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First published online July 21, 2006; 10.1104/pp.106.080283 Plant Physiology 143:50-59 (2007) © 2007 American Society of Plant Biologists
Characterization of a Novel Plant Promoter Specifically Induced by Heavy Metal and Identification of the Promoter Regions Conferring Heavy Metal Responsiveness1Department of Biology, Graduate University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China (X.Q., T.C.); and Department of Bio-Engineering, School of Chemical and Environmental Engineering, China University of Mining and Technology (Beijing), Beijing 100083, People's Republic of China (Y.Z.)
The bean (Phaseolus vulgaris) stress-related gene number 2 (PvSR2) gene responds to heavy metals but not to other forms of environmental stresses. To elucidate its heavy metal-regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region (–1623/+48) of PvSR2. Deletions from the 5' end revealed that a sequence between –222 and –147 relative to the transcriptional start site was sufficient for heavy metal-specific induction of the promoter region of PvSR2. Detailed analysis of this 76-bp fragment indicated that heavy metal-responsive elements were localized in two regions (–222/–188 and –187/–147), each of which could separately confer heavy metal-responsive expression on the  -glucuronidase gene in the context of a minimal cauliflower mosaic virus 35S promoter. Region I (–222/–188) contains a motif (metal-regulatory element-like sequence) similar to the consensus metal-regulatory element of the animal metallothionein gene, and mutation of this motif eliminated the heavy metal-inducible function of region I. Region II (–187/–147) had no similarity to previously identified cis-acting elements involved in heavy metal induction, suggesting the presence of a novel heavy metal-responsive element. Transformed tobacco (Nicotiana tabacum) seedlings expressing -glucuronidase under control of the PvSR2 promoter region (–687/+48) showed heavy metal-specific responsive activity that depended on the type and concentration of the heavy metal and the type of organ. These findings further our understanding of the regulation of PvSR2 expression and provide a new heavy-metal-inducible promoter system in transgenic plants.
In plants, a number of heavy metals are essential for normal growth as cofactors and as structural and catalytic components of proteins and enzymes. These micronutrients, as well as nonessential heavy metals such as cadmium, are toxic at high levels. Plants have evolved a suite of mechanisms that control and respond to the uptake and accumulation of both essential and nonessential heavy metals. These mechanisms include the chelation and sequestration of heavy metals by particular ligands including small peptides, organic acids, and amino acids, which bind free metal ions. They contribute to metal detoxification by buffering cytosolic metal ions (Clemens, 2001  ). The two best-characterized heavy metal-binding ligands in plant cells are the phytochelatins (PCs) and metallothioneins (MTs; for review, see Cobbett and Goldsborough, 2002). MTs are Cys-rich polypeptides encoded by a family of genes. In contrast, PCs are a family of enzymatically synthesized Cys-rich peptides. The induction of PC synthesis is based on the posttranscriptional activation of preformed PC synthase and is not under transcriptional control.
Several heavy metal-inducible genes have been reported in plants (Hagen et al., 1988
In yeast (Saccharomyces cerevisiae) and animals, heavy metal-responsive elements (HMREs) and their corresponding metal-induced transcription factors have been characterized in detail. In animals, MT genes are regulated transcriptionally by metal-regulatory elements (MREs), which are present in multiple copies (Stuart et al., 1985
We have previously isolated a novel heavy metal-specific responsive gene, Phaseolus vulgaris stress-related gene number 2 (PvSR2), from bean (Phaseolus vulgaris), which is different from previously identified plant MT-like genes (Zhang et al., 2001
The PvSR2 Promoter Contains Regulatory Elements in Response to Environmental Signals
We have previously reported a 5', 3' truncated cDNA (GenBank accession no. U54704) of the PvSR2 gene (Zhang et al., 2001
Analysis of the PvSR2 promoter using TRANSFAC (http://www.transfac.gbf.de/cgi-bin/) showed that a number of potential cis-acting elements to respond to environmental signals were present. An MRE-like sequence (5'-TGCAGGC-3'), similar to the conserved core MRE sequence involved in heavy metal-induced expression of MT genes in animals (Stuart et al., 1985 ), was located at position –212/–206. Two putative GATA boxes (–859/–856 and –807/–804) and one I box (–1,480/–1,475) thought to be involved in light response were also found (Giuliano et al., 1988; Lam and Chua, 1989). Nine putative basic motifs (5'-NGAAN-3') of heat shock elements were present in –1,501/–1,496, –1,373/–1,368, –1,219/–1,214, –1,072/–1,067, –1,019/–1,014, –993/–988, –463/–458, –259/–254, and –191/–186. However, a functional heat shock element contains at least three basic repeats that must be arranged in alternating orientations (Amin et al., 1988; Xiao and Lis, 1988).Thus, none of them appears to be functional. Lastly, an abscisic acid-response element-like motif (5'-ACGTG-3') required for gene expression during dehydration stress (Simpson et al., 2003) was located at –134/–130. These putative regulatory elements suggest that the promoter region of PvSR2 (P-PvSR) may respond to a variety of environmental signals, including heavy metal stress, dehydration, and light. The sequence also contains a putative TATA box (TATAAATT) at –24/–31 relative to the TSS and a putative CAAT box at –226/–223.
