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.     

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce (Picea abies (L.) H. Karsten) and silver birch (Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-m cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.Abbreviation CLSM confocal laser-scanning fluorescence microscopy     

Effects of vapour pressure deficit on growth of temperate and tropical evergreen rainforest trees of Australia

Little is known about the effect of vapour pressure deficit (VPD) on the growth of trees. Rainforest trees of eastern Australia provide an opportunity to investigate responses to VPD in species that occur in high precipitation areas but have contrasting dry seasons—summer in the temperate south and winter in the tropical north. Growth responses to VPD were measured in eight species of Australian rainforest trees from different latitudes to investigate possible differences in their response to atmospheric drought. Previous work on these species found that the tropical species have large reductions in gas exchange with increasing VPD whereas the temperate species were mainly unresponsive to increasing VPD. Plants were grown in glasshouses for a year under either low VPD or ambient conditions of a temperate climate. All species had non-significant increases in growth rates (1–9%) of plants grown under low VPD compared with plants grown under ambient VPD. In addition, growing the species under low VPD had no effect on allocation of biomass (leaf area ratio, leaf weight ratio and root/shoot ratio). Therefore, the high sensitivity of gas exchange to increasing VPD found in the tropical rainforest trees did not have a significant, long-term effect on growth under high VPD.     

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Seasonal dynamics of phloem and xylem formation in silver fir and Norway spruce as affected by drought

The dynamics of phloem growth ring formation in silver fir (Abies alba Mill.) and Norway spruce (Picea abies Karst.) at different sites in Slovenia during the droughty growing season of 2003 was studied. We also determined the timing of cambial activity, xylem and phloem formation, and counted the number of cells in the completed phloem and xylem growth rings. Light microscopy of cross-sections revealed that cambial activity started on the phloem and xylem side simultaneously at all three plots. However, prior to this, 1–2 layers of phloem derivatives near the cambium were differentiated without previous divisions. The structure of the early phloem was similar in silver fir and Norway spruce. Differences in the number of late phloem cells were found among sites. Phloem growth rings were the widest in Norway spruce growing at the lowland site. In all investigated trees, the cambium produced 5–12 times more xylem cells than phloem ones. In addition, the variability in the number of cells in the 2003 growth ring around the stem circumference of the same tree and among different trees was higher on the xylem side than on the phloem side. Phloem formation is presumably less dependent on environmental factors but is more internally driven than xylem formation.     

Cell Wall Lignin is Polymerised by Class Ⅲ Secretable Plant Peroxidases in Norway Spruce

Class Ⅲ secretable plant peroxidases occur as a large family of genes in plants with many functions and probable redundancy. In this review we are concentrating on the evidence we have on the catalysis of lignin polymerization by class Ⅲ plant peroxidases present in the apoplastic space in the xylem of trees. Some evidence exists on the specificity of peroxidase isozymes in lignin polymerization through substrate specificity studies, from antisense mutants in tobacco and poplar and from tissue and cell culture lines of Norway spruce (Picea abies) and Zinnia elegans. In addition, real time (RT-)PCR results have pointed out that many peroxidases have tissue specific expression patterns in Norway spruce. Through combining information on catalytic properties of the enzymes, on the expression patterns of the corresponding genes, and on the presence of monolignols and hydrogen peroxide in the apoplastic space, we can show that specific peroxidases catalyze lignin polymerization in the apoplastic space of Norway spruce xylem.     

Cloning, characterization and localization of three novel class III peroxidases in lignifying xylem of Norway spruce (Picea abies)

Plant class III peroxidases (POXs) take part in the formation of lignin and maturation of plant cell walls. However, only a few examples of such peroxidases from gymnosperm tree species with highly lignified xylem tracheids have been implicated so far. We report here cDNA cloning of three xylem-expressed class III peroxidase encoding genes from Norway spruce (Picea abies). The translated proteins, PX1, PX2 and PX3, contain the conserved amino acids required for heme-binding and peroxidase catalysis. They all begin with putative secretion signal propeptide sequences but diverge substantially at phylogenetic level, grouping to two subclusters when aligned with other class III plant peroxidases. In situ hybridization analysis on expression of the three POXs in Norway spruce seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm of young, developing tracheids within the current growth ring where lignification is occurring. Function of the putative N-terminal secretion signal peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions with EGFP (enhanced green fluorescent protein) and expressing them in tobacco protoplasts. Full-length coding region of px1 was also heterologously expressed in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1 peroxidase is processed via the endoplasmic reticulum (ER) most likely for secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal localization for participation in the maturation of the spruce tracheid secondary cell wall.     

