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.

[Factors influencing induction and in vitro culture of hairy roots in Phytolacca americana L.]

To use hairy roots for producing medicinal ingredients of Phytolacca americana L. we studied the factors influencing the induction and in vitro culture. Hairy roots could be incited from the veins of cut surface (morphological lower) of P. americana L. leaf explants around 18 days after infection with the strain of Agrobacterium rhizogenes ATCC15834. The highest rooting rate, 70%, was obtained when leaf explants were pre-cultured for 1 day, infected for 20 min, and co-cultured for 4 days. The transformation was confirmed by PCR amplification of rolC of Ri plasmid and silica gel thin-layer chromatography of opines from P. americana L. hairy roots. All the hairy root lines could grow rapidly on solid exogenous phytohormone-free MS medium. Among the 9 hairy root lines, the hairy root line 2 had most rapid growth, most branched lateral roots and most intensive root hair; the root surface of some hairy root lines seemed purple or red, while that of the other hairy root line appeared white. Among liquid media MS, 1/2MS, B5 and 6,7-V tested, the best growth for hairy root lines was attained in liquid exogenous phytohormone-free MS medium. Compared with exogenous phytohormone-free MS medium, 6,7-V medium was better for synthesis and accumulation of esculento side A in hairy roots. The established optimal conditions for induction and in vitro culture of P. americana hairy roots had laid an experimental and technological foundation for production of medicinal constituents esculento side A from large scale culture of hairy roots.

Effects of generalist herbivory on resistance and resource allocation by the invasive plant, Phytolacca americana.

Successful invasions by exotic plants are often attributed to a loss of co-evolved specialists and a re-allocation of resources from defense to growth and reproduction. However, invasive plants are rarely completely released from insect herbivory because they are frequently attacked by generalists in their introduced ranges. The novel generalist community may also affect the invasive plant's defensive strategies and resource allocation. Here, we tested this hypothesis using American pokeweed (Phytolacca americana L.), a species that has become invasive in China, which is native to North America. We examined resistance, tolerance, growth and reproduction of plant populations from both China and the USA when plants were exposed to natural generalist herbivores in China. We found that leaf damage was greater for invasive populations than for native populations, indicating that plants from invasive ranges had lower resistance to herbivory than those from native ranges. A regression of the percentage of leaf damage against mass showed that there was no significant difference in tolerance between invasive and native populations, even though the shoot, root, fruit and total mass were larger for invasive populations than for native populations. These results suggest that generalist herbivores are important drivers mediating the defensive strategies and resource allocation of the invasive American pokeweed.

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.

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.

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.

[Prevention and control of invaded plant Phytolacca americana in sandy coastal shelter forests].

The invasion of Phytolacca americana has produced serious damage to the coastal shelter forests in China. In order to search for the effective measures for controlling the growth of P. americana, several plots in the Robinia pseudoacacia forest invaded by P. Americana to the relatively same extent were installed, and the measures of physical control (mowing and root cutting) and chemical control (spraying herbicides) were adopted to control the invasion of P. Americana, taking the site with good growth of Amorpha fruticosa in the forest and without any control measures as the comparison. The results showed that mowing could rapidly decrease the growth of P. americana in the same year, but the growth recovered in the next year. 1/3 root cutting only reduced the aboveground growth of P. americana in the same year, and the growth was recovered in the third year; while 2/3 root cutting and whole cutting could effectively cleanup the P. americana plants all the time. Spraying quizalofop-p-ethyl and paraquat only killed the aboveground part of P. americana in the same year, but this part of P. americana recovered to the normal level in the next year; while spraying 45 g x L(-1) of glyphosate could completely kill the whole P. americana plants till the third year. The growth of P. americana at the site with good growth of A. fruticosa and without any control measures maintained at a low level all the time, suggesting that planting A. fruticosa in R. pseudoacacia forest would be an effective approach to prevent and control the P. americana invasion.

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.

Growth and cesium uptake responses of Phytolacca americana Linn. and Amaranthus cruentus L. grown on cesium contaminated soil to elevated CO2 or inoculation with a plant growth promoting rhizobacterium Burkholderia sp. D54, or in combination.

