Structure of lignins in developing xylem of Norway spruce

The developing xylem in a Norway spruce (Picea abies) clone was investigated during a growth season and compared to lignin from sapwood of the same tree clone. Klason and acid-soluble lignin contents were determined as well as the carbohydrate monomer distribution and protein content. By analyzing lignin thioacidolysis products, it was shown that only guaiacyl units could be detected in the materials, and the relative amount of beta-O-4' bonds was assessed. Monomeric and selected dimeric lignin products were identified by mass spectrometry. The specimens were embedded and thin sections examined by microscopy to determine the state of cell differentiation in the samples. In the spring and early summer, growth was very rapid and the intention was to collect tissue in which exclusively the middle lamella/primary cell wall had begun to lignify. Combining data regarding Klason lignin, protein content and carbohydrate monomer distribution with microscopy, it was found that the developing xylem sample from mid-June contained lignin from exclusively middle lamella/primary wall. The Klason lignin content in the developing xylem during the growth season was 20%, 5% and 10% in April, June and August, respectively. Thioacidolysis showed that the lignin had more condensed structures than lignin from the reference Norway spruce clone wood. Mass spectrometry showed that the developing xylem specimens from June and August 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 more in a bulk fashion during the summer.     

Concomitant analysis of cambial abscisic acid and cambial growth activity in poplar

Endogenous levels of cambial region abscisic acid (ABA) were quantified by immunoassay and assessed together with cambial growth activity in poplar (Populus nigra L. × P. maximowiczii Henry, clone Kamabuchi) over the course of a growing season. The level of cambial region ABA increased from spring to late-summer but decreased sharply in autumn. Cambial growth activity, measured as the radial number of undifferentiated cambial cells and enlarging xylem cells, also increased from spring to summer and decreased sharply in autumn, indicating the onset of cambial dormancy. Exogenous ABA, applied laterally to poplar stems at two times within the growing season, enhanced cambial growth activity, as the radial number of undifferentiated cambial cells increased in ABA-treated trees subsequent to the two application times. Xylem cell development was also affected by exogenous ABA as fibre length increased significantly in ABA-treated trees at both application times. The positive correlation of cambial region ABA and cambial growth activity as well as the positive effects of exogenous ABA application thereon sheds new light on the role of this hormonal growth regulator.     

杜仲次生木质部分化过程中木质素与半纤维素组分在细胞壁中分布的动态变化

利用紫外光显微镜、透射电子显微镜结合免疫胶体金标记,研究了杜仲（Eucommia ulmoides Oliv.)次生木质部分化过程中木质素与半纤维素组分（木葡聚糖和木聚糖）在细胞壁分布的动态变化。在形成层及细胞伸展区域,细胞壁具有木葡聚糖的分布,而没有木聚糖和木质素沉积,随着次生壁S1层的形成,木质素出现在细胞角隅和胞间层,木聚糖开始出现在S1层中,此时木葡聚糖则分布在初生壁和胞间层;随着次生,壁S2层及S3层的形成和加厚,木质逐逐步由细胞角隅和胞间层扩展到S1、S2和S3层,其沉积呈现出不均匀的块状或片状沉积模式,在次生壁各层形成与其木质化的同时,木聚糖逐渐分布于整个次生壁中,而木糖聚糖仍局限分布于初生壁和胞间层。结果表明,随着细胞次生壁的形成与木质化,细胞壁结构发生较大变化。细胞壁的不同区域,如细胞角隅、胞间层、初生壁和次生壁各层,具有不同的半纤维素组成,其与木质等细胞壁组分结构构成不同的细胞壁分子结构。     

Dynamic Changes in Distribution of Lignin and Hemicelluloses in Cell Walls During Differentiation of Secondary Xylem in Eucommia ulmoides (in English)

The dynamic changes in the distribution of lignin and hemicelluloses (xylans and xyloglucans) in cell walls during the differentiation of secondary xylem in Eucommia ulmoides Oliv. were studied by means of ultraviolet light microscopy and transmission electron microscopy combined with immunogold labelling. In the cambial zone and cell expansion zone, xyloglucans were localized both in the tangential and radial walls, but no xylans or lignin were found in these regions. With the formation of secondary wall S1 layer, lignin occurred in the cell corners and middle lamella, while xylans appeared in S1 layer, and xyloglucans were localized in the primary walls and middle lamella. In pace with the formation of secondary wall S2 and S3 layer, lignification extended to S1, S2 and S3 layer in sequence, showing a patchy style of lignin deposition. Concurrently, xylans distributed in the whole secondary walls and xyloglucans, on the other hand, still localized in the primary walls and middle lamella. The results indicated that along with the formation and lignification of the secondary wall, great changes had taken place in the cell walls. Different parts of cell walls, such as cell corners, middle lamella, primary walls and various layers of secondary walls, had different kinds of hemicelluloses, which formed various cell wall architecture combined with lignin and other cell wall components.     

