Hyperaccumulator extracts promoting the phytoremediation of rare earth elements (REEs) by Phytolacca americana: Role of active microbial community in rhizosphere hotspots.

The increased usage of rare earth elements (REEs) leads to the extensive exploitation of rare earth mines, and the REEs pollution in soil caused by the legacy mine tailings has brought great harm to environment and human health. Although Phytolacca americana can remove REEs from contaminated soil to some extent, there is still an urgent problem to improve its efficiency. Hyperaccumulator extract is a new organic material with potential in metal phytoextraction, but its role in REEs phytoremediation and the related underlying processes remain unclear. In this study, hyperaccumulator extracts from P. americana root (PR), stem (PS), leaf (PL) and EDTA were used to improve the phytoremediation efficiency of REEs with P. americana. Soil zymography was applied to assess the enzyme hotspots' spatial distribution in the rhizosphere, and the hotspots' microbial communities were also identified. The results indicated that the application of hyperaccumulator extracts improved the biomass and REEs uptake of P. americana, and the highest REEs content in plant was observed in the treatment of PS, which increased 299% compared to that of the control. Hotspots area of ß-glucosidase, leucine aminopeptidase and acid phosphatase were concentrated in the pant rhizosphere along the roots and increased 2.2, 5.3 and 2.2 times after PS application compared to unamended soils. The PS application increased the relative abundance of Proteobacteria, Cyanobacteria, Bacteroidota and Firmicutes phyla in rhizosphere. Soil fungi have a higher contribution on promoting REEs activation than that of bacteria. Available P and extractable REEs were leading predictors for the plant biomass and REEs concentrations. The co-occurrence network showed that the application of PS creates a more efficient and stable microbial network compared to other treatments. In conclusion, stem-derived hyperaccumulator extract is excellent in stimulating REEs phytoremediation with P. americana by improving hotspots microbial activities and form a healthy rhizosphere microenvironment.

Enolase, a cadmium resistance related protein from hyperaccumulator plant Phytolacca americana, increase the tolerance of Escherichia coli to cadmium stress.

Phytolacca americana is a Cd hyperaccumulator plant that accumulates significant amounts of Cd in leaves, making it a valuable phytoremediation plant species. Our previous research found enolase (ENO) may play an important part in P. americana to cope with Cd stress. As a multifunctional enzyme, ENO was involved not only in glycolysis but also in the response of plants to various environmental stresses. However, there are few studies on the function of PaENO (P. americana enolase) in coping with Cd stress. In this study, the PaENO gene was isolated from P. americana, and the expression level of PaENO gene significantly increased after Cd treatment. The enzymatic activity analysis showed PaENO had typical ENO activity, and the 42-position serine was essential to the enzymatic activity of PaENO. The Cd resistance assay indicated the expression of PaENO remarkably enhanced the resistance of E. coli to Cd, which was achieved by reducing the Cd content in E. coli. Moreover, both the expression of inactive PaENO and PaMBP-1 (alternative translation product of PaENO) can improve the tolerance of E. coli to Cd. The results indicated PaENO may be alternatively translated into the transcription factor PaMBP-1 to participate in the response of P. americana to Cd stress.

The expression of the Cd resistance related protein PaENO can significantly increase the tolerance of E. coli to Cd, which was achieved by reducing the content of Cd in E. coli cells, and was independent of the enzymatic activity of PaENO. Moreover, PaENO may be alternatively translated into the transcription factor PaMBP-1 to participate in the response of P. americana to Cd stress.

Atomically Dispersed Manganese on Biochar Derived from a Hyperaccumulator for Photocatalysis in Organic Pollution Remediation.

