Metabolic differences between gymnosperms and angiosperms in the formation of syringyl lignin

Sliced xylem tissue from shoots of both poplar and cherry reduces ferulic and sinapic acids to the corresponding aldehydes and alcohols, while tissue from gymnosperms such as Japanese red pine and ginkgo can reduce only ferulic acid. In young, less differentiated, xylem tissue and callus tissue of angiosperms the ability to reduce sinapic acid is markedly lower than that of the fully differentiated xylem.Both gymnosperm and angiosperm tissues reduced coniferyl and sinapyl aldehydes to the corresponding alcohols and, further, the peroxidases from both classes gave similar dehydrogenation polymers from a mixture of coniferyl and sinapyl alcohols. In agreement with these findings, sinapyl aldehyde and sinapyl alcohol, when fed to living plants and tissue cultures of gymnosperms, were shown to be readily converted to syringyl lignin which was not originally present.     

Effect of Pine Callus Elicitation by the Fusarium Strains of Various Pathogenicity on the Content of Phenolic Compounds

Pine (Pinus sylvestris L.) callus culture was treated with the mycelium extracts from six Fusarium strains. Previously, pine seedlings were infected with a spore suspension in order to test the pathogenicity of the used strains. Callus culture infection resulted in a decrease in the free proanthocyanidin (PA) and an increase in bound PA content. After treating the calli with all strains except F. oxysporum var. orthoceras, the lignin content became lower than the control one. The most considerable changes involved the p-hydroxybenzoic acid (HBA) content, and its greatest change was observed after treating the calli with F. nivale, when the HBA concentration (1229 g/g dry wt) exceeded fourfold the control one. There was a positive correlation (R = 0.81) between the HBA content in the callus culture cells treated with a fungal extract and the virulence of Fusarium strains. At the same time, there was an inverse correlation (R = –0.80) between the lignin content in a callus culture and the fungal virulence; the latter did not affect the contents of both free and bound PA.     

Embryogenic callus culture of Tribulus terrestris L. a potential source of harmaline, harmine and diosgenin

In the present study, a simple one medium formulation protocol for callus culture, somatic embryogenesis and in vitro production of β-carboline alkaloids and diosgenin in Tribulus terrestris L. was developed. Extensive callus induction and proliferation was obtained in leaf explant on Murashige and Skoog (MS) medium supplemented with 5.0 μM 6 benzyl adenine (BA) and 2.5 μM α-naphthaleneacetic acid (NAA). The embryogenic callus was maintained on subculture to fresh parental medium at 4-week intervals over a period of 28 months. The frequency of embryo formation was at a maximum (18.1 ± 0.9 per g of callus) on MS medium containing 5.0 μM BA and 2.5 μM NAA together with 75 mg l⁻¹ casein hydrolysate. Globular embryo developed into torpedo stage embryo under the influence of starvation. The accumulation of β-carboline alkaloids (harmaline and harmine) and steroidal saponin (diosgenin) in non-embryogenic and embryogenic callus culture derived from leaf explant was compared with root, leaf, stem, and fruit of the mother plant. The embryogenic callus accumulated equivalent amounts of harmaline (66.4 ± 0.5 μg/g dry weight), harmine (82.7 ± 0.6 μg/g dry weight), and diosgenin (170.7 ± 1.0 μg/g dry weight) to that of the fruit of T. terrestris. The embryogenic callus culture of this species might offer a potential source for production of important pharmaceuticals.     

Comparison of fermentation reactions in different regions of the human colon

Colonic contents were obtained from two human sudden-death victims within 3 h of death. One of the subjects (1) was methanogenic, the other (2) was a non-CH, producer. Measurements of bacterial fermentation products showed that in both individuals short-chain fatty acids, lactate and ethanol concentrations were highest in the caecum and ascending colon. In contrast, products of protein fermentation, such as ammonia, branched chain fatty acids and phenolic compounds, progressively increased from the right to the left colon, as did the pH of gut contents. In Subject 1, cell population densities of methanogenic bacteria (MB) increased distally through the gut and methanogenic activity was lower in the right (0.78–1–18 μmol CH₄ produced/h/g dry wt contents) than in the left colon (1.34 μmol CH₄ produced/h/g dry wt contents). Methane production rates did not correlate with MB numbers.
Sulphate-reducing bacteria (SRB) were not found and dissimilatory sulphate reduction was not detected in any region of the colon. Methanogenic bacteria did not occur in subject 2, but high numbers of SRB were present throughout the gut ( ca 10⁹/g dry wt contents). Sulphate reduction rates were maximal in the ascending and transverse colons (0.24 and 0.22 μmol ³⁵SO^2–₄ reduced/h/g dry wt contents, respectively). Short-chain fatty acid production by caecal contents was up to eight-fold higher than contents from the sigmoid/rectum. These findings demonstrate significant differences in fermentation reactions in different regions of the large gut.     