Molecular cloning and characterization of a cDNA encoding poplar UDP-glucose dehydrogenase,a key gene of hemicellulose/pectin formation

Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of newly formed cell walls. A cDNA clone (Ugdh) corresponding to UGDH was isolated from a cDNA library prepared from cambial zone of poplar (Populus tremula x tremuloides). Within the 1824-nucleotide (nt)-long clone, an open reading frame encoded a protein of 481 amino acids (aa), with a calculated molecular weight of 53.1 kDa. The derived aa sequence showed 90% and 63% identity with UGDHs from soybean and bovine liver, respectively, and had highly conserved aa motifs believed to be of importance for nt binding and catalytic efficiency. In poplar, the Ugdh corresponds to one or two genes, as found by genomic Southern analysis. The gene was expressed predominantly in differentiating xylem and young leaves, with little expression in the phloem zone of the stem. The expression pattern matched that of UGDH protein, as found by immunoblotting. In leaves, the Ugdh expression was upregulated by a short-term feeding with sucrose, sorbitol and polyethylene glycol, and this effect was to some extent mimicked by light exposure. The data suggest that Ugdh is regulated via an osmoticum-dependent pathway, possibly related to the availability of osmotically active carbohydrate precursors to UDP-glucose, a substrate of UGDH.     

Wood ontogeny during the first year of hybrid poplar development

During the first year of hybrid poplar development, we assessed radial growth dynamics quantified by the proportion of secondary xylem tissue within the stem area, the vessel area percentage, the content of both lignin and cellulose, the lignin monomeric composition, and the macromolecular properties of cellulose. The intraannual radial growth dynamics in the proportion of secondary xylem tissue was fitted by the Gompertz regression line whereas changes in the vessel area percentage were fitted maximally by a cubic regression line. Under constant temperature and photoperiod, this study revealed that nonlinear patterns of radial growth dynamics are the result of a developmental programme which drives cambial activity and ageing. The increased proportion of guaiacyl units found may be important for the greater stability of the lignin structure in the first year of hybrid poplar development. The tensile strength of juvenile wood was ensured by the trade-off between a slight increase in the degree of polymerization of cellulose and a slight decrease in the content of cellulose during ageing.     

Topochemical studies on modified lignin distribution in the xylem of Poplar (Populus spp.) after wounding

BACKGROUND AND AIMS: Information on the influence of wounding on lignin synthesis and distribution in differentiating xylem tissue is still scarce. The present paper provides information on cell modifications with regard to wall ultrastructure and lignin distribution on cellular and subcellular levels in poplar after wounding. METHODS: Xylem of Populus spp. close to a wound was collected and processed for light microscopy, transmission electron microscopy and cellular UV microspectrophotometry. Cell wall modification with respect to lignin distribution was examined at different stages of wound tissue development. Scanning UV microspectrophotometry and point measurements were used to determine the lignin distribution. KEY RESULTS: Xylem fibres within a transition zone between differentiated xylem laid down prior to wounding and the tissues formed after wounding developed distinctively thickened secondary cell walls. Those modified walls and cell corners showed, on average, a higher lignin content and an inhomogeneous lignin distribution within the individual wall layers. CONCLUSIONS: The work presented shows that wounding of the xylem may induce a modified wall architecture and lignin distribution in tissues differentiating at the time of wounding. An increasing lignin content and distinctively thickened walls can contribute to improved resistance as part of the compartmentalization process.     

Potassium-dependent cambial growth in poplar

To study the involvement of potassium in wood formation, poplar plants ( Populus tremula L. x Populus tremuloides Michx.) were grown over a period of one growing season, under different potassium regimes. Seasonal changes in cambial potassium content, osmotic potential, and cambial activity correlated strongly throughout the season, increasing from spring to summer and decreasing from summer to autumn. Moreover, changing the potassium supply during the growing season affected the seasonal changes of these parameters in a similar way. Low potassium supply markedly reduced cambial activity, the number of expanding cambial cell derivatives, the seasonal rate of radial wood increment, and the vessel frequency. The possible effect of hormones on potassium-dependent cambial growth was investigated and revealed that abscisic acid (ABA) strongly decreased the potassium content within the cambial zone and reduced cambial activity, as well as the number of expanding cambial cell derivatives. In summary, our results indicate a key role for potassium in the regulation of cambial growth and wood formation due to its strong impact on osmoregulation in expanding cambial cells. They also demonstrate involvement of ABA in regulation of potassium-dependent cambial growth.     

