Genes Induced in Response to Mercury-Ion-Exposure in Heavy

Hg ions readily accumulate in plants and strongly interact with sulfhydryl groups of vital enzymes and proteins in apoplast of root cells. Hg2+ binds ...
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Environ. Sci. Technol. 2009, 43, 843–850

Genes Induced in Response to Mercury-Ion-Exposure in Heavy Metal Hyperaccumulator Sesbania drummondii P. VENKATACHALAM,† A. K. SRIVASTAVA,‡ K . G . R A G H O T H A M A , † A N D S . V . S A H I * ,‡ Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907, and Department of Biology, Western Kentucky University, Bowling Green, Kentucky 42101-1080

Received May 12, 2008. Revised manuscript received September 8, 2008. Accepted September 15, 2008.

Sesbania drummondii plants have been recognized as a potential mercury (Hg) hyperaccumulator. To identify genes modulated by Hg, two suppressive subtraction hybridization (SSH) cDNA libraries (forward and reverse) were constructed. A total of 348 differentially expressed clones were isolated and 95 of them were identified as Hg responsive. Reverse Northern results showed that 31 clones from forward library were down-regulated and 64 clones from reverse library were upregulated in Hg-treated plants. Sixty-seven of them showed high homology to genes with known or putative function, and 28 were uncharacterized genes. Two full-length cDNAs coding for a putative metallothionein type 2 protein (SdMT2) and an auxinresponsiveprotein(SdARP)wereisolatedandcharacterized. The expression levels of SdMT2 and SdARP increased 3and 5-fold, respectively. Results suggest that up-regulated expression of SdARP may contribute to the survival of Sesbania plants under mercury stress, whereas SdMT2 is likely to be involved in alleviation of Hg toxicity. The possible correlation between gene expression and heavy metal tolerance of Sesbania plants is discussed.

Introduction Heavy metals are dangerous environmental pollutants and many of them are toxic even at very low concentrations. Mercury (mercury ions-Hg2+) contamination is one of the world’s most serious environmental problems (1, 2). It is released into the environment as a result of gold mining, industrial pollution, medical wastes, burning fossil fuels, and electronics. Once in the environment, these forms of Hg are converted by sulfate reducing bacteria to the extremely toxic compound methylmercury, which bioaccumulates in the food chain (2). In general, heavy metals cannot be destroyed biologically but are only transformed from one oxidation state or organic complex to another (3). Several of plant species have developed mechanisms to accumulate one or more heavy metals such as Hg, Pb, Cd, Cr, Ag, and Se from soil and water without leading to toxicity (4). * Corresponding author phone: 270-745-6012; fax: 270-745-6856; e-mail: [email protected]. † Purdue University. ‡ Western Kentucky University. 10.1021/es801304n CCC: $40.75

Published on Web 01/05/2009

 2009 American Chemical Society

In general, plants cannot detoxify methylmercury, and plant tolerance to mercury is quite low. Mercury accumulation has been studied in both nontransformed and transformed plant species including Arabidopsis (5-9). The toxic methylmercury can be catalyzed into volatile mercury by overexpression of MerA and MerB genes together in Arabidopsis transgenic plants (2, 8, 9). Overexpression of AtATM3 (Arabidopsis thaliana ABC transporter) resulted in an increased tolerance to both Cd and Pb, whereas the gene knockout rendered the plants more sensitive to the heavy metals (10). Similarly, heavy metal ATPase of Thlaspi caerulescens (TcHMA4) increased heavy metal tolerance in complemented yeast by increasing the efflux of Cd, Cu, Zn, and Pb (11). Among different forms of mercury, Hg2+ is highly watersoluble and reactive. Hg ions readily accumulate in plants and strongly interact with sulfhydryl groups of vital enzymes and proteins in apoplast of root cells. Hg2+ binds to water channel proteins of root cells causing a physical obstruction to water flow and consequently affect transpiration (12). Hg can induce expression of genes encoding superoxide dismutase, peroxidase, and catalase (6). Though Hg belongs to the group of transition metals, which are well-known to induce oxidative stress via Fenton-type reaction, the molecular mechanisms of Hg-induced oxidative damage and tolerance in plants have not been fully understood (12). There is a significant interest in identification of heavy metal hyperaccumulator plants worldwide (4, 13). Even though, different plant species have evolved mechanisms to hyperaccumulate heavy metals (14), none of them has been identified as mercury hyperaccumulator (15). In particular, Sesbania drummondii is characterized by its rapid growth, high biomass, and an appreciable capacity to take up various toxic metals including Hg, therefore, it is recognized as a potential hyperaccumulator (16). Interestingly, at 100 mg L-1 HgCl2, Sesbania accumulated 998 mg Hg kg-1 dry weight of shoots whereas roots accumulated 41 400 mg kg-1 (17). To improve phytoremediation of heavy metal polluted sites, a detailed molecular investigation on mercury ion induced/ regulated genes and their role in hyperaccumulation and/or tolerance in S. drummondii is needed. Molecular tools such as suppressive subtraction hybridization (SSH), mRNA differential display and cDNA-AFLP have been successfully used to study the heavy metal regulated gene expression in various plants such as Arabidopsis, Brassica, Datura, and Sesbania (6, 11, 13, 18-20). SSH is a powerful technique that enables combining the suppression PCR technique with the normalization and subtraction steps in a single reaction (21). In the present study, we employed SSH technique to identify genes that are induced by mercury ion in Sesbania drummondii. Differentially expressed genes were further validated by Northern blot analysis and a set of mercury ion responsive genes were cloned and characterized to understand the tolerance and accumulation mechanisms in this species.

