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First published online March 31, 2006; 10.1104/pp.105.074815 Plant Physiology 141:288-298 (2006) © 2006 American Society of Plant Biologists
The Shoot-Specific Expression of
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ABSTRACT |
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 TOP ABSTRACT RESULTSDISCUSSIONCONCLUSIONMATERIALS AND METHODSLITERATURE CITED |
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-glutamylcysteine synthetase (ECS) gene, S1pt::ECS, was expressed in the shoots of the ECS-deficient, heavy-metal-sensitive cad2-1 mutant of Arabidopsis (Arabidopsis thaliana). S1pt::ECS directed strong ECS protein expression in the shoots, but no ECS was detected in the roots of transgenic plant lines. The S1pt::ECS gene restored full mercury tolerance and partial cadmium tolerance to the mutant and enhanced arsenate tolerance significantly beyond wild-type levels. After arsenic treatment, the root concentrations of -glutamylcysteine (EC), PC2, and PC3 peptides in a S1pt::ECS-complemented cad2-1 line increased 6- to 100-fold over the mutant levels and were equivalent to wild-type concentrations. The shoot and root levels of glutathione were 2- to 5-fold above those in wild-type plants, with or without treatment with toxicants. Thus, EC and perhaps glutathione are efficiently transported from shoots to roots. The possibility that EC or other PC pathway intermediates may act as carriers for the long-distance phloem transport and subsequent redistribution of thiol-reactive toxins and nutrients in plants is discussed.
-glutamylcysteine (EC) may play important roles in the distribution and processing of nutrients and toxins. Phytochelatins (PCs) and the low-Mr peptide intermediates in PC biosynthesis, EC, and glutathione (GSH) can chelate or covalently bond to various elemental nutrients and toxins and are required for the transport of many toxins into the vacuole. Besides being rich sources of nitrogen and sulfur, these peptides might be expected to act as carriers for the long-distance phloem transport of small molecules between shoots and roots. This latter supposition is supported by data showing that, when plants are iron starved, the levels of nicotianamine, an endogenous chelator of iron, are modulated in leaves and roots, and believed to enhance both xylem and phloem mobility and redistribution of nicotianamine-chelated iron (Inoue et al., 2003
). A similar role can be proposed for the EC-containing peptides as long-distance transport and redistribution carriers of thiol-reactive nutrients, such as zinc and copper, and toxicants, such as arsenic, mercury, and cadmium. Supporting this hypothesis indirectly are data showing that the root-specific expression of Arabidopsis (Arabidopsis thaliana) PC synthase (PCS) in Arabidopsis resulted in increased levels of PCs and cadmium in shoots, presumably via long-distance xylem transport (Gong et al., 2003). In this article, we examine the long-distance movement of thiol-peptides from shoots down to roots by expressing a bacterial ECS protein in the shoots of an Arabidopsis ECS-deficient mutant using a shoot-specific, light-induced regulatory cassette.
The EC-containing peptides, GSH and PCs, play important roles in detoxifying thiol-reactive metals, such as cadmium and mercury, and the metalloid arsenic (Cobbett, 2000). The PCs are a family of peptides derived from EC and GSH, as shown in Figure 1
(Cobbett and Goldsbrough, 2002). The first enzyme in the PC biosynthetic pathway, EC synthetase (ECS), catalyzes the ATP-dependent formation of the unusual peptide bond between the -carboxyl group of Glu and the 
-amino group of Cys to make EC (Jez et al., 2004). This reaction is believed to be the rate-limiting and committed step in PC biosynthesis (Fig. 1; Noctor et al., 1998; Zhu et al., 1999b). GSH synthetase (GS) catalyzes a second ATP-dependent step combining EC with Gly to make GSH (Jez and Cahoon, 2004). The role of GS in making PCs is complicated by potential feedback inhibition of ECS enzyme activity by GSH in some organisms (Richman and Meister, 1975), but the role of GS is unclear in organisms like plants, where PC synthesis itself can remove excess GSH. Thus, the functional activity levels of both ECS and perhaps GS may limit the production of GSH (Zhu et al., 1999a). PCs are synthesized in a third and reiterative enzymatic step in the pathway catalyzed by PCS (Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al., 1999).
