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Jan 6, 2015 - esculentum Seedling Roots in Response to Cadmium, Chromium,. Mercury, and Lead. Jing Hou, Xinhui Liu,* Juan Wang, Shengnan Zhao, and ...
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Microarray-Based Analysis of Gene Expression in Lycopersicon esculentum Seedling Roots in Response to Cadmium, Chromium, Mercury, and Lead Jing Hou, Xinhui Liu,* Juan Wang, Shengnan Zhao, and Baoshan Cui State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, China S Supporting Information *

ABSTRACT: The effects of heavy metals in agricultural soils have received special attention due to their potential for accumulation in crops, which can affect species at all trophic levels. Therefore, there is a critical need for reliable bioassays for assessing risk levels due to heavy metals in agricultural soil. In the present study, we used microarrays to investigate changes in gene expression of Lycopersicon esculentum in response to Cd-, Cr-, Hg-, or Pb-spiked soil. Exposure to 1/10 median lethal concentrations (LC50) of Cd, Cr, Hg, or Pb for 7 days resulted in expression changes in 29 Cd-specific, 58 Cr-specific, 192 Hgspecific and 864 Pb-specific genes as determined by microarray analysis, whereas conventional morphological and physiological bioassays did not reveal any toxicant stresses. Hierarchical clustering analysis showed that the characteristic gene expression profiles induced by Cd, Cr, Hg, and Pb were distinct from not only the control but also one another. Furthermore, a total of three genes related to “ion transport” for Cd, 14 genes related to “external encapsulating structure organization”, “reproductive developmental process”, “lipid metabolic process” and “response to stimulus” for Cr, 11 genes related to “cellular metabolic process” and “cellular response to stimulus” for Hg, 78 genes related to 20 biological processes (e.g., DNA metabolic process, monosaccharide catabolic process, cell division) for Pb were identified and selected as their potential biomarkers. These findings demonstrated that microarray-based analysis of Lycopersicon esculentum was a sensitive tool for the early detection of potential toxicity of heavy metals in agricultural soil, as well as an effective tool for identifying the heavy metal-specific genes, which should be useful for assessing risk levels due to heavy metals in agricultural soil.



INTRODUCTION In farmland ecosystems, heavy metals have received special attention because they can accumulate in agricultural soil as a result of wastewater irrigation and the use of fertilizer containing sewage sludge.1 Importantly, such metals can affect species at all trophic levels.2−4 Once accumulated in the human body through the food chain, heavy metals can cause a variety of adverse effects, including behavioral disturbances, carcinogenesis, biochemical changes, reproductive abnormalities, neurological deficits, and immunological dysfunctions.5,6 Therefore, there is a critical need for reliable tools to assess the risk levels of heavy metals in agricultural soil. Cadmium (Cd), chromium (Cr), mercury (Hg), and lead (Pb) are four of the most toxic heavy metals commonly found in agricultural soil, and these metals pose considerable threats to the environment and human health.7−11 Crops are the primary source of human exposure to heavy metal pollutants, as they can accumulate and pass toxic metals into the food supply. Lycopersicon esculentum, a crop recommended by the U.S. EPA for soil toxicity assessments,12 is a good candidate test species for ecotoxicology due to its large-scale cultivation on farmlands, short growth cycle, simple genomic background, and ability to adapt to a wide range of environmental conditions. Plant roots are © XXXX American Chemical Society

generally used as samples in phytotoxicity studies because they come into direct contact with toxicants in the soil.13 Previous studies have shown that both the levels of accumulated chemicals and the inhibitory effects of such chemicals are much higher in the roots than in the aerial parts of plants.14−16 The ecotoxicological effects of heavy metals on plants are routinely assessed using conventional bioassays, such as the seed germination and root-elongation tests recommended by the U.S. EPA.12 However, toxicity effects caused by trace contaminants are difficult to detect using such conventional bioassays,17,18 therefore, determining the effects of low levels of toxicants requires more sensitive biological approaches. Recent studies have demonstrated that changes in gene expression associated with low levels of chemical toxicants are more informative and can be observed earlier than changes in physiology, biochemistry or morphology.14,19,20 These observations suggest that gene expression data may allow for the Received: August 24, 2014 Revised: December 31, 2014 Accepted: January 6, 2015

