The enzyme hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is involved in the production of methoxylated monolignols that are precursors to guaiacyl and syringyl lignin in angiosperm species. We identified and cloned a putative HCT gene from Pinus radiata, a coniferous gymnosperm that does not produce syringyl lignin. This gene was up-regulated during tracheary element (TE) formation in P. radiata cell cultures and showed 72.6% identity to the amino acid sequence of the Nicotiana tabacum HCT isolated earlier. RNAi-mediated silencing of the putative HCT gene had a strong impact on lignin content, monolignol composition, and interunit linkage distribution. AcBr assays revealed an up to 42% reduction in lignin content in TEs. Pyrolysis-GC/MS, thioacidolysis, and NMR detected substantial changes in lignin composition. Most notable was the rise of p-hydroxyphenyl units released by thioacidolysis, which increased from trace amounts in WT controls to up to 31% in transgenics. Two-dimensional 13 C-1 H correlative NMR confirmed the increase in p-hydroxyphenyl units in the transgenics and revealed structural differences, including an increase in resinols, a reduction in dibenzodioxocins, and the presence of glycerol end groups. The observed modifications in silenced transgenics validate the targeted gene as being associated with lignin biosynthesis in P. radiata and thus likely to encode HCT. This enzyme therefore represents the metabolic entry point leading to the biosynthesis of methoxylated phenylpropanoids in angiosperm species and coniferous gymnosperms such as P. radiata.lignin ͉ HCT ͉ tracheary elements T he global trend toward a biomaterials-based economy makes plant cell walls increasingly important as renewable resources for the production of biofuels and biocomposites. Lignin is the second most abundant terrestrial biopolymer after cellulose and a major structural component of cell walls in woodforming tissues (1). The content, composition, and structure of lignin all have considerable impact on the utilization of plantderived materials and have therefore been the subject of intensive research (1, 2). Lignins are heterogeneous cell wall polymers derived primarily from hydroxycinnamyl alcohols via combinatorial radical coupling reactions (3). Typically, they make up 20-30% of the cell wall material in woody tissue of both angiosperm and gymnosperm species. Lignin in coniferous gymnosperms such as Pinus radiata does not contain syringyl (S) components, which makes it different from lignin of many other vascular plants including angiosperms (4).We have developed a P. radiata callus culture system to better assign function to genes associated with cell wall-related processes such as lignification in conifers. These callus cultures can be transformed and subsequently induced to differentiate into tracheary elements (TEs), the main cellular components of wood in conifer species (5). The biochemical composition of cell wall polymers in differentiated TEs is similar to those produced in P. radiata wood...

A chemistry-based parameter has been sought for determining the gradation of compression wood (CW), i.e., the severity, in tissues of Pinus radiata wood. Fluorescence microscopy was the reference for characterisation of the tissues containing CW. The collected material contained CW of varying severity, beginning with normal wood (NW containing no CW), continuing with material with some features of CW (CW of mild severity, MCW) and ending up with a material with pronounced features of CW (CW of high severity, SCW). Matching opposite wood (OW) was also included in the study. The chemical analyses included lignin determination, sugar analysis in the acid hydrolysate, thioacidolysis, 31 P-NMR spectroscopic analysis and steric exclusion chromatography of thioacidolysis products. As the severity of CW changed progressively from NW through MCW to SCW, all chemical parameters changed concurrently. In particular, levels of galactose and lignin increased, while those of glucose and mannose decreased. The amounts of p-hydroxyphenyl b-ethers released by chemical degradation and uncondensed p-hydroxyphenyl C-9 units also increased at elevated CW severity levels. The amounts of galactose and the p-hydroxyphenyl content of the lignin correlated linearly with lignin for CW samples. The chemical differences between CW and OW in the stem, branch and seedling were similar, i.e., they are independent of the morphological origin of the sample. Parameters based on the p-hydroxyphenyl unit content appear the most suitable chemical indicators of CW severity, as they are least sensitive to the sample's morphological origin and their response to CW severity is high.

