Novel Binding Mechanisms of Fusion Broad Range Anti-Infective Protein Ricin A Chain Mutant-Pokeweed Antiviral Protein 1 (RTAM-PAP1) against SARS-CoV-2 Key Proteins in Silico.

The deadly pandemic named COVID-19, caused by a new coronavirus (SARS-CoV-2), emerged in 2019 and is still spreading globally at a dangerous pace. As of today, there are no proven vaccines, therapies, or even strategies to fight off this virus. Here, we describe the in silico docking results of a novel broad range anti-infective fusion protein RTAM-PAP1 against the various key proteins of SARS-CoV-2 using the latest protein-ligand docking software. RTAM-PAP1 was compared against the SARS-CoV-2 B38 antibody, ricin A chain, a pokeweed antiviral protein from leaves, and the lectin griffithsin using the special CoDockPP COVID-19 version. These experiments revealed novel binding mechanisms of RTAM-PAP1 with a high affinity to numerous SARS-CoV-2 key proteins. RTAM-PAP1 was further characterized in a preliminary toxicity study in mice and was found to be a potential therapeutic candidate. These findings might lead to the discovery of novel SARS-CoV-2 targets and therapeutic protein structures with outstanding functions.

Invasive Plants: Turning Enemies into Value.

In this review, a brief description of the invasive phenomena associated with plants and its consequences to the ecosystem is presented. Five worldwide invasive plants that are a threat to Portugal were selected as an example, and a brief description of each is presented. A full description of their secondary metabolites and biological activity is given, and a resume of the biological activity of extracts is also included. The chemical and pharmaceutical potential of invasive species sensu lato is thus acknowledged. With this paper, we hope to demonstrate that invasive species have potential positive attributes even though at the same time they might need to be controlled or eradicated. Positive attributes include chemical and pharmaceutical properties and developing these could help mitigate the costs of management and eradication.

Identification of phytolaccosides in biological samples from pokeweed intoxication patients using liquid chromatography-tandem mass spectrometry.

In Phytolaccaceae family, Phytolacca americana L. (American pokeweed) and P. esculenta Van Houtte (Chinese pokeweed) are the two representative species among the genus. Pokeweeds have triterpenoid saponins as toxic compounds in every part of the plant. The saponins phytolaccoside A, B, D, E, and G were isolated from P. americana, and esculentoside H, J, L, K, M, I, and N were isolated from P. esculenta. Along with saponins, their aglycones (phytolaccagenin, phytolaccagenic acid, esculentic acid and jaligonic acid) were also isolated from P. americana and P. esculenta. Two people who unknowingly ate misidentified pokeweed plant roots were transferred to the emergency room. Urine and gastric content after irrigation were collected from the first patient (patient 1), and blood and urine were collected from the second patient (patient 2). The samples were analyzed to identify toxic substances with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the blood sample, 1.9 ng/mL of esculentoside A and 1.5 ng/mL of esculentoside C were detected, while the concentration of esculentoside B and H were below the LLOQ. In gastric contents and ingested roots, esculentoside A, B, C, and H were identified. Esculentoside A, C, and H were identified in the urine of patient 1, and esculentoside A and C were identified in the urine sample of patient 2. The developed analytical method was validated for parameters such as linearity, limit of detection, precision, accuracy, matrix effect, recovery, and process efficiency, and they showed clear and unbiased results.

Pterostilbene and Its Glucoside Induce Type XVII Collagen Expression.

The glycosylation of pterostilbene by cultured plant cells of Phytolacca americana gave pterostilbene 4'-O-ß-D-glucoside. Both pterostilbene and its 4'-0-ß-D- glucoside induced type XVII collagen expression in the EpiDermFT EFT-400 human skin cell model. Pterostilbene 4'-O-ß-D-glucoside strongly induced type XVII collagen expression rather than pterostilbene.

Inhibitory effects of geranium essential oil and its major component, citronellol, on degranulation and cytokine production by mast cells.

