These authors contributed equally.
Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri
Article first published online: 4 DEC 2003
The Plant Journal
Volume 37, Issue 2, pages 251–268, January 2004
How to Cite
Becher, M., Talke, I. N., Krall, L. and Krämer, U. (2004), Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri. The Plant Journal, 37: 251–268. doi: 10.1046/j.1365-313X.2003.01959.x
- Issue published online: 4 DEC 2003
- Article first published online: 4 DEC 2003
- Received 1 April 2003; revised 8 October 2003; accepted 14 October 2003.
- metal tolerance;
- Cardaminopsis halleri;
- P-type metal ATPase
- Top of page
- Experimental procedures
- Supplementary Material
- Supporting Information
Arabidopsis halleri ssp. halleri (accession Langelsheim) is a naturally selected zinc (Zn)- and cadmium-tolerant Zn hyperaccumulator. This plant differs strikingly from its close relative A. thaliana by accumulating Zn specifically in above-ground tissues. A. thaliana GeneChips were used in order to identify, on a transcriptome-wide scale, genes with a potential involvement in cellular metal uptake or detoxification in the shoots of A. halleri. Compared to A. thaliana, transcript abundance of several genes was found and confirmed to be substantially higher in A. halleri after 4 days of exposure to low as well as high Zn concentrations in the hydroponic culture medium. The identified candidate genes encode proteins closely related to the following A. thaliana proteins: AtZIP6, a putative cellular Zn uptake system and member of the zinc-regulated transporter (ZRT)-iron regulated transporter (IRT)-like protein (ZIP)-family of metal transporters, the putative P-type metal ATPase AtHMA3, the cation diffusion facilitator ZAT/AtCDF1, and the nicotianamine synthase AtNAS3. Heterologous expression in mutant strains of the yeast Saccharomyces cerevisiae suggested that AhHMA3, AhCDF1-3, and AhNAS3 can function in cellular Zn detoxification. Our data indicate that, at the transcript level, the Zn tolerance strategy of A. halleri involves high constitutive expression of metal homeostasis genes in the shoots to accommodate higher basal levels of Zn accumulation, and possibly to prepare for sudden increases in Zn influx into shoot cells. Furthermore, profiling of metal homeostasis gene transcripts in shoot and root tissues by real-time RT-PCR indicated that A. halleri and A. thaliana respond differently to changes in plant Zn status.
- Top of page
- Experimental procedures
- Supplementary Material
- Supporting Information
In the post-genome era, one of the great challenges is to uncover the molecular basis of physiological diversity allowing plants to thrive in diverse and often extreme environments. Among the vegetation on highly metal-enriched soils, some so-called metal hyperaccumulator plants commonly accumulate, for example, more than 1% of their above-ground dry biomass in nickel or zinc (Zn), concentrations that are at least one order of magnitude higher than those found in non-accumulator plants (Baker and Brooks, 1989). In all plants, a complex metal homeostatic network exists for a regulated, specific, and targeted supply of micronutrient metal cations to metal-requiring processes and for the detoxification of excess or non-essential metal cations that have entered the plant (Clemens et al., 2002). Metal hyperaccumulators, which are highly metal tolerant, are likely to exhibit numerous alterations in their metal homeostasis network throughout the entire plant. In recent years, researchers have begun to identify and characterize individual candidate genes implicated in these alterations, mainly by screening yeast expression libraries or through educated guesses. The candidates that have been implicated in metal tolerance or hyperaccumulation are members of the cation diffusion facilitator (CDF) and zinc-regulated transporter (ZRT)-iron regulated transporter (IRT)-like protein (ZIP) families of transition metal membrane transport proteins.
In Arabidopsis thaliana, the expression of ZIP family genes AtZIP1, AtZIP3, and AtZIP4 is induced under conditions of Zn depletion, and AtZIP1 and AtZIP3 have been shown to mediate cellular Zn uptake across the plasma membrane (Grotz et al., 1998). In the Zn hyperaccumulator Thlaspi caerulescens J & C Presl (ecotype Prayon), endogenous overexpression of the Zn transporter gene TcZNT1, which is most closely related to AtZIP4, was found to be associated with an approximately sixfold higher Vmax for Zn uptake into the roots, when compared to the non-accumulator T. arvense L., in plants grown at 1 µm Zn in a hydroponic medium (Pence et al., 2000). In T. caerulescens, downregulation of TcZNT1 expression only occurred at Zn concentrations of 50 µm and above. Subsequently, expression of another member of the ZIP family, TcZNT2, which is 80% identical to TcZNT1 at the cDNA level, was shown to parallel high expression levels of TcZNT1 in the T. caerulescens ecotypes Lellingen, La Calamine, and Monte Prinzera (Assuncao et al., 2001). Furthermore, Assuncao et al. (2001) isolated the TcZTP1 cDNA, which is most closely related to A. thaliana ZAT, a member of the CDF family that has been proposed to mediate cellular Zn detoxification (Van der Zaal et al., 1999). In several T. caerulescens ecotypes, TcZTP1 transcript levels were found to be constitutively elevated when compared to T. arvense (Assuncao et al., 2001).
Each of the above studies assessed the expression of only between one and three individual genes in the Zn hyperaccumulator species T. caerulescens. However, in A. thaliana, 12 genes encode putative proteins of the CDF family and 15 genes encode putative proteins of the ZIP family (Mäser et al., 2001). In T. caerulescens, the CDF and ZIP families are expected to be of similar sizes. This highlights the importance of determining whether related genes in the same gene families are also highly expressed or even dominant under the respective conditions, or whether other, yet unidentified, gene families might play an important role in metal hyperaccumulation or tolerance. Transcript profiling approaches, if applicable to a metal hyperaccumulator species, are capable of providing a broader, unbiased picture of the molecular basis for metal homeostatic networks in metal-tolerant plant species. However, to date, neither collections of expressed sequence tags nor comprehensive sequence data are available for any hyperaccumulator taxon.
A. halleri (L.) O'Kane and Al-Shehbaz ssp. halleri (accession Langelsheim) is a naturally selected Zn- and cadmium-tolerant Zn hyperaccumulator occurring on metal-contaminated soils deposited as sediments on the banks of the river Innerste in Germany (Ernst, 1974; Küpper et al., 2000; Zhao et al., 2000). A. halleri was originally termed Cardaminopsis halleri (L.) Hayek, but later re-classified (O'Kane and Al-Shehbaz, 1997). Other populations of this species occur at a number of metal-contaminated and non-contaminated sites, mostly across Europe. Between-population differences have been described in metal accumulation and tolerance by Bert et al. (2000, 2002) and Macnair (2002). A. halleri is an outcrossing stoloniferous perennial, which flowers early in summer. Both A. halleri and a closely related non-hyperaccumulator species, A. lyrata, have 2n = 16 chromosomes. Crosses have been successfully generated of A. halleri and A. lyrata ssp. petraea, and segregation of Zn and cadmium tolerance has been analyzed in the F1 and back-cross 1 (BC1) generations (Macnair et al., 1999). These resources will allow a genetic analysis of metal hyperaccumulation and metal tolerance in A. halleri. According to chalcone synthase and alcohol dehydrogenase as well as nuclear ribosomal DNA sequences, A. lyrata and A. halleri are the two species that are most closely related to A. thaliana (Koch et al., 1999, 2000). Based on the sequence of 20 cDNAs cloned in our laboratory, A. thaliana and A. halleri share, on an average, about 94% identity at the nucleotide level within coding regions. We hypothesized that A. halleri may be amenable to transcript profiling using oligonucleotide microarray chips based on A. thaliana nucleotide sequences.
The shoots of metal hyperaccumulators are a site of extreme metal accumulation. For example, up to 1.5% Zn, on a dry-biomass basis, is often detected in shoots of field-grown A. halleri (Bert et al., 2002), whereas soil-grown A. thaliana commonly contain up to around 0.015% Zn in shoot dry biomass (Krämer et al., unpublished observations). The shoots of metal hyperaccumulator plants must thus possess highly effective mechanisms for metal accumulation and detoxification (Krämer et al., 2000). Here, we present the results of oligonucleotide microarray chip-based comparative transcript profiling in shoots of A. halleri and A. thaliana, which were cultivated under low and high Zn supplies for 4 days. In an interspecies comparison, this approach is an example of assaying transcript levels on a transcriptome-wide scale to investigate the molecular basis and mechanisms of a complex, naturally selected physiological trait.
