The differentially-expressed proteome in Zn/Cd hyperaccumulator Arabis paniculata Franch. in response to Zn and Cd

The Zn/Cd hyperaccumulator Arabis paniculata is able to tolerate high level of Zn and Cd. To clarify the molecular basis of Zn and Cd tolerance, proteomic approaches were applied to identify proteins involved in Zn and Cd stress response in A. paniculata. Plants were exposed to both low and high Zn or Cd levels for 10 d. Proteins of leaves in each treatment were separated by 2-DE (two-dimensional electrophoresis). Nineteen differentially-expressed proteins upon Zn treatments and 18 proteins upon Cd treatments were observed. Seventeen out of 19 of Zn-responsive proteins and 16 out of 18 of Cd-responsive proteins were identified using MALDI-TOF/TOF-MS (matrix-assisted laser desorption/ionization time of flight mass spectrometry). The most of identified proteins were known to function in energy metabolism, xenobiotic/antioxidant defense, cellular metabolism, protein metabolism, suggesting the responses of A. paniculata to Zn and Cd share similar pathway to certain extend. However, the different metal defense was also revealed between Zn and Cd treatment in A. paniculata. These results indicated that A. paniculata against to Zn stress mainly by enhancement of energy metabolism including auxin biosynthesis and protein metabolism to maintain plant growth and correct misfolded proteins. In the case of Cd, plants adopted antioxidative/xenobiotic defense and cellular metabolism to keep cellular redox homeostasis and metal-transportation under Cd stress.     

Effects of cadmium on the photosynthetic activity in mature and young leaves of soybean plants

Cadmium (Cd) is a widely spread pollutant and can be easily taken up by crop from soil, resulting in a serious health issue for humans. The objective of this study was to comparatively investigate the photosynthetic activity, chlorophyll a fluorescence, chlorophyll contents, and spectral reflectance in mature and young leaves of soybean plants after being treated with different concentrations of Cd for 10 days. The photosynthetic rate, chlorophyll contents, actual photochemical efficiency of PSII, and photochemical quenching in the young leaves decreased more significantly with increasing concentrations of Cd in the nutrient solution, compared with those in the mature leaves, though the young leaves had less Cd concentrations. Thus, there was more excessive excited energy produced in the young leaves than that in the mature leaves. In the young leaves, due to more excessive excited energy, more reactive oxygen species may be generated, which further damaged the photosynthetic apparatus. It was supported by the fact that the decrease of reflectance in near-infrared wavelengths of the young leaves was more noticeable than that of the mature leaves. In addition, the chlorophyll a fluorescence transients of the young leaves was significantly different from that in the mature leaves, indicating that the electron transport of young leaves were inhibited much more severely than that of the mature leaves. These observations imply that the responses of photosynthetic activity of soybean leaves to Cd stress depend on their growth stage, and the Cd-induced inhibition of photosynthetic activity might be attributed to the decrease in chlorophyll contents and the decrease in mesophyll CO₂ assimilation ability cause by the Cd, which further decreased the consumption of ATP and NADPH, leading to accumulation of NADPH on the acceptor sides of the PSI, and then feedback inhibited electron transport in chloroplasts.     

Subcellular distribution and toxicity of cadmium in Potamogeton crispus L

The submerged macrophyte Potamogeton crispus L. was subjected to varying doses of cadmium (0, 20, 40, 60 and 80 μM) for 7 d, and the plants were analyzed for subcellular distribution of Cd, accumulation of mineral nutrients, photosynthesis, oxidative stress, protein content, and ultrastructural distribution of calcium (Ca). Leaf fractionation by differential centrifugation indicated that 48-69% of Cd was accumulated in the cell wall. At all doses of Cd, the levels of Ca and B rose and the level of Mn fell; the levels of Fe, Mg, Zn, Cu, Mo, and P rose initially only to decline later. Exposure to Cd caused oxidative stress as evident by increased content of malondialdehyde and decreased contents of chlorophyll and protein. Photosynthetic efficiency, as indicated by the quenching of chlorophyll a fluorescence (Fv/Fm, Fo and Fm), decreased significantly, the extent of decrease being directly proportional to the concentration of Cd. Increased amounts of precipitates of calcium were noticed in the treated plants, located either outside the cell membrane or in chloroplasts, mitochondria, the nucleus, and the cytoplasm whereas control plants showed small deposits of the precipitates around surface of the vacuole membrane and in the intercellular space but rarely in the cytoplasm. Photosynthetic efficiency and oxidative stress could be used as indicators of physiological end-points in determining the extent of Cd phytotoxicity.     