Transformation of opium poppy (Papaver somniferum L.) with Agrobacterium rhizogenes MAFF 03-01724

Summary Transformed cultures of opium poppy (Papaver somniferum L.) were established by infecting hypocotyl segments with Agrobacterium rhizogenes MAFF 03-01724. Undifferentiated calli formed on the infected site grew satisfactorily on phytohormone-free solid medium in the dark and produced opine, mikimopine, which could not be detected in a normal culture. Numerous adventitious shoots developed from transformed calli during subculture. The transformed shoots separated individually were cultured on phytohormone-free MS solid medium at 22 ° C under 14 h/day light. They displayed wider leaves and longer internodes than shoots established from seeds or non-transformed root culture. The content of morphinan alkaloids in the cultures and regenerated shoots were quantitatively analyzed by enzyme-linked immunosorbent assay and high performance liquid chromatography. HPLC analysis revealed that non-transformed shoots contained much more codeine (1310 gmg/g dry wt.) than morphine (50 g/g dry wt.), while the transformed shoot cultures did not contain morphine, although the level of morphinan alkaloids in the transformed shoots (213 g morphine equivalents/g fr. wt.) was comparable to that in non-transformed shoots (182 g morphine equivalents/g fr. wt.) by ELISA.Abbreviations MS Murashige-Skoog (Murashige and Skoog 1962) - 1/2 MS half strength MS - HF phytohormone-free - NAA 1-naphthaleneacetic acid - ELISA enzyme-linked immunosorbent assay - HPLC high performance liquid chromatography     

Lignin dehydrogenative polymerization mechanism: a poplar cell wall peroxidase directly oxidizes polymer lignin and produces in vitro dehydrogenative polymer rich in beta-O-4 linkage

An investigation was performed to determine whether lignin dehydrogenative polymerization proceeds via radical mediation or direct oxidation by peroxidases. It was found that coniferyl alcohol radical transferred quickly to sinapyl alcohol. The transfer to syringaresinol was slower, however, the transfer to polymeric lignols occurred very slightly. This result suggests that the radical mediator theory does not sufficiently explain the mechanism for dehydrogenative polymerization of lignin. A cationic cell wall peroxidase (CWPO-C) from poplar (Populus alba L.) callus showed a strong substrate preference for sinapyl alcohol and the sinapyl alcohol dimer, syringaresinol. Moreover, CWPO-C was capable of oxidizing high-molecular-weight sinapyl alcohol polymers and ferrocytochrome c. Therefore, the CWPO-C characteristics are important to produce polymer lignin. The results suggest that CWPO-C may be a peroxidase isoenzyme responsible for the lignification of plant cell walls.     

Bio-oxidation of aliphatic and aromatic high molecular weight alcohols by Pichia pastoris alcohol oxidase

Summary The alcohol-oxidase-mediated oxidation of hexanol to hexanal was conducted by whole cells of Pichia pastoris in a biphasic reaction medium consisting of 3% water and 97% (v/v) water-saturated hexane. At substrate levels of ca. 10 g/l, hexanal was produced at a rate of 0.2 g/g cell dry wt. per hour with product yields and carbon recoveries of 96% or greater. Although the substrate range of P. pastoris alcohol oxidase has been documented as C₁–C₅ aliphatic alcohols and benzyl alcohol, the use of a biphasic organic reaction medium showed that this enzyme can also oxidize higher molecular weight aliphatic alcohols of C₆–C₁₁, as well as the aromatic alcohols phenethyl alcohol and 3-phenyl-1-propanol. The ability of alcohol oxidase to oxidize low-water-soluble alcohols greatly extends the utility of this enzyme.Issued as NRCC no. 30955 Offprint requests to: W. D. Murray     

HALOGENATED MONOTERPENE PRODUCTION IN REGENERATED PLANTLET CULTURES OF OCHTODES SECUNDIRAMEA (RHODOPHYTA, CRYPTONEMIALES)