Structural Alterations of Lignins in Transgenic Poplars with Depressed Cinnamyl Alcohol Dehydrogenase or Caffeic Acid O-Methyltransferase Activity Have an Opposite Impact on the Efficiency of Industrial Kraft Pulping

We evaluated lignin profiles and pulping performances of 2-year-old transgenic poplar (Populus tremula × Populus alba) lines severely altered in the expression of caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) or cinnamyl alcohol dehydrogenase (CAD). Transgenic poplars with CAD or COMT antisense constructs showed growth similar to control trees. CAD down-regulated poplars displayed a red coloration mainly in the outer xylem. A 90% lower COMT activity did not change lignin content but dramatically increased the frequency of guaiacyl units and resistant biphenyl linkages in lignin. This alteration severely lowered the efficiency of kraft pulping. The Klason lignin level of CAD-transformed poplars was slightly lower than that of the control. Whereas CAD down-regulation did not change the frequency of labile ether bonds or guaiacyl units in lignin, it increased the proportion of syringaldehyde and diarylpropane structures and, more importantly with regard to kraft pulping, of free phenolic groups in lignin. In the most depressed line, ASCAD21, a substantially higher content in free phenolic units facilitated lignin solubilization and fragmentation during kraft pulping. These results point the way to genetic modification of lignin structure to improve wood quality for the pulp industry.     

Seasonal development of secondary xylem and phloem in <Emphasis Type="Italic">Schizolobium parahyba</Emphasis> (Vell.) Blake (Leguminosae: Caesalpinioideae)

The cambial activity and periodicity of secondary xylem and phloem formation have been less studied in tropical tree species than in temperate ones. This paper describes the relationship between seasonal cambial activity, xylem and phloem development, and phenology in Schizolobium parahyba, a fast growing semideciduous seasonal forest tree from southeastern Brazil. From 2002 to 2003, wood samples were collected periodically and phenology and climate were recorded monthly in the same period. S. parahyba forms annual growth increments in wood, delimited by narrow initial parenchyma bands. The reduction of the cambial activity to a minimum correlates to the dry season and leaf fall. The higher cambial activity correlates to the wet season and the presence of mature leaves. In phloem, a larger conductive region was observed in the wet season, when the trees were in full foliage. The secondary phloem did not exhibit any incremental zone marker; however, we found that the axial parenchyma tends to form irregular bands.     

毛白杨维管形成层带细胞超微结构的季节性变化

木材(次生木质部)是树木形成层细胞分化的产物,形成层的活动方式不仅影响木材的产量,而且影响木材的结构和性质.利用透射电子显微镜观察了生长在北京地区的毛白杨(Populus tomentosa Carr.)枝条形成层带细胞一个完整活动周期的超微结构变化.在木质部母细胞完全恢复活动之前,形成层纺锤状原始细胞的分裂和韧皮部细胞的分化已经开始.枝条上芽的展开和幼叶的生长可能决定了形成层带细胞的这种活动方式.透射电镜观察更清楚地揭示了树木形成层细胞在活动初期的分化特点.活动期形成层细胞中的大液泡在进入休眠期后逐渐分成许多小液泡分散在细胞质中.随着液泡融合逐渐消失的深色蛋白类物质又重新充满了大部分液泡.油滴和淀粉颗粒的年变化情况同液泡中的蛋白类物质基本相似.无论在活动期还是休眠期,形成层纺锤形细胞的质膜上都发现有许多可能与物质运输有关的小泡状内折.由核膜、内质网和高尔基体及其分泌小泡组成的细胞内膜系统,在形成层活动周期的不同阶段,其形态和分布明显不同,尤其在形成层细胞的恢复活动及其衍生木质部细胞次生壁的沉积过程中发挥着重要作用.整个活动周期中,形成层纺锤形细胞的径向壁都比弦向壁厚,处在休眠期的形成层带细胞,其径向壁与弦向壁的差别则更明显.形成层恢复活动时,径向壁上特别是与弦向壁相连的角隅处出现部分自溶现象.细胞壁特别是径向壁的变薄是形成层细胞恢复活动的重要特征.     

