Gene expression in tension wood and bast fibres

Tension wood is produced in the xylem of some angiosperm trees, such as poplar (Populus spp.), whereas bast fibers are phloem-derived cells best known from annual crops, such as flax (Linum usitatissimum L.). Despite their different origins, secondary walls of both tension wood and bast fibers share distinctive properties, including an abundance of axially oriented, crystalline cellulose produced in a distinctive gelatinous-type layer. Because of these unique properties, tension wood and phloem fibers have separately been the subject of at least nine previously published gene or protein profiling studies. Here we review these experiments with a focus on those genes, whose expression distinguishes both tension wood and bast fibers from the more predominant types of xylem found elsewhere in the stem. Notable among these is an evolutionarily distinctive group of fasciclin-like arabinogalactan proteins (FLA) and a putative rhamnogalacturonan lyase.     

A new cellulose synthase gene (PtrCesA2) from aspen xylem is orthologous to Arabidopsis AtCesA7 (irx3) gene associated with secondary cell wall synthesis

We report here the molecular cloning and characterization of a new full-length cellulose synthase (CesA) cDNA, PtrCesA2 from aspen (Populus tremuloides) trees. The predicted PtrCesA2 protein shows a high degree of identity/similarity (87%/91%) to the predicted gene product of Arabidopsis AtCesA7 gene that has been associated with secondary cell wall development. Previously, a mutation in AtCesA7 gene (irx3) was correlated with a significant decrease in the amount of cellulose synthesized (about 70%) and genetic complementation of irx3 mutant with a wild-type AtCesA7 gene restored the normal phenotype. This is the first report of a full-length AtCesA7 ortholog from any non-Arabidopsis species. Interestingly, PtrCesA2 shares only 64% identity with our earlier reported PtrCesA1 from aspen suggesting its structural distinctness from the only other known CesA member from the aspen genome. PtrCesA1 is a xylem-specific and tension stress responsive gene that is highly similar to another Arabidopsis gene, AtCesA8 which also has been associated with secondary wall development. Moreover, AtCesA7 and AtCesA8 are suggested to be part of the same cellulose synthase complex. Isolation of PtrCesA2 from a xylem library enriched in cells with active secondary wall synthesis, PtrCesA2 expression levels similar to PtrCesA1 and high similarity of PtrCesA1 and PtrCesA2 to AtCesA8 and AtCesA7, respectively, suggest that both these aspen genes might be involved in the secondary wall development in aspen woody tissues. Availability of two aspen CesA orthologs will now enable us to examine if PtrCesA1 and PtrCesA2 functionally interact during aspen wood development that has long-term implications on genetic improvement of forest trees.     

Hierarchical structure of juvenile hybrid aspen xylem revealed using X-ray scattering and microtomography

X-ray scattering and microtomography (μCT) are useful techniques to reveal the structure of wood at the nano- and micrometer scales. The nanostructure of xylem in greenhouse-grown 2.5- to 3.5-month-old Populus tremula L.?×?tremuloides Michx. trees was characterized using wide-angle X-ray scattering (WAXS), and the cellular structure was investigated using μCT. For comparison, the nanostructure of wood in 2-year-old silver birch, Norway spruce and Scots pine saplings was determined. Based on the μCT results, the lengths of fiber lumina of the hybrid aspen saplings were shorter than any previous results on the lengths of wood fibers. The mean microfibril angles of the hybrid aspen saplings were significantly lower (8°–14°) than those of the birch, spruce and pine saplings (27°–35°) implicating that cellulose microfibrils were oriented nearly parallel to the cell axis in the young hybrid aspen saplings. Hybrid aspen saplings were found to contain tension wood based on the histochemical analysis and μCT images. However, typical tension wood properties, i.e. larger crystallite width and higher crystallinity than in normal wood, were detected only in a few hybrid aspen samples, while in most of the hybrid aspen saplings, the crystallite widths (3.0?±?0.1?nm) and the crystallinities (30?±?5?%) corresponded to those of normal wood. The deformations of cellulose crystallites were determined using WAXS in situ upon dehydration of the never-dried samples. In all the species studied, the cellulose unit cell dimension decreased and disorder of cellulose chains increased parallel to the chains upon drying. Also, the transverse disorder of chains increased in birch, spruce and pine, while no changes were detected in this direction in hybrid aspen. The crystallite widths and drying deformation results might indicate that the gelatinous layer has not fully developed in the young hybrid aspen saplings.     

