Synthetic Phytochelatin Surface Display in Cupriavidus metallidurans CH34 for Enhanced Metals Bioremediation

This work describes the effects of the cell surface display of a synthetic phytochelatin in the highly metal tolerant bacterium Cupriavidus metallidurans CH34. The EC20sp synthetic phytochelatin gene was fused between the coding sequences of the signal peptide (SS) and of the autotransporter beta-domain of the Neisseria gonorrhoeae IgA protease precursor (IgA beta), which successfully targeted the hybrid protein toward the C. metallidurans outer membrane. The expression of the SS-EC20sp-IgA beta gene fusion was driven by a modified version of the Bacillus subtilis mrgA promoter showing high level basal gene expression that is further enhanced by metal presence in C. metallidurans. The recombinant strain showed increased ability to immobilize Pb2+, Zn2+, Cu2+, Cd2+, Mn2+, and Ni2+ ions from the external medium when compared to the control strain. To ensure plasmid stability and biological containment, the MOB region of the plasmid was replaced by the E. coli hok/sok coding sequence.

Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro-Tom) plants to cadmium-induced stress

The effects of varying concentrations of cadmium (Cd) on the development of Lycopersicon esculentum cv. Micro-Tom (MT) plants were investigated after 40 days (vegetative growth) and 95 days (fruit production), corresponding to 20 days and 75 days of exposure to CdCl(2), respectively. Inhibition of growth was clearly observed in the leaves after 20 days and was greater after 75 days of growth in 1 mM CdCl(2), whereas the fruits exhibited reduced growth following the exposure to a concentration as low as 0.1 mM CdCl(2). Cd was shown to accumulate in the roots after 75 days of growth but was mainly translocated to the upper parts of the plants accumulating to high concentrations in the fruits. Lipid peroxidation was more pronounced in the roots even at 0.05 mM CdCl(2) after 75 days, whereas in leaves, there was a major increase after 20 days of exposure to 1 mM CdCl(2), but the fruit only exhibited a slight significant increase in lipid peroxidation in plants subjected to 1 mM CdCl(2) when compared with the control. Oxidative stress was also investigated by the analysis of four key antioxidant enzymes, which exhibited changes in response to the increasing concentrations of Cd tested. Catalase (EC 1.11.1.6) activity was shown to increase after 75 days of Cd treatment, but the major increases were observed at 0.1 and 0.2 mM CdCl(2), whereas guaiacol peroxidase (EC 1.11.1.7) did not vary significantly from the control in leaves and roots apart from specific changes at 0.5 and 1 mM CdCl(2). The other two enzymes tested, glutathione reductase (EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1), did not exhibit any significant changes in activity, apart from a slight decrease in SOD activity at concentrations above 0.2 mM CdCl(2). However, the most striking results were obtained when an extra treatment was used in which a set of plants was subjected to a stepwise increase in CdCl(2) from 0.05 to 1 mM, leading to tolerance of the Cd applied even at the final highest concentration of 1 mM. This apparent adaptation to the toxic effect of Cd was confirmed by biomass values being similar to the control, indicating a tolerance to Cd acquired by the MT plants.

Resposta antioxidante, formação de fitoquelatinas e composição de pigmentos fotoprotetores em Brachiaria decumbens Stapf submetida à contaminação com Cd e Zn

In order to evaluate the response of Brachiaria decumbens Stapf to Cd and Zn, plants were hydroponically exposed to 50 and 100 mmol L-1 of Cd and 500 and 2000 mmol L-1 of Zn. Metal content of shoots and roots was determined, as well as alterations in photosynthetic and photoprotective pigments, antioxidant metabolites and phytochelatin synthesis. Plants concentrated elevated levels of Cd and Zn, especially in roots. Zinc exposure negatively affected chlorophyll and β-carotene content, whereas the highest dose of Cd reduced VAZ cycle pigments and tocopherol levels in plant shoots. Cadmium was the maximum inducer of the phytochelatin synthesis pathway.

