Degradation of benzo[a]pyrene by the litter-decomposing basidiomycete Stropharia coronilla: role of manganese peroxidase

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn(2+)) supplied at a concentration of 200 micro M stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn(2+) than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn(2+). Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter(-1) was converted in an in vitro reaction solution containing 1 U of MnP ml(-1) within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter(-1)) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).     

Removal of Benzo[a]pyrene by a fungus Aspergillus sp. BAP14

A fungal strain BAP14 isolated from marine sediments of coast in Xiamen city, was found to have the ability to degrade benzo[a]pyrene (BaP), and identified as Aspergillus sp. based on 18S rRNA gene sequence. Aspergillus sp. BAP14 was able to remove about 30 and 60% of BaP with initial concentration of 10 mg l⁻¹ in 3 and 12 days of incubation, respectively. Addition of saccharides and low molecular weight polycyclic aromatic hydrocarbons appeared to have effect on the degradation ability, in particularly the addition of lactose and naphthalene. Furthermore, we demonstrated that lipidic particles could be observed in the presence of benzo[a]pyrene based on the morphologic performance of Aspergillus sp. BAP14 through scanning electronic microscopy (SEM) and atomic force microscopy (AFM), respectively.     

Degradation of Benzo[a]pyrene by the Litter-Decomposing Basidiomycete Stropharia coronilla: Role of Manganese Peroxidase

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn²⁺) supplied at a concentration of 200 μM stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn²⁺ than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn²⁺. Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter⁻¹ was converted in an in vitro reaction solution containing 1 U of MnP ml⁻¹ within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter⁻¹) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).     

Removal and mineralization of polycyclic aromatic hydrocarbons by litter-decomposing basidiomycetous fungi

Nine strains of litter-decomposing fungi, representing eight species of agaric basidiomycetes, were tested for their ability to remove a mixture of three polycyclic aromatic hydrocarbons (PAHs) (total 60 mg l(-1)) comprising anthracene, pyrene and benzo(a)pyrene (BaP) in liquid culture. All strains were able to convert this mixture to some extent, but considerable differences in degradative activity were observed depending on the species, the Mn(II) concentration, and the particular PAH. Stropharia rugosoannulata was the most efficient degrader, removing or transforming BaP almost completely and about 95% of anthracene and 85% of pyrene, in cultures supplemented with 200 micro M Mn(II), within 6 weeks. In contrast less than 40, 18, and 50% BaP, anthracene and pyrene, respectively, were degraded in the absence of supplemental Mn(II). In the case of Stropharia coronilla, the presence of Mn(II) led to a 20-fold increase of anthracene conversion. The effect of manganese could be attributed to the stimulation of manganese peroxidase (MnP). The maximum activity of MnP increased in S. rugosoannulata cultures from 10 U l(-1) in the absence of Mn(II) to 320 U l(-1) in Mn(II)-supplemented cultures. The latter degraded about 6% of a (14)C-labeled BaP into (14)CO(2) whereas only 0.7% was mineralized in the absence of Mn(II). In solid-state straw cultures, S. rugosoannulata, S. coronilla and Agrocybe praecox mineralized between 4 and 6% of (14)C-labeled BaP within 12 weeks.     

Light Regulation of Hypocotyl Elongation and Greening in Transgenic Tobacco Seedlings That Over-Express Rice Phytochrome A

Previous analysis of a transgenic tobacco line (BN1) that over-expressedrice phytochrome A (PhyA) indicated that the introduced PhyAwas spectrally and biologically active [Kay et al. (1989) PlantCell 1: 775, Nagatani et al. (1991) Proc. Natl. Acad. Sci. USA88: 5207]. In the present study, we have further investigatedresponses of the BN1 plants to light. Fluence rate dependenceanalysis of the inhibition of hypocotyl elongation indicatedthat the response is biphasic. The amplitude of the low fluencerate component increased by 2 to 3 fold in the BN1 plants comparedto the wild type. In contrast, the presence of rice PhyA didnot alter the level of chlorophyll in the BN1 seedlings grownunder the same light conditions. Ultrastructure studies showedthat chloroplasts in the BN1 plants were not significantly differentfrom those in the wild type plants, except that chloroplastsin the guard cells of the BN1 plants appeared to be more developedthan those of the wild type plants. The fluence response analysisof the potentiation of chlorophyll accumulation indicated nosignificant difference between the BN1 and the wild type plants.Thus, the introduced rice PhyA greatly influenced hypocotylelongation but did not significantly affect the greening process. ⁴Present address: NSFC Center for Biological Timing, Universityof Virginia Charlottesville, VA 22901, U.S.A. ⁵Present address: Advanced Research Laboratory, Hitachi Ltd.Hatoyama, Saitama, 350-03 Japan     