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was thoroughly mixed and then aged for 2 weeks under natural conditions to allow for adsorption and equilibration of the heavy metals. A preliminary test was carried out to calculate the median lethal concentrations (LC50, test chemicals concentrations that caused 50% mortality), which are shown in SI and Figure S1. The 1/10 LC50 test concentrations selected for this study were 3.3 mg Cd/kg soil, 1.2 mg Cr/kg soil, 2.3 mg Hg/kg soil and 194.4 mg Pb/kg soil, as these levels were below those that cause acute toxicity, which could lead to nonspecific gene expression responses.34 Thirty sterilized seeds were germinated in an 18 cm glass Petri dish filled with 500 g spiked soil. Water content of the soil was 70% of the maximum water-retaining capacity. The seeds were then incubated in a growth chamber at 25 ± 1 °C with 70% humidity in complete darkness for 7 days. Analysis of Heavy Metals in Roots. After 7 days of exposure, the root samples were separated from the seedlings, washed with deionized water, dried at 70 °C for 24 h, digested with nitric acid in closed polytetrafluoroethylene vessels at room temperature for 4 h, and then digested at 165 °C for 4 h. After filtration through a 0.2-μm filter, the solutions were analyzed using inductively coupled plasma mass spectrometry (ICP-MS). Thirty seedlings were used for each treatment. Root samples from 10 seedlings were pooled together to obtain three biological replicates that were analyzed separately. Morphological and Physiological Analysis. At the time of harvest, root length was measured, and biomass was determined after drying to a constant weight at 75 °C. The heavy metal-induced malondialdehyde content was determined using the thiobarbituric acid reaction protocol.35 Total amino acid content was quantified using the ninhydrin method.36 Root tips viability was determined by staining with triphenyltetrazolium-chloride (TTC).37,38 Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison tests at the 95% confidence level (p < 0.05). Microarray Analysis. A total of 15 microarrays (4 × 44 K tomato gene expression microarray; G2519F-022270) were purchased from Agilent (Palo Alto, CA), which were used to hybridize with RNA extracted from Cd-, Cr-, Hg-, Pb-treated, and the untreated roots (control). Each treatment was carried out for three replicates. A total of 30 seedlings were used for each treatment. Root samples from 10 seedlings randomly selected from each Petri dish were pooled together to obtain three biological replicates. RNA extraction, purification, amplification, labeling, hybridization and data acquisition were conducted using the protocols for Agilent one-color microarray-based gene expression analysis (see SI). Genes that were differentially expressed between each treatment group and the control were identified using t tests and significance analysis of microarrays. Genes with p-values 2 in either direction were identified as differentially expressed and were subjected to further functional analyses. Annotation analysis was performed using the NCBI Entrez Gene database (http://www.ncbi.nlm.nih.gov/gene/). The Venn diagram was constructed using the online tool VENNY (http://bioinfogp. cnb.csic.es/tools/venny/index.html). Gene ontology (GO) assignment and functional clustering were performed using the online program DAVID (http://david.abcc.ncifcrf.gov/ tools.jsp). In addition, differentially expressed genes were identified using one-way ANOVA (p < 0.05), and the most significant genes (p < 0.05; fold change >2) were subjected to