Three genera of macrophytic red algae ( Ochtodes , Plocamium , and Portieria ) contain novel halogenated monoterpenes. To develop an in vitro system for studying halogenated monoterpene production, a laboratory tissue culture was established for Ochtodes secundiramea (Montagne) Howe (Cryptonemiales, Rhizophyllidaceae). Specifically, callus cells were induced from thallus explants of O. secundiramea plants. Shoot primordia regenerated from callus cells and developed into plantlets that released tetraspores. A sporeling from one of these tetraspores was selected for further culture. Axenic plantlets were cultivated in ESS-enriched natural seawater. Thallus tissue was cut into small pieces before subculture. Each plantlet grew as a symmetrical array of highly branched shoot tissues emanating from a common center, ultimately assuming a spherical shape of 20 mm diameter 4 weeks after subculture. Specific growth rates of over 20% per day were attained in bubble-aerated flask culture at an optimal temperature of 26° C and photosynthetic saturation light intensity of 200 μmol photons·m ^{− 2}·s ^{− 1}. The cultured plantlets contained myrcene and seven halogenated monoterpenes, based on gas chromatography–mass spectroscopy analysis of dichloromethane extracts. Although bromomyrcene was the dominant acyclic halogenated monoterpene, the cyclic halogenated monoterpenes chondrocole C and ochtodene were also produced by the O. secundiramea plantlet cultures. Halogenated monoterpene production at light-saturated growth conditions increased with decreasing nitrogen availability below 1.0 mM medium nitrate concentration (N:P ratios of 1.6:1 to 32:1). The halogenated monoterpene yield was insensitive to medium nitrate concentrations above 1.0 mM (N:P ratios of 32:1 to 320:1), where the bromomyrcene yield was 1700 μg per gram of dry cell mass.     

Comparison of photomixotrophic and heterotrophic callus and suspension cultures of Pinus elliottii. 1. Photosynthetic properties and ultrastructural evidence for coexistence of starch granules and secondary metabolites

Photomixotrophic callus and suspension cultures of salsh pine (Pinus elliottii var. elliottii Engelm.) have been established. Callus tissues contained up to 2.76 g chlorophyll mg^-1 dry wt and suspensions 2.98 g chlorophyll mg^-1 dry wt. Maximum photosynthetic oxygen evolution was 25–32 mol O₂ h^-1 mg^-1 chlorophyll for callus and 35–39 mol O₂h^-1 mg^-1 chlorophyll for suspension, respectively. Photomixotrophic callus was friable with a high moisture content during early and exponential growth, but evolved into a compact and dense tissue during the latter stage of growth. Compact photomixotrophic callus accumulated and deposited secondary metabolites in the central vacuole and developed large starch granules in the chloroplasts. Secondary metabolites were not observed in photomixotrophic suspensions or in heterotrophic calli and suspensions. Photomixotrophic callus contained numerous mitochondria closely associated with well-developed chloroplasts containing 2–6 thylakoids per granum. Heterotrophic callus was characterized by a poorly developed cytoplasm and cup-shaped mitochondria.     

Nickel uptake and its localization in a cyanobacterium

Abstract Nickel bioconcentration in different cell preparations of the cyanobacterium Nostoc muscorum was examined. A two- or three-fold increase in phosphate concentration over that prescribed in growth medium (58 μM), favoured nickel accumulation restricted to a threshold limit. Intact cells showed highest nickel bioconcentration (8.41 μmol mg⁻¹ dry wt) over spheroplasts (6.19 μmol mg⁻¹ dry wt) or polyphosphate bodies (5.88 μmol mg⁻¹ dry wt). Such preparations derived from similar cells indicate that the cyanobacterial cell wall could accommodate around 14–19% of the total nickel taken in by the cell with the overall nickel-bioconcentration sequence as: intact cells > spheroplasts > polyphosphate bodies > cell wall. The data suggest that polyphosphate bodies are the main sink for nickel.     

