Comparative transcriptome analysis of cadmium responses in Solanum nigrum and Solanum torvum

Authors

  • Jin Xu,

    1. Key Laboratory of Agricultural Water Resources, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 286 Huaizhong RD, Shijiazhuang 050021, China
    2. Hebei Province Engineering Laboratory for Plant Breeding and Germplasm Enhancement of Stress-Tolerant Plants, 286 Huaizhong RD, Shijiazhuang 050021, China
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  • Jianhang Sun,

    1. Key Laboratory of Agricultural Water Resources, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 286 Huaizhong RD, Shijiazhuang 050021, China
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  • Liguo Du,

    1. Key Laboratory of Agricultural Water Resources, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 286 Huaizhong RD, Shijiazhuang 050021, China
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  • Xiaojing Liu

    1. Key Laboratory of Agricultural Water Resources, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 286 Huaizhong RD, Shijiazhuang 050021, China
    2. Hebei Province Engineering Laboratory for Plant Breeding and Germplasm Enhancement of Stress-Tolerant Plants, 286 Huaizhong RD, Shijiazhuang 050021, China
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Author for correspondence:
Jin Xu
Tel: +86 311 85871745
Email: xujin@sjziam.ac.cn

Summary

  • Solanum nigrum is a cadmium (Cd) accumulator, whereas Solanum torvum is a low Cd-accumulating plant. The molecular mechanisms that are responsible for differential cadmium (Cd) accumulation in the two Solanum species are poorly understood.
  • Here, grafting experiments confirmed that increased Cd loading into the root xylem was responsible for the differential Cd accumulation in the two Solanum species. An iron (Fe) supply assay indicated that low Fe accumulation in S. torvum leaves is related to its Cd sensitivity.
  • Transcriptome analyses revealed higher expression of the genes that encode several metal transporters as well as antioxidant-related genes, and several organic and amino acid biosynthesis/metabolism-related genes in Cd-treated S. nigrum. Our data also indicated that the different responsive mechanisms of the transporter genes to Fe deficiency might be responsible for differential uptake and redistribution of metals in the two Solanum species
  • These results form a basis upon which to further explore the molecular mechanisms of Cd accumulation and tolerance, and provide an insight into novel strategies that can be used for phytoremediation and food safety.

Introduction

Cadmium (Cd) is one of the most toxic nonessential elements, and it has a strong inhibitory effect on plant growth and reproduction (Macek et al., 2002). In leaves, concentrations of Cd that are higher than 5–10 μg g−1 DW are toxic to most plants (White & Brown, 2010; Lux et al., 2011). By contrast, some species can hyperaccumulate Cd to concentrations in excess of 100 μg g−1 DW in their leaves without showing any negative symptoms. Another strategy that allows plants to avoid Cd toxicity has emerged through natural evolution; some plants sequester Cd in their roots and prevent the translocation of Cd into the shoots, or remobilize Cd from shoots to roots by excluding Cd from leaves through the phloem (Chen et al., 2006; Mendoza-Cózatl et al., 2011). These plants are referred to as low Cd-accumulating plants (Yamaguchi et al., 2010). The typical characteristics of hyperaccumulator plants include: the high efficient uptake of Cd in roots; xylem loading and transport from the roots to the shoots; and the capacity to effectively detoxify Cd. Many studies regarding the molecular mechanisms of Cd hyperaccumulation have been reported; however, the molecular mechanisms that are responsible for low Cd accumulation in plants are poorly understood (Yamaguchi et al., 2010).

Cadmium is chemically similar to certain metal elements, including iron (Fe), zinc (Zn) and calcium (Ca), and, therefore, could displace these elements from metalloproteins (Clemens et al., 1998; Cohen et al., 1998; Verbruggen et al., 2009). Cd toxicity can be attributed to its competition with essential metals, especially Fe, for metal-binding molecules (Schutzendubel & Polle, 2002). Cd enters plant cells through Fe, Ca and Zn transporters/channels (Wu et al., 2012). Several studies have shown that Cd toxicity led to Fe deficiency in plants (Lombi et al., 2002; Yoshihara et al., 2006; Besson-Bard et al., 2009). However, the molecular mechanism of Fe accumulation on Cd tolerance and accumulation is still not fully understood.

Solanum nigrum is a Cd accumulator that is widely grown in Asia, Europe, and America. In a pot-culture experiment, S. nigrum accumulated 125 μg Cd g−1 of leaf DW without showing any phytotoxic symptoms or visible growth reduction (Sun et al., 2006). Most Cd hyperaccumulators grow slowly and have a low biomass; however, S. nigrum has a faster growth rate and higher biomass, and therefore S. nigrum shows more promise for use in phytoremediation (Wei et al., 2004). Solanum torvum cv. Torubamubiga is a low Cd-accumulating plant. Arao et al. (2008) found that, although the Cd concentration in S. torvum shoots was lower than that found in S. melongena, there was no difference in the Cd concentration that was measured in the roots of S. torvum and S. melongena. Further studies indicated that although all of the root uptake, xylem loading, and sequestration are responsible for the Cd accumulation in plants, the low loading rate of Cd into the xylem sap in the roots is the major contributor for the low Cd translocation to the above-ground parts of S. torvum (Mori et al., 2009).

The distinct types of Cd accumulation in the two Solanum species make Solanum an ideal genus in which to compare the physiological and molecular mechanisms that are involved in differential Cd accumulation. In this study, we compare Cd tolerance and accumulation between the Cd accumulator S. nigrum and the low Cd accumulator S. torvum. We investigated the molecular mechanisms that are responsible for Cd tolerance and accumulation using a combination of physiological and transcriptome analyses. These results form a basis upon which to further explore the molecular mechanisms of Cd accumulation and tolerance, and provide an insight into novel strategies that can be used for phytoremediation and food safety.

Materials and Methods

Plant materials and growth conditions

The seeds of S. nigrum L. and S. torvum Sw. were kindly provided by the Germplasm Bank of Wild Species in Southwest China. To obtain seedlings, the seeds were sown under sterile conditions in Petri dishes that contained MS medium (Murashige & Skoog, 1962) and were solidified with 0.8% (w/v) agar (Sigma). Seven-day-old seedlings were transferred into Hoagland solution (Hoagland & Arnon, 1950) and were grown in a sterilized, pathogen-free glasshouse. The cultures were maintained at 22–25°C under a 16 h photoperiod. The treatment with CdCl2 and/or 50 μM Fe-EDDHA (ethylendiamine-di (o-hydroxyphenylacetic) acid) was applied to 4-wk-old seedlings that were grown in Hoagland solution. The culture solution was replaced every 3 d.

Phenotypic analysis

Four-week-old plants that were grown in Hoagland solution were treated with CdCl2 (20, 50, or 100 μM) for 7 d. The relative root growth was calculated as the root length grown in the presence of Cd divided by the mean root length under control conditions as described by Freeman et al. (2010). Nine replicate plants were measured for each plant species and treatment.

Inductively coupled plasma mass spectroscopy (ICP-MS) analysis

Four-week-old plants that were grown in Hoagland solution were treated with 50 μM CdCl2 for 12 h, or 1, 3, or 5 d. The treated roots were immersed in a solution that contained 1 mM EDTA for 2 h and then thoroughly rinsed with distilled water. The samples were oven-dried at 75°C for 48 h. The dried plant tissues were ground and digested in concentrated nitric acid for 2–3 d at room temperature. The samples were then boiled for 1–2 h until they were completely digested. After adding 4 ml of Millipore-filtered deionized water and a brief centrifugation, the contents of Cd, Zn, Fe, Mn and Cu were determined using ICP-MS. Each experiment was repeated at least five times.

