Biochemical characterization of xylan xylosyltransferases involved in wood formation in poplar

The major polysaccharides in dicot wood biomass are cellulose and xylan. Although wood-associated cellulose synthase genes responsible for cellulose biosynthesis have been characterized, wood-associated xylan synthase genes have not been biochemically identified. A recent report by Lee et al. (2012) provides the first biochemical evidence that two functionally non-redundant Arabidopsis GT43 members are xylosyltransferases (XylTs) that function cooperatively in the elongation of the xylan backbone. We further extend this finding in the current report demonstrating that two poplar (Populus trichocarpa) GT43 glycosyltransferases, PtrGT43B and PtrGT43C, are xylan XylTs involved in wood formation. We show that microsomes from transgenic tobacco BY2 cells coexpressing PtrGT43B and PtrGT43C exhibited a high XylT activity capable of generating β-(1,4)-linked xylooligosaccharides, whereas little XylT activity was detected in microsomes with expression of PtrGT43B or PtrGT43C alone. These findings indicate that poplar GT43 members are XylTs that act cooperatively in catalyzing the successive transfer of xylosyl residues during xylan backbone biosynthesis, which provides further support of the hypothesis that the biochemical functions of GT43 members in vascular plants are evolutionarily conserved.     

Wood composition and energy content in a poplar short rotation plantation on fertilized agricultural land in a future CO2 atmosphere

To study the influence of elevated CO₂ and nitrogen (N) fertilization on wood properties and energy, Populus × euramericana trees were exposed to ambient CO₂ (about 370 μmol mol⁻¹ CO₂) or elevated CO₂ (about 550 μmol mol⁻¹ CO₂) using Free Air CO₂ Enrichment (FACE) technology in combination with two N levels. Elevated CO₂ was maintained for 5 years. After three growing seasons, the plantation was coppiced, one half of each experimental plot was fertilized and secondary sprouts were harvested after two growing seasons. Fourier transform infrared (FT-IR) spectra of wood revealed significant effects of both elevated CO₂ and N fertilization on wood chemistry, in particular, significant increases in lignin and decreases in N content. These results were corroborated by chemical analysis. Neither elevated CO₂ nor N fertilization affected the calorific value of wood, which was 19.3 MJ kg⁻¹. N fertilization enhanced the energy production per land area by 16–69% because of higher aboveground woody biomass production than on nonfertilized land. Estimates indicate that high yielding poplar short rotation cultivation may significantly contribute as an alternative feedstock for energy production.     

Comparative study of liquefaction process and liquefied products from bamboo using different organic solvents

The effects of various solvents, including phenol, ethylene glycol (EG) and ethylene carbonate (EC), and different liquid ratios on the liquefaction of bamboo, have been studied systematically in this paper. The processes were catalyzed by hydrochloride acid at 180 °C in autoclaves for different reaction times. The results show that phenol is the optimum solvent for bamboo liquefaction with a yield up to 99%. The Fourier transform-infrared (FT-IR) analyses of the residues show that cellulose, hemicelluloses and lignin are almost decomposed when using phenol as solvent. The gel permeation chromatography (GPC) results of the liquid products show that the high molecular weight of bamboo decreases significantly to around 1800 g mol⁻¹ after liquefaction. The gas chromatography and mass spectrometry (GC–MS) analysis shows that low boiling point products of liquefied bamboo are similar regardless of the type of solvent used.     

Nitrogen cycling in dense plantings of hybrid poplar and black alder

Summary Nitrogen cycling was studied during the third growing season in pure and mixed plantings (33×33 cm spacing) of hybrid poplar and black alder in southeastern Canada. After 3 years, hybrid poplar growth and N content of living tissues in a plot and of individual hybrid poplar plants increased with the proportion of black alder in a planting. No differences were detected among N contents of individual alder plants regardless of plot treatment. Black alder allocated a larger portion of its N to roots than hybrid poplar. Symbiotic nitrogen fixation was estimated to account for 80% of the nitrogen in aboveground alder tissues in the pure treatment using natural¹⁵N dilution. N return in leaf litter was estimated to be 70kg ha^–1 in the pure alder treatment and decreased to a minimum of 20 kg ha^–1 in the pure hybrid poplar plots. No difference was detected among treatments for throughfall N content. Nitrogen concentration in roots and leaf litterfall of black alder was higher than hybrid poplar. Significant soil N accretion occurred in mixed plantings containing two alders to one poplar and pure black alder plantings. Nitrogen availability (NO₃–N) increased with the amount of black alder in a plot. Results suggest that the early increase in nitrogen accumulation of hybrid poplar in mixed treatments can be attributed to an increase of total soil N availability resulting from the input of large amounts of N from easily mineralizable alder tissue.     

