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First published online April 4, 2008; 10.1104/pp.107.110247 Plant Physiology 147:719-731 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
AtOSA1, a Member of the Abc1-Like Family, as a New Factor in Cadmium and Oxidative Stress Response1,[W],[OA]University of Zurich, Institute of Plant Biology, 8008 Zurich, Switzerland (M.J., D.S., M.S., E.M.); University of Bern, IPS-Plant Nutrition, 3012 Bern, Switzerland (D.S., S.C., L.B.); University of Geneva, Bioenergetics Laboratory, 1254 Jussy/Lullier, Switzerland (G.S.); University of Fribourg, Department of Biology-Plant Biology, 1700 Fribourg, Switzerland (L.B.); and Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61–704 Poznan, Poland (M.J.)
The analysis of gene expression in Arabidopsis (Arabidopsis thaliana) using cDNA microarrays and reverse transcription-polymerase chain reaction showed that AtOSA1 (A. thaliana oxidative stress-related Abc1-like protein) transcript levels are influenced by Cd2+ treatment. The comparison of protein sequences revealed that AtOSA1 belongs to the family of Abc1 proteins. Up to now, Abc1-like proteins have been identified in prokaryotes and in the mitochondria of eukaryotes. AtOSA1 is the first member of this family to be localized in the chloroplasts. However, despite sharing homology to the mitochondrial ABC1 of Saccharomyces cerevisiae, AtOSA1 was not able to complement yeast strains deleted in the endogenous ABC1 gene, thereby suggesting different function between AtOSA1 and the yeast ABC1. The atosa1-1 and atosa1-2 T-DNA insertion mutants were more affected than wild-type plants by Cd2+ and revealed an increased sensitivity toward oxidative stress (hydrogen peroxide) and high light. The mutants exhibited higher superoxide dismutase activities and differences in the expression of genes involved in the antioxidant pathway. In addition to the conserved Abc1 region in the AtOSA1 protein sequence, putative kinase domains were found. Protein kinase assays in gelo using myelin basic protein as a kinase substrate revealed that chloroplast envelope membrane fractions from the AtOSA1 mutant lacked a 70-kD phosphorylated protein compared to the wild type. Our data suggest that the chloroplast AtOSA1 protein is a new factor playing a role in the balance of oxidative stress.
Heavy metals like Cu2+, Zn2+, and Mn2+ in trace amounts play an essential role in many physiological processes but can be toxic if accumulated at high concentrations. In contrast, other heavy metals such as Cd2+ and Pb2+ have no biological functions and can be extremely toxic. Cadmium is a nonessential heavy metal widespread in the environment, being an important pollutant and known to be toxic for plants not only at the root level where Cd2+ is taken up but also in the aerial part. It can be transported from root to shoot via the xylem (Salt et al., 1995  ; Verret et al., 2004). Cadmium has been reported to interfere with micronutrient homeostasis (Clemens, 2001; Cobbett and Goldsbrough, 2002). It might replace Zn2+ in the active site of some enzymes, resulting in the inactivation of the enzymatic activity. Cadmium also strongly reacts with protein thiols, potentially inactivating the corresponding enzymes. To overcome this problem, cells produce excess quantities of chelating compounds containing thiols, such as small proteins called metallothioneins (Cobbett and Goldsbrough, 2002) or peptides like glutathione and phytochelatins (Clemens et al., 2002), which limit the damage induced by Cd2+. In addition, several types of transport systems have been shown to contribute to heavy metal resistance, including P-type ATPases and ABC transporters. They transport either free or ligand-bound heavy metals across biological membranes, extruding them into the apoplast or into the vacuole (Kim et al., 2007).
