Geoffrey Daniel

Microbial decay of waterlogged archaeological wood found in Sweden : Applicable to archaeology and conservation

Abstract Waterlogged archaeological wood suffers from microbial degradation. Sections from 92 archaeological samples from seven marine and terrestrial sites in Sweden were investigated using light- and electron microscopy to determine the major forms of microbial decay. Decay was related to sample age, wood species and environmental factors. The results confirmed earlier observations that erosion bacteria were the main degraders of archaeological waterlogged soft- and hardwoods, whereas soft rot and tunnelling bacterial decay were less frequently observed. Characteristic slime material, derived from attack by erosion bacteria and left behind in the cell lumen, may affect the penetration of consolidants used to conserve waterlogged archaeological wood. Advanced decay by erosion bacteria had only a slight effect on the original colour, form and appearance of the wood. Different oxygen levels influence the type of microbes and the extent of decay. Examining the decay can reveal a great deal about the past history of a site and provide valuable information to archaeologists.

The influence of hemicellulose on fibril aggregation of kraft pulp fibres as revealed by FE-SEM and CP/MAS 13C-NMR

Three Norway spruce pulps were produced using different kraft pulping methods, in order to obtain large differences in cellulose and hemicellulose proportions at a similar lignin content. The hemicellulose content in the three pulps varied between 10% and 22%. The aim of the study was to evaluate the influence of cellulose and hemicellulose on fibre ultrastructure and correlate this with the differences observed in the mechanical properties between the pulps. The ultrastructure of the pulp fibres were studied using Field Emission Scanning Electron Microscopy (FE-SEM) and Solid-State Cross Polarisation Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance (CP/MAS 13C-NMR) in combination with spectral fitting. CP/MAS 13C-NMR measured the average bulk properties of the pulp fibres, while FE-SEM allowed for observations on the ultrastructure of fibre surfaces. The ultrastructure of the fibres varied with varying hemicellulose content. The pulp with a high hemicellulose content had a porous surface structure. In fibres with a low hemicellulose content, the fibril aggregates (macrofibrils) formed a much more compact surface structure. With CP/MAS 13C-NMR this change was reflected by an increase in average fibril aggregate width with decreasing hemicellulose content. Results from FE-SEM and CP/MAS 13C-NMR correlated well. The changes recorded in ultrastructure may explain the very different mechanical properties reported previously for pulps with different hemicellulose content.

Programmed cell death eliminates all but one embryo in a polyembryonic plant seed

Development of multiple embryos from a single zygote, the phenomenon called monozygotic polyembryony, is a widespread reproductive strategy found in higher plants and especially in gymnosperms. The enigma of plant monozygotic polyembryony is that only one embryo in a polyembryonic seed usually survives while the others are eliminated at an early stage. Here we report that programmed cell death (PCD) is the major mechanism responsible for elimination of subordinate embryos in a polyembryonic seed. Using post-fertilized pine (Pinus sylvestris) ovules, we show that once the dominant embryo is selected and, subsequently, the entire female gametophyte is affected by PCD, the cells of subordinate embryos initiate an autolytic self-destruction program. The progression of embryonic PCD follows a rigid basal-apical pattern, first killing the most basally situated cells, adjacent to the suspensor, and then proceeding towards the apical region until all cells in the embryonal mass are doomed. Our data demonstrate that during polyembryony, PCD serves to halt competition among monozygotic embryos in order to ensure survival of one embryo.

Characteristics of Gloeophyllum trabeum Alcohol Oxidase, an Extracellular Source of H2O2 in Brown Rot Decay of Wood

ABSTRACT A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (Km = 2.3 mM, kcat = 15.6 s−1). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the proteins different translocation. The extracellular distribution and the enzymes abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H2O2, a component of Fentons reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.

Defence related reactions of seedling roots of Norway spruce to infection by Heterobasidion annosum (Fr.) Bref.

Norway spruce seedlings were cultivated in sterile conditions. Roots were infected with a concentration series of germinating conidiospores of Heterobasidion annosu m (10 1 –10 6 ml −1 ). In other experiments, roots were treated with either mycelial preparations of H. annosum , other wood inhabiting fungi, with protein fractions of culture filtrates of H. annosum , or with chemical elicitors. Successive steps observed during infection were necrosis, formation of phenolics and increasing lignification of cortex and endodermis, colonization of meristem and finally of the stele. High spore concentrations caused necrosis and invasion within 48–72 h; these processes were delayed at low spore concentrations. Lyophilized culture filtrates of H. annosum caused a greater hypersensitive response than protein fractions but less than NaCl, Polygalacturonic acid or ethephon. Mycelial homogenate from nine other wood inhabiting fungi (saprophytes/parasites) induced a hypersensitive response to various extents but this was not correlated to their degree of cross-reactivity with a polyclonal antibody to H. annosum [enzyme-linked immunosorbent assay (ELISA)]. Peroxidase activity increased (two-threefold) in roots infected with H. annosum and one acidic isozyme was considered responsible for the increase in peroxidase. Using immunohistochemical and enzyme staining, peroxidase was found mainly in the cortical/endodermal regions of roots. Cytochemical labelling using anti-peroxidase and immunogold demonstrated increased peroxidase activity in cell walls, papillae and uninvaded middle lamellar cell corners of infected roots.