Effet du calcium, plomb et cuivre sur la bioaccumulation du cadmium et la production des phytochélatines par Chlamydomonas reinhardtii

Dans les milieux contaminés par les métaux, les organismes vivants sont exposés à plusieurs d’entre eux en même temps. Les modèles courants de prédiction des effets biologiques des métaux sur les organismes (p. ex., modèle du ligand biotique, BLM ; modèle de l’ion libre, FIAM), sont des modèles d’équilibre chimique qui prévoient, en présence d'un deuxième métal, une diminution de la bioaccumulation du métal d’intérêt et par la suite une atténuation de ses effets. Les biomarqueurs de toxicité, tels que les phytochélatines (PCs), ont été utilisés comme étant un moyen alternatif pour l’évaluation des effets biologiques. Les phytochélatines sont des polypeptides riches en cystéine dont la structure générale est (γ-glu-cys)n-Gly où n varie de 2 à 11. Leur synthèse semble dépendante de la concentration des ions métalliques ainsi que de la durée de l’ exposition de l’organisme, aux métaux. L'objectif de cette étude était donc de déterminer, dans les mélanges binaires de métaux, la possibilité de prédiction de la synthèse des phytochélatines par les modèles d’équilibres chimiques, tel que le BLM. Pour cela, la quantité de phytochélatines produites en réponse d’une exposition aux mélanges binaires : Cd-Ca, Cd-Cu et Cd-Pb a été mesurée tout en surveillant l’effet direct de la compétition par le biais des concentrations de métaux internalisés. En effet, après six heures d’exposition, la bioaccumulation de Cd diminue en présence du Ca et de très fortes concentrations de Pb et de Cu (de l’ordre de 5×10-6 M). Par contre, avec des concentrations modérées de ces deux métaux, le Cd augmente en présence de Cu et ne semble pas affecté par la présence de Pb. Dans le cas de la compétition Cd-Cu, une bonne corrélation a été observée entre la production de PC2, PC3 et PC4 et la quantité des métaux bioaccumulés. Pour la synthèse des phytochélatines et la bioaccumulation, les effets étaient considérés comme synergiques. Dans le cas du Cd-Ca, les quantités de PC3 et PC4 ont diminué avec le métal internalisé (effet antagoniste), mais ce qui était remarquable était la grande quantité de cystéine (GSH) et PC2 qui ont été produites à de fortes concentrations du Ca. Le Pb seul n’a pas induit les PCs. Par conséquent, il n’y avait pas de variation de la quantité de PCs avec la concentration de Pb à laquelle les algues ont été exposées. La détection et la quantification des PCs ont été faites par chromatographie à haute performance couplée d’un détecteur de fluorescence (HPLC-FL). Tandis que les concentrations métalliques intracellulaires ont été analysées par spectroscopie d’absorption atomique (AAS) ou par spectrométrie de masse à source plasma à couplage inductif (ICP-MS).

Transcript levels of AtMRPs after cadmium treatment: induction of AtMRP3

In yeasts, the ABC-type transporters are involved in vacuolar sequestration of cadmium. In plants, transport experiments with isolated vacuoles indicate that this is also true. In order to know more about the response of AtMRPs, a subclass of Arabidopsis ABC transporters, to cadmium, their expression pattern was analysed using the microchip technology and semi-quantitative reverse transcriptase-polymerase chain reaction. From 15 putative sequences coding for AtMRPs, transcript levels were detected for 14. All were expressed in the roots as well as in the shoots, although at a different level. In 4-week-old Arabidopsis, transcript levels of four AtMRPs were up-regulated after cadmium treatment. In all cases up-regulation was exclusively observed in the roots. The increase of transcript levels was most pronounced for AtMRP3. A more detailed analysis revealed that induction of AtMRP3 could also be observed in the shoot when leaves were cut and cadmium allowed to be taken up in the shoot. In young plantlets, a far higher portion of Cd2+ was translocated in the aerial part compared with adult plants. Consequently, AtMRP3 transcript levels increased in both root and shoot of young plants. This suggests that 7-day-old seedlings do not exhibit such a strict root–shoot barrier as 4-week-old plants. Expression analysis with mutant plants for glutathione and phytochelatin synthesis as well as with compounds producing oxidative stress indicate that induction of AtMRP3 is likely due to the heavy metal itself.