Measurement of Cd2+ flux with the scanning ion-selective electrode technique (SIET)

The net Cd2+ flux was measured noninvasively using SIET (BIO-001A; Younger USA Sci. & Tech. Corp., Beijing, China). SIET is a technique that specifically detects ion or molecule flow and velocity. Ion-selective microelectrodes with an external tip diameter of c. 3 μm were manufactured and silanized with tributylchlorosilane, and the tips were backfilled with a commercially available ionselective cocktail (Cd Ionophore I, 20909, Fluka, Switzerland). Previous study has proved that the Cd2+ electrode was highly discriminatory against other cations and demonstrated the utility of an ion-selective Cd2+ microelectrode as a research tool to study heavy-metal transport in biological systems (Piñeros et al., 1998). The microelectrodes were calibrated in 50 and 500 μM Cd2+ before the flux measurement. Only the electrodes with Nernstian slopes > 25 mV per decade were used (Ma et al., 2010). After exposure to 50 μM CdCl2 for 24 h, the root segments were sampled for the Cd2+ flux measurement. The measuring solution contained 50 μM CdCl2, 100 μM KCl, 20 μM CaCl2, 20 μM MgCl2, 500 μM NaCl, 100 μM Na2SO4 and 300 μM 2(N-morpholino)ethane sulfonic acid (MES), pH 5.7. SIET measures the concentration gradient of Cd2+ by means of a Cd2+-specific microelectrode vibrated between two different detection points on the root surface at a distance of 1.5 μm. Ion flux data were subsequently calculated. The Cd2+ flux data were recorded for a period of 10–15 min. The flux data were obtained with the ASET software, which is part of the SIET system. The three-dimensional ionic fluxes were calculated using MageFlux (http://xuyue.net/mageflux). The negative values in the figure represent the cation influx or anion efflux and vice versa.

Grafting experiment

Grafting was performed in 3-wk-old plants, and this included reciprocal and self-grafting. In this experiment, the S. torvum plants were grafted onto S. nigrum (St/Sn) or S. torvum (St/St, self-grafting), and the S. nigrum plants were grafted onto S. torvum (Sn/St) or S. nigrum (Sn/Sn, self-grafting). After allowing the graft to establish for 2 wk, the plants were transferred to a fresh hydroponic medium containing 50 μM CdCl2 for 24 h, and the subsequent determination of Cd accumulation in the scion shoots was performed as described earlier. At least six replicate plants were measured for each plant species and treatment.

Digital transcriptomics

Four-week-old S. nigrum and S. torvum plants were treated with 50 μM CdCl2 for 24 h. The RNA was extracted from the roots of the control and the two treated Solanum species using TRIzol (Gibco/BRL, Life Technologies, Grand Island, NY, USA). The detailed experimental procedure and bioinformatics analysis for the digital gene expression (DGE) profiling are described in the Supporting Information (Methods S1 and Figs S3–S5).

Reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression

The plant sample preparation, RNA extraction and RNA quality and integrity check have already been described. For the semiquantitative RT-PCR, we performed control reactions using the 18S rRNA and UBQ14 primers to ensure that an equal amount of RNA was used in each set of reactions. We optimized the cycle numbers to ensure that the amplification reaction was performed in the exponential phase. The transcriptome results were also verified using RT-quantitative PCR (RT-qPCR) according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al., 2009). The detailed experimental procedure for the RT-qPCR analysis is described in Methods S1.

Measurement of photosynthesis

The photosynthetic rate (Pn) was recorded on fully expanded leaves of the second youngest node at 0, 1, and 5 d separately after 50 μM CdCl2 treatment using an intelligent portable photosynthesis system (LCpro+, ADC, UK). These observations were recorded on six to eight plants per treatment.

Detection of reactive oxygen species (ROS) concentrations

To measure the inline image content, the treated plant materials (0.5 g) were ground in liquid nitrogen. The obtained powder was suspended in 50 mM phosphate-buffered saline (PBS) buffer (pH 7.8). After centrifugation (15 min, 12 000 g), the supernatant was used for inline image content measurements as previously described (Verma & Mishra, 2005).

For H2O2 content determination, the tissue powder was suspended in 100 mM PBS buffer (pH 7.8) that contained 1% (w/v) polyvinylpyrrolidone (PVP). After centrifugation (20 min, 12 000 g), the supernatant was used for H2O2 content measurements as described by Verma & Mishra (2005).

Measurement of oxidative damage

The Cd-induced oxidative damage (membrane liquid peroxidation) was estimated by measuring the malondialdehyde (MDA) concentrations. Fresh plant tissues were homogenized in 0.1% (w/v) trichloroacetic acid (TCA) solution. After centrifugation (15 min, 12 000 g), an aliquot of the supernatant was added to 0.5% thiobarbituric acid (TBA) in 20% TCA and heated at 90°C for 30 min. After cooling on ice, the mixture was centrifuged at 8000 g for 5 min. The absorbance was recorded at 532 and 600 nm. The MDA concentration was calculated from the difference between the absorbance values at 532 and 600 nm (Ben Amor et al., 2005).

The degree of membrane integrity was also assessed by the percentage of electrolyte leakage. Leaves and roots were immersed in 10 ml of ddH2O (double-distilled water) and incubated at 25°C for 2 h. The suspension medium was measured for the initial electrical conductivity (EC1). The samples were then boiled at 100°C for 15 min to release all the electrolytes, cooled and the final electrical conductivity (EC2) was measured. The electrolyte leakage was calculated using the formula inline image (Wang et al., 2008).

Statistical analysis

For each treatment, at least eight plants were analyzed; all the experiments were repeated at least three times. The results are presented as means ± SD. For statistical analysis, we used ANOVA and Tukey’s test with the SPSS 16.0 software package (SPSS, Chicago, Illinois, USA). Differences between the treatments were tested by the least significant difference (LSD) test at a 0.05 probability level.

All sequence data for this study were archived at the National Center for Biotechnology Information’s Short Read Archive (SRA) under accession no SRA053699.

Results

Cd tolerance assay

To compare the Cd tolerance in S. nigrum and S. torvum, 28-d-old plants were exposed to three Cd concentrations, 20, 50, and 100 μM, for 7 d. When the plants were grown without Cd, the roots of S. nigrum grew 1.14-fold longer than those of S. torvum (data not shown). Therefore, we used relative root growth to evaluate the Cd tolerance of the two species (Fig. 1a). At 20 μM Cd, the relative root growth of the two Solanum species was nearly identical. When the plants were exposed to 50 and 100 μM Cd, the root growth rate of S. nigrum was 1.34- and 1.27-fold higher, respectively, than that of S. torvum; these results indicated that S. nigrum is more tolerant to Cd stress than S. torvum. No obvious stress phenotype was observed in either of the plants after 7 d of 50 μM Cd treatment; however, 100 μM Cd led to visible leaf chlorosis and necrosis (data not shown). Therefore, we selected a Cd concentration of 50 μM for use in this study.

Figure 1.

(a) Relative root growth of Solanum nigrum and Solanum torvum plants that were exposed to Cd2+ for 7 d. The Cd content in the 4-wk-old (b) leaves, (c) roots, and (d) the Cd leaf : root ratios of the plants that were grown in Hoagland solution and treated with 50 μM CdCl2. The asterisks indicate the values that were significantly different from those of S. torvum (< 0.05). Error bars indicate ± SD.