Cloning and expression analysis of a wood-associated xylosidase gene (PtaBXL1) in poplar tension wood

In stems of woody angiosperms responding to mechanical stress, imposed for instance by tilting the stem or formation of a branch, tension wood (TW) forms above the affected part, while anatomically distinct opposite wood (OW) forms below it. In poplar TW the S3 layer of the secondary walls is substituted by a “gelatinous layer” that is almost entirely composed of cellulose and has much lower hemicellulose contents than unstressed wood. However, changes in xylan contents (the predominant hemicelluloses), their interactions with other wall components and the mechanisms involved in TW formation have been little studied. Therefore, in the study reported here we determined the structure and distribution of xylans, cloned the genes encoding the xylan remodeling enzymes β-xylosidases (PtaBXLi), and examined their expression patterns during tension wood, normal wood and opposite wood xylogenesis in poplar. We confirm that poplar wood xylans are substituted solely by 4-O-methylglucuronic acid in both TW and OW. However, although glucuronoxylans are strongly represented in both primary and secondary layers of OW, no 4-O-methylGlcA xylan was found in G-layers of TW. Four full-length BXL cDNAs encoding putative β-xylosidases were cloned. One, PtaBXL1, for which xylosidase activity was confirmed by heterologous expression in Escherichia coli, exhibited a wood-specific expression pattern in TW. In conclusion, xylan as PtaBXL1, encoding β4-xylosidase activity, are down-regulated in TW.     

Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar

The polyphenoloxidase (PPO) from black poplar senescent leaves has been purified to almost complete homogeneity by a combination of ammonium sulphate precipitation, Sephadex G75 filtration and DEAE-cellulose chromatography. The purified enzyme has a MW of 60 000 and is probably a Cu⁺ enzyme. Peroxidase (PO) activity co-purifies with PPO and has the same MW as it. The two enzymes differ in pH optimum and in response to the effect of ionic strength. Natural phenols are either substrates, inhibitors or activators of black poplar PPO. This enzyme is an o-diphenoloxidase which binds substrates with K_m in the millimolar range. With caffeic and chlorogenic acids inhibition by excess substrate is observed. Benzoic acid phenols and cinnamic acid phenols are either competitive or non-competitive inhibitors of PPO. Hydroquinone is a highly potent non-competitive inhibitor of the enzyme (K_i  90 μM). Ferulic acid is a potent activator of the PPO-catalysed oxidation of catechol (K_a  0.34 mM, ν_sat/ν_o  7.7).     

Impact of warming and drought on carbon balance related to wood formation in black spruce

Background and Aims

Wood formation in trees represents a carbon sink that can be modified in the case of stress. The way carbon metabolism constrains growth during stress periods (high temperature and water deficit) is now under debate. In this study, the amounts of non-structural carbohydrates (NSCs) for xylogenesis in black spruce, Picea mariana, saplings were assessed under high temperature and drought in order to determine the role of sugar mobilization for osmotic purposes and its consequences for secondary growth.

Methods

Four-year-old saplings of black spruce in a greenhouse were subjected to different thermal conditions with respect to the outside air temperature (T0) in 2010 (2 and 5 °C higher than T0) and 2011 (6 °C warmer than T0 during the day or night) with a dry period of about 1 month in June of each year. Wood formation together with starch, NSCs and leaf parameters (water potential and photosynthesis) were monitored from May to September.

Key Results

With the exception of raffinose, the amounts of soluble sugars were not modified in the cambium even if gas exchange and photosynthesis were greatly reduced during drought. Raffinose increased more than pinitol under a pre-dawn water potential of less than –1 Mpa, presumably because this compound is better suited than polyol for replacing water and capturing free radicals, and its degradation into simple sugar is easier. Warming decreased the starch storage in the xylem as well the available hexose pool in the cambium and the xylem, probably because of an increase in respiration.

Conclusions

Radial stem growth was reduced during drought due to the mobilization of NSCs for osmotic purposes and due to the lack of cell turgor. Thus plant water status during wood formation can influence the NSCs available for growth in the cambium and xylem.     