- Top of page
- Experimental procedures
- Supplementary Material
- Supporting Information
Physiological characterization of plant material
Four-week-old plants of A. halleri and A. thaliana, which had been pre-cultured in the presence of 1 µm ZnSO4, were exposed to a hydroponic solution containing 0, 100, and 300 µm added ZnSO4 for 4 days. Compared to low-Zn controls, Zn concentrations were significantly increased in shoots of all plants exposed to 100 or 300 µm Zn (P < 0.05; Table 1). At equivalent Zn concentrations in the medium, shoot Zn concentrations were between 2.4- and 5-fold higher in the Zn hyperaccumulator A. halleri than in A. thaliana. By contrast, root Zn concentrations were equivalent or lower in A. halleri, when compared to A. thaliana (data not shown). After 4 days of exposure to 300 µm Zn, a maximum concentration of approximately 85 µmol Zn g−1 shoot dry biomass (equivalent to 5551 µg Zn g−1 shoot dry biomass) was reached in A. halleri. Compared to controls, shoot biomass production was not reduced in A. halleri exposed to 100 or 300 µm Zn for 4 days. In A. thaliana, shoot biomass production was reduced to 79 and 51% of the low-Zn controls at 100 and 300 µm Zn, respectively (Table 1).
|Added Zn (µm)||A. halleri||A. thaliana|
|Shoot Zn (µmol g−1 DW)|
|0||8.0 ± 2.4||1.6 ± 0.6|
|100||43.2 ± 15.3||18.1 ± 2.2|
|300||84.9 ± 22.5||22.8 ± 10.2|
|Shoot fresh biomass (%)|
|100||108.9 ± 3.9||78.5 ± 13.3|
|300||101.0 ± 10.5||51.4 ± 22.2|
In order to perform transcript profiling under physiologically comparable conditions, 0 and 300 µm added Zn were selected as Zn treatments for the highly Zn-tolerant A. halleri plants, and 0 and 100 µm added Zn were selected as Zn treatments in the non-tolerant species A. thaliana (for shoot Ca, Cu, Fe, Mg, Mn, and S concentrations; see Table S1 in Supplementary Material). RT-PCR analysis of transcript levels of the stress marker genes GST1 (At1g02930), GST6 (At2g47730; Wagner et al., 2002), and HSP17.6B (At2g29500; Scharf et al., 2001) in shoot tissues indicated constant transcript levels of these genes across the two species and all selected treatment conditions (data not shown), suggesting that none of these treatments induced substantial general stress.
Three independent replicate experiments were conducted under the selected conditions for subsequent transcript profiling. Biotin-labeled cRNA was prepared from total RNA extracted from shoot tissues of 4-week-old plants of the two Arabidopsis species exposed to low and high Zn in a hydroponic culture medium for 4 days. Hybridization was carried out on oligonucleotide GeneChips featuring probe sets of approximately 8300 Arabidopsis genes (Zhu et al., 2001). For A. thaliana, robust signals, i.e. ‘present’ calls, were obtained for 63.9 ± 4.6% of the genes represented on the chip. For A. halleri, 36.8 ± 7.9% of the represented genes yielded a ‘present’ call.
Gene expression following 4 days of supply of high Zn concentrations was compared to gene expression following supply of low Zn in the culture medium for 4 days. This identified 339 genes (327 upregulated in response to high Zn and 12 downregulated; Table S2, Supplementary Material) in A. thaliana, and 136 genes (100 upregulated in response to high Zn and 36 downregulated; Table S3, Supplementary Material) in A. halleri, which responded by an at least twofold change in the average normalized signal intensities (Kreps et al., 2002). Among the genes found to be more highly expressed under high Zn exposure than under low Zn exposure in A. thaliana or A. halleri, 37–46% are of unknown function, between 29 and 30% are predicted to be involved in photosynthesis, metabolism and housekeeping, 16–19% in regulation and signaling, and 5–10% in stress protection or pathogen defense (Figure 1a,b). Among the transcripts that increased in abundance in response to high Zn, there were only 1–2% with a potential function in metal homeostasis. In A. thaliana, these encoded two late-embryogenesis-abundant proteins (At1g52690, 10-fold; At5g06760, 2.2-fold), two blue copper-binding proteins (At2g44790, 2.4-fold; At5g20230, 2.9-fold), the multispecific ATP-binding cassette (ABC) transporter AtMRP2 (At2g34660, 2.3-fold), a putative Zn-binding protein (At1g60190, 4.6-fold), the ferric-chelate reductase FRO2 (At1g01580, 2.8-fold; Robinson et al., 1999) and an FRO2-related protein (At1g23020, 7.1-fold). In A. halleri, only one transcript encoding a potential component of the metal homeostasis network, a protein with similarity to Cu,Zn-superoxide dismutases (At1g12520, 2.5-fold), was upregulated in response to high Zn. Thus, a 4-day exposure of A. halleri to 300 µm Zn, with a concomitant increase in shoot Zn concentrations by a factor of about 8.5 compared to plants grown under low Zn supply (Table 1), did not result in major alterations of the metal homeostasis network in the shoots at the transcript level.
After 4 days of growth at low Zn supply, shoots of A. halleri contained significantly higher concentrations of Zn than shoots of A. thaliana (see above, Table 1). This suggested a possible activation of cellular Zn uptake, sequestration, and detoxification systems in A. halleri under low Zn supply. Therefore, genes were identified that displayed at least eightfold higher average ratios of normalized signal intensities in A. halleri versus A. thaliana under control (low Zn) conditions. This stringent and conservative cut-off was applied in order to compensate for possible variation in sequence conservation between transcripts of A. halleri genes and their respective orthologs in the target species A. thaliana (see Data analysis in Experimental procedures). The resulting list of 50 genes contained 34% genes with putative functions in nutrition, metabolism, and housekeeping, about 26% regulatory genes, 14% genes with unknown function, about 10% genes with a predicted function in pathogen defense or stress protection, 10% with a putative role in metal homeostasis, and 6% genes in sulfur (S) and glutathione (GSH) metabolism (Figure 1c; Table 2).