Cd uptake and accumulation

We measured the Cd content in both species. As shown in Fig. 1(b,c), the Cd accumulation in the plants increased with the duration of the treatment. After 1 d exposure to 50 μM CdCl2, the Cd in the leaves reached a concentration that was c. 50% of the Cd accumulation level observed after 3–5 d. Compared with S. torvum, the Cd concentration that was observed in S. nigrum was threefold higher in the leaves and 1.2-fold higher in the roots. S. nigrum also showed a higher Cd leaf : root ratio than S. torvum (Fig. 1d). The low Cd leaf : root ratio and Cd concentrations in the S. torvum leaves indicated that S. torvum has a comparatively low Cd-accumulating capacity.

To compare the root Cd uptake in the two Solanum species, we used SIET to investigate the Cd2+ flux in the roots of S. nigrum and S. torvum. The CdCl2 treatment caused a steady net Cd2+ influx at the region that was located 0–2900 μm from the apex in both of the root tips (Fig. 2a). We also measured the site that was located 500 μm from the root apex, in which a vigorous Cd2+ flux was often observed. As shown in Fig. 2(b,c), there were marked differences in the Cd2+ fluxes between the two species. The S. nigrum roots exhibited a 2.78-fold higher Cd2+ influx than that of S. torvum, which indicated that a greater Cd uptake capacity existed in the S. nigrum roots than in the S. torvum roots.

Figure 2.

The net Cd2+ fluxes in the roots of Solanum nigrum and Solanum torvum. (a) Effects of Cd treatment on the net Cd2+ fluxes in S. nigrum and S. torvum roots. (b) Net Cd2+ fluxes in S. nigrum and S. torvum roots. (c) The mean fluxes of Cd2+ within the measuring periods. The asterisks indicate the values that were significantly different from those of S. torvum (< 0.05). Each point represents the mean of five to six individual roots, and the bars represent the standard error of the mean.

Effects of Cd on micronutrient accumulation

Cadmium toxicity interferes with the accumulation of micronutrients in plants. Because of this, the concentrations of Zn, Fe, and Cu in S. nigrum and S. torvum were compared (Fig. 3). There were no significant differences in the Cu concentrations between the two species with or without Cd treatment. When the plants were grown without Cd, there were no significant differences in the leaf Zn concentrations between the two species, and the accumulation of Zn in the root of S. nigrum was higher than in the S. torvum roots, whereas the accumulation of Fe was lower in the roots and higher in the leaves of S. nigrum compared with S. torvum.

Figure 3.

Metal contents in the cadmium (Cd)-treated Solanum nigrum and Solanum torvum plants. Symbols: purple triangles, S. nigrum roots; white squares, S. torvum roots; blue diamonds, S. nigrum leaves; white circles, S. torvum leaves. The asterisks indicate the values that were significantly different from those of S. torvum (< 0.05). Error bars indicate ± SD.

Both species showed reduced accumulations of Zn, Fe, and Cu under Cd stress. After 5 d exposure to 50 μM CdCl2, there were no significant differences in the Zn concentrations between the two plant species. However, the Fe concentrations in the S. nigrum roots were 1.54-fold lower than those in the S. torvum roots, whereas the Fe concentrations in the S. nigrum leaves were 2.92-fold higher than those in the S. torvum leaves.

Grafts confirmed the efficient root-to-shoot Cd transport in S. nigrum roots

It has recently been demonstrated that the low loading rate of Cd into the xylem sap is responsible for the low Cd accumulation in S. torvum shoots (Mori et al., 2009). We therefore assumed that the higher Cd accumulation that was observed in the S. nigrum leaves was not only the result of the greater Cd uptake capacity of the S. nigrum roots, but also possibly the result of a more efficient loading capacity of Cd into the root xylem. Furthermore, we wondered whether Cd loading in the roots was dependent on the shoot genotype. To test these hypotheses, a grafting experiment and subsequently determination of the scions’ Cd content were performed in S. nigrum and S. torvum to analyze the long-distance transport of Cd in whole plants (Fig. 4). After 24 h of treatment, the nongrafted and self-grafted plants showed a virtually identical Cd accumulation. Compared with the self-grafted plants (S. torvum/S. torvum plants), the S. torvum scions from the S. torvum/S. nigrum plants accumulated a 2.89-fold higher concentration of Cd, whereas the S. nigrum scions from the S. nigrum/S. torvum plants accumulated only 33.4% of the Cd concentration. It was clear that the Cd content of the scions largely depended on the rootstocks, which suggests that both the Cd uptake capacity in roots and the differential Cd loading capacity into the root xylem are responsible for the differential Cd accumulation between S. nigrum and S. torvum.

Figure 4.

The effect of grafting on the scions’ cadmium (Cd) accumulation in Solanum nigrum and Solanum torvum. The left side of the division sign (/) represents the scions, and the right side represents the rootstocks. The columns that are labeled with different letters were significantly different at < 0.05. Error bars indicate ± SD.

Gene expression analysis

The results in the previous sections indicate that both the Cd uptake capacity and its loading into the root xylem are primarily responsible for Cd accumulation. To further elucidate the mechanisms underlying the differential Cd accumulation in S. nigrum and S. torvum, we investigated the transcriptional regulation profiles in the two Solanum species roots using a tag-based DGE system. DGE analysis is an extremely sensitive method for the detection of differences in gene expression and facilitates the utilization of nonmodel species. We used a 24 h time course of Cd exposure to evaluate the Cd-responsive genes, because this time course accounted for 50% of the maximum Cd accumulation in the above-ground portions of both species, and the exponential increase stage implied an intense metabolism modulation in plants; and thereby induced a large amount of Cd-responsive gene expression. Twelve DGE libraries were created from three independent biological samples of four treatments and sequenced using Solexa (Illumina) Technology (San Diego, California, USA). After the 3′ adaptor fragments, a few low-quality sequences and several types of impurities were filtered. The sequencing quality evaluation is presented in Fig. S1. The saturation analysis showed that when the sequencing amount reached 2 M or higher, the number of detected genes almost ceased to increase (data not shown). The expressed tags that were obtained from the control and Cd-treated S. nigrum and S. torvum roots and that aligned to the reference genes generated expression data for 9387, 9211, 10 159, and 10 084 genes, respectively. The genes in all four groups of differential expression are listed in Table S2.

Genes involved in heavy metal transport and detoxification The transcriptional regulation of metal transport and detoxification-related genes that might be involved in the Cd uptake, transport and sequestration processes during Cd treatment were examined (Tables 1, 2). Two metal transporters, an Mg transporter MGT and an HMA gene, showed constitutively and inducibly higher expression levels in S. nigrum roots. A PDR-type ABC transporter PDR2 showed constitutively higher expression levels in S. torvum roots, whereas, it was more highly expressed in S. nigrum roots with Cd treatment. Members of the ZIP family are involved in the transport of Zn/Fe (Kobae et al., 2004). A ZIP transporter, IRT1, was more highly expressed in Cd-treated roots of S. nigrum; whereas IRT2 and a Zn transporter, ZIP11, were more highly expressed in the roots of S. torvum. The other differentially expressed metal ion transport gene that displayed higher expression levels in S. torvum was a Cu transport protein, COPT5.

Table 1.   Constitutive differences (−Cd; Solanum nigrum/Solanum torvum) of 4-wk-old plants that were grown in Hoagland solution for 1 d
UnigeneTPM-SnckTPM-StckStck/Snck P-valueFDRAnnotationMost related Arabidopsis gene
  1. Snck, control for S. nigrum roots; Stck, control for S. torvum roots; TPM, normalized expression level of genes, mean ± SE, = 3; Stck/Snck, log2 (multiples of differentially expressed); P-value, P-value from difference test; FDR, false discovery rate.

