Yong Woo Park

Enhancement of growth and cellulose accumulation by overexpression of xyloglucanase in poplar

Because the loosening of xyloglucan in the cell wall promotes plant growth (Takeda et al. (2002) Proc. Natl. Acad. Sci. USA 99, 9055–9060; Park et al. (2003) Plant J. 33, 1099–1106), we expressed Aspergillus xyloglucanase constitutively in Populus alba. The expression increased the length of stem even in the presence of sucrose. Increased stem growth was accompanied by a decrease in Youngs elastic modulus in the growth zone but an increased elasticity in mature tissue. The increased internode length corresponded to an increase in cellulose content as well as specific gravity, showing that the removal of xyloglucan might cause an increase in cellulose density in the secondary xylem.

Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts

Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba. A vector for plastid transformation of poplar (Populus alba) was constructed, which carried the spectinomycin resistance gene and the green fluorescence protein gene as marker genes. In the regenerated shoots, the site-specific integration of foreign genes and the establishment of a high homoplastomic state were confirmed. Immunoblot analysis and histological observations corroborated the accumulation of green fluorescence protein in chloroplasts. The establishment of a plastid transformation system in poplar provides a novel tool for tree biotechnology.

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.

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.

Biosafety assessment of transgenic poplars overexpressing xyloglucanase (AaXEG2) prior to field trials

We performed biosafety assessments of transgenic poplars prior to field trials. Constitutive expression of the Aspergillus aculeatus xyloglucanase in Populus alba increased the cellulose content and specific gravity of its stem, the leaves of which were visibly greener, thicker, and smaller than those of the wild-type plant. Although the young transgenic poplars grew faster than the wild type in a growth chamber, there was no distinguishable difference in growth between the poplars when they were placed in a special screened greenhouse. Allelopathic tests showed that the transgenic poplars do not produce harmful substances. Based on all the biosafety assessments and the scientific literature on poplar species, we came to the conclusion that transgenic poplars probably do not disturb the biological diversity of the surrounding environment, even when they are submitted to field trials.