Environ. Sci. Technol. 2006, 40, 6792-6798
Genes Associated with Heavy Metal Tolerance and Accumulation in Zn/Cd Hyperaccumulator Arabidopsis halleri: A Genomic Survey with cDNA Microarray HUAI-CHIH CHIANG, JING-CHI LO, AND KUO-CHEN YEH* Agriculture Biotechnology Research Center, Academia Sinica, 128 Academia Road Section 2, Taipei, Taiwan 11529, ROC
To survive in variable soil conditions, plants possess homeostatic mechanisms to maintain a suitable concentration of essential heavy metal ions. Certain plants, inhabiting heavy metal-enriched or -contaminated soil, thus are named hyperaccumulators. Studying hyperaccumulators has great potential to provide information for phytoremediation. To better understand the hyperaccumulating mechanism, we used an Arabidopsis cDNA microarray to compare the gene expression of the Zn/Cd hyperaccumulator Arabidopsis halleri and a nonhyperaccumulator, Arabidopsis thaliana. By analyzing the expression of metal-chelators, antioxidationrelated genes, and transporters, we revealed a few novel molecular features. We found that metallothionein 2b and 3, APX and MDAR4 in the ascorbate-glutathione pathway, and certain metal transporters in P1B-type ATPase, ZIP, Nramp, and CDF families, are expressed at higher levels in A. halleri than in A. thaliana. We further validated that the enzymatic activity of ascorbate peroxidase and class III peroxidases are highly elevated in A. halleri. This observation positively correlates with the higher ability of A. halleri to detoxify H2O2 produced by cadmium and paraquat treatments. We thus suggest that higher peroxidase activities contribute to the heavy metal tolerance in A. halleri by alleviating the ROS damage. We have revealed genes that could be candidates for the future engineering of plants with large biomass for use in phytoremediation.
Introduction Extensive mining and industrial activities in human society have released high amounts of heavy metals to the environment. Some heavy metals, such as zinc, copper, and iron, are essential for the growth of organisms, but they become toxic in excess. Heavy metals such as cadmium or mercury are generally considered nonessential in plants and are potentially highly toxic to plants because of their reactivity with S and N atoms in amino acid side chains. Plants have developed different strategies that allow them to grow on soils rich in heavy metals. A regulated network of metal transport, chelating, trafficking, and sequestration activities controls the uptake, distribution, and detoxification of metal ions (1). * Corresponding author phone: 886-2-2789-8630; fax: 886-2-26515600; e-mail:
[email protected]. 6792
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Brooks introduced the term “hyperaccumulator” to describe plants that accumulate various essential and nonessential heavy metals in exceedingly high levels in the aboveground parts of the plant (2). About 400 different species belonging to a wide range of taxa have been described as hyperaccumulators (3, 4). The use of hyperaccumulators offers an attractive approach for cleaning up the contaminated field. However, the slow-growing and small shoot biomasses of many hyperaccumulators have restricted their direct application in large-scale decontamination of polluted soils. To circumvent this limitation, it has been proposed that more efficient phytoremediation could be achieved by expressing selected genes in more favorable plant species (5, 6). Two important traits, tolerance and hyperaccumulation of special heavy metals, are considered to be crucial and urgent to learn from hyperaccumulators. Hyperaccumulators, Thlaspi species and Arabidopsis halleri, have recently become model plants for the study of heavy metal tolerance and accumulation. Some of the genes involved in heavy metal homeostasis have been revealed from the molecular studies of these hyperaccumulators (7-12). Overexpression of homologs of these genes in transgenic plants enhanced the capability for heavy metal tolerance or accumulation (1317). Recently, the Terry group has shown that