  2. Orange, up-regulated genes; green, down-regulated genes.

Metal transport and detoxicity
 gnl|UG|Les#S508683300.51 ± 0.11191.93 ± 25.46 8.56 7.44E–050.000120447Iron-regulated transporter 2 (IRT2)IRT2 (AT4G19680.1)
 YFR02I11A2.96 ± 0.1111.88 ± 1.44 1.99 7.95E–091.78E–08Zinc transporter ZIP11ZIP11 (AT1G55910.1)
 MLF03E07A1.66 ± 0.688.62 ± 3.07 9.82 00Copper transporter 5 (COPT5)COPT5 (AT5G20650.1)
 TVR05N17C58.50 ± 6.75180.73 ± 34.94 2.75 00PDR-type ABC transporter 2 (PDR2)PDR12 (AT1G15520.1)
 gnl|UG|Les#S5086582419.44 ± 6.520.76 ± 0.03 −4.6 2.55E–301.05E–29Magnesium transporter (MGT)MGT2 (AT1G16010.1)
 PLA08N18C21.52 ± 0.762.13 ± 0.52 −3.35 2.26E–289.13E–28CPx-type heavy metal ATPases (Cu-HMA)HMA mRNA (AT5G24580.1)
Antioxidant-related genes
 gnl|UG|Les#S198729851.12 ± 0.459.62 ± 3.09 9.99 00Pericarp peroxidase 3PCA (AT3G49110.1)
 TVL29A05A50.51 ± 20.53853.98 ± 234.31 4.11 2.60E–137.63E–13Secretory peroxidasePRXR1 (AT4G21960.1)
 gnl|UG|Les#S508686210.25 ± 0.1121.14 ± 2.84 11.06 00Cell wall peroxidasePOD mRNA (AT1G05240.1)
 SmFL26N14A19.89 ± 8.1675.21 ± 9.60 1.85 1.58E–134.69E–13Anionic peroxidase swpa7POD mRNA (AT4G33420.1)
 TVR09D09C7.70 ± 2.9888.70 ± 10.77 3.44 4.33E–141.36E–13Peroxidase 16 precursorPOD 16 (AT2G18980.1)
 TVR20O05A24.31 ± 9.77490.21 ± 68.92 4.24 1.10E–133.33E–13PeroxidasePOD mRNA (AT4G26010.1)
 TVR02B24C0.57 ± 0.332337.14 ± 344.27 17.86 00PeroxidaseRCI3 (AT1G05260.1)
 OVS01L14C22.27 ± 1.335.81 ± 0.36 −1.91 6.03E–162.05E–15L-ascorbate peroxidase 1 (APX1)APX3 (AT4G35000)
 gnl|UG|Les#S529556038.32 ± 14.252.41 ± 1.72 −11.84 1.43E–727.06E–72gp91-phoxRBOH F (AT1G64060)
 gnl|UG|Les#S5087040947.97 ± 4.765.39 ± 0.82 −3.13 5.29E–572.52E–56Peroxiredoxin-2EPeroxiredoxin-2E (AT3G52960)
 gnl|UG|Les#S5086957044.09 ± 13.343.90 ± 1.42 −3.4 4.77E–552.26E–54Cationic peroxidase 1POD 52 (AT5G05340)
 gnl|UG|Les#S19873013666.81 ± 204.3621.27 ± 4.42 −4.89 00Peroxidase 27 (POD27)POD 27 (AT3G01190)
 gnl|UG|Les#S5086832116.48 ± 3.531.73 ± 0.70 −3.2 2.83E–211.05E–20Peroxidase isoform 1PRXR1 (AT4G21960)
 gnl|UG|Les#S5087266838.78 ± 8.7210.24 ± 2.63 −1.84 6.57E–252.56E–24Peroxidase 31-likePOD 63 (AT5G40150)
 gnl|UG|Les#S508700021877.96 ± 229.810.90 ± 0.16 −10.98 00Nonsymbiotic hemoglobin class 1HB1 (At2g16060)
Amino acid biosynthesis and metabolism processes
 gnl|UG|Les#S508667828.90 ± 0.812.72 ± 1.45 −9.82 2.19E–187.79E–18Carbamoyl phosphate synthase (CARA)CARA (AT3G27740)
 gnl|UG|Les#S5087559142.71 ± 14.313.90 ± 0.92 −3.36 3.51E–521.64E–51Dehydroquinate dehydratase (MEE)MEE32 (AT3G06350)
 gnl|UG|Les#S5087555457.65 ± 2.630.36 ± 0.03 −7.33 1.62E–1108.42E–110Cysteine desulfuraseNFS1 (AT5G65720)
 SmFL26A01A7.76 ± 1.500.77 ± 0.19 −3.32 1.40E–103.46E–10Dihydroxy-acid dehydratase (DHAD)DHAD (AT3G23940)
 gnl|UG|Les#S5086806718.15 ± 3.091.55 ± 0.41 −3.54 1.92E–257.51E–25Aspartate aminotransferase-likeASP5 (AT4G31990)
 gnl|UG|Les#S5087636211.35 ± 1.600.33 ± 0.16 −10.13 1.41E–225.32E–22SAT1SERAT2;1 (AT1G55920)
 MLF01C11A62.14 ± 9.313.52 ± 1.51 −4 3.71E–881.88E–87DAHP synthase 1 (DHS1)DHS1 (AT4G39980)
 gnl|UG|Les#S3543102158.24 ± 17.547.64 ± 2.98 −2.9 1.25E–666.11E–66Chloroplast threonine deaminase 1 (OMR1)OMR1 (AT3G10050)
 SmFL04D23A15.63 ± 4.792.60 ± 0.52 −2.61 5.44E–171.90E–16Serine racemaseSR (AT4G11640)
Amino acid transporter
 SmFL25G05A25.36 ± 11.522.47 ± 0.48 −3.25 4.81E–301.98E–29Amino acid transporterCAT6 (AT5G04770)
 gnl|UG|Les#S5087564311.85 ± 4.032.67 ± 0.32 −2.16 7.67E–111.93E–10Amino acid permease 2AAP2 (AT5G09220)
 SmFL12E21A47.92 ± 9.543.38 ± 0.61 −3.83 8.35E–704.10E–69Amino acid transporterAAP (AT5G41800)
Carbohydrate and Cell wall metabolism processes
 PLA07L14C21.13 ± 4.791.60 ± 0.24 −3.69 3.48E–291.42E–28ATP-citrate synthase (CSY)CSY4 (AT2G44350.1)
 gnl|UG|Les#S19872200125.64 ± 11.094.44 ± 1.82 −4.74 2.06E–2031.13E–202ATP citrate synthase activity (CLA)ACLA-3 (AT1G09430.1)
 SmFL11P19A27.18 ± 8.842.92 ± 0.46 −3.26 7.32E–363.15E–35Malate dehydrogenase (PMDH)PMDH1 (AT2G22780)
 SmFL29H15A51.70 ± 12.01211.68 ± 11.09 2.02 4.45E–131.27E–12RGP2RGP2 (AT5G15650.1)
 TVR04L12C15.39 ± 1.56251.57 ± 23.35 4.04 00XTH9XTH9 (AT4G03210.2)
 TVR31P16A27.67 ± 10.50249.83 ± 40.49 3.24 3.21E–139.31E–13O-methyltransferase (OMT)OMT1 (AT5G54160)
 TVR34M06C1.87 ± 1.0614.48 ± 4.27 10.56 00Expansin (EXPA)EXLB1 (AT4G17030)
Table 2.   Induced differences (+ Cd; Solanum nigrum/S. torvum) of 4-wk-old plants that were grown in Hoagland solution and treated with 50 μM CdCl2 for 1 d
UnigeneTPM-SntrRTPM-SttrRSttr/Sntr P-valueFDRAnnotationMost related Arabidopsis gene
  1. Sntr, Cd-treated S. nigrum roots; Sttr, Cd-treated S. torvum roots; TPM, normalized expression level of genes, mean ± SE, = 3; Sttr/Sntr, log2 (multiples of differentially expressed); P-value, P-value from difference test; FDR, false discovery rate.

