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.     

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.     

Overexpression of a developing xylem cDNA library in transgenic poplar generates high mutation rate specific to wood formation

We investigated feasibility of the Full‐length complementary DNA OvereXpression (FOX) system as a mutagenesis approach in poplar, using developing xylem tissue. The main goal was to assess the overall mutation rate and if the system will increase instances of mutants affected in traits linked to the xylem tissue. Indeed, we found a high mutation rate of 17.7%, whereas 80% of all mutants were significantly affected in cellulose, lignin and/or hemicellulose. Cell wall biosynthesis is a major process occurring during xylem development. Enrichment of mutants affected in cell wall composition suggests that the tissue source for the FOX library influenced the occurrence of mutants affected in a trait linked to this tissue. Additionally, we found that FLcDNAs from mutants affected in cell wall composition were homologous to genes known to be involved in cell wall biosynthesis and most recovered FLcDNAs corresponded to genes whose native expression was highest in xylem. We characterized in detail a mutant line with increased diameter. The phenotype was caused by a poplar homolog of LONELY GUY 1 (LOG1), which encodes an enzyme in cytokinin biosynthesis and significantly increased xylem proliferation. The causative role of LOG1 in the observed phenotype was further reaffirmed by elevated cytokinin concentration in the mutant and recapitulation overexpression experiment wherein multiple independent lines phenocopied the original FOX mutant. Our experiments show that the FOX approach can be efficiently used for gene discovery and molecular interrogation of traits specific to woody perennial growth and development.     

Ethylene in relation to compression wood formation in Abies balsamea shoots

 The terminal (1-year-old) shoot of dormant, 2-year-old balsam fir [Abies balsamea (L.) Mill.] seedlings was either left vertically oriented or tilted to an angle of 60° from the vertical (tilting experiment), or was ringed with N-1-naphthylphthalamic acid (NPA), an inhibitor of indole-3-acetic acid transport, at a concentration of 0, 1 or 10 mg g⁻¹ lanolin (NPA experiment). After 6 weeks of growth, ethylene evolution from the cambial region was measured by gas chromatography – flame ionization detection, and tracheid production and compression wood formation were determined by microscopy. In vertical seedlings of the tilting experiment and in 0 mg g⁻¹-treated seedlings of the NPA experiment, compression wood was not formed and neither ethylene evolution nor tracheid production varied longitudinally or circumferentially within the stem. Tilting induced compression wood formation and increased ethylene evolution and tracheid production on the lower side of the stem, while decreasing tracheid production on the upper side. Compression wood formation was induced and tracheid production and ethylene evolution were stimulated at and above the point where 1 or 10 mg NPA g⁻¹ was applied, whereas below this point compression wood was not formed and tracheid production was inhibited. In both tilting and NPA experiments, there was a positive correlation between ethylene evolution and tracheid production when data from all seedlings were analyzed, but not when data from seedlings forming compression wood were excluded. The results indicate that cambial region ethylene evolution is enhanced when compression wood is being formed, and that the enhancement is related to compression wood formation per se rather than the associated increase in tracheid production. Received: 19 February 1998 / Accepted: 26 October 1998     

Stress generation in the tension wood of poplar is based on the lateral swelling power of the G-layer

The mechanism of active stress generation in tension wood is still not fully understood. To characterize the functional interdependency between the G-layer and the secondary cell wall, nanostructural characterization and mechanical tests were performed on native tension wood tissues of poplar (Populus nigra x Populus deltoids) and on tissues in which the G-layer was removed by an enzymatic treatment. In addition to the well-known axial orientation of the cellulose fibrils in the G-layer, it was shown that the microfibril angle of the S2-layer was very large (about 36 degrees). The removal of the G-layer resulted in an axial extension and a tangential contraction of the tissues. The tensile stress-strain curves of native tension wood slices showed a jagged appearance after yield that could not be seen in the enzyme-treated samples. The behaviour of the native tissue was modelled by assuming that cells deform elastically up to a critical strain at which the G-layer slips, causing a drop in stress. The results suggest that tensile stresses in poplar are generated in the living plant by a lateral swelling of the G-layer which forces the surrounding secondary cell wall to contract in the axial direction.     