Exposure to elevated ozone levels differentially affects the antioxidant capacity and the redox homeostasis of two subtropical Phaseolus vulgaris L. varieties

Ozone (O₃) has become one of the most toxic air pollutants to plants worldwide. However, investigations on O₃ impacts on crops health and productivity in South America countries are still scarce. The present study analyzed the differences on the enzymatic and non-enzymatic antioxidant system in foliar tissue of two subtropical Phaseolus vulgaris varieties exposed to high O₃ concentration. Both varieties were negatively impacted by the pollutant, but the responses between each variety were quite distinct. Results revealed that Irai has higher constitutive levels of reactive oxygen species (ROS) and ascorbate (AsA) concentration, but lower total thiol levels and catalase immunocontent. In this variety catalase protein concentration was increased after O₃ exposure, indicating a better cellular capacity to reduce hydrogen peroxide. On the opposite, Fepagro 26-exposed plants increased ROS generation and AsA concentration, but had the levels of total thiol content and catalase protein unchanged. Furthermore, O₃ treatment reduced the levels of chlorophylls a and b, and the relationship analysis between the chlorophyll ratio (a/b) and protein concentration were positively correlated indicating that photosynthetic apparatus is compromised, and thus probably is the biomass acquisition on Fepagro 26. Differently, O₃ treatment of Irai did not affect chlorophylls a and b content, and loss on the protein content was lower. Altogether, these data suggest that early accumulation of ROS on Fepagro 26 are associated with an insufficient leaf antioxidant capacity, which leads to cell structure disruption and impairs the photosynthesis. Irai seems to be more tolerant to O₃ toxic effects than Fepagro 26, and the observed differences on O₃ sensitivity between the two varieties are apparently based on constitutive differences involved in the maintenance of intracellular redox homeostasis.     

Molecular mechanism for cadmium-induced anthocyanin accumulation in Azolla imbricata

Anthocyanins inducibly synthesized by Cd treatment showed high antioxidant activity and might be involved in internal detoxification mechanisms of Azolla imbricata against Cd toxicity. In order to understand anthocyanin biosynthesis mechanism during Cd stress, the cDNAs encoding chalcone synthase (CHS) and dihydroflavonol reductase (DFR), two key enzymes in the anthocyanin synthesis pathway, were isolated from A. imbricata. Deduced amino acid sequences of the cDNAs showed high homology to the sequences from other plants. Expression of AiDFR, and to a lesser extent AiCHS, was significantly induced in Cd treatment plant in comparison with the control. CHS and DFR enzymatic activities showed similar pattern changes with these genes expression during Cd stress. These results strongly indicate that Cd induced anthocyanin accumulation is probably mediated by up-regulation of structural genes including CHS and DFR, which might further increase the activities of enzymes encoded by these structural genes that control the anthocyanin biosynthetic steps.     

Differences in EDTA-assisted metal phytoextraction between metallicolous and non-metallicolous accessions of Rumex acetosa L.