We investigated the effects of geranium essential oil (GEO) on anaphylaxis. GEO can exert antioxidant and anti-inflammatory effects, but its roles in allergic reactions are incompletely understood. Here, we used mouse cells to show that GEO inhibited the degranulation of cultured mast cells (CMCs). Citronellol is the major component of GEO and inhibited CMC degranulation. The l-enantiomer of citronellol more effectively suppressed CMC degranulation than did d-citronellol. We also examined whether citronellol could inhibit the immunoglobulin (Ig) E-induced production of tumor necrosis factor (TNF)-α. Treatment with various concentrations of citronellol before CMC activation with IgE significantly inhibited the induction of TNF-α in a dose-dependent manner. Mechanistically, citronellol suppressed the phosphorylation of mitogen-activated protein kinase (ERK), which is critical for ERK activation and the production of inflammatory cytokines in mast cells. These findings suggest that citronellol may represent a candidate compound for the effective treatment of allergic diseases.

Rabbit conjunctivae edema and release of NO, TNF-α, and IL-1ß from macrophages induced by fractions and esculentosides isolated from Phytolacca americana.

CONTEXT: The roots of Phytolacca americana L. (Phytolaccaceae) may be toxic. Despite heated controversy over the toxic compounds of P. americana, especially esculentosides, relevant studies remain scarce. OBJECTIVE: The objective of this study is to screen the toxic fractions and compounds of P. americana, to determine the controlling indices, and to provide evidence for unraveling the mechanism. MATERIALS AND METHODS: Petroleum ether (PE), CH2Cl2, n-BuOH, and water fractions were isolated from 70% ethanol extract of P. americana. The n-BuOH fraction was dissolved in 50% ethanol and precipitated by adding ethyl ether. The resultant supernatants and precipitates were referred to as SUPs and SEDs fractions, respectively. SUPs fraction was separated by column chromatography into four main stimulating esculentosides that were identified by HR-ESI/MS and NMR as EsA, EsB, EsC, and EsF. The irritating effects of esculentosides on rabbit conjunctivae (500 µg/eye) was observed by pathological examination and those on macrophages (5, 25, 50 and 100 µg/mL) were evaluated by detecting changes of NO, TNF-α, and IL-1ß levels. RESULTS AND DISCUSSION: n-BuOH, SUP fractions, and EsC induced severe conjunctival edema. The four esculentosides induced dose-dependent releases of proinflammatory mediators NO, TNF-α, and IL-1ß from macrophages, and releasing amounts peaked after 2 h of treatment. EsC and EsF induced macrophages to release mediators most significantly. EsC (50 µg/mL) functioned more effectively than EsF did, and similarly n-BuOH and SUPs fractions functioned more effectively than the esculentoside mixture. Thus, the four esculentosides exerted proinflammatory effects synergistically. CONCLUSION: All extracted esculentosides, especially EsC, induced inflammatory stimulation. Phytolacca americana-induced irritation of the gastrointestinal tract may be associated with esculentosides such as EsC.

Antitumor Activity of Americanin A Isolated from the Seeds of Phytolacca americana by Regulating the ATM/ATR Signaling Pathway and the Skp2-p27 Axis in Human Colon Cancer Cells.

The antiproliferative and antitumor activities of americanin A (1), a neolignan isolated from the seeds of Phytolacca americana, were investigated in human colon cancer cells. Compound 1 inhibited the proliferation of HCT116 human colon cancer cells both in vitro and in vivo. The induction of G2/M cell-cycle arrest by 1 was concomitant with regulation of the ataxia telangiectasia-mutated/ATM and Rad3-related (ATM/ATR) signaling pathway. Treatment with 1 activated ATM and ATR, initiating the subsequent signal transduction cascades that include checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and tumor suppressor p53. Another line of evidence underlined the significance of 1 in regulation of the S phase kinase-associated protein 2 (Skp2)-p27 axis. Compound 1 targeted selectively Skp2 for degradation and thereby stabilized p27. Therefore, compound 1 suppressed the activity of cyclin B1 and its partner cell division cycle 2 (cdc2) to prevent entry into mitosis. Furthermore, prolonged treatment with 1 induced apoptosis by producing excessive reactive oxygen species. The intraperitoneal administration of 1 inhibited the growth of HCT116 tumor xenografts in nude mice without any overt toxicity. Modulation of the ATM/ATR signaling pathway and the Skp2-p27 axis might be plausible mechanisms of action for the antiproliferative and antitumor activities of 1 in human colon cancer cells.