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; Zhu et al., 1999a, 1999b; Xiang et al., 2001; Li et al., 2004, 2005), based on an underlying assumption that the increased expression of these peptides might result in both tolerance and hyperaccumulation (Cobbett and Goldsbrough, 2002). The practical goal of this research has been to use these thiol-peptides in engineering the phytoremediation of soil and water contaminated with thiol-reactive elements (Meagher and Heaton, 2005; Meagher et al., 2006). Molecular genetic evidence that this pathway is essential to toxic-element processing is provided by studies showing that ECS- and PCS-deficient mutants of Arabidopsis are cadmium hypersensitive (Howden and Cobbett, 1992; Howden et al., 1995a, 1995b). In addition, Indian mustard (Brassica juncea) expressing transgenic ECS from a constitutive viral promoter show small increases in EC, GSH, and PC peptide levels. These plants are more cadmium resistant than the wild type (Zhu et al., 1999b) and accumulate 40% to 90% more cadmium in their shoots. Furthermore, transgenic Arabidopsis expressing very high levels of bacterial ECS and PCS have severalfold higher concentrations of EC, GSH, and PCs than the wild type, and show tolerance to arsenic and mercury (Dhankher et al., 2002; Li et al., 2004, 2005). In addition, the root-specific expression of PCS complemented the cadmium sensitivity of a PCS-deficient Arabidopsis mutant (Gong et al., 2003). The following study examined the long-distance movement of EC-containing peptides from shoots to roots. The bacterial ECS gene was strongly expressed in shoots. The Arabidopsis ECS-deficient mutant, cad2-1, served as an excellent host for these experiments because it contains minimal levels of the thiol-peptide products from this pathway. Our results demonstrate unambiguously that EC was very efficiently transported from shoots to roots. Results on the tolerance to and accumulation of toxic elements by these complemented mutant plants are also presented.
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RESULTS |
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TOPABSTRACTRESULTSDISCUSSIONCONCLUSIONMATERIALS AND METHODSLITERATURE CITED |
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The Arabidopsis mutant cad2-1 has dramatically reduced levels of ECS enzymatic activity due to a 6-base deletion within the sequence of the ECS gene (Cobbett et al., 1998). Homozygous mutant plants are hypersensitive to cadmium, mercury, and arsenic (Howden et al., 1995a; Ha et al., 1999). To examine the possible long-distance transport of the thiol-peptide products produced by ECS, the mutant cad2-1 was engineered to specifically express the Escherichia coli ECS protein in shoot tissues under control of a light-induced, shoot-specific expression system with 5' and 3' regulatory sequences derived from the soybean (Glycine max) Rubisco small subunit SRS1 gene, as shown in Figure 1, B and C. The S1pt::ECS gene was transformed into ECS-deficient cad2-1 homozygous mutant Arabidopsis plants.
ECS protein expression levels were examined among T2 generation S1pt::ECS-complemented cad2-1 mutant plant lines with western assays on 3-week-old plants using a monoclonal antibody, mAbECS1a, specific for the 57-kD bacterial ECS protein (Li et al., 2001). As shown in Figure 2
, high levels of ECS protein were easily detected in shoots, but not roots, of the representative S1pt::ECS-expressing lines (e.g. CS1, CS4, and CS6) and not in wild-type controls. These results suggest that bacterial ECS protein was neither synthesized in roots nor transported from shoots to roots. Preliminary experiments demonstrated that these and other S1pt::ECS plant lines expressing high levels of ECS were similarly tolerant to arsenic. Furthermore, these three plant lines grew approximately as well as unchallenged wild-type control plants on Murashige and Skoog (MS) salt medium. Therefore, the CS1 line was chosen as representative and carried forward for a more detailed analysis.
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We quantified thiol-peptide and Cys levels in the shoots and roots of the cad2-1 mutant, the CS1 line, and wild-type plants to determine the effectiveness of expressing ECS in shoots and the ability of EC or other intermediates in the PC pathway to be transported to roots. Because thiol-peptide accumulation in plants can be greatly enhanced by elevated concentrations of toxic heavy metals and metalloids, 3-week-old plants grown in liquid culture were transferred to fresh media for 72 h with and without relatively low concentrations of arsenate, cadmium, and mercury (see "Materials and Methods"). Thiol-peptide compositions of shoots and roots from treated plants were compared to those from plants grown on normal medium alone. Monobromobimane (mBBr)-derivatized thiol-peptides from the leaves and roots of the various plants following different treatments were separated by HPLC and identified by their fluorescence, as shown for arsenic-treated samples in Figure 3 .