In response to heavy metals, diverse signal transduction pathways are activated, including mitogen-activated protein kinases, transcription factors, and stress-induced proteins (Jonak et al., 2004
The Abc1 protein family originates from the Saccharomyces cerevisiae ABC1 gene, which has been isolated as a suppressor of a cytochrome b mRNA translation defect (Bousquet et al., 1991 The data presented in this study suggest that the chloroplast AtOSA1 (A. thaliana oxidative stress-related Abc1-like protein), an Arabidopsis protein belonging to the Abc1 protein family, is implicated in the plant response to oxidative stress that can be generated by Cd2+, hydrogen peroxide (H2O2), and light. Our results show that AtOSA1 is functioning differently from Abc1; hence, the proteins of the Abc1 family can fulfill diverse functions.
AtOSA1 Transcript Levels Change in Response to Cadmium Exposure
The elucidation of the physiological functions of gene products that transcript levels are up- or down-regulated by Cd2+ in the model plant Arabidopsis is of major interest to understand response of plants to Cd2+. Several transcriptomic analyses have been performed using a subarray spotted with a large number of different cDNA sequences. cDNA microarrays from two independent experiments revealed that transcript levels of AtOSA1 (At5g64940) were down-regulated after the treatment with 0.2 µM CdCl2 for 21 d (Fig. 1A
). The microarray data were confirmed by reverse transcription (RT)-PCR using the same mRNA template used for the microarray analyses and RNA isolated from plants exposed to 0.5 and 1 µM CdCl2. After the 1 µM CdCl2 treatment, the transcript level of AtOSA1 was up-regulated (Fig. 1B). Additionally, a time-course experiment was carried out with 1-week-old plants exposed to 0.5 µM CdCl2 (Fig. 1C). The data showed that AtOSA1 was up-regulated in the leaves after 5 d of Cd2+ exposure, then stably expressed until day 12 and, finally, down-regulated. In the absence of Cd2+, an increase in the expression of AtOSA1 was found to be correlated with plant aging. The analysis of AtOSA1 transcript levels in the major plant organs of 6-week-old flowering plants revealed that this gene is expressed particularly in leaves, but also in flowers and slightly in stems (Fig. 1D). Under normal growth conditions, we found only a very low level of AtOSA1 transcripts in roots. Expression of AtOSA1 is in all likelihood related to the green tissues, because in this experiment the flowers were not dissected and still contained green sepals. However, we cannot exclude that AtOSA1 is also expressed in petals, stamen, and pistils. The data collected for the At5g64940 entry in the digital northern program Genevestigator (www.genevestigator.ethz.ch; Zimmermann et al., 2004
AtOSA1 Has Homology to the Abc1-Like Protein Family
The protein sequence of AtOSA1 possesses a conserved region of around 120 to 130 amino acids (according to the Conserved Domain Database for protein classification; Marchler-Bauer et al., 2005
The hydropathy plot made with TMpred (Hofmann and Stoffel, 1993 ) revealed the presence of two transmembrane spans within the C-terminal part of AtOSA1 (Supplemental Fig. S1). Similar results were obtained using the DAS transmembrane prediction server (http://www.sbc.su.se/ miklos/DAS/).
The members of Abc1 protein family have been identified in both pro- and eukaryota, for example, AarF from Escherichia coli (Macinga et al., 1998
In Arabidopsis, the sole ABC1-like protein (At4g01660) studied so far has been predicted to be localized in mitochondria and can partially restore the respiratory complex deficiency when expressed in S. cerevisiae (Cardazzo et al., 1998
Identification of the Abc1 domain within the AtOSA1 sequence prompted us to determine the functional homology of AtOSA1 with Abc1 proteins. For this purpose, we used the yeast S. cerevisiae deletion mutant W303-1A abc1::HIS3 deficient in the endogenous ABC1 activity (Hsieh et al., 2004 ). Deletion of the ABC1 gene in yeast disturbs the function of the respiratory chain and prevents growth of this mutant strain on media containing nonfermentable carbon sources such as glycerol (Bousquet et al., 1991). The expression of the entire AtOSA1 gene, including its targeting presequence in the W303-1A abc1::HIS3 strain, did not restore growth of this mutant on glycerol-containing media. Neither AtOSA1-EYFP nor AtOSA1 restored growth. As a control, the growth of the same strain was restored after complementation with yeast ABC1 gene (Fig. 4, A and B
), suggesting functional divergence between AtOSA1 and the yeast ABC1. We included the targeting presequence, because chloroplast proteins tend to be targeted to the mitochondria when expressed in fungal cells (Pfaller et al., 1989; Brink et al., 1994). We monitored the expression of AtOSA1-EYFP by confocal microscopy. The signal emitted by the strains expressing TPAtOSA1-YFP (Fig. 4C) was similar to that of the Rhodamine HexylB used for staining mitochondria (Fig. 4D), confirming localization of AtOSA1 in yeast mitochondria or mitochondrion in the presence of the chloroplast targeting presequence.