early detection of potential toxicity and may yield a better understanding of the associated mechanisms of these toxicants.21,22 Recent developments in gene expression analysis involving microarray-based techniques have yielded new and powerful methods for determining toxin potency and function.23,24 Microarray techniques, combined with highthroughput genomic technologies, have received a great deal of attention among environmental researchers as potential screening tools, as they show particular promise for identifying molecular biomarkers specific to individual contaminants.25−27 Moreover, agricultural soils often contain complex mixtures of chemicals, and predicting the toxicological effects of multiple chemical compounds using traditional methods can be quite challenging. However, microarrays are a comprehensive tool that can discriminate contaminants based on unique gene expression profiles. These resulting data can be used to link contaminants with corresponding biological processes and provide information concerning contaminant exposure levels and potential effects at higher biological levels.28 Several recent studies conducted on model plants, including Nicotiana tabacum,29 Populus deltoides,30 Arabidopsis thaliana,31 Elodea nuttallii,32 and Thlaspi caerulescens,33 have successfully used microarrays to classify contaminants and predict their modes of toxicity. In this study, we hypothesize that microarray-based analysis of L. esculentum is a sensitive approach for the early detection of potential toxicity of heavy metals in agricultural soil, as well as an effective tool for identifying the specific heavy metalregulated genes. To test the hypotheses, the Agilent wholegenome microarrays were used in the study to assess the effects of heavy metals (Cd, Cr, Hg, and Pb) on L. esculentum at relatively low concentrations, and identify specific heavy metalregulated genes.



MATERIALS AND METHODS Soil and Chemicals. The soil used for the experiment was collected from uncontaminated farmland (top 20 cm) in Baoding, China (38°47′29.94″N, 115°30′0.62″E). Prior to use, the soil was air-dried, ground and sieved through 2 mm mesh. The physicochemical characteristics of the soil were determined and are shown in Supporting Information (SI) Table S1. The soil texture was classified as a sandy loam according to the U.S. Department of Agriculture classification system. Analytical grade standards of cadmium chloride (CAS 10108−64−2), potassium dichromate (CAS 7778−50−9), mercuric chloride (CAS 7487−94−7) and lead nitrate (CAS 10099−74−8) used in this study were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Plant Species and Exposures Conditions. Tomato (Lycopersicon esculentum Mill. cv. Zhongza No.109) seeds were obtained from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Prior to use, the seeds were surface sterilized in a 10% sodium hypochlorite solution for 10 min to prevent fungal or other microbial contamination, then rinsed and soaked in distilled water for 1 h.12 Soil spiking was performed by adding stock solutions of Cd, Cr, Hg, and Pb directly to the background soil. Briefly, the stock solutions of the different heavy metals were prepared in deionized water, and then the appropriate amount of each stock solution was poured into 500 g soil to reach the target concentration. Additional, deionized water was combined with the treatments that required less or no metal stock solutions to equalize the total added volumes for all treatments. The soil B

