Isolation and expression analysis of cadmium-induced genes from Cd/Mn hyperaccumulator Phytolacca americana in response to high Cd exposure.

Phytolacca americana is recognised as a hyperaccumulator that accumulates cadmium (Cd) and manganese (Mn). Although most studies have provided abundant physiological evidence, little is known about the molecular mechanisms of Cd accumulation in P. americana. In this study, Cd-induced genes were isolated using suppression subtractive hybridisation (SSH) library construction, and gene expression patterns under Cd stress were quantified using real-time quantitative PCR. The functions of PaGST, PaMT and PaFe-SOD were confirmed in transformant yeast. Reactive oxygen species (ROS) formation and cell death in root tips were detected, and SOD and POD activities in leaf tissue were also analysed. There were about 447 expressed sequence tags (ESTs) identified and confirmed. GO analysis showed those genes were mainly involved in metabolism, cell stress and defence, transcription and translation, signal transduction, transport, energy and ion transport, which formed the basis for a molecular understanding of P. americana Cd tolerance mechanisms. Cd also stimulated ROS formation and modified the antioxidant systems. Taken together, our results indicate that ROS formation and Cd-induced gene expression favour P. americana tolerance by activating the defence system and permitting subsequent adaptation to Cd toxicity.

Phytoremediation of Mn-contaminated paddy soil by two hyperaccumulators (Phytolacca americana and Polygonum hydropiper) aided with citric acid.

The purpose of this study was to investigate the phytoremediation potential of two hyperaccumulator plants, Phytolacca americana L. and Polygonum hydropiper L., on manganese-contaminated paddy soils. The biomass growth, Mn concentrations in plant tissues, and potential Mn removal efficiency from soils of these two plants were studied with citric acid, and the mechanisms of citric acid on these two plants were analyzed by examining the root activity, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in leaves, as well as the concentrations of O2·- and H2O2 in leaves. The results showed that the biomass of these two plants were both promoted under low level of citric acid (3 mmol kg-1). The concentration of Mn in the plants and the amount of Mn removed from the soil by the plants through harvesting were enhanced at low and intermediate (10 mmol kg-1) citric acid application levels. The results also showed that root activity was enhanced at the low citric acid level and significantly inhibited under the intermediate and high levels (15 mmol kg-1), which indicates the facilitative function of the low level of citric acid and the inhibitive function of the high level of citric acid application on plant biomass growth. Under the low and intermediate levels of citric acid application, O2·- in the plant leaves increased sharply, and the SOD, POD, and CAT activities also increased sharply, which made the level of H2O2 very similar to that of the control, ensuring the health of the plants. At the high level of citric acid application, however, the O2·- continued to rise sharply, while the activity of the three antioxidant enzymes declined sharply, causing the concentration of hydrogen peroxide to be much higher than that in the control, thus endangering the plants. The present study shows the potential of P. hydropiper for use in the phytoremediation of soil contaminated with a relatively low level of manganese.

Transcriptome analysis of Phytolacca americana L. in response to cadmium stress.

Phytolacca americana L. (pokeweed) has metal phytoremediation potential, but little is known about its metal accumulation-related genes. In this study, the de novo sequencing of total RNA produced 53.15 million reads covering 10.63 gigabases of transcriptome raw data in cadmium (Cd)-treated and untreated pokeweed. Of the 97,502 assembled unigenes, 42,197 had significant matches in a public database and were annotated accordingly. An expression level comparison between the samples revealed 1515 differentially expressed genes (DEGs), 923 down- and 592 up-regulated under Cd treatment. A KEGG pathway enrichment analysis of DEGs revealed that they were involved in 72 metabolism pathways, with photosynthesis, phenylalanine metabolism, ribosome, phenylpropanoid biosynthesis, flavonoid biosynthesis and carbon fixation in photosynthetic organisms containing 24, 18, 72, 14, 7 and 15 genes, respectively. Genes related to heavy metal tolerance, absorption, transport and accumulation were also identified, including 11 expansins, 8 nicotianamine synthases, 6 aquaporins, 4 ZRT/IRT-like proteins, 3 ABC transporters and 3 metallothioneins. The gene expression results of 12 randomly selected DEGs were validated using quantitative real-time PCR, and showed different response patterns to Cd in their roots, stems and leaves. These results may be helpful in increasing our understanding of heavy metal hyperaccumulators and in future phytoremediation applications.

