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Stephan Clemens - One of the best experts on this subject based on the ideXlab platform.
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arabidopsis thaliana Phytochelatin synthase 2 is constitutively active in vivo and can rescue the growth defect of the pcs1 deficient cad1 3 mutant on cd contaminated soil
Journal of Experimental Botany, 2014Co-Authors: Tanja Kuhnlenz, Holger Schmidt, Shimpei Uraguchi, Stephan ClemensAbstract:Phytochelatins play a key role in the detoxification of metals in plants and many other eukaryotes. Their formation is catalysed by Phytochelatin synthases (PCS) in the presence of metal excess. It appears to be common among higher plants to possess two PCS genes, even though in Arabidopsis thaliana only AtPCS1 has been demonstrated to confer metal tolerance. Employing a highly sensitive quantification method based on ultraperformance electrospray ionization quadrupole time-of-flight mass spectrometry, we detected AtPCS2-dependent Phytochelatin formation. Overexpression of AtPCS2 resulted in constitutive Phytochelatin accumulation, i.e. in the absence of metal excess, both in planta and in a heterologous system. This indicates distinct enzymatic differences between AtPCS1 and AtPCS2. Furthermore, AtPCS2 was able to partially rescue the Cd hypersensitivity of the AtPCS1-deficient cad1-3 mutant in a liquid seedling assay, and, more importantly, when plants were grown on soil spiked with Cd to a level that is close to what can be found in agricultural soils. No rescue was found in vertical-plate assays, the most commonly used method to assess metal tolerance. Constitutive AtPCS2-dependent Phytochelatin synthesis suggests a physiological role of AtPCS2 other than metal detoxification. The differences observed between wild-type plants and cad13 on Cd soil demonstrated: (i) the essentiality of Phytochelatin synthesis for tolerating levels of Cd contamination that can naturally be encountered by plants outside of metal-rich habitats, and (ii) a contribution to Cd accumulation under these conditions.
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analysis of plant pb tolerance at realistic submicromolar concentrations demonstrates the role of Phytochelatin synthesis for pb detoxification
Environmental Science & Technology, 2014Co-Authors: Sina Fischer, Holger Schmidt, Tanja Kuhnlenz, Michael Thieme, Stephan ClemensAbstract:Lead (Pb) ranks first among metals with respect to tonnage produced and released into the environment. It is highly toxic and therefore an important pollutant of worldwide concern. Plant Pb uptake, accumulation, and detoxification mobilize Pb into food webs. Still, knowledge about the underlying mechanisms is very limited. This is largely due to serious experimental challenges with respect to Pb availability. In most studies, Pb(II) concentrations in the millimolar range have been used even though the toxicity threshold is in the nanomolar range. We therefore developed a low-phosphate, low-pH assay system that is more realistic with respect to soil solution conditions. In this system the growth of Arabidopsis thaliana seedlings was significantly affected by the addition of only 0.1 μM Pb(NO3)2. Involvement of Phytochelatins in the detoxification of Pb(II) could be demonstrated by investigating Phytochelatin synthase mutants. They showed a stronger inhibition of root growth and a lack of Pb-activated phyto...
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multi tasking Phytochelatin synthases
Plant Science, 2009Co-Authors: Stephan Clemens, Derek PersohAbstract:Abstract Phytochelatins are essential for cadmium and arsenic detoxification in plants, some fungi, and animals. It is mysterious, that the responsible enzymes, Phytochelatin synthases (PCS), are constitutively expressed and so widespread in nature. Phylogenetic analysis indicates multiple horizontal transfers of PCS genes, but a bacterial origin appears unlikely. Differences between bacterial and eukaryotic PCS proteins in structure and activity had indicated bi-functionality of Phytochelatin synthases as peptidases and transpeptidases. Recent observations indicate that PCS indeed serve physiological functions that most likely are much more prevalent than cadmium or arsenic detoxification. First, PCS-deficient Arabidopsis thaliana mutants are hypersensitive to zinc suggesting a role of Phytochelatin synthesis, i.e. the formation of metal-binding peptides from glutathione in a transpeptidase reaction, in Zn homeostasis. Second, these mutants are also impaired in defense responses conferring resistance to incompatible pathogens (= nonhost resistance). The latter is hypothesized to be attributable to an involvement of PCS as a peptidase in indole glucosinolate metabolism. Possibly, micronutrient homeostasis and nonhost resistance are closely connected as PCS are not the only proteins involved in both processes.