AtOSA1 Is Localized in the Chloroplast
Sequence analysis of the AtOSA1 protein with Target P (http://www.cbs.dtu.dk/services/TargetP/; Emanuelsson et al., 2000
Cadmium Effect on AtOSA1 Mutants The identification of mutants for AtOSA1 was possible from T-DNA insertion lines of the SALK Institute (SALK 045739) and GABI Kat (GABI, 132G06). To find the homozygote lines for both mutants, we screened the F3-F4 generation by PCR using RP, LP, and LB T-DNA primers designed by SIGnAL T-DNA Express (http://signal.salk.edu). The mutants were named atosa1-1 (SALK 045739) and atosa1-2 (GABI 132G06), respectively (Supplemental Fig. S2). In both mutants, T-DNA insertions are located toward the 3' end, thereby excluding the presence of a membrane anchor in case truncated transcripts are translated (Supplemental Fig. S3). Seedlings of both mutants accumulated less cadmium than the wild type at 10 and 20 µM CdCl2 (Fig. 6A ). Therefore, we investigated cadmium tolerance in AtOSA1 T-DNA insertion mutants in 1-week-old seedlings grown on bactoagar plates containing 20 µM CdCl2. Interestingly, roots of atosa1-1 and atosa1-2 mutant seedlings were longer than that of wild-type seedlings (Fig. 6B), thereby suggesting that root growth is less affected by cadmium toxicity in AtOSA1 than in the wild type. Under hydroponic culture conditions, leaves from wild-type Arabidopsis plants took up significantly more cadmium than atosa1-1, confirming the data obtained in seedlings (Fig. 6C). A similar picture could be observed in the autoradiograms from 4-week-old plants exposed to 0.04 MBq 109CdCl2 for 4 h (Fig. 6D), in which higher radioactivity was detected in wild-type plants. Surprisingly, despite the fact that the mutant plants took up less cadmium, they exhibited a marked chlorotic phenotype when exposed to 0.5 µM CdCl2 for 7 d (Fig. 6E).
Superoxide Dismutase Activity and Gene Expression in the AtOSA1 T-DNA Mutants Leaf chlorosis observed in the AtOSA1 T-DNA insertion mutants but not in wild-type plants after cadmium treatment prompted us to determine whether atosa1-1 is more sensitive to oxidative stress than wild type and whether some of the genes involved in reactive oxygen species (ROS) scavenging are regulated differently in mutants. A suitable approach to determine sensitivity to ROS is measurement of the activity of superoxide dismutase (SOD), an essential enzyme to attenuate plant oxidative stress. In the first approach, we determined the overall SOD activity in the leaves of wild type and atosa1-1 exposed or not to 1 µM CdCl2. The AtOSA1 mutant plants showed increased SOD activity compared to wild-type plants both in absence and presence of Cd2+. The effect was particularly marked in the absence of Cd2+ treatment (Fig. 7A ). To determine whether chloroplasts also exhibit an increased SOD activity, we isolated chloroplasts from plants grown in the presence or absence of Cd2+. The data showed that chloroplasts isolated from the AtOSA1 deletion mutant displayed a slight but consistently higher SOD activity compared to the wild-type chloroplasts. This effect was independent of whether the plants were exposed to Cd2+ or not (Fig. 7B).