Phytoremediation is a potentially cost-effective and environmentally friendly remediation method for environmental pollution. However, the safe treatment and resource utilization of harvested biomass has become a limitation in practical applications. To address this, a novel manganese-carbon-based single-atom catalyst (SAC) method has been developed based on the pyrolysis of a manganese hyperaccumulator, Phytolacca americana. In this method, manganese atoms are dispersed atomically in the carbon matrix and coordinate with N atoms to form a Mn-N4 structure. The SAC developed exhibited a high photooxidation efficiency and excellent stability during the degradation of a common organic pollutant, rhodamine B. The Mn-N4 site was the active center in the transformation of photoelectrons via the transfer of photoelectrons between adsorbed O2 and Mn to produce reactive oxygen species, identified by in situ X-ray absorption fine structure spectroscopy and density functional theory calculations. This work demonstrates an approach that increases potential utilization of biomass during phytoremediation and provides a promising design strategy to synthesize cost-effective SACs for environmental applications.

Regioselective hydroxylation and dehydrogenation of capsaicin and dihydrocapsaicin by cultured cells of Phytolacca americana.

The biotransformations of capsaicin and dihydrocapsaicin were investigated using cultured plant cells of Phytolacca americana as biocatalysts. Four products, ie 15-hydroxycapsaicin, dihydrocapsaicin, 15-hydroxydihydrocapsaicin, and capsaicin 4-ß-glucoside, were isolated from the suspension cultures of P. americana treated with capsaicin for 3 days, showing that capsaicin was regioselectively hydroxylated, reduced, and glucosylated by cultured P. americana cells. On the other hand, dihydrocapsaicin was regioselectively dehydrogenated, hydroxylated, reduced, and glucosylated to give four products, ie capsaicin, 15-hydroxycapsaicin, 15-hydroxydihydrocapsaicin, and capsaicin 4-ß-glucoside, by cultured P. americana cells. In this paper, it is reported, for the first time, that dihydrocapsaicin is converted into 15-hydroxydihydrocapsaicin by plant cultured cells.

Full-length transcriptome analysis of Phytolacca americana and its congener P. icosandra and gene expression normalization in three Phytolaccaceae species.

BACKGROUND: Phytolaccaceae species in China are not only ornamental plants but also perennial herbs that are closely related to human health. However, both large-scale full-length cDNA sequencing and reference gene validation of Phytolaccaceae members are still lacking. Therefore, single-molecule real-time sequencing technology was employed to generate full-length transcriptome in invasive Phytolacca americana and non-invasive exotic P. icosandra. Based on the transcriptome data, RT-qPCR was employed to evaluate the gene expression stability in the two plant species and another indigenous congener P. acinosa. RESULTS: Total of 19.96 Gb and 19.75 Gb clean reads of P. americana and P. icosandra were generated, including 200,857 and 208,865 full length non-chimeric (FLNC) reads, respectively. Transcript clustering analysis of FLNC reads identified 89,082 and 98,448 consensus isoforms, including 86,989 and 96,764 high-quality ones. After removing redundant reads, 46,369 and 50,220 transcripts were obtained. Based on structure analysis, total 1675 and 1908 alternative splicing variants, 25,641 and 31,800 simple sequence repeats (SSR) as well as 34,971 and 36,841 complete coding sequences were detected separately. Furthermore, 3574 and 3833 lncRNA were predicted and 41,676 and 45,050 transcripts were annotated respectively. Subsequently, seven reference genes in the two plant species and a native species P. acinosa were selected and evaluated by RT-qPCR for gene expression analysis. When tested in different tissues (leaves, stems, roots and flowers), 18S rRNA showed the highest stability in P. americana, whether infested by Spodoptera litura or not. EF2 had the most stable expression in P. icosandra, while EF1-α was the most appropriate one when attacked by S. litura. EF1-α showed the highest stability in P.acinosa, whereas GAPDH was recommended when infested by S. litura. Moreover, EF1-α was the most stable one among the three plant species whenever germinating seeds or flowers only were considered. CONCLUSION: Full-length transcriptome of P. americana and P. icosandra were produced individually. Based on the transcriptome data, the expression stability of seven candidate reference genes under different experimental conditions was evaluated. These results would facilitate further exploration of functional and comparative genomic studies in Phytolaccaceae and provide insights into invasion success of P. americana.

Glycosylation of Stilbene Compounds by Cultured Plant Cells.