Over-expression of a putative poplar glycosyltransferase gene, PtGT1, in tobacco increases lignin content and causes early flowering

Family 1 glycosyltransferases catalyse the glycosylation of small molecules and play an important role in maintaining cell homeostasis and regulating plant growth and development. In this study, a putative glycosyltransferase gene of family 1, PtGT1, was cloned from poplar (Populus tomentosa Carr.). Sequence analysis showed that this gene encodes a protein of 481 amino acid residues with a conserved PSPG box at its C-terminal, suggesting that it is active in the glycosylation of plant secondary products. The PtGT1 gene was expressed in poplar stems and leaves, with a particularly high expression level in elongating stems. Transgenic tobacco plants ectopically over-expressing PtGT1 were obtained and phenotypes were analysed. Wiesner and M?ule staining showed that stem xylem of transgenic tobacco plants stained more strongly than controls. Measurement of the Klason lignins showed much higher lignin content in the transgenic lines than in control plants. Furthermore, the ectopic over-expression of PtGT1 in tobacco resulted in an early flowering phenotype. These findings offer a possible starting point towards better understanding of the function of poplar PtGT1, and provide a novel strategy for lignin engineering and flowering control in plants through the genetic manipulation of a poplar glycosyltransferase gene.     

Fluctuations of cambial activity in relation to precipitation result in annual rings and intra-annual growth zones of xylem and phloem in teak (Tectona grandis) in Ivory Coast

Background and Aims Teak forms xylem rings that potentially carry records of carbon sequestration and climate in the tropics. These records are only useful when the structural variations of tree rings and their periodicity of formation are known. Methods The seasonality of ring formation in mature teak trees was examined via correlative analysis of cambial activity, xylem and phloem formation, and climate throughout 1·5 years. Xylem and phloem differentiation were visualized by light microscopy and scanning electron microscopy. Key Results A 3 month dry season resulted in semi-deciduousness, cambial dormancy and formation of annual xylem growth rings (AXGRs). Intra-annual xylem and phloem growth was characterized by variable intensity. Morphometric features of cambium such as cambium thickness and differentiating xylem layers were positively correlated. Cambium thickness was strongly correlated with monthly rainfall (R(2) = 0·7535). In all sampled trees, xylem growth zones (XGZs) were formed within the AXGRs during the seasonal development of new foliage. When trees achieved full leaf, the xylem in the new XGZs appeared completely differentiated and functional for water transport. Two phloem growth rings were formed in one growing season. Conclusions The seasonal formation pattern and microstructure of teak xylem suggest that AXGRs and XGZs can be used as proxies for analyses of the tree history and climate at annual and intra-annual resolution.     

EFFECT OF ISOLATION OF BARK ON CAMBIAL ACTIVITY AND DEVELOPMENT OF XYLEM AND PHLOEM IN TREMBLING ASPEN

Circular patches of bark were surgically isolated on the sides of trembling aspen (Populus tremuloides Michx.) trees at breast height at various times during the dormant and growing seasons. Subsequently, samples of wood and attached bark were taken from isolated and control sites to determine the effects of isolation of the bark on cambial activity and xylem and phloem development. In control trees cambial activity and xylem and phloem development occurred normally. Isolation of bark during the dormant season (in November, February, or March) did not prevent initiation of cambial activity and of phloem differentiation in spring but continued normal cambial activity and phloem developmented were prevent. Xylem differentiation was essentially prevented by isolation of tissues during the dormant season. The ultimate effect of isolation of the bark on the cambium, either during the dormant season or during the growing season, was subdivision of all fusiform cambial cells into strands of parenchymatous elements; the ultimate effect on the newly formed phloem was early death of the sieve elements. The most conspicuous effect of isolation of the bark after xylem differentiation had begun was the curtailment of secondary wall formation. Shortening of cells of the cambial region was reflected in the length of the vessel members which differentiated from such cells. These results indicate that normal cambial activity and xylem and phloem development require a supply of currently translocated regulatory substances from the shoots.     

Dirigent proteins and dirigent sites in lignifying tissues.