To test the heavy metal-inducible activity of the PvSR2 promoter and localize the heavy metal-regulatory region, the 1,671-bp promoter fragment (–1,623/+48) was transcriptionally fused to the promoterless reporter gene
To test whether the MRE-like sequence present in this 76-bp promoter region was responsible for heavy metal induction, we also tested the chimeric construct pBmMRP0.2, in which the MRE-like sequence was mutated with G to T or A to C. Mutation of the MRE-like sequence significantly decreased heavy metal-inducible GUS expression level by approximately 27% compared to the wild-type version (based on one-sided paired t test; P < 0.05), while GUS activity in the absence of heavy metals was reduced by only approximately 2%, showing no statistical difference between the MRE-like sequence and its mutated version (P > 0.05; Student's t test). The result indicated that the MRE-like sequence could contribute to efficient heavy metal induction, other important elements must also be present in the –222/–147 region of the promoter.
To test this, the 76-bp promoter fragment was subdivided into two pieces, one from –222/–188 (termed region I) and the other from –187/–147 (termed region II). A third construct, in which the MRE-like sequence in region I was mutated as before, was also prepared (Fig. 3A
). All three sequences were fused to a CaMV 35S minimal promoter (35S
The PvSR2 Promoter (–687/+48) Drives Heavy Metal-Inducible GUS Expression in Transgenic Tobacco Seedlings
To assess further heavy metal-inducible promoter activity in stably transformed plants, the PPvSR2 (–687/+48):GUS chimeric construct (pMRP0.7-GUS) was introduced into tobacco. T-DNA inheritance was scored by kanamycin segregation analysis in the T1 generation. The T1 seedlings of six T0 lines (T0-3, T0-5, T0-6, T0-8, T0-12, and T0-17) segregated as a single, dominant, Mendelian character, and the data is presented in Table I
. Among six lines, line T0-8 was randomly selected and its kanamycin-resistant T1 seedlings were employed to analyze GUS transcript accumulation following heavy metal stress by northern-blot analysis. The data showed significant levels of GUS mRNA in Cu2+-, Zn2+-, Cd2+-, and Hg2+-treated seedlings compared to the control seedlings grown in the absence of heavy metals (Fig. 4
). This pattern of induction is consistent with previous northern-blot assay of the PvSR2 gene (Zhang et al., 2001
The transformed seedlings of the above six lines were selected to further analyze the effects of type and concentration of the heavy metal or the type of organ on the promoter activity. The P-PvSR[–687/+48] responded to various heavy metal ions by a 4- to 10-fold induction of GUS activity (Fig. 5A ). After treatment with 20 µM toxic nonessential heavy metal ions for 24 h, the highest inducible GUS activity was observed in the seedlings treated with Ag+ (10.3-fold induction), followed by Cd2+ (8.4-fold induction), Hg2+ (6.3-fold induction), and Pb3+ (5.2-fold induction). After treatment with 100 µM essential heavy metals for 24 h, Zn2+ (8.7-fold induction) was found to be the most potent inducer of GUS activity, followed by Cu2+ (4.6-fold induction), Mn2+ (4.5-fold induction), and Co2+ (3.6-fold induction). The seedlings treated with 1% (w/v) NaCl showed no significant induction (1.1-fold) compared with the control seedlings (without heavy metal stress).
