JXB Advance Access originally published online on May 23, 2006
Journal of Experimental Botany 2006 57(10):2173-2182; doi:10.1093/jxb/erj176
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RESEARCH PAPER |
Effects of simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on thiol content and cadmium accumulation in tobacco plants
skiskaska
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawiskiego 5A, 02-106 Warsaw, Poland
*To whom correspondence should be addressed. E-mail: asirko{at}ibb.waw.pl
Received 11 October 2005; Accepted 27 February 2006
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Transgenic tobacco (Nicotiana tabacum cv. LA Burley 21) lines expressing three genes encoding enzymes thought to be critical for the efficient production of phytochelatins, (i) serine acetyltransferase (EC 2.3.1.30 [EC] ) involved in the production of O-acetylserine, the cysteine precursor, (ii)
-glutamylcysteine synthetase (EC 6.3.2.2
[EC]
) involved in the production of -glutamylcysteine, the precursor of glutathione, and (iii) phytochelatin synthase (EC 2.3.2.15
[EC]
), were obtained and analysed for non-protein thiol content and cadmium accumulation. After a 3 week exposure to 15 µM CdCl2, plants expressing transgenes (either separately or in combination) had increased cadmium concentration in roots but not in shoots compared with the wild type. Nearly all transgenic lines analysed had more non-protein thiols than the wild type. The greatest effects (about 8-fold elevation of thiols) were found in one of the lines simultaneously expressing the three transgenes. Despite the fact that a multi-transgene strategy described in this work resulted in a strong increase in the levels of several classes of non-protein thiols in transgenic plants, other factors appeared to restrict cadmium accumulation in shoots. Key words: Cadmium, glutathione, phytochelatin, thiols, tobacco, transgenic plants
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cadmium (Cd) is a widespread heavy metal that is mainly released to the environment by anthropogenic sources and is highly toxic to animals and plants. Cd decreases the growth rate of plants by affecting various aspects of metabolism (Sanita di Toppi and Gabbrielli, 1999). Toxic Cd levels inhibit the normal uptake and utilization of macro- and micronutrients (Metwally et al., 2005). Toxicity of Cd may also result from its binding to sulphydryl groups of proteins leading to inhibition of activity or disruption of structure, perturbations in the intracellular calcium level (Perfus-Barbeoch et al., 2002), disturbance of cellular redox control (Schutzendubel et al., 2002), and/or inducing the production of reactive oxygen species (Romero-Puertas et al., 2004).
Chelation of the metal by phytochelatins (PCs) and compartmentalization of the complex in vacuoles (Clemens, 2001; Clemens et al., 2002; Tong et al., 2004) are generally considered ‘first line’ defence mechanisms. The efflux of Cd and its immobilization within the cell wall (Carrier et al., 2003), as well as formation and excretion of crystals containing Cd and calcium (Ca) from trichomes (Choi et al., 2001), were also reported. The ‘second line’ defence mechanisms are related to the synthesis of stress-related compounds, including heat-shock proteins, proline, and ethylene (Sanita di Toppi and Gabbrielli, 1999; Sharma and Dietz, 2006).
Plant species significantly differ in tolerance to and uptake of Cd. Some plant species have developed metal hypertolerance and hyperaccumulation mechanisms. Moreover, multiple mechanisms could be responsible for efficient Cd accumulation even in the same plants species. For example, in chickpea roots, tolerance to Cd appeared to be controlled by two types of factors, PC-dependent and PC-independent (Gupta et al., 2002). Generally, it is assumed that in non-hyperaccumulator plants PCs are essential for tolerance, whilst in the Cd-hypertolerant ecotypes (Silene vulgaris, Thlaspi caerulescens) PCs do not contribute to the Cd tolerance. This suggests that PC-mediated chelation might not be the most effective strategy to cope with an exposure to toxic levels of this metal (Ebbs et al., 2002; Schat et al., 2002). Nevertheless, in non-metallicolous plants, PC deficiency is generally correlated with metal hypersensitivity, and enhanced PC production with increased tolerance (Hall, 2002). PCs are produced from glutathione by phytochelatin synthase (PCS), which is activated primarily post-translationally (Vatamaniuk et al., 2000). Transcriptional regulation of AtPCS1 by Cd appears only during the early stages of plant development (Lee and Korban, 2002). Interestingly, in barley roots, transcriptional regulation of PCS by Cd was observed only during simultaneous nitrogen deficiency (Finkemeier et al., 2003).