Molecular Cloning and Characterization of a cDNA Clone That Encodes a Cdc2 Homolog from Nicotiana tabacum

We have isolated a cDNA clone (cdc2Nt1) that encodes a homologof p34^cdc2/CDC28 kinase from tobacco (Nicotiana tabacum). Thecdc2Ntl protein showed extensive similarity to other homologsof Cdc2 from plants. Complementation studies showed that thecdc2Ntl gene was able to overcome cell cycle arrest at boththe G1/S and the G2/M transitions of cdc28^ts mutants of buddingyeast, demonstrating that the cdc2Ntl protein was able to replacethe Cdc28 kinase at both the G1/S and the G2/M transitions.Analysis of gene expression demonstrated that the cdc2Ntl genewas transcribed constitutively throughout the cell cycle butthat it was preferentially expressed in actively dividing tobaccoBY-2 cells. (Received July 13, 1995; Accepted February 15, 1996)     

Sorbitol Synthesis in Transgenic Tobacco with Apple cDNA Encoding NADP-Dependent Sorbitol-6-Phosphate Dehydrogenase

The apple (Malus domestica) cDNA encoding NADP-dependent sorbitol-6-phosphatedehydrogenase (S6PDH) was stably integrated and expressed intransgenic tobacco (Nicotiana tabacum cv. SR1). Expression ofthe cDNA in either a sense or antisense orientation was accomplishedusing cauliflower mosaic virus regulatory sequences (CaMV35S).Sorbitol synthesis was confirmed by gas-chromatography-mass-spectroscopy(GC-MS). Sorbitol concentration in the leaves of the transgenicplants expressing the sense orientation varied from 186 to 446nmol (g fr wt)^-1. The concentration positively correlates withS6PDH activity in leaves. Neither sorbitol nor S6PDH activitywas detected in the extracts of nontransformed tobacco or transgenictobacco expressing the antisense orientation. These resultsprovide key genetic evidence that S6PDH expression is sufficientfor the synthesis of sorbitol in tobacco, implicating it asa key enzyme in the sorbitol biosynthetic pathway in apple andperhaps other members of the woody Rosaceae. ¹Present address: Laboratory of Pomology, Faculty of Agriculture,Kyoto University, Sakyo, Kyoto, 606-01 Japan     

Ubiquitin Protein Ligase Ring2 Is Involved in S‐phase Checkpoint and DNA Damage in Cells Exposed to Benzo[a]pyrene

Previous studies in our laboratory demonstrated that Ring2 may affect DNA damage and repair through pathways other than through regulating the expression of the nucleotide excision repair protein. In a series of experiments using wild‐type cell (16HBE and WI38) and small interfering RNA (siRNA) Ring2 cells exposed to benzo[a]pyrene (BaP), we evaluated the cell cycle and DNA damage. The benzo(a)pyrene‐7,8‐dihydrodiol‐9,10‐epoxide (BPDE–DNA) adduct assay demonstrated that in vitro exposure to BaP increased DNA damage in a time‐ and dose‐dependent manner in wild‐type and siRNA Ring2 cells. Analysis of covariance showed that a decrease of Ring2 caused DNA hypersensitivity to BaP. Flow cytometry results and proliferating cell nuclear antigen levels indicated that inhibition of Ring2 attenuated the effect of BaP on S‐phase arrest. Taken together, these data implied that the lower proportion of cells in the S phase induced by inhibition of Ring2 may play an important role in DNA hypersensitivity to BaP.     