Materials and Methods Plant Growth Conditions and Mercury Treatment. Metaltolerant S. drummondii seeds were surface sterilized and germinated as described earlier (13). Seven-day-old seedlings were grown hydroponically in half-strength Hoagland’s nutrient solution containing 50 mg L-1 HgCl2 (Sigma) while medium without HgCl2 served as control. After 14 days of treatment, seedlings were removed and thoroughly washed with deionized water and 5 mM EDTA to remove Hg deposition on surface. Root and shoot tissues were then VOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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separated, quickly frozen in liquid nitrogen, and stored at -80 °C for RNA extraction, while rest were used for other studies. Growth characteristics such as shoot and root lengths, dry weight (biomass) of roots and shoots were measured. To determine biomass, six seedlings from Hg treatment as well as control were weighed in triplicate and dried in a forced air oven at 68 °C for 2 days. Total RNA Extraction and Poly (A)+ RNA Purification. Total RNA was isolated from both shoot and root tissues of Hg-treated as well as control plants using Trizol reagent. The RNA integrity was checked on 1.2% (w v-1) agarose/formaldehyde gel. Poly (A)+ RNA was purified from 5 µg of total RNA using Poly A mRNA isolation kit (Promega, MD). Construction of SSH Libraries. Two suppressive subtractive hybridization cDNA libraries were constructed with the PCR select cDNA subtraction kit (BD Biosciences, U.S.). For forward subtraction, mRNA from a mixture of shoot and root tissues from control and Hg-treated plants were used as a “tester” and as a “driver”, respectively. A reverse subtracted cDNA pool was made with Hg-treated plant mRNA as tester and control plant mRNA as driver. cDNA synthesis, digestion, adaptor ligation, subtractive hybridizations, and PCR amplification were essentially carried out according to the protocol described earlier (13). The subtracted and PCR amplified products were cloned into the TOPO-pCR 4 plasmid vector by using the TA cloning kit (Invitrogen, CA). The ligated products were then transferred into chemically competent E. coli (TOPO 10F) cells to generate SSH libraries, then incubated at 37 °C overnight to obtain a subtracted expressed sequence tag (EST) bank. Individual bacterial colonies were stored at -80 °C as glycerol stocks for screening and sequencing. Amplification of cDNA Inserts and Differential Screening. The cDNA inserts were amplified by PCR with T3 and T7 sequencing primers to check the presence and size of individual inserts. PCR reactions (20 µL) contained 13 µL distilled water, 1 µL of each T3 and T7 primers (1.5 µM each), 2 µL of 10 × buffer, 2 µL of dNTPs mix (2.5 mM each), 1 µL of Taq DNA polymerase and an aliquot of each bacterial clone was added as DNA template. Reaction samples were first denatured at 94 °C for 4 min followed by 30 cycles at 94 °C for 1 min, 50 °C for 1.5 min and 72 °C for 2 min with a final extension at 72 °C for 7 min. PCR products were analyzed by 1.5% (w v-1) agarose gel electrophoresis. cDNA fragments g200bp were denatured with 0.6 N NaOH (1:1 ratio) and 2 µL of each fragment was spotted onto nylon membranes. Two identical membranes with cDNA inserts spotted in duplicate were prepared. After air drying the membranes, DNA was UV cross-linked and stored at -20 °C for later use. To identify differentially expressed genes, reverse Northern blot analysis was performed with radio labeled (32P) cDNA probes. The tester and driver cDNAs from unsubtracted library were used in separate labeling reactions. Blots were hybridized and washed according to standard procedures (22). Membranes were exposed to X-ray film (X-Omat, Kodak) at -80 °C for 24 h. The fragments that hybridized only with the labeled tester cDNA or showed g2.5 fold higher signals on these membranes compared to the signals on the membrane hybridized with the labeled driver cDNA were selected for sequencing. DNA Sequencing, Analysis and Accession Numbers. Differentially expressed clones identified by dot-blot screening of forward and reverse SSH libraries were sequenced at the Purdue University Genome sequencing facility. Each sequence was edited to remove the adaptor-primer sequence contamination. Unique ESTs were selected to identify their putative functions and annotated on the basis of the existing annotation of nonredundant databases at the NCBI, EMBL, TAIR, TIGR using BLASTN and BLASTX algorithm (http:// 844

