, Neumann D, Vansuyt G, Curie C, Schroder A, Kramer U, et al ( 2005 ) Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron. EMBO J 24 : 4041 – 4051 Crossref Search ADS PubMed Lasat MM, Pence NS, Garvin DF, Ebbs SD, Kochian LV ( 2000 ) Molecular physiology of zinc transport in the Zn hyperaccumulator Thlaspi caerulescens. J Exp Bot 51 : 71 – 79 Crossref Search ADS PubMed Macnair MR, Bert V, Huitson SB, Saumitou-Laprade P, Petit D ( 1999 ) Zinc tolerance and hyperaccumulation are genetically independent characters. Proc R Soc Lond B Biol Sci 266 : 2175 – 2179 Crossref Search ADS Marquardt J, Wans S, Rhiel E, Randolf A, Krumbein WE ( 2000 ) Intron-exon structure and gene copy number of a gene encoding for a membrane-intrinsic light-harvesting polypeptide of the red alga Galdieria sulphuraria. Gene 255 : 257 – 265 Crossref Search ADS PubMed Mäser P, Thomine S, Schroeder JI, Ward JM, Hirschi K, Sze H, Talke IN, Amtmann A, Maathuis FJ, Sanders D, et al ( 2001 ) Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiol 126 : 1646 – 1667 Crossref Search ADS PubMed McKie AT, Barlow DJ ( 2004 ) The SLC40 basolateral iron transporter family (IREG1/ferroportin/MTP1). Pflugers Arch 447 : 801 – 806 Crossref Search ADS PubMed Mills RF, Francini A, Ferreira da Rocha PS, Baccarini PJ, Aylett M, Krijger GC, Williams LE ( 2005 ) The plant P1B-type ATPase AtHMA4 transports Zn and Cd and plays a role in detoxification of transition metals supplied at elevated levels. FEBS Lett 579 : 783 – 791 Crossref Search ADS PubMed Mirouze M, Sels J, Richard O, Czernic P, Loubet S, Jacquier A, François IEJA, Cammue BPA, Lebrun M, Berthomieu P, et al ( 2006 ) A putative novel role for plant defensins: a defensin from the zinc hyperaccumulating plant, Arabidopsis halleri, confers zinc tolerance. Plant J 47 : 329 – 342 Crossref Search ADS PubMed Narindrasorasak S, Yao P, Sarkar B ( 2003 ) Protein disulfide isomerase, a multifunctional protein chaperone, shows copper-binding activity. Biochem Biophys Res Commun 311 : 405 – 414 Crossref Search ADS PubMed Papoyan A, Kochian LV ( 2004 ) Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance: characterization of a novel heavy metal transporting ATPase. Plant Physiol 136 : 3814 – 3823 Crossref Search ADS PubMed Peleman J, Saito K, Cottyn B, Engler G, Seurinck J, Van Montagu M, Inze D ( 1989 ) Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana. Gene 84 : 359 – 369 Crossref Search ADS PubMed Pence NS, Larsen PB, Ebbs SD, Letham DL, Lasat MM, Garvin DF, Eide D, Kochian LV ( 2000 ) The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Proc Natl Acad Sci USA 97 : 4956 – 4960 Crossref Search ADS PubMed Persans MW, Nieman K, Salt DE ( 2001 ) Functional activity and role of cation-efflux family members in Ni hyperaccumulation in Thlaspi goesingense. Proc Natl Acad Sci USA 98 : 9995 – 10000 Crossref Search ADS PubMed Petit JM, Briat JF, Lobreaux S ( 2001 ) Structure and differential expression of the four members of the Arabidopsis thaliana ferritin gene family. Biochem J 359 : 575 – 582 Crossref Search ADS PubMed Pich A, Scholz G ( 1996 ) Translocation of copper and other micronutrient in tomato plants (Lycopersicon esculentum Mill.): nicotianamine-stimulated copper transport in the xylem. J Exp Bot 47 : 41 – 47 Crossref Search ADS Pich A, Scholz G, Stephan UW ( 1994 ) Iron-dependent changes of heavy metals, nicotianamine, and citrate in different plant organs and in the xylem exudate of two tomato genotypes: nicotianamine as possible copper translocator. Plant Soil 165 : 189 – 196 Crossref Search ADS Ramakers C, Ruijter JM, Deprez RH, Moorman AF ( 2003 ) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339 : 62 – 66 Crossref Search ADS PubMed Reeves RD, Baker AJM ( 2000 ) Metal-accumulating plants. In I Raskin, BD Ensley, eds, Phytoremediation of Toxic Metals: Using Plants to Clean Up the Environment. John Wiley & Sons, Hoboken, NJ, pp 193–230 Rensing C, Mitra B, Rosen BP ( 1997 ) Insertional inactivation of dsbA produces sensitivity to cadmium and zinc in Escherichia coli. J Bacteriol 179 : 2769 – 2771 Crossref Search ADS PubMed Rogers EE, Guerinot ML ( 2002 ) FRD3, a member of the multidrug and toxin efflux family, controls iron deficiency responses in Arabidopsis. Plant Cell 14 : 1787 – 1799 Crossref Search ADS PubMed Roosens NH, Bernard C, Leplae R, Verbruggen N ( 2004 ) Evidence for copper homeostasis function of metallothionein (MT3) in the hyperaccumulator Thlaspi caerulescens. FEBS Lett 577 : 9 – 16 Crossref Search ADS PubMed Salt DE, Krämer U ( 2000 ) Mechanisms of metal hyperaccumulation