Distribution and mobility of manganese in the hyperaccumulator plant Phytolacca acinosa Roxb. (Phytolaccaceae)

The distribution and mobility of manganese (Mn) in the hyperaccumulator plant species Phytolacca acinosa Roxb. (Phytolaccaceae) were investigated in a hydroponic system. The plants were exposed to 2 or 5 mM Mn for up to 28 days. For any given plant, the Mn content in the mature leaves (nos. 5–9) was always higher than that in the old (nos. 1–4) and young leaves (nos. 10–14). Within the different parts of a leaf, Mn was preferentially accumulated in the leaf marginal area, where the observed level was threefold higher than that in the midrib. Cross-sectional analysis of the leaf revealed that the concentration of Mn was higher in the leaf epidermis than in the mesophyll. Cell fractionation analysis with P. acinosa leaves showed that most of the Mn (78.4%) was present in the final supernatant fraction (following centrifugation at 20,000 g for 45 min). The distribution of Mn in the leaves of P. acinosa was controlled mainly by the transpiration rate. Our investigation demonstrated that Mn was readily transported from the roots to shoots of P. acinosa but that it could not be remobilized readily after it reached leaves.     

商陆对锰污染土壤的修复实验研究

商陆是在中国境内发现的多年生、草本型锰超积累植物。通过室内土培试验,评价商陆对土壤中锰的去除潜力,确定最佳收获时间,以期达到最佳的重金属污染土壤植物修复效果。结果表明:商陆能将土壤中的锰转运到地上部位,叶片中Mn含量最高,平均值为17 043 mg/kg DW,远远大于茎和根的锰含量均值;单株的平均富集量在浓度为500 mg/kg DW时达最高,一棵商陆可富集平均13 mg的Mn;动态修复中确定的最佳收获时间为60 d,不同时间收获的商陆地下部分生物量差异不明显(P0.05),地上部分则差异较大。连续收获不改变其锰生物富集能力。这表明商陆对锰有较强的富集能力,是一种优良的修复锰污染土壤的物种,对土壤重金属污染的治理及植物修复领域数据库的完善具有重要意义。     

Contribution of arginase to manganese metabolism of <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Aspects of manganese metabolism during normal and acidogenic growth of Aspergillus niger were explored. Arginase from this fungus was a Mn[II]-enzyme. The contribution of the arginase protein towards A. niger manganese metabolism was investigated using arginase knockout (D-42) and arginase over-expressing (ΔXCA-29) strains of A. niger NCIM 565. The Mn[II] contents of various mycelial fractions were found in the order: D-42 strain < parent strain < ΔXCA-29 strain. While the soluble fraction forms 60 % of the total mycelial Mn[II] content, arginase accounted for a significant fraction of this soluble Mn[II] pool. Changes in the arginase levels affected the absolute mycelial Mn[II] content but not its distribution in the various mycelial fractions. The A. niger mycelia harvested from acidogenic growth media contain substantially less Mn[II] as compared to those from normal growth media. Nevertheless, acidogenic mycelia harbor considerable Mn[II] levels and a functional arginase. Altered levels of mycelial arginase protein did not significantly influence citric acid production. The relevance of arginase to cellular Mn[II] pool and homeostasis was evaluated and the results suggest that arginase regulation could occur via manganese availability.     

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.     

Accumulation and detoxification of manganese in hyperaccumulator Phytolacca americana

Pokeweed ( Phytolacca americana ) has recently received much attention because of its ability to hyperaccumulate manganese (Mn). The internal mechanism of detoxification of Mn, however, is not fully understood. In the present study, we investigated Mn accumulation, subcellular distribution, chemical speciation and detoxification through oxalate in pokeweed. The plant accumulated excess Mn in the leaves, mainly in the water-soluble fraction, and over 80% of Mn was in a water-soluble form, while accumulation of excess Mn in the cellular organelle and membrane fraction caused phytotoxicity. In addition, pokeweed has an intrinsically high oxalate content. In all experiments, there was sufficient oxalate to chelate Mn in leaf water extracts at all different levels of Mn application. Phase analysis of X-ray diffraction detected oxalate–Mn chelate complexes, and gel chromatography further confirmed the chelation of Mn by oxalate. In conclusion, pokeweed accumulates excess Mn in the soluble fraction of leaf cells, most likely in vacuoles, in which detoxification of Mn could be achieved by chelation with oxalate.     