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FIGURE 1. Differential screening of cDNA clones from the Sesbania SSH libraries (Forward-untreated control plant specific library and Reverse Hg-treated plant specific library). Duplicate dot-blots were prepared and the membranes were hybridized with radiolabeled probes. The spots differing in their intensity between membranes were classified as either up- (circled by continuous line) or down-regulated (circled by dotted line). (a) An array representing 100 clones from forward SSH library hybridized with unsubtracted tester cDNA probe (control plant) (b) An array representing 100 clones from forward SSH library hybridized with unsubtracted driver cDNA probe (Hg-treated plant) (c) An array representing 100 clones from reverse SSH library hybridized with unsubtracted tester cDNA probe (Hg-treated plant). Arrow indicates the clones: SdMT2 (right diagonal arrow) and SdARP (up arrow) (d) An array representing 100 clones from reverse SSH library hybridized with unsubtracted driver cDNA probe (control plant). www.ncbi.nlm.nih.gov/BLAST). The cDNAs were named according to homologous sequences in the database and cDNAs with BLAST scores 50 bp) were designed as having “no similarity”. Functional classification of the ESTs was performed according to the functional categories of Arabisopsis, Oryza, and Medicago proteins. Development of cDNA Library. Sesbania seedlings were subjected to Hg treatment as described above and total RNA was extracted for cDNA library construction according to manufacturer’s instructions (Stratagene, U.S.). Briefly, cDNAs were synthesized from 5 µg of Poly (A)+ RNA and EcoRI adapters were ligated to blunt ended cDNAs. After XhoI digestion and size separation on Sepharose CL-2B column, cDNAs were ligated to the EcoRI-XhoI site of the Uni-ZAP XR express vector followed by packaging. The library prepared in lambda phage vector was amplified to a stable stock as described by the standard protocol (22). Isolation and Cloning of Full-Length cDNAs Specifically Induced by Hg. Based on partial EST sequences obtained in the SSH library, primers were designed for PCR amplification of SdMT2 (Forward, 5′-GAAAGTTCTTCCTTGGCATCA-3′ and Reverse, 5′-TCGAGCGGCCGCCGGGGCCA-3′) and SdARP (Forward, 5′-GAGCCACAGGTATGGTTTGTTC-3′ and Reverse, 5′-GATTGCAAGCTTCAGACTCTG-3′) genes. cDNA library stock was used as template for amplification of these genes in two separate PCR reactions. The first PCR reaction was performed with forward gene specific primer and T3 sequence primer as reverse and second PCR reaction was carried out with reverse gene specific primer and T7 sequence primer

as forward to amplify partial cDNA sequences. PCR amplified partial cDNA fragments were cloned into pGEM-T vector and sequenced. Using the cloned partial sequence information a set of gene specific primers (SdMT2 forward: 5′-GAGCGGCCGCCGGGGCCA-3′ and reverse: 5′-GGAGTAAAACAATTGAAT-3′andSdARP forward:5′-AGCGTGGTCGCGGCCGAG-3′ and reverse: 5′-TCGAGCGGCCGCCCGGGC3′) were designed for isolation of full-length cDNA. Amplicons were cloned and sequence characterized. Alignments of deduced amino acid sequences were carried out using the ClustalW program and the phylogenetic analysis was performed using the TreeView program. Northern Hybridization Analysis. Based on the sequence information, 7 and 10 gene contigs from forward and reverse SSH libraries respectively were selected for synthesizing radiolabeled probe to perform Northern hybridization analysis. The purified cDNA fragments were used for synthesis of radiolabeled probes using [R-32P]dCTP (specific activity >3000 Ci mol-1 10 µCi µL-1) as described above. Blot hybridization, and washing conditions are essentially same as described earlier (13, 33). Blots were then exposed to X-ray film (X-Omat AR, Kodak) for signal detection at -80 °C.