Couplings in cell differentiation kinetics mitigate air temperature influence on conifer wood anatomy

Conifer trees possess a typical anatomical tree‐ring structure characterized by a transition from large and thin‐walled earlywood tracheids to narrow and thick‐walled latewood tracheids. However, little is known on how this characteristic structure is maintained across contrasting environmental conditions, due to its crucial role to ensure sap ascent and mechanical support. In this study, we monitored weekly wood cell formation for up to 7 years in two temperate conifer species (i.e., Picea abies (L.) Karst and Larix decidua Mill.) across an 8°C thermal gradient from 800 to 2,200 m a.s.l. in central Europe to investigate the impact of air temperature on rate and duration of wood cell formation. Results indicated that towards colder sites, forming tracheids compensate a decreased rate of differentiation (cell enlarging and wall thickening) by an extended duration, except for the last cells of the latewood in the wall‐thickening phase. This compensation allows conifer trees to mitigate the influence of air temperature on the final tree‐ring structure, with important implications for the functioning and resilience of the xylem to varying environmental conditions. The disappearing compensation in the thickening latewood cells might also explain the higher climatic sensitivity usually found in maximum latewood density.     

Comparative sugar recovery and fermentation data following pretreatment of poplar wood by leading technologies

Through a Biomass Refining Consortium for Applied Fundamentals and Innovation among Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, leading pretreatment technologies based on ammonia fiber expansion, aqueous ammonia recycle, dilute sulfuric acid, lime, neutral pH, and sulfur dioxide were applied to a single source of poplar wood, and the remaining solids from each technology were hydrolyzed to sugars using the same enzymes. Identical analytical methods and a consistent material balance methodology were employed to develop comparative performance data for each combination of pretreatment and enzymes. Overall, compared to data with corn stover employed previously, the results showed that poplar was more recalcitrant to conversion to sugars and that sugar yields from the combined operations of pretreatment and enzymatic hydrolysis varied more among pretreatments. However, application of more severe pretreatment conditions gave good yields from sulfur dioxide and lime, and a recombinant yeast strain fermented the mixed stream of glucose and xylose sugars released by enzymatic hydrolysis of water washed solids from all pretreatments to ethanol with similarly high yields. An Agricultural and Industrial Advisory Board followed progress and helped steer the research to meet scientific and commercial needs. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Analyses of GA20ox- and GID1-over-expressing aspen suggest that gibberellins play two distinct roles in wood formation

Gibberellins (GAs) are involved in many aspects of plant development, including shoot growth, flowering and wood formation. Increased levels of bioactive GAs are known to induce xylogenesis and xylem fiber elongation in aspen. However, there is currently little information on the response pathway(s) that mediate GA effects on wood formation. Here we characterize an important element of the GA pathway in hybrid aspen: the GA receptor, GID1. Four orthologs of GID1 were identified in Populus tremula  ×  P. tremuloides ( PttGID1.1–1.4 ). These were functional when expressed in Arabidopsis thaliana , and appear to present a degree of sub-functionalization in hybrid aspen. PttGID1.1 and PttGID1.3 were over-expressed in independent lines of hybrid aspen using either the 35S promoter or a xylem-specific promoter ( LMX5 ). The 35S : PttGID1 over-expressors shared several phenotypic traits previously described in 35S:AtGA20ox1 over-expressors, including rapid growth, increased elongation, and increased xylogenesis. However, their xylem fibers were not elongated, unlike those of 35S:AtGA20ox1 plants. Similar differences in the xylem fiber phenotype were observed when PttGID1.1 , PttGID1.3 or AtGA20ox1 were expressed under the control of the LMX5 promoter, suggesting either that PttGID1.1 and PttGID1.3 play no role in fiber elongation or that GA homeostasis is strongly controlled when GA signaling is altered. Our data suggest that GAs are required in two distinct wood-formation processes that have tissue-specific signaling pathways: xylogenesis, as mediated by GA signaling in the cambium, and fiber elongation in the developing xylem.     

Nucleotide variation in genes involved in wood formation in two pine species

Nucleotide diversity in eight genes related to wood formation was investigated in two pine species, Pinus pinaster and P. radiata. The nucleotide diversity patterns observed and their properties were compared between the two species according to the specific characteristics of the samples analysed. A lower diversity was observed in P. radiata compared with P. pinaster. In particular, for two genes (Pp1, a glycin-rich protein homolog and CesA3, a cellulose synthase) the magnitude of the reduction of diversity potentially indicates the action of nonneutral factors. For both, particular patterns of nucleotide diversity were observed in P. pinaster (high genetic differentiation for Pp1 and close to zero differentiation associated with positive Tajima's D-value for CesA3). In addition, KORRIGAN, a gene involved in cellulose-hemicellulose assembly, demonstrated a negative Tajima's D-value in P. radiata accompanied by a high genetic differentiation in P. pinaster. The consistency of the results obtained at the nucleotide level, together with the physiological roles of the genes analysed, indicate their potential susceptibility to artificial and/or natural selection.     