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overexpression of Phytochelatin synthase in tobacco distinctive effects of atpcs1 and cepcs genes on plant response to cadmium
Journal of Experimental Botany, 2008Co-Authors: Sylwia Wojas, Henk Schat, Stephan Clemens, Jacek Hennig, Aleksandra Sklodowska, Edyta Kopera, Wojciech Bal, Danuta Maria AntosiewiczAbstract:Phytochelatins, heavy-metal-binding polypeptides, are synthesized by Phytochelatin synthase (PCS) (EC 2.3.2.15). Previous studies on plants overexpressing PCS genes yielded contrasting phenotypes, ranging from enhanced cadmium tolerance and accumulation to cadmium hypersensitivity. This paper compares the effects of overexpression of AtPCS1 and CePCS in tobacco (Nicotiana tabacum var. Xanthi), and demonstrates how the introduction of single homologous genes affects to a different extent cellular metabolic pathways leading to the opposite of the desired effect. In contrast to WT and CePCS transformants, plants overexpressing AtPCS1 were Cd-hypersensitive although there was no substantial difference in cadmium accumulation between studied lines. Plants exposed to cadmium (5 and 25 μM CdCl 2 ) differed, however, in the concentration of non-protein thiols (NPT). In addition, PCS activity in AtPCS1 transformants was around 5-fold higher than in CePCS and WT plants. AtPCS1 expressing plants displayed a dramatic accumulation of γ-glutamylcysteine and concomitant strong depletion of glutathione. By contrast, in CePCS transformants, a smaller reduction of the level of glutathione was noticed, and a less pronounced change in γ-glutamylcysteine concentration. There was only a moderate and temporary increase in Phytochelatin levels due to AtPCS1 and CePCS expression. Marked changes in NPT composition due to AtPCS1 expression led to moderately decreased Cd-detoxification capacity reflected by lower SH:Cd ratios, and to higher oxidative stress (assessed by DAB staining), which possibly explains the increase in Cd-sensitivity. The results indicate that contrasting responses to cadmium of plants overexpressing PCS genes might result from species-dependent differences in the activity of Phytochelatin synthase produced by the transgenes.
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evolution and function of Phytochelatin synthases
Journal of Plant Physiology, 2006Co-Authors: Stephan ClemensAbstract:Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of Phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding Phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of Phytochelatin synthases and our current knowledge about the enzymology of Phytochelatin synthesis are reviewed.
Christopher S Cobbett - One of the best experts on this subject based on the ideXlab platform.
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Phytochelatin synthase pcs protein is induced in brassica juncea leaves after prolonged cd exposure
Journal of Experimental Botany, 2003Co-Authors: Senta Heiss, Christopher S Cobbett, Andreas Wachter, Jochen Bogs, Thomas RauschAbstract:Higher plants respond to cadmium exposure with the production of Phytochelatins (PCn), small heavy metal binding peptides, which are synthesized from glutathione by Phytochelatin synthase (PCS). The isolation of a PCS cDNA clone from Brassica juncea L. cv. Vitasso, a candidate species for phytoremediation, is reported here. CLUSTAL analysis revealed a close relationship of BjPCS1 with PCS proteins from Arabidopsis thaliana and Thlaspi caerulescens. BjPCS1 expressed as recombinant protein in E. coli had PCS activity in vitro that was activated by 50 microM Cu and 200 microM Cd to a similar extent. Immunoblot analysis with an antiserum directed against recombinant BjPCS1 showed constitutive PCS expression during plant development. As a percentage of the total protein, the expression was higher in the roots, internodes and petioles in comparison with the leaf tissue. When B. juncea plants were treated with 25 microM cadmium, PCn accumulated increasingly over a 6 d period. Levels in shoots were about 3-fold higher than in roots. Prolonged cadmium exposure caused a significant increase of PCS protein in leaves, whereas in roots PCS protein levels were not affected.