Results Plant Growth Characteristics. Sesbania drummondii seedlings grown hydroponically for 14 days with 50 mg L-1 HgCl2, showed inhibition of root and shoot growth, with shoots being affected more by Hg than roots (Supporting Information (SI) Figure S1). Control plants exhibited increased dry biomass in both shoots and roots as compared to Hg-treated one (SI Figure S1a and b). After 14 days of Hg treatment, plants displayed toxicity symptoms such as chlorosis and drying at edges, whereas control plant did not show such symptoms. Identification of Hg Responsive Genes by Differential Screening of SSH Library. The “forward” subtraction hybridizations facilitated the identification of the clones downregulated in Hg-treated Sesbania plants, while the “reverse” set enabled identification of clones up-regulated in Hgtreated plants. To increase the specificity of Hg-responsive cDNAs prior to sequencing, differential screening steps were conducted. PCR amplified cDNA inserts of 348 clones from both the subtractive libraries were spotted onto nylon membranes. The forward SSH library blots displayed strong hybridization signals for many clones with the probe from control plant (Figure 1a), whereas most of them did not bind to the cDNA probe from Hg-treated one and those clones were selected as differentially regulated cDNAs specific to control plants (Figure 1b). On SSH blot from reverse subtraction library, the cDNA probe from Hg-treated plants was strongly annealed to many clones that were considered as specific cDNAs up-regulated by Hg-treatment (Figure 1c), whereas the probe from control plant hybridized weakly with few clones (Figure 1d). Reverse Northern blot results demonstrating g2.5-fold differences in transcript abundance of genes in subtracted library were selected for further analysis. Among 115 clones identified following differential screening in the forward SSH library, 94 clones were selected for sequencing. In the case of reverse SSH library, a total of 233 clones were obtained and 211 clones displaying differential expression specific to Hg-treated plant were sequenced. DNA sequence analysis indicated that 95 clones represent nonredundant cDNA inserts from both the libraries. Further bioinformatics analysis resulted in the identification of 31 clones from forward SSH library (SI Table 1) and 64 clones from reverse SSH library (SI Table 2) as consistently up-regulated genes. Functional Classification of Hg Responsive ESTs. The EST sequences were identified by sequence similarity searches (BLASTN and BLASTX) in different databases

FIGURE 2. Functional categories of Hg responsive genes isolated from SSH libraries. Sequences having homology to unknown protein sequences were classified as unknown functional genes. Sequences with no similarity to known DNA/gene/protein sequences in the database were classified as no homology. The bars represent the number of gene transcripts: (a) Down-regulated in Hg-treated plants (forward SSH library); (b) Up-regulated in Hg-treated plants (reverse SSH library). (nonredundant sequences in NCBI, EST sequences in NCBI, Medicago, and rice EST database at TIGR, Arabidopsis database at TAIR). This analysis revealed that 94 ESTs exhibited significant protein homology to previously identified or putative proteins in rice, wheat, barley, maize, Arabidopsis, and other plants. There was one novel ESTs with no significant homology to mRNAs or proteins deposited in the database. Data presented in SI Tables 1 and 2 show the functional classification of the differentially expressed genes identified from forward and reverse SSH libraries. There were differences in the distribution of expressed genes among functional classes between the control and Hg-treated plants (Figure 2a and b). Of the 95 mercury modulated genes, 67 of them have known or putative function which could be grouped into seven major functional categories: stress/ defense (18 clones), photosynthesis (10 clones), cellular metabolism and signaling (21 clones), transcription factors (6 clones), cell division and growth related proteins (6 clones), transporters (1 clone), protein synthesis/degradation (5 clones), unclassified proteins (27 clones), and finally sequences with no identity (1 clone). The sequences obtained have been deposited in the GenBank database (http:// www.ncbi.nlm.nih.gov) under the accession numbers given in SI Tables 1 and 2. Confirmation of Differentially Expressed Hg Responsive Genes by Northern Blot Analysis. Northern blot analysis was carried out to validate the up- and down-regulation evidenced in the cDNA macroarray analysis. Of the 95 modulated clones, expression of 17 gene contigs representing different functional categories selected randomly was examined using independently prepared RNA from shoot and root tissues of seedlings grown in the absence or presence of HgCl2. Northern analysis of genes corresponding to various ESTs with differential abundance in the SSH data sets is summarized in Figure 3. In general, the results of RNA gelVOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Differential expression pattern of (a) down- and (b) up-regulated genes in Hg-treated plants. Total RNA (15 µg) extracted from shoots and roots of Sesbania seedlings was subjected to Northern blot analysis. The candidate genes indicated on right side of the figures were selected to prepare 32 P labeled probes. Ethidium bromide stained rRNAs indicate uniform loading of samples. blot were consistent with the expression data obtained by macroarray (dot blot) analysis. The expression patterns of five genes (metal ion binding gene, putative phophatase, methionine overaccumulation 1 mRNA, heme activator protein 2 (HAP2) transcription factor, metallothionein-like gene, glutathione S-transferase, and cysteine proteinase precursor mRNA) from forward library were up-regulated in untreated plants compared with Hg-treated plants (Figure 3a). Abundance of transcripts for six genes namely oxidase reductase, β-1,3 glucanase, WRKY transcription factor, metallothionein type 2, nitrate transporter, and auxin repressed protein were strongly induced in both shoot and root tissues followed by Hg-exposure (Figure 3b). These genes may be considered as Hg-specific genes. Similarly accumulation of mRNA transcripts for translationally controlled tumor protein (TCTP), heat-shock protein (HSP) 70 kDa, Rieske FeS protein, and glyceraldehyde-3-phosphate dehydrogenase A were upregulated in Hg-treated plants (Figure 3b). Cloning and Characterization of Hg Responsive Genes. Two genes which were induced in both shoot and root tissues exposed to Hg coding for metallothionein type 2 (SdMT2) and auxin repressed protein (SdARP) were selected for isolation of full-length cDNAs and further characterization 846