Because Cu2+ is readily taken up by plants and is easy to apply, it was chosen for studying the effects of increasing heavy metal concentration on GUS activity. GUS activity increased as a function of Cu2+ concentration from 0 (control, untreated with Cu2+) to 50 µM and decreased thereafter (Fig. 5B). This decrease presumably reflects the toxic effect of Cu2+ at high concentrations. Thus, to compare the inductive effects of heavy metals in leaves and roots, seedlings were treated with copper at the optimal concentration (50 µM) for 24 h. The GUS activity in the young leaves was 3-fold higher than that in the young roots in response to copper. GUS activity in both young leaves and young roots was induced roughly 4-fold by 50 µM Cu2+, indicating the leaf is not more metal responsive than the root (Fig. 5C). We conclude that the P-PvSR[–687/+48] responds to various heavy metals, but its activity varies with the kind and concentration of heavy metal. To acquire more detailed information on the location of P-PvSR[–687/+48] expression, histochemical GUS staining was performed with seedlings of line T0-8 treated by several heavy metal ions. Compared to untreated seedlings, which showed little GUS staining (Fig. 6A ), GUS expression were strongly induced by Hg2+ (Fig. 6B), Cd2+ (Fig. 6C), Cu2+ (Fig. 6D), and Zn2+ (Fig. 6E). A magnified view of GUS staining after treatment by Zn2+ (Fig. 6F) clearly shows the distribution through the vasculature.
Two HMREs Contribute to the Heavy Metal Responsiveness of the PvSR2 Promoter In this study, we have characterized a heavy metal-specific promoter both in a transient expression system and transgenic tobacco plants. Our analysis indicated HMREs were found localized in two regions (–222/–188 and –187/–147), each of which could separately confer heavy metal-responsive expression on the GUS reporter gene. The 35-bp region I contains a MRE-like sequence that exhibited a weak heavy metal induction (about 2-fold) in the context of a minimal CaMV 35S promoter (Fig. 3C). The PvSR2 MRE-like sequence differs from animal MRE core sequence in the fifth position, in which a G is replaced by a C and by flanking sequences that are AT rich rather than GC rich. Since mutation of the MRE-like sequence attenuated the heavy metal-inducible function of region I, we conclude that the MRE-like sequence is required for the heavy metal-inducible function of this region.
Of greater interest, however, was the 41-bp region II, which showed a greater heavy metal induction (4- to 5.5-fold) in the context of a minimal CaMV 35S promoter (Fig. 3C). Previously identified cis-acting elements involved in heavy metal-induced gene expression include MRE of animal MT genes (Stuart et al., 1985
The choice of promoter is often of great importance in regulating transgene in plants. The use of a heavy metal-inducible promoter to control the target gene expression could avoid potential harmful effects due to overexpression of the target gene under control of constitutive promoters in transgenic plants. For example, the yeast copper-inducible promoter system has been well characterized in transgenic tobacco plants for controlling GUS (Mett et al., 1993
Although originally identified in the promoter of all mammalian MT genes, MRE or MRE-like sequences have also been found in the promoters of plant metal-responsive or regulatory genes such as some MT-like genes (Evans et al., 1990
The MRE-like sequence (5'-TGCAGGC-3') present in –212/–206 of P-PvSR, can, in the context of a minimal CaMV 35S promoter, induce GUS expression by about 2-fold in the presence of heavy metals. This is in agreement with the findings that a single copy of MRE from mouse MT gene (Culotta and Hamer, 1989
A comprehensive understanding of the detailed mechanisms for metal-regulated transcription will ultimately require an understanding of how eukaryotic cells sense, transport, distribute, and remove metals from their environment. Metal-regulated transcription in higher plants is not well documented. In higher eukaryotes, MT genes are the most intensively studied and best-understood examples of metal-regulated transcription units. An MTF-1 binds to MREs and regulates animal MT gene transcription (Westin and Schaffner, 1988