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Phytochelatin synthase pcs protein is induced in brassica juncea leaves after prolonged cd exposure
Journal of Experimental Botany, 2003Co-Authors: Senta Heiss, Christopher S Cobbett, Andreas Wachter, Jochen Bogs, Thomas RauschAbstract:Abstract Higher plants respond to cadmium exposure with theproduction of Phytochelatins (PCn), small heavymetal binding peptides, which are synthesized fromglutathione by Phytochelatin synthase (PCS). Theisolation of a PCS cDNA clone from Brassica junceaL. cv. Vitasso, a candidate species for phytoremedia-tion, is reported here. CLUSTAL analysis revealed aclose relationship of BjPCS1 with PCS proteins fromArabidopsis thaliana and Thlaspi caerulescens.BjPCS1 expressed as recombinant protein in E. colihad PCS activity in vitro that was activated by 50 mMCu and 200 mM Cd to a similar extent. Immunoblotanalysis with an antiserum directed against recombi-nant BjPCS1 showed constitutive PCS expressionduring plant development. As a percentage of thetotal protein, the expression was higher in the roots,internodes and petioles in comparison with the leaftissue. When B. juncea plants were treated with25 mM cadmium, PCn accumulated increasingly over a6 d period. Levels in shoots were about 3-fold higherthan in roots. Prolonged cadmium exposure caused asignificant increase of PCS protein in leaves,whereas in roots PCS protein levels were notaffected.Key words: Brassica juncea, cadmium, heavy metal,Phytochelatin synthase.
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Phytochelatin biosynthesis and function in heavy metal detoxification
Current Opinion in Plant Biology, 2000Co-Authors: Christopher S CobbettAbstract:Plants respond to heavy-metal toxicity via a number of mechanisms. One such mechanism involves the chelation of heavy metals by a family of peptide ligands, the Phytochelatins. Molecular genetic approaches have resulted in important advances in our understanding of Phytochelatin biosynthesis. In particular, genes encoding the enzyme Phytochelatin synthase have been isolated from plant and yeast species. Unexpectedly, genes with similar sequences to those encoding Phytochelatin synthase have been identified in some animal species.
Thomas Rausch - One of the best experts on this subject based on the ideXlab platform.
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Phytochelatin synthase pcs protein is induced in brassica juncea leaves after prolonged cd exposure
Journal of Experimental Botany, 2003Co-Authors: Senta Heiss, Christopher S Cobbett, Andreas Wachter, Jochen Bogs, Thomas RauschAbstract:Abstract Higher plants respond to cadmium exposure with theproduction of Phytochelatins (PCn), small heavymetal binding peptides, which are synthesized fromglutathione by Phytochelatin synthase (PCS). Theisolation of a PCS cDNA clone from Brassica junceaL. cv. Vitasso, a candidate species for phytoremedia-tion, is reported here. CLUSTAL analysis revealed aclose relationship of BjPCS1 with PCS proteins fromArabidopsis thaliana and Thlaspi caerulescens.BjPCS1 expressed as recombinant protein in E. colihad PCS activity in vitro that was activated by 50 mMCu and 200 mM Cd to a similar extent. Immunoblotanalysis with an antiserum directed against recombi-nant BjPCS1 showed constitutive PCS expressionduring plant development. As a percentage of thetotal protein, the expression was higher in the roots,internodes and petioles in comparison with the leaftissue. When B. juncea plants were treated with25 mM cadmium, PCn accumulated increasingly over a6 d period. Levels in shoots were about 3-fold higherthan in roots. Prolonged cadmium exposure caused asignificant increase of PCS protein in leaves,whereas in roots PCS protein levels were notaffected.Key words: Brassica juncea, cadmium, heavy metal,Phytochelatin synthase.