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Shoot-Specific Expression of ECS Suppressed the Arsenic, Mercury, and Cadmium Hypersensitivity of the cad2-1 Mutant
Seed germination and plant growth in media without and with toxicants were compared among the mutant cad2-1, wild-type, and CS1 plants, using T2 generation CS1 seeds. Seeds of all three genotypes germinated efficiently and all plants grew similarly on half-strength MS salts (Fig. 5A ). The S1pt::ECS transgene in the CS1 plants suppressed the sensitivity phenotype of the mutant and resulted in significant levels of arsenate tolerance (Fig. 5B), although they did not grow nearly as well as unchallenged control plants (Fig. 5A). The CS1 plants were more resistant than wild type to arsenate concentrations ranging from 150 to 250 µM, as quantified in Figure 5C. The cad2-1 mutant seeds did not germinate on media containing 100 µM to 250 µM arsenate; therefore, no quantitative data for growth of the mutant on arsenic are shown.
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). The cad2-1, CS1, and wild-type plants were able to germinate on media containing 50 and 75 µM CdCl2, but not on higher concentrations of the toxicant shown in Figure 7
. Based on fresh shoot weight, the CS1 transgenic plants consistently showed significant Cd(II) tolerance relative to the cad2-1 mutant on media with 75 to 100 µM cadmium (Fig. 7, B and C). However, the CS1 plants grew slowly and showed more sensitivity to Cd(II) than wild type. The sensitivity of the CS1 and cad2-1 plants compared to the wild-type plants was statistically significant following 75 and 100 µM cadmium treatments (P < 0.0005–0.05).
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It seemed reasonable to expect that EC, GSH, and PCs would complex with thiol-reactive elements and result in increased accumulation of these toxicants in aboveground tissues. In other words, thiol-peptides provide a chemical sink for these reactive elements, and perhaps the thiol-peptide-element complexes would be stored at high levels in vacuoles as they are in yeast (Saccharomyces cerevisiae; Li et al., 1997; Sharma et al., 2002). To test for element accumulation, cad2-1 mutant, wild-type, and CS1 seeds were germinated in media with 25, 50, and 150 µM arsenate; 15 and 30 µM CdCl2; or 15 and 30 µM HgCl2. Plants were grown for 3 weeks, and the arsenic, cadmium, and mercury concentrations were quantified in the shoot tissues of these plants using inductively coupled plasma-optical mass spectroscopy, as shown in Figure 8
. Growing in media containing 25 µM arsenate for 3 weeks, the mutant cad2-1 accumulated 2-fold more arsenic in aboveground tissues than did wild-type or CS1 plants. Only minor differences in accumulation were found between wild-type and CS1 plants on higher concentrations (Fig. 8A). Growing on media with 15 and 30 µM HgCl2 for 3 weeks, the CS1 plants accumulated significantly more mercury in aboveground tissues than wild-type or mutant cad2-1 plants (Fig. 8B). When the plants were exposed to 15 µM Cd(II), there were no differences in cadmium accumulation among the cad2-1 mutant, the CS1, or wild-type plants (Fig. 8C). Similarly, there was no difference between the CS1 or wild-type plants on 30 µM CdCl2.
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DISCUSSION |
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TOPABSTRACTRESULTSDISCUSSIONCONCLUSIONMATERIALS AND METHODSLITERATURE CITED |
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This study examined the long-distance transport of the thiol-peptide EC from shoots, where it was synthesized, to roots. Recent publications have begun to identify the molecular genetic basis for systems controlling the long-distance transport of amino acids and nitrogen (Hirner et al., 1998; Fischer et al., 2002), purines (Burkle et al., 2003), sodium (Shi et al., 2002), and phosphate (Hamburger et al., 2002; Wang et al., 2004). EC, GSH, and PCs are rich sources of both nitrogen and sulfur, and may be efficiently transported for the redistribution of these nutrients and to supply them as substrates in various stress responses. The S1pt::ECS transgene, encoding the first enzymatic step in the PC biosynthetic pathway (Fig. 1), was specifically expressed in the shoots of the Arabidopsis ECS-deficient cad2-1 mutant. The S1pt::ECS plants expressed the bacterial ECS protein at high levels in the shoots, but not in roots, demonstrating the organ-specific, aboveground ECS expression from the transgene and, as expected, no phloem movement of this relatively large 57-kD protein (Fig. 2). The roots of the cad2-1 mutant had extremely low levels of EC, GSH, and PCs, establishing a low background in which to examine ECS enzyme expression. The majority of the molecular peptide phenotypes of the cad2-1 mutant were complemented by expression of the S1pt::ECS transgene. For example, the roots of the S1pt::ECS transgenic plants had much higher levels of EC and GSH than the mutant, levels that were as high or higher than wild type under all growth conditions. After 48 h of treatment with arsenic, the root, but not shoot, levels of PC2 and PC3 were also enhanced to wild-type levels. These results suggest that shoot-specific expression of ECS was sufficient to supply EC and GSH thiol-peptides to shoots and roots.