transgenic Indian mustard overexpressing adenosine triphosphate sulfurylase (APS), γ-glutamyl-cysteine synthetase (ECS), or glutathione synthetase (GS) could successfully take up and accumulate selenium in field conditions (18). These studies clearly suggested that the application of transgenic plants in phytoremediation is a beneficial and promising approach (5). For this approach to be practically feasible, it is crucial to have access to more molecular markers associated with heavy metal tolerance and accumulation. Although molecules related to heavy metal transport, chelation, and sequestration such as transporters and heavy metal chelators are good candidates for the function of tolerance and accumulation, knowledge about networks of global gene expression remains limited. For future genetic engineering of efficient hyperaccumulators with large biomass, studies of the coordinative gene expression in these hyperaccumulators are imperative. Genomics tools provide us with an opportunity to obtain an overview of the global molecular events involved in hyperaccumulation. The constitutive expression pattern of genes may contribute, at least in part, to molecular adaptation in hyperaccumulators. In recent studies of gene expression in A. halleri, Affymetrix AtGenome1 (8k) was employed (19, 20). However, the cross-species utilization of Affymetrix GeneChip may underestimate the number of genes overrepresented in A. halleri because of the sequence variation among orthologous genes in Arabidopsis thaliana and A. halleri. A 94% DNA sequence homology has been estimated between orthologs of genes in A. halleri and A. thaliana (20). The high homology between orthologs enables the efficient hybridization of orthologous genes from both species through cDNA microarray analysis (21). In this study, we used an AFGC Arabidopsis cDNA microarray containing 12 000 DNA elements (22) to compare the gene expression pattern of A. halleri and A. thaliana. We have paid special attention to genes related to heavy metal transport, chelation, and sequestration such as transporters and heavy metal chelators. The expression of genes related to antioxidation that may contribute to heavy metal tolerance was also examined. The findings should allow the identification of genes responsible for Zn/Cd tolerance and hyperaccumulation. We expect these 10.1021/es061432y CCC: $33.50
2006 American Chemical Society Published on Web 09/27/2006
TABLE 1. Heavy Metal-Related Transporters with Predominant Expression in A. halleri gene name (locus) ABC Transporter WBC11 Efflux Transporters P1B-ATPasee HMA3 (At4g30120) HMA4 (At2g19110) CDFe ZAT (At2g46800)
MTPa1 (At3g61940) MTPa2 (At3g58810) Uptake Transporters ZIPe IRT3 (At1g60960) ZIP3 (At2g32270) ZIP6 (At2g30080) ZIP9 (At4g33020) ZIP12 (At5g62160) Nrampe Nramp3 (At2g23150) Nramp5 (At4g18790)
expression (ratio)a Ah-G Ah-JF Ah-JT
hyperaccumulator with predominant expression
related reports
4.749
2.391
3.394
3.343 1.585
3.874 8.973
3.034 11.050
A. halleri T. caerulescens
(Becher et al., 2004)b (Bernard et al., 2004; Papoyan and Kochian, 2004)c
4.112
5.174
A. halleri T. caerulescens T. goesingense
(Becher et al., 2004; Drager et al., 2004)b (Assuncao et al., 2001)c (Persans et al., 2001; Kim et al., 2004)d
6.026 6.051
2.972 3.404
7.362 6.091
4.158 1.737 3.494 2.497 4.445
6.072 9.700 2.272 2.531 9.482
4.154 6.665 3.938 3.923 16.430
A. halleri A. halleri
(Becher et al., 2004)b (Weber et al., 2004)b
2.677 2.492
2.595 2.436
3.815 2.978
A. halleri
(Weber et al., 2004)b
10.65
a The ratio (A. halleri to A. thaliana) of gene expression data in each EST is averaged and presented with A. halleri collected from Germany (Ah-G); Fumuro, Japan (Ah-JF); and Mt. Tenjin, Japan (Ah-JT). The gene expression data are listed in the Supporting Information Table 2. b The study of Zn/Cd hyperaccumulator, A. halleri. c The study of Zn/Cd hyperaccumulator, T. caerulescens. d The study of Ni hyperaccumulator, T. goesingens. e Family.
genes will serve as molecular tools for future expression in transgenic plants for phytoremediation applications.