Transcript levels of genes (AtAPX1, At1g07890; AtFSD1, At4g25100; AtFSD2, At5g51100) responding to oxidative stress (Kliebenstein et al., 1998 ; Ball et al., 2004) were investigated. AtAPX1, AtFSD1, AtFSD2, as well as AtOSA1 were found to be up-regulated in wild type after 1 µM cadmium treatment. In atosa1-1, only AtFSD2 was comparatively induced by cadmium. Interestingly, AtFSD1 was more expressed in atosa1.1 under control conditions when compared to wild type and induction of AtFSD2 was stronger in the mutant (Fig. 7C). H2O2, known as an ROS inducer, reduced the growth of the seedling roots more in the mutants than in the wild type (Fig. 8A ). The effect of H2O2 was also more pronounced in atosa1-1 leaves compared to the wild-type leaves. Indeed, after spraying leaves of wild-type and mutant plants with 300 µM H2O2 in 0.2% (v/v) Tween 20, we observed a rapid appearance of necrotic spots in the mutant, already 1 d after spraying (Fig. 8, B and C). In contrast, only a very few or no spots were found in the wild-type plants 4 d after spraying with H2O2 (Fig. 8B). No necrotic spots were detected when both the wild type and AtOSA1 T-DNA-inserted mutant were sprayed with 0.2% (v/v) Tween 20 only (data not shown).
The Effect of Light on AtOSA1 T-DNA-Inserted Mutants Light has a complex effect on AtOSA1 mutants depending on light intensities. At a low light regime (50 µmol m–2 s–1) for 8 h during 4 weeks, the shoot growth of atosa1-1 and atosa1-2 was significantly altered compared to the wild type (Fig. 9, A and B ). After an additional 4 weeks of growth in the same experimental conditions, leaf sizes were still different, and based on fresh weight, chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoid contents were higher in the mutants compared to the wild type (Fig. 9, C and D). Under a light regime of 120 to 150 µmol m–2 s–1 for 8 and 16 h, no visible phenotypes were found in the AtOSA1 mutants. Surprisingly, under 16 h of high light (350 µmol m–2 s–1), atosa1-1 exhibited a pale-green phenotype (Fig. 9E). In this case, the analyses of pigments showed slightly less chlorophyll and carotenoids in atosa1-1 compared to the wild type (Fig. 9F).
Analysis of photosynthetic activities in terms of net CO2 assimilation rate also revealed differences between Atosa1 mutants and the wild type depending on the light intensities. Under higher light intensities, mutants were more affected than the wild type (Fig. 10A ). Increasing light intensities from 50 to 150 µmol m–2 s–1 led to a reduction of AtOSA1, AtAPX1, AtFSD1, and AtFSD2 transcript levels in wild-type plants. A similar reduction of AtAPX1, AtFSD1, and AtFSD2 could be observed in the atosa1-1 mutant, but this effect was visible only under higher light intensities (Fig. 10B).
No significant differences were found by the electron microscopic analysis in chloroplast structures (stroma lamellae, grana stacks, and envelope membranes) between the atosa1-1 and wild type. In addition, the inductively coupled plasma mass spectrometry data showed that the content in essential metals and heavy metals was not changed by the AtOSA1 T-DNA insertion (data not shown).