杨树刺槐混交林及纯林枯落叶分解

研究了杨树（Ｐｏｐｕｌｕｓｓｐｐ．）、刺槐（Ｒｏｂｉｎｉａｐｓｅｕｄｏａｃａｃｉａ）纯林及混交林枯落叶一年中的分解及养分动态变化规律．结果表明,杨树枯落叶分解速度较慢,刺槐较快,而混交林则较杨树有较大提高．在分解过程中,杨树枯落叶Ｎ、Ｐ释放困难,需经过长达１０个月的养分富积过程;刺槐枯落叶Ｎ、Ｐ元素释放较为容易;混交林枯落叶养分富积时间缩短、幅度下降,分解速度加快,说明杨树和刺槐混交有利于枯落叶分解和Ｎ、Ｐ循环．     

Bioresistance of poplar wood compressed by combined hydro-thermo-mechanical wood modification (CHTM): Soft rot and brown-rot

This work studied fungal bioresistance of combined hydro-thermo-mechanically modified (CHTM) poplar wood. The CHTM technique, introduced by Mohebby et al. (2009), is a combination of two wood modification techniques-hydrothermal wood modification and densification of wood. Blocks of poplar wood were initially treated hydrothermally at temperatures of 120, 150, and 180 °C for holding times of 0, 30, and 90 min. Afterwards, the treated blocks were compressed by a hot press (160 and 180 °C) for 20 min with a compression set of 60%. After the CHTM-treated blocks were dried, small specimens were cut for soft-rot and brown-rot decay tests according to ENV 807 and EN 113. Mass losses as well as metabolic moisture contents were determined in the decayed samples. Results revealed that the combination of wood modification techniques showed fungal suppression. It was also found that the hydrothermal treatment step could significantly reduce fungal attack in comparison with densification. Reduction of the mass losses was associated with the hydrothermal treatment temperature. Also, the level of metabolic moisture content was correlated with the mass losses for both fungi. Any reduction of the mass loss decreased the moisture content in the wood.     

Structural and physico-chemical characterization of hemicelluloses from ultrasound-assisted extractions of partially delignified fast-growing poplar wood through organic solvent and alkaline solutions

One organic and three alkaline hemicellulosic fractions were isolated by an ultrasound-assisted extraction which partially delignified the fast-growing poplar wood. Successive treatments were conducted with dimethyl sulfoxide under ultrasonic irradiation at 570 W, 25 °C for 30 min, 70% ethanol containing 1% NaOH, 3% NaOH and 6% NaOH at 75 °C for 3 h, respectively. The four hemicellulosic fractions obtained were comparatively studied by sugar analysis, alkaline nitrobenzene oxidation of bound lignin, GPC, FT-IR, 1D and 2D NMR spectroscopy as well as TGA and DTA. The results showed that the ultrasonic treatment and sequential extractions with three different concentrations of NaOH led to a release of 75.5% of the original hemicelluloses and 96.2% of the lignin. All four purified hemicellulose fractions contained relatively low amounts of associated lignin, ranging between 0.96 and 3.10%. In addition, the hemicellulosic fraction H₄ isolated with 6% NaOH is formed by a linear backbone of four (β-1 → 4)-xylopyranosyl residues and at least one of the xylose residues is monosubstituted at C-2 by a 4-O-methylglucuronic acid, giving a typical ratio of 4-O-methyl glucuronic acid to Xyl of 1 to 4.     

Decomposition of roots of black alder and hybrid poplar in short-rotation plantings: Nitrogen and lignin control

The decomposition of the roots (0–2 mm, 2–5 mm and 5–10 mm) of black alder (Alnus glutinosa (L.) Gaertn.) and hybrid poplar (Populus nigra L. X Populus trichocarpa Torr & Gray) was followed over a 462-day period in pure and mixed plantings in southern Quebec. Small roots of alder had the highest initial concentrations of nitrogen and lignin, and lost 9 and 10% less mass than medium and large roots, respectively. Large roots of poplar had the highest lignin-to-nitrogen ratio and showed the smallest loss of mass over the total incubation period. Slow root decomposition of black alder and hybrid poplar was characterized by a greater proportion of initial root nitrogen immobilized per unit of carbon respired. Lignin concentration in roots of alder and poplar increased rapidly at the beginning of the incubation. Our results suggest that high levels of nitrogen in roots of alder could contribute in slowing the rate of decomposition by allowing the formation of nitrogen-lignin derivatives and low levels of nitrogen in roots of poplar may limit the growth of microorganisms and the rate of root decomposition. A multiple regression was developed using initial nitrogen, lignin concentration and the ratio of lignin to nitrogen to produce an index of the rate of root decomposition. The correlation between the index values and the percentage of residual root mass was significant (r=0.98, p<0.01).     