in plants. In I Raskin, BD Ensley, eds, Phytoremediation of Toxic Metals: Using Plants to Clean Up the Environment. John Wiley & Sons, Hoboken, NJ, pp 231–246 Sambrook J, Russel DW ( 2001 ) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Sarret G, Saumitou-Laprade P, Bert V, Proux O, Hazemann JL, Traverse A, Marcus MA, Manceau A ( 2002 ) Forms of zinc accumulated in the hyperaccumulator Arabidopsis halleri. Plant Physiol 130 : 1815 – 1826 Crossref Search ADS PubMed Sentenac H, Bonneaud N, Minet M, Lacroute F, Salmon JM, Gaymard F, Grignon C ( 1992 ) Cloning and expression in yeast of a plant potassium ion transport system. Science 256 : 663 – 665 Crossref Search ADS PubMed Shikanai T, Muller-Moule P, Munekage Y, Niyogi KK, Pilon M ( 2003 ) PAA1, a P-type ATPase of Arabidopsis, functions in copper transport in chloroplasts. Plant Cell 15 : 1333 – 1346 Crossref Search ADS PubMed Shin H, Shin HS, Dewbre GR, Harrison MJ ( 2004 ) Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. Plant J 39 : 629 – 642 Crossref Search ADS PubMed Small RL, Wendel JF ( 2000 ) Copy number lability and evolutionary dynamics of the Adh gene family in diploid and tetraploid cotton (Gossypium). Genetics 155 : 1913 – 1926 Crossref Search ADS PubMed Taji T, Seki M, Satou M, Sakurai T, Kobayashi M, Ishiyama K, Narusaka Y, Narusaka M, Zhu JK, Shinozaki K ( 2004 ) Comparative genomics in salt tolerance between Arabidopsis and Arabidopsis-related halophyte salt cress using Arabidopsis microarray. Plant Physiol 135 : 1697 – 1709 Crossref Search ADS PubMed Thomine S, Lelievre F, Debarbieux E, Schroeder JI, Barbier-Brygoo H ( 2003 ) AtNRAMP3, a multispecific vacuolar metal transporter involved in plant responses to iron deficiency. Plant J 34 : 685 – 695 Crossref Search ADS PubMed Thomine S, Wang R, Ward JM, Crawford NM, Schroeder JI ( 2000 ) Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes. Proc Natl Acad Sci USA 97 : 4991 – 4996 Crossref Search ADS PubMed Usadel B, Nagel A, Thimm O, Redestig H, Bläsing OE, Palacios-Rojas N, Selbig J, Hannemann J, Piques MC, Steinhauser D, et al ( 2005 ) Extension of the visualization tool MapMan to allow statistical analysis of arrays, display of corresponding genes, and comparison with known responses. Plant Physiol 138 : 1195 – 1204 Crossref Search ADS PubMed Verret F, Gravot A, Auroy P, Leonhardt N, David P, Nussaume L, Vavasseur A, Richaud P ( 2004 ) Overexpression of AtHMA4 enhances root-to-shoot translocation of zinc and cadmium and plant metal tolerance. FEBS Lett 576 : 306 – 312 Crossref Search ADS PubMed von Wiren N, Klair S, Bansal S, Briat JF, Khodr H, Shioiri T, Leigh RA, Hider RC ( 1999 ) Nicotianamine chelates both Fe(III) and Fe(II): implications for metal transport in plants. Plant Physiol 119 : 1107 – 1114 Crossref Search ADS PubMed Weber M, Harada E, Vess C, von Roepenack-Lahaye E, Clemens S ( 2004 ) Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors. Plant J 37 : 269 – 281 Crossref Search ADS PubMed Wintz H, Fox T, Wu YY, Feng V, Chen W, Chang HS, Zhu T, Vulpe C ( 2003 ) Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal novel transporters involved in metal homeostasis. J Biol Chem 278 : 47644 – 47653 Crossref Search ADS PubMed Yogeeswaran K, Frary A, York TL, Amenta A, Lesser AH, Nasrallah JB, Tanksley SD, Nasrallah ME ( 2005 ) Comparative genome analyses of Arabidopsis spp.: inferring chromosomal rearrangement events in the evolutionary history of A. thaliana. Genome Res 15 : 505 – 515 Crossref Search ADS PubMed Author notes 1 This work was supported by the Deutsche Forschungsgemeinschaft (Kr 1967/3), the European Union (Research Training Network METALHOME, HPRN–CT–2002–00243), the German Federal Ministry of Education and Research (Biofuture 0311877), and the Max Planck Institute of Molecular Plant Physiology. 2 These authors contributed equally to the paper. * Corresponding author; e-mail kraemer@mpimp-golm.mpg.de; fax 49–331–5678–408. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ute Krämer (kraemer@mpimp-golm.mpg.de). [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.105.076232 © 2006 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Zinc-Dependent Global Transcriptional Control, Transcriptional Deregulation, and Higher Gene Copy Number for Genes in Metal Homeostasis of the Hyperaccumulator Arabidopsis halleri  
JF - PLANT PHYSIOLOGY
DO - 10.1104/pp.105.076232
DA - 2006-09-06
UR - https://www.deepdyve.com/lp/oxford-university-press/zinc-dependent-global-transcriptional-control-transcriptional-SajUbK14YD
SP - 148
EP - 167
VL - 142
IS - 1
DP - DeepDyve
ER -