Because possible connections between Abc1 proteins, electron transport, and ubiquinone (plastoquinone and phylloquinone) synthesis have been postulated (Poon et al., 2000
In addition to the Abc1 protein family, AtOSA1 contains motifs found in eukaryotic-type protein kinases. Therefore, we decided to examine protein kinase activities in the AtOSA1 mutant by in gelo phosphorylation assays using myelin basic protein as a substrate. Because we localized AtOSA1 in chloroplasts and the proteomic analysis identified AtOSA1 in the envelope fraction (Froehlich et al., 2003
We performed microarray chip analyses to identify genes up- and down-regulated in response to cadmium stress. Among the genes exhibiting an altered transcript level in response to Cd2+, we identified AtOSA1 (At5g64940) as a member of the Abc1 family. In Arabidopsis, 17 genes contain a typical Abc1 motif and hence constitute a small gene family. The sole Abc1 representative described so far in plants (At4g01660) is a homolog to the yeast ABC1 (Cardazzo et al., 1998 ). Both are localized in mitochondria (Bousquet et al., 1991; Cardazzo et al., 1998), in contrast to AtOSA1, which is targeted to the chloroplast and does not subcluster with them. AtOSA1 transcript level followed a complex kinetics in response to Cd2+ during dose-dependent and time-course experiments. In the absence of cadmium treatment, its expression in leaves increased during the life of Arabidopsis, and it has been reported that plant aging increases oxidative stress in chloroplasts (Munne-Bosch and Alegre, 2002). Two independent T-DNA insertion mutants, lacking functional AtOSA1, exhibited a complex behavior toward cadmium. Indeed, the seedling roots of AtOSA1 deletion mutants were less affected by Cd2+ than those of the wild-type plants, possibly due to a reduced Cd2+ uptake.
The increased cadmium tolerance of wild type compared to atosa1 mutants is very likely not supported by the direct binding of cadmium to AtOSA1. Indeed, AtOSA1 lacks of sequence motifs containing cysteins, involved in the binding of heavy metal ions (Zn2+, Cd2+, Pb2+, Co2+, Cu2+, Ag+, or Cu+), like CXXC and CPC. Such motifs have been found, for example, in members of the subclass of heavy metal-transporting P-type ATPases (P1B-type ATPases; Eren and Argüello, 2004
The pale phenotype of leaves was more pronounced in the case of mutant plants exposed even to a low dose of Cd2+ despite the fact that lack of AtOSA1 results in lower Cd2+ uptake rates in shoots. Such a chlorotic phenotype of leaves was not correlated with an elevated accumulation of cadmium and was also observed under high light conditions. This pale phenotype might be a consequence of Cd2+ toxic effect due to a modification of the cellular cadmium distribution (Ranieri et al., 2001
Although the mechanism of oxidative stress induction by Cd2+ is still obscure, Cd2+ can inhibit electron transfer and induces ROS formation (Wang et al., 2004 Besides cadmium, the AtOSA1 T-DNA-inserted mutants actually showed a phenotype illustrated by a reduced tolerance to H2O2 and light. At 150 µmol m–2 s–1, we observed the same transpiration rate for wild type, atosa1-1, and atosa1-2. Nevertheless, stomatal conductance and CO2 assimilation were higher in wild type than in mutants (data not shown). This observation suggests that, at this light intensity (150 µmol m–2s–1), transpiration occurs not only at the stomatal level but also directly through the epidermis. This hypothesis is supported by the experiments showing increased sensitivity of atosa1 toward H2O2 (Fig. 8B). Indeed, it is still possible that the AtOSA1 mutation also affects the epidermal cell wall and the cuticule. At low light intensity and period, atosa1 exhibited retardation in growth correlated with an increase in pigment production (Chla, Chlb, and carotenoids). Under higher light intensity and period, a pale-green phenotype correlated with a decrease in pigment contents when compared with the wild type. In addition, changes of light intensities influenced photosynthetic activities. These data suggest participation of the chloroplast AtOSA1 in light-generated stress (ROS) and pigment response.