The increased demand for sulphur-containing metabolites, reflected by both increased activities of sulphate assimilation enzymes and an elevated expression of genes encoding these proteins, seems to be a common response in plants exposed to Cd stress. In Arabidopsis thaliana, genes encoding several isoforms of serine acetyltransferase (SAT) (Howarth et al., 2003; Kawashima et al., 2005), Atcys-3A encoding cytosolic O-acetylserine (thiol) lyase (OASTL) (Dominguez-Solis et al., 2001), ATP sulphurylase, APS reductase, and sulphite reductase (Harada et al., 2002), as well as -glutamylcysteine synthetase (-ECS) and glutathione synthase (GS) (Xiang and Oliver, 1998), were induced by Cd. Modulation of activities of ATP sulphurylase and APS reductase, as well as mRNA levels encoding the above activities and a potential sulphate transporter, in roots and shoots of Brassica juncea were also reported (Heiss et al., 1999; Lee and Leustek, 1999). Cd was found to induce sulphate uptake through activation of the transcription of a high-affinity sulphate transporter in maize (Nocito et al., 2002). In addition, an elevation of activity of -ECS was reported for maize seedlings upon Cd treatment (Ruegsegger and Brunold, 1992) and for tomato cells selected for Cd tolerance (Chen and Goldsbrough, 1994).
Several reports about increasing accumulation and tolerance to Cd in transgenic plants due to expression of genes involved in PC biosynthesis can be found in the literature. Arabidopsis thaliana plants with overexpression of ectopically located Atcys-3A, encoding cytosolic OASTL from A. thaliana (Dominguez-Solis et al., 2001, 2004), as well as B. juncea plants overexpressing Escherichia coli genes encoding either -ECS (Zhu et al., 1999b) or GS (Zhu et al., 1999a), were more tolerant of Cd and could accumulate higher amounts than the control non-transgenic plants.
Interestingly, two sets of contrasting reports concerning the effects of PCS overproduction on heavy metal accumulation and tolerance can be found in the literature. The first set is composed of two independent reports on increased metal accumulation in the above-ground tissues due to expression of a wheat gene, TaPCS1: (i) in the cad1-3 mutant of A. thaliana that has an inactivated PCS1 gene and shows an undetectable level of PCs (Gong et al., 2003) and (ii) in Nicotiana glauca (Gisbert et al., 2003). In the second set of papers are included two reports on increasing Cd sensitivity in A. thaliana due to ectopic overexpression of the Arabidopsis gene AtPCS1 (Lee et al., 2003; Li et al., 2004). As suggested by the authors, a possible reason for such an increased hypersensitivity might be a limitation of glutathione due to extensive utilization of this compound for the synthesis of PCs. A recently published report (Pomponi et al., 2006), demonstrating that external glutathione has a positive impact on both tolerance and accumulation of Cd in transgenic tobacco overexpressing AtPCS, seems to support the above conclusion.
To learn more about the role of non-protein thiols in Cd accumulation in tobacco plants, and to test the hypothesis that an increased potential for PC synthesis is a suitable approach for increasing Cd accumulation in such plants, a multi-transgene approach resulting in simultaneous expression of genes encoding enzymes catalysing the presumed ‘bottle-neck’ steps of PC biosynthesis was undertaken.
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Materials and methods |
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Tobacco transformation
Transgenic lines of tobacco, Nicotiana tabacum, were obtained by Agrobacterium-mediated transformation of LA Burley 21 (Legg et al., 1970) with the binary T-DNA. Tobacco seedlings were transformed according to the procedure described by Rossi et al. (1993) using overnight cultures of Agrobacterium tumefaciens (LBA4404) containing appropriate plasmids. Regenerating plantlets were excised and transplanted to plates with MS medium (Murashige and Skoog, 1962) supplemented with the selection marker (150 µg ml–1 kanamycin) to allow root formation. Rooted plantlets were transferred to soil, checked for the presence of the transgenes, self-pollinated, and maintained in a greenhouse for seed production.