A small RNA targets pokeweed antiviral protein transcript.

Ribosome-inactivating proteins (RIPs) are a class of plant defense proteins with N-glycosidase activity (EC 3.2.2.22). Pokeweed antiviral protein (PAP) is a Type I RIP isolated from the pokeweed plant, Phytolacca americana, thought to confer broad-spectrum virus resistance in this plant. Through a combination of standard molecular techniques and RNA sequencing analysis, we report here that a small RNA binds and cleaves the open reading frame of PAP mRNA. Additionally, sRNA targeting of PAP is dependent on jasmonic acid (JA), a plant hormone important for defense against pathogen infection and herbivory. Levels of small RNA increased with JA treatment, as did levels of PAP mRNA and protein, suggesting that the small RNA functions to moderate the expression of PAP in response to this hormone. The association between JA and PAP expression, mediated by sRNA299, situates PAP within a signaling pathway initiated by biotic stress. The consensus sequence of sRNA299 was obtained through bioinformatic analysis of pokeweed small RNA sequencing. To our knowledge, this is the first account of a sRNA targeting a RIP gene.

Fungal inoculation and elevated CO2 mediate growth of Lolium mutiforum and Phytolacca americana, metal uptake, and metal bioavailability in metal-contaminated soil: evidence from DGT measurement.

Fungal inoculation and elevated CO2 may mediate plant growth and uptake of heavy metals, but little evidence from Diffusive Gradients in Thin-films (DGT) measurement has been obtained to characterize the process. Lolium mutiforum and Phytolacca americana were grown at ambient and elevated CO2 on naturally Cd and Pb contaminated soils inoculated with and without Trichoderma asperellum strain C3 or Penicillium chrysogenum strain D4, to investigate plant growth, metal uptake, and metal bioavailability responses. Fungal inoculation increased plant biomass and shoot/root Cd and Pb concentrations. Elevated CO2 significantly increased plants biomass, but decreased Cd and Pb concentrations in shoot/root to various extents, leading to a metal dilution phenomenon. Total Cd and Pb uptake by plants, and DGT-measured Cd and Pb concentrations in rhizosphere soils, were higher in all fungal inoculation and elevated CO2 treatments than control treatments, with the combined treatments having more influence than either treatment alone. Metal dilution phenomenon occurred because the increase in DGT-measured bioavailable metal pools in plant rhizosphere due to elevated CO2 was unable to match the increase in requirement for plant uptake of metals due to plant biomass increase.

Elevated CO2 increases Cs uptake and alters microbial communities and biomass in the rhizosphere of Phytolacca americana Linn (pokeweed) and Amaranthus cruentus L. (purple amaranth) grown on soils spiked with various levels of Cs.

General concern about increasing global atmospheric CO(2) levels owing to the ongoing fossil fuel combustion and elevated levels of radionuclides in the environment, has led to growing interest in the responses of plants to interactive effects of elevated CO(2) and radionuclides in terms of phytoremediation and food safety. To assess the combined effects of elevated CO(2) and cesium contamination on plant biomass, microbial activities in the rhizosphere soil and Cs uptake, Phytolacca americana Linn (pokeweed, C3 specie) and Amaranthus cruentus L. (purple amaranth, C4 specie) were grown in pots of soils containing five levels of cesium (0, 100, 300, 500 and 1000 mg Cs kg(-1)) under two levels of CO(2) (360 and 860 µL L(-1), respectively). Shoot and root biomass of P. americana and Amaranthus crentus was generally higher under elevated CO(2) than under ambient CO(2) for all treatments. Both plant species exhibited higher Cs concentration in the shoots and roots under elevated CO(2) than ambient CO(2). For P. americana grown at 0, 100, 300, 500 and 1000 mg Cs kg(-1), the increase magnitude of Cs concentration due to elevated CO(2) was 140, 18, 11, 34 and 15% in the shoots, and 150, 20, 14, 15 and 19% in the roots, respectively. For A. cruentus, the corresponding value was 118, 28, 21, 14 and 17% in the shoots, and 126, 6, 11, 17 and 22% in the roots, respectively. Higher bioaccumulation factors were noted for both species grown under elevated CO(2) than ambient CO(2). The populations of bacteria, actinomycetes and fungi, and the microbial C and N in the rhizosphere soils of both species were higher at elevated CO(2) than at ambient CO(2) with the same concentration of Cs. The results suggested that elevated CO(2) significantly affected plant biomass, Cs uptake, soil C and N concentrations, and community composition of soil microbes associated with P. americana and A. cruentus roots. The knowledge gained from this investigation constitutes an important advancement in promoting utilization of CO(2) fertilization for improvement of phytoextraction of soils contaminated with radionuclides.