Obtained results suggest that AtOSA1 mutants have a hypersensitivity to broad abiotic stresses, including photooxidative stress. RT-PCR analyses in atosa1 plants showed different behavior for transcripts of genes responding to oxidative stress. For instance, it was shown that AtFSD1 transcript in Arabidopsis is high at 60 µmol m–2 s–1 and then down-regulated under increasing light fluences (Kliebenstein et al., 1998
AtOSA1 is probably not directly induced by external oxidative stress but acts in a more complex manner, for example, as a part of a signal transduction pathway related to oxidative stress. Indeed, the Abc1 family has been described as a family of putative kinases (Leonard et al., 1998
Based on the phylogenetic tree, cell localization, and involvement in oxidative stress response, AtOSA1 is rather not a functional homolog of the yeast ABC1 and At4g01660 (Cardazzo et al., 1998
In Arabidopsis, AtOSA1 (At5g64940) clusters together with Abc1-like gene At3g07700. Interestingly, a homolog of this gene in B. juncea is also cadmium regulated and possibly localized in the chloroplast (Fusco et al., 2005
cDNA Microarrays
The mRNAs were isolated as described at http://www.unil/ibpv. Fluorescent labeling of cDNAs, hybridization on homemade DNA microarray slides spotted with ESTs and 3' end coding sequences (corresponding to putative ABC transporter proteins [124 of 127] and other protein families), and fluorescence analyses (Scanarray 4000) were performed as described by Bovet et al. (2005)
For semiquantitative RT-PCR, the housekeeping genes AtACT2 (actin; At3g18780) and AtS16 (At5g18380) were amplified using the primers actin2-S (5'-TGGAATCCACGAGACAACCTA-3') and actin2-AS (5'-TTCTGTGAACGATTCCTGGAC-3') and S16-S (GGCGACTCAACCAGCTACTGA) and S16-AS (CGGTAACTCTTCTGGTAACGA), respectively. For the ascorbate peroxidase 1 (AtAPX1) gene (At1g07890), Fe-SOD 1 (AtFSD1) gene (At4g25100), and Fe-SOD 2 (AtFSD2) gene (At5g51100), we designed the following primers: APX1-S (5'-GCATGGACATCAAACCCTCTA-3') and APX1-AS (5'-TTAAGCATCAGCAAACCCAAG-3'); FSD1-S (5'-GGAGGAAAACCATCAGGAGAG-3') and FSD1-AS (5'-TCCCAGACATCAATGGTAAGC-3'); and FSD2-S (5'-CCACTCCCTCGTCTCTCTTG-3') and FSD2-AS (5'-CCACCTCCAGGTTGGATAGA-3'). The primers for AtOSA1 were AtOSA1-S (5'-GACAGGCAATCACAAGCATTC-3') and AtOSA1-AS (5'-CGATTAGAACTTGGAGGCTGA-3'), respectively. For the selection of the atosa1-1 T-DNA insertion homozygote lines (SALK 045739), the primers were: RP (5'-AACGCGTTGAAATGCCCTCTC-3'), LP (5'-CTTGCTTCTTATCCATCGAGC-3'), and LB T-DNA (5'-GCGTGGACCGCTTGCTGCAACT-3'). For the selection of the atosa1-2 T-DNA insertion homozygote lines (GABI 132G06), the primers were: RP (5'-TTTGTTGGAGGCATTTTATGG-3'), LP (5'-GAATGCTTGTGATTGCCTGTC-3'), and LB T-DNA (5'-ATTTGGACGTGAATGTAGACA-3'). The primers for the verification of truncated transcript were: 1-S (5'-AATCGCCGGGATCTTCTTAC-3') and 1-AS (5'-TTGTCACTTCCTCCGTTTCC-3'), 2-S (5'-TTTGTTGGAGGCATTTTATGG-3') and 2-AS (5'-AACGCGTTGAAATGCCCTCTC-3'), and 3-S (5'-GACAGGCAATCACAAGCATTC-3') and 3-AS (5'-CGATTAGAACTTGGAGGCTGA-3'). The PCR reactions were performed in a final volume of 25 µL containing the following mixture: PCR buffer, 0.2 mM dNTPs, 0.5 µM of both 5' and 3' primers, 1 unit Taq DNA polymerase (Promega), and adjusted amounts of cDNA. DNA was isolated using NUCLOSPIN plant (Macherey-Nagel). Total RNA was purified from the plants using the RNeasy Plant Mini kit (Qiagen) and stored at –80°C following quantification by spectrophotometry. After DNAse treatment (DNase, RQ1, RNase free, Promega), cDNAs were prepared using Moloney murine leukemia virus reverse transcriptase, RNaseH minus, point mutant (Promega) as indicated by the manufacturer and stored at –20°C. cDNAs were diluted approximately 10 times for the PCR reaction. After denaturation at 95°C for 3 min, 35 PCR cycles (94°C for 45 s, 58°C for 45 s, and 72°C for 1 min) were run.