A cytoskeletal basis for wood formation in angiosperm trees: the involvement of cortical microtubules

Rearrangements of cortical microtubules (CMTs) during the differentiation of axial secondary xylem elements within taproots and shoots of Aesculus hippocastanum L. (horse-chestnut) are described. A correlative approach was employed using indirect immunofluorescence microscopy of α-tubulin in 6- to 10-μm sections and transmission electron microscopy of ultrathin sections. All cell types – fibres, vessel elements and axial parenchyma – derive from fusiform cambial cells which contain randomly oriented CMTs. At the early stages of development, fibres and axial parenchyma cells possess helically arranged CMTs, which increase in number as secondary wall thickening proceeds and simple pits develop. In contrast, incipient vessel elements are distinguished by the marking out of sites of bordered pits; these sites first appear as microtubule-free regions within the reticulum of randomly oriented CMTs that characterises their precursor fusiform cambial cells. Subsequently, the ring of CMTs which develops at the periphery of the microtubule-free region decreases in diameter as the over-arching pit border is formed. Like bordered pits, large-diameter, non-bordered pits (contact pits) which develop between vessel elements and adjacent contact ray cells originate as microtubule-free regions and are also associated with development of a ring of CMTs at the periphery. In the case of contact pits, however, there is no reduction in the diameter of the CMT ring during pit development. Tertiary cell wall thickenings are also a feature of vessel elements and appear to form at sites where bands of laterally associated, transversely oriented CMTs, separated from each other by microtubule-free zones, are found. Later, these bands of CMTs become narrower, and separate into pairs of microtubule bundles located on each side of the developing wall thickening. Development of perforations between vessel elements is also associated with the presence of a ring of CMTs at their periphery. Received: 13 July 1998 / Accepted: 30 November 1998     

Calcium dependence and contraction in somite formation

The existence of a calcium-dependent contractile process in the formation of somites from segmental plate mesoderm was investigated using a Ca2+ agonist and Ca2+ and calmodulin antagonists. The contribution of cell movement and apical constriction in the segmentation process were assessed using SEM of normal and drug-treated somite and segmental plate tissue. Explants that contained segmental plates of stage 14-15 chick embryos were cultured on vitelline membranes in calcium- and magnesium-free (CMF) Hands' solution, liquid culture medium, and medium containing drugs. Ca2+ ionophore A23187 promoted the rapid completion of one new somite pair. CMF halted segmentation. The Ca2+ antagonists verapamil and papaverine reversibly inhibited segmentation. Theophylline did not inhibit segmentation, suggesting that the effects of the Ca2+ antagonists are not due to inhibition of phosphodiesterase activity. These results suggest that somitogenesis is Ca2+-dependent. Two drugs that inhibit the binding of calmodulin, chlorpromazine and trifluoperazine (TFP), halted segmentation. The inhibitory effect of TFP was reversible. The effects of TFP on somites were compared with those of cytochalasin D. The contribution of microtubules to cell shape and movement in somitogenesis was examined by incubation with nocodazole, a reversible inhibitor of tubulin polymerization. Cell elongation and somitogenesis were inhibited.     

Ectopic expression of a wood-abundant expansin PttEXPA1 promotes cell expansion in primary and secondary tissues in aspen

Expansins are primary agents inducing cell wall extension, and are therefore obvious targets in biotechnological applications aimed at the modification of cell size in plants. In trees, increased fibre length is a goal of both breeding and genetic engineering programmes. We used an α-expansin Ptt EXPA1 that is highly abundant in the wood-forming tissues of hybrid aspen ( Populus tremula L. ×  P. tremuloides Michx.) to evaluate its role in fibre elongation and wood cell development. Ptt EXPA1 belongs to Subfamily A of α-expansins that have conserved motifs at the N- and C-termini of the mature protein. When PttEXPA1 was over-expressed in aspen, an extract of the cell wall-bound proteins of the transgenic plants exhibited an increased expansin activity on cellulose–xyloglucan composites in vitro , indicating that Ptt EXPA1 is an active expansin. The transgenic lines exhibited increased stem internode elongation and leaf expansion, and larger cell sizes in the leaf epidermis, indicating that Ptt EXPA1 protein is capable of increasing the growth of these organs by enhancing cell wall expansion in planta . Wood cell development was also modified in the transgenic lines, but the effects were different for vessel elements and fibres, the two main cell types of aspen wood. Ptt EXPA1 stimulated fibre, but not vessel element, diameter growth, and marginally increased vessel element length, but did not affect fibre length. The observed differences in responsiveness to expansin of these cell types are discussed in the light of differences in their growth strategies and cell wall composition.     

