Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.Suchita Bhandari and Takeshi Fujino contributed equally to this research.     

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.     

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.     

Overexpression of Poplar Xylem Sucrose Synthase in Tobacco Leads to a Thickened Cell Wall and Increased Height

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue.     

Wood cell walls: biosynthesis, developmental dynamics and their implications for wood properties

Progress has been made toward understanding the biosynthesis and modifications of the cellulose and the hemicellulose/pectin matrix of woody cell walls (and hence wood properties) by identifying 1600 carbohydrate active enzymes (CAZYmes) in Populus, and pinpointing key candidates involved in various developmental stages of wood formation. Transgenic modifications of primary wall modifying enzymes have demonstrated on the possibility of shaping the dimension of wood cells. Candidates for the biosynthesis of secondary wall matrix have been identified, and the cellulose microfibril angle of wood fibers has been modified. In addition, molecular analysis of the plastic development of wood cell walls has provided further information regarding the mechanisms regulating their structure.     

实时荧光定量分析小黑杨应拉木相关基因及转录因子的表达

利用实时定量RT-PCR技术,比较了不同时期小黑杨应拉木中与木材形成相关的基因及其转录因子的表达模式。研究结果表明,与纤维素合成相关的基因如纤维素合成酶基因（CesA）、蔗糖合成酶基因（SUSY）、ACC氧化酶基因（ACO）等基因胁迫后在弯曲茎的上侧中表达量高于其在弯曲茎的下侧中的表达;尤其是FLA基因家族在弯曲茎的上侧中大量表达,而在弯曲茎的下侧中几乎不表达。与之相反,与木质素合成相关的基因如咖啡酰辅酶A-O-甲基转移酶（CCoAOMT）、苯丙氨酸裂解酶（PAL）、漆酶（laccase）、过氧化物酶（POD）等基因在弯曲茎的上侧中表达量低而在弯曲茎的下侧中表达量高,转录因子MYB基因在弯曲茎的上侧中表达量高于其在弯曲茎的下侧中的。     

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.     

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.     

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.     

CAMBIUM, a process-based model of daily xylem development in Eucalyptus

In hardwoods such as Eucalyptus spp., xylem (wood) is a heterogeneous tissue consisting of multiple cell types. As such, xylem development involves multiple complex interactions. To describe and understand xylem development, and ultimately predict the resultant wood properties, a process-based approach to modelling wood property variation is potentially very useful. In this paper, a new model (CAMBIUM), which incorporates concepts of these processes, is described. CAMBIUM predicts how wood density and fibre and vessel anatomical properties vary from pith-to-bark at a daily time step as a function of changing environmental conditions and a set of simulated physiological processes. Simulations from an existing process-based model of stand development (CABALA) are used as inputs. A key feature of CAMBIUM is a model of the interaction between different xylem cell types. Some weaknesses were identified in the ability of the model to simulate vessel spatial patterns and frequencies, emphasizing the complexities inherent in this aspect of angiosperm wood formation. The model was, however, able to provide realistic estimates of short-term variation and temporal ranges in eucalypt fibre diameter and secondary wall development and wood density.     

Overexpression of a pectin methylesterase inhibitor in Arabidopsis thaliana leads to altered growth morphology of the stem and defective organ separation

The methylesterification status of cell wall pectins, mediated through the interplay of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), influences the biophysical properties of plant cell walls. We found that the overexpression of a PMEI gene in Arabidopsis thaliana plants caused the stems to develop twists and loops, most strongly around points on the stem where leaves or inflorescences failed to separate from the main stem. Altered elasticity of the stem, underdevelopment of the leaf cuticle, and changes in the sugar composition of the cell walls of stems were evident in the PMEI overexpression lines. We discuss the mechanisms that potentially underlie the aberrant growth phenotypes.     

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.