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imported into the cell and metal bound by biosorption on the cell surface. Effects of Heavy Metals on Root Morphology and Physiology. As shown in SI Table S3, none of the Cd-, Cr-, or Hg-spiked soils at 1/10 LC50 had significant effects (p < 0.05) on root elongation or biomass compared with controls. By contrast, Pb significantly (p < 0.05) increased both root elongation and biomass by 24% and 20%, respectively. According to the physiological tests, the malondialdehyde contents of L. esculentum roots exposed to Cd-, Cr-, Hg-, and Pb-spiked soils at 1/10 LC50 were not significantly different from the control (p < 0.05). The malondialdehyde contents were selected as an indicator of heavy metal stress because it is a widely used biomarker of oxidative stress.41−43 Positive correlation between heavy metal concentrations and malondialdehyde contents has been observed in many previous studies.44,45 No significant differences (p < 0.05) in total amino acid content were observed between the treated and untreated roots for any of the four treatments. Similarly, root tip viability was not significantly affected (p < 0.05) for any of the four treatments compared with the control. The above results are consistent with previous studies. For example, at concentrations less than 8 μM, Cd did not significantly affected root length in L. esculentum.46 Another previous study focusing on the impact of heavy metals on L. esculentum found that phytochelatins production was not detected in either the roots or leaves of plants exposed to Cr(VI) at 5 mg/L or 10 mg/L.47 Furthermore, treatment of L. esculentum with 25 μM Cd had no effect on glutathione reductase activity.48 The results of these morphological and physiological bioassays indicate that conventional toxicology methods are not suitable for predicting or detecting the early effects of heavy metal exposure in L. esculentum roots. Therefore, the detection of early biological responses of plants to heavy metals may require the use of molecular methods. Characteristic Gene Expression Profiles Induced by Heavy Metals. We investigated changes in genomic expression profile of L. esculentum in response to heavy metal treatment using Agilent whole-genome cDNA microarrays. All data are publicly available at Gene Expression Omnibus (GSE63024). Statistical analysis revealed that exposure to Cd-, Cr-, Hg-, and Pb-spiked soil caused the differential expression (p < 0.05; fold change >2.0) of 252 genes (194 upregulated and 58 down-regulated), 394 genes (214 up-regulated and 180 down-regulated), 605 genes (322 up-regulated and 283 down-regulated) and 1366 genes (832 up-regulated and 534 down-regulated), respectively (Figure 2a). A full list of the differentially expressed genes for each treatment is shown in SI File S1. These results suggested that exposure to heavy metals at 1/10 LC50 can strongly affect gene expression in L. esculentum, whereas conventional morphological and physiological bioassays did not indicate any toxicant stresses. To visualize the differences in the gene expression profiles for all four heavy metal treatments, the microarray data were analyzed using one-way ANOVA, and the most significant genes (p < 0.05; fold change >2) were subjected to the hierarchical clustering analysis. As shown in Figure 3, the gene expression profiles induced by Cd, Cr, Hg, and Pb were divided into two subclusters, the left subcluster consisting of the samples exposed to Hg- and Cr-spiked soil, and the right subcluster consisting of the samples exposed to Cd- and Pbspiked soil. No individual heavy metal-treated samples grouped with the control or other subclusters. These results indicated

the hierarchical clustering analysis and principal component analysis (PCA) using the R-software program. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). To validate the microarray results, four significantly down-regulated genes (fold change 2.0) from the Cd, Cr, Hg, and Pb exposure groups were selected for RT-qPCR analysis. The samples were treated as described above, and primer pairs were designed using Primer3 (http://primer3.ut.ee/). The probeID, geneID, gene symbol, primer sequences and product sizes for the RT-qPCR analyses are shown in SI Table S2. Total RNA was extracted from the samples using TRIZOL Reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions, and then reverse transcribed using the Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. PCR amplification was performed using SYBR Premix Ex TaqII (Takara, Japan), according to the manufacturer’s instructions, and the accumulation of fluorescent products was detected using a 7500 fast real-time PCR system (Applied Biosystems, Foster, CA). The LeACTIN gene (Accession number: U60480) was used as a reference housekeeping gene, and the 2−ΔΔCT method was used to calculate the gene expression fold changes relative to the control.39



RESULTS AND DISCUSSION Root Heavy Metal Content. As shown in Figure 1, the heavy metal contents of metal-exposed roots were significantly

Figure 1. Heavy metal content in the roots of Lycopersicon esculentum exposed to Cd-, Cr-, Hg-, and Pb-spiked soil. The error bars represent the standard deviations of three biological replicates.

higher than in the control. At the selected exposure levels, the heavy metal concentrations were 8.39, 5.68, 7.13, and 6.30 times greater than in controls for the Cd-, Cr-, Hg-, and Pbexposed roots, respectively. These results indicate that considerable amounts of all four heavy metals can accumulate in and on the roots of L. esculentum at 1/10 LC50. It can be observed that large amount of Pb accumulated in the roots of L. esculentum. However, a previous study similar to our study showed that 44 μg/g Pb in tomato roots did not cause any significant effects on the biomass of roots.40 It can be inferred from the results that our measurement of the Pb accumulated in roots may be the total concentration, consisting of metal C

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Figure 3. Hierarchical clustering of the differentially expressed genes in Lycopersicon esculentum exposed to Cd-, Cr-, Hg-, and Pb-spiked soil. Each row represents a single gene, and each column represents one chemical treatment. The microarray data were analyzed using one-way ANOVA and the most significant genes (p < 0.05; fold change >2) were included in the hierarchical clustering analysis.