Tracing Intracellular Localization and Chemical Forms of Copper in Elsholtzia splendens with Cluster Analysis

Copper (Cu) accumulation in soils is becoming a major environmental problem. Elsholtzia splendens, a Cu-tolerant plant growing in Cu mine areas, can accumulate high levels of Cu. This study develops a systematic method of detecting the intracellular localization and different chemical forms of Cu to comprehensively understand the mechanisms involved in Cu tolerance and detoxification of E. splendens. Various chemical forms of Cu were absorbed by E. splendens, the highest proportion of which was stored in its cell walls (68 %) and vacuoles (42 %). Most of the Cu in the roots, stems and leaves were extracted by 2 % hydrate acetic acid (HAc) in controls. The distribution characteristics of Cu, particularly in the roots, were closely related to the Cu tolerance of E. splendens. In addition, the binding capacity of Cu to soluble salts or proteins decreased and its binding capacity to insoluble oxalate increased with the increasing Cu supply. These results indicated that Cu ions were integrated with compounds of low bioavailable, such as undissolved phosphate or oxalate, which contributed to the metal tolerance and detoxification of E. splendens. Cluster analysis, based on Cu abundance levels, reveals that correlations exist between the chemical forms of Cu and their intracellular locations, which in turn indicates a relationship between their migration activity and toxicity.     

Phytolacca acinosa Roxb. with Arthrobacter echigonensis MN1405 enhances heavy metal phytoremediation

The growth and metal-extraction efficiency of plants when exposed to toxic metals can be enhanced by inoculating with certain bacteria, but the mechanisms of this process remain unclear. We report results from glasshouse experiments on the effect of Arthrobacter echigonensis MN1405 in promoting Phytolacca acinosa Roxb. growth when exposed to 100 mg/L Mn solution. Mn removal efficiency in solution was significantly enhanced by bacterial inoculation; Mn was accumulated in the root of P. acinosa Roxb. plant. The bacteria oxidized the Mn on root surface, which formed a Mn plaque to serve as a barrier or a containment to prevent metal toxicity. In this process, pH condition was an important factor on the effects of microbial-assisted heavy metal phytoremediation. Our finding suggests that A. echigonensis MN1405 assisted P. acinosa to achieve high remediation efficiency of Mn removal and accumulation in Mn contamination area.     

Correlation between the production of exopolysaccharides and oxalic acid secretion by Ganoderma applanatum and Tyromyces palustris

The secretion of exopolysaccharides and oxalic acid in cultures of a white rot Ganoderma applanatum strain and a brown rot Tyromyces palustris strain were tested in terms of culture time, pH range, and temperature. The high yield of exopolysaccharides (EPS) required a moderate temperature of 28 °C for G. applanatum and 20 °C for T. palustris. G. applanatum and T. palustris accumulated more EPS when the concentration of the carbon source (maltose for G. applanatum and fructose for T. palustris) was 30 g/L. The results indicate that the production of oxalic acid by G. applanatum is correlated with the initial pH value of the culture medium and the concentration of oxalic acid increased to 1.66 ± 0.2 mM at the initial pH of 6.5 during the fungal growth. During the growth of T. palustris, the reduction of the initial pH value of the growing medium lowered the oxalic acid concentration from 7.7 ± 0.6 mM at pH 6.0 to 1.99 ± 0.2 mM at pH 3.5. T. palustris accumulated considerably more oxalic acid than G. applanatum and its presence did not affect significantly the production of exopolysaccharides. We also observed that the maximum amounts of exopolysaccharides secreted during cultivation of G. applanatum and T. palustris were 45.8 ± 1.2 and 19.1 ± 1.2 g/L, respectively.     

Increase of ascorbic acid content and nutritional quality in spinach leaves during physiological acclimation to low temperature

The effect of acclimation to 10 °C on the leaf content of ascorbic and oxalic acids, was investigated in spinach (Spinacia oleracea L.). At 10 °C the content of ascorbic acid in leaves increased and after 7 days it was about 41% higher than in plants remaining under a 25 °C/20 °C day/night temperature regime. In contrast, the content of oxalate, remained unchanged. Transfer to 10 °C increased the ascorbic but not the oxalic acid content of the leaf intercellular washing fluid (IWF). Oxalate oxidase (OXO EC 1.2.3.4) activity was not detected in extracts of leaf blades. Therefore, oxalic acid degradation via OXO was not involved in the control of its content. Our results show that low temperature acclimation increases nutritional quality of spinach leaves via a physiological rise of ascorbic acid that does not feed-forward on the content of oxalic acid.     