Plant growth conditions and preparation of plant material
Seeds obtained from the self-pollinated primary transformants (T0) were surface-sterilized and germinated on MS plates containing 200 µg ml–1 kanamycin. The kanamycin-resistant seedlings (T1 generations) were subsequently transferred (3–4 weeks after sowing) to hydroponic conditions and cultivated in a growth chamber (photoperiod 16 h light/8 h dark, 24 °C) in the following liquid medium [3.5 mM KNO3, 5 mM Ca(NO3)2, 1.7 mM Mg(NO3)2, 10 mM NH4NO3, 1 mM KH2PO4, 1 mM MgSO4, 0.9 mM MgCl2, 2 mM CaCl2, 0.1 mM NaCl, 50 µM FeNaEDTA, 0.64 µM Cu(NO3)2, 10 µM Mn(NO3)2, 0.82 µM (NH4)2Mo4O13, 0.096 µM (CH3COO)2Zn, 0.11 µM CoCl2, 25 µM H3BO4; the medium buffered with 1 mM MES to pH 5.8]. Every week the plants were transferred to fresh medium. Plants of each line, about 2 months old, were divided into two groups: (i) the plants grown in the presence of 15 µM CdCl2 for 3 weeks and (ii) the plants grown without Cd for the same time. Each plant was cultivated in a separate jar with aeration and transferred to fresh medium once a week. At the end of the experiment, plants were weighed and the shoots and roots of five or six individuals from each line were pooled. In the case of the wild type, four independent portions of the pooled material (five or six individuals in each case) were prepared. Before pooling, plants were washed in distilled water and dried in paper towels; the shoots were separated from the roots, weighed, and frozen in liquid nitrogen. The pooled material, homogenized and kept until needed at –80 °C, was subsequently used to determine Cd and thiol contents as well as transcript levels of the transgenes.
DNA cloning and plasmid construction
Standard molecular cloning techniques, such as restriction endonuclease digestion, DNA gel electrophoresis, and DNA ligation, were performed as described by Sambrook et al. (1989). The following oligonucleotides were used for amplification of the Escherichia coli cysE (CYSE1: 5'-CGG GAT CCA TGT CGT GTG AAG AAC TGG AAA-3' and CYSE2: 5'-GCG GTA CCT TAG ATC CCA TCC CCA TAC TCA-3') and gshA (GSH1: 5'-CGG GAT CCA TGG TCC CGG ACG TAT CA GAG-3' and GSH2: 5'-AAC TGC AGT CTA GAT CAG GCG TGT TTT TCC AG-3'), and Schizosaccharomyces pombe PCS (PCS1: 5'- CCG GGA TCC ATG GAC ATT GTT AAA CGA GCA GTC-3' and PCS2: 5'-CGG GTA CCA TGC ATT CAC GTA TTT TTA CAG CAG CTT GA-3') genes using the respective genomic DNA. The sequences underlined at the 5'-ends are restriction enzyme binding sites introduced to facilitate cloning of the respective PCR products into plasmid vectors. The PCR product (0.8 kb) containing cysE was cloned into BamHI/KpnI sites of pRok2 (Baulcombe et al., 1986) between the 35S RNA promoter and terminator of cauliflower mosaic virus (CaMV). The PCR product (1.6 kb) containing gshA was cloned into NcoI/BamHI sites of pRTL2 (Carrington et al., 1991) between the 35S promoter with a double enhancer and terminator. The gshA gene was preceded by the translational enhancer from tobacco etch virus (TEV leader). The PCR product (1.2 kb) containing PCS was cloned into BamHI/KpnI sites of pUC19 and subsequently recloned with SalI and KpnI into pRTL2 between the 35S promoter and terminator. Insertion of the PCS gene replaced the TEV leader. The pPC plasmid was obtained by cloning of the HindIII–HindIII fragment (2.4 kb) with PCS expression cassette into the HindIII site of pGreenII0029 (Hellens et al., 2000). The pDUO plasmid was obtained in two steps: (i) cloning of the EcoRI–HindIII fragment (2.4 kb) containing cysE expression cassette into EcoRI/HindIII sites of pGreenII0029 and (ii) subsequent cloning of the PstI–PstI fragment (3.2 kb) containing the gshA expression cassette into the PstI site of the plasmid obtained in the previous step of cloning. The pTRI plasmid was obtained by cloning the HindIII–HindIII fragment (2.4 kb) containing the PCS expression cassette into the HindIII site of pDUO. Orientation of the inserts was checked by restriction analysis.