Figure 2. Differentially expressed genes induced by Cd, Cr, Hg, and Pb treatment. (a). The number of differentially expressed genes relative to the control (p < 0.05 and fold change >2) as a result of the various heavy metal treatments (Cd, Cr, Hg, and Pb) are shown. The percentages of up- and down-regulated genes for each of the heavy metal treatments are indicated on the bars. (b). Venn diagram showing the number of differentially expressed genes in each treatment (Cd, Cr, Hg, and Pb) that were significantly different from the control (p < 0.05 and fold change >2). The numbers in the overlapping regions represent genes common to multiple treatments, and the numbers in nonoverlapping regions represent genes unique to the indicated treatment.

that characteristic gene expression profiles can be used to discriminate each of the heavy metal treatment groups from the control, and furthermore, that they can discriminate all four heavy metal treatments from one another. We also performed PCA to validate the clustering analysis. This analysis also showed that the heavy metal-treated groups and the control could be successfully separated. Furthermore, the gene expression profiles of the Cd-, Cr-, Hg-, and Pb-treated roots were distinct from one another, whereas and the replicate samples were situated near one another, consistent with the results of the hierarchical clustering analysis (Figure 4). These results demonstrate that microarray analysis of L. esculentum is an effective tool for discriminating the effects of the heavy metals (Cd, Cr, Hg, and Pb) from one another at relatively low concentrations. Selection of Genes Expressed Specifically for the Heavy Metal Treatments. To test the hypothesis that microarray-based analysis of L. esculentum is an effective tool for identifying the specific heavy metal-regulated genes, we identified the genes that were specifically altered in L. esculentum due to exposure to Cd, Cr, Hg, and Pb. As shown in the Venn diagram, the overlapping regions represent differentially expressed genes that were common to multiple

Figure 4. Principal component analysis (PCA) based on the differentially expressed genes (p < 0.05; fold change >2) in Lycopersicon esculentum exposed to Cd-, Cr-, Hg-, Pb-spiked soil and the control. The points with the same color represent the same treatment. Each treatment consists of three replicates.

treatments, and the nonoverlapping regions represent differentially expressed genes that were unique to single treatments. Excluding the genes that were common to the heavy metal treatments, a total of 1143 genes were identified, consisting of 29 Cd-specific, 58 Cr-specific, 192 Hg-specific, and 864 Pbspecific genes (Figure 2b and SI File S2). To validate the results of the microarray analysis, eight genes (FLS1, MLP31, NF-YB3, AT4G02450, SULTR1;2, HSP60, AT3G60750, and AT5G13200) were selected and verified using RT-qPCR. Figure 5 shows plots of gene expression levels as measured by RT-qPCR against gene expression levels as measured by the microarrays. A highly significant correlation D