  2. Orange, up-regulated genes; green, down-regulated genes.

Metal transport and detoxicity
 gnl|UG|Les#S508683301.04 ± 0.6211.25 ± 1.83 10.17 00Iron-regulated transporter 2 (IRT2)IRT2 (AT4G19680.1)
 YFR02I11A4.16 ± 0.95123.19 ± 5.33 4.8 3.42E–128.33E–12Zinc transporter ZIP11ZIP11 (AT1G55910.1)
 MLF03E07A11.38 ± 5.3139.60 ± 15.91 1.78 00Copper transporter 5 (COPT5)COPT5 (AT5G20650.1)
 gnl|UG|Les#S5086582414.14 ± 0.942.31 ± 0.85 −2.54 3.31E–161.02E–15Magnesium transporter (MGT)MGT2 (AT1G16010.1)
 gnl|UG|Les#S529542756.70 ± 6.364.69 ± 1.64 −3.52 1.06E–815.08E–81Iron-regulated transporter 1 (IRT1)IRT1 (AT4G19690.1)
 TVR05N17C390.34 ± 33.17180.90 ± 55.27 −1.6 1.49E–123.71E–12PDR-type ABC transporter 2 (PDR2)PDR12 (AT1G15520.1)
 PLA08N18C13.90 ± 1.951.07 ± 0.46 −3.63 3.19E–211.08E–20CPx-type heavy metal ATPases (Cu-HMA)HMA mRNA (AT5G24580.1)
Antioxidant-related genes
 gnl|UG|Les#S198729854.43 ± 1.9158.12 ± 21.08 3.69 6.88E–131.75E–12Pericarp peroxidase 3PCA (AT3G49110.1)
 TVL29A05A58.13 ± 32.64624.28 ± 204.68 3.38 6.72E–131.72E–12Secretory peroxidasePRXR1 (AT4G21960.1)
 gnl|UG|Les#S508686214.21 ± 1.4538.43 ± 6.99 3.16 3.42E–128.34E–12Cell wall peroxidasePOD mRNA (AT1G05240.1)
 TVR09D09C6.62 ± 3.1696.94 ± 9.89 13.22 00Peroxidase 16 precursorPOD 16 (AT2G18980.1)
 TVR20O05A8.46 ± 3.4676.27 ± 4.20 12.9 00PeroxidasePOD mRNA (AT4G26010.1)
 TVR02B24C0.40 ± 0.121841.69 ± 267.36 12.35 0.0001205890.000178528PeroxidaseRCI3 (AT1G05260.1)
 gnl|UG|Les#S5087405664.82 ± 26.528.39 ± 3.23 −2.97 1.05E–855.05E–85PeroxidasePOD 59 (AT5G19890)
 gnl|UG|Les#S5086881620.19 ± 5.196.68 ± 1.13 −1.67 4.40E–141.25E–13Peroxidase 21POD mRNA (AT2G37130)
 OVS01L14C17.60 ± 4.602.96 ± 0.10 −2.5 3.26E–181.05E–17L-ascorbate peroxidase 1 (APX1)APX3 (AT4G35000)
 gnl|UG|Les#S529556061.25 ± 9.865.53 ± 2.04 −3.35 4.15E–811.97E–80gp91-phoxRBOH F (AT1G64060)
 gnl|UG|Les#S5087040944.37 ± 3.563.84 ± 1.07 −3.44 1.10E–614.95E–61Peroxiredoxin-2EPeroxiredoxin-2E (AT3G52960)
 gnl|UG|Les#S50869570171.31 ± 14.454.92 ± 1.40 −5.05 00Cationic peroxidase 1POD 52 (AT5G05340)
 gnl|UG|Les#S198730131129.83 ± 221.740.68 ± 0.20 −10.56 00Peroxidase 27 (POD27)POD 27 (AT3G01190)
 gnl|UG|Les#S5086832114.70 ± 2.620.75 ± 0.33 −10.58 1.47E–335.71E–33Peroxidase isoform 1prxr1 (AT4G21960)
 gnl|UG|Les#S508700021810.39 ± 279.170.60 ± 0.24 −17.44 00Nonsymbiotic hemoglobin class 1HB1 (At2g16060)
Amino acid biosynthesis and metabolism processes
 gnl|UG|Les#S5087559162.47 ± 10.468.83 ± 3.65 −12.61 4.03E–1342.07E–133Dehydroquinate dehydratase (MEE)MEE32 (AT3G06350)
 gnl|UG|Les#S5087555453.72 ± 3.990.65 ± 0.15 −6.33 2.59E–1081.29E–107Cysteine desulfuraseNFS1 (AT5G65720)
 SmFL26A01A12.89 ± 3.140.87 ± 0.15 −3.94 1.44E–214.89E–21Dihydroxy-acid dehydratase (DHAD)DHAD (AT3G23940)
 gnl|UG|Les#S5086806720.30 ± 7.421.91 ± 0.64 −3.43 8.41E–313.19E–30Aspartate aminotransferase-likeASP5 (AT4G31990)
 gnl|UG|Les#S5087636252.83 ± 7.545.79 ± 2.32 −3.05 5.03E–652.29E–64SAT1SERAT2;1 (AT1G55920)
 MLF01C11A31.22 ± 1.703.54 ± 1.17 −3.03 3.76E–391.52E–38DAHP synthase 1 (DHS1)DHS1 (AT4G39980)
 gnl|UG|Les#S3543102160.23 ± 23.739.98 ± 4.07 −2.59 3.60E–681.66E–67Chloroplast threonine deaminase 1 (OMR1)OMR1 (AT3G10050)
 SmFL04D23A18.57 ± 4.784.27 ± 0.17 −2.19 4.30E–181.38E–17Serine racemaseSR (AT4G11640)
Amino acid transporter
 SmFL25G05A18.81 ± 5.801.25 ± 0.17 −3.75 7.60E–272.75E–26Amino acid transporterCAT6 (AT5G04770)
 gnl|UG|Les#S508756439.91 ± 3.700.78 ± 0.21 −3.72 2.00E–176.34E–17Amino acid permease 2AAP2 (AT5G09220)
 SmFL12E21A28.21 ± 3.234.03 ± 1.10 −2.76 1.21E–334.70E–33Amino acid transporterAAP (AT5G41800)
Carbohydrate and cell wall metabolism processes
 PLA07L14C73.60 ± 12.574.25 ± 1.60 −4.18 1.39E–1167.01E–116ATP-citrate synthase (CSY)CSY4 (AT2G44350.1)
 gnl|UG|Les#S19872200434.54 ± 69.5343.53 ± 17.01 −3.25 00ATP citrate synthase activity (CLA)ACLA-3 (AT1G09430.1)
 TVR36E09W4.62 ± 1.271.19 ± 0.22 −1.97 5.01E–057.65E–05Malate dehydrogenase (MDH)MDH (AT5G58330)
 gnl|UG|Les#S51712555307.67 ± 15.05104.26 ± 12.76 −1.58 4.09E–1672.15E–166Malate dehydrogenase (mMDH)mMDH1 (AT1G53240)
 SmFL29H15A45.34 ± 17.77147.96 ± 27.76 1.65 4.60E–131.19E–12RGP2RGP2 (AT5G15650.1)
 TVR04L12C9.57 ± 1.4167.15 ± 6.27 2.76 00XTH9XTH9 (AT4G03210.2)
 TVR34M06C43.72 ± 24.37659.61 ± 237.82 3.9 8.44E–142.34E–13Expansin (EXPA)EXLB1 (AT4G17030)