Correlation of cytokinins and abscisic acid with monocarpic senescence in soybeans

The involvement of cytokinins and abscisic acid (ABA) in themonocarpic senescence (foliar yellowing following fruit development)of soybeans was examined. Foliar sprays of cytokinin (10^–4M zeatin or 10^–5 M benzyladenine), begun when the plantsfirst set fruit and repeated every other day, significantlydelayed, but did not prevent, monocarpic senescence. Foliarsprays of 10^–4 M ABA, applied in the same manner, significantlyhastened senescence of fruiting soybeans but apparently hadno effect on depodded plants. Leaf and stem material from pre-senescentand senescent plants was extracted, chromatographed, and bioassayedfor cytokinin-lilce activity (Amaranthus betacyanin productionassay) and ABA-like activity (oat coleoptile straight growthassay for inhibitors). ABA-like activity increased, and cytokinin-likeactivity decreased in shoot tissue before the plants began tosenesce. Cytokinin-like activity in the fruit also declinedduring this period. These results implicate a decrease in cytokininsand an increase in ABA-like inhibitors in the control of monocarpicsenescence of soybeans, but neither alone is causal. ¹ Supported in part by Research Grant 416-15-79 from the USDACooperative State Research Service under PL 89–106. ² Present address: Biology Dept., College of St. Benedict'sSt. Joseph, Minn. 56374, U.S.A. (Received February 4, 1978; )     

Mechanism of action of benzo(a)pyrene and nicotine on hormone production by rat pituitary tumor cells

The effects of the cyclic aromatic hydrocarbon, benzo(a)pyrene (BaP) and that of the tobacco alkaloid, nicotine, on prolactin (PRL) and growth hormone (GH) synthesis by rat pituitary tumor cells in culture (GH cells) have been studied. Treatment of GH cells with nicotine (0.1–300 μg/ml) neither affected the growth, nor significantly altered the general pattern of hormone production in these cells. BaP at concentrations greater than 5 μg/ml irreversively inhibited the growth of these cells. The sublethal concentrations of BaP, which did not affect either 1) cell growth, or 2) amino acid transport or 3) total protein synthesis or degradation, did however inhibit specifically, hormone synthesis by these cells. More interestingly concentrations of nicotine which did not affect either cell growth or hormone synthesis, modulated both of these cellular processes in the presence of BaP. A concentration dependent stimulation of microsomal BaP monooxygenase activity was observed in nicotine or BaP treated cells. The effects of these drugs on stimulation of BaP monooxygenase activity seems to be additive. Nicotine also enhanced the association of radioactivity (presumably [³H] BaP metabolites) with DNA in [³H] BaP treated cells. It is concluded that nicotine by itself did not demonstrate any cytotoxic effect nor influence hormone synthesis in GH cells. However, this constituent of tobacco smoke stimulated BaP monooxygenase activity and the interaction of [³H] BaP metabolites with cellular DNA and also modulated BaP induced inhibition of hormone synthesis in GH cells.     

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

Activities of CaMV 35S and nos promoters in pollen: implications for field release of transgenic plants

The expression of foreign genes in pollen may pose potentialproblems in the field release of transgenic plants, since pollenrepresents a route whereby foreign genes and their productsmay escape into the wider environment. The possible risks posedby cross-hybridization with wild relatives have been extensivelyexplored, but problems that may arise due to the expressionof foreign gene products in pollen have not been so widely studied.The activities of the CaMV 35S and nos promoters in pollen inpopulations of stably transformed plants and in transient expressionanalysis are described. These promoters are commonly used inall areas of plant molecular biology research and their expressionpatterns will be of interest to those involved in field releasestudies. The results show that both promoters had no detectablepollen activity in Arabidopsis, but both showed activity intobacco pollen. The CaMV 35S-gus gene fusion showed heritableexpression levels in tobacco pollen of up to a maximum of 64.6pmol 4-MU min^–1 mg ^–1 total protein. nos promoteractivity in transgenic tobacco pollen was highly variable, withGUS activities ranging from undetectable levels up to 2561 pmol4-MU min^–1 mg^–1 total protein within the transgenicpopulation. Histochemical staining of anther sections from 10–12mm buds revealed that the CaMV 35S promoter had some activityin the vascular bundle, stomium and tapetum, while GUS expressionfrom the nos promoter in sporophytic tissues was confined entirelyto the stomium. Key words: CaMV 35S promoter, nos promoter, pollen, transgenic plant release     

