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Infrared spectroscopy coupled with partial least squares (PLS) regression has been shown to be a rapid alternative to wet chemical analytical methods for determining the cell-wall compositions of wood. Both near infrared (NIR) spectroscopy, and mid-infrared spectroscopy with attenuated total reflectance Fourier transform infrared (ATR FTIR) sampling, coupled with PLS regression, can be used to quickly and accurately predict the lignin contents and monosaccharide compositions of milled wood. However, milling wood can be time consuming and laborious. In this study we demonstrate that PLS-1 models built using NIR and ATR FTIR spectra of milled Pinus radiata wood, with different sized wood particles and different moisture contents, can rapidly and accurately predict the cell-wall compositions of solid wood. A robust assessment of the prediction accuracy was conducted using a separate test set of solid wood samples with both 'smooth' and 'rough' surface finishes. The lowest standard error (SE) values for most of the compositional predictions were obtained for the 'rough' solid wood samples, using PLS-1 models built from NIR spectra of 'large' milled wood particles (0.422 mm) with ambient moisture content. The SE achieved for NIR spectroscopy prediction of lignin for the 'rough' solid wood was 1.91%, and for the monosaccharides, arabinose (0.37%), xylose (1.25%), galactose (2.00%), mannose (1.54%), and 4-O-methyl glucuronic acid (0.24%). The powerful combination of NIR spectroscopy with PLS regression offers an attractive method for rapid prediction of cell-wall compositions of solid wood samples, thus avoiding milling. In addition, this technique highlights the different levels of these cell-wall components in opposite and compressed regions in solid wood. [ABSTRACT FROM AUTHOR]

Variable-pressure scanning electron microscopy was used to investigate the dimensional changes in longitudinal, tangential and radial directions, on wetting and drying, of tracheids of opposite wood (OW) and three grades of compression woods (CWs), including severe CW (SCW) and two grades of mild compression wood (MCW) (MCW1 and MCW2) in corewood of radiata pine (Pinus radiata) saplings. The CW was formed on the underside and OW on the upper side of slightly tilted stems. In the longitudinal direction, the shrinkage of SCW tracheids was ~300% greater than that of OW tracheids, with the shrinkage of the MCW1 and MCW2 tracheids being intermediate. Longitudinal swelling was also investigated and hysteresis was demonstrated for the tracheids of all corewood types, with the extent of hysteresis increasing with CW severity. A statistical association was found between longitudinal shrinkage and the content of lignin and galactosyl residues in the cell-wall matrix. The galactosyl residues are present mostly as (1→4)-β-galactans, which are known to have a high capacity for binding water and swell on hydration. The small proportions of (1→3)-β-glucans in the CWs have similar properties. These polysaccharides may play a functional role in the longitudinal shrinking and swelling of CW tracheids. Tangential shrinkage of tracheids was greater than radial shrinkage but both were greatest for OW and least for SCW, with the MCW1 and MCW2 being intermediate. [ABSTRACT FROM AUTHOR]

Background: Compression wood (CW) forms on the underside of tilted stems of coniferous gymnosperms and opposite wood (OW) on the upperside. The tracheid walls of these wood types differ structurally and chemically. Although much is known about the most severe form of CW, severe CW (SCW), mild CWs (MCWs), also occur, but less is known about them. In this study, tracheid wall structures and compositions of two grades of MCWs (1 and 2) and SCW were investigated and compared with OW in slightly tilted radiata pine (Pinus radiata) stems. Results: The four wood types were identified by the distribution of lignin in their tracheid walls. Only the tracheid walls of OW and MCW1 had a S3 layer and this was thin in MCW1. The tracheid walls of only SCW had a S2 layer with helical cavities in the inner region (S2i). Using immunomicroscopy, (1→4)-β-D-galactans and (1→3)-β-D-glucans were detected in the tracheid walls of all CWs, but in only trace amounts in OW. The (1→4)-β-D-galactans were located in the outer region of the S2 layer, whereas the (1→3)-β-D-glucans were in the inner S2i region. The areas and intensities of labelling increased with CW severity. The antibody for (1→4)-β-D-galactans was also used to identify the locations and relative amounts of these galactans in whole stem cross sections based on the formation of an insoluble dye. Areas containing the four wood types were clearly differentiated depending on colour intensity. The neutral monosaccharide compositions of the non-cellulosic polysaccharides of these wood types were determined on small, well defined discs, and showed the proportion of galactose was higher for CWs and increased with severity. Conclusion: The presence of an S3 wall layer is a marker for very MCW and the presence of helical cavities in the S2 wall layer for SCW. The occurrence and proportions of (1→4)-β-D-galactans and (1→3)-β-D-glucans can be used as markers for CW and its severity. The proportions of galactose were consistent with the labelling results for (1→4)-β-D-galactans. [ABSTRACT FROM AUTHOR]