Two common sorrel (Rumex acetosa) accessions, one from a Zn-Pb contaminated site (CS accession) and the other from an uncontaminated site (UCS accession), were hydroponically exposed to a mixture of heavy metals (Pb²⁺ + Zn²⁺ + Cd²⁺) with and without EDTA at an equimolar rate. The metallicolous CS accession showed a higher tolerance to metal treatment in the absence of the chelating agent, whereas the UCS accession was especially tolerant to EDTA treatment alone. Combination of metal and EDTA treatment resulted in a higher Pb accumulation in shoots of both accessions although plants hardly showed phytotoxic symptoms. Cd and Zn uptake was not augmented by EDTA addition to the polymetallic medium. Chelant-assisted Pb accumulation was 70% higher in the CS accession than in the UCS accession, despite the fact that the former accession evapotranspired less water than the UCS accession. These results support the existence of a non-selective apoplastic transport of metal chelates by R. acetosa roots, not related to transpiration stream.     

Analysis and characterization of cultivable heavy metal-resistant bacterial endophytes isolated from Cd-hyperaccumulator Solanum nigrum L. and their potential use for phytoremediation

This study investigates the heavy metal-resistant bacterial endophytes of Cd-hyperaccumulator Solanum nigrum L. grown on a mine tailing by using cultivation-dependent technique. Thirty Cd-tolerant bacterial endophytes were isolated from roots, stems, and leaves of S. nigrum L. and classified by amplified ribosomal DNA-restriction analysis into 18 different types. Phylogenetic analysis based on 16S rDNA sequences showed that these isolates belonged to four groups: Actinobacteria (43%), Proteobacteria (23%), Bacteroidetes (27%) and Firmicutes (7%). All the isolates were then characterized for their plant growth promoting traits as well as their resistances to different heavy metals; and the actual plant growth promotion and colonization ability were also assessed. Four isolates were re-introduced into S. nigrum L. under Cd stress and resulted in Cd phytotoxicity decrease, as dry weights of roots increased from 55% to 143% and dry weights of above-ground from 64% to 100% compared to the uninoculated ones. The total Cd accumulation of inoculated plants increased from 66% to 135% (roots) and from 22% to 64% (above-ground) compared to the uninoculated ones. Our research suggests that bacterial endophytes are a most promising resource and may be the excellent candidates of bio-inoculants for enhancing the phytoremediation efficiency.     

Application of phosphate-solubilizing bacteria for enhancing bioavailability and phytoextraction of cadmium (Cd) from polluted soil

In this study, phosphate-solubilizing bacteria (PSB), Bacillus megaterium, were used to enhance Cd bioavailability and phytoextractability of Cd from contaminated soils. This strain showed a potential for directly solubilizing phosphorous from soils more than 10 folds greater than the control without inoculation. The results of pot experiments revealed that inoculation with B. megaterium significantly increased the extent of Cd accumulation in Brassica juncea and Abutilon theophrasti by two folds relative to the uninoculated control. The maximum Cd concentrations due to inoculation were 1.6 and 1.8 mg Cd g⁻¹ plant for B. juncea and A. theophrasti after 10 wk, respectively. The total biomass of A. theophrasti was not significantly promoted by the inoculation treatment, yet the total biomass of B. juncea increased from 0.087 to 0.448 g. It is also worth to mention that B. juncea predominantly accumulates Cd in its stems (39%) whereas A. theophrasti accumulates it in its leaves (68%) after 10 wk. The change of the Cd speciation indicated that inoculation of B. megaterium as PSB increased the bioavailabilty of Cd and consequently enhanced its uptake by plants. The present study may provide a new insight for improving phytoremediation using PSB in the Cd-contaminated soils.     

Salicylic acid involved in the regulation of nutrient elements uptake and oxidative stress in Vallisneria natans (Lour.) Hara under Pb stress