For complementation of W303-1A abc1::HIS3 (Hsieh et al., 2004
The AtOSA1 cDNA was PCR amplified (AtOSA1-S, 5'-TGCTACCGGTGCGGCCGCATGGCGACTTCTTCTTCTTCATCG-3'; and AtOSA1-AS, 5'-TCGTCCATGGAAGCTGTTCCAGTGATTAGTTTTTCC-3') to introduce appropriate restriction sites and cloned into AgeI/NcoI from vector pEYFP (BD Biosciences) to fuse it with EYFP. We used cDNA prepared as described above as a template for the PCR. The resulting AtOSA1-EYFP was cut off by NotI and cloned into vector pRT (Überlacker and Werr, 1996
Arabidopsis (Arabidopsis thaliana) suspension cell cultures were grown as described in Millar et al. (2001)
First, the mesophyll protoplasts were prepared from leaves according to the protocol described in Cosio et al. (2004)
Arabidopsis (Col-0) called above wild-type and AtOSA1 T-DNA-inserted mutant (SALK 045739, GABI 132G06) plants were grown on soil in a growth chamber (8-h-light period, 22°C; 16-h-dark period, 21°C; 70% relative humidity) and at a light intensity of 140 to 160 µmol m–2 s–1. For sterile growth after sterilization, the seeds (approximately 20) were placed on 0.8% (w/v) agar plates containing one-half-strength Murashige and Skoog (MS; Duchefa) or MAMI and 1% (w/v) Suc. MAMI medium is: KH2PO4 (200 mg/L); MgSO4.7H2O (187.5 mg/L); Ca(NO3).4H2O (79.25 mg/L); KNO3 (22 mg/L); Fe-EDDHA sequestren (17.5 mg/L); MnCl2.4H2O (48.75 µg/L); H3BO3 (76.25 µg/L); ZnSO4.7H2O (12.25 µg/L); CuSO4.5H2O (6.875 µg/L); NaNoO4.2H2O (12.5 µg/L); and Ni(NO3)2.6H2O (3.75 µg/L). The plates were stored at 4°C for 24 h for synchronization of seed germination and then placed vertically in the phytotron (25°C, 16 h light, and 70% humidity) at light intensity of 80 to 120 µmol m–2 s–1. For treatments, seeds were germinated and grown vertically on one-half-strength MS bactoagar plates in the presence or absence of 1, 10, or 20 µM CdCl2 or 1 mM H2O2 at 16 h light for 7 d. Hydroponic culture: Seeds were first germinated and grown vertically on one-half-strength MAMI bactoagar plates at 8 h light for 2 weeks. Seedlings were then transferred in one-half-strength MAMI liquid medium under the same growth conditions for 2 weeks. Plants were finally cultivated for an additional 3 weeks in the presence or absence of 0.2, 0.5, or 1 µM CdCl2 in one-half-strength MAMI. Cadmium was desorbed after 10 min of root incubation in 1 mM CaCl2 cold solution. The cadmium content was determined by atomic absorption spectroscopy (AAS) in shoots and roots.
The plants were root labeled with 0.04 MBq 109CdCl2 in one-eighth-strength MAMI for 4 h. After washing with cold distilled water, plants were grown in one-half-strength MS for an additional 3 d, dried, and subjected to autoradiography.
