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.     

A novel heavy metal ATPase peptide from <Emphasis Type="Italic">Prosopis juliflora</Emphasis> is involved in metal uptake in yeast and tobacco

Heavy metal pollution of agricultural soils is one of the most severe ecological problems in the world. Prosopis juliflora, a phreatophytic tree species, grows well in heavy metal laden industrial sites and is known to accumulate heavy metals. Heavy Metal ATPases (HMAs) are ATP driven heavy metal pumps that translocate heavy metals across biological membranes thus helping the plant in heavy metal tolerance and phytoremediation. In the present study we have isolated and characterized a novel 28.9 kDa heavy metal ATPase peptide (PjHMT) from P. juliflora which shows high similarity to the C-terminal region of P_1B ATPase HMA1. It also shows the absence of the invariant signature sequence DKTGT, and the metal binding CPX motif but the presence of conserved regions like MVGEGINDAPAL (ATP binding consensus sequence), HEGGTLLVCLNS (metal binding domain) and MLTGD, GEGIND and HEGG motifs which play important roles in metal transport or ATP binding. PjHMT, was found to be upregulated under cadmium and zinc stress. Heterologous expression of PjHMT in yeast showed a higher accumulation and tolerance of heavy metals in yeast. Further, transgenic tobacco plants constitutively expressing PjHMT also showed increased accumulation and tolerance to cadmium. Thus, this study suggests that the transport peptide from P. juliflora may have an important role in Cd uptake and thus in phytoremediation.     

Mechanisms of cadmium detoxification in cattail (Typha angustifolia L.)

Cadmium (Cd) is a widespread heavy metal pollutant and environmental and human health hazard, which may be partially resolved using green and cost-effective phytoremediation techniques. However, the efficiency of phytoremediation is often limited by the small biomass of Cd-hyperaccumulator plants. Although cattail (Typha angustifolia L.) is tolerant of heavy metals and has a high biomass, there is little information available on its detoxification mechanisms for heavy metals, especially Cd. In the present study we investigated the tolerance of cattail to Cd and mechanisms involved in its Cd detoxification. Our results show that: (a) cattail is tolerant of Cd; (b) the root Casparian band, cell wall, vacuole, glutathione (GSH), and glutathione peroxidase (GPX) play important roles in Cd detoxification; and (c) mechanisms of Cd detoxification differ in leaf cell cytoplasm (mainly a GSH-related antioxidant defense system) and root cell cytoplasm (mainly a GSH-related chelation system). In summary, cattail possesses multiple detoxification mechanisms for Cd and is a promising species for phytoremediation of Cd-polluted environments.     

Cadmium uptake,localization and stress-induced morphogenic response in the fern Pteris vittata

Cadmium uptake, tissue localization and structural changes induced at cellular level are essential to understand Cd tolerance in plants. In this study we have exposed plants of Pteris vittata to different concentrations of CdCl₂ (0, 30, 60, 100 μM) to evaluate the tolerance of the fern to cadmium. Cadmium content determination and its histochemical localization showed that P. vittata not only takes up, but also transports and accumulates cadmium in the aboveground tissues, delocalizing it mainly in the less bioactive tissues of the frond, the trichomes and the scales. Cadmium tolerance in P. vittata was strictly related to morphogenic response induced by the metal itself in the root system. Adaptive response regarded changes of the root apex size, the developmental pattern of root hairs, the differentiation of xylem elements and endodermal suberin lamellae. All the considered parameters suggest that, in our experimental conditions, 60 μM of Cd may represent the highest concentration that P. vittata can tolerate; indeed this Cd level even improves the absorbance features of the root and allows good transport and accumulation of the metal in the fronds. The results of this study can provide useful information for phytoremediation strategies of soils contaminated by Cd, exploiting the established ability of P. vittata to transport, delocalize in the aboveground biomass and accumulate polluting metals.     