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could be proposed as a potential indication of Cd impacts, and its related genes may serve as its marker genes. Cr-Specific Biological Processes and Their Related Genes. As shown in SI File S3, five process groups were identified in Cr treatment. The “external encapsulating structure organization”, “reproductive developmental process”, “lipid metabolic process” and “response to stimulus” were unique to Cr treatment, while the “cellular process” was identified not only in Cr but also in Pb treatment. Although categorized in “cellular process” with a high degree of gene expression, the process group was not a Cr-specific biological process. Therefore, the other four process groups are proposed as potential indication of Cr impacts. Among them, only “lipid metabolic process” caused by Cr stress has been observed in germinating Actinidia deliciosa pollen.53 The related genes in the four selected Crspecific process groups and the corresponding fold changes were shown in SI Table S4 (b). Importantly, AT5G09760 and GH3.1 categorized in “external encapsulating structure organization” and “response to stimulus” showed a significant expression of 7.74-fold change and 7.67-fold change, respectively. AT5G09760, involved in the pectin methylesterase (PME) gene family, functions in the modification of cell walls via dimethylesterification of cell wall pectin. PMEs were shown to play a role in the separation of border cells from the root tip of pea,54 and found to be active in pollen tube elongation of maize.55 Moreover, PMEs have been proved to play an important role in hypocotyl elongation,56 cambial cell differentiation57 and microsporogenesis.58 GH3.1, a putative indole3-acetic acid (IAA)-amido synthetase, helps the plant to cope with the presence of excess auxin by catalyzing the synthesis of IAA-amino acid conjugates. Staswick et al.59 have proved that several GH3 genes, encoding IAA-amido synthetases, conjugates the excess IAA to amino acid to maintain auxin homeostasis. Hg-Specific Biological Processes and Their Related Genes. As shown in SI File S3, five process groups were identified in Hg treatment. The “cellular metabolic process” and “cellular response to stimulus” were unique to Hg treatment, while the “growth”, “macromolecule catabolic process” and “cellular macromolecule catabolic process” exhibited some overlaps of GO terms with the “growth” and “protein catabolic process” in Pb treatment. Therefore, the “cellular metabolic process” and “cellular response to stimulus” could be proposed as a potential indication of Hg impacts. Genes categorized in the two processes may serve as marker genes of Hg treatment, which were shown in SI Table S4 (c). It was worth noting that Hg treatment resulted in a 47.84-fold increase in the transcriptions of ACS2. ACS2 is a 1-aminocyclopropane-1-carboxylate synthase (ACS) thought to catalyze the conversion of Sadenosyl-L-methionine (SAM) into 1-aminocyclopropane-1carboxylate (ACC). ACC is the precursor of ethylene, which was a regulator of multiple plant processes. Recently, gemomewide expression analysis of Medicago sativa seedlings in early responses to Hg stress showed that genes from ethylene metabolism were highly represented, suggesting that ethylene may participate in the early perception of Hg stress.60 Pb-Specific Biological Processes and Their Related Genes. As shown in SI File S3, twenty-three process groups were identified in Pb treatment. All of them were unique to Pb treatment except for “growth”, “cellular process” and “protein catabolic process”, which were commonly identified in Cr and Hg treatment. Therefore, the 20 Pb-specific process groups (i.e., “DNA metabolic process”, “monosaccharide catabolic

Figure 5. Correlation analysis between microarray and RT-qPCR results. Eight differentially expressed genes (FLS1, MLP31, NF-YB3, AT4G02450, SULTR1;2, HSP60, AT3G60750, and AT5G13200) were analyzed by RT-qPCR. The microarray and RT-qPCR results are represented log2 fold change values.

(R2 = 0.949) was observed between the microarray and RTqPCR data sets, confirming that the microarrays generated reliable expression data. Functional Analysis of the Specifically Expressed Genes. To further identify the genetic markers and biological processes influenced by Cd, Cr, Hg, and Pb treatment, GO assignment and functional clustering were performed using DAVID. Functional analyses showed that the 29 Cd-specific, 58 Cr-specific, 192 Hg-specific, and 864 Pb-specific genes were categorized into 4, 24, 40, and 147 biological processes, respectively, which were further clustered into 1, 5, 5, and 23 biological process groups using the DAVID functional annotation clustering at the highest classification stringency (SI File S3). These process groups consisted of biological processes that have similar biological meaning due to sharing similar gene members. Cd-Specific Biological Processes and Their Related Genes. According to the results shown in SI File S3, only the “ion transport” process group was identified in Cd treatment, which was not found in Cr, Hg or Pb treatment. In this group, three genes with the symbol of AT3G07600, PAA1, and ZIP4 were shown in SI Table S4 (a). AT3G07600 is a gene associated with heavy metal transport and detoxification. There were few reports about the response of this gene to Cd stress. As a copper-transporting p-type ATPase of plant, PAA1 was probably involved in Cd fluxes into chloroplasts.49 ZIP4, a gene in the family of ZIP transporter, is considered as the zinc transporters that responsible for the uptake of zinc into cytosol. Up to now, no Cd-specific transporter has been found for plant cells. Cd and Zn were two metals with close chemical properties. The uptake of Cd is likely to occur through available members of the ZIP transporter family.50 Stephens et al.51 have demonstrated that MtZIP6 had measurable Cd transport capacity. Guerinot et al.52 also reported that Cd and Zn are most likely crossing the plasma membrane via metal uptake ZIP transporters. Responses of Elodea nuttallii transcriptome to Cd pollution demonstrated that the most strongly enriched category for Cd contamination was ‘ion transport and transport ATPases’ (e.g., heavy metal p-type ATPase),32 which was well consistent with our study. Therefore, “ion transport” E