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Phytochelatin synthase pcs protein is induced in brassica juncea leaves after prolonged cd exposure
Journal of Experimental Botany, 2003Co-Authors: Senta Heiss, Christopher S Cobbett, Andreas Wachter, Jochen Bogs, Thomas RauschAbstract:Higher plants respond to cadmium exposure with the production of Phytochelatins (PCn), small heavy metal binding peptides, which are synthesized from glutathione by Phytochelatin synthase (PCS). The isolation of a PCS cDNA clone from Brassica juncea L. cv. Vitasso, a candidate species for phytoremediation, is reported here. CLUSTAL analysis revealed a close relationship of BjPCS1 with PCS proteins from Arabidopsis thaliana and Thlaspi caerulescens. BjPCS1 expressed as recombinant protein in E. coli had PCS activity in vitro that was activated by 50 microM Cu and 200 microM Cd to a similar extent. Immunoblot analysis with an antiserum directed against recombinant BjPCS1 showed constitutive PCS expression during plant development. As a percentage of the total protein, the expression was higher in the roots, internodes and petioles in comparison with the leaf tissue. When B. juncea plants were treated with 25 microM cadmium, PCn accumulated increasingly over a 6 d period. Levels in shoots were about 3-fold higher than in roots. Prolonged cadmium exposure caused a significant increase of PCS protein in leaves, whereas in roots PCS protein levels were not affected.
Beth A. Ahner - One of the best experts on this subject based on the ideXlab platform.
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environmental cadmium levels increase Phytochelatin and glutathione in lettuce grown in a chelator buffered nutrient solution
Journal of Environmental Quality, 2003Co-Authors: Elizabeth A Maier, Rosalyn D Matthews, Jennifer A Mcdowell, Rebecca R Walden, Beth A. AhnerAbstract:: Phytochelatins are enzymatically synthesized peptides involved in metal detoxification and have been measured in plants grown at very high Cd concentrations, but few studies have examined the response of plants at lower environmentally relevant Cd concentrations. Using an ethylenediaminetetraacetic acid (EDTA)-buffered nutrient medium, we have varied Cd exposure and measured Phytochelatin and glutathione concentrations in romaine lettuce (Lactuca sativa L. var. longifolia Lam. var. Parris Island) grown in a flow-through hydroponic (FTH) system. Very low free ionic Cd (10(-9.6) M) increased average Phytochelatin concentrations above those of controls, and increasing Cd resulted in increased Phytochelatin production, though increases were tissue dependent. Glutathione concentrations also increased with increasing Cd. In other standard hydroponic experiments, the media were manipulated to vary total Cd concentration while the ionic Cd was fixed. We found that the total amount of Cd (primarily EDTA bound) in the medium altered thiol production in roots, whereas thiols in leaves remained constant. The Cd uptake into roots and translocation to old leaves was also influenced by the total concentration in the medium. Cadmium in all tissues was lower and in some tissues thiol concentrations were higher than in FTH-grown plants grown in identical medium, suggesting that nutrient delivery technique is also an important variable. Though Phytochelatin and glutathione production can be sensitive to changes in bioavailable Cd, thiol concentrations will not necessarily reflect the Cd content of the plant tissues.
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Role for heavy metals in forest decline indicated by Phytochelatin measurements
Nature, 1996Co-Authors: James E. Gawel, Beth A. Ahner, Andrew J. Friedland, François M. M. MorelAbstract:FOREST decline in the United States and Europe has been documented for a number of tree species1,2 and atmospheric pollutants from industrial sources, such as acids or oxidants, are thought to be partly responsible1–4. Heavy metals have also been implicated because their deposition pattern is correlated with forest decline5–11, but so far there has been no direct evidence for a physiological link between tree damage and exposure to metals. Here we use the concentrations of Phytochelatins, which are intracellular metal-binding peptides that act as specific indicators of metal stress12,13, to show that metals are indeed likely to be a contributing factor in the decline of forests in the north-eastern United States. Phytochelatin concentrations in red spruce, a species in decline, are higher than in balsam fir, a species which is not. Concentrations increase with altitude, as does forest decline, and they also increase across the region in forest stands that show increasing levels of tree damage.