In this study, the alterations in nutrient elements content, reactive oxygen species level and antioxidant response were studied in leaves of Vallisneria natans (Lour.) Hara exposed to salicylic acid (SA, 10 or 100 μM), or Pb (50 μM) or their combinations for 4 d. No significant alterations in Mn and Ca content were observed but content of Cu, Zn, Fe and P decreased in plants exposed to SA alone. SA application inhibited the uptake of Pb and partially reversed Pb-induced the alterations in Mn, Ca and Fe content in leaves of V. natans exposed to 50 μM Pb. The decreased chlorophyll (a + b) and increased malondialdehyde and O²⁻ and H₂O₂ content were detected in plants exposed to 100 μM SA, 50 μM Pb, 10 μM SA + 50 μM Pb or 100 μM SA + 50 μM Pb. Application SA partially inhibited Pb-induced the increase of malondialdehyde, O²⁻ and H₂O₂ content. 100 μM SA decreased the activity of NADH oxidase and the content of non-protein thiols, carotenoids and ascorbic acid and increased the content of dehydroascorbate in plants treated with or without Pb. SA alone decreased the ascorbate peroxidase activity and increased the catalase and peroxidase activity, while SA application increased catalase activity but had no significant effect on ascorbate peroxidase and peroxidase activity in V. natans exposed to Pb. The results indicate that SA involves in the regulation of Pb uptake, nutrient balance and oxidative stress.     

Characterization of cadmium (Cd) distribution and accumulation in Tagetes erecta L. seedlings: Effect of split-root and of remove-xylem/phloem

Tagetes erecta has a high potential for cadmium (Cd) phytoremediation. Through several hydroponic experiments, characteristics of ¹⁰⁸Cd distribution and accumulation were investigated in T. erecta with split -roots or removed xylem/phloem. The results showed that ¹⁰⁸Cd transport from roots to aboveground tissues showed the homolateral transport phenomenon in split-root seedlings. ¹⁰⁸Cd content of leaves on the +¹⁰⁸Cd side and the −¹⁰⁸Cd side was not significantly different, which implied that there was horizontal transport of ¹⁰⁸Cd from the +¹⁰⁸Cd side to the −¹⁰⁸Cd side in cut-root seedlings. Like ¹⁰⁸Cd transport, the transport of ⁷⁰Zn was homolateral. Reduction of water consumption in the removed xylem treatment significantly decreased ¹⁰⁸Cd accumulation; whereas, the removed phloem treatment had no significant effect on water consumption, but did decrease ¹⁰⁸Cd accumulation in leaves of the seedlings. The removal of phloem significantly reduced distal leaf ¹⁰⁸Cd content, which was significantly lower than that in the basal leaves in both the split-root and unsplit-root seedlings. Overall, the results presented in this study revealed that the root to aboveground cadmium translocation via phloem is as an important and common physiological process as xylem determination of the cadmium accumulation in stems and leaves of marigold seedlings.     

Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants.     

Cadmium-tolerant bacteria induce metal stress tolerance in cereals

Cadmium usually hampers plant growth, but bacterial inoculation may improve stress tolerance in plants to Cd by involving various mechanisms. The objective was to characterize and identify bacteria that improve plant growth under Cd stress and reduce Cd uptake. Cadmium-tolerant bacteria were isolated from rhizosphere soil, which was irrigated with tannery effluent, and six strains were selected as highly tolerant to Cd, showing minimum inhibitory concentration as 500 mg L^?1 or 4.45 mmol L^?1. These strains were identified by 16S rRNA gene analysis and functional analysis in regard to plant growth promotion characteristics. To determine their effect on cereal growth under Cd stress, seeds were inoculated with these strains individually and grown in soil contaminated with three Cd levels (0, 40 and 80 mg kg^?1). Biomass production, relative water content (RWC), electrolyte leakage (ELL) and tissue Cd concentration were measured. Biomass of both cereals was inhibited strongly when exposed to Cd; however, bacterial inoculation significantly reduced the suppressive effect of Cd on cereal growth and physiology. The bacterial isolates belonged to the genera Klebsiella, Stenotrophomonas, Bacillus and Serratia. Maize was more sensitive than wheat to Cd. Klebsiella sp. strain CIK-502 had the most pronounced effects in promoting maize and wheat growth and lowering Cd uptake under Cd stress.     