Although similar MRE core elements were found in the upstream region of some plant genes, no heavy metal-responsive functions of them were reported so far. Thus, heavy metal-responsive transcription factors binding to MRE or MRE-like sequences is not a main pathway in plants. Therefore, alternative mechanisms of sensing, such as signal transduction pathways, that involve protein kinases may be involved. Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, it has been shown that MAPKs play a role in the signaling of biotic and abiotic stresses, plant hormones, and cell cycle cues. Recently, the effect of cadmium on plant MAPKs in rice suggests that a MAPK cascade may function in the cadmium-signaling pathway (Yeh et al., 2004
Plant Materials and Bacterial Strains
Tobacco (Nicotiana tabacum L. cv W38) plants were grown in Murashige and Skoog (MS) medium under a 16-h-light (25°C)/8-h-dark (20°C) cycle. Surface-sterilized bean (Phaseolus vulgaris L. cv Saxa) seeds were grown in pots (in soil) with a photoperiod of 16 h at 22°C during the day and at 18°C during the night in the greenhouse. When two primary leaves were fully expanded, the plants were sprayed with 0.2% (w/v) HgCl2. Escherichia coli strain DH5
The 5'-flanking region of PvSR2 was isolated from bean genomic DNA according to the instructions of TaKaRa LA PCR in vitro cloning kit (TaKaRa). Briefly, genomic DNA isolated from young bean leaves was digested with restriction enzymes PstI and EcoRI and then ligated to a cassette DNA with corresponding restriction sites. A primary PCR to amplify the 5'-regulatory region was performed with a PvSR2 gene-specific primer (GSP; 5'-CTCCACTGTGTTAACGCCGGGCTTC-3') and cassette primer C1. Diluted primary PCR products were then amplified using a nested PvSR2 GSP (5'-GTTCCTTGGCGTAAGAGTAGAGGATGG-3') and cassette primer C2. The PCR and nested PCR amplifications were performed as described by the manufacturer. The nested PCR products were cloned into a pMD18-T vector (TaKaRa), sequenced, and designated as pUC-MRP.
Total RNA was isolated from the leaves of bean 6 h after spraying with 0.2% (w/v) HgCl2 using RNAgent Total RNA Isolation system (Promega). 5' RACE was carried out using a SMART RACE cDNA Amplification kit (CLONTECH) on adapter-ligated cDNA, synthesized from 1 µg of total RNA. 5'-RACE reaction was performed using a PvSR2-specific primer1 (GSP1; 5'-AGATGGAACCTGTCGTACACCGGA-3') by touchdown PCR program, as described by the manufacturer. The PCR fragments were cloned into a pMD18-T vector and sequenced. Four separated positive clones were sequenced for accurately mapping the transcription start site.
A series of nested 5' deletions of P-PvSR fragments (1.6, 1.2, 0.7, 0.2, and 0.15 kb) were amplified by PCR from pUC-MRP using the common antisense primer (ASP; 5'-GCTCTAGATGATGGAACTGTGAAGATTGT-3') and either the sense primers SP1 (5'-CCCAAGCTTCTGCAGACATCGTTTTGTATT-3'), SP2 (5'-CCCAAGCTTTGTTTTGAAATAGGAAAAAGTAAC-3' ), SP3 (5'-CCCAAGCTTTTCTTCCTACATCTCACCCA-3'), SP4 (5'-CCCAAGCTTAATTTAAATGCAGGCGTTT-3'), or SP5 (5'-CCCAAGCTTAGGAAGCAATAACGTGGAAAAT-3'), respectively. The 5'-deletion derivatives were cloned into the HindIII/XbaI sites of pBI221 (CLONTECH) upstream of GUS instead of the CaMV 35S promoter, producing constructs pBMRP1.6, pBMRP1.2, pBMRP0.7, pBMRP0.2, and pBMRP0.15, respectively. Similarly, the promoter fragment (–222/+48) with mutation of the MRE-like sequence was obtained by PCR from pBMRP0.2 using the ASP and the sense primers mSP4 (5'-CCCAAGCTTAATTTAAAGTACTTAGTTTTTATATTTGAAAGA-3'). Above PCR amplification was completed using 0.25 units of LA Taq DNA Polymerase (TaKaRa) and 1 ng template in a 25-µL reaction mixture for 25 cycles. PCR cycling parameters were denatured at 94°C for 30 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for 1 min, with a final elongation at 72°C for 10 min. The PCR products were inserted to the HindIII/XbaI sites of pBI221 to yield pBmMRP0.2. The promoter region of all constructs was confirmed by sequencing. The underlined sequences on all above primers indicate the HindIII and XbaI restriction endonuclease sequences that were included to facilitate the cloning into pBI221. The modified firefly gene (luc+) derived from pGL3-Basic (Promega) was amplified by PCR with the primer LucSP (5'-GCTCTAGAGCCACCATG GAAGACGCCA-3'; XbaI restriction site underlined) and the LucASP (5'-TACCGAGCTCTTACACGGCGATCTTTCCGC-3'; SacI restriction site underlined). The PCR amplification was performed at an annealing temperature of 58°C for 25 cycles. The PCR products were cloned into a pMD18-T vector, sequenced, and designated as pUC-Luc. The XbaI-SacI fragment containing the luc+ from pUC-Luc was inserted into the same sites of pBI221 instead of GUS to yield the plasmid pBI221-Luc. To generate construct pMRP0.7-GUS for the transformation of tobacco, a HindIII-EcoRI fragment containing the P-PvSR[–687/+48]-GUS cassette from pBMRP0.7 was inserted into the HindIII/EcoRI sites of pBI121 (CLONTECH).