Oxidation of hydroxycinnamic acid and hydroxycinnamyl alcohol derivatives by laccase and peroxidase. Interactions among p-hydroxyphenyl,guaiacyl and syringyl groups during the oxidation reactions

Fungal laccase oxidized derivatives of hydroxycinnamic acid. The rates decreased in the order sinapic acid > ferulic acid ≥p-coumaric acid. The laccase oxidized sinapyl alcohol faster than coniferyl alcohol. The rates of oxidation of the hydroxycinnamic acid derivatives by an isoenzyme of peroxidase from horseradish decreased in the order p-coumaric acid > ferulic acid ≥ sinapic acid. The peroxidase oxidized coniferyl alcohol much faster than sinapyl alcohol. The laccase and the peroxidase predominantly oxidized (a) ferulic acid in a reaction mixture that contained p-coumaric acid and ferulic acid, (b) sinapic acid in a mixture of p-coumaric acid plus sinapic acid, and (c) sinapic acid in a mixture of ferulic acid plus sinapic acid. In a reaction mixture that contained both coniferyl and sinapyl alcohols, both fungal laccase and horseradish peroxidase predominantly oxidized sinapyl alcohol. From these results, it is concluded (1) that the p-hydroxyphenyl radical can oxidize guaiacyl and syringyl groups and produce their radicals and (2) that the guaiacyl radical can oxidize the syringyl group under formation of its radical; and that (3) in both cases the reverse reactions are very slow.     

Solasodine production in rapidly proliferating tissue cultures of Solanum laciniatum ait

Callus and suspension cultures, established from seedling and leaf explants of Solanum laciniatum Ait were analysed for solasodine using a spectrophotometric assay. Solasodine concentration in both types of culture ranged from 0.5 – 1.0 mg/g dry wt., with a small number of callus explants containing higher concentrations. There was no overall fall in concentration as a result of serial subculture, and in suspension cultures the level remained constant throughout a single passage. Solasodine concentration was enhanced by the induction of organogenesis in both primary leaf explants and callus. ABA, added at 0.04 mg 1^?1, increased solasodine yield in callus cultures whilst CEPA, at concentrations of 10 mg 1^?1 and higher, inhibited production. Dark grown callus contained significantly more solasodine than light grown.     

Induced spawning of grey mullet Mugil cephalus L. with fractionated salmon pituitary extract

The efficacy of fractionated salmon pituitary gonadotropin as a spawning agent in Mugil cephalus L. was tested. Natural spawning was induced in all females with a total dose of 11.9–20.9 μg/g body wt. Spawning dose varied inversely with initial mean egg diameters of recipient females. A 'critical' mean egg diameter of 650–700 μ was observed to precede the hormone dose that induced spawning. A 'priming' effect was observed following the initial injection and is discussed. The 'latency period' was determined to be 10–15 h; fecundity was estimated at 648 eggs/g body wt. Courtship, spawning and fertilization occurred naturally with uninjected males.     

Plant regeneration from seedling cotyledons, leaves and petioles of Glycine clandestina

A B5-based culture medium containing 4.4 μ M N⁶-benzyladenine (BA) and 0.025 μ M indole-3-butyric acid (IBA) induced callus from seedling cotyledons, leaves and petioles of Glycine clandestina Wendl. Only hard, green, nodular callus tissues were capable of producing shoot buds and of five accessions examined, only two (G1231 and G1145) were morphogenetically competent. Callus that did not regenerate could often be induced to produce shoot buds after subculture to fresh regeneration medium. Buds developed into shoots following transfer of callus to a medium containing 0.9 μ M BA and 0.025 μ M IBA. Shoots were rooted in hormone-free, half-strength B5 medium supplemented with 0.2% activated charcoal. The application of these results is discussed in relation to somatic hybridisation between the cultivated soybean and wild Glycine species.     

Oxidation of n-alkanes: Growth of Pseudomonas putida

Summary The induction of alkane hydroxylase activity was investigated in two strains of Pseudomonas putida with a view to the production of primary alcohols. n-Nonanol production rates (16.0 mol/g dry wt/h) with an alcohol dehydrogenase negative mutant P. putida PpS173 were considerably lower than might be expected from the growth of a wild type on n-alkane. Production of cells by fed-batch culture on n-nonane, with a specific alkane hydroxylase activity of 3.9 mmol/g/h, was considered most suitable for isolation of the alkane hydroxylase.     