The effect of excess Zn on mineral nutrition and antioxidative response in rapeseed seedlings

Zinc (Zn) is a necessary element for plants, but excess Zn can be detrimental. To investigate Zn toxicity, rapeseed (Brassica napus) seedlings were treated with 0.07–1.12 mM Zn for 7 d. Inhibition of plant growth along with root damage, chlorosis and decreased chlorophyll (a and b) content in newly expanded leaves (the second and third leaves formed following cotyledons) were found under Zn stress. The Zn content increased in plants under external Zn stress, while concentrations of phosphorus, copper, iron, manganese and magnesium reduced significantly, especially in roots. Meanwhile, increased lipid peroxidation was detected biochemically and histochemically. Compared with controls, NADH oxidase and peroxidase (POD) activity increased in leaves and roots of plants under high Zn, but superoxide dismutase (SOD), catalase and ascorbate peroxidase activities decreased. The changes in glutathione S-transferase activity and in ascorbic acid, dehydroascorbate, non-protein thiols and glutathione contents were also measured under Zn stress. Isoforms of SOD and POD were separated using non-denaturing polyacrylamide gel electrophoresis and their activities were analyzed. Our results suggested that excess Zn exerts its toxicity partially through disturbing nutrient balance and inducing oxidative stress in plants. These data will be helpful for better understanding of toxicity of Zn and the adaptive mechanism in Zn non-hyperaccumulator plants.     

Screening Capsicum chinense fruits for heavy metals bioaccumulation

Elevated concentrations of heavy metals in edible plants could expose consumers to excessive levels of potentially hazardous chemicals. Sixty-three accessions (genotypes) of Capsicum chinense Jacq, collected from 8 countries of origin were grown in a silty-loam soil under field conditions. At maturity, fruits were collected and analyzed for seven heavy metals (Cd, Cr, Ni, Pb, Zn, Cu, and Mo) concentrations. The main objectives of this investigation were: 1) to determine the concentrations of seven heavy metals in the soil and monitor their accumulation in mature fruits, 2) to categorize the pepper accessions as low or high heavy metal accumulators, and 3) to determine if heavy metal content of the pepper fruit was lower than the permitted limits. Concentrations and relative proportions of heavy metals in pepper fruits of C. chinense varied among accessions. Fruits of Plant Introduction (PI) 355820 accumulated significant concentrations of Cd (0.47 μ g g^?1dry fruit). PI-260522 accumulated the highest concentration of Pb (2.12 μ g g^?1 dry fruit) among the 63 accessions tested. This accession (PI-260522) contained about twice the Pb limit on a fresh weight basis. Among the 63 accessions analyzed, PI-238051 contained the highest levels of Ni (17.2 μ g g^?1). We concluded that high accumulator genotypes may be useful for phytoremediation, while, low accumulator accessions might be appropriate selections for growing on Cd-, Pb-, or Ni-contaminated soils to prevent potential human exposure to heavy metals and health hazards through the food chain.     

Cd-tolerant Suillus luteus: A fungal insurance for pines exposed to Cd

Soil metal pollution can trigger evolutionary adaptation in soil-borne organisms. An in vitro screening test showed cadmium adaptation in populations of Suillus luteus (L.: Fr.) Roussel, an ectomycorrhizal fungus of pine trees. Cadmium stress was subsequently investigated in Scots pine (Pinus sylvestris L.) seedlings inoculated with a Cd-tolerant S. luteus, isolated from a heavy metal contaminated site, and compared to plants inoculated with a Cd-sensitive isolate from a non-polluted area. A dose-response experiment with mycorrhizal pines showed better plant protection by a Cd-adapted fungus: more fungal biomass and a higher nutrient uptake at high Cd exposure. In addition, less Cd was transferred to aboveground plant parts. Because of the key role of the ectomycorrhizal symbiosis for tree fitness, the evolution of Cd tolerance in an ectomycorrhizal partner such as S. luteus can be of major importance for the establishment of pine forests on Cd-contaminated soils.     