Selectivity and delignification kinetics for oxidative and nonoxidative lime pretreatment of poplar wood, part III: long-term

Lime pretreatment is an effective method for improving lignocellulose digestibility by removing lignin. For several weeks, mixtures of poplar wood, water, and calcium hydroxide (lime) were submitted to temperatures from 25 to 65°C, with and without aeration. Kinetic models for lignin and carbohydrate degradation were obtained as functions of temperature, time, and aeration using first-order kinetics in lignin and carbohydrates. Model 1 considered two reacting moieties (slow and fast), and Model 2 considered three (slow, medium, and fast). Model 1 was statistically better and was employed to determine differential and integral selectivities, which measure the ability of pretreatment to retain carbohydrates while removing lignin. During the first 2 weeks, when lignin content ≥ 0.80 g/g lignin in raw biomass, both glucan and xylan differential and integral selectivities decreased rapidly. Afterwards, selectivities were nearly constant ranging between 0 and 3 g lignin removed/g carbohydrate degraded.     

Short-term effects of nitrogen availability on wood formation and fibre properties in hybrid poplar

The application of nitrogen-containing fertilisers is one approach used to increase growth rates and productivity of forest tree plantations. However, the effects of nitrogen fertilisation on wood properties have not been systematically assessed. The aim of this work was to document the impacts of nitrogen fertilisation on wood formation and secondary xylem fibre properties. We used three fertilisation treatments in which the level of ammonium nitrate was adjusted to 0, 1 and 10 mM in a complete nutrient solution applied daily over a period of 28 days in standardised greenhouse experiments with clonal material of Populus trichocarpa (Torr and Gray) × deltoides (Bartr. ex Marsh). We showed that there was a short-term and repeatable response in which xylem fibre morphology and secondary cell wall structure adapt to a shift in N availability. Under high-nitrogen exposure, xylem fibres were 17% wider and 18% shorter compared to the adequate nitrogen treatment. A very significant thickening of the fibre cell walls was also observed throughout the stem of trees receiving the high-N treatment. It appeared that cell wall structure was greatly affected by the high-N treatment as fibres developed a modified inner cell wall layer. Histological observations indicated that the internal cell wall layer was enriched in cellulose and chemical determinations showed that wood contained more holocellulose. Together, these results indicate that the response of poplar to nitrogen availability may involve marked effects on secondary xylem formation.     

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.     

Concomitant proliferation and formation of a stratified epithelial sheet by explant outgrowth of epidermal keratinocytes from adult mice

Summary A chemically defined medium containing 1.2 mM Ca²⁺ has been developed for the culture of primary epidermal keratinocytes from untreated adult mice such that proliferation is accompanied by the formation of desmosomes and stratification. Cultured cutaneous explants of 1 mm² from the backs of untreated, control, and carcinogen-exposed mice all demonstrated epithelial outgrowth within 1 wk, and by 5 wk approached confluence with characteristics of terminal differentiation such as desmosomes and stratification. Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to the medium in concentrations of 0.001, 0.01, and 0.1 μg/ml resulted in a delay of approximately 1 wk in the outgrowth of the explants compared with the acetone controls and in a 30% decrease in the diameter of the epithelial outgrowth at 3 wk. The inhibition in outgrowth was overcome at higher concentrations (0.5, 1.0, and 10 μg/ml TPA). No obvious differences in morphology or in the rate of epidermal outgrowth within a 5-wk interval among explants from normal untreated epidermis, epidermis from mice treated with acetone, or epidermis from mice treated with an initiating application of 7,12-dimethylbenz[a]anthracene were observed. The defined composition of this medium and its ability to support reproducibly and conveniently both proliferation and differentiation of normal as well as treated primary adult murine epidermal cells suggest that it should be useful for a number of studies not previously possible that are relevent to the biology of the skin, to toxicology, and to carcinogenesis in the murine model system.     

