Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochelatin synthase (<Emphasis Type="Italic">AtPCS1</Emphasis>) exhibit enhanced As and Cd tolerance

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.     

Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae

Pro has been shown to play an important role in ameliorating environmental stress in plants and microorganisms, including heavy metal stress. Here, we describe the effects of the expression of a mothbean delta(1)-pyrroline-5-carboxylate synthetase (P5CS) gene in the green microalga Chlamydomonas reinhardtii. We show that transgenic algae expressing the mothbean P5CS gene have 80% higher free-Pro levels than wild-type cells, grow more rapidly in toxic Cd concentrations (100 microM), and bind fourfold more Cd than wild-type cells. In addition, Cd-K edge extended x-ray absorption fine structure studies indicated that Cd does not bind to free Pro in transgenic algae with increased Pro levels but is coordinated tetrahedrally by sulfur of phytochelatin. In contrast to P5CS-expressing cells, Cd is coordinated tetrahedrally by two oxygen and two sulfur atoms in wild-type cells. Measurements of reduced/oxidized GSH ratios and analyses of levels of malondialdehyde, a product of the free radical damage of lipids, indicate that free Pro levels are correlated with the GSH redox state and malondialdehyde levels in heavy metal-treated algae. These results suggest that the free Pro likely acts as an antioxidant in Cd-stressed cells. The resulting increased GSH levels facilitate increased phytochelatin synthesis and sequestration of Cd, because GSH-heavy metal adducts are the substrates for phytochelatin synthase.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein

The use of microalgal biomass for metal pollutant bioremediation might be improved by genetic engineering to modify the selectivity or capacity of metal biosorption. A plant cadmium (Cd) and zinc (Zn) transporter (AtHMA4) was used as a transgene to increase the ability of Chlamydomonas reinhardtii to tolerate 0.2 mM Cd and 0.3 mM Zn exposure. The transgenic cells showed increased accumulation and internalization of both metals compared to wild-type. AtHMA4 was expressed either as the full-length (FL) protein or just the C-terminal (CT) tail, which is known to have metal-binding sites. Similar Cd and Zn tolerance and accumulation was observed with expression of either the FL protein or CT domain, suggesting that enhanced metal tolerance was mainly due to increased metal binding rather than metal transport. The effectiveness of the transgenic cells was further examined by immobilization in calcium alginate to generate microalgal beads that could be added to a metal contaminated solution. Immobilization maintained metal tolerance, while AtHMA4-expressing cells in alginate showed a concentration-dependent increase in metal biosorption that was significantly greater than alginate beads composed of wild-type cells. This demonstrates that expressing AtHMA4 FL or CT has great potential as a strategy for bioremediation using microalgal biomass.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Subclinical response to cadmium in liver cells

Effects of cadmium (Cd) in vivo and in vitro were studied in the absence of enhanced metallothionein (MT) production and overt Cd toxicity. Such a condition was established by extended oral exposure of male rats of 0.2 μmol Cd/kg and by incubation of isolated hepatocytes with up to 25 μM for 30 min. Subsequently, mitochondrial and extramitochondrial responses to Cd were recorded. Cadmium diminished the activity of cytochrome c oxidase (CYT C OX) by 50% in vivo and by 35% in vitro. In hepatocytes, this was accompanied by increased Cd and decreased protoheme (PrH) in mitochondria. Extramitochondrial PrH and cytochrome P 450 were not significantly altered. In hepatocytes from phenobarbitone pretreated rats, 25 μM Cd decreased CYT C OX but not mitochondrial PrH. Moreover, simultaneous incubation of hepatocytes with 25 μM Cd and either 2.5 mM dithiothreitol or 5 mM reduced glutathione diminished cellular and mitochondrial Cd and prevented the decrease in CYT C OX but not that in PrH. In contrast, coincubation with either 250 μM l-buthionine-sulfoximine or diethylmaleate, which did not alter Cd uptake, prevented the decrease in PrH but not that in CYT C OX owing to Cd. These results show that Cd exerts mitochondrial alterations in vivo and in vitro in the absence of enhanced MT production. Moreover, Cd effects on CYT C OX and PrH do not seem to be firmly linked.     