Tolerance to cadmium in plants: the special case of hyperaccumulators

On sols highly polluted by trace metallic elements the majority of plant species are excluders, limiting the entry and the root to shoot translocation of trace metals. However a rare class of plants called hyperaccumulators possess remarkable adaptation because those plants combine extremely high tolerance degrees and foliar accumulation of trace elements. Hyperaccumulators have recently gained considerable interest, because of their potential use in phytoremediation, phytomining and biofortification. On a more fundamental point of view hyperaccumulators of trace metals are case studies to understand metal homeostasis and detoxification mechanisms. Hyperaccumulation of trace metals usually depends on the enhancement of at least four processes, which are the absorption from the soil, the loading in the xylem in the roots and the unloading from the xylem in the leaves and the detoxification in the shoot. Cadmium is one of the most toxic trace metallic elements for living organisms and its accumulation in the environment is recognized as a worldwide concern. To date, only nine species have been recognized as Cd hyperaccumulators that is to say able to tolerate and accumulate more than 0.01 % Cd in shoot dry biomass. Among these species, four belong to the Brassicaceae family with Arabidopsis halleri and Noccaea caerulescens being considered as models. An update of our knowledge on the evolution of hyperaccumulators will be presented here.     

黔西北铅锌矿区植物群落分布及其对重金属的迁移特征

重金属耐性植物和超富集植物的筛选、鉴定和驯化是植物修复技术研究与发展的关键。以黔西北4个不同恢复年限的铅锌矿为研究对象,通过群落生态调查利用聚类分析方法筛选出研究区域中重金属耐性植物优势种,并分析其对重金属Pb、Zn、Cu、Cd的迁移富集能力。结果表明:4个矿区共发现高等植物22种,分属13科21属,筛选出9种重金属耐性植物优势种,其中转运系数大于1的植物有:黄花蒿(Cu)、珠光香青(Zn)、大叶醉鱼草(Zn/Pb/Cd)、野艾蒿(Cu/Zn/Pb/Cd);没有富集系数大于1的植物。其中大叶醉鱼草具有耐贫瘠、耐旱、生物量大等优势,可将其作为典型的重金属耐性先锋植物,用于矿区废弃地的植物修复。     

Different Growth and Physiological Responses to Cadmium of the Three Miscanthus Species

Miscanthus has been proposed as a promising crop for phytoremediation due to its high biomass yield and remarkable adaptability to different environments. However, little is known about the resistance of Miscanthus spp. to cadmium (Cd). To determine any differences in resistance of Miscanthus to Cd, we examined plant growth, net photosynthetic rate (Pn), activities of anti-oxidant and C₄ photosynthetic enzymes, concentrations of Cd in leaves and roots, and observed the chloroplast structure in three Miscanthus species treated with 0, 10, 50, 100 or 200 μM Cd in solutions. Miscanthus sinensis showed more sensitivity to Cd, including sharp decreases in growth, Pn, PEPC activity and damage to chloroplast structure, and the highest H₂O₂ and Cd concentrations in leaves and roots after Cd treatments. Miscanthus sacchariflorus showed higher resistance to Cd and better growth, had the highest Pn and phosphoenolpyruvate carboxylase (PEPC) activities and integrative chloroplast structure and the lowest hydrogen peroxide (H₂O₂) and leaf and root Cd concentrations. The results could play an important role in understanding the mechanisms of Cd tolerance in plants and in application of phytoremediation.     

Cloning and Characterization of a Phragmites australis Phytochelatin Synthase (PaPCS) and Achieving Cd Tolerance in Tall Fescue

The production of phytochelatins (PCs) provides an important means for plants to achieve tolerance to cadmium (Cd) toxicity. A reed gene encoding PC synthase (PaPCS) was isolated and its function tested through its heterologous expression in a strain of yeast sensitive to Cd. Subsequently, the Cd sensitive and high biomass accumulating species tall fescue was transformed either with PaPCS or PaGCS (a glutamyl cysteine synthetase gene of reed) on their own (single transformants), or with both genes together in the same transgene cassette (double transformant). The single and double transformants showed greater Cd tolerance and accumulated more Cd and PC than wild type plants, and their Cd leaf/root ratio content was higher. The ranking in terms of Cd and PC content for the various transgenic lines was double transformants>PaGCS single transformants>PaPCS single transformants>wild type. Thus PaGCS appears to exert a greater influence than PaPCS over PC synthesis and Cd tolerance/accumulation. The double transformant has interesting potential for phytoremediation.     