Western blot analysis
Western blot analysis was performed according to the standard procedure (Sambrook et al., 1989). For protein electrophoresis, equal amounts of protein were loaded in each lane of the SDS-PAGE gels. The resolved proteins were electroblotted to nitrocellulose membranes. The serum from rabbits immunized with E. coli SAT, -ECS, or S. pombe PCS (all overproduced as His-tagged recombinant proteins in E. coli cells) served as primary antibodies. Goat anti-rabbit IgGs conjugated with alkaline phosphatase were used as secondary antibodies.
RT-PCR (reverse transcription–polymerase chain reaction)
Expression of gshA and PCS in transgenic plants was verified by non-quantitive RT-PCR. Total RNA extracted from frozen powdered material using the cold phenol method (Linthorst et al., 1993) served as templates. Each 20 µl RT reaction contained 5 µg RNA, 2 pmol of specific antisense primer, 1 mM dNTPs, 10 mM DTT, and 1 µl of PowerScriptTM Reverse Transcriptase (BD Biosciences Clontech) in buffer supplied by the manufacturer. The RNA and primers were preheated to 70 °C for 10 min and snap-cooled in iced water before adding the remaining components. The RT reactions proceeded for 1 h at 42 °C and were terminated by heating to 70 °C for 15 min. Then 1 µl aliquots of the reaction mixtures were used for PCR, with specific primers designed for fragments of genes, GSH5H (5'-GCG ATT CCT CGA CCT GTT TA-3') and GSHA3 (5'-AAC TGC AGT CTA GAT CAG GCG TGT TTT TCC AG-3') for gshA, and PCSH3 (5'-TTG CTA CTG GGT GGA TTT GA-3') and PCS3 (5'-CGG GTA CCA TGC ATT CAC GTA TTT TTA CAG CAG CTT GA-3') for PCS, using 27 cycles (94 °C 30 s; 65 °C 30 s; 72 °C 1.5 min) for gshA and 24 cycles (94 °C 30 s; 62 °C 30 s, 72 °C 1.5 min) for PCS. The PCR products (517 bp for gshA; 577 bp for PCS) were separated on agarose gels by electrophoresis and detected by ethidium bromide staining.
Determination of non-protein thiols in the plant samples
Thiols in shoots were determined independently by two methods. The amount of reduced thiol groups present in the low-molecular-weight compounds was measured according to the modified Ellman's test (Ellman, 1959). Approximately 100 mg of the leaf tissue was homogenized in liquid nitrogen and subsequently 500 µl of ice-cold 0.1 M HCl was added. After vigorous mixing, the samples were centrifuged (10 min, 20 000 g, 4 °C) and supernatants collected for analysis. Aliquots of 200 µl were added to 800 µl of the following buffer [775 µl of 0.5 M K2HPO4; 25 µl of 10 mM DTNB (5,5'-dithio-2-nitrobenzoic acid)] and reactions allowed to continue for 5 min. The absorbance of the samples was measured at 412 nm and corrected against the absorbance of a sample without added DTNB. The amount of soluble thiols was expressed as nanomoles of [SH] per gram fresh weight.
The total amounts of both reduced and oxidized forms of non-protein compounds containing thiol groups were determined by the modified HPLC method with UV detection (Bald et al., 2001). The frozen material was ground in a microfuge tube using a plastic pestle and, subsequently, 600 µl of ice-cold 0.1 M HCl was added. After vigorous mixing, all the samples were centrifuged (10 min, 20 000 g, 4 °C) and supernatants collected for analysis. To 200 µl of the extract, 28 µl of 1 M NaOH and 8 µl of 6 M NaBH4 were added. The mixture was stirred and stored for 2 min. Then, 20 µl of 3 M HCl was added to decompose the excess of NaBH4. The mixture was centrifuged (20 000 g, 1 min). Next, 400 µl of 0.2 M phosphate buffer, pH 7.6, and 10 µl of 0.1 M 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) were added, the mixture was stirred, put aside for 4 min, and acidified by adding 100 µl of 3 M HCl, followed by centrifugation (20 000 g, 3 min), and filtration through a 0.22 µm filter. Aliquots of 50 µl of the final solution were injected into the HPLC system (Breeze system; Waters, Milford, MA, USA) via an autosampler and separated on a C-18 column (Symmetry, 4.6 nm x150 nm; Waters). Elution buffer A was 0.1% (v/v) trifluoroacetic acid in H2O and buffer B was 90% (v/v) acetonitrile in H2O, 0.1% trifluoroacetic acid. The gradient used for these experiments was 0–50% buffer B in 25 min at a flow rate of 1 ml min–1, and the products derived were detected by absorption at 354 nm. The following thiol compounds were quantified: -Glu-Cys, GSH, PC2, PC3, PC4, and PC5. The very low levels of Cys in the samples measured did not allow reliable detection and quantification of this compound. Synthetic standards were obtained for all with the exception of PC5. Commercial samples of Cys, -Glu-Cys, and GSH were used as delivered. PC2, PC3, and PC4 were synthesized at the milligram scale in the solid state, using the Fmoc strategy, as described before for GSH derivatives (Krezel and Bal, 2003). The yield of the crude unprotected peptides, complexed with trifluoroacetic acid was between 20% and 30%. The final purification was done using HPLC on the Breeze system described above, using a Symmetry column from Waters.