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The potential of pyrolysis followed by gas-chromatography and mass-spectrometry (Py-GC/MS) was investigated for identifying compression wood (CW) in saplings of radiata pine ( Pinus radiata) by examining samples of CW and opposite wood (OW). Phenolic compounds and anhydrosugars were identified among the pyrolysis products that provided information about the cell-wall polymers. Sample preparation, such as coarse-milling, fine-milling, and fine-milling followed by calcium-chloride treatment was also investigated. Fine-milling typically decreased the total yield of phenolic compounds compared with coarse-milling. Fine-milling followed by calcium-chloride washing significantly increased the proportions of pyrolysis products from polysaccharides, specifically from (1→4)-β-D-galactans that were of interest in distinguishing CW from OW. Six pyrolysis products were identified that were unique to the CW samples examined, including derivatives of (1→4)-β-D-galactans and H-units of lignin. Other pyrolysis products were identified that had significantly different proportions between the two wood types, and sometimes among samples of the same wood type. [ABSTRACT FROM AUTHOR]

Using a functional genomics approach, four candidate genes ( PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda. These genes encode CAZy family GT34 glycosyltransferases that are involved in the synthesis of cell-wall xyloglucans and heteromannans. The full-length coding sequences of three orthologs ( PrGT34A, B and C) were isolated from a xylem-specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1→6)-α-xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a galactosyltransferase from fenugreek ( Trigonella foenum-graecum) that is involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell-culture system. The heterologous proteins were purified, and in vitro assays showed that, when incubated with UDP-xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and, when incubated with UDP-galactose and the same cello-oligosaccharides, PrGT34B showed some galactosyltransferase activity. The ratio of xylosyltransferase to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products using galactosidases showed the linkages formed were α-linkages. Analysis of the products of PrGT34B by MALDI-TOF MS showed that up to three xylosyl residues were transferred from UDP-xylose to cellohexaose. The heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity. [ABSTRACT FROM AUTHOR]

Polysaccharides were located in the walls of normal and compression wood tracheids of Pinus radiata (radiata pine), Picea sitchensis (Sitka spruce) and Picea abies (Norway spruce) by transmission electron microscopy using immunogold labelling with monoclonal antibodies to (1→4)-β-galactan (LM5), (1→3)-β-glucan, arabinogalactan proteins (AGPs) (MAC207) and heteroxylans (LM10 and LM11). In fully differentiated compression wood tracheids, (1→4)-β-galactan was found in the S2(L) layer and, to a smaller extent, at the interface between the compound middle lamella and the S1 layer. (1→4)-β-Galactan appeared to be displaced from, or modified in, the S1 layer during cell wall formation. (1→3)-β-Glucan (callose) was confined to the helical cavities in the inner S2 layer of severe compression wood. MAC207 AGP glycan epitope was found exclusively in the S1 and S3 layers of normal wood tracheids and in the S1 and inner S2 layers of compression wood tracheids. Binding of LM10, which specifically recognizes unsubstituted or low-substituted xylans, occurred at similar locations to the MAC207 epitope, whereas binding of LM11, which recognizes more highly substituted as well as unsubstituted xylans, occurred throughout the tracheid walls with the exception of the primary wall. Immunogold labelling showed that the different wall layers of softwood tracheids have different polysaccharide compositions which change abruptly during cell wall formation. [ABSTRACT FROM PUBLISHER]