镉在土壤-香根草系统中的迁移及转化特征

以无植物组处理为对照,采用盆栽试验方式探讨不同Cd浓度胁迫条件下香根草根际土壤中重金属Cd的积累、迁移及转化特征。土壤Cd处理设4个浓度梯度,分别为0、2、20、80 mg/kg土壤干重。结果表明:(1)香根草可以显著降低土壤中生物有效态Cd和总Cd含量。(2)香根草各部分Cd积累量随处理浓度的增加和处理时间的延长而增加,90 d时80 mg/kg处理组地上部分和根的Cd积累量分别高达180.42 mg/kg和241.54 mg/kg。(3)各浓度Cd处理下,富集系数随着Cd处理浓度的增加而显著降低,随处理时间的延长而升高。(4)香根草地上部分Cd含量小于根部,各处理转移系数均小于1。随着处理时间的延长,中低浓度处理组的转移系数稍有降低,高浓度处理组的转移系数则显著上升。(5)种植香根草使其根际土中残渣态的Cd转化为生物有效态Cd,提高Cd清除效率。研究结果表明,香根草能够有效地吸收土壤中的Cd,降低土壤中总Cd含量,提高土壤安全性,可作为Cd污染地区植物修复的备选物种。     

Arbuscular Mycorrhizal Colonization Alters Subcellular Distribution and Chemical Forms of Cadmium in Medicago sativa L. and Resists Cadmium Toxicity

Some plants can tolerate and even detoxify soils contaminated with heavy metals. This detoxification ability may depend on what chemical forms of metals are taken up by plants and how the plants distribute the toxins in their tissues. This, in turn, may have an important impact on phytoremediation. We investigated the impact of arbuscular mycorrhizal (AM) fungus, Glomus intraradices, on the subcellular distribution and chemical forms of cadmium (Cd) in alfalfa (Medicago sativa L.) that were grown in Cd-added soils. The fungus significantly colonized alfalfa roots by day 25 after planting. Colonization of alfalfa by G. intraradices in soils contaminated with Cd ranged from 17% to 69% after 25–60 days and then decreased to 43%. The biomass of plant shoots with AM fungi showed significant 1.7-fold increases compared to no AM fungi addition under the treatment of 20 mg·kg⁻¹ Cd. Concentrations of Cd in the shoots of alfalfa under 0.5, 5, and 20 mg·kg⁻¹ Cd without AM fungal inoculation are 1.87, 2.92, and 2.38 times higher, respectively, than those of fungi-inoculated plants. Fungal inoculation increased Cd (37.2–80.5%) in the cell walls of roots and shoots and decreased in membranes after 80 days of incubation compared to untreated plants. The proportion of the inactive forms of Cd in roots was higher in fungi-treated plants than in controls. Furthermore, although fungi-treated plants had less overall Cd in subcellular fragments in shoots, they had more inactive Cd in shoots than did control plants. These results provide a basis for further research on plant-microbe symbioses in soils contaminated with heavy metals, which may potentially help us develop management regimes for phytoremediation.     

Transgenic tobacco plants expressing ectopically wheat H⁺-pyrophosphatase (H⁺-PPase) gene TaVP1 show enhanced accumulation and tolerance to cadmium

Cadmium (Cd) is considered an extremely significant pollutant due to its high toxicity to many organisms. Plants have evolved several mechanisms to cope with Cd, the most important of which is vacuolar sequestration. Cadmium can be directly transported into vacuoles by cations/H⁺ exchangers, such as CAXs, which are energized by the pH gradient established by proton pumps. A cDNA (TaVP1) encoding wheat vacuolar H⁺-pyrophosphatase (V-H-PPase) was ectopically expressed in transgenic tobacco to evaluate whether this proton pump expression would enhance Cd tolerance and accumulation in planta. When TaVP1-expressing plants were exposed to various concentrations of Cd, they were found to be more tolerant to Cd compared to wild type plants. Cadmium accumulation in the plant biomass in transgenic plants was higher than that in wild type plants. To the best of our knowledge, this is the first report on the potential for enhancing proton pump expression as a strategy to improve Cd tolerance and accumulation in plants.     

