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.     

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.     

In vitro breeding of Brassica juncea L. to enhance metal accumulation and extraction properties

In vitro breeding and somaclonal variation were used as tools to improve the potential of Indian mustard (Brassica juncea L.) to extract and accumulate toxic metals. Calli from B. juncea were cultivated on a modified MS medium supplemented with 10–200 μM Cd or Pb. Afterwards, new B. juncea somaclones were regenerated from metal-tolerant callus cells. Three different phenotypes with improved tolerance of Cd, Zn and Pb were observed under hydroponic conditions: enhanced metal accumulation in both shoots and roots; limited metal translocation from roots to shoots; reduced accumulation in shoots and roots. Seven out of thirty individual variants showed a significantly higher metal extraction than the control plants. The improvement of metal shoot accumulation of the best regenerant (3× Cd, 1.6× Zn, 1.8× Pb) and metal extraction (6.2× Cd, 3.2× Zn, 3.8× Pb) indicated a successful breeding and selection of B. juncea, which could be used for phytoremediation purpose.     

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype.     

Antioxidant defense system in leaves of Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) and rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) under cadmium stress

Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from environment. Using two brassica species (Brassica juncea and Brassica napus), nutrient solution experiments were conducted to study variation in tolerance to cadmium (Cd) toxicity based on (1) lipid peroxidation and (2) changes in antioxidative defense system in leaves of both plants (i.e., superoxide dismutase (SOD EC 1.15.1.1), catalase (CAT EC 1.11.1.6), ascorbate peroxidase (APX EC 1.11.1.11), guaiacol peroxidase (GPX EC 1.11.1.7), glutathione reductase (GR EC 1.6.4.2), levels of phytochelatins (PCs), non-protein thiols (NP-SH), and glutathione. Plants were grown in nutrient solution under controlled environmental conditions, and subjected to increasing concentrations of Cd (0, 10, 25 and 50 μM) for 15 days. Results showed marked differences between both species. Brassica napus under Cd stress exhibited increased level of lipid peroxidation, as was evidenced by the increased malondialdehyde (MDA) content in leaves. However, in Brassica juncea treated plants, MDA content remained unchanged. In Brassica napus, with the exception of GPX, activity levels of some antioxidant enzymes involved in detoxification of reactive oxygen species (ROS), including SOD, CAT, GR, and APX, decreased drastically at high Cd concentrations. By contrast, in leaves of Brassica juncea treated plants, there was either only slight or no change in the activities of the antioxidative enzymes. Analysis of the profile of anionic isoenzymes of GPX revealed qualitative changes occurring during Cd exposure for both species. Moreover, levels of NP-SH and PCs, monitored as metal detoxifying responses, were much increased in leaves of Brassica juncea by increasing Cd supply, but did not change in Brassica napus. These results indicate that Brassica juncea plants possess the greater potential for Cd accumulation and tolerance than Brassica napus.     

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

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.     

Response to nickel in the proteome of the metal accumulator plant Brassica juncea

To gain a comprehensive understanding of the molecular mechanism of heavy metal accumulation in Brassica juncea, comparative proteomic approaches were used to analysis protein profiles in leaf tissues of 6-week-old B. juncea after exposure to 100 µM Ni. Proteomic analysis revealed that 61 protein spots showed 1.5-fold change in protein abundance after Ni exposure as compared to that of corresponding spots in control. Out of the 61 differentially expressed protein spots, 37 protein spots were ambiguously identified by matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS). The majority of these identified proteins were found to be involved in sulphur metabolism, protection against oxidative stress, clearly indicated that heavy metal sequestration and antioxidant system were activated by Ni treatment. The induced expression of photosynthesis and ATP generation-related proteins were also observed in plants exposed to metals, suggesting the tolerance and accumulation is an energy-demanding process. The identification of these proteins in response to Ni can lead a deep understanding of heavy metal accumulation and tolerance in B. juncea.     

