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.     

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.     

The cation-efflux transporter BjCET2 mediates zinc and cadmium accumulation in <Emphasis Type="Italic">Brassica juncea</Emphasis> L. leaves

Brassica juncea L. is a Zn/Cd accumulator. To determine the physiological basis of its metal accumulation phenotype, the functional properties and role of the metal efflux transporter BjCET2 were investigated using transgenic technology. Heterologous expression of BjCET2 in the double mutant yeast strain Δzrc1Δcot1 enhanced the metal tolerance of the yeast strain and led to decrease in Zn or Cd accumulation. Detection of green fluorescence from green fluorescent protein (GFP) in the root tip of transgenic tobacco further revealed that BjCET2::GFP is localized at the plasma membrane. Semi-quantitative RT-PCR analysis showed that BjCET2 was most abundant in the root and was weakly expressed in the stem and leaves. The expression of BjCET2 was up-regulated by heavy metals. However, exposure to low temperature, salt and drought did not affect the expression of BjCET2. Overexpression of BjCET2 in transgenic B. juncea plants conferred heavy metal tolerance and increased Cd/Zn accumulation in the leaves. BjCET2-deficient B. juncea mediated by antisense RNA resulted in hypersensitivity to heavy metals and decreased Zn/Cd accumulation in the plants. These results suggest that the heavy metal efflux of BjCET2 plays important roles in the metal tolerance of B. juncea and in Zn/Cd accumulation in B. juncea.     

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.     

Influence of zinc on cadmium-induced toxicity in nodules of pigeonpea (Cajanus cajan L. Millsp.) inoculated with arbuscular mycorrhizal (AM) fungi

Arbuscular mycorrhizal (AM) fungi are known to alleviate heavy-metal stress in plants. The intent of the present work was to analyze accumulation of heavy metals (Cd and Zn) in nodules of two Cajanus cajan (L.) Millsp. genotypes and their subsequent impact on nitrogen fixation, oxidative stress, and non-protein thiols (glutathione and phytochelatins) with and without AM fungus Glomus mosseae. Accumulation of Cd and Zn in nodules resulted in sharp reduction in nodule number, nodule dry mass as well as nitrogen fixation (leghemoglobin and nitrogenase (N₂ase)), although Cd had more pronounced effects than Zn. Cd-induced lipid peroxidation, H₂O₂ accumulation, and electrolyte leakage were largely reversed by Zn supplementation. Zn application significantly altered the negative effects of Cd on the synthesis of non-protein thiols, suggesting antagonistic behaviour of Zn. Higher concentration of Zn was more effective in lessening the negative effects of Cd than its lower concentration. Remarkable genotypic variation was found, with more severe effects of both the metals in P792 than Sel 85N. Glomus mosseae attenuated the phytotoxic effects of metals in nodules by decreasing metal uptake, oxidative stress, and by enhancing defense system ultimately leading to better nitrogen-fixing potential of pigeonpea nodules.     

Characterization of metal tolerance and accumulation in Grevillea exul var exul

Grevillea exul var exul (Proteaceae), a tree species native to serpentine soils in New Caledonia, is a reported manganese accumulator. Since the metal tolerance of this species remains unknown, its growth and metal accumulation were studied for seven heavy metals under controlled conditions. Brassica juncea, a popular species for metal phytoremediation, was used as a reference. G. exul seedlings were more tolerant to Cr, Zn, Ni, and Cu than B. juncea. There were no differences in Hg, and Cd tolerance between both species. B. juncea seedlings concentrated more Cd, Hg, and Cr in their shoot than G. exul seedlings, while Ni, Zn, and Mn levels were similar for both species. Comparison then focused on tolerance at toxic doses of Ni and Mn using older individuals of both species. No growth inhibition for G. exul plants was observed, whereas the growth of B. juncea was significantly inhibited at the higher metal concentrations. Shoot Mn and Ni concentrations were again lower in G. exul plants as compared to B. juncea, suggesting a mechanism of partial Ni and Mn exclusion in G. exul. In a subsequent study, 1-year-old G. exul plants favored Ni accumulation in roots while Mn accumulated preferentially in shoots.     

Plant growth regulators improve growth,photosynthesis, mineral nutrient and antioxidant system under cadmium stress in menthol mint ( Mentha arvensis L.)

Menthol mint (Mentha arvensis L.) cultivation is significantly affected by the heavy metals like cadmium (Cd) which also imposes severe health hazards. Two menthol mint cultivars namely Kosi and Kushal were evaluated under Cd stress conditions. Impact of plant growth regulators (PGRs) like salicylic acid (SA), gibberellic acid (GA3) and triacontanol (Tria) on Cd stress tolerance was assessed. Reduced growth, photosynthetic parameters, mineral nutrient concentration, and increased oxidative stress biomarkers like electrolyte leakage, malondialdehyde, and hydrogen peroxide contents were observed under Cd stress. Differential upregulation of proline content and antioxidant activities under Cd stress was observed in both the cultivars. Interestingly, low electrolyte leakage, lipid peroxidation, hydrogen peroxide and Cd concentration in leaves were observed in Kushal compared to Kosi. Among all the PGRs tested, SA proved to be the best in improving Cd-stress tolerance in both the cultivars but Kushal responded better than Kosi.     