|Chip id.||MIPS/AGI code||Annotation||Name||Average ratio low Zn||P < 0.05||Average ratio high Zn||P < 0.05|
|19521_s_at||At2g27880||Argonaute (AGO1)-like protein||AGO5a||218.9||*||18.2||*|
|19296_at||At4g30120||Cd2+-transporting P-type ATPase-like protein (3.A.3)||HMA3b||175.2||*||124.1||*|
|17762_s_at||At2g14580||Putative pathogenesis-related protein-like (PR1)||PR1-like||110.0||*||121.6||*|
|19045_at||At2g46950||Putative cytochrome P450||CYP709B2c||44.8||*||32.6||*|
|15437_at||At4g33550||Contains Pfam protease inhibitor/seed storage/LTP family domain PF00234||COR12d||32.4||*||49.5||*|
|16845_at||At2g27420||Putative cysteine proteinase||29.4||*||27.4||*|
|17133_at||At1g24370||F21J9.24 unknown protein||25.3||*||46.4||*|
|17046_s_at||At2g46800||Cation diffusion facilitator family Zn transporter (2.A.4)||ZATe||22.6||*||13.5||*|
|17019_s_at||At2g46830||MYB-related transcription factor (CCA1)||CCA1f||20.1||*||–|
|17501_s_at||At5g10140||MADS box protein FLOWERING LOCUS F||FLFg||16.9||*||13.9||*|
|13211_s_at||At3g12500||Basic endochitinase, glycosyl hydrolase family 19||CHI-Bh||14.8||*||–|
|18751_f_at||At4g38620||Putative MYB transcription factor||MYB4i||14.5||*||3.6||*|
|12267_at||At1g09420||Similar to glucose-6-phosphate dehydrogenases, e.g. gi|2276344, gi|2829880, gi|2352919||13.6||*||11.8||*|
|20228_s_at||At4g21940||Calcium-dependent protein kinase-like protein||CPK15||13.5||*||–|
|15299_s_at||At1g30660||T5I8.11 hypothetical protein||13.3||*||4.0||*|
|19704_i_at||At5g24160||Squalene monooxygenase 1,2 (squalene epoxidase 1,2)||SQP1; 2j||13.3||*||–|
|16007_at||At2g30790||Putative photosystem II oxygen-evolving complex 23K protein||OEC23||11.1||*||8.2||*|
|12332_s_at||At3g12500||Basic endochitinase, glycosyl hydrolase family 19 (see above)||CHI-B||11.0||*||3.1|
|17871_at||At2g16360||40S ribosomal protein S25||RPS25A||10.7||*||12.0||*|
|20525_at||At2g31380||B-box Zn finger protein; salt-tolerance-like protein||STHk||10.1||*||–|
|20044_at||At4g27310||B-box Zn finger protein||9.8||–|
|15125_f_at||At5g24780||Vegetative storage protein||VSP1l||9.4||*||–|
|18910_i_at||At2g22920||Putative serine carboxypeptidase I||9.1||*||–|
|18045_at||At3g22460||Similar to OAS-TL from Brassica juncea||OASA2m||9.0||*||–|
|15073_at||At2g01890||Putative purple acid phosphatase||PAP8n||8.7||*||7.7||*|
|19193_at||At4g16590||Cellulose synthase like protein||CSLA01||8.4||*||–|
|13085_i_at||At1g78820||Strong similarity to glycoprotein EP1 gb|L16983 Daucus carota and a member of S locus glycoprotein family PF|00954||8.3||11.8||*|
|17666_at||At1g09020||Contains similarity to Rattus AMP-activated protein kinase (gb|X95577)||8.0||*||5.2||*|
|13003_at||At1g09240||Nicotianamine synthase 3||NAS3o||8.0||*||5.2||*|
|17822_s_at||At5g55260||Serine/threonine protein phosphatase X isoform 2||PPX2||7.8||*||7.9||*|
|15807_at||At2g30080||ZRT-IRT-like protein (2.A.5)||ZIP6p||7.2||*||5.5||*|
|19522_at||At2g48020||Major facilitator superfamily, putative sugar transporter (2.A.1)||7.2||*||4.1||*|
|17941_at||At2g18720||Putative translation initiation factor eIF-2 gamma subunit||7.1||*||–|
|13023_at||At4g00050||Putative bHLH transcription factor||bHLH016||7.1||*||3.8|
|12732_r_at||At3g08500||R2R3-MYB transcription factor||MYB83i||7.0||*||–|
|13377_s_at||At2g18480||Major facilitator superfamily, putative sugar transporter (2.A.1)||6.7||*||–|
|18930_at||At1g23730||Similar to gb|L19255 carbonic anhydrase from Nicotiana tabacum and a member of the prokaryotic-type carbonic anhydrase family PF|00484. EST gb|Z235745 comes from this gene||CA3||6.5||*||5.5||*|
|17361_s_at||At4g10120||Sucrose-phosphate synthase-like protein||6.0||*||–|
|18596_at||At1g62570||T3P18.13, putative flavin-containing monooxygenase||5.8||*||5.3||*|
|12457_at||At3g21670||Nitrate transporter of proton-dependent oligopeptide transporter family 2.A.17||NTP3||5.8||–|
|18966_at||At2g29420||Putative glutathione S-transferase||GST25||5.2||*||–|
|12270_at||At4g27300||Putative receptor protein kinase||PPC:1.7.2||5.1||*||–|
|19720_at||At1g22690||Contains similarity to gibberellin-regulated protein 2 precursor (GAST1) homolog gb|U11765 from A. thaliana||5.1||*||5.4||*|
|12744_at||At3g16470||Jasmonate inducible protein isolog, jacalin lectin family, myrosinase-binding protein-like||5.0||*||–|
|18187_at||At2g32860||Putative beta-glucosidase, glycosyl hydrolase family 1||4.7||*||3.8||*|
|17507_at||At3g13320||Low-affinity Ca2+/proton antiporter CAX2, Ca2+:cation antiporter family (2.A.19)||CAX2q||4.6||*||–|
|12953_at||At4g01080||A_IG002N01.14 hypothetical protein||4.5||*||–|
The responses of A. halleri and A. thaliana to high Zn supply overlapped by only nine genes, which were upregulated in both species after 4 days of exposure to high Zn when compared to control conditions (Figure 1d). No putative candidate gene for metal homeostasis or tolerance was identified within this group of nine genes. The transcriptional responses of the two species to varying Zn supplies were, thus, distinct. Interestingly, for 32% of the genes more highly expressed in A. halleri than in A. thaliana under low-Zn conditions, expression increased in A. thaliana in response to high Zn supply (Figure 1d).
Identification of candidate genes and confirmation of microarray results
Following analysis of the GeneChip data, we selected candidates putatively involved in metal homeostasis or tolerance and other genes in order to confirm their expression using alternative techniques. In A. halleri, normalized signals of the GeneChip hybridizations were constitutively about 100-fold higher for the probe set corresponding to AtHMA3 (Axelsen and Palmgren, 2001), which encodes a putative P-type metal ATPase (At4g30120, error-free sequence accession AY055217), and 10–20-fold higher for a gene encoding the CDF family Zn transporter ZAT (At2g46800; Van der Zaal et al., 1999), when compared to A. thaliana (Table 2). On the microarrays probed with A. halleri cRNA, elevated signals were also detected for the probe set representing the transcript encoding the ZRT-IRT-like protein AtZIP6 (At2g30080), a putative Zn transporter, both at control and high Zn supplies (Mäser et al., 2001). RNA gel blots confirmed the results for the genes encoding the transport proteins HMA3, ZAT (data not shown; manuscript in preparation) and ZIP6 (Figure 2). Microarray signals for the probe set corresponding to the tonoplast low-affinity Ca2+(Mn2+)/H+ antiporter AtCAX2 (At3g13320) were higher in A. halleri than in A. thaliana under low-Zn conditions only (Table 2; Hirschi et al., 1996). This result, as well as the expression profile of a gene encoding a putative lectin-like protein with no expected role in metal homeostasis (At3g16470), was also confirmed by RNA gel blot analysis (Figure 2).
Apart from ZIP6, the microarray chip featured probes for six further ZIP family genes, namely IRT2, IRT3, ZIP2, ZIP4, ZIP5, and ZIP9. Out of these, only ZIP4 displayed a 3.8-fold higher normalized signal in A. halleri shoots compared to A. thaliana. This was below the cut-off of 8 applied in chip data evaluation. Real-time RT-PCR was performed to specifically analyze transcript levels of ZIP6 in comparison to two other ZIP genes that have been proposed to encode functional plasma membrane Zn uptake systems (Assuncao et al., 2001; Grotz et al., 1998). Compared to A. thaliana, ZIP6 transcript levels were clearly higher in shoots (23–24-fold) and roots (4–9-fold) of A. halleri under both low- and high-Zn conditions (Figure 3a), confirming the microarray data for shoots. In contrast, shoot transcript levels of ZIP1 and ZIP4 were more similar between the two species. This suggests that in A. halleri, transcript levels of ZIP6 are regulated distinctly from transcript levels of other ZIP family members. Generally, under high-Zn conditions, ZIP1 and ZIP4 transcript levels were downregulated in A. thaliana, but remained at higher levels in A. halleri. Under low-Zn conditions, root transcript levels of ZIP4 were sixfold higher in A. halleri than in A. thaliana, whereas ZIP1 transcript levels were equivalent in both species. These data suggest that gene expression of ZIP1 and ZIP4 is also regulated distinctly between A. halleri and A. thaliana. Of the three ZIP family genes analyzed by real-time RT-PCR, ZIP6 expression was predominant in the shoots of A. halleri.
According to the microarray hybridizations, exposure to high Zn caused an increase in transcript levels of the ferric chelate reductase gene FRO2 in A. thaliana (Robinson et al., 1999), suggesting that excess Zn may trigger iron deficiency responses. However, no significant decrease in iron concentration was detected in shoots (Table S1) or roots (data not shown) of A. thaliana exposed to high Zn. Compared to plants grown under low-Zn conditions, root transcript levels of FRO2 and IRT1, which encodes the primary root iron uptake system of A. thaliana (Vert et al., 2002), were confirmed to be dramatically increased under exposure to high Zn (Figure 3b). This correlated with ferric chelate reductase activity, which was 5.2-fold higher in seedlings exposed to 100 µm Zn for 4 days than in plants grown under low-Zn conditions (Figures 3c; P < 0.001). For comparison, growth of Arabidopsis seedlings on an iron-deficient medium has been reported to result in an approximately 11-fold increase in ferric chelate reductase activity (Yi and Guerinot, 1996).