Overexpression of Glutathione Synthetase in Indian Mustard Enhances Cadmium Accumulation and Tolerance1

An important pathway by which plants detoxify heavy metals is through sequestration with heavy-metal-binding peptides called phytochelatins or their precursor, glutathione. To identify limiting factors for heavy-metal accumulation and tolerance, and to develop transgenic plants with an increased capacity to accumulate and/or tolerate heavy metals, the Escherichia coli gshII gene encoding glutathione synthetase (GS) was overexpressed in the cytosol of Indian mustard (Brassica juncea). The transgenic GS plants accumulated significantly more Cd than the wild type: shoot Cd concentrations were up to 25% higher and total Cd accumulation per shoot was up to 3-fold higher. Moreover, the GS plants showed enhanced tolerance to Cd at both the seedling and mature-plant stages. Cd accumulation and tolerance were correlated with the gshII expression level. Cd-treated GS plants had higher concentrations of glutathione, phytochelatin, thiol, S, and Ca than wild-type plants. We conclude that in the presence of Cd, the GS enzyme is rate limiting for the biosynthesis of glutathione and phytochelatins, and that overexpression of GS offers a promising strategy for the production of plants with superior heavy-metal phytoremediation capacity.

Mechanistic study of arsenic uptake, transformation and tolerance in arsenic hyperaccumulator Pteris vittata

Pteris vittata, the first reported arsenic hyperaccumulating plant, is potentially used in phytoremediation of arsenic, as it can accumulate up to 2.3% of arsenic in its fronds. In this study, the mechanisms of arsenic tolerance, uptake and transformation were studied in the plant. Arsenic species were analyzed by HPLC-AFS. Results showed that arsenic was mainly accumulated in leaflets, and inorganic arsenate and arsenite were only species in P. vittata. Arsenite was the predominant species in leaflets, whereas arsenate was the predominant species in roots. Arsenic induced the synthesis of thiol containing compounds in P. vittata. As-induced thiol was purified by a novel method: covalent chromatography following preparative HPLC. The purified thiol was characterized as a phytochelatin with two units (PC2). ^ In P. vittata, enhanced tolerance likely results from unusual intracellular detoxification mechanisms. Although PC-dependent sequestration of arsenic into vacuoles is essential for nonhyperaccumulators, this sequestration is not the major arsenic tolerance mechanisms in this arsenic hyperaccumulator. PC-independent sequestration of arsenic is likely the major arsenic tolerance mechanism. PC-dependent arsenic detoxification is probably a supplement to this major mechanism. ^ Interactions between arsenic and phosphate were studied. Under hydroponic condition, arsenic supply decreased the concentrations of phosphate in roots. In soil, arsenic increased the concentrations of phosphate in roots. Arsenic concentrations in rachises and leaflets were not affected by arsenic supply in either hydroponic or soil system. Phosphate decreased arsenic accumulation in roots, rachises and leaflets in the hydroponic system. ^ The uptake kinetics of arsenate, arsenite, monomethyl arsinic acid (MMA), dimethyl arsonic acid, and phosphate were studied in P. vittata. Phosphate uptake systems in Pteris vittata cannot distinguish phosphate and As(V), resulting in As hyperaccumulation. Arsenic hyperaccumulation in this plant is an inevitable consequence during phosphate acquisition. Arsenate, arsenite and MMA are transported via the phosphate uptake systems. The co-transport of arsenite/phosphate and MMA/phosphate is reported for the first time in plants. These unique phenomena are useful for understanding arsenic hyperaccumulation and the evolution of this capacity in P. vittata. ^