Cadmium induced oxidative stress and changes in soluble and ionically bound cell wall peroxidase activities in roots of seedling and 3–4 leaf stage plants of <Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) czern

Metabolic adaptations to heavy metal toxicity in plants are thought to be related with developmental growth stage and the type of metal by which plant is affected. In the present study, changes in ionically bound CWP, soluble peroxidase activity, H(2)O(2) level and Malonaldehyde content in roots of cadmium and copper stressed seedlings and cadmium stressed 3-4 leaf stage plants of Brassica juncea were investigated. Cadmium inhibits root growth and reduces fresh biomass. The reduction in root growth and fresh biomass is correlated with increased lipid peroxidation and reduced tolerance. Treatment with cadmium resulted in an increase in ionically bound CWP activity in roots of seedlings but no significant change in its activity was found in roots of 3-4 leaf stage plants. Increased level of H(2)O(2) in roots of cadmium and copper treated seedlings, show a direct correlation with increased activity of ionically bound CWP. H(2)O(2) level in 3-4 leaf stage plant roots was found to be very low. Soluble peroxidase activity decreased in cadmium (50 and 100 mu-icroM) treated seedlings but it was ineffective to cause any change in its activity in 3-4 leaf stage plants. Copper treated seedlings showed an increase in ionically bound CWP activity, H(2)O(2) level and MDA content. Ascorbic acid (50 mM) pretreated seedlings shows significant decrease in ionically bound CWP activity when exposed to 50 muM cadmium. Hence, it is concluded that inhibition of root growth in Brassica juncea seedlings by cadmium, is associated with CWP catalyzed H(2)O(2) dependent reactions which are involved in metabolic adaptations to heavy-metal stress.     

Overexpression of<Emphasis Type="Italic">Arabidopsis</Emphasis> phytochelatin synthase (<Emphasis Type="Italic">atpcsi</Emphasis>) does not change the maximum capacity for non-protein thiol production induced by Cadmium

Phytochelatins (PCs) play an important role in heavy-metal homeostasis and detoxification. However, we previously reported that the overexpression of PC synthase inArabidopsis does not lead to increased tolerance of cadmium but, rather, plants show higher Cd sensitivity. Here, we compared the maximum capacity for non-protein thiol (NPT) production at various concentrations of Cd in order to estimate PC synthesis indirectly for both transgenic (pcs9) and wild-type plants. The pcs9 line produced the highest level of NPT when treated with 200 p.M Cd for 3 d. In comparison, the maximum productivity by the wild type was in response to 500 μM Cd. Nevertheless, the absolute amounts of NPT produced did not differ significantly between those two genotypes. Furthermore, exogenous application of 1 mM GSH did not dramatically change the capacity for either pcs9 or wild-type plants. These results suggest that Cd hypersensitivity in the transgenic pcs9 may not be caused by supraoptimal intracellular concentrations of PC, but may, instead, be due to overexpressed PC synthase itself because that enzyme can bind metals. This action, therefore, may lead to some unknown disruption in cellular metal homeostasis under Cd stress.     

南通沿海滩涂耐盐植物重金属抗性内生细菌的筛选及生物多样性

【目的】沿海滩涂耐盐植物重金属抗性内生细菌的筛选及其促生长潜在能力的研究有助于我们获得一些能够耐受并促进耐盐植物在被Cd2+、Pb2+、Hg2+、Cu2+,Zn2+等重金属离子污染的贫瘠的沿海滩涂上正常生长的菌株,达到既能够利用广袤的滩涂生物资源产生经济价值又能够净化生态环境的目的。【方法】以江苏南通沿海滩涂地区的4种耐盐植物为材料,采用稀释平板涂布法从中分离得到45株内生细菌,从中挑取23株代表性的菌株,对其进行抗重金属Cu2+、Pb2+、Cd2+、Zn2+,Hg2+的活性筛选;固氮、解磷、吲哚乙酸(IAA)的产生、1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性等促生指标以及NaCl耐受能力的筛选。【结果】发现分离所得的大多数细菌能够耐受高浓度的Cu2+以及Pb2+,但是对Cd2+、Zn2+,Hg2+的耐受能力则较弱;26.1%的细菌具有固氮能力,21.7%的细菌具有解磷能力,60.9%的细菌能够产生IAA,39.1%的细菌含有ACC脱氨酶。对他们进行16S rRNA基因鉴定后发现,他们分属于芽胞杆菌属(Bacillus)、喜盐芽胞杆菌属(Halobacillus)、海洋芽胞杆菌属(Oceanobacillus)、微小杆菌属(Exiguobacterium)、沙雷氏菌属(Serratia)、短波单胞菌属(Brevundimonas)、弧菌属(Vibrio)、葡萄球菌属(Staphylococcus)共8个属,显示了丰富的多样性。其中菌株KLBMP 2432以及菌株KLBMP 2447为潜在的新种。【结论】沿海滩涂地区的耐盐植物内生细菌具有丰富多样的生物多样性以及促生长能力,且存在潜在的新种资源,并对重金属Cu2+,Pb2+具有较强的抗性。     