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Export of Cadmium and Phytochelatin by the Marine Diatom Thalassiosira weissflogii
Environmental Science & Technology, 1996Co-Authors: Jennlfer G. Lee, Beth A. Ahner, François M. M. MorelAbstract:Cadmium is one of the most toxic trace metals and induces high concentrations of the metal-binding polypeptide Phytochelatin, (γ-Glu-Cys)nGly where n ≥ 2, in the marine diatom Thalassiosira weissflogii. Here we show that at high inorganic cadmium concentrations there is an efflux of cadmium from T. weissflogii so large that over half the cadmium taken up by the cell is returned to the medium. At high inorganic cadmium, there is also an efflux of Phytochelatin from the cell. The efflux of both cadmium and Phytochelatin stops when the external inorganic cadmium concentration is reduced. The efflux of Phytochelatin and cadmium occurs at a molar ratio of approximately 4 γ-Glu-Cys subunits per cadmium, a stoichiometry similar to that measured in vivo for the cadmium−Phytochelatin complex. We hypothesize that T. weissflogii exports the Phytochelatin− cadmium complex as a detoxification mechanism. The cadmium−Phytochelatin complex does not appear to be very stable in seawater once outside the cell since the cadm...
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Phytochelatin production in marine algae 1 an interspecies comparison
Limnology and Oceanography, 1995Co-Authors: Beth A. Ahner, Shing Kong, Fracois M M MorelAbstract:Phytochelatins are metal-binding peptides produced enzymatically by higher plants, fungi, and algae in response to many metals, particularly Cd. We have studied Phytochelatin production in several marine phytoplankton exposed to a range of free Cd ion concentrations. As a result of increased analytical resolution, we have found that all the species contain Phytochelatin, even when there is no added Cd, and that elevated Phytochelatin concentrations are induced by Cd, even at very low and environmentally relevant concentrations (as low as 10 -12 M free ion concn). In some but not all species, intracellular Cd and Phytochelatin concentrations are maintained at a fixed stoichiometric ratio at high Cd concentrations. Phytochelatin production and accumulation appear to be regulated in a manner that varies among phytoplankton species.
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Phytochelatin production in marine algae 2 induction by various metals
Limnology and Oceanography, 1995Co-Authors: Beth A. Ahner, Fracois M M MorelAbstract:Phytochelatin has been quantified in Thalassiosira weissflogii, a marine diatom after exposure to a series of trace metals (Cd, Pb, Ni, Cu, Zn, Co, Ag, and Hg) at concentrations similar to those in the marine environment. Within the range of concentrations relevant to natural waters, Cd, and to a lesser extent Cu and Zn, are the most effective inducers of Phytochelatins. The generality of this result was confirmed by short-term experiments with two other phytoplankton species. Quantification of intracellular Cd, Ni, and Zn shows that Phytochelatin production does not follow a simple stoichiometric relationship to the metal quotas. The rapid formation of Phytochelatin in T. weissflogii after Cd exposure and the fast elimination when metal exposure is alleviated reveal a dynamic pool of Phytochelatin which is tightly regulated by the cell. Many trace metals have been shown to induce Phytochelatin production in plants (Grill et al. 1987), although the concentration necessary to stimulate the response as well as the magnitude of the response depend on the particular metal. Although it is believed that production of this peptide is a general metal detoxification system, Cd has been found to be the most effective inducer of Phytochelatin synthase (Grill et al. 1989). Our goal is to elucidate the factors that control Phytochelatin production by phytoplankton in the laboratory in order to better understand what stimulates Phytochelatin production in the field (Ahner et al. 1994). In a companion paper (Ahncr et al. 1995), we investigated Phytochelatin production by several phytoplankton species in response to Cd. In this study we examine the response of Thalassiosira weissflogii to a variety of metals (Cd, Pb, Cu, Ni, Zn, Co, Ag, and Hg), all of which have been found to stimulate Phytochelatin production in higher plants. As in our experiments with Cd, we tested free metal concentrations that would be encountered in natural seawater in order to evaluate which metals may stimulate this response in natural populations of algae. We performed short-term assays with two other phytoplankton species to compare the patterns of the Phytochelatin response to various metals. Finally, to assess how changing environmental conditions might affect cellular concentrations of Phytochelatin, we examined the kinetics of Phytochelatin production and elimination upon changes in metal exposure.