Cadmium induction of metallothionein synthesis in Mytilus galloprovincialis

1. The accumulation and subcellular distribution of cadmium and resulting induction of metallothionein has been studied in mussels, Mytilus galloprovincialis, exposed to 400 μg Cd 1⁻¹.2. Cadmium accumulated by mussels was primarily associated with metallothionein: a maximum four-fold increase in concentrations of the metal-binding protein (3 mg g⁻¹ to 12 mg g⁻¹) was observed after 30 days.3. Results for M. galloprovincialis confirm the importance of metallothionein in the detoxification of intracellular cadmium and indicate that induction rates are identical to those observed in the closely-related M. edulis.4. Intercomposition of data between these species may therefore be valid when employing metallothionein assays to determine sub-lethal response to cadmium contamination.     

Phytochelatin synthesis and cadmium localization in wild type of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

AlthoughArabidopsis thaliana is known as a model plant, in molecular studies, as well as heavy metal tolerance of higher plants, there have been no detailed studies of its cadmium accumulation, tolerance and cellular distribution in a wild type of this species. In hydroponic experiments the wild type of A. thaliana (L.) Heynh cv. Columbia plants grew at cadmium concentrations varying from 5 to 100 M with phytotoxicity symptoms depending on the concentration and time of application. The concentration of cadmium in roots and shoots increased from 0.28 and 0.08 mg g^–1 d.wt at 5 M Cd treatment after 7 days to 0.82 and 0.85 mg g^–1 d.wt at 100 M Cd treatment after 14 days, respectively. Most of the cadmium (69–88% of its total pool) was found in shoot. Cd application induced the biosynthesis of phytochelatins (PCs) in root and shoot tissues. Studies with buthionine sulfoximine [BSO, specific inhibitor of glutathione (GSH) synthesis] supported the presence of Cd–phytochelatin complexes and their role in Cd detoxification and tolerance in wild type of A. thaliana. Cellular distribution of cadmium was examined using energy-dispersive X-ray micro-analysis. Particularly interesting was the observation of cadmium localized in the root pericycle.     

Effects of Cadmium Stress on Growth,Morphology, and Protein Expression in Rhodobacter capsulatus B10

The effects of cadmium stress on growth, morphology, and protein expression were investigated in Rhodobacter capsulatus B10 using two-dimensional polyacrylamide gel electrophoresis and a scanning electron microscope with an energy dispersive X-ray spectrometer. The bacterium grew in the presence of 150 μM CdCl₂ and highly induced heat-shock proteins (GroEL and Dnak), S-adenosylmethionine synthetase, ribosomal protein S1, aspartate aminotransferase, and phosphoglycerate kinase. Interestingly, the ribosomal protein S1 was proportionally expressed as the amount of cadmium in the medium, suggesting that S1 may be required for the repair of cadmium-mediated cellular damage. On the other hand, we identified five cadmium-binding proteins: 2-methylcitrate dehydratase, phosphate peripalsmic binding protein, inosine-5′-monophosphate dehydrogenase/guanosine-5′-monophosphate reductase, inositol monophosphatase, and lytic murein transglycosylase. The cadmium-treated cells had a filamentous structure and contained less phosphorous than the untreated cells. We propose that these characteristics of the cadmium-treated cells may be due to the inactivation of the phosphate peripalsmic binding protein and lytic murein transglycosylase by cadmium.     

Effect of exposure to cadmium on the tropical freshwater prawn Macrobrachium rosenbergii

The effect of cadmium (Cd) on mortality, resistance and bioaccumulation in the tropical freshwater prawn Macrobrachium rosenbergii from Egypt were studied. Survival of prawns exposed to Cd doses over 60 μg l^?1 were significantly lower than that of prawns exposed to lower doses. After 96 h prawns exposed to >40 μg l^?1 of Cd had a greater reduction in total haemocyte count and phagocytic activity than those exposed to lower concentrations. Bioaccumulation of Cd in the gills, hepatopancreas and muscles was variable. Cadmium accumulated significantly in the gills and hepatopancreas, but Cd accumulation in the muscles increased only marginally. Macrobrachium rosenbergii manifested histopathological alterations in the gills, hepatopancreas and muscles when exposed to various concentrations of cadmium.     