Structural analysis of a type 1 ribosome inactivating protein reveals multiple L­asparagine­N­acetyl­D­glucosamine monosaccharide modifications: Implications for cytotoxicity.

Pokeweed antiviral protein (PAP) belongs to the family of type I ribosome­inactivating proteins (RIPs): Ribotoxins, which function by depurinating the sarcin­ricin loop of ribosomal RNA. In addition to its antibacterial and antifungal properties, PAP has shown promise in antiviral and targeted tumor therapy owing to its ability to depurinate viral RNA and eukaryotic rRNA. Several PAP genes are differentially expressed across pokeweed tissues, with natively isolated seed forms of PAP exhibiting the greatest cytotoxicity. To help elucidate the molecular basis of increased cytotoxicity of PAP isoenzymes from seeds, the present study used protein sequencing, mass spectroscopy and X-ray crystallography to determine the complete covalent structure and 1.7 Å X­ray crystal structure of PAP­S1aci isolated from seeds of Asian pokeweed (Phytolacca acinosa). PAP­S1aci shares ~95% sequence identity with PAP­S1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP­S1aci. A rare proline substitution (Pro174) was identified in the active site of PAP­S1aci, which has no effect on catalytic Glu175 positioning or overall active­site topology, yet appears to come at the expense of strained main­chain geometry at the pre­proline residue Val173. Notably, a rare type of N­glycosylation was detected consisting of N­acetyl­D­glucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAP­S1aci. Of note, our modeling studies suggested that the ribosome depurination activity of seed PAPs would be adversely affected by the N­glycosylation of Asn44 and Asn255 with larger and more typical oligosaccharide chains, as they would shield the rRNA­binding sites on the protein. These results, coupled with evidence gathered from the literature, suggest that this type of minimal N­glycosylation in seed PAPs and other type I seed RIPs may serve to enhance cytotoxicity by exploiting receptor­mediated uptake pathways of seed predators while preserving ribosome affinity and rRNA recognition.

Regioselective Glycosylation of 3-, 5-, 6-, and 7-Hydroxyflavones by Cultured Plant Cells.

Regioselective glycosylation of 3-, 5-, 6-, and 7-hydroxyflavones was investigated using cultured plant cells of Eucalyptus perriniana and Phytolacca americana as biocatalysts. 3- and 7-Hydroxyflavones were practically glycosylated into the corresponding ß-D-glucosides by E. perriniana and P. americana.

Biochemical analysis of Phytolacca DOPA dioxygenase.

The biochemical analysis of Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) was carried out. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. A mutated PaDOD1 protein with a change of 177 Asn to Gly showed reduced specific activity compared with PaDOD1, while DOPA dioxygenase activity was not observed for a mutated PaDOD2 protein which had its conserved motif replaced with that of PaDOD. A three-dimensional (3D) structural model of PaDOD1 and PaDOD2 showed that the conserved motif in DODs was located in the N-terminal side of a loop, which was found close to the putative active site. The difference in stability of the loop may affect the enzymatic activity of PaDOD2.

Molecular binding mechanisms of manganese to the root cell wall of Phytolacca americana L. using multiple spectroscopic techniques.

The root cell wall (RCW) of Mn hyperaccumulator Phytolacca americana L. (P. americana) plays an important role in immobilizing and detoxifying excessive Mn, but the molecular binding mechanism of Mn to RCW has been little studied. This study investigated the effect of varied pH on Mn adsorption by the isolated RCW from P. americana in batch experiments, and explored the binding mechanisms of Mn to RCW using Fourier transform infrared spectroscopy (FTIR), synchrotron-based X-ray absorption near-edge structure (XANES), and extended X-ray fine structure spectroscopy (EXAFS). Results showed that Mn binding capacity depends on solution pH, with an optimal pH of 5.0-6.0. Experimental isotherm data could be successfully modeled by the Langmuir and Freundlich equations; the estimated maximum Mn adsorption capacity was 5.446 mg g(-1) according to the established Langmuir isotherm. FTIR spectroscopy demonstrated hydroxyl and carboxyl groups were probably involved in the Mn binding process. XANES results showed that Mn remained as Mn(II) after adsorption on RCW, without any change of oxidation state; EXAFS analysis further revealed that Mn was complexed to RCW via bidentate inner-sphere coordination with carboxyl, which provides new structure information of Mn adsorbed on biomaterials and accounted for high Mn accumulation on RCW of P. americana.