This paper describes a method for the two-dimensional mapping of chemical composition on the transverse face of cross-sectional discs from trees. The method uses an imaging spectrograph coupled to a near infrared (NIR) camera (900-1700 nm) to obtain NIR hyper spectral data sets which are processed using partial least squares regression to visualise the distribution and variation of lignin, galactose and glucose in Pinus radiata discs with R 2 /standard error of performance values of 0.84/1.48 (lignin), 0.87/0.68 (galactose) and 0.87/0.95 (glucose). The hardware design and software control are described along with a method for calibration based on one dimension spatially resolved predictions of chemical composition from conventional NIR spectroscopy. The NIR imaging system was designed as a rapid and cost-effective means of mapping chemical composition over the entire disc at a spatial resolution of ~4 mm 2 /pixel. The resulting maps of chemical composition clearly indicate, at high spatial resolution, the extent of heterogeneity that occurs in logs.

Enzymatic and topochemical aspects of lignification were studied in a Pinus radiata D. Don cell culture system that was induced to differentiate tracheary elements and sclereids with lignified secondary cell walls. The activities of the lignin-related enzymes phenylalanine ammonia lyase (PAL; EC 4.3.1.5) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) increased concomitantly with cell differentiation, indicating that the increase in enzyme activity was related to lignification of the cell walls and was not induced by stress. This result also indicates that PAL and CAD are suitable markers for tracheary element differentiation in coniferous gymnosperms. To further characterize lignification in this cell culture system, cellular UV-microspectrophotometry and thioacidolysis were employed. Typical UV-absorption spectra of lignin were obtained from the secondary cell walls of the tracheary elements and sclereids and from the compound middle lamella connecting differentiated cells, and the presence of lignin was confirmed by thioacidolysis. Certain aspects of lignin topochemistry in the cell walls of the tracheary elements were similar to cell walls of P. radiata wood, such as the high lignin concentration in the compound middle lamella connecting adjacent cells and the lower lignin concentration in the secondary cell walls. Therefore, the P. radiata cell culture system appears to be well suited to study the formation of lignified secondary cell walls in coniferous gymnosperms.

Mild hydrogenolysis has been compared with thioacidolysis as a method for degrading lignins in situ and in isolated form before analysis by gas chromato graphy/ mass spectrometry and quantitative 31 P nuclear magnetic resonance (NMR) spectroscopy. Both degradation methods gave similar levels of β-aryl ether-linked phenylpropane units that were released as monomers. Degradation by hydrogenolysis generally gave lower levels of total phenylpropane units when analyzed by 31 P NMR, especially in the case of lignins with high levels of condensed units. Overall, these results indicate that mild hydrogenolysis could offer an alternative to thioacidolysis for probing lignin structure.

A near infrared (NIR) imaging spectrograph was used to generate maps of chemical composition distribution on the surface of transverse wood discs taken from tree stems. The measured chemical components were lignin, galactose, glucose and mannose as well as cellulose and hemicelluloses, which were calculated from monomeric sugars. These components were determined using NIR-based chemistry models, which had been developed specifically for the imaging spectrograph. Explained test-set variation for key constituents ranged between 60% (galactan) and 78% (lignin). Day-today variability was 1-2% (standard deviation/range) depending on the chemical property. Various operational parameters such as room temperature, sample temperature, sample surface preparation and sample thickness were found to have a non-negligible, but manageable, influence on predicted results. The influence of room and sample temperatures could be reduced by incorporating temperature changes into the chemistry model. Extractives, transported to, and concentrated at, the disc surface during drying, needed to be physically removed from the surface to avoid an unpredictable influence on chemical results. Wood fibre angles at the disc surface needed to be aligned in a consistent manner to the camera. NIR information was found to derive from a sample depth of up to 10 mm. This distance was consequently chosen as the minimal sample thickness.

No abstract

The sound speed of wood is related to important wood quality properties such as the microfibril angle of the S2 layer in the cell wall, stiffness, and shrinkage propensity. Measuring the sound speed of seedling stems has benefits to the forestry industry, potentially enabling early selection of trees that yield better quality wood. A nondamaging longitudinal-wave time-of-flight (LWToF) acoustic technique was used to determine the sound speed of 10 cm long sections of 2-year-old Pinus radiata D. Don seedlings. The measured sections were harvested and acoustic resonance used to determine the sound speed of the sections before and after the bark was removed and after the remaining xylem was dried. A linear relationship between the acoustic resonance sound speed of the dry xylem and the LWToF sound speed of the seedling stem was found (R2 = 0.89). To demonstrate a potential application using the LWToF acoustic technique, it was used as a tool for investigating the effect of various applied stresses on wood properties of a clone of P. radiata. The LWToF sound speed measurements of phytohormone stressed stems were significantly lower than the control stems, indicating the negative impact on stiffness and shrinkage propensity imposed by this treatment.