Luigi Sanità Di Toppi - One of the best experts on this subject based on the ideXlab platform.
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Evolution and functional differentiation of recently diverged Phytochelatin synthase genes from Arundo donax L.
Journal of Experimental Botany, 2019Co-Authors: Mingai Li, Luca Stragliati, Ada Ricci, Luigi Sanità Di Toppi, Erika Bellini, Alessandro Saba, Claudio VarottoAbstract:Phytochelatin synthases (PCSs) play pivotal roles in the detoxification of heavy metals and metalloids in plants; however, little information on the evolution of recently duplicated PCS genes in plant species is available. Here we characterize the evolution and functional differentiation of three PCS genes from the giant reed (Arundo donax L.), a biomass/bioenergy crop with remarkable resistance to cadmium and other heavy metals. Phylogenetic reconstruction with PCS genes from fully sequenced monocotyledonous genomes indicated that the three A. donax PCSs, namely AdPCS1-3, form a monophyletic clade. The AdPCS1-3 genes were expressed at low levels in many A. donax organs and displayed different levels of cadmium-responsive expression in roots. Overexpression of AdPCS1-3 in Arabidopsis thaliana and yeast reproduced the phenotype of functional PCS genes. Mass spectrometry analyses confirmed that AdPCS1-3 are all functional enzymes, but with significant differences in the amount of the Phytochelatins synthesized. Moreover, heterogeneous evolutionary rates characterized the AdPCS1-3 genes, indicative of relaxed natural selection. These results highlight the elevated functional differentiation of A. donax PCS genes from both a transcriptional and an enzymatic point of view, providing evidence of the high evolvability of PCS genes and of plant responsiveness to heavy metal stress.
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a cd fe zn responsive Phytochelatin synthase is constitutively present in the ancient liverwort lunularia cruciata l dumort
Plant and Cell Physiology, 2014Co-Authors: Francesca Degola, Alessandro Petraglia, Maria De Benedictis, Alberto Massimi, Laura Fattorini, Sergio Sorbo, Adriana Basile, Luigi Sanità Di ToppiAbstract:Lunularia cruciata occupies a very basal position in the phylogenetic tree of liverworts, which in turn have been recognized as a very early clade of land plants. It would therefore seem appropriate to take L. cruciata as the startingpoint for investigating character evolution in plants' metal(loid) response. One of the strongest evolutionary pressures for land colonization by plants has come from potential access to much greater amounts of nutritive ions from surface rocks, compared to water. This might have resulted in the need to precisely regulate trace element homeostasis and to minimize the risk of exposure to toxic concentrations of certain metals, prompting the evolution of a number of response mechanisms, such as synthesis of Phytochelatins, metal(loid)-binding thiol-peptides. Accordingly, if the ability to synthesize Phytochelatins and the occurrence of an active Phytochelatin synthase are traits present in a basal liverwort species, and have been even reinforced in 'modern' tracheophytes, e.g. Arabidopsis thaliana, then such traits would presumably have played an essential role in plant fitness over time. Hence, we demonstrated here that: (i) L. cruciata compartmentalizes cadmium in the vacuoles of the phototosynthetic parenchyma by means of a Phytochelatin-mediated detoxification strategy, and possesses a Phytochelatin synthase that is activated by cadmium and homeostatic concentrations of iron(II) and zinc; and (ii) A. thaliana Phytochelatin synthase displays a higher and broader response to several metal(loid)s [namely: cadmium, iron(II), zinc, copper, mercury, lead, arsenic(III)] than L. cruciata Phytochelatin synthase.