Development and validation of a HPLC-MS/MS method for the determination of phytolaccagenin in rat plasma and application to a pharmacokinetic study.

Radix Phytolaccae (the dried root of Phytolacca acinosa Roxb. or Phytolacca americana L.) is widely used in East Asian countries for the treatment of inflammation-related diseases. The active component of Radix Phtolaccae is Phytolcaccagenin a triterpenoid saponin. Phytolcaccagenin has anti-inflammatory activities that exceed those of Esculentoside A and its derivatives regarding suppression of LPS-induced inflammation, and has a lower toxicity profile with less hemolysis. To date, no information is available about analytical method and pharmacokinetic studies of phytolaccagenin. To explore PK profile of this compound, a HPLC-MS/MS assay of phytolaccagenin in rat plasma was developed and validated. The method was fully validated according to FDA Guidance for industry. The detection was performed by a triple-quadrupole tandem mass spectrometer with multiple reactions monitoring (MRM) in positive ion mode via electrospray ionization. The monitored transitions were m/z 533.2>515.3 for Phytolcaccagenin, and 491.2>473.2 for I.S. The analysis was performed on a Symmetry C18 column (4.6 mm × 50 mm, 3.5 µm) using gradient elution with the mobile phase consisting of acetonitrile and 0.1% formic acid water at a flow rate of 1 ml/min with a 1:1 splitter ratio. The method was validated with a LLOQ of 20 ng/ml and an ULOQ of 1000 ng/ml. The response versus concentration data were fitted with 1/x weighting and the correlation coefficient (r) were greater than 0.999. The average matrix effect and the average extraction recovery were acceptable. This validation in rat plasma demonstrated that phytolaccagenin was stable for 30 days when stored below -20°C, for 6h at room temperature (RT, 22°C), for 12 h at RT for prepared control samples in auto-sampler vials, and during three successive freeze/thaw cycles results at -20°C. The validated method has been successfully applied to an intravenous bolus pharmacokinetic study of phytolaccagenin in male Sprague-Dawley rats (10 mg/kg, i.v.). Blood samples taken from 0 to 24h after injection were collected, and data analyzed with WinNonlin. The half-life and clearance were 1.4±0.9 h and 2.1±1.1 L/h/kg, respectively.

Antibacterial effect of crude extract and metabolites of Phytolacca americana on pathogens responsible for periodontal inflammatory diseases and dental caries.

BACKGROUND: The oral cavity is the store house of different species of microorganisms that are continuously engaged in causing diseases in the mouth. The present study was conducted to evaluate the antibacterial potential of crude extracts of the aerial parts of Phytolacca americana and its natural compounds against two oral pathogens, Porphyromonas gingivalis and Streptococcus mutans, which are primarily responsible for periodontal inflammatory diseases and dental caries, as well as a nonpathogenic Escherichia coli. METHODS: Crude extract and fractions from the aerial parts of P. americana (0.008-1.8 mg/mL) were evaluated for their potential antibacterial activity against two oral disease causing microorganisms by micro-assays. The standard natural compounds present in P. americana, kaempferol, quercetin, quercetin 3-glucoside, isoqueritrin and ferulic acid, were also tested for their antibacterial activity against the pathogens at 1-8 µg/mL. RESULTS: The crude extract was highly active against P. gingivalis (100% growth inhibition) and moderately active against S. mutans (44% growth inhibition) at 1.8 mg/mL. The chloroform and hexane fraction controlled the growth of P. gingivalis with 91% and 92% growth inhibition at a concentration of 0.2 mg/mL, respectively. Kaempferol exerted antibacterial activity against both the pathogens, whereas quercetin showed potent growth inhibition activity against only S. mutans in a concentration dependent manner. CONCLUSION: The crude extract, chloroform fraction, and hexane fraction of P. americana possesses active natural compounds that can inhibit the growth of oral disease causing bacteria. Thus, these extracts have the potential for use in the preparation of toothpaste and other drugs related to various oral diseases.

Impact of Phytolacca americana extracts on gene expression of colon cancer cells.

