Kei'ichi Baba

Xyloglucan for Generating Tensile Stress to Bend Tree Stem

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase (XET) activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide (XXXG) for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer microfibrils.

Loosening xyloglucan accelerates the enzymatic degradation of cellulose in wood.

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrils was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.

Overexpression of Poplar Cellulase Accelerates Growth and Disturbs the Closing Movements of Leaves in Sengon

In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.

Cellulose metabolism in plants.

Many bacterial genomes contain a cellulose synthase operon together with a cellulase gene, indicating that cellulase is required for cellulose biosynthesis. In higher plants, there is evidence that cell growth is enhanced by the overexpression of cellulase and prevented by its suppression. Cellulase overexpression could modify cell walls not only by trimming off the paracrystalline sites of cellulose microfibrils, but also by releasing xyloglucan tethers between the microfibrils. Mutants for membrane-anchored cellulase (Korrigan) also show a typical phenotype of prevention of cellulose biosynthesis in tissues. All plant cellulases belong to family 9, which endohydrolyzes cellulose, but are not strong enough to cause the bulk degradation of cellulose microfibrils in a plant body. It is hypothesized that cellulase participates primarily in repairing or arranging cellulose microfibrils during cellulose biosynthesis in plants. A scheme for the roles of plant cellulose and cellulases is proposed.

Cloning of a lectin cDNA and seasonal changes in levels of the lectin and its mRNA in the inner bark ofRobinia pseudoacacia

A cDNA clone encoding a lectin was isolated by immunological screening of an expression library prepared from poly(A)+ RNA from the inner bark ofRobinia pseudoacacia. The cDNA clone (RBL104) had an open reading frame of 858 bp that encoded a polypeptide with a predicted molecular weight of 31210. This molecular weight corresponded closely to that of a polypeptide immunoprecipitated from products of translationin vitro of the poly(A)+ RNA. Thus, RBL104 appeared to be a full-length cDNA. The N-terminal amino acid sequence of the purified lectin protein matched a portion of the predicted amino acid sequence. It appeared that the lectin was synthesized as a precursor that consisted of a putative signal peptide of 31 amino acids and a mature polypeptide of 255 amino acids. Southern blot analysis of the genomic DNA revealed that the lectin was encoded by a small multigene family. The lectin was mostly localized in the axial and ray parenchymal cells of the inner bark. A small amount of lectin was also found in the axial and ray parenchymal cells of the xylem. The lectin accumulated in the inner bark in September, remained at high levels during the winter and disappeared in May. The mRNA for the lectin was detected from August to the following March. The appearance and disappearance of the mRNA were observed prior to those of the lectin protein.

Developmental changes in the bark lectin of Sophora japonica L.

Lectin is the major protein in the phloem tissue of S. japonica. By immunohistochemistry using anti-seed lectin antibody it was demonstrated that the lectin was localized in the ray and the axial parenchyma. Neither lectin nor other cross-reactive materials were observed in the cambium, sieve tubes and companion cells. The distribution and localization changed in relation to tissue development. Lectin content in the bark changed during the year, the average in summer being about 50% of that in winter. The distribution of lectin in the bark in winter was similar from the innermost (youngest) to the outermost (oldest) region. In contrast, in summer the innermost region hardly contained any lectin, and the outermost region contained less lectin than the middle. Lectin localization in tissues and cells differed also depending on tissue age. In new tissue, produced in the current year, lectip was absent in summer, was located in the cytoplasmic layer between cell wall and vacuole in autumn, and sequestered in the vacuoles in winter. On the other hand, lectin in old tissue (formed in the previous year) was located throughout the year mainly within the vacuoles, with only very small contents in the cytoplasmic layer in autumn. Within the outermost (oldest) region, in which the lectin content was low in summer, the cells which bordered the outer bark never contained any lectin in summer. The intracellular localization in autumn in new tissue, determined by immunogold electron microscopy, was in the lumen of the endoplasmic reticulum and vesicles, with gold particles hardly present in the cytoplasm. From these findings we conclude that lectin is synthesized on the endoplasmic reticulum and most vigorously in the new tissue in autumn, and that it is mainly consumed in the outermost bark regions, where dilatation occurs and-or where cork cambium is differentiated.

Relation between developmental changes on anatomical structure and on protein pattern in differentiating xylem of tension wood

Tension wood was induced inEucalyptus camaldulensis L. by fixing the stem at an angle. Proteins in the differentiating tissue of tension wood were compared to those of normal wood on sodium dodecyl sulfate polyacrylamide gel electrophoresis. An obvious difference was found in the salt-soluble fraction of 14 days after inclination. At least five bands (19,22,37,41, and 55kDa) were specific in the differentiating tissue of tension wood. These proteins were bound to the cell wall, plasma membrane, or both by their electric charge; they were undetectable until 14 days after inclination. Mature tension wood was observed in the tissue at 14 days. Thus, all differentiating tissue at 14 days was produced after inclination. On the other hand, the differentiating zone at 7 days contained the same tissue, as in tension wood estimated by the vessel number and diameter during the early phase; and the tissue was indistinguishable from normal wood during the late phase. The proteins found here were related to the phenomenon occurring in the late stage of xylem differentiation.

Wood identification of JapaneseCyclobalanopsis species (Fagaceae) based on DNA polymorphism of the intergenic spacer betweentrnT andtrnL 5′ exon

DNA was extracted from wood samples of six representativeCyclobalanopsis species (Fagaceae) growing in Japan that cannot be distinguished from one another by conventional microscopy. A part of the intergenic spacer region betweentrnT andtrnT 5′ exon was amplified and sequenced. The sequences obtained from wood samples were grouped into three DNA types by a single nucleotide polymorphism as reported previously in leaf samples: I (Quercus acuta, Q. sessilifolia, Q. salicina), II (Q. myrsinaefolia, Q. glauca), and III (Q. gilva). Thus,Q. gilva can be distinguished from the otherQuercus species, and the others are separated in two subgroups based on DNA polymorphism. The present findings support the possibility of wood identification based on DNA polymorphism.

Loosening xyloglucan prevents tensile stress in tree stem bending but accelerates the enzymatic degradation of cellulose

In response to environmental variation, xyloglucan could fix the microfibrils to the inner surface of the wall to withstand the tensile stress generated within the G-layer. This would explain why the basal regions of stems of transgenic poplars overexpressing xyloglucanase could not bend upward. This finding has ramifications for the production of bioethanol, which requires tree cellulose to be enzymatically hydrolyzed. The level of cellulose degradation with enzymes was markedly increased in the xylem overexpressing xyloglucanase. We propose that xyloglucan serves as a key hemicellulose and a tightening tether of cellulose microfibrils in the secondary walls.

Enlargement of individual cellulose microfibrils in transgenic poplars overexpressing xyloglucanase

Holocellulose samples prepared from transgenic poplars overexpressing xyloglucanase had crystal widths of 3.2–3.5 nm as a result of the (2 0 0) plane, based on their X-ray diffraction patterns, and crystal widths were greater than those of the wild type (3.0 nm). Cellulose microfibril widths in the holocellulose samples were further determined from transmission electron microscopic (TEM) images of individualized fibrils prepared by 2,2,6,6-tetramethylpiperidine-1-oxy radical-mediated oxidation of the holocelluloses and the successive disintegration of the oxidized products in water. The TEM images also supported the finding that cellulose microfibril widths of transgenic poplars were larger than those of the wild type. The cellulose microfibril widths of transgenic poplars were approximately 6 nm, whereas those of the wild type were about 5 nm. However, such enlargement of cellulose microfibril widths could not be explained by the increased cellulose contents of the transgenic poplars alone.