Cloning and characterization of cellulose synthase-like gene, PtrCSLD2 from developing xylem of aspen trees

Genetic improvement of cell wall polymer synthesis in forest trees is one of the major goals of forest biotechnology that could possibly impact their end product utilization. Identification of genes involved in cell wall polymer biogenesis is essential for achieving this goal. Among various candidate cell wall-related genes, cellulose synthase-like D (CSLD) genes are intriguing due to their hitherto unknown functions in cell wall polymer synthesis but strong structural similarity with cellulose synthases (CesAs) involved in cellulose deposition. Little is known about CSLD genes from trees. In the present article PtrCSLD2, a first CSLD gene from an economically important tree, aspen (Populus tremuloides) is reported. PtrCSLD2 cDNA was isolated from an aspen xylem cDNA library and encodes a protein that shares 90% similarity with Arabidopsis AtCSLD3 protein involved in root hair tip growth. It is possible that xylem fibers that also grow by intrusive tip growth may need expression of PtrCSLD2 for controlling the length of xylem fibers, a wood quality trait of great economical importance. PtrCSLD2 protein has a N-terminal cysteine-rich putative zinc-binding domain; eight transmembrane domains; alternating conserved and hypervariable domains; and a processive glycosyltransferases signature, D, D, D, QXXRW; all similar to aspen CesA proteins. However, PtrCSLD2 shares only 43-48% overall identity with the known aspen CesAs suggesting its distinct functional role in cell wall polymer synthesis perhaps other than cellulose biosynthesis. Based on Southern analysis, the aspen CSLD gene family consists of at least three genes and this gene copy estimate is supported by phylogenetic analysis of available CSLDs from plants. Moreover, gene expression studies using RT-PCR and in situ mRNA hybridization showed that PtrCSLD2 is expressed at a low level in all aspen tissues examined with a slightly higher expression level in secondary cell wall-enriched aspen xylem as compared to primary cell wall enriched tissues. Together, these observations suggest that PtrCSLD2 gene may be involved in the synthesis of matrix polysaccharides that are dominant in secondary cell walls of poplar xylem. Future molecular genetic analyses will clarify the functional significance of CSLD genes in the development of woody trees.     

巴西橡胶树HbMYB52基因的克隆及其在拟南芥中的表达

为揭示Hb MYB52在巴西橡胶树(Hevea brasiliensis)木材发育过程中的功能,从其转录组中分离克隆到1个MYB转录因子G21亚组成员基因,命名为Hb MYB52,开放阅读框为726 bp,编码242个氨基酸的蛋白,在木质部中高度表达。在拟南芥(Arabidopsis thaliana)中过表达Hb MYB52,虽未改变转基因植株株型,但植株维管束间纤维细胞壁明显增厚,同时抑制了木质纤维、导管次生壁形成。转基因拟南芥株系3和株系6中纤维素和木质素含量减少,相应各组分合成的关键酶基因的表达量也不同程度下降;株系8产生了木质素异位沉积,且木质素合成关键酶基因表达活跃。因此,推测Hb MYB52参与了植物次生壁形成调控,在拟南芥次生壁形成中可能发挥了双重功能:一方面负调控维管束次生壁形成以及各组分的生物合成,另一方面具有促进束间纤维次生壁增厚的作用。     

Induction of Tension Wood in GA3-treated Branches of the Weeping Type of Japanese Cherry, Prunus spachiana

GA₃ prevented the bending of branches in the weeping type ofJapanese cherry, Prunus spachiana. Eccentric growth of GA₃-treatedbranches was observed. In the xylem on the upper side of suchbranches the presence of gelatin fibers, which stained stronglywith fast green, was demonstrated. Moreover, a less dense distributionof vessels and a steeper angle of cellulose microfibrils inthe secondary walls of fibers were also observed on this side.Similar features were noted in the xylem of the branches ofcherry trees of the upright type, but they were not found inGA₃-untreated control branches of trees of the weeping type.These results suggest that GA₃ induces tension wood on the upperside of branches of Prunus spachiana of the weeping type, sothat the branches become to grow upwards, resembling branchesof the upright type. (Received December 19, 1994; Accepted May 29, 1995)     

Gene expression in Eucalyptus branch wood with marked variation in cellulose microfibril orientation and lacking G-layers

In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G-layer). Gene expression was studied in Eucalyptus nitens branches oriented at 45 degrees using microarrays containing 4900 xylem cDNAs, and wood fibre characteristics revealed by X-ray diffraction, chemical and histochemical methods. Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G-layers and had higher cellulose and decreased Klason lignin compared with lower branch wood. Expression of two closely related fasciclin-like arabinogalactan proteins and a beta-tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches. Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G-layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified.     