In addition to RNA gel blot, real-time RT-PCR also confirmed highly elevated expression levels of HMA3 in A. halleri when compared to A. thaliana (Figure 4). The microarray also featured probe sets for the three members of the CPX-motif-containing P-type ATP-dependent metal transporter family that are most closely related to HMA3. These are HMA1, HMA2, and HMA4, which at the nucleotide level are 47, 67, and 67% identical, respectively, to the HMA3 target represented on the microarray chip. All target sequences for HMA genes represented on the chip are within the same divergent region at the 3′ end of the open-reading frame of aligned HMA genes, suggesting similar hybridization efficiencies of A. halleri cRNAs with HMA probe sets. The average ratios of A. halleri versus A. thaliana normalized signals under low Zn supply were 4.6 for HMA1, 0.25 for HMA2, and 1.3 for HMA4. To assess a possible overexpression of HMA1 in A. halleri, which may have gone undetected when using stringent criteria during data evaluation, RT-PCR was performed to analyze transcript levels of HMA1 in A. thaliana and A. halleri. This confirmed a 2.6–4-fold relative overexpression of HMA1 in A. halleri, which was much less pronounced than the 220–270-fold relative overexpression of HMA3 in A. halleri (Figure 4).
The enzyme cysteine synthase or OAS-TL catalyzes the final step in the biosynthesis of cysteine. Cysteine is an intermediate in the biosynthetic pathways of important metal chelating molecules, namely phytochelatins and other thiols, nicotianamine and phytosiderophores. In barley, iron deficiency was reported to induce an increase in transcript levels encoding a cytosolic OAS-TL (Negishi et al., 2002), which was proposed to contribute to an increased production of phytosiderophores (see also Weber et al., this issue). According to real-time RT-PCR, compared to A. thaliana, transcript levels of OASA2 (At3g22460), a gene encoding a putative cytosolic OAS-TL, were 2.6- and 2.1-fold higher in shoots and roots of A. halleri, respectively (Figure 4). As the homologous OASA1 has been proposed to encode the major functional cytosolic OAS-TL in A. thaliana (Jost et al., 2000), OASA1 transcript levels were also analyzed. Transcript levels of OASA1 were approximately equivalent in shoots of A. halleri and A. thaliana (Figure 4), and generally approximately 10-fold higher than OASA2 transcript levels. In total protein extracts from shoots of low-Zn-exposed plants, total OAS-TL enzyme activities were only very slightly, albeit significantly, elevated in A. halleri (101.4 ± 9.1 µmol mg−1 protein h−1, mean ± SD), when compared to A. thaliana (79.7 ± 6.0 µmol mg−1 protein h−1, mean ± SD; n = 3 independent experiments; P < 0.05; data not shown). On Western blots of total protein extracts from shoots, no significant differences could be demonstrated between protein levels in A. halleri and A. thaliana using two independently generated antibodies specific for the cytosolic isoform of OAS-TL (Jost et al., 2000; Stansilav Kopriva, unpublished; data not shown). The stop codon in the AtOASA2 cDNA, which has been proposed to result in a truncated and inactive OASA2 protein in A. thaliana, was not present at the corresponding position in a partial AhOASA2 cDNA.
Compared to A. thaliana, normalized microarray signals were also slightly elevated in A. halleri shoots for NAS3, but not for NAS1 or NAS2 (Table 2). AtNAS3 is one of three nicotianamine synthase isoforms, which have been reported to catalyze the final step in the biosynthesis of nicotianamine in A. thaliana (Suzuki et al., 1999). Nicotianamine is a low-molecular weight high-affinity transition metal chelator molecule that has previously been implicated in micronutrient nutrition and mobility (Ling et al., 1999). A fourth putative NAS gene was identified in the database and named NAS4 (At1g56430) based on the high similarity of the predicted gene product to NAS3 (77% identity at the amino acid level). Real-time RT-PCR confirmed 6.5–7-fold higher NAS3 transcript levels in shoots of A. halleri, compared to A. thaliana (Figure 4). In the same tissues, transcript levels of NAS1 were 4.6–16-fold lower in A. halleri than in A. thaliana. Thus, in the shoots, the sum totals of NAS gene transcript levels were of comparable magnitude in both species. Whereas NAS3 transcripts levels were very low in the roots of both species, NAS2 transcripts were virtually absent in the shoots of both species. NAS1 and NAS4 were expressed in both roots and shoots. NAS2 transcripts levels were extremely high in the roots of A. halleri and between 7.5- and 45-fold higher than in roots of A. thaliana (see Weber et al., this issue).
Microarray analysis suggested that, compared to A. thaliana, transcript levels of a gene encoding a putative carbonic anhydrase-like protein, termed here CA3, are elevated in A. halleri (At2g23730, Table 2). Proteins with a carbonic anhydrase signature contain a Zn2+ ion in their catalytic site. The carbonic anhydrase protein might be involved in Zn binding or possibly in the synthesis of carboxylate-containing ligands, which have been suggested to chelate Zn in A. halleri (Sarret et al., 2002). Real-time RT-PCR confirmed CA3 transcript levels to be more than 10-fold higher in A. halleri than in A. thaliana.
Between A. halleri and A. thaliana, the largest difference in expression of any gene on the microarrays was found for AGO5, a gene of unknown function related to PINHEAD (PNH)/ZWILLE (ZLL) and ARGONAUTE (AGO1) in a gene family implicated in post-transcriptional gene silencing (Table 2; Bohmert et al., 1998). According to real-time RT-PCR, AGO5 transcript levels were indeed between 500- and 1400-fold higher in A. halleri, compared to A. thaliana (Figure 4).
Functional analysis of candidate genes
For subsequent experiments, we focused on a subset of genes exhibiting higher transcript levels in A. halleri than in A. thaliana(Figures 2–4), and considered most likely to possess a direct role in shoot cellular metal accumulation or detoxification. We successfully cloned cDNAs comprising full-length open-reading frames of HMA3, CAX2, NAS3, and the ZAT-like CDF1-3 from A. halleri, but have so far been unable to clone an A. thaliana or A. halleri ZIP6 cDNA devoid of mutations. In order to determine whether the encoded proteins have a function in Zn or Cd detoxification in A. halleri, cDNAs were expressed in the Sacharomyces cerevisiae mutants zrc1 cot1 and YYA4, which are hypersensitive to Zn2+ (MacDiarmid et al., 2000) and Cd2+ (Gaedeke et al., 2001), respectively.
Expression of AtCAX2 or AhCAX2 did not alleviate the Cd-hypersensitive phenotype of S. cerevisiae YYA4 mutant cells. By contrast, Cd hypersensitivity was exacerbated in YYA4 cells expressing either of the two CAX2 cDNAs (Figure 5a, shown for AhCAX2 only). Similarly, expression of AtCAX2 or AhCAX2 did not improve growth of the Zn-hypersensitive zrc1 cot1 double mutant under exposure to high Zn concentrations (data not shown; Shigaki et al., 2003). Expression of the A. halleri cDNA AhCDF1-3 homologous to ZAT complemented the Zn-hypersensitive phenotype of the zrc1 cot1 mutant (Figure 5b). Similarly, expression of AhHMA3 improved growth of zrc1 cot1 cells on agarose media containing 300 and 400 µm Zn, when compared to empty vector transformants (Figure 5c). However, Zn tolerance of the zrc1 cot1 double mutant expressing AhHMA3 was lower than that of empty vector-transformed wild-type yeast cells, which are able to grow at Zn concentrations of 3 mm and more under our experimental conditions. Expression of AhHMA3 did not complement the Cd-hypersensitive yeast mutant YYA4 (data not shown). When AhNAS3 was expressed in the zrc1 cot1 mutant, the cells were able to grow at 500 µm Zn, a concentration that inhibited growth of the empty vector transformants (Figure 5d).
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Feasibility of interspecies hybridizations
To our knowledge, interspecies transcript profiling using A. thaliana oligonucleotide microarray chip technology has not previously been reported. Results of the microarray hybridizations were independently confirmed by RNA gel blot analysis, and classic (data not shown) and real-time RT-PCR (Table 2; Figures 3 and 4). This supports the general feasibility of using microarrays based on A. thaliana nucleotide sequences for comparative transcript profiling in A. halleri and A. thaliana (see also Weber et al., this issue). Independent support for this approach is provided by studies involving the hybridization of chimpanzee, orangutan and rhesus macaque cRNAs on human GeneChips (Chismar et al., 2002). Of the transcripts called present, i.e. reliably detectable (see Affymetrix user manual; Affymetrix, Santa Clara, CA, USA), in human samples, a proportion of 63% was found present in equivalent rhesus macaque samples (Chismar et al., 2002). A similar proportion of 58% of genes called present in A. thaliana were called present in A. halleri in this study. Microarray chip technology may prove very useful for exploring biological diversity in A. thaliana accessions and in plant species closely related to A. thaliana.