The levels of GSH were enhanced in shoots and roots dramatically above wild-type levels, with or without treatment with toxic elements. GSH levels were enhanced to similarly high levels in shoots and roots, suggesting GSH is more efficiently transported than EC from shoots to roots. However, EC peptide levels were significantly elevated in roots of the CS1 transgenic plants, making it possible that GSH synthesis was also enhanced in the roots of these plants. It should be noted that GSH synthesis and/or transport in the CS1 plants does not appear to be dependent upon other stress signals or other systems of induced phloem transport. Furthermore, the elevated GSH levels in transgenic plants suggest that EC substrate concentrations are all that limited GS-catalyzed synthesis of GSH (Fig. 1) in both the wild type and cad2-1 mutants, as suggested by previous studies in diverse prokaryotes and eukaryotes (Wu and Moye-Rowley, 1994; Grondin et al., 1997; May et al., 1998; Manna et al., 1999).
The fact that arsenic could more efficiently induce the biosynthesis of PCs in roots of these plants is not unexpected once the roots contain high levels of EC and GSH. Enhanced root PC synthesis may have occurred via the direct activation of PCS enzyme activity by arsenic (Fig. 1; Vatamaniuk et al., 1999, 2000). However, it is hard to explain why root PC levels were not similarly enhanced after cadmium treatment because Cd(II) is known to efficiently enhance PCS activities. Arsenic might have increased the stability of PC peptides more efficiently than Cd(II) and Hg(II). Withdrawing arsenic and cadmium for set time periods would allow an examination of the decay profiles of PC complexes. The possibility that arsenic more efficiently enhanced the shoot-to-root transport of these peptides than the mercury or cadmium treatments seems unlikely, because the arsenic-treated plants do not have more highly elevated GSH levels than the mercury- or cadmium-treated plants.
A previous study by Gong et al. (2003) used the root-specific expression of PCS to demonstrate the apparent xylem transport of thiol-peptides up to leaves. Our results using the leaf-specific expression of ECS demonstrate the existence of a complementary activity—EC can be synthesized in the shoots, and EC and apparently GSH are transported via the phloem down to roots. The long-distance phloem transport of EC and GSH has potential implications for the cotransport of bound thiol-reactive nutrients and toxic elements. Phloem transport of these thiol-peptides and their element complexes could provide an important mechanism for the lateral redistribution of nutrients like copper and zinc brought up from roots or the return of these nutrients to roots during leaf senescence in perennial plants. Several essential plant nutrient metal ions, including Zn(II), Cu(II), and Co(II), have reasonably strong affinities for Cys thiols. It has recently been demonstrated that a Zn-GSH or Zn-(GS)2 peptide complex acts as both the peptide donor and acceptor in the PCS-catalyzed formation of PCs (Vatamaniuk et al., 2000). However, our preliminary efforts to demonstrate the downward movement of arsenic applied to the shoots to the roots of CS1 plants did not detect any significant levels of root arsenic (data not shown). Future research will be needed to determine the precise roles of EC-containing peptides in conutrient or cotoxicant transport (Meagher and Heaton, 2005; Meagher et al., 2006). Finally, because phloem transport must involve many plasma membrane-to-plasma membrane intracellular transporters, our data suggests there is a great deal to learn about the molecular genetic basis for the rapid shoot-to-root movement of thiol-peptides in plants.