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.     

The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana

The irregular xylem 2 (irx2) mutant of Arabidopsis thaliana exhibits a cellulose deficiency in the secondary cell wall, which is brought about by a point mutation in the KORRIGAN (KOR) beta,1-4 endoglucanase (beta,1-4 EGase) gene. Measurement of the total crystalline cellulose in the inflorescence stem indicates that the irx2 mutant contains approximately 30% of the level present in the wild type (WT). Fourier-Transform Infra Red (FTIR) analysis, however, indicates that there is no decrease in cellulose in primary cell walls of the cortical and epidermal cells of the stem. KOR expression is correlated with cellulose synthesis and is highly expressed in cells synthesising a secondary cell wall. Co-precipitation experiments, using either an epitope-tagged form of KOR or IRX3 (AtCesA7), suggest that KOR is not an integral part of the cellulose synthase complex. These data are supported by immunolocalisation of KOR that suggests that KOR does not localise to sites of secondary cell wall deposition in the developing xylem. The defect in irx2 plant is consistent with a role for KOR in the later stages of secondary cell wall formation, suggesting a role in processing of the growing microfibrils or release of the cellulose synthase complex.     

Multilayered structure of tension wood cell walls in Salicaceae sensu lato and its taxonomic significance

Salicaceae have been enlarged to include a majority of the species formerly placed in the polyphyletic tropical Flacourtiaceae. Several studies have reported a peculiar and infrequently formed multilayered structure of tension wood in four of the tropical genera. Tension wood is a tissue produced by trees to restore their vertical orientation and most studies have focused on trees developing tension wood by means of cellulose‐rich, gelatinous fibres, as in Populus and Salix (Salicaceae s.s.). This study aims to determine if the multilayered structure of tension wood is an anatomical characteristic common in other Salicaceae and, if so, how its distribution correlates to phylogenetic relationships. Therefore, we studied the tension wood of 14 genera of Salicaceae and two genera of Achariaceae, one genus of Goupiaceae and one genus of Lacistemataceae, families closely related to Salicaceae or formerly placed in Flacourtiaceae. Opposite wood and tension wood were compared with light microscopy and three‐dimensional laser scanning confocal microscopy. The results indicate that a multilayered structure of tension wood is common in the family except in Salix, Populus and one of their closest relatives, Idesia polycarpa. We suggest that tension wood may be a useful anatomical character in understanding phylogenetic relationships in Salicaceae. Further investigation is still needed on the tension wood of several other putatively close relatives of Salix and Populus, in particular Bennettiodendron, Macrohasseltia and Itoa.     

Cellulose and lignin biosynthesis is altered by ozone in wood of hybrid poplar (Populus tremula × alba)

Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula × alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 l l(-1)). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.     

Ethylene evolution changes in the stems of<Emphasis Type="Italic"> Metasequoia glyptostroboides</Emphasis> and<Emphasis Type="Italic"> Aesculus turbinata</Emphasis> seedlings in relation to gravity-induced reaction wood formation

Two-year-old Metasequoia glyptostroboides and 3-month-old Aesculus turbinata seedlings were tilted at a 45° angle to induce compression wood formation on the lower side of the former species and tension wood on the upper side of the latter. Two weeks later, the seedlings were tilted in an opposite direction at 45° so that the upper and lower sides changed to each other. This reverse tilting was kept for 7 weeks for M. glyptostroboides and 6 weeks for A. turbinata. The seedlings were sampled and analyzed at intervals throughout each experimental period so that an ethylene evolution kinetic was monitored. Ethylene evolution from the cambial region of the upper and lower sides of tilted stems was measured separately by gas chromatography with a flame ionization detector. Xylem production expressed as wood area during each experimental period was microscopically determined. In both tilting and reverse tilting periods, the rates of ethylene evolution from the lower side of M. glyptostroboides and the upper side of A. turbinata, where xylem production was accelerated and compression or tension wood formation was induced, had increased to high levels, whereas those from the opposite sides had either remained low (in tilting period) or rapidly recovered to low levels (in reverse tilting period). The cambial activity quantified by wood formation, including reaction wood, in both species showed the same tendency as ethylene evolution. The stem side with vigorous ethylene evolution, xylem development and reaction wood formation reversed with the reversal of tilting orientation. The roles of accelerated ethylene evolution in reaction wood formation in the tilted seedlings of gymnosperm and angiosperm trees are compared and discussed.This work was presented at the 5th Pacific Region Wood Anatomy Conference, Yogyakarta, Indonesia, 9–14 September 2002