Protein Kinase Inhibitors Inhibit Stimulation of Inositol Phospholipid Turnover and Induction of Phenylalanine Ammonia-Lyase in Fungal Elicitor-Treated Tobacco Suspension Culture Cells

Elicitor prepared from Phytophthora nicotianae stimulated inositolphospholipid turnover and induced phenylalanine ammonia-lyaseactivity in tobacco suspension culture cells [Kamada and Muto(1994) Plant Cell Physiol. 35: 397]. Protein kinase inhibitors,K252a and staurosporine inhibited both responses. These resultssuggest that inositol phospholipid turnover plays an importantrole in PAL induction through protein kinases. In addition,their mode of inhibition were different, proposing that severaltypes of protein kinases are involved in these elicitor-inducedresponses. ¹Present address: The Johns Hopkins University School of Hygieneand Public Health, 615 N. Wolfe St., Baltimore, Maryland 21205,U.S.A. ²Present address: Nagoya University BioScience Center and GraduateSchool of Agricultural Sciences, Nagoya University, Chikusa-ku,Nagoya, 464-01 Japan.     

Interaction of benzo[a]pyrene, 2,3,3′,4,4′,5 hexachlorobiphenyl (PCB 156) and cadmium on biomarker responses in flounder (Platichthys flesus L.)

Interactive effects of a mixed pollutant exposure on biomarker responses were studied in European flounder (Platichthys flesus L.). The model chemicals, benzo[a]pyrene (BaP, 2.5 mg kg^-1), 2,3,3′,4,4′5 hexachlorobiphenyl (PCB-156, 2.5 mg kg^-1), and cadmium (cadmium, 1 mg kg^-1), were administered to fish by subcutaneous injections. Biomarker responses were quantified both following administration of single chemicals and sequential combinations of the chemicals in pairs. Significant induction of CYP1A protein levels and corresponding ethoxyresorufin-O-deethylase (EROD) activities was observed in BaP and PCB treated flounder after 2 and 8 days, respectively. The strongest induction (44 fold) was caused by BaP. No further induction was observed after additional treatment with PCB 156. CYP1A induction caused by BaP was inhibited (40% compared with BaP treatment alone) in flounder pre treated with cadmium, whereas induction by PCB 156 appeared to be unaffected by pre treatment with cadmium. Flounder treated with cadmium only had significantly elevated hepatic levels of metallothionein (MT) after 15 days. Pre treatment with BaP and PCB prior to cadmium inhibited the MT induction (30-50%) compared with cadmium alone. Furthermore, significantly higher glutathione S transferase activities were observed in flounder administered cadmium alone, and in flounder treated with BaP or PCB 156 prior to cadmium. GST selenium independent peroxidase activities appeared to be unaffected by any of the treatments in the present study. The results indicate that chemical mixtures may affect biomarker responses differently from compounds administered alone, and that the sensitivity of both CYP1A and MT are influenced by pollutants other than their primary inducers.     

The role of abscisic acid and salination in the adaptive response of plants to reduced root aeration

Cessation of root aeration to tobacco plants (Nicotiana rustica)growing in half-Hoagland nutrient solution resulted in rapidwilting of the shoot. Plants growing in salinated solutions,however, retained their turgor despite lack of aeration. Thisresistance to aeration stress was associated with increasedconcentration of abscisic acid in the leaves. Maximum amountsof abscisic acid in leaves were reached by the 4th day of salinationand coincided with the maximum resistance to lack of aeration.When the plants were returned to half-Hoagland, leaf-abscisicacid decreased to the presalination level. Resistance to lackof aeration also resulted when plants were pretreated with abscisicacid. We tentatively conclude that resistance to lack of rootaeration reflects adaptation to osmotic stress and is partlydue to an increase in the content of leaf-abscisic acid. ¹ Volcani Institute of Agricultural Research, Bet Dagan, Israel.Present address: Department of Horticulture, Michigan StateUniversity, East Lansing, Michigan 48823, U. S. A. (Received June 12, 1971; )     