Preparation of polyethylene glycol-modified streptokinase with disappearance of binding ability towards anti-serum and retention of activity

A 37 amino acid-peptide has been isolated from porcine jejuno-ileum on the basis of its glucagon-like activity in liver (interaction with glucagon-binding sites and activation of adenylate cyclase) using gel filtration, ion-exchange and high-performance liquid chromatography. Depending on the criteria chosen, this peptide is referred to as either ‘bioactive enteroglucagon’ (activity in liver), ‘oxyntomodulin’ (specific action in gastric oxyntic glands) or ‘glucagon-37’ (chemical structure).     

Phototropic bending of non-elongating and radially growing woody stems results from asymmetrical xylem formation

Active phototropic bending of non-elongating and radially growing portion of stems (woody stems) has not been previously documented, whereas negative gravitropic bending is well known. We found phototropic bending in woody stems and searched for the underlying mechanism. We inclined 1-year-old Quercus crispula Blume seedlings and unilaterally illuminated them from a horizontal direction perpendicular to ('normal' illumination) or parallel to ('parallel' illumination) the inclination azimuth. With normal illumination, active phototropic bending and xylem formation could be evaluated separately from the negative gravitropic response and vertical deflection resulting from the weight of the seedlings. One-year-old stems with normal illumination bent significantly, with asymmetrical xylem formation towards the illuminated upper surface and side of the stem, whereas those with parallel illumination showed non-significant lateral bending, with asymmetrical xylem formation only on the upper side. A mechanical model was built on the assumption that a bending moment resulted from the asymmetrical xylem formation during phototropic bending of the woody stems. The model fitted the relationship between the observed spatial distributions of the xylem and the observed lateral bending, and thus supported the hypothesis that phototropic bending of woody stems results from asymmetrical xylem formation, as such occurs during gravitropism.     

Novel gene expression profiles define the metabolic and physiological processes characteristic of wood and its extractive formation in a hardwood tree species,Robinia pseudoacacia

Wood is of critical importance to humans as a primary feedstock for biofuel, fiber, solid wood products, and various natural compounds including pharmaceuticals. The trunk wood of most tree species has two distinctly different regions: sapwood and heartwood. In addition to the major constituents, wood contains extraneous chemicals that can be removed by extraction with various solvents. The composition and the content of the extractives vary depending on such factors as, species, growth conditions, and time of year when the tree is cut. Despite the great commercial and keen scientific interest, little is known about the tree-specific biology of the formation of heartwood and its extractives. In order to gain insight on the molecular regulations of heartwood and its extractive formation, we carried out global examination of gene expression profiles across the trunk wood of black locust (Robinia pseudoacacia L.) trees. Of the 2,915 expressed sequenced tags (ESTs) that were generated and analyzed in the current study, 55.3% showed no match to known sequences. Cluster analysis of the ESTs identified a total of 2278 unigene sets, which were used to construct cDNA microarrays. Microarray hybridization analyses were then performed to survey the changes in gene expression profiles of trunk wood. The gene expression profiles of wood formation differ according to the region of trunk wood sampled, with highly expressed genes defining the metabolic and physiological processes characteristic of each region. For example, the gene encoding sugar transport had the highest expression in the sapwood, while the structural genes for flavonoid biosynthesis were up-regulated in the sapwood-heartwood transition zone. This analysis also established the expression patterns of 341 previously unknown genes.     

Alcohol and acid formation during the anaerobic decomposition of propylene glycol under methanogenic conditions

Intermediates formed during the anaerobic decomposition of propylene glycol under methanogenic conditions were studied using a serum bottle technique. The pathway is similar to the anaerobic decomposition of ethylene glycol as previously reported. For both compounds, the decomposition is believed to proceed via an initial disproportionation of the glycol to form equal molar amounts of the volatile fatty acid and normal alcohol of the same chain length. In the case of ethylene glycol, disproportionation results in the formation of acetate and ethanol, while disproportionation of propylene glycol produces propionate and n-propanol. Following disproportionation, the alcohols produced from glycol fermentation are oxidized to their corresponding volatile fatty acid with the reduction of protons to form hydrogen. Ethanol and propionate oxidation to acetate proceeds via a well-established syntrophic pathway that is favorable only under low hydrogen partial pressures. Subsequent degradation of acetate proceeds via acetoclastic methanogenesis with the production of carbon dioxide and methane. Despite the production of hydrogen in the initial steps of glycol degradation, both compounds are completely degradable under the methanogenic conditions tested in this study.