Selection, confirmation, and functional analysis of candidate genes
At the transcript level, the two Arabidopsis species responded quite distinctly to increased Zn concentrations supplied in the growth medium, with an overlap of only nine genes of mainly unknown functions (Figure 1). Compared to A. thaliana grown at low Zn supply, after 4 days of high Zn supply an increase in steady-state transcript levels occurred for only a very small number of genes with potential functions in metal homeostasis. These genes encode proteins including a late embryogenesis-abundant protein (Kruger et al., 2002), a glutathione conjugate transporter (Liu et al., 2001), and blue copper-binding and putative Zn-binding proteins. For several of these genes an increase in transcript levels has previously been reported in response to abiotic stress (Desikan et al., 2000; Kreps et al., 2002; Rossel et al., 2002), and it is also possible that they play a role in protecting A. thaliana from metal stress. A 4-day exposure of A. thaliana to high Zn concentrations resulted in increases in root transcript levels of FRO2 and IRT1 (Table S2, Figure 3b), which are known to be strongly induced after 3 days of growth in iron-deficient media (Robinson et al., 1999; Vert et al., 2003), but plant iron concentrations were not significantly changed (Table S1). Connolly et al. (2002) have also observed that IRT1 transcript levels increase in plants grown in an iron-deficient medium supplemented with excess Zn for 3 days. In their study, the IRT1 protein, which is present at high levels in iron-deficient plants, was undetectable under conditions of excess Zn supply. We observed that under high Zn supply, seedlings exhibited enhanced ferric chelate reductase activity, suggesting that iron deficiency responses are at least partially activated under these conditions (Figure 3c).
For some metal homeostasis genes we observed substantially higher steady-state transcript abundance in A. halleri shoots under low as well as high Zn supply, when compared to A. thaliana. These candidates include A. halleri genes highly similar to AtHMA3 of the P-type metal ATPase family, ZAT of the CDF family, AtZIP6 of the ZIP metal transporter gene family and AtNAS3, a nicotianamine synthase gene (Table 2; Figures 2–4; Suzuki et al., 1999). HMA3 is a member of the P1B or CPX group of P-type ATPases with eight predicted transmembrane domains (Axelsen and Palmgren, 2001), which has eight members in A. thaliana. AtHMA3 or AhHMA3 have not previously been functionally characterized, and belong to the subgroup of putative Zn2+, Co2+, Cd2+, and Pb2+-ATPases. The Staphylococcus aureus CADA, which is 29% identical to AhHMA3 at the amino acid sequence level, is one of the most closely related, functionally characterized proteins to date, and catalyzes ATP-dependent cellular efflux of Cd (Nucifora et al., 1989). AtHMA4, which shares about 45% identity with AhHMA3, has recently been reported to increase Cd tolerance of wild type yeast cells and to complement Zn hypersensitivity of the E. coli zntA mutant (Mills et al., 2003). Expression of the AhHMA3 cDNA slightly improved growth of the Zn-hypersensitive yeast double mutant strain zrc1 cot1 on an agarose medium containing 300 or 400 µm Zn (Figure 5a). These data suggest that the cellular function of AhHMA3 is the detoxification of Zn, consistent with an involvement of this protein in Zn tolerance in A. halleri. The regulation of AhHMA3 transcript levels suggests a role primarily in shoots, and also in A. halleri roots exposed to high Zn (Figure 4). Moreover, A. halleri can trigger a transcriptional response to high Zn supply, which for root HMA3 transcript levels is more drastic than in A. thaliana (compare ZIP1, Figure 3). So far, there is only a single conference report on the potential involvement of a P-type metal ATPase in hyperaccumulation in Thlaspi caerulescens (Papoyan et al., 2002). This P-type ATPase is homologous to AtHMA4, and is reported to be primarily expressed in roots.
A second major candidate gene revealed in the microarray analysis is ZAT, a member of the CDF protein family. Overexpression of the A. thaliana ZAT cDNA under the control of a CaMV 35S promoter has previously been shown to result in increased Zn tolerance and elevated Zn accumulation in roots (Van der Zaal et al., 1999). Moreover, compared to related non-accumulator species, constitutively elevated transcript levels of genes very closely related to ZAT have also been detected in the nickel hyperaccumulator Thlaspi goesingense (Persans et al., 2001) and in the Zn hyperaccumulator Thlaspi caerulescens (Assuncao et al., 2001). When expressed in the zrc1 cot1 yeast mutant, AhCDF1-3 restored Zn tolerance (Figure 5b). This suggests that AhCDF1-3 functions in the detoxification of Zn by transporting Zn2+ out of the cytoplasm, presumably into the vacuole.
In A. halleri, transcript levels of NAS3 were moderately higher than in A. thaliana under low- and high-Zn conditions (Table 1; Figure 4). The AtNAS3 protein has been demonstrated to catalyze the synthesis of a nicotianamine molecule from three molecules of S-adenosylmethionine (Suzuki et al., 1999). Nicotianamine, which forms highly stable chelates with Cu2+, Fe2+, Fe3+, and Zn2+ (pKS > 12), is essential for metal mobility in the phloem and intercellularly (Ling et al., 1999; Takahashi et al., 2003), and has been implicated in naturally selected Ni hyperaccumulation (Vacchina et al., 2003). Expression of AhNAS3 in yeast suggested that it can contribute to cellular Zn tolerance, presumably because heterologously produced nicotianamine chelates Zn2+ ions and thereby reduces Zn toxicity (Figure 5d; see also Weber et al., this issue). The approximated sums of shoot transcript levels of NAS1, NAS2, NAS3, and NAS4 did not differ substantially between A. halleri and A. thaliana (Figure 4). It remains to be investigated whether distinct localization, regulation, or biochemical properties of NAS1, NAS3, and NAS4 isoforms in the shoot of A. halleri contribute to Zn hyperaccumulation or hypertolerance.
Known ZIP proteins catalyze the transport of divalent transition metal cations, mainly Fe2+ or Zn2+, into the cytoplasm (Grotz et al., 1998). Transcript levels of a previously uncharacterized member of this gene family, ZIP6 (Mäser et al., 2001), were found to be constitutively high in shoots, and to a lesser extent in roots of A. halleri (Table 2; Figures 2 and 3). This was not observed for two other members of the ZIP gene family, ZIP1 and ZIP4. However, ZIP1 and ZIP4 transcript levels were also differentially regulated between A. halleri and A. thaliana. Transcript abundance of ZIP1 and ZIP4 was downregulated under high-Zn conditions in both shoots and roots of A. thaliana. Although A. halleri shoots exposed to high Zn contained about 4.7-fold higher total Zn concentrations than A. thaliana shoots (Table 1), the regulatory systems effecting a downregulation of transcript levels encoding the Zn uptake systems ZIP1 and ZIP4 were apparently less activated in roots and not activated in shoots of A. halleri. Either the involved Zn regulatory mechanisms may require considerably higher free Zn concentrations to activate in A. halleri, or effective Zn sequestration or chelation in A. halleri may result in lower free Zn concentrations at the sites where Zn regulation is triggered. In addition to being transcriptionally regulated by metals, members of the ZIP family have been reported to undergo post-transcriptional regulation (Connolly et al., 2002).
Under low Zn supply, transcript levels of CAX2 were elevated in shoots of A. halleri compared to A. thaliana, whereas they were equivalent in both species upon exposure to high Zn (Figure 2). AtCAX2 has been described as a low-affinity Ca2+ or Mn2+ proton antiport system in the tonoplast (Hirschi et al., 1996), which is also able to transport Cd2+ in vesicle transport assays. Overexpression of AtCAX2 in tobacco increased Mn2+ tolerance and Cd2+ and Ca2+ accumulation (Hirschi et al., 2000). Here, expression of AhCAX2 in the Cd-hypersensitive yeast double mutant YYA4, which is defective in vacuolar Cd sequestration, resulted in an exacerbated Cd-hypersensitivity (Figure 5a). This may be a consequence of the modification of cellular calcium, manganese, or proton homeostasis by CAX2 in YYA4 cells (Hirschi, 2001; Schaaf et al., 2002). Taken together, our data do not support a direct involvement of a CAX2-like protein in metal detoxification or accumulation in A. halleri.