The thiol standards were derived with CMQT as described above for the plant material and quantified by HPLC calibration curves. The derivatization procedure was controlled by ESI-MS (electrospray ionization-mass spectrometry) on a Q-Tofl mass spectrometer (Micromass, Manchester, UK). The linear dependence of absorption at 354 nm on the number of CMQT-derived thiol groups was found. Individual thiols in the plant material were identified and quantified by comparing retention times and peak areas with those of standards. Occasionally, appropriate fractions were collected and their identities confirmed by ESI-MS. PC5 in plant samples was identified solely by ESI-MS of its CMQT derivative and quantified by assuming the above-mentioned proportionality of its absorption at 354 nm. The amount of each thiol category was expressed as nanomoles of [SH] (GSH equivalent) per gram fresh weight.
Determination of Cd in the plant samples
Dried samples (ground plant tissue material, 50–100 mg), after suspension in 9 ml of 65% HNO3 supplemented with 0.9 ml 35% H2O2, were mineralized for 25 min (180 °C) in the laboratory microwave oven (ETHOS PLUS; Milestone). Then, the samples were cooled down and 0.1 ml of 35% H2O2 was added making the final volume of the sample to 10 ml. The Cd content in plant tissues was measured by monitoring absorption at 228.8 nm in a mixture of air/acetylene using flame atomic absorption spectrophotometry and SOLAAR M6 apparatus (TJA Solutions; now Thermo Electron Corporation, USA).
Statistical analysis
Statistical analyses were performed with Statistica 6.0 (StatSoft Polska Sp. Z o.o., Kraków). One-way ANOVA followed by the Tukey HSD test for unequal N used as a post-hoc test was used for determination of the significance of differences between the mean values. The correlation coefficients (Pearson r) were calculated using the linear correlation method.
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Results |
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Generation of transgenic tobacco lines
The plant expression cassettes constructed for the purposes of this study are shown schematically in Fig. 1. They are based on a binary vector pGreenII0029 (Hellens et al., 2000) and contain combinations of three genes: (i) cysE from E. coli encoding SAT involved in the production of O-acetylserine, the cysteine precursor; (ii) gshA from E. coli encoding
-ECS involved in the production of -glutamylcysteine (-EC), the precursor of glutathione; and (iii) PCS from Schizosaccharomyces pombe, encoding phytochelatin synthase (PCS). PCS is an intronless gene and therefore was amplified from genomic DNA. Functionalities of the cassettes were verified in the test of transient expression in planta after inoculation of leaves of Nicotiana benthamiana with cultures of Agrobacterium tumefaciens harbouring the appropriate plasmids. The presence of the expected transcripts (results not shown) in the leaves collected 2 d after inoculation indicated that the plasmids were correctly designed and constructed.
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The tobacco line Nicotiana tabacum cv. LA Burley 21 (Legg et al., 1970) was used for Agrobacterium-mediated transformation with pPC, pDUO, or pTRI constructs. Tobacco was chosen as a model plant for transformation since it is an easy, fast, and efficient transformation system. An additional advantage is the relative ease of genetic and biochemical analysis of the transformants. The LA Burley 21 cultivar produces a large mass of shoots which may be an important advantage for phytoremediation applications and extraction purposes. Three independent lines of PC and TRI and the only two existing DUO lines were arbitrarily selected for future analysis. Assays of the T1 generation of the selected transformants for segregation of the kanamycin marker revealed segregation ratios of sensitive to resistant individuals of about 1:3 (results not shown). These results suggest that the T-DNA was inserted into a single independent locus in each transformant. It was not determined, and cannot be excluded, whether single or multiple copies of T-DNA were integrated into these loci.
