G. Daniel1*, T. Nilsson 1 and B. Pettersson 2 Introduction Materials
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IAWA Bulletin n.s., Vol. 12 (1),1991: 70-83 POORLY AND NON-LIGNIFIED REGIONS IN THE MIDDLE LAMELLA CELL CORNERS OF BIRCH (BETIJLA VERRUCOSA) AND OTHER WOOD SPECIES by G. Daniel1*, T. Nilsson 1 and B. Pettersson 2 1 Department of Forest Products, Swedish University of Agricultural Sciences, Box 7008, S-750 07, Uppsala, Sweden 2Swedish Pulp and Paper Research Institute, Box 5604, S-114 86 Stockholm, Sweden Summary Middle lamella cell corners in birch wood determine the distribution of lignin in wood frequently show a non-homogeneous struc- cell walls principally by either ultraviolet mi- ture and the existence of less dense (i. e elec- croscopy (Fergus & Goring 1970), or bromi- tron-Iucent) regions. Using a variety of cyto- nation (Saka et al. 1978, 1982) and mercuri- chemical, immunological and mercurisation sation (Westermark et al. 1986; Eriksson techniques in conjunction with electron mi- et.al. 1988) in conjunction with scanning or croscopy, the distribution of lignin within transmission electron microscopy. Birch spe- these regions was studied. Results showed cies in particular have received considerable the regions to have a variable lignin content attention and numerous reports have discus- consistent with intermediate and incomplete sed the variations in both lignin level and stages of lignification. Observations on cor- lignin type between the various cellular ele- ner regions partially degraded by decay fungi ments (see Saka & Goring 1985 for review). further showed the electron-Iucent regions to The major method to compare lignin levels possess an elevated level of non-lignified across wood cell walls is to use the propor- components (presumably carbohydrates) with tions of lignin found in the various regions a fibrillar type structure. Examination of a range normally with reference to that found in the of other wood species (including hard- and middle lamella cell corner region. The present softwoods) by TEM showed similar struc- work reports on the frequent and appatent tural variations in middle lamella cell corner overlooked presence of poorly, or non-ligni- homogeneity suggesting a common feature. fied regions within the middle lamella cell It is considered that this natural variation in wall corners themselves as determined using cell corner density and lignification may lead cytochemical, TEM X-ray microanalysis and to errors when lignin concentrations of cell immunological techniques. It is considered corners are used in ratio estimates of lignin in that the presence of these regions may have a secondary cell walilayers. significant effect on methods of lignin deter- Key words: Birch wood, decay fungi, lignin mination and lead to subsequent errors in cal- distribution, middle lamella cell corner, lig- culation. nin antisera, immunocytochemistry, lignin mercurisation, TEM X-ray microanalysis. Materials and Methods SampIes of Betula verrucosa (Ehr.) were Introduction taken from the Department of Forest Products The location of lignin across wood cell wood collection, with the present material walls is of particular interest since its removal originally derived from trees grown in central during pulp production is significantly affect- Sweden. Additional sampies were obtained ed by its distribution. A considerable effort from Dr. U. Westermark (STFI, Stockholm). has been made over the last two decades to All samples had been air dried previously. * To whom correspondence should be addressed. Downloaded from Brill.com10/05/2021 12:32:54PM via free access Daniel, Nilsson & Pettersson - Middle lamella cell corners of Betula 71 For all studies, small sticks (c. 3.0 x 3.0 x lignin antigen used for immunisation came 0.5 mm) were removed from larger blocks from ball milled spruce wood in which the using a scalpel. Observations on lignin distri- polysaccharides had been hydrolysed using bution were performed using cytochemistry, cellulolytic enzymes from Trichoderma reesei chemicallabelling (mercurisation) and by use QM9414 (Nummi et al. 1983, 1986). of lignin antisera in conjunction with either transmission electron microscopy (TEM) or TEM immunocytochemicallabe/ling 0/ lignin TEM X-ray microanalysis. In addition, ex- For post-immunogold labelling of lignin, amples are shown from ultrastructural studies small sticks of solvent extracted B. verrucosa on the degradation of birch by basidiomycete were processed for TEM as for cytochemical rot fungi where variations in middle lamella studies, except that sampies were embedded cell corner structure were enhanced. directly after ethanol dehydration in London resin (London Resin Company, Basingstoke, Lignin staining U. K.). Ultrathin sections for immunolabel- For cytochemical studies, small sticks of ling were pretreated with 10% H202 (5 mins) B. verrucosa were placed in freshly prepared and washed in phosphate buffered saline 1 % w/v KMn04, and left for 1 hour (hr) at (PBS). After incubation in 0.1 