Antioxidant-related genes  Cadmium stress induced a marked accumulation of ROS and affected the antioxidant content in the plants (Xu et al., 2009). Fifteen antioxidative stress-related genes were differentially regulated between S. nigrum and S. torvum during treatment with Cd. Among these genes, six members of the peroxidase superfamily were up-regulated in the S. torvum roots when compared with S. nigrum under either constitutive (−Cd) or induced (+Cd) conditions. By contrast, five peroxidase family genes, an ascorbate peroxidase gene (APX1), an NADPH oxidase gene (gp91-phox), a peptidyl-cysteine S-nitrosylation-related gene (peroxiredoxin-2E), and a nonsymbiotic hemoglobin gene (ns-Hb1) showed higher expression levels in S. nigrum roots than in S. torvum.

When gene expression levels were compared between the S. nigrum and S. torvum roots, an antioxidative gene, ns-Hb1, was more up-regulated in S. nigrum than all of the other genes that were tested (17.4-fold higher expression in S. nigrum). The ns-Hb1 protein has a superior affinity for oxygen and its overexpression in plants was shown to increase the ROS scavenging capacity, thereby improving stress tolerance (Borisjuk et al., 2007; Thiel et al., 2011).

Genes involved in metabolic processes  The expression levels of eight genes that are involved in amino acid biosynthesis and metabolism were higher in the S. nigrum roots than in the S. torvum roots with or without Cd treatment. These genes are involved in the biosynthesis and metabolism of several amino acids, such as aromatic amino acids, aspartate, serine, and threonine. Three amino acid transporters also showed higher expression levels in S. nigrum roots with or without Cd treatment.

Cadmium toxicity markedly affected carbohydrate metabolism in these plants. Two citrate synthase CSY and CLA genes, and two malate dehydrogenase MDH genes were more highly expressed in the S. nigrum roots than in the S. torvum roots. Cd treatment also markedly modulated cell wall metabolism processes. Three genes (RGP9, XTH9, and EXPA) that are involved in cell wall metabolism and biosynthesis were more highly expressed in the S. torvum roots with or without Cd treatment.

Verification of the DGE results

To verify the gene expression patterns that were observed in the DGE studies by using an independent experimental approach, semiquantitative reverse transcription (RT)-PCR and RT-qPCR analyses were performed for selected genes. We selected UBQ14, 18S RNA, ACT, TUBST1, S23, RPL8, and UBI2 as the candidate reference genes for RT-qPCR normalization. The geNorm software analysis indicates that the expression of 18S RNA was the most stable in the two Solanum species, and the pairwise variation V value of 18S RNA, ACT, UBQ14 and UBI2 was 0.15 (Fig. S2), which suggested that these genes were suitable reference genes for RT-qPCR normalization. The gene expression levels were normalized using the geometric mean of the four most stable expressed reference gene quantities, as described in the geNorm manual (http://medgen.ugent.be/~jvdesomp/genorm/geNorm_manual.pdf). The results presented in Fig. 5 showed good agreement with the DGE data. The expression levels of MGT, IRT1, PDR2, HMA, ns-Hb1, MEE, and CYS genes were higher in S. nigrum, whereas IRT2, ZIP11, and COPT5 were more highly expressed in S. torvum under Cd stress conditions. The high confirmation rate indicates the reliability of our data.

Figure 5.

Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) (a) and quantitative RT-PCR (b) confirmation of the digital gene expression (DGE) data from Solanum nigrum and Solanum torvum. ck, 4-wk-old untreated control; Cd, treated with 50 μM CdCl2 for 24 h. Bars represent the 95% CI of the normalized expression.

Effect of Fe on leaf Cd tolerance

Cadmium toxicity strongly impaired the seedling growth and led to Fe deficiency. ICP-MS analysis indicated that the accumulation of Fe in S. torvum leaves is lower than in S. nigrum. To determine whether the lower Fe accumulation is related to the low Cd tolerance in S. torvum leaves, we analyzed the impact of Fe supply on leaf physiology in Cd-treated plants. Supplementation with Fe did not affect Cd accumulation in the leaves of the two species after 5 d of treatment. Fe deficiency increased Cd accumulation in S. nigrum leaves after 5 d of treatment; however, it did not affect Cd accumulation in S. torvum leaves (Fig. 6). The Pn of both species continued to decrease during the exposure to 50 μM Cd stress. Fe supplementation did not affect the Pn in the Cd-treated S. nigrum plant; however, the rate of reduction was lower in Fe-supplemented S. torvum plants (Fig. 7a). By contrast, Fe deficiency reduced the Pn to a greater extent in both S. nigrum and S. torvum than did Cd treatment alone. Similarly, the inline image and H2O2 accumulation in leaves of S. torvum supplemented with 50 μM Fe was lower than with Cd treatment alone, and Fe deficiency induced higher inline image and H2O2 accumulation in both S. nigrum and S. torvum leaves (Fig. 7b,c). Consistent with the phenomenon of ROS accumulation, Fe deficiency increased the MDA concentration and electrolyte leakage, whereas Fe supplementation effectively reduced the MDA concentration and electrolyte leakage in leaves of Cd-treated S. torvum (Fig. 8a,b), indicating that there is a positive effect of Fe supplementation on alleviating Cd-induced oxidative damage in S. torvum plants.

Figure 6.

Effects of Fe supplementation and deficiency on Cd accumulation in Solanum nigrum and Solanum torvum leaves. The leaf Fe accumulation (a) and the leaf Cd accumulation (b) were recorded separately at 1 and 5 d after Cd treatment. Asterisks indicate values significantly different from those of the plants treated with Cd alone (< 0.05). Error bars indicate ± SD.

Figure 7.

Effects of Fe supplementation and deficiency on Cd tolerance in Solanum nigrum and Solanum torvum leaves. Photosynthetic rate (Pn) (a), production of superoxide (inline image) (b) and hydrogen peroxide (H2O2) (c) were recorded separately at 0, 1, and 5 d after Cd treatment. Asterisks indicate values significantly different from those of the plants treated with Cd alone (< 0.05). Error bars indicate ± SD.

Figure 8.

Effects of Fe supply on Cd tolerance in Solanum nigrum (open bars) and Solanum torvum (closed bars). Malondialdehyde (MDA) content (a) and electrolyte leakage (b) in the leaves of 4-wk-old S. nigrum and S. torvum plants that were grown in Hoagland solution treated with 50 μM CdCl2 (Cd), 50 μM Fe (Fe), 50 μM CdCl2 plus 50 μM Fe (Cd+Fe) or Fe deficiency plus 50 μM CdCl2 (Cd–Fe) for 5 d. The different letters indicate the values that were significantly different from their appropriate controls (without Cd) at < 0.05. Error bars indicate ± SD.