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the capability to synthesize Phytochelatins and the presence of constitutive and functional Phytochelatin synthases are ancestral plesiomorphic characters for basal land plants
Journal of Experimental Botany, 2014Co-Authors: Alessandro Petraglia, Maria De Benedictis, Francesca Degola, G Pastore, Margherita Calcagno, Roberta Ruotolo, Alessio Mengoni, Luigi Sanità Di ToppiAbstract:Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as Phytochelatins. Moreover, very little information is available to date regarding Phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce Phytochelatins and possess constitutive and functional Phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 μM cadmium for 72 h, and then assayed for: (i) glutathione and Phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative Phytochelatin synthases by western blotting; and (iii) in vitro activity of Phytochelatin synthases. Of all the species tested, ten produced Phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional Phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to Phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize Phytochelatins, as well as the presence of active Phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants.
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increase in ascorbate glutathione metabolism as local and precocious systemic responses induced by cadmium in durum wheat plants
Plant and Cell Physiology, 2008Co-Authors: Annalisa Paradiso, Luigi Sanità Di Toppi, Rosalia Berardino, Maria Concetta De Pinto, M M Storelli, Franca Tommasi, Laura De GaraAbstract:Durum wheat plants (Triticum durum cv Creso) were grown in the presence of cadmium (0–40kM) and analysed after 3 and 7 d for their growth, oxidative stress markers, Phytochelatins, and enzymes and metabolites of the ascorbate (ASC)–glutathione (GSH) cycle. Cd exposure produced a dose-dependent inhibition of growth in both roots and leaves. Lipid peroxidation, protein oxidation and the decrease in the ascorbate redox state indicate the presence of oxidative stress in the roots, where H2O2 overproduction and Phytochelatin synthesis also occurred. The activity of the ASC–GSH cycle enzymes significantly increased in roots. Consistently, a dosedependent accumulation of Cd was evident in these organs. On the other hand, no oxidative stress symptoms or Phytochelatin synthesis occurred in the leaves; where, at least during the time of our analysis, the levels of Cd remained irrelevant. In spite of this, enzymes of the ASC–GSH cycle significantly increased their activity in the leaves. When ASC biosynthesis was enhanced, by feeding plants with its last precursor, L-galactono-c-lactone (GL), Cd uptake was not affected. On the other hand, the oxidative stress induced in the roots by the heavy metal was alleviated. GL treatment also inhibited the Cd-dependent Phytochelatin biosynthesis. These results suggest that different strategies can successfully cope with heavy metal toxicity. The changes that occurred in the ASC–GSH cycle enzymes of the leaves also suggest that the whole plant improved its antioxidant defense, even in those parts which had not yet been reached by Cd. This precocious increase in the enzymes of the ASC–GSH cycle further highlight the tight regulation and the relevance of this cycle in the defense against heavy metals.
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overexpression of arabidopsis Phytochelatin synthase in tobacco plants enhances cd2 tolerance and accumulation but not translocation to the shoot
Planta, 2006Co-Authors: M Pomponi, Luigi Sanità Di Toppi, Vincenzo Censi, Valentina Di Girolamo, Angelo De Paolis, Rita Aromolo, Paolo Costantino, Maura CardarelliAbstract:Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced Phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis Phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35S AtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd2+ tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd2+ accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd2+ accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd2+ translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd2+ tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of Phytochelatin synthase does not enhance long distance root-to-shoot Cd2+ transport.