The 35-bp region I promoter sequence (–222/–188) or its derivative containing a mutated MRE-like sequence was introduced upstream from the minimal CaMV 35S promoter (–64/+8, 35S
Isolation of tobacco mesophyll protoplasts and protoplasts were transiently transformed with polyethylene glycol as described by Negrutiu et al. (1987)
GUS activity was quantified as described by Jefferson (1987)
A. tumefaciens-mediated tobacco transformation with pMRP0.7-GUS was performed as described by Horsch et al. (1985) The sterilized six lines seeds were germinated in heavy metal-free MS agar medium (MnSO4-, ZnSO4-, Na2MoO4-, CuSO4-, and FeSO4-deficient MS medium) containing 200 µg/mL kanamycin for 10 d. For each treatment, ten 10-d-old kanamycin-resistant seedlings from each line were analyzed. To analyze the responsive type of heavy metal ions, the seedlings were transferred to the same MS medium supplemented with either 20 µM each HgCl2, CdCl2, AgNO3, and Pb(NO)3 or 100 µM each ZnSO4, CoCl2, CuSO4, and MnCl2 or 1% (w/v) NaCl. In the control groups, the seedlings and wild-type tobacco seedlings were grown in heavy metal-free MS agar medium with no heavy metal stress. For dose response curves, different concentrations (0, 5, 10, 50, 70, and 100 µM) CuSO4 were applied. After induction by heavy metal for 24 h, the seedling leaves were homogenized in 1 mL of chilled GUS extraction buffer. GUS extraction and GUS assay were carried out as described above. In addition, seedlings were induced by 50 µM CuSO4 for 24 h, and GUS activity in the leaves and roots of the seedlings were also analyzed, respectively.
Histochemical localization of GUS activity in transgenic plant seedlings of line T0-8 treated without (control) or with 50 µM each of HgCl2, CdCl2, ZnSO4, and CuSO4 for 24 h was performed as previously described by Jefferson et al. (1987)
Total RNA was isolated from the leaves of 10-d-old seedlings of line T0-8 grown in heavy metal-free MS agar medium either alone or supplemented with 20 µM each of CdCl2 or HgCl2, or 100 µM each of CuSO4 or ZnSO4. Fifteen micrograms total RNA was separated on a 1.2% (w/v) formaldehyde agarose gel electrophoresis, transferred onto nylon membranes (Millipore), and hybridized with [ Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers U54704 and DQ109992.
We are grateful to Professor Hans Lambers (University of Western Australia) and Professor David Morse (Université de Montréal) for comments on the manuscript. Received March 12, 2006; accepted July 10, 2006; published July 21, 2006.
1 This work was supported by the National Natural Science Foundation of China (grant nos. 39870078, 30370128, and 30570146) and by the National Program of Research and Development of Transgenic Plants of China (grant nos. J00–A–008–03 and JY–03–A–20–02) to T.C. 
2 Present address: College of Life Science, Capital Normal University, Beijing, People's Republic of China. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Tuanyao Chai (tychai{at}gucas.ac.cn). www.plantphysiol.org/cgi/doi/10.1104/pp.106.080283 * Corresponding author; e-mail tychai{at}gucas.ac.cn; fax 86–10–8825–6343.
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TOP
ABSTRACT
RESULTS
cells (Zhang et al., 2001
64, –64/+8 from the TSS) upstream of the GUS gene to generate pBMRE, pBmMRE, and PvSR[–187/–147]-GUS (
2 values were calculated with 1 degree of freedom.