A Protein Encoded by din1, a Dark-Inducible and Senescence-Associated Gene of Radish, Can Be Imported by Isolated Chloroplasts and Has Sequence Similarity to Sulfide Dehydrogenase and Other Small Stress Proteins

In an attempt to isolate cDNA clones for dark-inducible chloroplastproteins, we screened a cDNA library which was prepared fromradish cotyledons by a two-step method. The source plants weregrown under continuous light for 14 d and kept in darkness for24 h. One of the selected clones, S2D12, corresponded to thedin1 gene which we previously reported as a dark-inducible,senescence-associated gene [Azumi and Watanabe (1991) PlantPhysiol. 95: 577]. A 22 kDa polypeptide was produced from thecDNA in an in vitro expression system in the presence of [³⁵S]methionine.This polypeptide was capable of being imported by isolated chloroplasts,processed to a smaller mature form and localized in the stromalfraction. As the amino acid sequence of the putative matureprotein has no homology to any known chloroplast protein, din1was suggested to be the first gene for a chloroplast proteinwhich is negatively controlled by light. The putative matureprotein has similarity to sulfide dehydrogenase from Wolinellasuccinogenes and other small stress proteins; glpE and pspEfrom Escherichia coli and hsp67B2 from Drosophila melanogaster. ¹ The nucleotide sequence data in this paper has been submittedto EMBL, GenBank and DDBJ Data Libraries under the acces sionnumber AB004242 ² Present address: The Institute of Physical and Chemical Research(RIKEN), 2-1 Hirosawa, Wako-shi, Saitama, 351-01 Japan     

Hepatic CYP1A induction in rainbow trout (Oncorhynchus mykiss) after exposure to benzo[a]pyrene in water

Juvenile rainbow trout were exposed to unlabelled benzo[a]pyrene BaP and ³H benzo a pyrene (³H BaP), in a static exposure system for 2 days. The initial concentration was 30 μg l^-1 and 0.625 μCi l^-1, corresponding to 6 mg kg^-1 body weight and 125 μCi kg^-1 body weight. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity was measured during the exposure and depuration periods, elucidating the time course pattern of CYP1A induction. Maximum induction (11-fold) of EROD activity was observed on day 2 after addition of BaP to the water. Tissue distribution of ³H-BaP was studied by liquid scintillation counting and whole body autoradiography. The concentration of ³H-BaP-derived radioactivity was highest in the bile at all sampling times. High levels of radiolabelled compound were also present in the gills, liver and the olfactory organ. There was an overall decrease in all tissues during the depuration period. The elimination of ³H-BaP-derived radioactivity from the gills, however, was slow compared with liver and blood (6.2 days vs 2.7 and 2.9 days, respectively).     

Phytochemical studies on tobacco alkaloids XIV. The occurrence and properties of putrescine N-methyltransferase in tobacco roots

Putrescine N-methyltransferase, a new enzyme catalyzing theformation of N-methylputrescine from putrescine and S-adenosyl-L-methioninewas found in roots of tobacco plants. The enzyme was purified30-fold from crude extracts of tobacco roots. NMethylputrescinewas identified as the reaction product by comparison with theauthentic compound. The enzyme had a pH optimum between pH 8and 9, and a molecular weight of about 60,000, as determinedby gel filtration. Km values for putrescine and 5-adenosyl-L-methioninewere 4.0 x 10^–4 M and 1.1 x 10^–4 M, respectively.Enzyme activity was inhibited by N-chloromercuribenzoate andAg⁺. No cofactors were required. Of the various substrates tested,only putrescine served as a methyl acceptor. The enzyme waslocalized exclusively in the roots and its activity was greadyenhanced by decapitation. The presence of putrescine N-methyltransferase in tobacco rootsstrongly suggests that N-methylputrescine participates as anintermediate in nicotine biosynthesis. (Received March 2, 1971; )     