Growth and cesium uptake responses of plants to elevated CO(2) and microbial inoculation, alone or in combination, can be explored for clean-up of contaminated soils, and this induced phytoextraction may be better than the natural process. The present study used open-top chambers to investigate combined effects of Burkholderia sp. D54 inoculation and elevated CO(2) (860 µL L(-1)) on growth and Cs uptake by Phytolacca americana and Amaranthus cruentus grown on soil spiked with various levels of Cs (0-1000 mg kg(-1)). Elevated CO(2) and bacterial inoculation, alone or in combination, significantly increased biomass production with increased magnitude, ranging from 22% to 139% for P. americana, and 14% to 254% for A. cruentus. Total tissue Cs in both plants was significantly greater for bacterial inoculation treatment singly, and combined treatments of bacterial inoculation and elevated CO(2) than for the control treatment in most cases. Regardless of CO(2) concentrations and bacterial inoculation, A. cruentus had higher tissue Cs concentration, Cs transfer factors and concentration ratios than P. americana, but they had slightly different contents of antioxidant enzymes. It is concluded that combined effects of elevated CO(2) and microbial inoculation with regard to plant ability to grow and remove radionuclides from soil can be explored for CO(2)- and microbe-assisted phytoextraction technology.

Paired comparison of water, energy and carbon exchanges over two young maritime pine stands (Pinus pinaster Ait.): effects of thinning and weeding in the early stage of tree growth.

The effects of management practices on energy, water and carbon exchanges were investigated in a young pine plantation in south-west France. In 2009-10, carbon dioxide (CO(2)), H(2)O and heat fluxes were monitored using the eddy covariance and sap flow techniques in a control plot (C) with a developed gorse layer, and an adjacent plot that was mechanically weeded and thinned (W). Despite large differences in the total leaf area index and canopy structure, the annual net radiation absorbed was only 4% lower in plot W. We showed that higher albedo in this plot was offset by lower emitted long-wave radiation. Annual evapotranspiration (ET) from plot W was 15% lower, due to lower rainfall interception and transpiration by the tree canopy, partly counterbalanced by the larger evaporation from both soil and regrowing weedy vegetation. The drainage belowground from plot W was larger by 113 mm annually. The seasonal variability of ET was driven by the dynamics of the soil and weed layers, which was more severely affected by drought in plot C. Conversely, the temporal changes in pine transpiration and stem diameter growth were synchronous between sites despite higher soil water content in the weeded plot. At the annual scale, both plots were carbon sinks, but thinning and weeding reduced the carbon uptake by 73%: annual carbon uptake was 243 and 65 g C m(-2) on plots C and W, respectively. Summer drought dramatically impacted the net ecosystem exchange: plot C became a carbon source as the gross primary production (GPP) severely decreased. However, plot W remained a carbon sink during drought, as a result of decreases in both GPP and ecosystem respiration (R(E)). In winter, both plots were carbon sources, plots C and W emitting 67.5 and 32.4 g C m(-2), respectively. Overall, this study highlighted the significant contribution of the gorse layer to mass and energy exchange in young pine plantations.

[Effects of manganese on antioxidant system of manganese-hyperaccumulator Phytolacca americana L.].

A hydroponic experiment was conducted to study the growth, manganese (Mn) accumulation, lipid peroxidation, H2O2 concentration, and antioxidant system of Phytolacca americana L. exposed to different concentration Mn. With increasing Mn concentration in the medium, the plant Mn content increased significantly, and the Mn accumulation was in the sequence of leaf > stem > root. Comparing with the control, low concentration (5 mmol x L(-1)) Mn promoted the plant growth, decreased the leaf H2O2 concentration, and had less effects on the leaf malondialdehyde (MDA) content, while high concentration (> or = 10 mmol x L(-1)) Mn led to a remarkable increase of leaf H2O2 and MDA contents, indicating an evident oxidative damage occurred in leaves. The activities of ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase and the content of reduced ascorbate increased with increasing Mn concentration, while the SOD activity was inhibited significantly at 5 mmol x L(-1) of Mn but enhanced at > or = 10 mmol x L(-1) of Mn. The activities of catalase and peroxidase and the content of reduced glutathione increased at 5-10 mmol x L(-1) of Mn but dropped markedly at 20 mmol x L(-1) of Mn. All the results suggested that the Mn-induced oxidative damage and Mn accumulation might be responsible for the growth inhibition of P. americana plants at high Mn exposure, and the increase of antioxidative enzyme activities and low molecular antioxidant contents was, at least partly, contributed to the Mn tolerance and hyperaccumulation of P. americana. However, due to their different Mn concentration-dependent change modes, these antioxidants played different roles in the Mn tolerance of P. americana.