Oxyresveratrol and gnetol are naturally occurring stilbene compounds, which have diverse pharmacological activities. The water-insolubility of these compounds limits their further pharmacological exploitation. The glycosylation of bioactive compounds can enhance their water-solubility, physicochemical stability, intestinal absorption, and biological half-life, and improve their bio- and pharmacological properties. Plant cell cultures are ideal systems for propagating rare plants and for studying the biosynthesis of secondary metabolites. Furthermore, the biotransformation of various organic compounds has been investigated as a target in the biotechnological application of plant cell culture systems. Cultured plant cells can glycosylate not only endogenous metabolic intermediates but also xenobiotics. In plants, glycosylation reaction acts for decreasing the toxicity of xenobiotics. There have been a few studies of glycosylation of exogenously administrated stilbene compounds at their 3- and 4'-positions by cultured plant cells of Ipomoea batatas and Strophanthus gratus so far. However, little attention has been paid to the glycosylation of 2'-hydroxy group of stilbene compounds by cultured plant cells. In this work, it is described that oxyresveratrol (3,5,2',4'-tetrahydroxystilbene) was transformed to 3-, 2'-, and 4'-ß-glucosides of oxyresveratrol by biotransformation with cultured Phytolacca americana cells. On the other hand, gnetol (3,5,2',6'-tetrahydroxystilbene) was converted into 2'-ß-glucoside of gnetol by cultured P. americana cells. Oxyresveratrol 2'-ß-glucoside and gnetol 2'-ß-glucoside are two new compounds. This paper reports, for the first time, the glycosylation of stilbene compounds at their 2'-position by cultured plant cells.

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.

Diversity in cadmium accumulation and resistance associated with various metallothionein genes (type III) in Phytolacca americana L.

Metallothioneins (MTs) are known for their heavy metal deoxidation during phytoremediation. To estimate their roles in the cadmium (Cd) hyperaccumulator Phytolacca americana L., three MT genes, PaMT3-1, PaMT3-2 and PaMT3-3, belonging to the MT3 subfamily were cloned. They separately encoded 63, 65 and 65 amino acids, containing12, 10 and 11 cysteines (Cys), respectively. Each gene was individually transformed and expressed in Escherichia coli cells. A Cd-resistance assay showed that the recombinant strains had enhanced survival rates, especially those containing PaMT3-1 and PaMT3-3. Additionally, the recombinant strains were high Cd accumulators, with the recombinant PaMT3-1's maximum accumulation being 2.16 times that of the empty vector strains. The numbers of cysteines and the structures of MT proteins were associated with the Cd enrichment and resistance capabilities. PaMT3-1 could be an effective gene resource in future plant Cd remediation-related breeding programs.

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.

Cadmium phytoremediation potential of turnip compared with three common high Cd-accumulating plants.

Phytoextraction is a phytoremediation technique used for remediating polluted soils and it greatly relies on the plants' capacities to accumulate contaminants. Turnip is a high cadmium (Cd)-accumulating plant. We compared the Cd tolerance, growth, and Cd accumulation characteristics of two turnip landraces with three additional commonly known high Cd-accumulating species to systematically estimate its Cd phytoremediation potential. Results showed that the turnips could tolerate relatively lower Cd concentrations than other plants. Growth characteristics analyses indicated that the turnips initially grew rapidly and then gradually slowed down, and their photosynthetic parameters indicated that biomass accumulation was easily affected by light. However, the Cd uptake and translocation capacities of the two turnip landraces were higher than those of Phytolacca americana Linn. and Bidens pilosa Linn. but close to that of Brassica napus Linn.. Ultimately, large amounts of Cd accumulated in turnips during early growth and slightly increased as the fleshy roots increased in size. Based on these findings, the present turnip landraces have potential for soil remediation, but additional research is needed before these landraces can be practically used. Moreover, turnips are good candidates for studying the molecular mechanism of high Cd accumulation in plants.

Glycosylation and Methylation of Quercetin and Myricetin by Cultured Cells of Phytolacca americana.

The glycosylation and methylation of quercetin by cultured plant cells of Phytolacca americana gave quercetin 3-Ο-ß-D-glucoside and isorharnnetin 3-Ο-ß-D- glucoside. Myricetin was glycosylated and methylated to syringetin 3-Ο-ß-D-glucoside by cultured P. americana cells.