M glycine (35 room temperature (RT). After continuous mins), sections were washed in drops of dis- washing with distilled water, the sticks were tilled water (5 x 1 min) and then PBS (5 x 1 dehydrated using an ethanol (10% steps, 10 min) and finally incubated at 4°C overnight mins each) and then ethanol-acetone series in drops of spruce milled wood lignin (2: I, 2: 2, 1: 3; pure acetone, 15 mins each) (MWL) rabbit antisera (1 : 10 or 1 : 100 con- and finally infiltrated and flat embedded in taining PBS -1 % bovine serum alburnen Spurr's (1969) epoxy resin. After resin poly- (BSA) and 0.05% Tween 20). Following merisation at 70°C for 24 hrs, selected mate- primary antisera incubation, the sections were rial was sectioned using a Reichert FCD4 washed in PBS-1 % BSA-Tween 20 (pH 7.4; ultramicrotome with sections collected using 5 x 5 mins), TRIS-HCI-0.1%-BSA-Tween formvar coated copper grids. Further sampies 20 (pH 7.2, 3 x 5 mins), and subsequently were infiltrated and embedded directly in incubated with Goat anti-rabbit IgG conjugated Spurr's resin without pre-KMn04 staining or with gold (5 or 15 nm particles, Janssen, Life dehydration. After sectioning, these samples Science products, B-2340, Beerse, Belgium) were post-stained using 1% w Iv KMn04, in TRIS-HCI-1% BSA-Tween 20 (pH 8.4) for 10 mins. for 1 hr. Sampies were thereafter washed KMn04 is a weil known general stain for thoroughly in TRIS-HCI-O.l % BSA (pH lignin in wood. One of its main reactions is 8.4) and finally in distilled water (10 x 1 based on the reduction of permanganate to min). Post-staining when performed, was with manganese oxides by phenolic groups (Bland saturated 50% ethanolic uranyl acetate (10 et al. 1971). Regions possessing enhanced mins) or 1% aq KMn04. levels of phenolic groups such as lignin will Controls included use of lignin anti sera therefore react more strongly and produce previously cross absorbed with spruce milled greater contrast during TEM examination. wood lignin (i.e its antigen) and omission of Additional sticks were treated with 1% wIv the primary antibody step during labelling. aq osmium tetroxide (1 hr, RT), washed in dis- tilled water (3 x 15 mins) and then processed Chemicallabe/ling 0/ lignin (mercurisation) for TEM as outlined above without further and TEM X-ray microanalysis staining. Observations were made using a Birch sampies were initially extracted in Philips 201 TEM operated at 60 kV. benzene/akohol (3: 1) in a soxhlet apparatus over a 3-day period to remove any extrac- Production and preparation 0/ lignin antisera tive materials. Thereafter sampies were pro- Methods used for preparation of the lignin cessed according to Westermark et al. (1986). polyclonal antisera have been reported previ- In principle this involved refluxing the wood ously (Pettersson et al. 1988). The original sampies at 95°C in a methanol acetic acid, Downloaded from Brill.com10/05/2021 12:32:54PM via free access 72 IAWA Bulletin n.s., Vol. 12 (1),1991 mercuric acetate (11) mixture for 6 hrs, after mally results in a characteristic thinning of which sampies were boiled with methanol for cell walls of which the cell corners are the a further 8 hrs (2 changes) to remove unbound final regions to be degraded. In order to visu- mercury. Sampies were dehydrated in ethanol alise components within electron-lucent re- and then acetone and thereafter embedded in gions of cell corners, birch wood degraded Spurr' s resin and sectioned as outlined above. to high weight losses by the white rot Gold sections were collected on nickel grids fungus Phanerochaete chrysosporium P-127 and coated with a thin layer of carbon using (= ATCC 28326) (wild type) were studied. an Edwards 360A coating apparatus. For Small blocks of birch were placed in 125 ml- TEM X-ray rnicroanalysis, sections were Erlenmeyer flasks containing moist vermicu- analysed using a Philips 420T TEM using an lite, autoclaved and inoculated with a spore- accelerating voltage of 100 kV and a spot mycelium suspension of P. chrysosporium. diameter of c. 40 nm. X-ray microanalyses Flasks were incubated at 38°C and sampIes were performed in the STEM mode (speci- taken after 6 weeks when weight losses were men tilt 45°) using a LINK AN 10,000 series c. 70%. For further details see Daniel et al. energy dispersive X-ray analyser (EDXA) (1989). Small pieces from the degraded wood system over a live time of 60 secs with Hg sampies were processed for TEM as describ- recorded at the La (9.987 KeV) peak. Anal- ed above. yses were performed on regions of interest within rniddle lamella cell wall corners and Results for controls, background resin. General morphology 0/ birch cell corners Brown and white rot decayed birch sampies Figure 1 shows typical fibres of B. verru- Brown rot fungi are known to degrade cosa where both prominent electron-Iucent primarily the cellulose and hemicellulose (i.e. less den se) regions, and more structur- components of wood leaving a modified lig- ally homogeneous rniddle lamella cell corners nin skeleton (Kirk & Highley 1973).