Materials and Methods Plant Material and Growth Conditions. Sterile A. halleri seeds were placed on a wet filter paper in a Petri dish and incubated at 4 °C in the dark for a month. The seeds were then transferred to media containing 0.25% phytagel (SigmaAldrich Inc., St. Louis, MO), 1/2 MS salt (GibcoBRL, Life Technologies, Rockville, MD), 500 mg/L of 2-morpholinoethanesulfonic acid (MES, J. T. Baker, Phillipsburg, NJ), and 1% sucrose (Sigma-Aldrich, St. Louis, MO), at pH 5.7 for germination. Both A. halleri and A. thaliana (ecotype Columbia-0) were germinated in the growth chamber with light intensity at 70 µmol m-2 s-1 under a 16-h light/8-h dark cycle at 22 °C. After growing to the stage of two leaves, seedlings were transferred to liquid culture in flasks containing 1/2 MS salt, 500 mg/L MES, and 1% sucrose, pH 5.7, and grown under continuous light at 30 µmol m-2 s-1, with gentle shaking at 22 °C. Growth media were replaced once every week. Seedlings of A. halleri and A. thaliana at the 10-leaf stage were harvested for microarray experiments. For propagation, A. halleri spp. gemmifera (Fumuro, Japan) seedlings were transferred to a magenta box (Sigma-Aldrich) filled with phytagel media containing 0.25% phytagel, 1/2 MS salt, 500 mg/L MES, and 1% sucrose, pH 5.7. A. halleri cuttings were cultivated in either the same phytagel medium as described above in magenta boxes or in soil for asexual propagation. A. halleri cuttings at the 6-leaf stage were transferred to new phytagel media for 2 weeks to induce rooting. The transferred cuttings were cultured in a growth chamber with light intensity at 70 µmol m-2 s-1, for a 16-h light/8-h dark cycle at 22 °C. The A. halleri plants were further cultured for 2 weeks before the treatments. The tissue cultured A. halleri spp. gemmifera exhibits Zn/Cd tolerance and Zn hyperaccumulation (Figures 1 and 2 of the Supporting Information) The tissue cultured A. halleri (Ah-JF) samples were used for following experiments. Microarray Experiments and Data Analysis. Total RNA of A. thaliana and A. halleri were extracted from whole plant
tissue with use of total RNA extraction kits (Qiagen, Valencia, CA). The quality and quantity of total RNA samples were analyzed by use of the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). The T7-oligo dT primer (5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG (T)24VN-3′) was used to synthesize first-strand cDNA from 1 µg of total RNA with use of the cDNA synthesis system (Roche Applied Science, Penzberg, Germany). The resulting cDNA was used for in vitro transcription to amplify the aRNA with use of a microarray RNA target synthesis kit (Roche Applied Science). Amino allyl-dUTP was used for postlabeling Cy5 in A. halleri samples and Cy3 in A. thaliana samples according to the manufacturer’s instructions (Amersham Bioscienses, Piscataway, NJ). The AFGC 12K cDNA microarray (Arabidopsis Functional Genomic Consortium, AFGC (22, 23)) was used for the following hybridization. The hybridization procedure was modified from the AFGC protocol by adding 50% formamide into the hybridization buffer and reducing the hybridization temperature to 42 °C. The hybridization signals were acquired with use of Axon GenePix 4000B and analyzed with use of GenePix 4.0 software (Axon Instruments, Union City, CA). For additional transporter examination, we designed gene specific primers to PCR out 207 transporter genomic-DNA and printed these on the slides for microarray experiments. Microarray data files were first imported into SMD, ASCC (http://bitora.sinica.edu.tw:7777/smd/MicroArray/SMD/), and data points flagged as bad were removed. The filtered data were normalized with use of “LOWESS Normalization” and analyzed with use of GeneSpring 6.1 software (Silicon Genetics, Redwood City, CA). Statistical analyses to examine the reproducibility of the three data sets were performed with use of Significance Analysis of Microarrays (SAM (24)). Only genes with an average of g2-fold up-regulation and a false discovery rate