The Shoot-Specific Expression of Bacterial ECS Complemented the Arsenic-, Mercury-, and Cadmium-Sensitive Phenotypes of the cad2-1 Mutant
The cad2-1 mutant is hypersensitive to arsenic, mercury, and cadmium due to a block in EC synthesis and, hence, the PC pathway (Fig. 1). EC, GSH, and PCs play critical roles in toxic-element detoxification and stress responses (Cobbett and Goldsbrough, 2002; Cobbett and Meagher, 2003). With high levels of shoot-specific expression of the bacterial ECS, the S1pt::ECS plants showed significant tolerance to arsenic, mercury, and cadmium compared to cad2-1 plants. Considering the toxicity of these elements, it is unlikely that root tissues would have remained alive and functional in the S1pt::ECS-complemented cad2-1 lines had it not been for the efficient phloem transport of EC and/or GSH to roots.
The increased arsenate resistance associated with ECS expression in a cad2-1 mutant background (e.g. the CS1 line) was most striking. In fact, arsenate resistance in CS1 exceeded that observed for the wild-type plants. The oxyanion arsenate (AsO4–3) is a chemical analog of phosphate (PO4–3). There is reasonable evidence that arsenate is erroneously pumped into both yeast and plant cells by phosphate transporters (Ullrich-Eberius et al., 1989; Bun-ya et al., 1996), most likely due to the structural similarity of the two compounds (Ullrich-Eberius et al., 1989; Bun-ya et al., 1996). Arabidopsis plants deficient in root phosphate transporters are moderately resistant to arsenate (Shin et al., 2004). Arsenate is efficiently reduced into arsenite (AsO3–3) by GSH-dependent arsenate reductase activities in bacteria, fungi, plants, and animals (Oden et al., 1994; Mukhopadhyay et al., 1998; Dhankher et al., 2002, 2006; Dong et al., 2005). Arsenite is highly thiol-reactive and may be chelated and sequestrated as As(S-R)3 thiol-peptide complexes during its detoxification (Dey et al., 1994; Rosen, 1999; Sharples et al., 2000). Thus, the significant levels of arsenic tolerance observed for the S1pt::ECS/cad2-1 transgenic CS1 plants examined in this study must be at least in part due to high levels of EC and its downstream products in both roots and shoots relative to wild-type plants. The GSH levels surpassed those in wild type, and this may account for the greater resistance of the CS1 line to arsenic than wild type. Finally, these abnormally high levels of GSH may enhance GSH-dependent oxidative stress pathways, above the levels that can commonly be induced by toxins, thus providing increased resistance (Xiang et al., 2001; Ball et al., 2004).
The CS1 plants showed significant tolerance to mercury and cadmium compared to the cad2-1 mutant plants. The leaf-specific expression of the S1pt::ECS transgene restored mercury resistance to near wild-type levels (Fig. 6). Hg(II) is highly thiol-reactive and forms extremely strong bonds with sulfur, but there are many other chemical activities resulting in mercury toxicity (Rugh et al., 1996; Bizily et al., 2003). In response to Hg(II) stress, the levels of EC and GSH in the roots and shoots of CS1 plants were higher than in wild-type plants, whereas mercury caused no significant increases in the levels of PCs. These results suggest that the levels of EC and GSH were essential to mercury resistance, but the levels of these peptides alone were not directly proportional to mercury resistance or the transgenic plants would have been more mercury resistant than wild type. Perhaps the GSH-dependent oxidative stress-response pathway indirectly protects cells from the toxic effects of mercury, and this pathway is not enhanced when GSH levels are increased beyond wild-type levels. Furthermore, the lack of observed increases in PCs suggests that mercury may not enhance the enzymatic activity of PCS as effectively as other thiol-reactive metal ions like Cd(II) (Vatamaniuk et al., 2000, 2004). Although, transgenic PCS expression did enhance the resistance of yeast to arsenic, mercury, and cadmium (Vatamaniuk et al., 1999). Consistent with the view that PCS is not activated by mercury are our data showing that the strong overexpression of Arabidopsis PCS only weakly increases mercury resistance in transgenic Arabidopsis (Li et al., 2004). In light of the CS1 line's resistance to arsenic and mercury, it might seem surprising these plants were relatively poorly complemented for Cd(II) resistance relative to the levels of resistance for wild type (Fig. 7). However, even wild-type Arabidopsis that are strongly and constitutively overexpressing either ECS or PCS in shoots and roots are hypersensitive to cadmium, suggesting that Arabidopsis may have special problems processing the cadmium-thiol-peptide complexes (Li et al., 2004, 2005).
