Sucrose cleavage pathways in aspen wood
Abstract: Cellulose is the main component of wood and one of the most important renewable raw materials. In several tree species including Populus species, carbon for cellulose biosynthesis is derived from the disaccharide sucrose. This thesis describes experimental work on the mechanism of sucrose cleavage in developing wood and subsequent production of UDP-glucose (UDP-Glc) for cellulose biosynthesis. Sucrose synthase (SUS) has been proposed previously to interact directly with cellulose synthase complexes (CSC) and specifically supply UDP-Glc for cellulose biosynthesis. To investigate the role of SUS in wood biosynthesis, transgenic lines of hybrid aspen (Populus tremula L. x tremuloides Michx.) with strongly reduced soluble SUS activity in developing wood were characterized. The reduction of soluble SUS activity to few percentage of wild type increased soluble sugar content but decreased wood density and consequently reduced the lignin, hemicellulose and cellulose content per volume of wood. The results demonstrate that SUS has an important role in carbon flux from sucrose to all wood polymers but has no specific role in supplying UDP-Glc to cellulose synthesis machinery. I also investigated the role of cytosolic neutral/alkaline invertases (cNINs) during cellulose biosynthesis in hybrid aspen by analysing transgenic lines where NIN activity was decreased during secondary cell wall formation. The decrease in NINs activity caused a reduction in UDP-Glc and consequently reduced crystalline cellulose content but increased amorphous cellulose in cellulose microfibrils of wood. The results in this study demonstrated that cNIN activity is a major rate-controlling step in the cellulose biosynthesis. There is a lack of global analytical methods to measure sugar phosphates linked to cell wall polymer biosynthesis. To address this problem, I worked with the UPSC metabolomics facility to develop a robust method based on chloroform/methanol extraction, two-step derivatization and detection using reverse phase liquid chromatography-mass spectrometry (RP-LC-MS) without adding ion-pairing reagent. The method could quantitatively identify 18 sugar phosphates including UDP-Glc and structural isomers in Populus leaf and wood extracts. The method can now be used to gain deeper understanding into wood metabolism and cell wall biosynthesis.
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