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.     

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.     

Effects of environmental factors on wood formation in Scots pine stems

Summary To find the optimal conditions for growth and development of tracheid walls in Scots pine stems the effects of temperature and precipitation on xylem cell production by the cambium, radial cell expansion and secondary wall thickening have been studied. The observations were carried out on 10 specially chosen 50 to 60-year-old trees, growing in central Siberia, over 2 seasons. The data on the number of cells in differentiation zones and mature xylem along radial rows of tracheids, radial and tangential sizes of tracheids and their lumens were used for calculating cambial activity, the rates and durations of cell development in the zones, and both the thickness and cross sectional areas of tracheid walls. The mean day, mean maximal diurnal and mean minimal nocturnal temperatures have been shown by correlation and regression analyses to affect differentially separate stages of cytogenesis. The temperature influenced the initial division the side of xylem and radial cell expansion mainly in May–June, while the influence of precipitation increased in July–August. Throughout all seasons it was the temperature that had the main influence on the biomass accumulation in cell walls. Optimal values of temperature and precipitation for cell production by cambium, radial cell expansion and secondary wall thickening have been calculated. The data are discussed in connection with productivity and quality of wood.     

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.     

beta-tubulin affects cellulose microfibril orientation in plant secondary fibre cell walls

Cellulose microfibrils are the major structural component of plant secondary cell walls. Their arrangement in plant primary cell walls, and its consequent influence on cell expansion and cellular morphology, is directed by cortical microtubules; cylindrical protein filaments composed of heterodimers of alpha- and beta-tubulin. In secondary cell walls of woody plant stems the orientation of cellulose microfibrils influences the strength and flexibility of wood, providing the physical support that has been instrumental in vascular plant colonization of the troposphere. Here we show that a Eucalyptus grandisbeta-tubulin gene (EgrTUB1) is involved in determining the orientation of cellulose microfibrils in plant secondary fibre cell walls. This finding is based on RNA expression studies in mature trees, where we identified and isolated EgrTUB1 as a candidate for association with wood-fibre formation, and on the analysis of somatically derived transgenic wood sectors in Eucalyptus. We show that cellulose microfibril angle (MFA) is correlated with EgrTUB1 expression, and that MFA was significantly altered as a consequence of stable transformation with EgrTUB1. Our findings present an important step towards the production of fibres with altered tensile strength, stiffness and elastic properties, and shed light on one of the molecular mechanisms that has enabled trees to dominate terrestrial ecosystems.     

Variable wood formation and adaptation to the alpine environment of Ephedra pachyclada (Gnetales: Ephedraceae) in the Mustang district, western Nepal

BACKGROUND AND AIMS: Plants of Ephedra normally have vessels, but are known to become nearly vessel-less in some alpine localities. Previous studies implied that wood formation in Ephedra differs fundamentally from that in dicotyledons in which vessel-bearing and vessel-less taxa are systematically distinct. Using E. pachyclada in the Mustang district of Nepal, growing in an altitudinal range of over 2000 m, variation in wood formation and adaptation to alpine environment was studied in this normally vessel-bearing species. METHODS: Variation in wood anatomy and wood formation was observed with conventional optical microscopy. The lengths of three kinds of tracheary elements were measured and statistically analysed against habitat altitude and plant size of the individuals studied. KEY RESULTS: In E. pachyclada three kinds of tracheary elements, vessel elements, tracheids and fibre-tracheids, were nearly equal in length within individuals showing no elongation after differentiation from cambial initials. Tracheary element lengths among individuals had a negative correlation with altitude and a positive correlation with plant size. Multivariate analyses showed that altitude has a stronger correlation with tracheary element lengths than plant height or stem diameter. Moreover, several individuals from high elevations completely lacked vessels, and vessel formation fluctuated even in individuals from lower elevations. CONCLUSIONS: Wood anatomical trends in E. pachyclada are considered as an adaptation to extremely dry conditions in high mountains. Fluctuation in vessel formation in individuals from low elevations indicated that vessels differentiate only when their lateral expansion is allowed. These results showed that E. pachyclada has a different system of wood formation from dicotyledons and supported the opinion that the wood structure of Gnetales is fundamentally different from that of angiosperms.