Characterization of transgenic plants grown with and without Cd
Eight-week-old individuals (T1 generation) of the selected transgenic lines and the wild type were grown for 3 weeks in liquid medium with 15 µM CdCl2 or in a control medium without Cd. Expression of transgenes was verified in the pooled material from the shoots (Fig. 2). The presence of E. coli SAT was confirmed for all lines transformed with the cysE expression cassette. As the presence of -ECS and PCS could not be demonstrated by western blots (not shown), the expression of the gshA and PCS transgenes was confirmed in the respective lines by RT-PCR. Growth in the presence of Cd did not influence the level of the expression of the transgenes (not shown).
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Regardless of the line tested, similar effects of Cd, such as yellowing of the leaves and darkening of the roots (not shown) were observed. All transgenic lines except PC40 were smaller than the wild type in the medium without Cd and all were apparently retarded by Cd (Fig. 3A–C). The differences in growth inhibition by Cd between the wild type and transgenic groups were not significant (in all cases P >0.1).
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The level of reduced non-protein thiol was evaluated in the leaves of individual plants using the modified Ellman's test in order to avoid large discrepancies between the pooled plants. Similar levels of thiols were found in the plants from the same lines (not shown).
Subsequently, individual categories of non-protein thiols were assessed in the shoots and in the roots of the plant material collected. Since the extracts were treated with strong reducing agents just before derivatization, the sum of both reduced and oxidized forms present in the tissues was assayed by this method. The results of the analysis performed in shoot and root tissues are shown in Fig. 4. As expected, in both conditions tested (absence and presence of 15 µM CdCl2), all transgenic lines, except the PC group, contained higher concentrations of total thiols than the control, in both shoots and roots. In the absence of Cd, the predominant thiol in all groups of plants was glutathione and only small amounts of -EC were detected in the roots of all three TRI lines, and both -EC and PC2 in the shoots of TRI2. Plants grown in the presence of Cd had from 1.3- to 3.7-fold more thiols in shoots than the same lines grown without Cd. In roots, Cd exposure resulted in over 10-fold higher total thiol levels in all lines except TRI, where the difference was only about 2.5-fold. Thus, in all lines, except TRI, the shoot-to-root ratio of thiols was much lower in the presence than in the absence of Cd. However, all groups of plants had several-fold higher levels of total thiols in the shoots compared with the roots, both in the control conditions and when grown with Cd. Furthermore, exposure to Cd resulted in a slight decrease of glutathione concentration in shoots that was accompanied by a strong increase in the concentrations of other categories of thiols (comparing within lines). Such a decrease of glutathione was observed in all lines except TRI2. All plant groups, except TRI, when grown in the presence of Cd, had slightly more glutathione in the roots than the respective group grown in the absence of Cd. Interestingly, in the shoots, the entire spectrum of phytochelatins (PC2, PC3, PC4, and PC5) was present, while in the roots small amounts of PC2 were present only in the DUO10 line and were totally absent in all other plants, including wild type.
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Testing selected transgenic lines for accumulation of Cd
The level of Cd accumulation in shoots and roots of the plants analysed is shown in Fig. 3D. The differences in Cd accumulation per gram of the leaf tissue were not significant between the wild type and transgenic groups while the concentration of Cd in the roots was higher in many of the transgenic groups compared with the wild type. For example, the level of Cd accumulation reached 153% in PC1, 162% in PC4, 167% in TRI2, 122% in TRI8, and 138% in TRI15 (Fig. 3D). Noticeably, the discrepancy between the concentration of Cd in the roots and shoots was higher in all transgenic groups compared with the wild-type plants. The root-to-shoot ratio of Cd concentration was, on average, about 1.9 in the wild type and for the transgenic groups it was 3.0, 2.7, and 3.3 for PC, DUO, and TRI, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Expression of transgenes encoding SAT,
-ECS, and PCS was expected to ‘push’ the pathway of phytochelatin biosynthesis during exposure to heavy metals. Essentially, this aim was achieved. The TRI group, particularly the TRI2 line, seemed to be the ‘most effective’ thiol-producer since it had about an 8-fold higher concentration of thiols in the shoots compared with the control. Positive correlations between the concentrations of -EC and other thiols in the shoots of the TRI group were observed (1, 0.99, 0.96, 0.94, and 0.98 for glutathione, PC2, PC3, PC4, and PC5, respectively, at P <0.05). These correlations were weaker in the shoots of the other lines (e.g. the correlations between -EC and PC2 and between -EC and PC5 were statistically insignificant in other groups of plants). This may suggest that the synthesis of longer phytochelatins is not limited by the availability of sulphur-containing substrates in the shoots of the TRI group.
