Regulation of secondary xylem formation in young hybrid poplars by modifying the expression levels of the <Emphasis Type="Italic">PtaGLIMa</Emphasis> gene

Due to the importance of wood in many industrial applications, a tremendous amount of research has focused on the regulation of secondary xylem formation and wood properties. In this study, we performed functional analysis of PtaGLIM1a, a LIM gene that is predominantly expressed in the differentiation of secondary xylem of the hybrid poplar (Populus tremula × P. alba). With no growth retardation, transgenic poplar plants with increased and reduced expression levels of PtaGlim1a exhibited enhanced and diminished secondary growth, respectively, accompanied by a corresponding change in their lignin abundance. This study demonstrates that the wood-associated PtaGlim1a acts as a positive regulator of secondary xylem formation in poplar trees and could potentially be utilized in modifying the synthesis of plant secondary wall lignin.     

Transgenic poplars with reduced lignin show impaired xylem conductivity, growth efficiency and survival

We studied xylem anatomy and hydraulic architecture in 14 transgenic insertion events and a control line of hybrid poplar (Populus spp.) that varied in lignin content. Transgenic events had different levels of down-regulation of two genes encoding 4-coumarate:coenzyme A ligase (4CL). Two-year-old trees were characterized after growing either as free-standing trees in the field or as supported by stakes in a greenhouse. In free-standing trees, a 20 to 40% reduction in lignin content was associated with increased xylem vulnerability to embolism, shoot dieback and mortality. In staked trees, the decreased biomechanical demands on the xylem was associated with increases in the leaf area to sapwood area ratio and wood specific conductivity (k(s)), and with decreased leaf-specific conductivity (k(l)). These shifts in hydraulic architecture suggest that the bending stresses perceived during growth can affect traits important for xylem water transport. Severe 4CL-downregulation resulted in the patchy formation of discoloured, brown wood with irregular vessels in which water transport was strongly impeded. These severely 4CL-downregulated trees had significantly lower growth efficiency (biomass/leaf area). These results underscore the necessity of adequate lignification for mechanical support of the stem, water transport, tree growth and survival.     

Wood ontogeny during ex vitro acclimatization in micropropagated hybrid poplar clones

Wood ontogeny patterns were determined during the ex vitro acclimatization period in micropropagated plantlets of hybrid poplar clones T-14 [Populus tremula × (Populus × canescens)] and T-50 [(Populus × canescens) × Populus tremula]. The temporal course of developmental changes in the woody tissue was characterized on a weekly basis starting from the day of transfer to the ex vitro environment until full acclimatization was achieved on day 28. In vitro rooted plantlets had already initiated lignification of secondary xylem cells. The greatest increase in the amount of woody tissue was observed on days 21 and 28. At the end of the acclimatization period, T-14 plantlets contained on average 41.4 % of secondary xylem tissue compared to 30.3 % found in T-50 plantlets. During the course of acclimatization, both clones displayed identical patterns of vessel lumen size distribution from small vessel lumen area to large vessel lumen area. This pattern differs from the characteristic diffuse-porous pattern of approximately evensized vessel lumen area distribution typical of mature wood. At the end of acclimatization, the differences in vessel lumen area and relative conductivity between the clones were negligible. Development of secondary xylem tissue during ex vitro acclimatization promotes the establishment of vigorous regenerants with stems that show increased bending strength and stiffness.     

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.     

Molecular characterization of PoGT8D and PoGT43B, two secondary wall-associated glycosyltransferases in poplar

Dicot wood is mainly composed of cellulose, lignin and glucuronoxylan (GX). Although the biosynthetic genes for cellulose and lignin have been studied intensively, little is known about the genes involved in the biosynthesis of GX during wood formation. Here, we report the molecular characterization of two genes, PoGT8D and PoGT43B, which encode putative glycosyltransferases, in the hybrid poplar Populus alba x tremula. The predicted amino acid sequences of PoGT8D and PoGT43B exhibit 89 and 75% similarity to the Arabidopsis thaliana IRREGULAR XYLEM8 (IRX8) and IRX9, respectively, both of which have been shown to be required for GX biosynthesis. The PoGT8D and PoGT43B genes were found to be expressed in cells undergoing secondary wall thickening, including the primary xylem, secondary xylem and phloem fibers in stems, and the secondary xylem in roots. Both PoGT8D and PoGT43B are predicted to be type II membrane proteins and shown to be targeted to Golgi. Overexpression of PoGT43B in the irx9 mutant was able to rescue the defects in plant size and secondary wall thickness and partially restore the xylose content. Taken together, our results demonstrate that PoGT8D and PoGT43B are Golgi-localized, secondary wall-associated proteins, and PoGT43B is a functional ortholog of IRX9 involved in GX biosynthesis during wood formation.     

