| 1. |
- Gerber, Lorenz, et al.
(författare)
-
Deficient sucrose synthase activity in developing wood does not specifically affect cellulose biosynthesis, but causes an overall decrease in cell wall polymers
- 2014
-
Ingår i: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 203, s. 1220-1230
-
Tidskriftsartikel (refereegranskat)abstract
- The biosynthesis of wood in aspen (Populus) depends on the metabolism of sucrose, which is the main transported form of carbon from source tissues. The largest fraction of the wood biomass is cellulose, which is synthesized from UDP-glucose. Sucrose synthase (SUS) has been proposed previously to interact directly with cellulose synthase complexes and specifically supply UDP-glucose for cellulose biosynthesis.To investigate the role of SUS in wood biosynthesis, we characterized transgenic lines of hybrid aspen with strongly reduced SUS activity in developing wood.No dramatic growth phenotypes in glasshouse-grown trees were observed, but chemical fingerprinting with pyrolysis-GC/MS, together with micromechanical analysis, showed notable changes in chemistry and ultrastructure of the wood in the transgenic lines. Wet chemical analysis showed that the dry weight percentage composition of wood polymers was not changed significantly. However, a decrease in wood density was observed and, consequently, the content of lignin, hemicellulose and cellulose was decreased per wood volume. The decrease in density was explained by a looser structure of fibre cell walls as shown by increased wall shrinkage on drying.The results show that SUS is not essential for cellulose biosynthesis, but plays a role in defining the total carbon incorporation to wood cell walls.
|
|
| 2. |
- Rende, Umut, et al.
(författare)
-
Cytosolic invertase contributes to the supply of substrate for cellulose biosynthesis in developing wood
- 2017
-
Ingår i: New Phytologist. - : Wiley-Blackwell. - 0028-646X .- 1469-8137. ; 214:2, s. 796-807
-
Tidskriftsartikel (refereegranskat)abstract
- Carbon for cellulose biosynthesis is derived from sucrose. Cellulose is synthesized from uridine 5'-diphosphoglucose (UDP-glucose), but the enzyme(s) responsible for the initial sucrose cleavage and the source of UDP-glucose for cellulose biosynthesis in developing wood have not been defined. We investigated the role of CYTOSOLIC INVERTASEs (CINs) during wood formation in hybrid aspen (Populus tremula × tremuloides) and characterized transgenic lines with reduced CIN activity during secondary cell wall biosynthesis. Suppression of CIN activity by 38–55% led to a 9–13% reduction in crystalline cellulose. The changes in cellulose were reflected in reduced diameter of acid-insoluble cellulose microfibrils and increased glucose release from wood upon enzymatic digestion of cellulose. Reduced CIN activity decreased the amount of the cellulose biosynthesis precursor UDP-glucose in developing wood, pointing to the likely cause of the cellulose phenotype. The findings suggest that CIN activity has an important role in the cellulose biosynthesis of trees, and indicate that cellulose biosynthesis in wood relies on a quantifiable UDP-glucose pool. The results also introduce a concept of altering cellulose microfibril properties by modifying substrate supply to cellulose biosynthesis.
|
|
| 3. |
- Rende, Umut
(författare)
-
Sucrose cleavage pathways in aspen wood
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
|
|
| 4. |
- Rende, Umut, et al.
(författare)
-
Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry
- 2019
-
Ingår i: Plant Methods. - : Springer Science and Business Media LLC. - 1746-4811. ; 15
-
Tidskriftsartikel (refereegranskat)abstract
- Background Sugar phosphates are important intermediates of central carbon metabolism in biological systems, with roles in glycolysis, the pentose-phosphate pathway, tricarboxylic acid (TCA) cycle, and many other biosynthesis pathways. Understanding central carbon metabolism requires a simple, robust and comprehensive analytical method. However, sugar phosphates are notoriously difficult to analyze by traditional reversed phase liquid chromatography. Results Here, we show a two-step derivatization of sugar phosphates by methoxylamine and propionic acid anhydride after chloroform/methanol (3:7) extraction from Populus leaf and developing wood that improves separation, identification and quantification of sugar phosphates by ultra high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS). Standard curves of authentic sugar phosphates were generated for concentrations from pg to ng/mu l with a correlation coefficient R-2 > 0.99. The method showed high sensitivity and repeatability with relative standard deviation (RSD) < 20% based on repeated extraction, derivatization and detection. The analytical accuracy for Populus leaf extracts, determined by a two-level spiking approach of selected metabolites, was 79-107%. Conclusion The results show the reliability of combined reversed phase liquid chromatography-tandem mass spectrometry for sugar phosphate analysis and demonstrate the presence of two unknown sugar phosphates in Populus extracts.
|
|