Abstract: One full length (PrCESA10) and seven other cDNA clones (PrCESA2, 3, 5–8, 11) encoding cellulose synthases (CESAs) were isolated from the coniferous gymnosperm Pinus radiata. PrCESA10 encodes a protein predicted to contain the same domains and regions as angiosperm CESA genes: a zinc finger domain, a hypervariable region 1 (HVR1), a plant-conserved region (CR-P), a class-specific region or hypervariable region 2 (HVR2), in addition to the four conserved domains U1–U4 that are characteristic of the family 2 processive β-glycosyltransferases. The P. radiata protein is also predicted to contain eight transmembrane domains. The zinc finger domain, the CR-P and the C-terminal portion of the proteins, are highly conserved between P. radiata and the nearest angiosperm CESA protein from Solanum tuberosum. Reverse transcriptase-PCR showed that all the P. radiata genes were expressed in all organs tested, although to different extents. In situ hybridization studies with PrCESA10 in stems of 2- and 12-month-old seedlings showed that it was expressed in the secondary xylem in the two-to-three most recently developed tracheids, which were laying down secondary cell walls. [Copyright &y& Elsevier]

Tracheid and sclereid differentiation was induced in callus cultures of Pinus radiata D. Don by culturing on a basal medium containing activated charcoal but no phytohormones; sclereids differentiated in callus derived from xylem strips, but not in callus derived from hypocotyl segments. The tracheids differentiated in hypocotyl-derived callus had helical, scalariform, reticulated or pitted secondary cell-wall patterns, but those differentiated in xylem-derived callus had a reticulate or pitted pattern. The thickened tracheid and sclereid walls contained lignin as indicated by the red colour reaction given with phloroglucinol-HCI. The presence of lignin in the cell walls of differentiated callus was confirmed using pyrolysis gas chromatography- mass spectrometry by the detection of phenylpropanoid components derived from lignin. Lignin was also detected using solid-state 13C cross-polarisation/magic-angle spinning nuclear magnetic resonance spectroscopy and quantified as thioglycolic acid lignin. Monosaccharide analyses of the cell walls isolated from differentiated and undifferentiated calli showed that the cell walls of the differentiated calli contained higher proportions of glucose and mannose, consistent with the presence of greater proportions of gluco- and/or galactoglucomannans in the secondary cell walls of the differentiated cells. A protocol for the stable transformation of undifferentiated, xylem-derived cultures was successfully developed. Transgenic cell lines were established following Biolistic particle bombardment with a plasmid containing the coding region of the nptII gene and the coding region of the cad gene from P. radiata. Expression of the nptII gene in transgenic lines was confirmed by an NPTII-enzyme-linked immunosorbent assay. The overexpression of cad in the transgenic lines resulted in a down-regulation of cinnamyl alcohol dehydrogenase (EC 1.1.1.195) expression. [ABSTRACT FROM AUTHOR]

Tilted stems of softwoods form compression wood (CW) and opposite wood (OW) on their lower and upper sides, respectively. More is known about the most severe form of CW, severe CW (SCW), but mild CWs (MCWs) also occur widely. Two grades of MCWs, MCW1 and MCW2, as well as SCW and OW were identified in the stems of radiata pine ( Pinus radiata ) that had been slightly tilted. The four wood types were identified by the distribution of lignin in the tracheid walls determined by fluorescence microscopy. A solution of the fluorescent dye acridine orange (AO) (0.02% at pH 6 or 7) was shown to metachromatically stain the tracheid walls and can also be used to determine lignin distribution. The lignified walls fluoresced orange to yellow depending on the lignin concentration. Microscopically well-characterized discs (0.5 mm diameter) of the wood types were used to determine lignin concentrations and lignin monomer compositions using the acetyl bromide method and thioacidolysis, respectively. Lignin concentration and the proportion of p- hydroxyphenyl units (H-units) relative to guaiacyl (G-units) increased with CW severity, with <1% H-units in OW and up to 14% in SCW. Lignin H-units can be used as a marker for CW and CW severity. Similar discs were also examined by Raman and FTIR micro-spectroscopies coupled with principal component analysis (PCA) to determine if these techniques can be used to differentiate the four different wood types. Both techniques were able to do this, particularly Raman micro-spectroscopy. [ABSTRACT FROM AUTHOR]