The protein product of the PCS transgene was not detected in any of the transgenic groups (PC, DUO, TRI) despite the fact that the plants showed the presence of the PCS transcript. Since there are no critical differences between S. pombe and N. tabacum with respect to codon usage in the PCS gene (not shown), non-optimal codon usage cannot explain the low amount of SpPCS. The PC lines (expressing PCS) indicated very limited enhancement of phytochelatin contents in the shoots, when compared with the non-transformed control. Therefore, the possibility that the vast overproduction of thiols in TRI2 is solely due to the simultaneous overproduction of the two limiting enzymes of the pathway, SAT and -ECS, cannot be excluded. Problems with efficient production of PCS from S. pombe in transgenic A. thaliana have been reported previously (Li et al., 2001). In control conditions, when PCS protein was apparently inactive as PCs were not produced (Fig. 4), the huge elevation of glutathione observed in TRI lines resulted from co-action of bacterial SAT and -ECS due to simultaneous expression of cysE and gshA. Therefore, it may be that higher PCs levels in TRI lines resulted mainly from an increased supply of sulphur-containing substrates and not an enhanced activity of PCS. Unfortunately, the lines co-expressing two transgenes, cysE and gshA, were unavailable for the comparison with the TRI lines (expressing cysE, gshA, and PCS). The problem of factual contribution of the product of the transgene (ScPCS) to the total PCS activity in the transformants will be addressed in future experiments.
The observations in this paper that, in the absence of Cd, the predominant thiol was glutathione and that -EC was almost undetected in DUO and TRI lines (Fig. 4) is in agreement with the previous observations (Noctor et al., 2002) that the activity of GS, in contrast to the activity of -ECS, is not limiting the amount of glutathione in plants.
An increased root-to-shoot ratio of Cd in transgenic plants indicated that the metal was more strongly retained in the roots of these plants than in the wild type. In these experiments the roots were not desorbed in CaCl2 to remove the extracellular Cd (Rauser, 1987). Here, the total amount of Cd present in the root tissue, consisting of a form bound in the apoplasts of the rhizodermis or root cortex (extracellular) and a form transported to the symplast of the roots (intracellular), was measured. It may be that an increased level of thiols could change the proportion between these two forms of Cd.
According to the literature, Cd occurs both inside the vacuole and in the cell wall of T. caerulescens (Cosio et al., 2005) and B. napus (Carrier et al., 2003), in cell walls of Zea mays (Khan et al., 1984), and within vacuoles of N. tabacum (Vogeli-Lange and Wagner, 1990). Additionally, it is generally assumed that transport of Cd–PC complexes to the vacuoles might be an important limiting factor for Cd accumulation at the level of the single cells (Clemens, 2001, 2006; Clemens et al., 2002; Tong et al., 2004). It remains to be seen whether any differences in Cd distribution at the subcellular level exist between wild-type plants and the transgenic lines with the high thiol contents analysed in this study.
Interestingly, all transgenics, except the PC4 line, had slightly reduced Cd concentrations in the shoots compared with the wild type (Fig. 3D). Although these differences were in most cases not significant and, apparently, no correlation between Cd accumulation and thiol levels was found, it may be speculated that, in the transformants, less Cd was transported to the shoots because it was retained in the roots due to its sequestration by an excess of thiols. Such effects would be in an agreement with a previous report that tobacco plants expressing mammalian metallothionein had a 60–70% lower Cd concentration in their shoots compared with controls (Elmayan and Tepfer, 1994).
It has been shown for A. thaliana plants that seedlings do not have barriers blocking the transport of Cd from roots to shoots, while in older plants Cd cannot be efficiently transported to the shoots and, therefore, is not accumulated in the aerial parts to the same extent as in the roots (Bovet et al., 2003, 2005). Cd in the xylem sap has oxygen and/or nitrogen ligands, such as hydrated cations or small organic acids (Salt et al., 1995, 2002). As Cd prefers sulphur ligands over oxygen and nitrogen ligands, the sulphur-co-ordinated Cd can be either stored in the roots or it must be actively moved from storage for transport (Salt et al., 2002). The increased root-to-shoot ratio of Cd found in this work in most transgenic plants (as compared with the wild type) suggests the presence of similar barriers in tobacco or at least in the cultivar tested, LA Burley 21. Then, the amount of Cd in the shoots is limited most probably by the transport of Cd through the xylem from the roots to the shoots.