A comparative study of heavy metal accumulation and antioxidant responses in Vaccinium myrtillus L. leaves in polluted and non-polluted areas

The purpose of this study was to explore a possible relationship between the availability of metals in soil (Cd, Fe, Mn, Pb and Zn) and their concentrations in leaves of Vaccinium myrtillus L. as a species which has been reported to be a successful colonist of acid-and-heavy metal-contaminated soil. Analysis also concerned the antioxidant response of plants from three heavily polluted (immediate vicinity of: zinc smelter, iron smelter and power plant) and three relatively clean sites (nature reserve, ecological site and unprotected natural forest community) in southern Poland. The contents of glutathione, non-protein thiols, protein, proline and activity of guaiacol peroxidase in leaves of bilberry were measured. Generally, the concentrations of metals in the HNO₃ and CaCl₂ extracants of the soil from the polluted sites were higher. Moreover, the antioxidant responses were also elevated in bilberries in the polluted sites. Significant positive relationships between Cd, Pb and Zn concentrations in soil and in the plants were found. In the leaves of V. myrtillus from the polluted sites, higher concentrations of Cd, Pb and Zn were noted (In Miasteczko ?l?skie respectively 6.26, 157.09 and 207.17 mg?kg^?1?d.w.). We found a positive correlation between the increase in the NPTs and protein contents as well as the Cd, Pb and Zn concentrations in V. myrtillus. Cd, Pb and Zn also decreased guaiacol peroxidase activity. However, the activity of this enzyme increased under Fe. A decreasing trend in glutathione contents was observed with increasing iron and manganese concentrations in bilberry leaves. Parameters such as protein, non-protein –SH groups and changes in GPX activity seem to be universal, sensitive and correlated well with heavy metal stress.     

Correlations between cadmium and mineral nutrients in absorption and accumulation in various genotypes of rice under cadmium stress

The absorption and accumulation of Cd2+, Fe3+, Zn2+, Mn2+, Cu2+ and Mg2+ in the roots and leaves of 20 rice cultivars (Oryza sativa L.) with different genotypes under cadmium (Cd) stress were investigated with pot experiments. The results showed that there existed significant differences among the rice cultivars in the contents of six mineral elements in both roots and leaves at both heading and ripening periods. The statistical analysis showed that, for their contents in roots, significant and positive correlations between Cd2+ and Fe3+, Cd2+ and Zn2+, Cd2+ and Mn2+, Cd2+ and Cu2+ existed, but no significant correlation between Cd2+ and Mg2+, at the two periods. In the leaves, Cd also showed significant and positive correlations with Fe3+, Zn2+ and Cu2+ at the both periods, but a significant and negative correlation with Mn2+ and no significant correlation with Mg2+ at heading, a significant and positive correlation with Mg2+ and no significant correlation with Mn2+ at ripening. These results suggested that there were cooperative absorption between Cd2+ and Fe3+, Mn2+, Cu2+, Mn2+ in rice plants. Genotypic differences in Cd uptake and translocation among the rice cultivars suggested that paddy field of some rice cultivars may be irrigated with partially treated sewage water.     

Influence of extracellular polymeric substances (EPS) on Cd adsorption by bacteria

The role of extracellular polymeric substances (EPS) in Cd adsorption by Bacillus subtilis and Pseudomonas putida was investigated using a combination of batch adsorption experiments, potentiometric titrations, Fourier transform infrared spectroscopy (FTIR). An increased adsorption capacity of Cd was observed for untreated bacteria relative to that for EPS-free bacteria. Surface complexation modeling of titration data showed the similar pK_a values of functional groups (carboxyl, phosphate and hydroxyl) between untreated and EPS-free bacteria. However, site concentrations on the untreated bacteria were found to be higher than those on the EPS-free bacteria. FTIR spectra also showed that no significant difference in peak positions was observed between untreated and EPS-free bacteria and carboxyl and phosphate groups were responsible for Cd adsorption on bacterial cells. The information obtained in this study is of fundamental significance for understanding the interaction mechanisms between heavy metals and biofilms in natural environments.