Under low-Zn conditions, transcript levels of OASA2 encoding a putative cytosolic o-acetylserine thiol lyase (OAS-TL) were higher in A. halleri than in A. thaliana (Table 2; Figure 4). Assuming comparable amplification efficiencies of OASA1 and OASA2 cDNA fragments in real-time RT-PCR (Figure 4), the OASA2 transcript is estimated to contribute a minor fraction of the total OASA transcript levels. This interpretation is consistent with similar levels of total OAS-TL enzyme activity and cytosolic OAS-TL protein levels in A. halleri and A. thaliana, as determined in this study. In situ hybridization experiments have previously shown that the highest levels of OASA1 transcript are localized specifically in the trichomes of A. thaliana (Gutierrez-Alcala et al., 2000), but protein levels were not determined. It remains to be investigated whether a functional OASA2 protein is made in A. halleri, and whether OASA1 and OASA2 transcripts display a distinct localization in leaves of A. halleri.
A ‘proactive’ strategy for Zn hypertolerance in A. halleri?
The microarray data indicated that a relatively high proportion of about 32% of the genes found to be upregulated in A. thaliana in response to high Zn were more highly expressed in A. halleri than in A. thaliana under low Zn supply (Figure 1d). These included genes known to be regulated by abiotic stress (e.g. CCA1, At3g31380, At4g27310, and At3g21670; Desikan et al., 2000; Fowler and Thomashow, 2002; Kreps et al., 2002), and the chitinase B gene CHI-B (At3g12500) known to be upregulated in response to jasmonate and ethylene (Ellis and Turner, 2001). It has been proposed that an excess of Zn can cause general and moderate oxidative stress in plants, for example by uncontrolled binding to and thus inactivating proteins and thiols or by damaging membranes (Clemens et al., 2002; Van Assche and Clijsters, 1990). Increased expression of genes involved in stress protection may therefore be important for optimum performance of plant cells under exposure to high levels of Zn, or may have a specific, yet unidentified, role in metal homeostasis or metal responses.
Compared to A. thaliana, A. halleri maintains increased transcript levels of several metal homeostasis genes, mainly HMA3, CDF1-3, NAS3, and ZIP6 when external Zn supply is low (Table 2; Figures 2–4). What is the significance of an increased abundance of transcripts encoding cellular metal detoxification systems under low Zn supply in a metal hyperaccumulator? The situation is reminiscent of Zn homeostasis in the yeast S. cerevisiae (MacDiarmid et al., 2003). Zinc-limited yeast cells upregulate transcription of ScZRT1 and ScZRT2, which encode plasma membrane transporters that mediate cellular Zn uptake and, like AhZIP6, belong to the ZIP family of proteins. When external Zn concentrations increase rapidly, large amounts of Zn enter the yeast cell before Zn uptake systems are downregulated at both the transcriptional and post-transcriptional levels, resulting in a so-called ‘Zn shock’. When growing under Zn-limiting conditions, in order to guard against the deleterious effects of a ‘Zn shock’, yeast cells ‘proactively’ induce transcription of the Zn detoxification system ScZRC1, which, like AhCDF1, belongs to the CDF family (MacDiarmid et al., 2003). By analogy, A. halleri may have evolved to pursue a ‘proactive strategy’ of metal hypertolerance. Compared to A. thaliana, elevated rates of Zn accumulation in the shoot of A. halleri are associated with higher transcript levels of genes encoding putative root and shoot cellular Zn uptake systems, predominantly ZIP6 in the shoot, over a wide range of Zn supplies (Figure 3). A high constitutive abundance of transcripts of metal detoxification genes like CDF1/ZAT and HMA3 may confer the ability to instantly detoxify metal ions under fluctuating Zn supply (Figure 4).
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Comparative transcript profiling of metal hyperaccumulator and non-accumulator shoots and roots (Weber et al., this issue) has identified a distinct and tissue-specific regulation of different members of the ZIP and NAS metal homeostasis gene families in the Zn hyperaccumulator A. halleri. We propose that this reflects cellular tasks common to roots and shoots of hyperaccumulators. These tasks are an elevated cellular Zn uptake rate, with a predominant role of ZIP9 in roots and ZIP6 in shoots, and the necessity for low-molecular weight chelator biosynthesis to achieve cytoplasmic Zn buffering and intercellular metal mobility, with major roles for NAS2 in roots and possibly NAS3 in shoots. We propose that the striking differences between root and shoot transcript profiles reflect the different functions of the root and the shoot in metal hyperaccumulation. In the roots of A. halleri, NAS2 and natural resistance-associated macrophage protein family (Nramp3), which encodes a natural resistance-associated macrophage protein, are predominant and likely to have a role in sustaining root-to-shoot mobility of Zn (Thomine et al., 2003; Weber et al., this issue). Our data suggest that ZAT/CDF1 and HMA3, which are predominant in the shoot transcript profiles, are important for metal sequestration and detoxification, thereby generating a metal sink in the shoot that could be one of the driving forces in metal hyperaccumulation. Taken together, the two papers in this issue comprehensively add a molecular dimension to the complex network of physiological processes, generating the metal hyperaccumulator phenotype in a single hyperaccumulator species (Chaney et al., 1997; Salt and Krämer, 2000, specifically summarized in Clemens et al., 2002). This will provide a starting point for a genetic analysis of metal hyperaccumulation in A. halleri, as well as for the identification of upstream regulatory genes.
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Plant material, growth conditions, and experimental treatments
Seeds of A. thaliana (L.) Heynhold (accession Columbia) and A. halleri (L.) O'Kane and Al-Shehbaz ssp. halleri (accession Langelsheim) were placed on a layer of 0.8% (w/v) solidified Noble agar (Bio101, Vista, CA, USA) in clipped black 0.5-ml Eppendorf tubes, and germinated and cultivated in a hydroponic culture solution. After germination, solutions were exchanged weekly. The hydroponic solution contained 0.28 mm KH2PO4, 1.25 mm KNO3, 1.5 mm Ca(NO3)2, 0.75 mm MgSO4, 5 µm of a complex of Fe(III) and N,N′-di-(2-hydroxybenzoyl)-ethylenediamine-N,N′-diacetate (HBED, Strem Chemicals, Inc., Newburyport, MA, USA) prepared according to Chaney (1988), 25 µm H3BO3, 5 µm MnCl2, 1 µm ZnSO4, 0.5 µm CuSO4, 50 µm KCl, and 0.1 µm Na2MoO4, buffered at pH 5.7 with 3 mm 2-(N-morpholino) ethanesulfonate (MES). Plants were grown in a climate-controlled growth chamber at a photon flux density of 120 µmol m−2 sec−1 during the day, a photoperiod of 14-h light and 10-h dark, day and night temperatures of 20 and 16°C, and 60 and 75% constant relative humidity during the day and night, respectively. When plants were 4 weeks old, the hydroponic solutions were replaced by the experimental treatments, i.e. a culture solution composed as described above, with either no added ZnSO4 (‘low Zn’), or 100 or 300 µm added ZnSO4 (‘high Zn’), as indicated. Each treatment comprised at least three replicate culture vessels of 400 ml, containing three individual plants each. Four days after initiation of treatments, roots and shoots were carefully separated, pooled, frozen in liquid nitrogen, and stored at −80°C until further processing. Three independent replicate experiments were conducted for the microarray hybridizations. Subsequently, two additional replicate experiments were conducted for confirmation of results by real-time RT-PCR, in which plants were cultivated at a photoperiod of 11-h light and 13-h dark, a photon flux density of 145 µmol m−2 sec−1 during the day, and day and night temperatures 20 and 18°C, respectively. In these experiments, the treatments were initiated 6 weeks after initiation of germination.
Determination of biomass production and nutrient concentrations
After harvest, biomass of fresh shoot material was determined. Subsequently, shoot material was dried at 60°C for 3 days and weighed, and each tissue sample was digested in 1 ml of 65% (w/v) nitric acid (Suprapur, Merck, Darmstadt, Germany) in a MARS 5 microwave (CEM GmbH, Kamp-Lintford, Germany) at 200°C and 15 bar for 10 min. After digestion, the volume was adjusted to 10 ml with water, and nutrient concentrations were determined by inductively coupled plasma optical emission spectroscopy using an IRIS Advantage Duo ER/S (Thermo Jarrell Ash, Franklin, MA, USA).
For microarray expression profiling, total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Subsequently, genomic DNA was digested (DNAfree, Ambion, Austin, TX, USA), and the RNA was purified using RNeasy spin columns (Qiagen, Hilden, Germany). Quality and quantity of RNA were checked using a Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany) and formaldehyde gel electrophoresis.