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(Figures 4-6). The nucleotide sequence of SdMT2 and the putative amino acid sequence of the SdMT2 protein were depicted in Figure 4a (GenBank accession No. EF564345). The alignment suggested that the SdMT2 was a full length cDNA including a 73 bp 5′ noncoding domain, a 237 bp ORF (termination codon excluded), and a 201 bp 3′ region. The coding region of SdMT2 represented a putative 79 amino acid protein with a molecular weight of 7.8 kDa and a theoretical pI value is 5.1. Notably, 14 of the 79 residues in the SdMT2 sequence were cysteines, comprising 17% of the total amino acids. The positions of the 14 cysteine residues at the N- and C-terminal domains were completely conserved (CCXXXCXCXXXCXCXXXCXXC and CXCXXXCXCXXCXC) with those in the plant type-2 MTs, pointing to the functional importance of these amino acid residues. This MT protein is therefore a member of the type 2 MTs and it contains a metallothionein conserved domain in C-terminal half (KMYPDLSYAE LVMGVAPVKV). The deduced alignment of the putative proteins of SdMT2 with MT type 2 reported for other plants is presented in Figure 4b. The N- and C-terminus of the SdMT2 protein shared a high amino acid homology with other plants MT type 2 proteins; viz., 89% with Vigna, 86% with Arachis and Glycine, and 82% with Cicer and Lablab. Phylogenetic analysis showed that SdMT2 is grouped with Arachis spp. in the leguminoceae family in the type-2 MT clade (Figure 4c). SdARP cDNA is 632 bp in size containing 348 bp ORF (termination codon excluded), a 24 bp 5′-flanking sequence, and a 260 bp 3′-flanking sequence (GenBank accession no. EF564346) (Figure 5a). The predicted protein consists of 115 amino acids with a calculated molecular mass of 12.7 kDa and a theoretical pI value of 9.7. Multiple alignments of the deduced amino acid sequences of the orthologs revealed the presence of highly conserved amino acid domains at both the N- and C-terminal ends and showed 82%, 74%, and 73% identity to the ARP sequences from Robinia, Arachis, and Malus and Fragaria, respectively (Figure 5b). To examine the evolutionary relatedness of the five putative ARP proteins, an unrooted phylogenetic tree with the deduced amino acid sequences was generated (Figure 5c). The tree showed that plant ARPs separated into two distinct clades. To examine the effect of length of Hg treatment on expression of MT2 and ARP, plants were grown hydroponically and treated with 50 mg L-l HgCl2 for 10 days, and total RNA isolated from shoots and roots was used for Northern analysis. Abundance of mRNA transcripts for SdMT2 and SdARP was 3- and 5-fold higher than that of control (Figure 6). SdMT2 and SdARP were preferentially expressed in both shoots and roots.