These results indicate that Fe deficiency increased Cd accumulation only in S. nigrum leaves after 5 d of treatment. IRT1 and IRT2 are two important Fe transporter genes involved in the Cd accumulation and tolerance in plants (Vert et al., 2002, 2009). To explore the possible involvement of these genes in modulating the differential Fe/Cd accumulation in the two species, we examined the gene expression of IRT1 and IRT2 in the roots of plants treated with Cd or without Fe supply. As shown in Fig. 9, both Cd treatment and Fe deficiency up-regulated the expression of IRT1 in S. nigrum. By contrast, Cd treatment and Fe deficiency down-regulated the expression of IRT1 and IRT2 in the roots of S. torvum. The results of these experiments are discussed in the following section.

Figure 9.

The expression profile of IRT1 and IRT2 were monitored by RT-qPCR in the 4-wk-old Solanum nigrum and Solanum torvum plants grown under conditions without (ck) and with 50 μM CdCl2 (Cd) as well as without Fe supply (Fe−) at 1 d. The asterisks indicate the values that were significantly different from their appropriate controls (ck) (< 0.05) Error bars indicate ± SD.

Discussion

The transcriptional regulation of genes plays an important role in metal homeostasis (Wintz et al., 2003). The majority of transcriptomics analyses of differential Cd accumulation are based on the comparative analysis between hyperaccumulator and related nonhyperaccumulator species. The reported Cd hyperaccumulators are also Zn hyperaccumulators, which suggests that Cd and Zn accumulation at least partially rely on common genetic determinants (Xing et al., 2008; Verbruggen et al., 2009). However, in this study, we found that although S. torvum accumulated lower Cd, the Zn concentration was unaffected, implying that a specific mechanism exists in S. torvum for low Cd accumulation. Therefore, in this study, we chose the Cd accumulator S. nigrum and its low Cd-accumulating relative S. torvum for comparative transcriptome analysis. Also, to the best of our knowledge, this study represents the first transcriptome analysis of S. nigrum using a tag-based DGE system, which allowed us to identify new genes that may contribute to the differential Cd accumulation and tolerance phenotype of the two Solanum species.

The enhanced expression of heavy metal transporter genes in hyperaccumulators is universal and is regarded as a central characteristic of heavy metal hyperaccumulation (Weber et al., 2006; Verbruggen et al., 2009). However, in this study, we found that several metal transporters showed higher expression in S. torvum, which suggests that these genes are involved in low Cd accumulation. These results are discussed in the following. Our study supports the idea that the modulation of Cd distribution in plants and intracellular redistribution (via sequestrate metal in vacuoles or organelles in roots or long-distance transport from root to shoot and storage in leaf vacuoles) play a vital role in differential Cd accumulation.

The Cd accumulator S. nigrum was more tolerant to Cd stress than S. torvum (Figs 1a, 7, 8). The leaf Cd accumulation was threefold higher in S. nigrum than in S. torvum, which demonstrates the high Cd detoxification capability of S. nigrum. A previous study indicated that, although S. torvum accumulated similar Cd concentrations in its roots, it accumulated lower Cd concentrations in its shoots compared with eggplant (S. melongena; Arao et al., 2008), which suggested that the roots contained a vital factor that controlled the Cd loading capacity into the xylem. In support of this result, by using horticultural and physiological approaches, we demonstrated that the root-to-shoot translocation plays a major role in the differential Cd accumulation between S. nigrum and S. torvum. Subsequently, our transcriptomic studies offered some insight into the molecular mechanisms that potentially mediate these differences in Cd accumulation.

Several studies have indicated that a high antioxidative capacity is responsible for heavy metal hyperaccumulation in plants (Schutzendubel & Polle, 2002; Cho & Seo, 2005; Wang et al., 2008). S. nigrum accumulated lower ROS concentrations than S. torvum in the presence of Cd (Fig. 7b,c), which suggests that S. nigrum has a high free radical scavenging capacity. However, in this study, we found that most differentially expressed antioxidant-related genes are peroxidase family genes. Other several important antioxidative enzyme genes, such as SOD and CAT, did not show differential expression between the two species. The transcriptome analysis also identified an ns-Hb1 gene, which may function as an antioxidant. Of all of the genes that were tested, ns-Hb1 was the most up-regulated gene in the S. nigrum roots compared with that in the S. torvum roots. Plant ns-Hbs function in a variety of cellular processes and its overexpression in plants were shown to enable the cell to maintain high quantities of ATP when under stress (Borisjuk et al., 2007; Thiel et al., 2011). Previous studies indicated that purified recombinant Arabidopsis ns-Hbs displayed intrinsic peroxidase-like activity (Sakamoto et al., 2004). Overexpression of GhHb1 gene decreased the concentration of cellular NO in Arabidopsis seedlings (Qu et al., 2006), indicating that ns-Hbs plays a role in modulating the NO concentrations and the ratio of H2O2/NO in the defense process (Igamberdiev et al., 2004; Qu et al., 2006). In this study, we found that S. nigrum roots accumulated a higher level of ns-Hb1 transcript than that in S. torvum. Moreover, we found that Cd treatment further reduced ns-Hb1 expression in S. torvum roots but did not have a significant effect on ns-Hb1 expression in S. nigrum roots (Table S1). These findings imply that this Hb1 gene may play an important role in the Cd stress-response process of S. nigrum seedlings. Interestingly, we also found that a peptidyl-cysteine S-nitrosylation-related gene, peroxiredoxin-2E, was more highly expressed in S. nigrum roots. The post-translational modification (S-nitrosylation) of cysteine thiol to form nitrosothiol (SNO) is a key feature of NO and is coupled to the stimulation of all isoforms of NO synthase (Stamler, 1994; Stamler et al., 2001; Liu et al., 2004). The majority of all NO-affected proteins seem to be regulated by the S-nitrosylation of a single critical Cys residue (Lindermayr et al., 2005). Our previous studies have indicated that NO is associated with long-term Zn and Cd tolerance and accumulation in S. nigrum seedlings (Xu et al., 2010, 2011). The enhanced expression of the peroxiredoxin-2E gene in S. nigrum suggests that the gene may be involved in modulating Cd tolerance and accumulation by the NO signaling pathway and requires further investigation.

Transcriptome analysis also revealed that an Asp biosynthesis-related gene (ASP), a cysteine desulfurase gene (NFS) that can catalyze Cys to Ala, an aromatic amino acid biosynthesis-related gene (MEE), and other five amino acid metabolism-related genes showed higher expression in S. nigrum than in S. torvum, implying that the Cd response in amino acid biosynthesis and metabolism is greater in the roots of S. nigrum than in the roots of S. torvum. Amino acid accumulation is a central plant response to heavy metal exposure. Further metabolomic analysis of amino acids will help to elucidate the roles of amino acids in controlling Cd tolerance and accumulation in hyperaccumulating and low Cd-accumulating plants.

The ZIP family transporters are the important Zn/Fe transporters (Talke et al., 2006; Plaza et al., 2007) and the COPT family transporters are the important Cu transport proteins (Sancenon et al., 2003; Penarrubia et al., 2010). Two ZIP transporters (IRT2 and ZIP11) and a COPT transporter (COPT5) displayed a lower level of gene expression in S. nigrum than in S. torvum. IRT2 may compartmentalize Fe/Cd into vesicles, to prevent toxicity by excess free Fe/Cd in the cytosol (Vert et al., 2009). The results were consistent with the observed phenotype of high Fe accumulation in S. torvum roots and implied that S. nigrum may rely on other transporters to acquire Cd. ZIP11 is an endomembrane system-located Zn/Cd transporter (Plaza et al., 2007; Antosiewicz et al., 2008). COPT5 is located in the tonoplast and functions as a vacuolar Cu transporter (Klaumann et al., 2011). An Arabidopsis copt5 mutant showed markedly reduced root growth under Cd toxicity (Klaumann et al., 2011), which implies that COPT5 is also involved in Cd tolerance in plants. High expression of IRT2, ZIP11, and COPT5 in S. torvum roots may promote Cd into the vacuolar or endomembrane systems, thereby increasing root Cd sequestration and reducing Cd transport from the roots to the shoots.