Localization of dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission electron microscopy–immunogold labeling

The lignification process in mature Norway spruce [Picea abies (L.) H. Karsten] xylem cell walls was studied using transmission electron microscopy (TEM)–immunogold detection with a polyclonal antibody raised against a specific lignin substructure, dibenzodioxocin. The study reveals for the first time the exact location of this abundant eight-ring structure in the cell wall layers of wood. Spruce wood samples were collected in Southern Finland at the time of active growth and lignification of the xylem cell walls. In very young tracheids where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown at all. During secondary cell wall thickening, the dibenzodioxocin structure was more abundant in the secondary cell wall layers than in the middle lamella. The highest number of gold particles revealing dibenzodioxocin was in the S2+S3 layer. Statistically significant differences were found in the frequency of gold particles present in various cell wall layers. For comparison, wood sections were also cut with a cryomicrotome for light and fluorescence microscopy.     

Lignin isolated from primary walls of hybrid aspen cell cultures indicates significant differences in lignin structure between primary and secondary cell wall.

Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.     

Cloning of cDNA Encoding CCoAOMT from Populus tomentosa and Down-regulation of Lignin Content in Transgenic Plant Expressing Antisense Gene (in English)

cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT) from Chinese white poplar ( Populus tomentosa Carr.) was cloned by RT-PCR and sequenced. Northern analysis displayed that the CCoAOMT was expressed specifically in the developing secondary xylem and its expression was coincident with lignification. The antisense CCoAOMT cDNA was transformed into P. tremula×P. alba mediated by Agrobacterium tumefaciens (Smith et Townsend) Conn. Transgenic plants were identified with PCR, PCR-Southern and Southern analysis. Lignin content in 5- to 6-month-old transgenic plants was measured. One of the transgenic lines had significant reduction of 17.9% in Klason lignin content as compared with that of untransformed poplar. The results demonstrate that antisense repression of CCoAOMT is an efficient way to reduce lignin content for improving pulping property in engineered trees.     

Cloning of cDNA Encoding COMT from Chinese White Poplar (Populus tomentosa), Sequence Analysis and Specific Expression

cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT) from Chinese white poplar ( Populus tomentosa Carr.) was cloned by RT-PCR and sequenced. Northern analysis displayed that the CCoAOMT was expressed specifically in the developing secondary xylem and its expression was coincident with lignification. The antisense CCoAOMT cDNA was transformed into P. tremula×P. alba mediated by Agrobacterium tumefaciens (Smith et Townsend) Conn. Transgenic plants were identified with PCR, PCR-Southern and Southern analysis. Lignin content in 5- to 6-month-old transgenic plants was measured. One of the transgenic lines had significant reduction of 17.9% in Klason lignin content as compared with that of untransformed poplar. The results demonstrate that antisense repression of CCoAOMT is an efficient way to reduce lignin content for improving pulping property in engineered trees.     

川西亚高山不同年龄紫果云杉径向生长对气候因子的响应

运用树木年轮气候学的基本方法,建立王朗自然保护区紫果云杉在集中分布上限区域的年轮宽度年表,选取差值年表分析不同年龄云杉的径向生长同逐月气候因子的相关及响应关系,结果显示:幼龄组云杉年表的敏感度高于中龄组和老龄组云杉,幼龄组云杉对生长季前及生长季的气温状况显著正相关;中龄组云杉年表仅与当年4月份和7月份的月平均最低气温显著正相关;老龄组云杉的年轮宽度指数同上年生长季(上年8月份)的月平均气温和月平均最低温显著负相关,上年生长季高温的"滞后效应"在老龄组云杉体现的更为突出;幼龄组与中龄组云杉对当年6月份降水持续增加显示出明显的负相关关系,上年12月份的降水会对幼龄组和老龄组云杉径向生长不利。研究表明幼龄组云杉包含的气候信息要优于中龄组和老龄组云杉,在该区域进行相关研究时应根据研究需要选取不同年龄跨度的云杉年表。     