Discussion Metal hyperaccumulator plants have evolved mechanism of hyper-tolerance and thus are able to accumulate various heavy metals such as Hg, Pb, Ni, Cd, Cs, or Zn (11, 23). Sesbania drummondii, a fast-growing plant with high biomass is known to accumulate high levels of Hg in both roots and shoots (17). Though, it has much higher tolerance to Hg, the molecular basis of Hg tolerance and accumulation of this plant is not known. The main goal of this study was to identify and isolate genes that might play a role in the hyperaccumulation mechanism of S. drummondii by SSH analysis. This is the first description on global analysis of the transcriptional responses of Sesbania plants to Hg ion-exposure. S. drummondii grown hydroponically in the presence of Hg for 14 days showed certain degree of toxicity than that of untreated control. Such phytotoxic effects, may be consequence of general disturbance of physiological and biochemical status of the plant due to oxidative stress (6, 18). The earlier study indicated that mercury at 50 mg L-1 had no or little toxic effect on photosynthesis of Sesbania (17). In contrast, Hg exposures to plants have been shown to affect photosynthesis

FIGURE 4. (a) The cDNA sequence (514 bp) and deduced amino acids (240 bp ORF) of Sesbania drummondii Metallothioneins type 2 (SdMT2). (b) Comparison of the amino acid sequences of MT2 from different plants. The numbering starts with the predicted N-terminal amino acids of MT2 Sesbania and with the N-terminal amino acids of other species. Identical residues between the MT2s are presented as asterisks. The positions of the 14 cysteine residues at the N- and C-terminal domains were completely conserved (CCXXXCXCXXXCXCXXXCXXC and CXCXXXCXCXXCXC) with those in the plant type-2 MTs (boxed). (c) Phylogenetic tree of genes in the MT2 family. Analysis was performed using the CLUSTALW using default parameters. of spinach (24). Similar observations were also made in plants treated with CsCl (18) and Cd (25). The Hg content analysis revealed that S. drummondii grown in hydroponic solution accumulated more Hg in roots than shoots (17). Identification of Hg Modulated Genes by SSH Analysis. A number of genes playing potential roles in Hg tolerance were identified in this study. We screened approximately 348 clones obtained from two SSH libraries by DNA macro array analysis to identify 95 transcripts that were up- or downregulated by Hg, and a total of 64 were confirmed to be Hginduced. The BLAST results revealed that most of the ESTs (67) had protein homology to genes from other plants deposited in the database and several of them were previously identified as heavy metal induced/regulated genes such as metallothioneins type 2 and 1 (6, 26), β-1,3 glucanase, nitrate transporter (18), DnaJ and heat-shock proteins (19, 20), translationally controlled tumor protein (27), and auxinresponsive protein (19). The induction of these genes in this study suggested that multiple signaling pathways may be activated in response to heavy metal stresses including Hg exposure in plants. This result appears to support the robustness of the subtracted cDNA libraries.

Broad Functional Categories of the Hg Responsive Genes. As indicated from the results, a large number of genes are involved in heavy metal accumulation, detoxification and tolerance during this stress response. The genes isolated in this study were assigned to broad functional categories based on the database similarity results and the possible role of each functional category with respect to Hg regulation is discussed below. Stress/Defense Related Genes. A total of 18 genes had homology to known genes that have been involved in biotic and abiotic stresses including heavy metal stress. Eleven cDNAs of this category were identified as up-regulated in response to Hg exposure, while seven including metal ion binding (ATFP6) mRNA, methionine overaccumulation 1 mRNA, metallothionein-like mRNA, papain-like cysteine protease mRNA, calreticulin-1 mRNA, and glutathione Stransferase (GST) were down-regulated. One of the genes up-regulated by Hg is metallothionein (MT2) which is apparently involved in the intracellular metal binding and detoxification under heavy metal stress (26, 28). Northern analysis results indicated that mRNA transcript accumulation was induced in both shoots and roots from Hg-treated plants VOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. (a) The cDNA sequence (632 bp) and deduced amino acids (348 bp ORF) of Auxin Repressed Protein (SdARP). (b) Comparison of the amino acid sequences of Sesbania ARP, Robinia ARP, Arachis ARP, Malus ARP, and Fragaria ARP. The numbering starts with the predicted N-terminal amino acids of ARP Sesbania and with the N-terminal amino acids of other species. Identical residues between the ARPs are presented as asterisks. (c) Phylogenetic tree of genes in the ARP family. Analysis was performed using the CLUSTALW using default parameters.