The Potential of Two Perennial C4 Grasses and a Perennial C4 Sedge as Ligno-cellulosic Fuel Crops in N.W. Europe. Crop Establishment and Yields in E. England

Three perennial C₄ rhizomatous species, Cyperus longus L., Spartinacynosuroides and Spartina pectinata Link, were examined as potentialrenewable energy crops. These species are unusual among C₄ plantsin showing natural distributions which extend into cool temperateregions. This study examined whether these species could beestablished in the cool temperate climate of eastern Englandand whether they could consistently attain the relatively highdry matter yields associated with C₄ plants of warmer regions.Clonally produced material was planted in 1986, on two siteswith contrasting soil types in Essex, eastern England. Plantingwas within a randomized-block design incorporating replicatedplots of each species, both with and without fertilizer. Survivorshipand stem demography were monitored at monthly intervals from1986 to Jun. 1989 for stem recruitment and to Dec. 1991 forstem density. Yields were determined from 1987 (the year followingestablishment) to 1993. Survivorship of the planted propagules over the first 12 monthswas 92% for S. pectinata , 96% for S. cynosuroides and 100%for C. longus. Recruitment of new stems peaked in Apr. of mostyears, although a significant number of new stems appeared asearly as Feb. Stem death peaked in Sep. or early Oct. and allabove-ground stems had died by mid-Nov. Stem density trendsindicated that 2-4 years were required to reach a steady-statedensity, depending upon species. The stem density of the twoSpartina species had reached more than 1000 m^-2 in 1989 althoughthat of S. pectinata fluctuated considerably in the subsequentyears. C. longus stem density rose to approx. 600 m^-2 by 1988and did not change significantly in the subsequent years. In the 6 years following establishment, annual yields averagedacross all fertilizer treatments and both sites were 1·0,1·1 and 1·3 kg m^-2 for C. longus, S. cynosuroidesand S. pectinata, respectively. The average annual yield ofall three species at the site with the heavier soil was 1·3kg m^-2. This was significantly greater than the 1·0 kgm^-2 on the lighter soil. Nitrogen addition did not significantlyincrease yield. Even in the absence of any nitrogen addition,the annual yield of S. pectinata averaged 1·2 kg m^-2over the 6 years, with no evidence of any decline with the increasingage of the stands.Copyright 1995, 1999 Academic Press Cyperus longus, Spartina cynosuroides, Spartina pectinata, energy crop, dry matter yield     

Effect of 10-aza-substitution on benzo[a]pyrene mutagenicity in vivo and in vitro

Benzo[a]pyrene (BaP), an environmental carcinogen, shows genotoxicity after metabolic transformation into the bay-region diol epoxide, BaP-7,8-diol 9,10-epoxide. 10-Azabenzo[a]pyrene (10-azaBaP), in which a ring nitrogen is located in the bay-region, is also a carcinogen and shows mutagenicity in the Ames test in the presence of the rat liver microsomal enzymes. In order to evaluate the effect of aza-substitution on in vivo genotoxicity, BaP and 10-azaBaP were assayed for their in vivo mutagenicity using the lacZ-transgenic mouse (Muta™Mouse). BaP was potently mutagenic in all of the organs examined (liver, lung, kidney, spleen, forestomach, stomach, colon, and bone marrow), as described in our previous report, whereas, 10-azaBaP was slightly mutagenic only in the liver and colon. The in vitro mutagenicities of BaP and 10-azaBaP were evaluated by the Ames test using liver homogenates prepared from several sources, i.e. CYP1A-inducer-treated rats, CYP1A-inducer-treated and non-treated mice, and humans. BaP showed greater mutagenicities than 10-azaBaP in the presence of a liver homogenate prepared from CYP1A-inducer-treated rodents. However, 10-azaBaP showed mutagenicities similar to or more potent than BaP in the presence of a liver homogenate or S9 from non-treated mice and humans. These results indicate that 10-aza-substitution markedly modifies the nature of mutagenicity of benzo[a]pyrene in both in vivo and in vitro mutagenesis assays.