[Antioxidative enzymes play key roles in cadmium tolerance of Phytolacca americana].

Phytolacca americana L. has the capacity to take up and accumulate to very high levels heavy metals such as Mn and Cd, and is used for phytoextraction of heavy metal contaminated soils. The role of antioxidative enzyme of Phytolacca americana in response to Cd stress is unknown. The 6-week-old seedlings of Phytolacca americana were exposed to half strength Hoagland solution with 200 micromol/L CdCl2 or 400 micromol/L CdCl2 for 4 days. The content of H2O2 and MDA, and electrolyte leakage increased, while the photosynthetic rate decreased, indicated that the oxidative damage induced by Cd stress in Phytolacca americana was one of the metal toxicity mechanism. The activities of SOD and POD increased rapidly with elevated Cd concentration and exposure time, CAT activity was stable in response to 200 micromol/L CdCl2 stress, and increased only at 3 d later upon 400 micromol/L CdCl2, treatment. Suggested that the enzymatic antioxidation capacity played important role in Cd tolerance of hyperaccumulator plant.

Cadmium accumulation and distribution in populations of Phytolacca americana L. and the role of transpiration.

The concentrations of heavy metals in Phytolacca americana L. and corresponding soil samples from three contaminated sites and an uncontaminated site were studied. Hydroponic experiments were also conducted to investigate the Cd uptake ability and mechanism of P. americana. The field results showed that the average Cd concentration was 42 mg kg(-1) in P. americana leaves, with the highest concentration of 402 mg kg(-1) found at Datianwan. A significant relationship was observed between the concentrations of Cd in leaves and those of corresponding soils on a logarithmic scale. Under laboratory hydroponic conditions, the maximum Cd concentration in aerial tissues of P. americana was 637 mg kg(-1), under treatment with 100 microM Cd. The population from the uncontaminated site (Zijinshan) also had a remarkable ability to accumulate Cd in shoots to concentrations well in excess of 100 microM in the hydroponic experiment, similar to the population from contaminated site, suggesting that Cd accumulation is a constitutive trait of P. americana. In the presence of 100 microM Cd, the addition of polyethylene glycol decreased leaf transpiration, the shoot Cd concentration, and the shoot/root Cd concentration ratio. There was a significantly positive relationship between the shoot Cd concentration and the leaf transpiration of P. americana. A similar significant positive correlation was also obtained between the shoot/root Cd concentration and leaf transpiration. Moreover, pretreatment with 5 microM abscisic acid or 5 microM HgCl(2) significantly decreased the Cd concentration in P. americana shoots. These results suggest that transpiration has an important role in Cd accumulation in shoots of P. americana.

[Studies on the physiological response of Phytolacca americana to manganese toxicity by FTIR spectroscopy].

A technique based on Fourier transform infrared (FTIR) spectrometry was used to detect the physiological changes in chemical composition of Phytolacca americana under a wide range of Mn2+ treatments (0, 0.125, 0.25, 0.5 and 1 g x kg(-1)). Using the roots, stems and leaves of phytolacca americana, absorption bands corresponding to carbohydrates, ester and proteins varied differently. The absorption band heights at 3 000 and 2 916 cm(-1) of stem tissues rose firstly and then decreased, indicating the exudation and transporting situation of organic substances which served as organic osmotic contents to enhance manganese tolerance under low treatments; Meanwhile, the band heights at 2 922 cm(-1) (roots) and 1 606 cm(-1) (leaves) which have different tendency in accordance with Mn treatments showed that the capability to chelate Mn decreased under higher manganese concentration. In addition, the changes of the bands at 1 732 and 1 026 cm(-1) (roots), 1 028 cm(-1) (stems), 1 052 and 967 cm(-1) (leaves) differed from each other, suggesting that under the conditions of severe Mn stress the ex-oxidation of membrane lipid increased; Otherwise, the band heights at 1 375 cm(-1) increased firstly and then decreased, representing that the cell wall improved Mn resistance by increasing cation-exchange capacity (CEC). The result also implied that it is practical to apply FTIR to the research comparing the chemical differences of phytoaccumulators under metal treatments.