Dark septate endophyte (DSE) fungi isolated from metal polluted soils: Their taxonomic position, tolerance, and accumulation of heavy metals In Vitro

To understand the possible role of the plant root associated fungi on metal tolerance, their role in the uptake of heavy metals and the potential transfer of these metal ions to the plant, three strains of dark septate endophytic (DSE) fungi were isolated from a waste smelter site in southwest China, and one strain was isolated from a non-contaminated site. According to molecular phylogenetic analysis of the ITS 1-5.8S rDNA-ITS 2 gene regions and morphological characteristics, one is identified as Exophiala pisciphila, and the other three are non-sporulating fungi under the experiment condition with the nearest phylogenetic affinities to the Thysanorea papuana strain EU041814. Tolerance and accumulation abilities of the three DSE strains for metals were investigated in liquid culture. Minimum inhibitory concentrations (MIC) of Pb, Zn, and Cd were determined. It was demonstrated that the tolerance of the DSE strains varied between metal species and strains. The E. pisciphila strain is able to accumulate lead and cadmium over 20% and 5% of dry weight of biomass, respectively. Partial of the sequestrated metals can be washed with CaCh. Morphological and enzyme activity changes taking place in the presence of excessive Pb, Cd, and/or Zn also indicate that the mechanism of heavy metal tolerance and accumulation of the DSE strains would be a complex process. The findings indicated promising tolerance and accumulation of the DSE strains with potential values in metal cycling and restoration of soil and water system.     

Functional analyses of TaHMA2, a P1B‐type ATPase in wheat

Currently, there are few studies concerning the function of heavy metal ATPase 2 (HMA2), particularly in monocotyledons, and the potential application of this protein in biofortification and phytoremediation. Thus, we isolated and characterized the TaHMA2 gene from wheat (Triticum aestivum L.). Our results indicate that TaHMA2 is localized to the plasma membrane and stably expressed, except in the nodes, which showed relatively high expression. Zinc/cadmium (Zn/Cd) resistance was observed in TaHMA2‐transformed yeast. The over‐expression of TaHMA2 increased the elongation and decreased the seed‐setting rate in rice (Oryza sativa L.), but not Arabidopsis thaliana, tobacco (Nicotiana tabacum L.) or wheat. TaHMA2 over‐expression also improved root‐shoot Zn/Cd translocation, especially in rice. The seeds of transgenic rice and wheat, not tobacco, showed decreased Zn concentrations. The Zn concentration was decreased in all parts of the transgenic rice seeds, but was decreased only in the ventral endosperm of wheat, which showed an increased Zn concentration in the embryo and aleurone. The over‐expression of TaHMA2 improved plant tolerance under moderate Zn stress and Zn deficiency, but Zn and Cd resistance decreased under high levels of Zn and Cd stress, respectively. The Cd concentration in transgenic rice seedlings was dramatically increased under Zn deficiency. Thus, over‐expression of TaHMA2 showed a more obvious phenotype in monocotyledons than in dicotyledons. These findings provide important information for TaHMA2, and more efforts should be made in the future to characterize the reduced Zn concentration in TaHMA2 transgenic grains and the diversity of TaHMA2 substrate specificity.     

Effect of bacterial inoculation of strains of pseudomonas aeruginosa,alcaligenes feacalis and bacillus subtilis on germination,growth and heavy metal (cd,cr, and ni) uptake of brassica juncea

Bacterial inoculation may influence Brassica juncea growth and heavy metal (Ni, Cr, and Cd) accumulation. Three metal tolerant bacterial isolates (BCr3, BCd33, and BNi11) recovered from mine tailings, identified as Pseudomonas aeruginosa KP717554, Alcaligenes feacalis KP717561, and Bacillus subtilis KP717559 were used. The isolates exhibited multiple plant growth beneficial characteristics including the production of indole-3-acetic acid, hydrogen cyanide, ammonia, insoluble phosphate solubilization together with the potential to protect plants against fungal pathogens. Bacterial inoculation improved seeds germination of B. juncea plant in the presence of 0.1 mM Cr, Cd, and Ni, as compared to the control treatment. Compared with control treatment, soil inoculation with bacterial isolates significantly increased the amount of soluble heavy metals in soil by 51% (Cr), 50% (Cd), and 44% (Ni) respectively. Pot experiment of B. juncea grown in soil spiked with 100 mg kg^?1 of NiCl₂, 100 mg kg^?1 of CdCl₂, and 150 mg kg^?1 of K₂Cr₂O₇, revealed that inoculation with metal tolerant bacteria not only protected plants against the toxic effects of heavy metals, but also increased growth and metal accumulation of plants significantly. These findings suggest that such metal tolerant, plant growth promoting bacteria are valuable tools which could be used to develop bio-inoculants for enhancing the efficiency of phytoextraction.     

