O-acetylglucuronoxylans (AcGX) are the major hemicelluloses found in the secondary cell wall of dicotyledon species. The backbone is formed by β(1→4)-linked xylopyranosyl (Xylp) residues, which are substituted by α(1→2)-linked (4-O-methyl)glucopyranosyluronic acid ((Me)GlcpA). The AcGX are also highly acetylated on the 2-O or 3-O; or both positions of Xylp units. Notably, acetylation patterns in AcGX are not well understood since they are typically destroyed during the alkaline isolation. Accurate quantitation of MeGlcpA is also challenged by the lack of commercially available MeGlcpA sources, thus the accuracy of MeGlcpA content determined with the GlcpA standard is unknown.
The current thesis established new procedures of detailed characterization of AcGX. The xylan OLIgosaccharide Mass Profiling (OLIMP) method encompassed endoxylanase hydrolysis and mass spectrometry detection. The endoxylanase cleaves the xylan backbone into acetylated xylooligosaccharides (AcXOS). As the action is hindered by the side groups, endoxylanase can be a selective tool to liberate AcXOS from plant tissues for fingerprinting the acetylation pattern in AcGX. Additionally, mass fragmentation analyses were performed to elucidate the spatial distribution of acetyl residues. The accuracy of MeGlcpA quantitation using the GlcpA standard was examined by comparing it to the in-house purified MeGlcpA in acid methanolysis and gas chromatography (GC) analysis.
Several of the genes responsible for the biosynthesis of AcGX were previously identified using Arabidopsis thaliana as the model plant. Herein, the structures of AcGX in Arabidopsis wild-type and biosynthetic mutant plants that are defective in reducing end tetrasaccharide sequence or backbone synthesis, irx7, irx9, irx10 and irx14; and (Me)GlcpA addition, gux1gux2 were analyzed using methods established for structural characterization of AcGX. Mono-acetylations (2-O or 3-O position) were reduced in irx7, irx9 and irx14, whereas 2-O acetylation was elevated in the (Me)GlcpA deficient mutant, gux1gux2, indicating that the addition of (Me)GlcpA residues is taking place before acetylation of xylans. Structural elucidation on the major AcXOS liberated from wild-type plant suggests that the acetyl residues are added in every other Xylp residue in AcGX. Interestingly, a novel pentose substitution on the GlcpA side group in AcGX was identified.
In acid methanolysis, the GlcpA standard was partially lactonized, thus yielding six derivatives in GC chromatogram. When all six GlcpA derivatives were used in the calibration curve, the MeGlcpA content was underestimated by nearly 30%. The MeGlcpA content can be closely estimated by choosing the appropriate GlcpA derivatives in the calibration curve. The method was used to investigate the impact of Schizophyllum commune glycoside hydrolase family 115 α-glucuronidase (AGU) transgene expressed in Arabidopsis. The (Me)GlcpA content in the ScAGU115 expressing plants was surprisingly unchanged despite the active recombinant enzyme present within the cell walls.
In this work, the acetylation pattern in the AcGX of Arabidopsis wild-type and mutant plants was studied in detail. The methods developed herein revealed that the acetylation of AcGX was reduced in Arabidopsis lines that encode a defective biosynthetic gene related to backbone or reducing end sequence synthesis, suggesting pleiotropic effect of a single gene mutation in xylan biosynthesis. The MeGlcA substituents in AcGX can be effectively reduced by disruption of the glucoronyltrasnferases; however, the substitution in the AcGX of gux1gux2 was compensated by acetylation. On the other hand, constitutive transgenic expression of ScAGU115 α-glucuronidase did not remove the (Me)GlcpA substituents in planta. The reason may be ascribed to the shielding by neighbouring acetyl substituents, or limited accessibility of the recombinant enzyme to cell wall substrates. Therefore, a viable approach for the effective tailoring of AcGX substituents in planta could be co-expression with an acetyl xylan esterase to obtain synergism between these side-group removing enzymes.Plant biomass represents a plentiful source of carbon on Earth that can be exploited as a renewable raw material for producing fuels, chemicals and materials. However, harnessing the greatest benefits from the structurally complex plant cell walls remains a major challenge. Secondary cell walls, the major component of plant biomass, are mainly formed by complex polysaccharides and polyphenols. These biomolecules are interacting to strengthen wall support, and also contributes to recalcitrance against extraction or decomposition by enzymatic hydrolysis. Cell wall modification via in planta engineering can be utilized to design the constituents of plant cell walls, to thereby improve the extractability of sugars and polymers, or tailor the cell wall properties. This goal is approachable either by manipulating biosynthetic genes or by expressing microbial polysaccharides-modifying enzymes in plants.
O-acetylglucuronoxylans (AcGX) are the second most abundant polysaccharides found in the secondary cell wall of dicotyledon species. The model plant, Arabidopsis thaliana has been utilized for the studies of AcGX biosynthesis and modifications. Detailed studies on the compositions and structures of AcGX are needed to detect the outcomes resulted from cell wall modification. Therefore, the current thesis established new procedures of detailed characterization of AcGX. Using the established methods, AcGX in Arabidopsis wild-type and mutant plants were examined. The results showed that the mutation of single gene involved in AcGX biosynthesis could cause multiple effects to the AcGX structures. Additionally, expressing single side group removing enzyme was ineffective to modify the substituents in AcGX. The findings contribute to the understanding of AcGX biosynthesis and modification that will help to improve the strategies devised for plant improvements