Vibrational spectroscopy and X-ray diffraction methods to establish the differences between hardwood and softwood

FT-IR spectrometry and X-ray diffraction were applied to probe the differences between pulp fibers from Eucalyptus wood (hardwood) and Norway spruce wood (softwood). Wood processing was found to induce certain structural alterations within its components depending on the type of wood and the applied procedure. These differences were established by using techniques such as; spectral comparison of wood samples with those of individual component fractions, derivative spectroscopy, bands deconvolution, etc. FT-IR spectroscopy was shown to be an important tool that provided details about the structural characteristics of hardwood and softwood samples. Using second-derivative spectra and deconvolution processes small differences between spectra became apparent that allowed correlations to be made related to wood composition. In addition a correlation was established between the integral absorptions for the various bands and lignin content as well as the lignin/carbohydrate content. Relations between various spectral characteristics and the degree of crystallinity and sample composition were established.     

Overexpression of the endogenous peroxidase-like gene spi 2 in transgenic Norway spruce plants results in increased total peroxidase activity and reduced growth

Peroxidases constitute a large family of proteins found in all higher plants. Owing to the complexity of the peroxidase isoenzyme family it has been difficult to assess the precise function of individual peroxidase enzymes. In this work we have studied the effects of an endogenous peroxidase-like gene from Norway spruce [Picea abies (L.) Karst], spi 2, on the development and growth of Norway spruce somatic embryo plants. Embryogenic cells of Norway spruce transformed with spi 2 under control of the maize ubi-1 promoter showed up to 40 times higher total peroxidase activity than the control cells; regenerated plants overexpressing spi 2 showed an increased total peroxidase activity. Based on these results and the overall sequence similarity with cationic peroxidases we conclude that spi 2 encodes a peroxidase. Overexpression of spi 2 resulted in increased sensitivity to stress, leading to a reduction in epicotyl formation and in height growth compared with control plants. The plants overexpressing spi 2 also showed a deeper phloroglucinol staining but similar levels of Klason lignin.     

Developmental disorders in buds and needles of mature Norway spruce, Picea abies (L.) Karst., in relation to needle boron concentrations

Dieback of the terminal shoot and consequently bushy growth induced by boron deficiency have been reported widely throughout the world in several tree species. Recently, similar growth damage was documented in half of the young spruce stands in eastern Finland. To clarify the role of B deficiency, the light microscopic structure of emerging buds and of developing and previous-year needles of mature Norway spruce (Picea abies L. Karst.) from damaged (D stand), partly damaged (PD stand) and healthy (H stand) stands were analysed. The samples, on which needle nutrient concentrations were also determined, were taken seven times between early spring (April) and early winter (November). Cell death characterized by precipitation of the cell content, possibly due to the release of tannins after membrane rupture, was seen in the apex of emerging buds, and this led to fatal damage in about half of the buds in the trees from the D stand, where the needle B concentration was well below the deficiency level of 4–5 mg kg⁻¹. Furthermore, an increase in living cells that accumulated tannins in the vacuoles, which is a common stress and/or defense reaction, was found in the primordial shoots of buds and in the differentiating needles in the PD and D stands. The increase in the areas of the central cylinder and of the xylem found in the needles indicate structural plasticity during needle differentiation to drought. The time frame for bud emergence from late May up to mid-September means that an adequate B supply is necessary throughout the summer in order to avoid fatal bud damage and thus bushy growth of the trees.     

Cytomixis in shoot apex of Norway spruce [<Emphasis Type="Italic">Picea abies</Emphasis> (L.) Karst.]

Atypical cell walls and nuclei were observed in the apex of Norway spruce shoots from late April to early May on the material collected from a few grafts of a clone of Norway spruce growing on an experimental area. Images of ultrastructure attest to cytomixis. The phenomenon of cytomixis has previously been described in various plant material, both in the meiotic and mitotic cells, but this is the first report of cytomixis in gymnosperms.     