Degradation of poplar wood by Fomes sclerodermeus: production of ligninolytic enzymes in sawdust of poplar and cedar

Fomes sclerodermeus is a white-rot fungus. Its production of laccase, manganese peroxidase and lignin peroxidase on sawdust-based media was evaluated. No lignin peroxidase activity was measured in any media tested. The higher production of laccase and manganese peroxidase were found on media containing poplar sawdust. F. sclerodermeus was grown on wood blocks of poplar during six months. Dry weight losses of the blocks reached a mean value of 51%. The quantification of cellulose and lignin in the 6-months incubated blocks showed losses of up to 58 and 56% for cellulose and lignin, respectively. The decay examined under microscope revealed mycelium colonizing the lumen of vessel elements, cell wall thinning and entire degradation of the radial parenchyma.     

Lignin composition in cambial tissues of poplar

The cambial tissues of a Populus balsamifera, Balsam poplar clone were studied during a growth season. The Klason and acid-soluble lignin contents were determined as well as the carbohydrate monomer distribution and the protein content. Both the phloem and the xylem sides of the cambial region were examined. The samples were analyzed by thioacidolysis and structures of dimeric products were determined by mass spectrometry after desulphuration. Chemical analysis of samples during the growth season was combined with microscopy of embedded specimens that showed the state of cell differentiation at the time of sampling. In spring and early summer, growth is very rapid and the intention was to collect tissue in which exclusively the middle lamella/primary cell wall had begun to lignify. The Klason lignin, protein content and carbohydrate monomer distribution showed that all the specimens from the cambial tissues sampled during a growth season contained predominantly middle lamella and primary walls; except for the developing xylem sampled in August where the carbohydrate composition showed that secondary walls were present. Thioacidolysis showed that the lignin from the cambial tissues had more condensed structures than the lignin from the reference balsam poplar clone wood. More guaiacyl than syringyl units were detected and mass spectrometry showed that the cambial tissues contained more lignin structures with end-groups than the reference sample. These results suggest that lignification in the cambial layer and early developing xylem may take place predominantly in a bulk fashion during the summer.     

Influence of over-expression of the FLOWERING PROMOTING FACTOR 1 gene (FPF1) from Arabidopsis on wood formation in hybrid poplar (Populus tremula L. × P. tremuloides Michx.)

Constitutive expression of the FPF1 gene in hybrid aspen (Populus tremula L. × P. tremuloides Michx.) showed a strong effect on wood formation but no effect on flowering time. Gene expression studies showed that activity of flowering time genes PtFT1, PtCO2, and PtFUL was not increased in FPF1 transgenic plants. However, the SOC1/TM3 class gene PTM5, which has been related to wood formation and flowering time, showed a strong activity in stems of all transgenic lines studied. Wood density was lower in transgenic plants, despite significantly reduced vessel frequency which was overcompensated by thinner fibre cell walls. Chemical screening of the wood by pyrolysis GC/MS showed that FPF1 transgenics have higher fractions of cellulose and glucomannan products as well as lower lignin content. The latter observation was confirmed by UV microspectrophotometry on a cellular level. Topochemical lignin distribution revealed a slower increase of lignin incorporation in the developing xylem of the transgenics when compared with the wild-type plants. In line with the reduced wood density, micromechanical wood properties such as stiffness and ultimate stress were also significantly reduced in all transgenic lines. Thus, we provide evidence that FPF1 class genes may play a regulatory role in both wood formation and flowering in poplar.     

Wood formation during ex vitro acclimatisation in micropropagated true service tree (<Emphasis Type="Italic">Sorbus domestica</Emphasis> L.)