Investigating heavy-metal hyperaccumulation using Thlaspi caerulescens as a model system

Background

Metal-hyperaccumulating plant species are plants that are endemic to metalliferous soils and are able to tolerate and accumulate metals in their above-ground tissues to very high concentrations. One such hyperaccumulator, Thlaspi caerulescens, has been widely studied for its remarkable properties to tolerate toxic levels of zinc (Zn), cadmium (Cd) and sometimes nickel (Ni) in the soil, and accumulate these metals to very high levels in the shoot. The increased awareness regarding metal-hyperaccumulating plants by the plant biology community has helped spur interest in the possible use of plants to remove heavy metals from contaminated soils, a process known as phytoremediation. Hence, there has been a focus on understanding the mechanisms that metal-hyperaccumulator plant species such as Thlaspi caerulescens employ to absorb, detoxify and store metals in order to use this information to develop plants better suited for the phytoremediation of metal-contaminated soils.

Scope

In this review, an overview of the findings from recent research aimed at better understanding the physiological mechanisms of Thlaspi caerulescens heavy-metal hyperaccumulation as well as the underlying molecular and genetic determinants for this trait will be discussed. Progress has been made in understanding some of the fundamental Zn and Cd transport physiology in T. caerulescens. Furthermore, some interesting metal-related genes have been identified and characterized in this plant species, and regulation of the expression of some of these genes may be important for hyperaccumulation.

Conclusions

Thlaspi caerulescens is a fascinating and useful model system not only for studying metal hyperaccumulation, but also for better understanding micronutrient homeostasis and nutrition. Considerable future research is still needed to elucidate the molecular, genetic and physiological bases for the extreme metal tolerance and hyperaccumulation exhibited by plant species such as T. caerulescens.Key words: Zn, Cd, Ni, Thlaspi caerulescens, hyperacumulator, phytoremediation, heavy metal     

Root foraging for zinc and cadmium requirement in the Zn/Cd hyperaccumulator plant Sedum alfredii

Positive root response to metals may enhance metal accumulation for greater requirement in hyperaccumulators. The effects of spatially heterogeneous Zn/Cd addition on root allocation, metal accumulation, and growth of the Zn/Cd hyperaccumulator Sedum alfredii were assessed in a pot experiment. Young shoots of S. alfredii were grown with or without supplied Zn/Cd. Two concentrations were used of each metal, and each metal concentration had one homogeneous and two heterogeneous treatments. Growth increased by 1.6–3.2 times with the increasing overall dose of Zn/Cd addition, and shoot biomass was positively correlated with shoot Zn/Cd concentration (P?<?0.001). In all heterogeneous treatments, the plants consistently allocated approximately 90% of root biomass to the metal-enriched patches, and shoot Zn/Cd contents were greater than or similar to those in the homogeneous treatment at each metal concentration. Plants in the control treatment showed symptoms of Zn deficiency, although their shoots had Zn concentrations 100-fold higher than the critical deficiency value for most plants. We conclude that S. alfredii has evolved root foraging mechanisms associated with its greater requirements for Zn/Cd. These results could have important implications both for phytoremediation and for investigation of positive role of Cd in higher plants.     