For the SOD activity measurements without any treatment, we used 4-week-old plants grown on soil. For measurement following a Cd2+ application, plants were germinated on 0.8% (w/v) agar plates containing one-half-strength MS (Duchefa) and 1% (w/v) Suc. The plates were stored at 4°C for 16 h for synchronization of seed germination, then placed vertically in the phytotron (22°C, 8 h light, and 70% humidity). Two-week-old seedlings were transferred to liquid medium and cultivated under hydroponic conditions for 3 weeks on MAMI medium. CdCl2 was added to the medium to a final concentration of 1 µM and the samples were taken 24 h later. The activity of SOD was measured as described by Hacisalihoglu et al. (2003)
Photosynthetic gas exchange measurements were performed on attached leaves before plants flowered using an open infrared gas analyzer system (CIRAS-1; PP-Systems). Measurements were made on plants grown at 8 h light at a photosynthetic photon flux density of 50, 100, or 150 µmol m–2 s–1, and CO2 concentration of 350 µmol. Leaf temperature was adjusted to the desired level using the internal heating/cooling system of the analyzer.
The method for detecting protein kinases in gelo was adapted from Mori and Muto (1997)
The plants were grown at 8 h light (50 µmol m–2 s–1) for 8 weeks. From these plants, leaf samples (50 mg) were collected and analyzed for the content of Chla and Chlb, as well as carotenoids (n = 10). Plants were grown at 16 h light (350 µmol m–2 s–1) for 5 weeks. From these plants, leaf samples (50 mg) were collected and analyzed for the content of Chla and Chlb, as well as carotenoids. Pigments were measured using the method described by Pruzinska et al. (2005)
Each value represents the mean of n replicates. Error bars represent SE. Significant differences from wild type as determined by Student's t test are indicated as follows: *, P < 0.1; **, P < 0.05; and ***, P < 0.001, respectively.
The following materials are available in the online version of this article.
We thank Prof. F. Kessler for providing us with Tic110-GFP construct, and E. Hsieh for the kind gift of W303-1A abc1::HIS3 and W303-1A strains as well as p3HN4 plasmid. We acknowledge Amélie Fragnière, Regis Mark, and Esther Vogt for technical assistance; Dr. Daniel Studer, University of Bern, for electron microscopy; Prof. Detlef Günther, Swiss Federal Institute of Technology, for inductively coupled plasma mass spectrometry measurements; Dr. Stefan Hortensteiner, University of Bern, for phylogenetic tree; and Prof. Urs Feller, University of Bern, and Dr. Sonia Plaza, University of Fribourg, for AAS measurements. Received October 1, 2007; accepted March 20, 2008; published April 4, 2008.
1 This work was supported by the Bundesamt für Bildung und Wissenschaft (grant nos. 01.0599 and EU HPRNT–CT–2002–00269 to E.M. and to L.B. under COST Action E28 [Genosylva: European Forest Genomic Network] and COST 859 [Phytotechnologies to promote sustainable land use management and improve food chain safety]). M.J. was a Marie Curie fellow (HPRN–CT–2002–00269). 
2 These authors contributed equally to the article.
3 Present address: Philip Morris Products S.A., PMI Research & Development, Quai Jeanrenaud 56, 2000 Neuchâtel, Switzerland. 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: Lucien Bovet (lucien.bovet{at}pmintl.com).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.110247 * Corresponding author; e-mail lucien.bovet{at}pmintl.com.
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ABSTRACT
RESULTS
-32P]ATP (AA0068; Amersham-Bioscience) in 5 mL of 50 mM MOPS-KOH, pH 7.6, 10 mM MgCl2, 0.5 mM CaCl2 for 3 h following a 45-min preincubation in the same buffer without the labeled ATP. The gel was then rapidly washed with deionized water and incubated in 100 mL of 50 mM MOPS-KOH, pH 7.6, containing 10 g of a strong basic anion exchanger (Amberlit IRA-410; Sigma) for 3 h. The removal of unbound 32P was terminated by incubation of the gel in 50 mM MOPS-KOH, pH 7.6, supplemented with 1% (w/v) sodium pyrophosphate for 3 h. The polypeptides were then fixed in the gel in 10% (v/v) 2-propanol, 5% (v/v) acetic acid, and 1% (w/v) sodium pyrophosphate. The gel was finally dried and subjected to autoradiography.