Tissue-specific dirigent protein gene expression and associated dirigent (site) localization were examined in various organs of Forsythia intermedia using tissue printing, in situ mRNA hybridization and immunolabeling techniques, respectively. Dirigent protein gene expression was primarily noted in the undifferentiated cambial regions of stem sections, whereas dirigent protein sites were detected mainly in the vascular cambium and ray parenchyma cell initials. Immunolocalization also revealed cross-reactivity with particular regions of the lignified cell walls, these being coincident with the known sites of initiation of lignin deposition. These latter regions are considered to harbor contiguous arrays of dirigent (monomer binding) sites for initiation of lignin biopolymer assembly. Dirigent protein mRNA expression was also localized in the vascular regions of roots and petioles, whereas in leaves the dirigent sites were primarily associated with the palisade layers and the vascular bundle. That is, dirigent protein mediated lignan biosynthesis was initiated primarily in the cambium and ray cell initial regions of stems as well as in the leaf palisade layers, this being in accordance with the occurrence of the lignans for defense purposes. Within lignified secondary xylem cell walls, however, dirigent sites were primarily localized in the S(1) sublayer and compound middle lamella, these being coincident with previously established sites for initiation of macromolecular lignin biosynthesis. Once initiation occurs, lignification is proposed to continue through template polymerization.     

Non-uniform distribution and seasonal variation of endogenous indol-3yl-acetic acid in the cambial region of Pinus contorta Dougl.

Endogenous, free indol-3yl-acetic acid (IAA) levels were measured in the main stem in the 10-year-old cambial zone, in the adjoining differentiating xylem, and in the adjoining mature xylem of 15–20-year-old Pinus contorta Dougl. by single-ion-current monitoring, combined gas chromatography — mass spectrometry, on several dates from early spring to early winter. Microscopy was used to determine the state of cambial activity on each harvest date. The IAA levels were found to be nearly constant at 1 g g^-1 DW in the cambial zone from March to July, then to increase to near 2 g g^-1 DW during the remainder of the growth season. No clear correlation was evident between number of fusiform cells per radial file and IAA content in the cambial zone. By contrast, the IAA content in differentiating xylem was higher than that in the adjoining meristematic zone on all harvest dates and also exhibited marked seasonal variation, peaking near 16 g g^-1 DW in mid summer, and declining to 1 g g^-1 DW in autumn. In mature xylem, IAA levels were very low and showed negligible variation. The fresh weight to dry weight ratio of differentiating xylem was greater than that of the cambial zone, and greater in the cambial zone than in mature xylem.     

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.     

Seasonal changes in carbohydrate reserves in mature northern<Emphasis Type="Italic"> Populus tremuloides</Emphasis> clones

To assess the changes in seasonal carbohydrate status of Populus tremuloides, sugar and starch concentrations were monitored in roots, stem xylem and phloem and branches of ten different clones. Time of root growth was assessed by extraction of roots from in-growth cores collected five times during the season. Overall the results showed that the main period of root growth in these northern clones was shifted from spring to late summer and fall likely due to the microclimatic conditions of the soil. This increase in root growth was associated with a decline in total non-structural carbohydrate content in the roots during this period. This study also found that the carbohydrate reserves in these clones were being stored as close as possible to the organs of annual growth (leaves and roots). At the time of leaf flush, the largest reduction in stored carbohydrates (3% of dry weight) was observed in the branches of the trees, compared to a slight decline in the stem and roots. Starch and sugar reserves in most tissues were very low in early summer. This suggests that reserves that might be used for the regrowth of foliage after insect defoliation or other disturbances, are relatively small compared to the portion that is needed for maintenance and typical growth developments such as leaf flush.     

Effect of local heating and cooling on cambial activity and cell differentiation in the stem of Norway spruce (Picea abies)

BACKGROUND AND AIMS The effect of heating and cooling on cambial activity and cell differentiation in part of the stem of Norway spruce (Picea abies) was investigated. METHODS: A heating experiment (23-25 degrees C) was carried out in spring, before normal reactivation of the cambium, and cooling (9-11 degrees C) at the height of cambial activity in summer. The cambium, xylem and phloem were investigated by means of light- and transmission electron microscopy and UV-microspectrophotometry in tissues sampled from living trees. KEY RESULTS: Localized heating for 10 d initiated cambial divisions on the phloem side and after 20 d also on the xylem side. In a control tree, regular cambial activity started after 30 d. In the heat-treated sample, up to 15 earlywood cells undergoing differentiation were found to be present. The response of the cambium to stem cooling was less pronounced, and no anatomical differences were detected between the control and cool-treated samples after 10 or 20 d. After 30 d, latewood started to form in the sample exposed to cooling. In addition, almost no radially expanding tracheids were observed and the cambium consisted of only five layers of cells. Low temperatures reduced cambial activity, as indicated by the decreased proportion of latewood. On the phloem side, no alterations were observed among cool-treated and non-treated samples. CONCLUSIONS: Heating and cooling can influence cambial activity and cell differentiation in Norway spruce. However, at the ultrastructural and topochemical levels, no changes were observed in the pattern of secondary cell-wall formation and lignification or in lignin structure, respectively.