Biochemical and RAPD Analysis of Hibiscus rosa sinensis Induced by Heavy Metals

The present study was designed to assess the effects of three different metals (cadmium, lead, and zinc) at the same concentrations on Hibiscus rosa sinensis during metal uptake. The effects of different metals at the same concentrations were assessed on biomass, root-shoot length, and biochemical parameters like chlorophyll and antioxidant enzymes like SOD and CAT to establish the tolerance potential and toxic effects on plants in different metals. The accumulation of metals by plants was found to be in the following order: Zn > mixed metals > Cd > Pb, where Zn was accumulated approximately 79.6% in plant tissues. Plants removed from Cd showed more enzyme activities than the other two metals. DNA stability was investigated by a Random Amplified Polymorphic DNA (RAPD) technique, which demonstrated that the samples in Cd and mixed metal showed similar trends, whereas samples in Zn and Pb showed similar band intensity to the control. Results suggested that Cd and/or heavy metal stress influences antioxidant status and also induces DNA changes during remediation. Therefore, these studies could be a useful biomarker assay for better treatment for metals’ remediation from soil by means of phytoremediation.     

Cadmium accumulation and tolerance of Lagerstroemia indica and Lagerstroemia fauriei (Lythraceae) seedlings for phytoremediation applications

Contamination by heavy metals is one of the most serious environmental problems generated from human activities. Because phytoremediation utilizes plants to uptake contaminants, it could potentially be used to remediate metal-contaminated areas. A pot culture experiment with four levels of cadmium (Cd) (0, 20, 40, and 80 mg of Cd/kg dry soil) was conducted to investigate Cd accumulation and tolerance of roots, shoots, and leaves of Lagerstroemia indica and Lagerstroemia fauriei as well as their potential for phytoremediation. Experimental results indicated that Cd inhibited seedling growth only at the higher Cd exposure concentration (40 and 80 mg/kg). The tolerance index revealed that on average L. indica is more tolerant of Cd than L. fauriei. Moreover, plants in the experiment accumulated Cd differentially. In comparisons between L. indica and L. fauriei, the leaves of the former had higher concentrations of Cd, while the roots of latter had higher concentrations of Cd. Furthermore, the roots, shoots, and leaves had very high bioaccumulation factors that markedly exceeded 1.0 (exceptional only in shoots of 80 mg/kg for L. fauriei), indicating that the seedlings extracted Cd from the soil. The leaves' translocation factor of L. indica was greater than 1.0, being significantly higher than that of L. fauriei. Chlorophyll a, Chlorophyll b and total declined in both species significantly as Cd concentrations exceeded 40 mg/kg in the soil. In contrast, lipid peroxidation and proline content was found to increase with increasing Cd concentration. From the assessments of biomass production, Cd tolerance and uptake L. indica and L. fauriei could stand as excellent species for remediating Cd-contaminated soils.     

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.     

Phytoremediation of heavy metal contaminated soil potential by woody plants on Tonglushan ancient copper spoil heap in China

Fast-growing metal-accumulating woody plants are considered potential candidates for phytoremediation of metals. Tonglushan mining, one of the biggest Cu production bases in China, presents an important source of the pollution of environment. The sample was collected at Tonglushan ancient copper spoil heap. The aims were to measure the content of heavy metal in the soil and woody plants and to elucidate the phytoremediation potential of the plants. The result showed that soil Cu, Cd and Pb were the main contamination, the mean contents of which were 3166.73 mg/kg, 3.66 mg/kg and 137.06 mg/kg respectively, which belonged to severe contamination. Fourteen species from 14 genera of 13 families were collected and investigated; except for Ligutrum lucidum, the other 13 woody plants species were newly recorded in this area. In addition, to assess the ability of metal accumulation of these trees, we proposed accumulation index. Data suggested that Platanus × acerilolia, Broussonetia papyrifera, Ligutrum lucidum, Viburnum awabuki, Firmiana simplex, Robina pseudoacacia, Melia azedarach and Osmanthus fragrans exhibited high accumulated capacity and strong tolerance to heavy metals. Therefore, Platanus × acerilolia and Broussonetia papyrifera can be planted in Pb contaminated areas; Viburnum awabuki, Firmiana simplex, Robina pseudoacacia and Melia azedarach are the suitable trees for Cd contaminated areas; Viburnum awabuki, Melia azedarach, Ligutrum lucidum, Firmiana simplex, Osmanthus fragrans and Robina pseudoacacia are appropriate to Cu, Pb and Cd multi-metal contaminated areas.