Micropropagated plantlets derived from a superior 90+-year-old slow-growing true service tree (Sorbus domestica L.) have been successfully acclimatised to the ex vitro environment. The temporal pattern of developmental changes was examined in relation to secondary xylem growth during ex vitro acclimatisation. In vitro rooted plantlets already initiated lignification of secondary xylem cells. During early days after transfer to ex vitro conditions, the growth of woody tissue was slow. The most prominent increase in woody tissue development occurred between days 7 and 35. From days 35 to 63, sizes of vessel lumen areas significantly increased. In developing woody tissue, a characteristic diffuse-porous pattern of roughly even vessel area distribution throughout the growing season, typical for mature wood, was not followed. The proportion of woody area occupied by vessels was significantly higher in stems of fully acclimatised plantlets than in stems sampled 35 days after transfer. On day 63 after transfer, a proportion of woody tissue area in fully acclimatised plantlets represented up to 14.8% of the stem area. Early formation of wood during ex vitro acclimatisation provides brittle plantlets with a mechanical support to cope better with deformations and mechanical injuries during handling at the subsequent transplantations.     

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).     

Gene expression profiles in different stem internodes reveal the genetic regulation of primary and secondary stem development in <Emphasis Type="Italic">Betula platyphylla</Emphasis>

Secondary growth of stems is an important process for the radial increase of trees. To gain an insight into the molecular mechanisms underlying stem development from primary to secondary growth and to provide information for molecular research and breeding in Betula platyphylla (birch), the gene expression profiles of material from the first, third, and fifth internodes (IN) of 3-month-old seedlings were analyzed. Compared with the first IN, 177 genes were up-regulated and 157 genes down-regulated in the third IN; in the fifth IN, 180 genes were up-regulated and 275 genes were down-regulated. The expressions of 24 genes were up-regulated and 6 genes were down-regulated in the fifth IN relative to the third IN. The differentially expressed genes were annotated as having roles in cambium, xylem, and phloem development and formation; including cell wall expansion, cellulose biosynthesis, lignin biosynthesis and deposition, xylem extension, cell wall modification, and growth hormone responses. The expressions of genes related to cell wall expansion and cellulose biosynthesis in the primary cell wall were down-regulated in the third and fifth IN relative to the first IN. Genes involved in lignin biosynthesis, xylem extension, and cellulose synthesis in the secondary cell wall were up-regulated in the third and fifth IN relative to the first IN. These results described the patterns of gene expression during stem development in birch and provided candidate genes for further functional characterization.     

海桑次生木质部导管解剖特征与土壤理化因子年内动态变化的关系研究

为揭示海桑次生木质部导管解剖特征随土壤理化因子年内动态变动而变化的适应机制,该研究利用海桑具有生长轮的特点,通过显微技术界定了海桑采样枝条一年内10个不同连续时间段形成的新"生长层"(新形成的次生木质部),观测了10个不同连续时间段新"生长层"的导管解剖特征,并对10个新"生长层"形成阶段所对应的土壤理化因子数量特征进行了测定,用逐步回归法分析了10个不同连续时间段海桑新"生长层"管孔数量解剖特征与对应土壤理化因子数量特征之间的关系。结果显示:(1)10个不同连续时间段海桑形成新"生长层"管孔数量特征指标,除相邻管孔间接触壁长占比无显著差异外,其他8项指标包括管孔径向直径、管孔弦向直径、导管壁厚、导管长度、管孔密度、单孔率、导管聚合度和相邻管孔间接触壁长等均具有显著差异(P0.05);多重比较显示,10个不同连续时间段海桑形成新"生长层"管孔数量特征8项指标具不同程度的变动。(2)海桑新"生长层"形成阶段(10个不同连续时间段)土壤理化因子,包括土壤有机质含量、土壤全氮含量、土壤全磷含量、土壤pH值和土壤全盐量等5项指标均具有显著差异(P0.05);多重比较显示,海桑新"生长层"形成阶段(10个不同连续时间段)土壤理化因子指标均具有不同程度变动。(3)10个不同连续时间段海桑形成新"生长层"的管孔数量解剖特征,与所对应新"生长层"形成阶段土壤理化因子数量特征的逐步回归分析表明,随着土壤全盐含量的升高,海桑导管弦向直径和导管聚合度同时呈显著增大趋势(P0.05)。研究表明,海桑在一年内不同连续时间段,随土壤全盐量增加,土壤渗透势将增大,水分在次生木质部导管中输导的安全性将下降,而海桑导管分子随一年内不同连续时间段土壤全盐量的增加呈增大趋势,根据木材生态解剖学的观点,水分输导的安全性将进一步降低,但导管聚合度随一年内不同连续时间段土壤全盐量的增加而增大,具有增进水分输导安全性的作用,这可能是海桑对土壤盐含量变化的生态适应策略。     

Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-flight secondary ion mass spectrometry

Lignin, one of the main structural polymer of plant cell walls, varies in amount and monomeric composition among tissue and cell types, as well as among plant species. However, few analytical methods are available that can conveniently and accurately determine the morphological distribution of lignin units at the cellular level. In this report, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly map guaiacyl (G) and syringyl (S) lignin units in several successive growth rings of the maple xylem. TOF-SIMS imaging and a semiquantitative approach revealed clear difference in the annual distribution of lignins between the fiber and vessel. While the vessel walls were constantly G-rich with varied S/G ratios through a growth ring, the fibers showed fairly regular annual distribution of lignins in which the earlywood was S-rich with an almost constant S/G ratio and the latewood was G-rich resulting from a decrease of the S unit. The reliability of TOF-SIMS results was demonstrated by its high correlation with the results of thioacidolysis on radial distribution of the S/G ratio in several contiguous tree rings and also in the latewood and earlywood of each ring. These results indicate that TOF-SIMS allows direct visualization of lignin composition in plant tissues.     

Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall.

Recessive mutations at three loci cause the collapse of mature xylem cells in inflorescence stems of Arabidopsis. These irregular xylem (irx) mutations were identified by screening plants from a mutagenized population by microscopic examination of stem sections. The xylem cell defect was associated with an up to eightfold reduction in the total amount of cellulose in mature inflorescence stems. The amounts of cell wall-associated phenolics and polysaccharides were unaffected by the mutations. Examination of the cell walls by using electron microscopy demonstrated that the decreases in cellulose content of irx lines resulted in an alteration of the spatial organization of cell wall material. This suggests that a normal pattern of cellulose deposition may be required for assembly of lignin or polysaccharides. The reduced cellulose content of the stems also resulted in a decrease in stiffness of the stem material. This is consistent with the irregular xylem phenotype and suggests that the walls of irx plants are not resistant to compressive forces. Because lignin was implicated previously as a major factor in resistance to compressive forces, these results suggest either that cellulose has a direct role in providing resistance to compressive forces or that it is required for the development of normal lignin structure. The irx plants had a slight reduction in growth rate and stature but were otherwise normal in appearance. The mutations should be useful in facilitating the identification of factors that control the synthesis and deposition of cellulose and other cell wall components.     

Xylem cell length under drought and its value for predicting radial growth of SRF poplar cultivars (Populus spp.)

Xylem cell length of juvenile tree rings was investigated in poplars in order to check the hypotheses that fiber length or vessel element length are indicative of drought tolerance and have predictive value for final stem base diameter at the end of rotation. The radial increment in the drought year 2003 served as the reference indicator for quantifying drought tolerance. All nine investigated cultivars suffered severely. In terms of their moderately decreased radial increment in 2003, the two aspen cultivars were clearly less drought susceptible than the seven hybrid poplar cultivars. The variance components of xylem cell length data explained by the two genetic factors ‘cultivar’ and ‘botanic section’ as well as the ‘tree ring’ (of the years 2002 and 2003) were compared by means of ANOVAs. The cultivar effects were superior to the effects of the critical precipitation status in 2003 and the botanic section. Fiber and vessel element length were found to be less sensitive to the drought compared with radial increment. They did neither correlate with radial increment in the drought year 2003 nor in 2002. Therefore, higher xylem cell length cannot indicate drought tolerance in poplars. However, a linear relationship between fiber length of both juvenile tree rings and the stem base diameter proved to be highly significant to a linear mixed effect model. Higher fiber length of a juvenile tree ring was considered to be predictive of larger stem base diameter at the end of rotation.