Hydroxylation and Glycosylation of Phenylpropanoids by Cultured Cells of Phytolacca americana.

Hydroxylation and glycosylation of cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid were investigated using cultured plant cells of Phytolacca americana as biocatalysts. Regioselective hydroxylation at the 4-position of cinnamic acid and 3-position of p-coumaric acid was observed. Although cinnamic acid was transformed to mono-glucoside products, di-glycosylation occurred in the case of the biotransformation of p-coumaric acid, caffeic acid, and ferulic acid.

Optical Resolution of (RS)-Denopamine to (R)-Denopamine P-D- Glucoside by Glucosyltransferase from Phytolacca americana Expressed in Recombinant Escherichia coli.

The optical resolution of racemic compounds by stereoselective glucosylation was investigated using plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. The glucosyltransferase glucosylated chemoselectively the phenolic hydroxyl group of phenol compounds. The (R)-stereoselective glucosylation of (RS)-denopamine by glucosyltransferase occurred to give (R)-denopamine ß-D-glucoside.

Synthesis and pharmacological evaluation of glycosides of resveratrol, pterostilbene, and piceatannol.

To enhance their water solubility and pharmacological activities, the stilbenes resveratrol, pterostilbene, and piceatannol were glycosylated to their monoglucosides (ß-glucosides) and diglycosides (ß-maltosides) by cultured cells and cyclodextrin glucanotransferase (CGTase). Cultured cells of Phytolacca americana and glucosyltransferase (PaGT) were capable of glucosylation of resveratrol to its 3- and 4'-ß-glucosides. Pterostilbene was slightly transformed into its 4'-ß-glucoside by P. americana cells. Piceatannol was readily converted into piceatannol 4'-ß-glucoside, with the highest yield among the three substrates. The 3- and 4'-ß-glucosides of resveratrol were subjected to further glycosylation by CGTase to give 3- and 4'-ß-maltoside derivatives. The inhibitory action of resveratrol and pterostilbene toward histamine release induced with compound 48/80 from rat peritoneal mast cells was improved by ß-glucosylation and/or ß-maltosylation (i.e., the inhibitory activity for histamine release of the 3- and 4'-ß-glucosides of resveratrol, the 3- and 4'-ß-maltosides of resveratrol, and the 4'-ß-glucoside of pterostilbene was higher than that of the corresponding aglycones, resveratrol and pterostilbene, respectively). In addition, the phosphodiesterase (PDE) inhibitory activity of resveratrol and pterostilbene was enhanced by ß-glucosylation and/or ß-maltosylation (i.e., the PDE inhibitory activities of the 3- and 4'-ß-glucosides of resveratrol, the 4'-ß-maltoside of resveratrol, and the 4'-ß-glucoside of pterostilbene were higher than those of the corresponding aglycones, resveratrol and pterostilbene, respectively).

Glycosylation of artepillin C with cultured plant cells of Phytolacca americana.

Biotransformation of artepillin C was investigated using cultured plant cells as biocatalysts. Artepillin C was converted into its 4- and 9-beta-D-glucosides, and 4,9-beta-D-diglucoside by cultured cells of Phytolacca americana. In contrast, cultured lpomoea batatas cells glucosylated artepillin C to only its 4- and 9-beta-D-glucosides.

Cadmium and manganese accumulation in Phytolacca americana L. and the roles of non-protein thiols and organic acids.