Distribution of Cd within plant organs not only varies between plant species but also depends on duration and concentration of exposure as well as on the presence of other metals (Bittsanszky et al., 2005; Koprivova et al., 2002). Thus, the adaptation mechanisms may develop during 3 weeks of Cd exposure while a completely different Cd distribution may be found in plants subjected to a shorter-term Cd stress. This could explain differences in root-to-shoot distribution of Cd observed between these results and those recently published by Pomponi et al. (2006).
Despite being at a similar stage of development, most transgenic plants had a smaller mass of shoots and roots than the wild type (Fig. 3A, B). It is possible that the lower performance of the plants was a consequence of transgene expression. On the other hand, an initial period of selection of the transgenic plantlets in the medium containing kanamycin might also result in differences in the yield, despite an apparently similar size of the seedlings transferred to hydroponic conditions.
By contrast to the total fresh weights (Fig. 3A, B), the total dry weights of the individual plants were not documented during the experiment. Therefore, it is difficult to calculate and compare the total amount of Cd accumulated in the shoots and roots of an average plant of each line. Interestingly, the concentration of Cd in the roots (Fig. 3D) of all lines is negatively correlated with the yield (weights) of the shoots (Fig. 3A) and roots (Fig. 3B) of an average plant (–0.88 and –0.90, respectively; P <0.05). This result suggests that the increased PC content in transgenic lines does not protect plants against harmful influences of Cd. Similar conclusions could be drawn from the preliminary testing of the tolerance to Cd of the seedlings of the selected transgenic lines. Regardless of the line tested, the appearance of seedlings (length of the roots and shoot colour) was similar in response to exposure to CdCl2 in all of the concentrations tested (100–300 µM; not shown).
In summary, increasing the potential for phytochelatin biosynthesis in tobacco plants due to expression of heterologous genes encoding crucial enzymes involved in thiol biosynthesis resulted in the elevation of Cd concentration in the roots but not in the shoots of the transgenic plants upon a 3 week exposure to 15 µM CdCl2. No effect of increased PC levels on tolerance to Cd was observed. Then, mechanisms other than the availability of precursors for phytochelatin biosynthesis limit efficient Cd accumulation in shoots and must be responsible for Cd tolerance in the tobacco lines studied. The plants characterized in this study may be a good starting point for subsequent studies to understand better the still unclear physiological function of PCS (Clemens, 2006) and the involvement of sulphur-containing compounds in accumulation and transport of heavy metals and other pollutants.
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Acknowledgements |
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We gratefully acknowledge Dr Grazyna Chwatko and Professor Edward Bald from the Department of Enviromental Chemistry, University of Lodz, for providing us with a batch of CMQT and for sharing their expertise regarding the application of CMQT in the HPLC analysis of thiols, Dr Artur Kr
el (currently at the University of Texas, USA) for help with peptide synthesis and Professor Aleksandra Sk
odowska (Laboratory of Environmental Pollution Analysis, Faculty of Biology, Warsaw University) for performing the assays of Cd contents. Dr Phil Mullineaux and Dr Roger Hellens (John Innes Centre and the Biotechnology and Biological Sciences Research Council, Norwich) are acknowledged for pGreen. Special thanks are given to Dr Malcolm Hawkesford (Rothamsted Research, Harpenden) for revising the English of the final version. This work was partly supported by the by EU Commission through funding of FP5 project PHYTAC (QLRT-2001-00429 and QLRT-2001-02778 [NAS]) and by Polish Ministry of Education and Science through funding of the project SPB/COST/112/2005. |
Abbreviations |
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CaMV, cauliflower mosaic virus CMQT, 2-chloro-1-methylquinolinium tetrafluoroborate;
-EC, -glutamylcysteine; -ECS, -glutamylcysteine synthetase; GS, glutathione synthase; GSH, glutathione; OASTL, O-acetylserine (thiol) lyase; PC, phytochelatin; PCS, phytochelatin synthase; RT-PCR, reverse transcription–polymerase chain reaction; SAT, serine acetyltransferase; TEV, tobacco etch virus. |
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