Role of EC-Containing Peptides in Accumulation of Toxicants
The S1pt::ECS complemented Arabidopsis cad2-1 mutants in the expression of high levels of several thiol-peptides aboveground, but did not accumulate more arsenic or cadmium aboveground compared to the cad2-1 mutant or wild-type plants and only accumulated about 30% more mercury. One possible explanation for these results is that toxicants taken up by roots were chelated to form the thiol-peptide-metal complexes, which themselves do not move to the aboveground tissues; only the toxicants in the form of free ions or in complexes with other non-thiol-peptide carriers like His may be transported to the shoots. By this proposal, the amount of the shoot-transportable toxicant available would be inversely proportional to the levels of thiol-peptides in roots. This inverse relationship is supported by the fact that, at a very low arsenic concentration where the cad2-1 mutant grew and had extremely low levels of these thiol-peptides, it accumulated 2-fold more arsenic aboveground than the CS1 line or the wild type (Figs. 4 and 8). Similar results were reported for the cad2-1 mutant relative to wild type (Howden et al., 1995a; Cobbett et al., 1998). Contrary to the view of thiol-peptides sequestering the toxicants in roots are the recent data of Gong et al. (2003). Using the root-specific expression of Arabidopsis PCS to enhance PC levels, they demonstrate the apparent xylem transport of thiol-peptide metal-ion complexes up to leaves, and increased accumulation of cadmium over what occurs in the cad1-3 PCS mutant. Our preliminary attempts to show altered rates of arsenic movement from shoots to roots of the CS1 line did not reveal any differences relative to wild type or the cad2-1 mutant (data not shown).
The levels of EC peptide in the transgenic CS1 plants were 2- to 10-fold greater than in cad2-1 plants under various growth conditions. The CS1 plant levels of GSH were 2- to 10-fold higher than in the mutant and 2- to 5-fold higher than wild type with or without mercury treatment. And yet, the level of mercury accumulation in the aboveground tissues of the CS1 plants was only 65% higher than in wild-type plants (Fig. 8B). As with arsenic and cadmium, there is little correlation between mercury accumulation and the thiol-peptide content of these plants.
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CONCLUSION |
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TOPABSTRACTRESULTSDISCUSSIONCONCLUSIONMATERIALS AND METHODSLITERATURE CITED |
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MATERIALS AND METHODS |
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TOPABSTRACTRESULTSDISCUSSIONCONCLUSIONMATERIALS AND METHODSLITERATURE CITED |
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The coding sequence of the Escherichia coli SK1592 ECS gene was cloned previously by Li et al. (2001). A light-induced, leaf-specific expression vector with the promoter region and terminator from SRS1 (termed collectively S1pt) was used to express the ECS gene in plants (Fig. 1A). The SRS1 promoter region was previously described for the S1p vector by Dhankher et al. (2002). The S1pt cassette was constructed by adding the terminal 3'-untranslated region (UTR) and polyadenylation signals to S1p (Shirley et al., 1987, 1990; Fig. 1B). The ECS coding sequence was cloned into the NcoI/HindIII replacement region of the S1pt cassette in pBluescript SK(II), and then the assembled construct was moved as a SacI/XhoI fragment into the replacement region of the binary vector pBin19 to make S1pt::ECS. The above construct was introduced into the cad2-1 mutant of Arabidopsis (Arabidopsis thaliana; ecotype Columbia) by Agrobacterium-mediated transformation according to Ye et al. (1999). The sterilized T1 transgenic seeds were plated onto MS medium (Murashige and Skoog, 1962) and supplemented with kanamycin (50 µg/mL) and timentin (300 µg/mL) for 3 weeks before transplanting the surviving seedlings into soils.