FIGURE 6. Confirmation of differential expression pattern of metallothionein 2 and auxin repressed protein genes by Northern blot analysis with RNA extracted from shoots (S) and roots (R) of control. Hg-treated Sesbania plants grown for 10 days. Ethidium bromide stained gel is shown as loading control. compared to untreated control (Figure 3b). Several identified clones including thaumatin like protein PR-5a, DnaJ-like protein, β-1,3 glucanase, HSP 70, aluminum induced Sali5848

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4a mRNA and early light induced protein also have potential to be involved in cellular defense response, stress tolerance and detoxification. Induced expression for HSP70 and β-1,3 glucanase genes was observed in both shoots and roots of Hg-treated plants (Figure 3b). This suggests that Sesbania plant has potential to grow and hyperaccumulate heavy metals naturally. This molecular data support our earlier study (17) that Sesbania has great potential for hyperaccumulation of heavy metals. It is possible that the increased expression of β-1,3 glucanase and HSP70 play a critical role in protection against heavy metal oxidative stress by metal detoxification process (18, 20, 29, 30). Photosynthesis Related Genes. Interestingly, a set of three genes related to photosynthesis showed up-regulated expression by Hg treatments, indicating that the presence of Hg in plant tissues had little effect on photosynthetic efficiency. RNA blot results display that transcript accumulation level for Rieske Fe S protein precursor was 7 and 2 fold higher in shoots and roots from Hg-treated plants than that

of untreated control (Figure 3b). Earlier studies demonstrated that heavy metal toxicity is generally associated with inhibition of chlorophyll biosynthesis and damage to photosynthetic apparatus (6). In contrast, Fusco et al. (19) showed up-regulation of several photosynthetic genes in heavy metal accumulator Brassica juncea, and this finding is consistent with our results. The induction or up-regulation of these genes under Hg exposure may assist to compensate for oxidative damages caused by mercury ion. Cellular Metabolism and Signaling Related Genes. Several up-regulated cDNAs (17) represent proteins that are essential for maintaining enhanced cellular metabolism during heavy metal stress. These include oxidoreductase mRNA, alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and sucrose synthase mRNA. Northern analysis data showed that GAPDH was constitutively expressed in both Hg-treated and untreated plants, having an increase in GAPDH transcripts in shoots of Hgtreated plants while accumulation of transcripts for putative phosphatase was reduced in both roots and shoots (Figure 3). Increased transcripts of cytidine, adenine nucleotide translocator, asparagine synthetase, serine hydroxymethyltransferase, lipid protein precursor, geranylgeranyl reductase, and isopentenyl pyrophosphate isomerase were observed in Hg-treated plants thus pointing to changes in amino acid, nucleic acid, and fatty acid metabolism during the adaptation of Sesbania to Hg. A gene coding for oxidoreductase was up-regulated by Hg exposure and it is presumed to be involved in cellular respiration during heavy metal oxidative stress. In Brassica, similar types of gene transcripts were induced upon 6 weeks of Cd treatment (19). The downregulation of the gene encoding putative phosphatase may play an important role in signal transduction cascade. It has been reported that induced expression of genes in this category may be implicated to participate in different cellular process under heavy metal stress (19, 29). Taken together these data suggests that many of the genes expressed under Hg treatment may also play a role in alleviation of heavy metal toxicity. The ability of Sesbania to activate sets of genes associated with metal tolerance is probably the main reason for observed tolerance against other heavy metals such as Cd. Transcription Factors and Transporters. Six transcription factors including HAP2, AAC2 binding, CP12 putative domain, C2H2 type zinc finger, WRKY-82, and TF gene and a putative low-affinity nitrate transporter were also identified in this study. Northern analysis showed that expression of HAP2 transcription factor was down-regulated (Figure 3a), while WRKY82 was induced under Hg treatment (Figure 3b). The induction of WRKY gene transcripts was 3 fold higher in roots of Hg-treated plant than that of untreated control (Figure 3b). Accumulation of mRNA transcripts of low-affinity nitrate transporter was increased in roots grown hydroponically with Hg compared to control (Figure 3b). Both WRKY and bZIP have been induced in response to Cd exposure suggesting an important role for them in the transcriptional control of plants under heavy metal stress (19, 29). Upregulation of nitrate transporter under Hg exposure is not surprising because a similar transporter was also induced by cesium treatment (18). Cell Division and Growth Related Genes. Six up-regulated genes including auxin repressed protein (ARP), TCTP, Actin, cell wall type 2 proline rich protein isolated in this category showed significant similarity to proteins and enzymes associated with cell division, growth and development. RNA blot analysis indicates that transcript accumulation for ARP was induced in roots and shoots by Hg treatment (Figure 3b). This supports the notion that induced expression of ARP was positively correlated with shoot growth in Sesbania under Hg treatment (31). Another induced gene represents a