Cadmium induced oxalic acid secretion and its role in metal uptake and detoxification mechanisms in <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

This study examines the role of oxalic acid in the uptake of Cd and participation in detoxification process in Phanerochaete chrysosporium. Cd-induced oxalic acid secretion was observed with growth inhibition and enzyme inactivation (LiP and MnP) of P. chrysosporium. The peak value of oxalic acid concentration was 16.6 mM at initial Cd concentration of 100 mg L^?1. During the short-term uptake experiments, the uptake of Cd was enhanced and accelerated in the presence of oxalic acid and resulted in alleviated growth and enzyme inhibition ratios. The formation of a metal-oxalate complex therefore may provide a detoxification mechanism via effect on metal bioavailability, whereby many fungi can survive and grow in environments containing high concentrations of toxic metals. The present findings will advance the understanding of fungal resistance to metal stress, which could show promise for a more useful application of microbial technology in the treatment of metal-polluted waste.     

Interaction of nickel and manganese in accumulation and localization in leaves of the Ni hyperaccumulators Alyssum murale and Alyssum corsicum

The genus Alyssum contains >50 Ni hyperaccumulator species; many can achieve >2.5% Ni in dry leaf. In soils with normal Mn levels, Alyssum trichome bases were previously observed to accumulate Ni and Mn to high levels. Here we report concentration and localization patterns in A. murale and A. corsicum grown in soils with nonphytotoxic factorial additions of Ni and Mn salts. Four leaf type subsets based on size and age accumulated Ni and Mn similarly. The greatest Mn accumulation (10 times control) was observed in A. corsicum with 40 mmol Mn kg^?1 and 40 mmol Ni kg^?1 added to potting soil. Whole leaf Ni concentrations decreased as Mn increased. Synchrotron X-ray fluorescence mapping of whole fresh leaves showed localized in distinct high-concentration Mn spots associated with trichomes, Ni and Mn distributions were strongly spatially correlated. Standard X-ray fluorescence point analysis/mapping of cryofractured and freeze-dried samples found that Ni and Mn were co-located and strongly concentrated only in trichome bases and in cells adjacent to trichomes. Nickel concentration was also strongly spatially correlated with sulfur. Results indicate that maximum Ni phytoextraction by Alyssum may be reduced in soils with higher phytoavailable Mn, and suggest that Ni hyperaccumulation in Alyssum species may have developed from a Mn handling system.     

Accumulation and interaction of calcium and manganese in Phytolacca americana

To further understand the hyperaccumulation of Mn, the present study investigated the accumulation of Ca and Mn and their interaction in Mn hyperaccumulator pokeweed (Phytolacca americana Linn.). Exogenous Ca was observed to have a distinctive impact on the Mn phytotoxicity and accumulation in pokeweed, but exogenous Mn had little influence on the accumulation of Ca. Both Ca and Mn accumulated in pokeweed were detected to be mainly in the form of oxalate. Investigation with SEM and TEM found there were two kinds of crystals in the leaves, Ca oxalate crystals and Mn-containing crystals. Further detection showed that there was no inclusion of Mn inside the Ca oxalate crystals, and that other elements, such as C, O and P, were present in the Mn-containing crystals. These results suggest that Ca oxalate crystals in pokeweed have no direct effect on the detoxification of Mn. In addition, the finding of element P and O in the Mn-containing crystals indicates that excess Mn could be deposited by phosphate, which could contribute to Mn accumulation and detoxification in pokeweed.     

Isolation of Oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10?mM of oxalic acid whereas only 15 isolates were grow at 50?mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23?%) after 60?min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents.     

Antimicrobial and antiviral activity-guided fractionation from Scutia buxifolia Reissek extracts

Antimicrobial and antiviral activities of the fractions from Scutia buxifolia stem bark and leaves were evaluated. Best antimicrobial results occurred with the ethyl acetate (EA) and n-butanolic (NB) fractions from the leaves against Micrococcus sp. (minimal inhibitory concentration—MIC = 62.5 μg/ml), and NB fraction from stem bark and leaves against Klebsiella pneumoniae and Enterococcus faecalis (MIC = 62.5 μg/ml). The most active fractions were selected and fractioned into silica column to perform an in vitro antibiofilm assay, which evidenced subfractions EA2 and EA3 as the more active against Candida albicans (biofilm inhibitory concentration—BIC = 582 ± 0.01 μg/ml) and Staphylococcus aureus (BIC = 360 ± 0.007 μg/ml), respectively. The NB (selectivity index—SI = 25.78) and the EA (SI = 15.97) fractions from the stem bark, and the EA (SI = 14.13) fraction from the leaves exhibited a potential antiviral activity towards Herpes Simplex Virus type 1 whereas EA2 and EA3 subfractions from leaves (SI = 12.59 and 10.06, respectively), and NB2 subfraction from stem bark (SI = 12.34) maintained this good activity. Phenolic acids and flavonoids (gallic acid, chlorogenic acid, caffeic acid, rutin, isoquercitrin, quercitrin and quercetin) were identified by HPLC and may be partially responsible for the antimicrobial and antiherpes activities observed. The results obtained in this study showed that Scutia buxifolia has antibiofilm and anti-herpetic activities and that these properties are reported for the first time for this species.     

