Enzymatic saccharification of Eucalyptus bark using hydrothermal pre-treatment with carbon dioxide.

In this study, saccharification of the inner bark of Eucalyptus was carried out by enzymatic hydrolysis to produce bioethanol from non-food biomass. To enhance the accessibility of the enzyme to the polysaccharides such as cellulose and holocellulose in the cell wall of the bark, the bark was subjected to hydrothermal pre-treatment with carbon dioxide. This pre-treatment considerably influenced enzymatic hydrolysis. The main component (over 90%) of the generated monosaccharide was glucose, and the yield of glucose on the basis of alpha-cellulose reaches about 80%. This result suggests that the secondary wall, whose main component is cellulose, was effectively hydrolyzed by the enzyme. Microscopic analysis revealed that after pre-treatment, the phloem parenchyma cell had a considerably swollen primary wall and the phloem fibre showed many nano-clefts within its secondary wall. These structural changes appeared to promote enzymatic hydrolysis, because of high accessibility of enzymes to cellulose in the secondary wall.

Pectin may hinder the unfolding of xyloglucan chains during cell deformation: implications of the mechanical performance of Arabidopsis hypocotyls with pectin alterations.

Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on mur1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants (Col-0) and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels (approximately tensile strength) of the hypocotyls of the mutants with pectin alterations (mur1, qua2) were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for mur1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.

Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component.

Xyloglucans are the main hemicellulosic polysaccharides found in the primary cell walls of dicots and nongraminaceous monocots, where they are thought to interact with cellulose to form a three-dimensional network that functions as the principal load-bearing structure of the primary cell wall. To determine whether two Arabidopsis thaliana genes that encode xylosyltransferases, XXT1 and XXT2, are involved in xyloglucan biosynthesis in vivo and to determine how the plant cell wall is affected by the lack of expression of XXT1, XXT2, or both, we isolated and characterized xxt1 and xxt2 single and xxt1 xxt2 double T-DNA insertion mutants. Although the xxt1 and xxt2 mutants did not have a gross morphological phenotype, they did have a slight decrease in xyloglucan content and showed slightly altered distribution patterns for xyloglucan epitopes. More interestingly, the xxt1 xxt2 double mutant had aberrant root hairs and lacked detectable xyloglucan. The reduction of xyloglucan in the xxt2 mutant and the lack of detectable xyloglucan in the xxt1 xxt2 double mutant resulted in significant changes in the mechanical properties of these plants. We conclude that XXT1 and XXT2 encode xylosyltransferases that are required for xyloglucan biosynthesis. Moreover, the lack of detectable xyloglucan in the xxt1 xxt2 double mutant challenges conventional models of the plant primary cell wall.

The regulation from guaiacyl to syringyl lignin in the differentiating xylem of Robinia pseudoacacia.

13C- and deuterium (D)-labeled ferulic acid and sinapic acid ([8-(13)C, 3-OCD3]-ferulic acid and [8-(13)C, 3,5-OCD3]-sinapic acid) were administered to robinia (Robinia pseudoacacia L.) shoots. To estimate the distribution of the label from administrated ferulic or sinapic acid, continuous 50-microm-thick tangential sections cut from the cambium of robinia were subjected to lignin chemical analysis by the DFRC method. Labeled ferulic acid was incorporated into guaiacyl and syringyl lignin. The incorporation of labeled ferulic acid into syringyl units was observed only in the later stage of lignification. Labeled sinapic acid was incorporated into syringyl lignin in the early stage and the later stage of lignification. In general, syringyl lignin was deposited in the later stage of cell wall lignification. Thus, the incorporation of sinapic acid to syringyl lignin in the early stage of lignification was abnormal. Taken together, the aromatic ring-modifying reactions (the conversion from guaiacyl to syringyl moiety, including the hydroxylation and methylation) were more important for the regulation of the sinapyl alcohol biosynthesis than the reducing reactions (the reduction of acids to alcohols) in the differentiating xylem.

4-Coumarate:coenzyme A ligase in black locust (Robinia pseudoacacia) catalyses the conversion of sinapate to sinapoyl-CoA.

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.

Multiform biosynthetic pathway of syringyl lignin in angiosperms.

To clarify the pathway for biosynthesis of sinapyl alcohol in angiosperms, tracer experiments using stable isotopes were performed on robinia ( Robinia pseudoacacia L.), oleander ( Nerium indicum Mill.), magnolia ( Magnolia kobus DC.) and Arabidopsis thaliana (L.) Heynh. Precursors used in the experiment were (13)C- and (2)H ( D)-labeled [8-(13)C, 3-OCD(3)]ferulic acid and [8-(13)C, 3,5-OCD(3)]sinapic acid. The incorporation of labeled precursor into lignin was confirmed by gas chromatography-mass spectrometry of the products of derivatization followed by reductive cleavage. Crude extracts of differentiating xylem or stems from these plants were also assayed for 4-coumarate-CoA ligase (4CL; EC 6.2.1.12) activity using sinapic acid and ferulic acid as substrates. In robinia and oleander, 4CL activity toward sinapic acid was detected, and labeled sinapic acids were incorporated into syringyl lignin. These results indicate that robinia and oleander have a pathway that produces sinapyl alcohol from sinapic acid via sinapoyl-CoA. By contrast, in magnolia and Arabidopsis, 4CL activity toward sinapic acid could not be detected, and labeled sinapic acid was not incorporated into lignin. These results suggest that syringyl lignin biosynthesis in angiosperms operates via multiple pathways that depend on the species.

Evidence for the biosynthetic pathway from sinapic acid to syringyl lignin using labeled sinapic acid with stable isotope at both methoxy groups in Robinia pseudoacacia and Nerium indicum.

A tracer experiment using synthesized labeled lignin precursors was designed to confirm the actual biosynthetic pathway for syringyl lignin. Tetradeuteroferulic acid-[8-D, 3-OCD(3)] and heptadeuterosinapic acid-[8-D, 3,5-OCD(3)] were synthesized and fed to shoots of robinia (Robinia pseudoacacia) and oleander (Nerium indicum) trees. The incorporation of each labeled precursor into lignin was traced by gas chromatography-mass spectrometry. The synthesized sinapic acid, in which both methoxy groups were labeled, was useful in monitoring the conversion of sinapic acid into syringyl lignin. When heptadeuterosinapic acid was fed, syringyl units containing seven deuterium labels were detected. The results of this study support the traditionally accepted pathway that sinapic acid is converted to sinapyl alcohol via sinapoyl-CoA in robinia and oleander.