Isolation of cellular membranes from lignin‐producing tissues of Norway spruce and analysis of redox enzymes

There are no earlier reports with successful isolation of plasma membranes from lignin‐forming tissues of conifers. A method to isolate cellular membranes from extracellular lignin‐producing tissue‐cultured cells and developing xylem of Norway spruce was optimized. Modifications to the homogenization buffer were needed to obtain membranes from these phenolics‐rich tissues. Membranes were separated by aqueous polymer two‐phase partitioning. Chlorophyll a determination, marker enzyme assays and western blot analyses using antibodies for each membrane type showed that mitochondrial, chloroplastic and to a certain extent also ER and Golgi membranes were efficiently diminished from the upper phase, but tonoplast and plasma membranes distributed evenly between the upper and lower phases. Redox enzymes present in the partially purified membrane fractions were assayed in order to reveal the origin of H₂O₂ needed for lignification. The membranes of spruce contained enzymes able to generate superoxide in the presence of NAD(P)H. Besides members of the flavodoxin and flavodoxin‐like family proteins, cytochrome b5, cytochrome P450 and several stress responsive proteins were identified by nitroblue tetrazolium staining of isoelectric focusing gels and by mass spectrometry. Naphthoquinones juglone and menadione increased superoxide production in activity‐stained gels. Some juglone‐activated enzymes were preferentially using NADH. With NADH, menadione activated only some of the enzymes that juglone did, whereas with NADPH the activation patterns were identical. Duroquinone, a benzoquinone, did not affect superoxide production. Superoxide dismutase, ascorbate peroxidase, catalase and an acidic class III peroxidase isoenzyme were detected in partially purified spruce membranes. The possible locations and functions of these enzymes are discussed.     

Downregulation of caffeoyl-CoA O-methyltransferase (CCoAOMT) by RNA interference leads to reduced lignin production in maize straw

Lignin is a major cell wall component of vascular plants that provides mechanical strength and hydrophobicity to vascular vessels. However, the presence of lignin limits the effective use of crop straw in many agroindustrial processes. Here, we generated transgenic maize plants in which the expression of a lignin biosynthetic gene encoding CCoAOMT, a key enzyme involved in the lignin biosynthesis pathway was downregulated by RNA interference (RNAi). RNAi of CCoAOMT led to significantly downregulated expression of this gene in transgenic maize compared with WT plants. These transgenic plants exhibited a 22.4% decrease in Klason lignin content and a 23.3% increase in cellulose content compared with WT plants, which may reflect compensatory regulation of lignin and cellulose deposition. We also measured the lignin monomer composition of the RNAi plants by GC-MS and determined that transgenic plants had a 57.08% higher S/G ratio than WT plants. In addition, histological staining of lignin with Wiesner reagent produced slightly more coloration in the xylem and sclerenchyma than WT plants. These results provide a foundation for breeding maize with low-lignin content and reveal novel insights about lignin regulation via genetic manipulation of CCoAOMT expression.     

Co-inoculation of Ceratocystis polonica and Heterobasidion annosum with callus of two Norway spruce clones with different in vivo susceptibility

We have studied how callus cultures from two clones of Norway spruce influence the growth of two pathogens, Ceratocystis polonica and Heterobasidion annosum, when co-cultivated in vitro. In field experiments, trees of clone 409 were susceptible to both fungi, whereas clone 589 was less affected. Callus was cultured on medium containing cytokinins (benzylaminopurine, kinetin) and with or without auxin (2,4-dichlorophenoxyacetic acid). For co-cultivation with fungus, one piece of callus was placed towards the edge of each Petri dish. One and 14 days after inoculation with callus the dishes were co-inoculated with the fungus. Both clones strongly stimulated the initial growth of both fungi. Clone 589 inhibited the growth of both fungi when the fungi were inoculated one day after the callus. When the callus was cultured on medium without auxin for 14 days before co-inoculation clone 589 strongly inhibited the growth of both fungi, whereas clone 409 inhibited H. annosum only. This revised version was published online in June 2006 with corrections to the Cover Date.