Microarray expression profiling
For each cDNA synthesis (cDNA Synthesis System, F. Hoffmann-La Roche Ltd., Basel, Switzerland), 20 µg total RNA were used. Biotin-labeled cRNA was synthesized using the T7-Megascript kit (Ambion). The quality of cRNA was tested using the Bioanalyzer and a spectrophotometric analysis. After fragmentation of cRNAs according to the Affymetrix technical manual, one cRNA sample per experiment was tested on the Test 3 array (Affymetrix). Only high-quality sets of labeled cRNAs were processed further. For hybridization, 15 µg of cRNA were used on each Arabidopsis GeneChip Microarray (Affymetrix), which contained probes for approximately 8300 Arabidopsis genes (Zhu et al., 2001). Hybridization, washing, staining (Fluidics Station 400, Affymetrix), and scanning (G2500A Gene Array Scanner, Agilent Technologies, Palo Alto, CA, USA) were performed according to standard procedures using the Microarray Suite (mas) 5.0 software (Affymetrix).
The trimmed mean intensity of all raw probe set signals was lower for chips hybridized with A. halleri labeled cRNA by a factor of 4.0 ± 1.5 (average ± SD), when compared to chips hybridized with A. thaliana labeled cRNA. This difference is a consequence of the slight sequence divergence between the two Arabidopsis species (see Introduction). In order to be able to compare different chips, raw signal intensities were scaled to obtain an identical trimmed mean intensity for each chip, according to standard procedures described in the user manual (Affymetrix Microarray Suite User's Guide, version 5.0 (mas 5.0)). For each chip, the median signal of all probe sets was, thus, scaled to a target intensity of 500 in the mas 5.0 software. Subsequently, data were evaluated using the genespring™ software (version 5.0, Silicon Genetics, Redwood City, CA, USA). Data were normalized per chip to the median of each chip, and subsequently per gene to the respective gene on the control chip from the same experiment. Arithmetic means of expression values were calculated from the three replicate experiments using the ratio interpretation. Statistical significance of expression changes was determined using a non-parametric test (Wilcoxon two-sample rank test) and the Benjamini and Hochberg procedure to control false discovery rates. This was done because the most highly expressed genes showed highest numerical variance in expression, even after signals were log-transformed. In intraspecies comparisons, genes were required to be called present and have a scaled signal intensity of at least 50 in at least 50% of the chips under comparison, and genes were considered to be differentially expressed when exhibiting at least an average twofold difference in normalized signal across all replicate experiments (Figure 2a,b; Kreps et al., 2002). In interspecies comparisons, genes were required to be called present on all A. halleri chips. This was done because in cross-species hybridizations of rhesus macaque cRNA on human GeneChips, probe sets that were called present on all chips hybridized with rhesus macaque cRNA were considered reliable (Chismar et al., 2002). In order to compensate for possible variability in sequence conservation between A. thaliana and A. halleri, genes were considered more highly expressed in A. halleri when they exhibited at least an average eightfold normalized signal intensity in comparison to A. thaliana, across all replicate experiments. This cut-off value is composed of a twofold cut-off, multiplied by a fourfold average difference in trimmed means of raw signals per chip (see above).
For the comparison between A. halleri and A. thaliana under low-Zn conditions, a second data evaluation was performed using the mas 5.0 software. Comparisons were performed for each of the three replicate experiments independently, using cut-offs and requirements as above, and data were transferred into microsoft access and excel for further analysis. Arithmetic mean values were calculated from the three replicate signal log ratios derived from chip pair comparisons in the replicate experiments, and means were back-transformed. Genes that displayed a mean A. halleri:A. thaliana signal log2 ratio of at least 3 (i.e. back-transformed ratio of at least 8) were included in the list of genes more highly expressed in A. halleri (Table 2; Figure 1), but values for ratios were given based on the data evaluation performed using genespring™ software. For the generation of Figure 1, duplicates were eliminated, as in some cases, several probe set identifiers correspond to identical Arabidopsis Genome Initiative (AGI) codes. For annotation, existing annotations were compared for each probe set (Ghassemian et al., 2001; http://genetics.mgh.harvard.edu/sheenweb/search_affy.html), and when ambiguous, target sequences provided by the manufacturer were used in blast search queries against the Arabidopsis genome on the Munich Information Center for Protein Sequences (MIPS) and The Arabidopsis Information Resource (TAIR) websites.
Cloning and sequence analysis
Full-length open-reading frames were amplified by RT-PCR using a proofreading DNA polymerase (Pfu Turbo, Stratagene, La Jolla, CA, USA), following cDNA synthesis using the SuperScript™ first-Strand Synthesis System (Invitrogen). For AhCAX2 and AtCAX2, cDNAs were cloned, which encode an N-terminally truncated protein lacking the autoinhibitory domain (Pittman and Hirschi, 2001) using the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGAGCAAGGATCACTTT-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCATATACCATCTTGGTGAGGA-3′ and the following PCR programme: 4 min at 95°C, followed by 40 cycles of 45 sec at 94°C, 30 sec at 58°C, 5.5 min at 72°C, and a final extension step of 10 min at 72°C. The PCR product was cloned into a GATEWAY (Invitrogen) entry vector and subcloned into target vectors by site-specific recombination according to the manufacturer's instructions. The A. thaliana At3g16470 cDNA and a homologous sequence from A. halleri were cloned using the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGCGAAAAAGTTGGAAGCTCA-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCATTTGGCTATGGGCGCAAC-3′ and the following PCR programme: 2 min at 94°C, followed by 5 cycles of 15 sec at 94°C, 30 sec at 47°C, 3 min at 72°C and 35 cycles of 15 sec at 94°C, 30 sec at 63°C, 3 min at 72°C, and a final extension step of 10 min at 72°C. The PCR product was cloned as described for AhCAX2. Cloning of AhCDF1-3 will be described elsewhere. For cloning of AhHMA3, the primers 5′-CACCATGGCGGAAGGTGAAGAGT-3′ and 5′-TCACTTTTGTTGATTGTCCTTAG-3′ were used in the following PCR protocol: 4 min at 95°C, followed by 35 cycles of 60 sec at 94°C, 30 sec at 55°C, 4.5 min at 72°C, and a final extension step of 10 min at 72°C. For cloning of the AhNAS3 cDNA, the primers 5′-CACCACCTCATAGTGTCGACATGGG-3′ and 5′-TTGGGAAACAAGAAACGTCCTC-3′ were used in a standard PCR reaction, with extension at 72°C for 2.5 min. The PCR products were cloned into a GATEWAY entry vector using the pENTR directional TOPO cloning kit (Invitrogen) and subcloned into target vectors by site-specific recombination according to the manufacturer's instructions. Other cDNAs were cloned using primers as listed in Table S4 (Supplementary Material) and the following default protocol: 30 sec at 95°C, followed by 32 cycles of 30 sec at 95°C, 30 sec at 55°C, and 60 sec at 72°C.
RNA gel blot analysis
To generate specific probes for RNA gel blot analysis, a 570-bp fragment was excised from the cloned AtCAX2 by digestion with CfoI, and a 400-bp fragment was excised with NcoI and XhoI from the cloned At3g16470 cDNA. Fragments of 500 bp were PCR-amplified from A. thaliana cDNA for AtHMA3 and AtZIP6 using the primers 5′-GCAACGCTATGTATGCAGGA-3′ and 5′-TGCTGCTGACACAACAACAG-3′, and 5′-TTACCGGAAGCGTTTGAGTC-3′ and 5′-CCGCGAACATTAAGCACATA-3′, respectively (2 min at 94°C, 40 cycles of 45 sec at 94°C, 30 sec at 55°C, 1 min at 72°C, and 10 min at 72°C), cloned using the TOPO TA cloning kit (Invitrogen) according to the manufacturer's instructions and verified by sequencing. Radiolabeled probes were generated by random priming using 32P-dCTP according to the manufacturer's instructions (ReadyPrime™ labeling kit, Amersham Pharmacia Biotech, Little Chalfont, UK). For RNA gel blot analysis, 30 µg of total RNA were resolved on a formaldehyde MOPS gel, blotted onto a Hybond N+ membrane (Amersham Pharmacia), hybridized overnight at 60°C and washed in 5× SSC, 0.5% SDS at 60°C for 20 min and in 1× SSC, 0.5% SDS at 60°C for 10 min (Sambrook and Russel, 2001). Results are from one experiment representative of a total of two.