Phytolacca americana L. can accumulate large amounts of heavy metals in its aerial tissues, especially cadmium (Cd) and manganese (Mn). It has great potential for use in phytoextraction of metals from multi-metal-contaminated soils. This study was conducted to further investigate the Cd- and Mn-tolerance strategies of this plant. Concentrations of non-protein thiols (NPTs) and phytochelatins (PCs) in leaves and roots increased significantly as the concentration of Cd in solution increased. The molar ratios of PCs:soluble Cd ranged from 1.8 to 3.6 in roots and 8.1 to 31.6 in leaves, suggesting that the cellular response involving PC synthesis was sufficient to complex Cd ions in the cytosol, especially that of leaves. In contrast, excess Mn treatments did not result in a significant increase in NPT or PC concentrations in leaves or roots. Oxalic acid concentrations in leaves of plants exposed to 2 or 20 mM Mn reached 69.4 to 89.3 mg (0.771 to 0.992 mmol) g(-1) dry weight, respectively, which was approximately 3.7- to 8.6-fold higher than the Mn level in the 0.6 M HCl extract. Thus, oxalic acid may play an important role in the detoxification of Mn.

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.

[Preliminary analysis of manganese uptake mechanism in the hyperaccumulator Phytolacca americana L].

Phytolacca americana L. (P. americana) is a manganese (Mn) hyperaccumulator plant discovered in southern China, and knowledge of Mn uptake characteristics and mechanisms on this plant may provide essential and critical information for phytoremediation. Synchrotron radiation X-ray fluorescence spectroscopy (SRXRF) microprobe was empolyed in this study to explore the Mn distribution in the root cross-section of P. americana, and effects of metabolic inhibitors (DNP and Na3VO4) and Ca-channel inhibitor (LaCl3) on Mn uptake of P. americana was also investigated under laboratory conditions. Results showed that P. americana has strong abilities for absorpting and accumulating Mn, and the Mn concentration in root, stem, and leaf of P. americana may reach up to 402, 208, and 601 mg x kg(-1) DW, respectively, even only treated with 5 micromol x L(-1) Mn. The highest Mn content can be found in the vascular bundle of root, and then the epidermis, while the lowest Mn content can be observed in the cortex. The Mn content increased when shifted from cortex to vascular bundle, indicating that there was an active transportation in Mn absorption of P. americana root, and the inhibitory effect of DNP and Na3VO4 on Mn uptake further verified the possibilities of active absorption. The Mn uptake was inhibited by 30% with LaCl3, suggesting that Mn uptake in P. americana also closely related to the Ca-channel.

Inhibition of pokeweed antiviral protein (PAP) by turnip mosaic virus genome-linked protein (VPg).

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.

The effects of copper, manganese and zinc on plant growth and elemental accumulation in the manganese-hyperaccumulator Phytolacca americana.

Synchrotron radiation X-ray fluorescence (SRXRF) and inductively coupled plasma mass spectrometry were used to estimate major, minor and trace elements in Cu-, Zn- and Mn-treated Phytolacca americana. The effects of the addition of Cu, Zn and Mn on morphological parameters, such as root length, shoot height, and fresh and dry weights of shoots and roots, were also examined. In addition, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidases (GPX) and catalase (CAT) and the expression of Fe-SOD, Cu/Zn-SOD, metallothionein-2 and glutathione S-transferase (GST) exposed to the highest amounts of Cu, Zn or Mn were detected. Our results confirmed the following: (1) Zn supplementation leads to chlorosis, disturbed elemental homeostasis and decreased concentrations of micro- and macroelements such as Fe, Mg, Mn, Ca and K. Cu competed with Fe, Mn and Zn uptake in plants supplemented with 25 µM Cu. However, no antagonistic interactions took place between Cu, Zn, Mn and Fe uptake in plants supplemented with 100 µM Cu. Mn supplementation at various concentrations had no negative effects on elemental deficits. Mn was co-located with high concentrations of Fe and Zn in mature leaves and the concentrations of macro elements were unchanged. (2) P. americana supplemented with increased concentrations of Zn and Cu exhibited lower biomass production and reduced plant growth. (3) When plants were supplemented with the highest Zn and Cu concentrations, symptoms of toxicity corresponded to decreased SOD or CAT activities and increased APX and GPX activities. However, Mn tolerance corresponded to increased SOD and CAT activities and decreased POD and APX activities. Our study revealed that heavy metals partially exert toxicity by disturbing the nutrient balance and modifying enzyme activities that induce damage in plants. However, P. americana has evolved hyper accumulating mechanisms to maintain elemental balance and redox homeostasis under excess Mn.