Phytolacca americana from contaminated and noncontaminated soils of South Korea: effects of elevated temperature, CO(2) and simulated acid rain on plant growth response.

Chemical analyses performed on the invasive weed Phytolacca americana (pokeweed) growing in industrially contaminated (Ulsan) and noncontaminated (Suwon) sites in South Korea indicated that the levels of phenolic compounds and various elements that include some heavy metals (Al, As, B, Cd, Co, Cu, Fe, Mn, Ni, Pb, and Zn) were statistically higher in Ulsan soils compared to Suwon soils with Al being the highest (>1,116 mg/l compared to 432 mg/l). Analysis of metals and nutrients (K, Na, Ca, Mg, Cl, NH(4), N, P, S) in plant tissues indicated that accumulation occurred dominantly in plant leaves with Al levels being 33.8 times higher in Ulsan plants (PaU) compared to Suwon plants (PaS). The ability of PaU and PaS to tolerate stress was evaluated under controlled conditions by varying atmospheric CO(2) and temperature and soil pH. When grown in pH 6.4 soils, the highest growth rate of PaU and PaS plants occurred at elevated (30 degrees C) and non-elevated (25 degrees C) temperatures, respectively. Both PaU and PaS plants showed the highest and lowest growth rates when exposed to atmospheric CO(2) levels of 360 and 650 ppm, respectively. The impact of soil pH (2-6.4) on seed germination rates, plant growth, chlorophyll content, and the accumulation of phenolics were measured to assess the effects of industrial pollution and global-warming-related stresses on plants. The highest seed germination rate and chlorophyll content occurred at pH 2.0 for both PaU and PaS plants. Increased pH from 2-5 correlated to increased phenolic compounds and decreased chlorophyll content. However, at pH 6.4, a marked decrease in phenolic compounds, was observed and chlorophyll content increased. These results suggest that although plants from Ulsan and Suwon sites are the same species, they differ in the ability to deal with various stresses.

[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.

[Tissue culture and rapid propagation of Phytolacca americana].

OBJECTIVE: To explore the technique of rapid propagation for Phytolacca americana. METHODS: Aseptic seedling were used as explants. RESULTS: The best explants were the stems from strong aseptic seedling. The optimal culture media were MS + NAA (0.2 mg/L) +6-BA (1.0 mg/L) for primarily culture, MS + NAA (0.2 mg/L) +6-BA (2.0 mg/L) for the induction of clustered shoots 1/2MS with NAA 0.4 mg/L for rooting. CONCLUSION: The propagating coefficient of Phytolacca americana can be improved by inducing the clustered shoots from aseptic seedling.

Expression of pokeweed antiviral proteins in creeping bentgrass.

Fungal diseases of creeping bentgrass, an important amenity grass used extensively on golf courses, are a serious problem in golf course management. Transgenic approaches to improving disease resistance to fungal diseases are being explored in many species, and in some cases ribosome-inactivating proteins have been found to be effective. We have generated transgenic creeping bentgrass plants expressing three forms of ribosome-inactivating proteins from pokeweed, which are termed pokeweed antiviral proteins (PAP). PAP-Y and PAP-C are nontoxic mutants of PAP; PAPII is the native form of another ribosome-inactivating protein from pokeweed. In creeping bentgrass, PAP-C transformants did not accumulate the protein, suggesting that it is unstable, and in a field test these plants were not protected from infection by the fungal pathogen Sclerotinia homoeocarpa, the causal agent of dollar spot disease. PAPII transformants could accumulate stable levels of the protein but had symptoms of toxicity; one low-expressing line exhibited good disease resistance. PAP-Y transformants accumulated stable levels of protein, and under greenhouse conditions they appeared to be phenotypically normal.

Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots.

Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, beta-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.