Total RNA was isolated from shoot and root tissues of plants using the Qiagen RNeasy plant RNA kit (Qiagen) and treated with DNAse to eliminate any genomic DNA (Dna-free™, Ambion). All kits were used according to the manufacturer's instructions. Synthesis of cDNA was carried out with poly-dT oligonucleotide primers using the Ambion RETROscript™ kit. Primers for real-time RT-PCR were designed using primer express software (v 2.0, Applied Biosystems, Foster City, CA, USA) for amplicon lengths of between 60 and 100 bp (Table S4, Supplementary Material). Primers were designed according to database genome sequence information for A. thaliana and according to the sequence of cloned cDNA fragments for A. halleri. Isoform specificity and A. thaliana/A. halleri species compatibility of primers were ensured in silico through sequence alignments, and experimentally by analysis of the dissociation curves and agarose gel electrophoresis of the PCR products. The primer pair for the constitutively expressed control gene, elongation factor (EF)1α (At5g60390, Vert et al., 2003), was chosen to span an intron. The absence of genomic DNA was confirmed by analysis of dissociation curves and agarose gel electrophoresis of the PCR products.
PCR reactions were performed in a 96-well plate with an Applied Biosystems ABI Prism 5700 Sequence Detection System, using SYBR Green to monitor cDNA amplification. Equal amounts of cDNA, corresponding to approximately 1 ng of mRNA, were used in each PCR reaction. In addition, a PCR reaction contained 10 µl of qPCR mastermix (Eurogentec, Liège, Belgium), 0.6 µl of SYBR Green and 5 pmol of forward and reverse primers (Eurogentec) in a total volume of 20 µl. The following standard thermal profile was used: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 60 sec at 60°C. Data were analyzed using geneamp 5700 sds software (v 1.3, Applied Biosystems). At a threshold value of the normalized reporter Rn of 0.1, the average threshold cycle (CT) value for EF1α was 16. Between different cDNAs, the mean CT for EF1α varied by less than 4.4% (±0.7 amplification cycles or less), a variability that was indistinguishable from the variability between technical replicates performed on identical cDNAs. For data evaluation, the difference was calculated between the CT of the target gene and the CT of EF1α for the respective template, thus normalizing target gene expression to EF1α expression.
Determination of enzyme activities and protein levels
Extraction of total protein and determination of OAS-TL enzyme activity was carried out according to Harms et al. (2000). For Western blotting, proteins were extracted, resolved, blotted, and detected according to the standard procedures (Lämmli, 1970; Towbin et al., 1979; ECL Western Blotting System, Amersham Biosciences, Little Chalfont, UK). For analysis of ferric chelate reductase activity, seedlings germinated on plates containing 0.5× MS salts, 0.8% agar (Invitrogen), and 1% sucrose for 10 days were transferred aseptically to plates containing different concentrations of Zn and composed as described above (Plant material, growth conditions, and experimental treatments), solidified with 0.8% agarose (Seakem LE, BMA, Rockland, ME, USA), for 4 days. Seedlings were transferred from plates into the assay buffer 3 h before the end of the light period, and ferric chelate reductase activity was determined immediately as described by Yi and Guerinot (1996).
Yeast complementation assays
Cloned cDNAs for AtCAX2, AhCAX2, AhNAS3, AhCDF1-3, and AhHMA3 were subcloned into the yeast expression vector pFL61 (Minet et al., 1992). The following yeast strains were used for complementation assays: YYA4 (Mat a, ycf1::loxP-KAN-loxP, yhl035::HIS3, ade2-1, his3-11, -15, leu2-3, -112, trp1-1, ura3-1, and can1-100; Gaedeke et al., 2001) and its parental wild-type strain W303-1A (Mat a, ade2-1, his3-11, -15, leu2-3, -112, trp1-1, ura3-1, and can1-100), and zrc1 cot1 (Mat a, zrc1::natMX3, cot1::kanMX4, his3Δ1, leu2Δ0, met15Δ0, and ura3Δ0; Brachmann et al., 1998) and its parental wild-type strain BY4741 (Mat a, his3Δ1, leu2Δ0, met15Δ0, and ura3Δ0; Goldstein and McCusker, 1999). Competent yeast cell preparation and transformation were carried out using the polyethyleneglycol method as described by Dohmen et al. (1991). Each mutant yeast strain was transformed with pFL61 empty vector (ev), pFL61-AtCAX2, pFL61-AhCAX2, pFL61-AhCDF1-3, pFL61-AhHMA3, and pFL61-AhNAS3. As controls, the respective wild-type strains were transformed with pFL61 (ev). Transformants were selected for and maintained on plates containing synthetic complete (SC) medium lacking uracil (Sherman et al., 1986). For experimental plates, a solid low-sulphate/phosphate (LSP) medium was used (modified after 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 as in SC, 2% (w/v) d-glucose, 1.5% (w/v) agarose (Seakem, BMA, Rockland, ME, USA). In experiments using the zrc1 cot1 mutant, LSP without added Zn was supplemented with various concentrations of ZnSO4 (1.4 µm for controls and between 50 µm and 2.5 mm for experimental treatments). In experiments using the YYA4 mutant, LSP with added basal Zn (1.4 µm) was supplemented with various concentrations of CdSO4 (25–300 µm). For drop assays, transformed yeast strains were grown overnight in 5 ml of liquid LSP to early stationary phase (OD600 of approximately 1.0). Cells were washed once with Zn-free LSP and diluted to equal OD600. Volumes of 2.5 µl of each of the serial dilutions were spotted onto experimental plates, and plates were incubated at 30°C for 7 days. All results are from one yeast transformant out of a total of at least three independent transformants.
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We thank Anne-Garlonn Desbrosses-Fonrouge and Christian Krach for mutant and vector constructions, and Astrid Schröder for technical support. We are grateful to Yasmin Mamnun, Department of Molecular Genetics, Vienna Biocenter, for providing the YYA4 double mutant and to Stephan Clemens, IPB Halle, for the cDNA sequences of AhNAS1 and AhNAS2. We thank Rüdiger Hell, University of Heidelberg, and Stanislav Kopriva, University of Freiburg, for kindly providing the anti-cytOAS-TL antibodies. We thank Michael Udvardi for critical reading of the manuscript and Lothar Willmitzer for his support. This work was supported by the German Federal Ministry of Education and Research, grant 0311877 (M.B., I.N.T., U.K.) and by the European Union, contract QLKT-CT-2000-00479 (L.K.).
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The following material is available from http://www.blackwellpublishing.com/products/journals/suppmat/TPJ/TPJ1959/TPJ1959sm.htm
Table S1 Concentrations of cations and sulfur in shoot tissues of A. halleri and A. thaliana exposed to different Zn concentrations in the root medium for 4 days
Table S2 Genes that are differentially expressed in shoots of A. thaliana in response to exposure to 100 µm Zn for 4 days, compared to low Zn
Table S3 Genes that are differentially expressed in shoots of A. halleri in response to exposure to 300 µm Zn for 4 days, compared to low Zn
Table S4 Nucleotide sequences of primers used for cloning candidate gene fragments from A. halleri and for real-time RT-PCR
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- Supporting Information
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Table S1. Concentrations of cations and sulphur in shoot tissues of A. halleri and A. thaliana exposed to different zinc concentrations in the root medium for 4 d. Four-week-old plants were supplied with a hydroponic culture solution containing 0, 100 or 300 μM added ZnSO4 for 4 days. Given are arithmetic means ± SD of values determined for four independent replicates from one experiment representative of a total of two experiments. For each replicate, tissues were pooled from three individual plants. Asterisks indicate that mean concentration is significantly different (P < 0.05) from the mean concentration in the respective low-zinc control treatment.
Table S2. Genes which are differentially expressed in shoots of A. thaliana after exposure to 100 μM Zn for 4 days (H), compared to low zinc (L). For each gene the "average ratio" corresponds to the signal intensity under high zinc conditions normalized to the signal intensity under low zinc conditions, averaged over three independent experiments.
Table S3. Genes which are differentially expressed in shoots of A. halleri after exposure to 300 μM Zn for 4 days (H), compared to low zinc (L).For each gene the "average ratio" corresponds to the signal intensity under high zinc conditions normalized to the signal intensity under low zinc conditions, averaged over three independent experiments.
Table S4. Nucleotide sequences of primers used for cloning candidate gene fragments from A. halleri and for real-time RT-PCR.
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