Cadmium resistance in tobacco plants expressing the MuSI gene

MuSI, a gene that corresponds to a domain that contains the rubber elongation factor (REF), is highly homologous to many stress-related proteins in plants. Since MuSI is up-regulated in the roots of plants treated with cadmium or copper, the involvement of MuSI in cadmium tolerance was investigated in this study. Escherichia coli cells overexpressing MuSI were more resistant to Cd than wild-type cells transfected with vector alone. MuSI transgenic plants were also more resistant to Cd. MuSI transgenic tobacco plants absorbed less Cd than wild-type plants. Cd translocation from roots to shoots was reduced in the transgenic plants, thereby avoiding Cd toxicity. The number of short trichomes in the leaves of wild-type tobacco plants was increased by Cd treatment, while this was unchanged in MuSI transgenic tobacco. These results suggest that MuSI transgenic tobacco plants have enhanced tolerance to Cd via reduced Cd uptake and/or increased Cd immobilization in the roots, resulting in less Cd translocation to the shoots.     

In vitro shoot tip multiplication of cowpea Vigna unguiculata (L.) walp

Summary Shoot multiplication was induced in cowpea, cv. Georgia-21, from shoot tip explants. Shoot tips, 5 mm long, were isolated from in vitro-grown seedlings and cultured on MS medium containing N⁶-benzyladenine (BA) at 1, 2.5, or 5 mg/liter (4.4, 11.1, or 22.2 μM) or 6-furfurylaminopurine (kinetin) at 1, 2.5, or 5 mg/liter (4.6, 11.6, or 23.2 μM) combined with 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.3 μM) or naphthaleneacetic acid (NAA) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.7 μM). Cultures were maintained at a 12-h photoperiod (40 μmol·m⁻²·s⁻¹) and 23 ± 2° C. Treatments with BA induced greater shoot proliferation than those with kinetin. The highest number of shoots was produced on 5 mg (22.2 μM) BA per liter in combination with NAA or 2,4-D at 0.01 mg/liter (0.05 μM). Callus proliferated from the basal ends of shoot pieces in all treatments. The cultures also formed roots in the presence of kinetin, but not on BA-containing medium. To produce whole plants, the shoots were separated and rooted on 0.1 mg (0.5 μM) NAA per liter. Resulting plants grew normally under greenhouse conditions. Shoot tips provide an excellent explant source for cowpea micropropagation and can be used for callus induction.     

The binding of gold(I) to metallothionein

The binding of gold(I) to metallothionein, MT, has been unambiguously established by the reaction of Na₂AuTM with purified horse kidney MT. Zinc was displaced more readily than cadmium although the latter could be displaced using large Au/Cd ratios. The metal exchange reactions were complete within 2 hr of mixing. Further evidence that such reactions might be physiologically significant were obtained by studying in vitro metal displacements in the liver cytosol of in vivo metal treated rats: When Na₂AuTM was added to the cytosol of rats administered CdCl₂ in vivo, zinc, copper and cadmium were displaced in 2/1/1 ratios from the metallothionein fraction. The zinc and cadmium displacement provide direct evidence that the gold was binding to MT. Addition of Cd⁺² to liver cytosol of gold-treated rats resulted in displacement of copper and zinc, but not gold, from the MT fractions. When liver MT is prepared from rats exposed to Au or Cd, the Cd/protein ratio increased during the preparation, but the Au/protein ratio decreased. The Mt-bound metals account for 95% of the cytosolic Cd but only 15%–30% of the cytosolic gold in these studies. Thus, the nonspecific binding of gold to MT in vivo should be considered as one aspect in its equilibration among protein binding sites, which include, inter alia, metallothionein. Gold was found to coelute with zinc and cadmium in the MT fraction of rat kidney cytosol, when both Cd and Na₂AuTM were administered to the rats. The possible significance of gold binding to MT in the treatment of rheumatoid arthritis-chrysotherapy-is briefly discussed.     

Development of surface‐engineered yeast cells displaying phytochelatin synthase and their application to cadmium biosensors by the combined use of pyrene‐excimer fluorescence

The development of simple, portable, inexpensive, and rapid analytical methods for detecting and monitoring toxic heavy metals are important for the safety and security of humans and their environment. Herein, we describe the application of phytochelatin (PC) synthase, which plays a critical role in heavy metal responses in higher plants and green algae, in a novel fluorescent sensing platform for cadmium (Cd). We first created surface‐engineered yeast cells on which the PC synthase from Arabidopsis (AtPCS1) was displayed with retention of enzymatic activity. The general concept for the sensor is based on the Cd level‐dependent synthesis of PC₂ from glutathiones by AtPCS1‐displaying yeast cells, followed by simple discriminative detection of PC₂ via sensing of excimer fluorescence of thiol‐labeling pyrene probes. The intensity of excimer fluorescence increased in the presence of Cd up to 1.0 μM in an approximately dose‐dependent manner. This novel biosensor achieved a detection limit of as low as 0.2 μM (22.5 μg/L) for Cd. Although its use may be limited by the fact that Cu and Pb can induce cross‐reaction, the proposed simple biosensor holds promise as a method useful for cost‐effective screening of Cd contamination in environmental and food samples. The AtPCS1‐displaying yeast cells also might be attractive tools for dissection of the catalytic mechanisms of PCS. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1197–1202, 2013     