Simultaneous detection of 35S- and 32P-labeled proteins on electrophoretic gels

A new method for the determination of oxalic acid in urine, which does not require isolation of oxalic acid, was developed by derivatizing oxalic acid and separating and quantitating the product by automated liquid chromatography. Oxalic acid in urine was reacted with o-phenylenediamine to form the strongly uv-absorbing compound 2,3-dihydroxyquinoxaline. Isolation and quantitation of this derivative were accomplished using a reverse-phase C₈ column, 5% methanol in 0.1 m ammonium acetate buffer (pH 6.6) as eluant, and absorption at 314 nm. The method was linear from 1 to 151 μg oxalic acid/ml of sample and the conversion of oxalic acid to the dihydroxyquinoxaline over this concentration range was 94.9%. The precision of duplicates averaged ±1.1%. Analyses of urine before and after treatment with oxalate decarboxylase were employed to differentiate actual urinary oxalic acid from oxalogenic compounds. Under the conditions employed, no urine was found to contain inhibitors of oxalate decarboxylase. No significant contribution to the method was found in a study of 19 potentially interfering urinary constituents. Levels of oxalic acid found in 27 urine samples from patients by this method averaged 71% of levels found using an earlier colorimetric method.     

Diversity of low-molecular weight organic acids synthesized by <Emphasis Type="Italic">Salix</Emphasis> growing in soils characterized by different Cu,Pb and Zn concentrations

The aim of the study was to evaluate the biosynthesis and exudation of 10 low-molecular weight organic acids (LMWOAs) into the rhizosphere with a simultaneous analysis of the acid contents in the roots and leaves of 9 Salix taxa growing on two experimental areas, differing in their concentrations of copper (Cu), lead (Pb) and zinc (Zn) in the soil (Area 1—low, Area 2—high concentration). The obtained results reveal a significant difference in the phytoextraction of the tested Salix taxa for the analysed metals in both areas. The highest contents of Cu, Pb and Zn were observed for all Salix collected from Area 2, especially in S. × smithiana roots (116 ± 8.76, 87.84 ± 7.30 and 203.42 ± 14.62 mg kg^?1 DW, respectively). The results obtained in Area 2 also revealed acidification of the rhizosphere and a higher concentration of acids, mainly oxalic, malic, malonic, acetic and citric acids. Contents of oxalic, malic, acetic and citric acids increased in the roots of Salix taxa from Area 2, while in the leaves formic and succinic acids were also present. S. × smithiana was the taxon with the highest concentration of acids in the rhizosphere and roots (73.48 ± 6.77 and 49.79 ± 2.65 μM 100 g^?1 DW, respectively), while in leaves a higher content was observed for S. alba and S. viminalis ‘PR’ taxa (78.12 ± 3.95 and 71.12 ± 3.75 μM 100 g^?1 DW, respectively).     

Kinetics of manganese transport and gene expressions of manganese transport carriers in Caco-2 cell monolayers

Two experiments were conducted to investigate the kinetics of manganese (Mn) transport in Caco-2 cell monolayers and the gene expressions of Mn transport carriers in apical (AP) and basolateral (BL) membranes. In experiment 1, the cells were treated with the medium containing 146 μmol/L of Mn (MnSO₄·H₂O). Both the uptake and transport of Mn from AP–BL or from BL–AP at different time-points were assessed to determine the optimal time for kinetics of Mn transport. The transport of Mn increased linearly with higher efficiency values in AP–BL than in BL–AP direction, however, the uptake of Mn revealed an asymptotic pattern within 120 min. In experiment 2, the kinetics of Mn transport in AP–BL was determined with media containing Mn concentrations from 0 to 2,500 μmol/L at 40 and 120 min, respectively, and mRNA levels of divalent metal transporter 1 (DMT1) and ferroportin (FPN1) were determined in Caco-2 cells treated with the medium containing 0 or 800 μmol/L of Mn for 120 min. The kinetics of Mn transport showed a carrier-mediated process when Mn concentrations were lower than 1,000 μmol/L and a linear increment when Mn concentrations exceeded 1,000 μmol/L at either 40 or 120 min. Mn treatment decreased (P < 0.01) DMT1 mRNA level and increased (P < 0.01) FPN1 mRNA level. The results from the present study suggested that Mn transport in AP–BL fit both carrier-mediated saturable and non-saturable diffusion processes, and Mn transport carriers DMT1 and FPN1 mediate the apical uptake and basolateral exit of Mn in Caco-2 cells.     