Enhanced Heavy Metal Tolerance and Accumulation by Transgenic Sugar Beets Expressing Streptococcus thermophilus StGCS-GS in the Presence of Cd,Zn and Cu Alone or in Combination

Phytoremediation is a promising means of ameliorating heavy metal pollution through the use of transgenic plants as artificial hyperaccumulators. A novel Streptococcus thermophilus γ-glutamylcysteine synthetase-glutathione synthetase (StGCS-GS) that synthesizes glutathione (GSH) with limited feedback inhibition was overexpressed in sugar beet (Beta vulgaris L.), yielding three transgenic lines (s2, s4 and s5) with enhanced tolerance to different concentrations of cadmium, zinc and copper, as indicated by their increased biomass, root length and relative growth compared with wild-type plants. Transgenic sugar beets accumulated more Cd, Zn and Cu ions in shoots than wild-type, as well as higher GSH and phytochelatin (PC) levels under different heavy metal stresses. This enhanced heavy metal tolerance and increased accumulation were likely due to the increased expression of StGCS-GS and consequent overproduction of both GSH and PC. Furthermore, when multiple heavy metal ions were present at the same time, transgenic sugar beets overexpressing StGCS-GS resisted two or three of the metal combinations (50 μM Cd-Zn, Cd-Cu, Zn-Cu and Cd-Zn-Cu), with greater absorption in shoots. Additionally, there was no obvious competition between metals. Overall, the results demonstrate the explicit role of StGCS-GS in enhancing Cd, Zn and Cu tolerance and accumulation in transgenic sugar beet, which may represent a highly promising new tool for phytoremediation.     

Expression of a Brassica napus heme oxygenase confers plant tolerance to mercury toxicity

Plant heme oxygenases (HOs) regulate biosynthesis of phytochrome which accounts for photo‐acceptance and ‐morphogenesis. Recent studies have demonstrated that plant HOs also regulate many other physiological processes including response to environmental stimuli. To elucidate the mechanism by which HOs regulate plant adaptation to heavy metal exposure, three novel HOs genes were isolated from rapeseed (Brassica napus) and their expression patterns were analysed. Alignment of deduced protein sequences revealed that the three BnHOs share high identity with their corresponding orthologos (AtHO1‐3) from Arabidopsis. To investigate whether the BnHO regulates plant tolerance to Hg toxicity, we constructed B. napus transgenic plants overexpressing BnHO‐1. Under Hg stress, the transgenic plants had 1.41–1.59 folds higher biomass than the untransformants. However, overexpression of BnHO‐1 resulted in less accumulation of Hg in some lines of transformants than in untransformants. The transgenic plants show lower abundance of reactive oxygen species and attenuated oxidative injury compared with the untransgenic plants. We cloned the promoter sequences of BnHO‐1 from B. napus. Analysis revealed that the 1119 bp fragment contains a conserved Cd responsive element (CdRE) and others responding to multiple environmental stimuli. Transient expression in tobacco leaves showed differential responses to heavy metals (Zn, Cu, Pb, Hg and Cd).     

Heavy Metal Tolerance and Accumulation in Indian Mustard (Brassica Juncea L.) Expressing Bacterial γ-Glutamylcysteine Synthetase or Glutathione Synthetase

The overexpression of either γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase (GS) in Brassica juncea transgenics was shown previously to result in higher accumulation of glutathione (GSH) and phytochelatins (PCs), as well as enhanced Cd tolerance and accumulation. The present study was aimed at analyzing the effects of γ-ECS or GS overexpression on tolerance to and accumulation of other metal/loids supplied individually in agar medium (seedlings) or in hydroponics (mature plants). Also, as pollution in nature generally consists of mixtures of metals, glutamylcysteine synthetase (ECS) and GS seedlings were tested on combinations of metals. Compared to wild-type plants, ECS and GS transgenics exhibited a significantly higher capacity to tolerate and accumulate a variety of metal/loids (particularly As, Cd, and Cr) as well as mixed-metal combinations (As, Cd, Zn/As, Pb, and Zn). This enhanced metal tolerance and accumulation of the ECS and GS transgenics may be attributable to enhanced production of PCs, sustained by a greater availability of GSH as substrate, as suggested by their higher concentrations of GSH, PC2, PC3, and PC4 as compared to wild-type plants. Overexpression of GS and γ-ECS may represent a promising strategy for the development of plants with an enhanced phytoremediation capacity for mixtures of metals.