In this study, three heavy metal transporters, PDR2, IRT1, and an HMA transporter, showed higher expression in Cd-treated S. nigrum roots than in S. torvum roots. IRT1 is the main route of Fe entry into the plant and mediates the accumulation of additional metal ions (Vert et al., 2002). Because irt1 mutation is lethal, it is clear that no other ZIP gene can substitute for its loss. Even overexpression of IRT2, which is the closest homolog of IRT1, cannot compensate for the loss of IRT1 (Varotto et al., 2002). IRT1 may also be involved in the high-affinity Cd uptake in Thlaspi caerulescens Ganges roots (Lombi et al., 2002). PDR2 is a member of the pleiotropic drug resistance (PDR) subfamily of the ATP-binding cassette (ABC) family of transporters. In Arabidopsis, two PDR genes, AtPDR8 and AtPDR12, have been suggested to transport heavy metal ions and confer Cd and Pb resistance in Arabidopsis (Lee et al., 2005; Kim et al., 2007). The most closely related Arabidopsis gene to the Solanum PDR2 is AtPDR12, which suggests that the Solanum PDR2 gene may be involved in heavy metal resistance and transmembrane transport in plants. Members of the HMA family are thought to be involved in the transport of heavy metals (Axelsen & Palmgren, 2001), and several HMA genes have been shown to be involved in heavy metal long-distance transport and detoxification (Hussain et al., 2004; Wong & Cobbett, 2009). HMA2 has been suggested to transport Zn and Cd in Arabidopsis (Wong et al., 2009). TcHMA3 plays a key role in the extreme Cd tolerance in T. caerulescens (Ueno et al., 2011). HMA4 is responsible for the efficient xylem loading of Cd (Bernard et al., 2004; Verret et al., 2004) and has been implicated as a key gene in Cd hyperaccumulator species. However, several similar papers failed to phenocopy Cd hyperaccumulation by overexpressing HMA4 (Hanikenne et al., 2008; Barabasz et al., 2010, 2012; Siemianowski et al., 2011), which suggests that Cd hyperaccumulation and tolerance are the results of many gene networks working simultaneously. In this study, an HMA gene showed higher expression in S. nigrum roots than in S. torvum roots under Cd toxicity, which suggests that the gene is linked to Cd accumulation and tolerance in S. nigrum. The most closely related Arabidopsis gene of the Solanum HMA is a heavy metal transport/detoxification domain-containing protein (AT5G24580). However, the characterization of the Arabidopsis HMA gene remains unclear and requires further investigation.

Compared with S. torvum, S. nigrum roots accumulated only 20% more Cd; however, the leaves accumulated 179% more Cd, which indicates that a low Cd long-distance transport rate is an important mechanism for low Cd accumulation in S. torvum. Yamaguchi et al. (2011) reported that the barrier function of the root endodermal Casparian band is one mechanism that is responsible for low Cd loading into the stele in S. torvum. In this study, several Casparian band biosynthesis-related genes, including RGP2 (UDP-glucose: protein transglucosylase), XTH9 (xyloglucan endotransglucosylase-hydrolase), and OMT1 (O-methyltransferase) that is involved in Casparian band suberin biosynthesis (Held et al., 1993), displayed higher constitutive expression levels in S. torvum roots. A metabolite analysis also identified an amino acid that is an important component of the Casparian band, hydroxy-L-proline (Hyp), and shows greater abundance in the Cd-treated S. torvum roots (data not shown). Although our present results, which were obtained from the transcriptome and metabolite analyses, supported this hypothesis, we did not observe any morphological differences between the roots’ endodermal Casparian bands of the two Solanum species (data not shown). In addition, it is difficult to explain why only Cd and Fe transport was restrained, while Zn and Cu transport was unaffected. Our study showed that during Cd treatment, no significant differences in Zn accumulation existed in S. torvum and S. nigrum. However, S. torvum accumulated a higher Fe concentration in the roots and a lower Fe in the leaves. These results suggest that S. torvum has a lower capacity of Fe loading into the xylem, which thereby limited its long-distance root-to-shoot transport. A similar phenotype of low Cd and Fe accumulation in S. torvum leaves implies that the modulating or transport pathways that were responsible for Cd and Fe loading into the xylem were similar.

Cadmium toxicity led to Fe deficiency in plants. Decreased Fe accumulation in leaves magnified the malfunction of the photosynthetic system and subsequent oxidative damage (Siedlecka et al., 1997; Solti et al., 2008). In this study, we found that the accumulation of Fe in S. torvum leaves is lower than that in S. nigrum. Therefore, we hypothesize that low Fe accumulation may be related to the high Cd sensitivity in S. torvum leaves compared with S. nigrum. Further study on the supplementation with Fe in Cd-treated plants supported the assumption. Supplementation with Fe effectively improved the Pn and reduced ROS accumulation in S. torvum plants, thereby increasing Cd tolerance in S. torvum leaves. However, we found that Fe supplementation did not affect Cd accumulation in leaves after 5 d of treatment; we therefore propose that the Fe-alleviated oxidative damage induced by Cd is a direct effect of Fe supply.

Iron deficiency increased Cd accumulation solely in S. nigrum leaves. This result is in agreement with previous reports by Lombi et al. (2002), who found that Cd uptake was significantly enhanced by Fe deficiency in the hyperaccumulator T. caerulescens Ganges, but not in the nonaccumulator T. caerulescens Prayon ecotype. The quantitative RT-PCR analysis of two Fe transporters, IRT1 and IRT2, indicated that the abundance of these mRNAs were higher in S. nigrum roots under Fe-deficient conditions than in S. torvum roots. Fe deficiency markedly induced the expression of IRT1 and IRT2 in S. nigrum roots, whereas it depressed the expression of these genes in S. torvum roots. IRT1 is localized in the plasma membrane, and the high expression of IRT1 increased Cd/Fe uptake in S. nigrum roots. However, the elevated expression level of IRT2 may result in Cd/Fe sequestration in root vacuoles to maintain a relatively low cellular Cd concentration in the shoots of S. torvum. Additionally, the different responsive mechanisms of the transporter genes to Fe deficiency may be responsible for the differential uptake and redistribution of metals in the two Solanum species. This topic requires extensive further analysis.

In summary, our data indicated that S. nigrum, which is a Cd accumulator, has a higher ability to promote root-to-shoot Cd translocation than its relative, the low Cd accumulator S. torvum. Differential responsive mechanisms of the transporter genes to Fe deficiency induced by Cd might be responsible for differential uptake and redistribution of Cd/Fe in the two Solanum species. On the other hand, Cd accumulation is a complex biological process that is involved in the integrated gene modulation network and coordinated regulation between roots and leaves. In the present study, we only focused on Cd uptake and translocation in roots. The remobilization of Cd through the phloem has been demonstrated to play a role in modulating Cd distribution and accumulation in plants (Reid et al., 2003; Tanaka et al., 2003; Grant et al., 2008). Further studies regarding the molecular mechanisms of Cd exclusion through the phloem would provide further insight into reducing Cd accumulation in plants.

Acknowledgements

The authors would like to thank the Germplasm Bank of Wild Species in Southwest China for providing the S. nigrum and S. torvum seeds. This work was supported by the China National Natural Sciences Foundation (31170228).

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