Immunological characterization of metallothioneins in mouse LMTK cells and in a variant resistant to cadmium

A cadmium-resistant variant isolated from mouse fibroblast LMTK cells can grow in the presence of 40 muM Cd2+. This variant retained its properties in the absence of selecting agent. Induction of metallothionein was measured in cell extracts by radioimmunoassay. The maximum amount of metallothioneins in the cells was reached after 36 hours. The cadmium resistant variant produced two times more metallothionein than the wild-type when exposed to 10-20 muM Cd2+. By Ouchterlony double diffusion, the metallothioneins from cultured cells formed a line of partial identity with the mouse liver serotype and a line of complete identity with one of the two mouse kidney serotypes. These observations raise the possibility of a tissue-specific expression of metallothionein genes.     

Effects of Metal Combinations on the Production of Phytochelatins and Glutathione by the Marine Diatom Phaeodactylum tricornutum

Copper, Cd and Zn can be found at elevated concentrations in contaminated estuarine and coastal waters and have potential toxic effects on phytoplankton species. In this study, the effects of these metals on the intracellular production of the polypeptides phytochelatin and glutathione by the marine diatom Phaeodactylum tricornutum were examined in laboratory cultures. Single additions of Cu and Cd (0.4 μM Cu² and 0.45 μM Cd²⁺) to the culture medium induced the production of short-chained phytochelatins ((γ-Glu-Cys)_n-Gly where n = 2–5), whereas a single addition of Zn (2.2 μM Zn²⁺) did not stimulate phytochelatin production. Combination of Zn with Cu resulted in a similar phytochelatin production compared with a single Cu addition. The simultaneous exposure to Zn and Cd led to an antagonistic effect on phytochelatin production, which was probably caused by metal competition for cellular binding sites. Glutathione concentrations were affected only upon exposure to Cd (85% increase) or the combination of Cd with Zn (65% decrease), relative to the control experiment. Ratios of phytochelatins to glutathione indicated a pronounced metal stress in response to exposures to Cu or Cd combined with Zn. This study indicates that variabilities in phytochelatin and glutathione production in the field can be explained in part by metal competition for cellular binding sites.     

Characterization of a cadmium-zinc complex in lettuce leaves

Vegetable food contributes a higher amount of daily cadmium (Cd) intake in humans than food of animal origin. The bioavailability of plant Cd depends on the content of plant zinc (Zn). The mechanism by which increased plant Zn lowers the intestinal absorption of plant Cd could be mediated by changes in the chemical speciation of Cd or Zn in plant edible tissues, including Zn-induced phytochelatin synthesis. To test this hypothesis we investigated the chemical speciation of Cd and Zn in leaf extracts of lettuce grown under 10 ΜM of Cd accompanied by 0.32 or 31.6 ΜM Zn in nutrient solution. Gel filtration chromatography of the low- or high-Zn leaf extracts yielded a major low molecular weight Cd-Zn complex that eluted at similar elution volume. Compared to low-Zn leaf extracts, high-Zn leaf extracts contained a higher proportion of Zn incorporated into high molecular weight components, and higher content of the amino acids Cys, Glu, Gly, and Asp in the low molecular weight Cd-Zn complex. The peptides isolated by high performance liquid chromatography (HPLC) of the Cd-Zn complex from the low- or high-Zn leaf extracts did not have an amino acid composition identical to phytochelatins. We concluded that

1. Sequestration of Cd or Zn via phytochelatin does not occur in leaves of lettuce containing levels of those metals representatives of Zn-Cd or Cd-only contaminated crops; and
2. Higher Cys, Glu, Gly, and Asp content in high-Zn than low-Zn leaves could lower Cd absorption in animals fed high-Zn crop diets, by enhancing metallothionein synthesis or changing Cd or Zn speciation in the animal gut.