putative TCTP which is one of the recently identified meristematic cell growth-related proteins that are actively expressed in rapidly dividing cells (32, 33). Two novel TCTP genes were also found to be preferentially up-regulated under oxidative stress caused by aluminum (Al) in the root tips of Al-tolerant soybean cultivars (27). Up-regulation of cell growth related genes is presumed to be involved in metal stress signaling response (18, 26, 27). Protein Synthesis/Degradation Associated Genes. In this category, three up-regulated cDNAs encoding peptides with homology to ribosomal proteins were isolated. Up-regulation of ribosomal and other proteins would indicate a requirement for increased protein turnover in Hg-treated plants. Similar expression pattern was previously noticed in Brassica upon exposure to Cd (19). Tissue Specific Expression of Hg Modulated Genes. To determine the tissue specificity of Hg induced gene expression, we used RNA samples from shoots and roots grown hydroponically with (50 mg L-1) or without (0 mg L-1) HgCl2. Results of Northern analysis of Hg down-regulated genes showed that transcript abundance for four genes (HAP2A transcription factor, methionine over accumulation gene, glutathione S-transferase, and cysteine protease) was increased in roots compared to shoots of untreated control plants (Figure 3a). The accumulation of mRNA transcripts for metallothionein like protein, metal ion binding protein, putative phosphatase, and glutathione S-transferase was slightly suppressed in Hg exposed roots and shoots compared to control (Figure 3a). Furthermore, RNA blot analysis of Hg up-regulated genes indicated the induced expression of six genes (oxidoreductase, β-1,3 glucanase, MT2, ARP, WRKY transcription factor, and nitrate transporter) in both shoots and roots exposed to Hg. Although, six genes were induced in response to Hg exposure, the abundance of mRNA transcripts for β-1, 3 glucanase, MT2, ARP genes were higher in shoots as compared to roots (Figure 3b). Four genes (HSP70, GAPDH, TCTP, and Rieske FeS protein) were expressed constitutively, the abundance of these gene transcripts were slightly higher in Hg treated plants. This result indicates that shoots and roots respond differently to Hg stress. Interestingly, SdMT2 had homology to metallothionein type 2 (MT2) genes that have been implicated in heavy metal stress tolerance and detoxification. Metallothioneins type 2 are low molecular weight metal binding proteins playing a pivotal role in heavy metal detoxification and homeostasis in plants (28, 34). It is possible that up-regulated expression of metallothioneins in Sesbania leads to reduced Hg toxicity. Overexpression of members of the type 2 metallothionein protein class has been shown to protect plants against toxic levels of heavy metals (35, 36). On the other hand, it is reported that metallothionein like protein genes showed enhanced expression in tissues where active cell division occurs (nonstressed). In these cells metallothioneins function could be related to balancing the redox status rather than a general antioxidant function (37). Similarly down-regulation of metallothionein like protein gene could be correlated with decrease in cell division and growth of Hg-treated Sesbania plant compared to that of control. The metallothionein like protein genes of Sesbania display different gene sequences and gene expression patterns, which may be associated with the diverse biological functions. Also an auxin repressed protein gene was isolated and characterized from Sesbania drummondii and its deduced amino acid sequence was highly conserved with that of other plants (Figure 5b). Expression of MT2 and ARP was strongly induced by Hg and the transcript abundance was higher in shoots than roots (Figure 6). These results together suggest that Sesbania drummondii plants have evolved tolerance mechanism to mercury that involves changes in several biochemical processes as reflected by VOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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changes in the expression of diverse groups of genes. The potential candidate genes identified in this study could play an important role in heavy metal tolerance and/or hyperaccumulation of Hg in plants; furthermore, they could be used as molecular markers for Hg tolerance.

Acknowledgments Financial support from the Sponsored Programs, the Graduate School, and the Applied Research and Technology Program of Ogden College of Science and Engineering at Western Kentucky University are greatly acknowledged. P.V. and A.K.S. contributed equally to this research.

Supporting Information Available Figure S1 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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