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Environmental Science & Technology process”, “cell division”, “cellular component organization”, and “cellular catabolic process”) could be proposed as a potential indication of Pb impacts. Among these processes, some of them have been proved to show responses to Pb stress.61 Genes categorized in the selected process groups and their fold changes are shown in SI Table S4 (d). From SI Table S4 (d), it can be observed that some of the genes are shared in the process groups. For example, GLYR1 were shared between “monosaccharide catabolic process” and “metabolic process”. As a result, a total of 78 genes related to the 20 Pb-specific processes were selected as potential genetic markers of Pb. In addition, we can also observe that several genes showed a high degree of differential expression. For example, FZR3 (8.80-fold change), categorized in “cell division”, is highly expressed in the cell cycle during mitosis.62,63 This gene is especially activated in early growth stage of plants, which has been demonstrated by the significant expression of FZR3 in root or immature leave of tomato, as well as other plants.64−66 SYP111 (5.33-fold change), categorized in “cellular macromolecule localization”, is highly expressed in mitotic cells and required for the formation of the cell plate.67 It is reported that mutations of SYP111 affected the rate of cell divisions and consequently resulted in growth defects.68 In this paper, our study presents the first microarray-based whole-genome expression analysis of L. esculentum exposure to different heavy metals. The results demonstrated that microarray-based analysis of L. esculentum was a sensitive assay for the early detection of potential toxicity of heavy metals, as well as an effective tool for identifying the specific heavy metalregulated genes. It is anticipated to be useful for the development of sensitive and specific bioassays for assessing risk levels in agricultural soil. Although heavy metal-specific genes and biological processes were identified at the relatively low concentrations, further analyses of these specific genes at different concentrations in response to different heavy metal contamination are indispensible.





ACKNOWLEDGMENTS



REFERENCES

The research was financially supported by the Fund for Innovative Research Group of the National Natural Science Foundation of China (Grant No. 51421065), National Basic Research Program of China (2013CB430405), National Natural Science Foundation of China (21377013), and Fundamental Research Funds for the Central Universities and the Public Sector Special Scientific Research Program of National Environmental Protection Ministry (201309049).

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ASSOCIATED CONTENT

S Supporting Information *

Table S1: Physicochemical characteristics of the soil used in the study; Table S2: Primer sequences used for the RT-qPCR confirmations of the microarray results; Table S3: Morphological and physiological responses of L. esculentum to Cd-, Cr-, Hg-, and Pb-spiked soil; Table S4: Cd-, Cr-, Hg-, and Pbspecific biological process groups and their related genes; Figure S1: Determination of the median lethal concentration (LC50); File S1: List of the differentially expressed genes for Cd, Cr, Hg, and Pb treatment, respectively; File S2: List of genes expressed specifically for Cd, Cr, Hg, and Pb treatment, respectively; File S3: List of process groups of terms having similar biological meaning for Cd, Cr, Hg, and Pb, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.



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Corresponding Author

*Phone: 86-10-58802996; fax: 86-10-58802996; e-mail: xhliu@ bnu.edu.cn. Notes

The authors declare no competing financial interest. F

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