ECS expression was compared among transgenic, mutant, and control plants on western blots (Li et al., 2001) using an E. coli ECS-specific monoclonal antibody as described previously (Li et al., 2001). Primary antibody was followed by horseradish peroxidase-conjugated goat antimouse or antirabbit antisera, respectively (Sigma), and enhancement using an ECL kit from Amersham following the manufacturer's instructions. Protein concentrations of the plant extracts were determined by a Bradford assay (Bradford, 1976). Equal loading of each sample was further confirmed by Coomassie Brilliant Blue G staining of aliquots run previously on separate gels (data not shown). The concentration of ECS in shoots was similar to that determined for the ECS gene expressed from the constitutive actin ACT2 promoter (Li et al., 2005), or approximately 0.1% of total shoot protein (data not shown).
Plant Growth, and Heavy-Metal and Metalloid Treatments
The Arabidopsis wild type, its mutant cad2-1, and the transgenic S1::ECS/cad2-1 plants were grown for all assays with cycles of 16 h light and 8 h darkness at 22°C (±1°C) on agar or in liquid media (Li et al., 2005). To test metal and metalloid resistance, the sterilized seeds (Li et al., 2001) were plated onto solid 0.8% plant tissue culture grade agar (Caisson Laboratories) containing half-strength MS (Murashige and Skoog, 1962) medium and various levels of arsenate (Na3AsO4), mercury (HgCl2), or cadmium (CdCl2; Figs. 5–7
). After the seeds germinated, the plates were vertically positioned and grown for 3 weeks before the shoot fresh weight was quantified. Three sets of 10 plants were weighed for each plant genotype and treatment. To determine thiol-peptide levels, 3-week-old plants that were grown on platforms in liquid MS media and were prepared for HPLC assays after 72-h exposure to 25 µM CdCl2 or 100 µM Na3AsO4 as described by Li et al. (2005). The Cd(II)-treated cad2-1 mutant roots turned brown, but the tissues and cells appeared intact. A lower concentration of 5 µM HgCl2 was used instead of the 25 µM concentration used in previous studies (Li et al., 2004, 2005). In initial experiments, exposing cad2-1 plants to 25 µM Hg(II) in liquid for 48 to 72 h caused the plant roots to turn black, lose cellular integrity, and lose more than 50% of their weight. Exposing cad2-1 plants to 5 µM Hg(II) resulted in root tissues turning gray, suggesting they were still taking in mercury, but these tissues appeared intact and viable during the treatment.
HPLC Analysis of Thiol-Peptides
Cys and thiol-containing peptides EC, GSH, PC2, and PC3 were analyzed using fluorescence-detection HPLC as described (Fahey and Newton, 1987). Peptides were extracted and derivatized with mBBr as described previously (Sneller et al., 2000; Cazale and Clemens, 2001; Sauge-Merle et al., 2003) with minor modifications (Li et al., 2005). In vitro-labeled Cys and peptides were prepared as described by Li et al. (2004).
Quantification of Arsenic, Mercury, and Cadmium in Shoot Tissues
Assays on the accumulation of arsenic, cadmium, or mercury in shoot tissues were conducted in liquid culture. The mutant cad2-1 or transgenic S1::ECS/cad2-1 plants were grown for 3 weeks in half-strength solid MS medium containing various low concentrations of arsenate (25, 50, and 150 µM), or Hg(II) (10 and 30 µM HgCl2), or Cd(II) (15 and 30 µM CdCl2). Shoots were harvested, and washed three to four times with deionized water to remove any traces of surface contamination. For mercury assay, the shoot tissues were lyophilized at –34°C for more than 72 h. . For arsenic or cadmium assays, the shoot tissues were dried at 80°C incubator instead of lyophilized. Shoot samples were ground to a powder prior to acid digestion, and elemental analysis was preformed as described by Suszcynsky and Shann (1995) and modified by Li et al. (2004).
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers 08X900 and P00865.
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ACKNOWLEDGMENTS |
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Received November 29, 2005; returned for revision February 25, 2006; accepted March 6, 2006.
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FOOTNOTES |
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2 Present address: Emory School of Medicine, Department of Human Genetics, Emory University, Atlanta, GA 30345.
3 Present address: Department of Plant, Soil, and Insect Sciences, University of Massachusetts, Amherst, MA 01002.
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: Richard B. Meagher (meagher{at}uga.edu).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.074815.
* Corresponding author; e-mail meagher{at}uga.edu; fax 706–542–1387.
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-Glutamylcysteine Synthetase Directs the Long-Distance Transport of Thiol-Peptides to Roots Conferring Tolerance to Mercury and Arsenic