Autophagy and metacaspase determine the mode of cell death in plants.

Metacaspase-dependent autophagy in plants promotes cell disassembly during vacuolar cell death and inhibits necrosis.

Depth of burial, an important factor in controlling bacterial decay of waterlogged archaeological poles.

Abstract Wood discs from two waterlogged softwood poles were investigated for variations in bacterial decay. Exterior and interior degradation was observed using light, transmission and scanning electron microscopy. Maximum water content ( U max ) was measured and extent of decay from the surface towards the wood interior was investigated. Despite a well preserved appearance, the majority of the tracheids were heavily degraded. Erosion bacteria were identified as the main degraders and a significant difference in degree of decay was found between the upper and lower parts of each pole. Small differences in oxygen concentration, derived from depth of burial, are suggested as the controlling factor. Active erosion bacterial decay was found in the inner parts of the 1200-year old wooden poles.

Fibril angle variability in earlywood of Norway spruce using soft rot cavities and polarization confocal microscopy

The main purpose of this study was to investigate the variability of the fibril angle of tracheids in earlywood of Norway spruce (Picea abies L. Karst.). Polarization confocal microscopy was chosen and compared with the method utilizing the orientation of soft rot cavities. There was a significant correlation between the soft rot and polarization confocal microscopy methods, which showed the same trend of high fibril angles in the first part of the earlywood followed by a decrease toward the end of earlywood. This declining trend was less pronounced in annual rings containing compression wood. Moreover, large variations in fibril angle occurred between neighboring tracheids. The investigation also emphasized the differences between X-ray diffraction and microscopic methods, as the large variation seen by the latter methods is not seen by the X-ray diffraction approach because of its large area of measurement. No correlation was found between fiber morphology (i.e., average length, width, density) and the average fibril angle in the investigated annual rings.

Pyranose 2-dehydrogenase, a novel sugar oxidoreductase from the basidiomycete fungus Agaricus bisporus

Abstract A novel C-2-specific sugar oxidoreductase, tentatively designated as pyranose 2-dehydrogenase, was purified 68-fold to apparent homogeneity (16.4 U/mg protein) from the mycelia of Agaricus bisporus, which expressed maximum activity of the enzyme during idiophasic growth in liquid media. Using 1,4-benzoquinone as an electron acceptor, pyranose 2-dehydrogenase oxidized d-glucose to d-arabino-2-hexosulose (2-dehydroglucose, 2-ketoglucose), which was identified spectroscopically through its N,N-diphenylhydrazone. The enzyme is highly nonspecific. d-,l-Arabinose, d-ribose, d-xylose, d-galactose, and several oligosaccharides and glycopyranosides were all converted to the corresponding 2-aldoketoses (aldosuloses) as indicated by TLC. d-Glucono-1,5-lactone, d-arabino-2-hexosulose, and l-sorbose were also oxidized at significant rates. UV/VIS spectrum of the native enzyme (λmax 274, 362, and 465 nm) was consistent with a flavin prosthetic group. In contrast to oligomeric intracellular pyranose 2-oxidase (EC 1.1.3.10), pyranose 2-dehydrogenase is a monomeric glycoprotein (pI 4.2) incapable of reducing O2 to H2O2 (> 5 × 104-fold lower rate using a standard pyranose oxidase assay); pyranose 2-dehydrogenase is actively secreted into the extracellular fluid (up to 0.5 U/ml culture filtrate). The dehydrogenase has a native molecular mass of ∼79 kDa as determined by gel filtration; its subunit molecular mass is ∼75 kDa as estimated by SDS-PAGE. Two pH optima of the enzyme were found, one alkaline at pH 9 (phosphate buffer) and the other acidic at pH 4 (acetate buffer). Ag+, Hg2+, Cu2+, and CN– (10 mM) were inhibitory, while 50 mM acetate had an activating effect.

Use of a fluorescence labelled, carbohydrate-binding module from Phanerochaete chrysosporium Cel7D for studying wood cell wall ultrastructure

The α-amino group of the carbohydrate-binding module (CBM) from Phanerochaete chrysosporium cellulase Cel7D was covalently labelled with fluorescein isothiocyanate. The fluorescein-labelled CBM was characterised regarding substrate binding, showing specificity only to cellulose and not to mannan and xylan. Conjugation of fluorescein isothiocyanate to CBM did not affect its binding to cellulose. The labelled CBM was successfully used as a probe for detecting cellulose in lignocellulose material such as never dried spruce and birch wood as well as pulp fibres.