Effects of Cadmium,Chromium and Lead on Growth,Metal Uptake and Antioxidative Capacity in <Emphasis Type="Italic">Typha angustifolia</Emphasis>

This study investigates the modulation of antioxidant defence system of Typha angustifolia after 30 days exposure of 1 mM chromium (Cr), cadmium (Cd), or lead (Pb). T. angustifolia showed high tolerance to heavy metal toxicity with no visual toxic symptom when exposed to metal stress, and Cd/Pb addition also increased plant height and biomass especially in Pb treatment. Along with increased Cr, Cd, and Pb uptake in metal treatments, there was enhanced uptake of plant nutrients including Ca and Fe, and Zn in Pb treatment. A significant increase in malondialdehyde (MDA) content and superoxide dismutase (SOD) and peroxidase (POD) activities were recorded in plants subjected to Cr, Cd, or Pb stress. Furthermore, Pb stress also improved catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX) activities; whereas Cr stress depressed APX and GPX. The results indicate that enzymatic antioxidants and Ca/Fe uptake were important for heavy metal detoxification in T. angustifolia, stimulated antioxidative enzymes, and Ca, Fe, and Zn uptake could partially explain its hyper-Pb tolerance.     

Variation in response to heavy metals during vegetative growth in <Emphasis Type="Italic">Dorycnium pentaphyllum</Emphasis> Scop.

Seeds, young plants and adult plants of the perennial Mediterranean leguminous shrub Dorycnium pentaphyllum Scop. were exposed to Cd (1–100 μM) or Zn (10–10,000 μM) on nutrient solution. This species is resistant to Cd and Zn at different phenological stages. The lowest doses of Zn and Cd improved seed germination and young seedling growth, while only the highest doses of both heavy metals inhibited germination and decreased growth. High doses of Cd reduced seed imbibition and young seedling water content, while Zn did not. Osmotic adjustment was more efficient in Zn-treated young plants than in Cd-treated ones, while chlorophyll concentrations decreased in the former but not in the latter. Those differences were not observed anymore in adult plants. Exclusion processes were more efficient at the adult stage than at the young seedling stage and were more marked in response to Zn than to Cd. It is concluded that D. pentaphyllum could be used for phytostabilization of heavy metal-contaminated areas. The physiological strategies of tolerance, however, differ according to the age of the plants and the nature of the metal.     

Organic Acids Accumulation and Antioxidant Enzyme Activities in Thlaspi caerulescens under Zn and Cd Stress

Growth, organic acid and phytochelatin accumulation, as well as the activity of several antioxidative enzymes, i.e. superoxide dismutase (SOD), ascorbate peroxidase (APX) guaiacol peroxidase (POX) and catalase (CAT) were investigated under Zn and Cd stress in hydroponically growing plants of Thlaspi caerulescens population from Plombières, Belgium. Tissue Zn and Cd concentration increased (the highest concentration of both was in roots) as the concentration of these metals increased in the nutrient solution. Increasing Zn concentration enhanced plant growth, while with Cd it declined compared to the control. Both metals stimulated malate accumulation in shoots, Zn also caused citrate to increase. Zn did not induce phytochelatin (PC) accumulation. In plants exposed to Cd, PC concentration increased with increasing Cd concentration, but decreased with time of exposure. Under Zn stress SOD activity increased, but APX activity was higher at 500 and 1000 μM Zn and CAT activity only at 500 μM Zn in comparison with the control. CAT activity decreased in Cd- and Zn-stressed plants. The results suggest that relative to other populations, a T. caerulescens population from Plombières, when grown in hydroponics, was characterized by low Zn and Cd uptake and their translocation to shoots and tolerance to both metals. The accumulation of malate and citrate, but not PC accumulation was responsible for Zn tolerance. Cd tolerance seems to be due to neither PC production nor accumulation of organic acids.     

Cadmium tolerance and its phytoremediation by two oil yielding plants Ricinus communis (L.) and Brassica juncea (L.) from the contaminated soil

The effect of increasing level of cadmium in soil was investigated on biomass production, antioxidants, Cd bioaccumulation and translocation in Ricinus communis vis-à-vis a commonly studied oil crop Brassica juncea. The plants were exposed to 25, 50, 75, 100, and 150 mg Cd/Kg soil for up to 60 days. It was found that R. communis produced higher biomass at all the contamination levels than that of B. juncea. Proline and malondialdehyde in the leaves increased with increase in Cd level in both the species, whereas soluble protein decreased. The bioaccumulation of Cd was higher in B. juncea on the basis of the per unit biomass, total metal accumulation per plant was higher in R. communis. The translocation of Cdfrom roots to shoot was also higher in B. juncea at all Cd concentrations. R. communis appeared more tolerant and capable to clean Cd contaminated soil for longer period in one sowing than B. juncea and the former can grow in wasteland soil also in which later cannot be cultivated.     

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.