Native Americans have used Phytolacca americana to treat breast ailments, gastrointestinal disorders, rashes, and inflammation. Some anti-cancer and anti-viral research has been reported on this perennial herb, but none has been published concerning the effects of its extracts on cancer cell genes. In this study, changes in gene expression at the transcription level were evaluated in HCT-116 colon cancer cells after exposure to P. americana ethanol extract and its water fraction using the Human Cancer Pathway Finder PCR Array. Of the genes significantly affected in HCT-116 cells exposed to the ethanol extract at 3200 µg/ml, changes in expression of MYC, PLAU, and TEK may benefit the treatment of colon cancer. Exposing the cells to 1600 µg/ml of the water fraction resulted in several gene changes that may also be beneficial in the treatment of colon cancer: NME4, TEK, and THBS1. A few genes on this array that are known to play a specific role in colon cancer had activities changed in a way that may be detrimental in the treatment of colon cancer. Further studies should be performed to understand how these changes would impact colon cancer treatment.

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.

Differential cell-protective function of two resveratrol (trans-3,5,4'-trihydroxystilbene) glucosides against oxidative stress.

Resveratrol (trans-3,5,4'-trihydroxystilbene; RSV), a natural polyphenol, exerts a beneficial effect on health and diseases. RSV targets and activates the NAD(+)-dependent protein deacetylase SIRT1; in turn, SIRT1 induces an intracellular antioxidative mechanism by inducing mitochondrial superoxide dismutase (SOD2). Most RSV found in plants is glycosylated, and the effect of these glycosylated forms on SIRT1 has not been studied. In this study, we compared the effects of RSV and two glycosyl RSVs, resveratrol-3-O-ß-d-glucoside (3G-RSV; polydatin/piceid) and resveratrol-4'-O-ß-d-glucoside (4'G-RSV), at the cellular level. In oxygen radical absorbance capacity and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays, the antioxidant activity of 3G-RSV was comparable to that of RSV, whereas the radical-scavenging efficiency of 4'G-RSV was less than 50% of that of RSV. However, 4'G-RSV, but not 3G-RSV, induced SIRT1-dependent histone H3 deacetylation and SOD2 expression in mouse C2C12 skeletal myoblasts; as with RSV, SIRT1 knockdown blunted these effects. RSV and 4'G-RSV, but not 3G-RSV, mitigated oxidative stress-induced cell death in C2C12 cells and primary neonatal rat cardiomyocytes. RSV and 4'G-RSV inhibited C2C12 cell proliferation, but 3G-RSV did not. RSV was found in both the intracellular and extracellular fractions of C2C12 cells that had been incubated with 4'G-RSV, indicating that 4'G-RSV was extracellularly deglycosylated to RSV, which was then taken up by the cells. C2C12 cells did not deglycosylate 3G-RSV. Our results point to 4'G-RSV as a useful RSV prodrug with high water solubility. These data also show that the in vitro antioxidative activity of these molecules did not correlate with their ability to protect cells from oxidative stress-induced apoptosis.

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.

[Molluscicidal effect of Phytolacca americana linn leaf against Oncomelania hupensis and its acute toxicity].

OBJECTIVE: To study the molluscicidal activity, the influence on glycogen content of Oncomelania hupensis and the acute toxicity to zebra fish of the extract from Phytolacca americana Linn leaf. METHODS: The different polar factions of the extract of Phytolacca americana Linn leaf were separated by using the systemic solvent segregation method, and then the molluscicidal activity of the fractions was detected according to the Laboratory Final Milluscicides Screening Method issued by WHO. The glycogen content of soft tissues of Oncomelania hupensis treated by the ethyl acetate polar fraction was determined by the anthrone method. Finally, the acute toxicity of the ethyl acetate polar fraction to non-targets was studied with zebra fish. RESULTS: The ethyl acetate polar fraction was the best active components against the snails. Its 48 h LC50 and LC90 were 6.0 mg/100 ml and 26.1 mg/ 100 ml, respectively. The glycogen content of soft tissues of the snails decreased by 20% after treated with the fraction. The fish treated by the concentration of LC50 (48 h) of the ethyl acetate polar fraction survived for 12 h. CONCLUSION: The Phytolacca americana Linn leaf possesses an adequate molluscicidal activity and a significant acute toxicity to the zebra fish.

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.