Acid phosphatase activity in maize leaves as related to their evolution and phosphorus deficiency

The effect of phosphorus deficiency on the activity of acid phosphatase of the first, second and third leaves of maize plants was followed. The supernatant obtained by centrifuging the homogenate of plant tissue at 1500 ×g was further centrifuged at 18 000 ×g, the sediment marked as fraction II and the supernatant as fraction III. Acid phosphatase activity of fraction II of the first to third leaves was for the whole period of culture higher in plants grown in the nutrient solution without phosphate. In fraction III this relation was established in the first leaf, after 3 days of culture in the second leaf and after 5 days in the third leaf. In all leaves higher enzyme activity was unambiguously determined in fraction III when compared with fraction II. Higher acid phosphatase activity was established in those leaves which were younger in their development, particularly in the first days of culture. With the ageing of leaves the enzyme activity decreased.     

Influence of manganese deficiency and toxicity on isoprenoid syntheses

Twenty-eight day old wheat (Triticum aestivum L. cv Stacy) response to varying Mn concentration (10.1-10,000 micromolar) in nutrient solution was measured. Manganese concentrations in the most recently matured leaves (blade 1) were 0.21 to 19.03 mmol Mn per kilogram dry weight, respectively. Fresh and dry weights increased to a maximum at the 5 micromolar Mn nutritional level (0.37 millimole Mn per kilogram dry weight) and were decreased at Mn above and below this concentration. Blade 1 chloroplast pigment concentrations increased up to the 20 micromolar Mn nutritional level (1.98 millimole Mn per kilogram dry weight) and decreased at higher Mn concentrations. Thylakoid Mn content was above 1 mole Mn/100 mole chloroplast at Mn nutrition levels which resulted in greatly decreased plant growth. Total phytoene biosynthesis was decreased by Mn deficiency and toxicity. In vitro ent- kaurene synthesis was greatly influenced by Mn concentration with a maximal biosynthesis at 1 micromolar Mn and decreases at Mn levels above and below this concentration. In vivo blade 1 gibberellic acid equivalent concentrations were maximal at 20 parts per million Mn nutrition solution levels (1.98 millimole Mn per kilogram dry weight) and decreased at Mn tissue concentrations above and below this value; additionally, gibberellic acid concentrations were reciprocal to extracted C₂₀ alcohol concentrations. Mn influence on gibberellin and chloroplast pigment biosyntheses exactly matched the measured changes in growth.     

Pseudomonas fluorescens ATCC 13525 Containing an Artificial Oxalate Operon and Vitreoscilla Hemoglobin Secretes Oxalic Acid and Solubilizes Rock Phosphate in Acidic Alfisols

Oxalate secretion was achieved in Pseudomonas fluorescens ATCC 13525 by incorporation of genes encoding Aspergillus niger oxaloacetate acetyl hydrolase (oah), Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla hemoglobin (vgb) in various combinations. Pf (pKCN2) transformant containing oah alone accumulated 19 mM oxalic acid intracellularly but secreted 1.2 mM. However, in the presence of an artificial oxalate operon containing oah and FpOAR genes in plasmid pKCN4, Pf (pKCN4) secreted 13.6 mM oxalate in the medium while 3.6 mM remained inside. This transformant solubilized 509 μM of phosphorus from rock phosphate in alfisol which is 4.5 fold higher than the Pf (pKCN2) transformant. Genomic integrants of P. fluorescens (Pf int1 and Pf int2) containing artificial oxalate operon (plac-FpOAR-oah) and artificial oxalate gene cluster (plac-FpOAR-oah, vgb, egfp) secreted 4.8 mM and 5.4 mM oxalic acid, released 329 μM and 351 μM P, respectively, in alfisol. The integrants showed enhanced root colonization, improved growth and increased P content of Vigna radiata plants. This study demonstrates oxalic acid secretion in P. fluorescens by incorporation of an artificial operon constituted of genes for oxalate synthesis and transport, which imparts mineral phosphate solubilizing ability to the organism leading to enhanced growth and P content of V. radiata in alfisol soil.