Purification and properties of an alcohol dehydrogenase (HUADHII) from Hanseniaspora uvarum K5

An alcohol dehydrogenase HUADHII was purified 43.2-fold from Hanseniaspora uvarum K₅. The enzyme was trimeric with subunits of mol. wt 42 kDa. The N -terminal amino acid sequence of HUADHII has between 45 and 75% identity with part of the sequence of isoenzymes related to group I from Saccharomyces cerevisiae and Kluyveromyces lactis. C2–C4 alcohols and aldehydes were the preferred substrates. The presence of an'α'double bond increased the enzyme activity both for alcohols and aldehydes. It was significantly inhibited by metal-binding agents and thiol reagents. Kinetic studies suggested that HUADHII catalyses the oxidation of ethanol by a random sequential mechanism. It appears that HUADHII, a cytoplasmic fermentative enzyme, is structurally and functionally similar to members of the group I alcohol dehydrogenases.     

Effect of picloram and methyl jasmonate on growth and taxane accumulation in callus culture of Taxus × media var. Hatfieldii

We have analysed the effect of some culture conditions and media components on callus growth rate and production of taxanes in callus of Taxus × media var. Hatfieldii. For callus induction and maintenance a Gamborg B5 medium and a White - Rangaswamy medium (WR) with different modifications were used. On an improved WR medium (containing 10 μM picloram) the callus growth factor increased up to 5.8 fold (fresh weight). Picloram only enhanced the growth of callus, but not taxane production. On WR medium with (100 μM) methyl jasmonate the paclitaxel content increased from 2.37 μg g-1 to 90 μg g-1 and cephalomannine from 5.14 μg g-1 to 29.14 μg g-1 (dry weight), whereas growth of the cultures ceased. The presence of paclitaxel and cephalomannine was established by high performance liquid chromatography. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fungal and bacterial biomass in tundra soils along an arctic transect from Taimyr Peninsula, central Siberia

Microscopic analyses of tundra soils from northern central Siberia, Taimyr Peninsula (74.5°N, 98.5°E) were performed in order to investigate spatial variation of fungal and bacterial biomass. Biomass figures of fungi and bacteria (µg C g^-1 dry wt.) were measured from 11 permafrost soil pits. Fungal biovolume of up to 3.5 mm³ g^-1 dry wt. (median 0.19 mm³ g^-1 dry wt.) and a maximum hyphal length of 393 m g^-1 dry wt. (median 21 m g^-1 dry wt.) were determined. Fungal biomass was found up to 455 µg C g^-1 dry wt. (median 24 µg C g^-1 dry wt.). The amounts generally decreased with depth but increased within organic horizons. Little fungal biomass was found in the unvegetated soils or deep horizons above the permafrost table. Bacterial counts ranged from 0.16 to 7.38*10⁹ g^-1 dry wt. and bacterial biomass ranged from 0.68 to 20.38 µg C g^-1 dry wt. (median 6.19 µg C g^-1 dry wt.) because of small cell volume (median 0.04 µm³). Microbial biomass was generally dominated by fungi as shown by the ratio of fungal to bacterial biomass, which was between 0 and 174.1 (median 4.5). Plant cover and soil organic matter content were found to be the important keys in understanding microbial ecology in arctic tundra soils.     

Restriction pattern analysis of deoxyribonucleic acid isolated from callus and cell suspension of actinorhizal and non-actinorhizal Betulaceae

Using cell suspensions, a method was elaborated to isolate high-molecular-weight genomic deoxyribonucleic acid (DNA; 65 MDa or more) from members of the Betulaceae: Alnus incana (L.) Moench, Alnus glutinosa (L.) Gaertn. and Betula papyrifera Marsh. The method was also effective for isolation of DNA from callus cells. Based on the chemical lysis of protoplasts, this procedure yielded 130 μg (callus) to 250 μg (cell suspension) of DNA (g fresh cells)⁻¹, with a ratio A₂₀₀/A₂₈ of 1.7–2.0. The purified DNA obtained, formed distinct bands when restricted fragments were electrophoresed. Among the 10 endonucleases used for restriction analysis of Alnus glutinosa, Alnus incana and Betula papyrifera genomes, PvuI1 (EC 3.1.23.33) was unique in giving identical patterns for the two Ainus species. An unusual pattern occurred when Al-2 DNA was restricted with Ava II (EC 3.1.23.4). It formed a ladder with a repeating fragment unit of 181 base pairs long. With the enzymes tested, no differences in restriction patterns were observed among clones of Alnus incana (AI-2 vs AI-2), Betula papyrifera (BP-4 vs BP-8) and subclones of Ainus glutinosa AG-1 (PLFJ709 vs LF1709), suggesting genetic stability of the Betulaceae cultures.     

Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD.