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    Targeted and non-targeted effects in cell wall polysaccharides from transgenetically modified potato tubers
    Huang, J.H. - \ 2016
    Wageningen University. Promotor(en): Harry Gruppen; Henk Schols. - Wageningen : Wageningen University - ISBN 9789462576292 - 126
    potatoes - cell walls - polysaccharides - transgenic plants - pectins - tubers - xyloglucans - genetically engineered foods - galactans - characteristics - nontarget effects - effects - aardappelen - celwanden - polysacchariden - transgene planten - pectinen - knollen - xyloglucanen - genetisch gemanipuleerde voedingsmiddelen - galactanen - karakteristieken - onbedoelde effecten - effecten

    The plant cell wall is a chemically complex network composed mainly of polysaccharides. Cell wall polysaccharides surround and protect plant cells and are responsible for the stability and rigidity of plant tissue. Pectin is a major component of primary cell wall and the middle lamella of plants. However, pectin biosynthesis in planta and the mechanisms underlying the influence of structural differences arising from a modified biosynthesis machinery on functional properties remain poorly understood. In our research, the changes in the chemical structures of cell wall polysaccharides after transgenic modification of potato tuber polysaccharides were examined. The cell wall material from potato wild-type varieties, from known and from new potato transgenic lines targeting changes of the homogalacturonan or rhamnogalacturonan I backbone were isolated and characterized. The modified cell wall polysaccharides were examined by determining their individual monosaccharide levels on fresh weight base and their cell wall characteristic parameters, and levels of acetylation and methyl esterification of cell wall pectin. Data for both targeted and non-targeted structures of cell wall polysaccharides from wild-type and transgenic potatoes were obtained. A shorter galactan side chain was found from the buffer soluble pectin and calcium bound pectin of β-galactosidase (β-Gal) transgenic lines. All pectin fractions from rhamnogalacturonan lyase (RGL) transgenic lines had less galactan chains attached to their rhamnogalacturonan I backbones. Two uridine diphosphate-glucose 4-epimerase (UGE) transgenic lines, UGE 45 and UGE 51, had diverse effects on length of the galactan side chain. The xyloglucans from the RGL and UGE transgenic lines retained its XXGG building blocks but differed in the proportion of repeating units compared to the respective wild-type varieties. In contrast, the β-Gal transgenic lines predominantly consisted of XXXG-type xyloglucan in the 4 M alkali extract, but showed XXGG-type building blocks in 1 M alkali extract. In addition, a quick-screening method was validated and used to analyze 31 transgenic lines and their respective wild-type potato varieties. An overall comparison of pectin backbone, pectin side chains, acetylation and methyl-esterification of pectin, pectin content and (hemi)cellulose content of cell wall polysaccharides from these transgenic lines provided a better insight in the frequency, level and combination of both targeted and non-targeted structural changes compared to that of their respective wild-type varieties. The same evaluation method was used to correlate cell wall composition in wild-type and selected transgenic lines and their established gene expression with the texture of corresponding cooked potato cubes. Changed physical properties for the genetically modified tubers could be connected to specific cell wall characteristics. Tubers from transgenic lines containing cell wall pectin with short galactan side chains were less firm after cold processing compared to wild-type tubers. The enhanced understanding of transgenic modifications of potato tubers resulting into significant targeted and non-targeted modifications in cell wall polysaccharides will lead to a better selection of potato lines with tailored cell wall characteristics and desired properties of the tubers during processing.

    Potato cell walls are composed of pectin, hemicellulose and cellulose. Cell wall polysaccharides are responsible for the stability, rigidity and flexibility of plant tissue. Pectin, a major component of primary plant cell walls, primarily consists of homogalacturonan (HG) and rhamnogalacturonan I (RG-I). To understand the structure–function relationships of potato cell wall pectin, this study aimed to identify the characteristics of both pectin and other polysaccharides as present in cell wall material (CWM) and of individual polysaccharide populations from wild-type potato varieties and their respective transgenic potato lines.

    Chapter 1 gives a general introduction to the fine chemical structures of potato cell wall polysaccharides, the main models of cell wall architecture and the cell wall-degrading enzymes, which include pectinases, hemicellulases and cellulases. In addition, transgenic modification of the cell wall through the heterologous expression of various enzymes from fungal or plant origin that could modify potato cell wall polysaccharides in planta is addressed. Transgenic modifications of potato cell wall polysaccharides that targeted pectin structures and cellulose levels are summarised. However, due to unsuccessful starch removal during CWM isolation and incomplete analysis of CWM yield and composition, characteristics regarding the different cell wall polysaccharides from previously-studied transgenic potato lines are hardly available.

    CWMs were extracted from the Karnico (wild-type) potato and its transgenic lines that expressed either β-galactosidase or rhamnogalacturonan lyase (Chapter 2). Improved starch removal procedures proved to be successful. Pectic polysaccharides were fractionated from CWMs of wild-type potato and its transgenic lines β-Gal-14 and RGL-18. Most β-Gal-14 pectin populations had less galactose (Gal) than wild-type, indicating that the transgenic line had shorter galactan side chains, although the side chain length differed for individual pectin populations. The ratio of HG:RG-I was introduced to evaluate the pectin backbone structure. High HG:RG-I ratios were consistently found in RGL-18 pectic polysaccharide populations. A low level of RG-I segments in combination with lower Gal contents indicated the removal of the galactan-rich RG-I segments in all pectin populations of RGL transgenic lines. In addition, RGL-18 transgenic modification increased the methyl-esterification and lowered the acetylation of pectins present in hot buffer extracts, when compared to wild-type. No effect on pectin esterification was found for β-Gal transgenic lines. Side effects of the mutation generated unexpected changes in the various pectin populations.

    The xyloglucan structure was extensively modified after transgenic modification of the pectin structure. Two xyloglucan extracts were obtained from the Karnico and its β-Gal-14 and RGL-18 transgenic lines (Chapter 3). The extracts of the Karnico and RGL-18 lines were mainly comprised of the XXGG-type xyloglucan as represented by XXGG and XSGG as predominant repeating units. In contrast, the XXXG-type xyloglucan was primarily present in the β-Gal-14 4 M alkali extract built up by LLUG repeats, although XXGG type of xyloglucan was present in the 1M alkali extract. Both the RGL and β-Gal transgenic lines had different proportions of xyloglucan building blocks (XSGG/XXGG ratios) than wild-type. After transgenic modification of pectin backbone or pectin side chains, the xyloglucan structures has been biosynthetically modified by plant itself.

    Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyses the conversion of UDP-glucose into UDP-galactose, which hypothetically should lead to more galactose being built into the cell wall polysaccharides. CWMs from the Kardal (wild-type) potato and its UGE45-1 and UGE51-16 transgenic lines were isolated, fractionated and characterised (Chapter 4). Both the UGE45 and UGE51 genes encoded for UGE enzymes, but the corresponding transgenic lines exhibited different modifications of the galactan side chains and of other cell wall structures. The Gal content of CWM from the UGE45-1 transgenic line was 38% higher than that of the wild-type and resulted in longer pectin side chains. The Gal content present in CWM from UGE51-16 was 17% lower than that of wild-type, which resulted in a slightly shorter galactan side chains for most pectin populations. Both UGE transgenic lines showed a decreased acetylation and an increased methyl-esterification of the cell wall pectin. Side effects were found in the xyloglucan structures of the transgenes as reflected by different proportions of XSGG/XXGG repeating units in comparison to wild-type. Pectin side chain biosynthesis had not only a varying level of galactan side chain modification, but also influenced the structure and possibly the interaction of other cell wall polysaccharides.

    In Chapter 5, a new screening strategy is introduced to evaluate higher numbers of transgenic potato tubers via CWM yield and sugar composition. A total of four wild-type potato varieties and 31 transgenic lines were evaluated to determine the effects on targeted structures including RG-I or HG pectin backbone elements, galactan or arabinogalactan side chains, acetyl groups of pectin and cellulose levels. Modification of the pectin backbone or pectin side chains in the transgenic lines has either a simultaneous increase or simultaneous decrease of HG:RG-I ratio, side chain length and methyl-esterification of pectin. The pectin esterification transgenic line exhibited only limited side effects. The cellulose level targeted lines had also high HG:RG-I ratios, longer galactan chains and similar pectin content compared to the wild-type, indicative for a less branched pectin backbone with longer side chains. From the monosaccharide composition data, various pectin and cell wall characteristics parameters are suggested as powerful indicators of cell wall polysaccharide structure.

    In Chapter 6, the achievements of this research are summarised and discussed in the context of potato cell wall architecture. The strategy and outcome of a quick screening method for multiple transgenic lines and an in-depth analysis of individual pectin and xyloglucan populations for the evaluation of potato CWMs is discussed. Furthermore, the texture of steam-cooked potatoes and the stability of potato cubes after freeze-thaw cycles are correlated with gene expression and cell wall composition in wild-type and selected transgenetically modified potato tubers. CWMs from transgenetically modified potatoes showed different physical properties during processing. In isolated CWMs, acetylation of cell wall pectin, molar Gal levels and starch content were the main parameters that could be related to the texture or firmness of tubers. Tubers from transgenic lines that resulted in shorter pectin side chains felt apart more easily after several freeze-thaw cycles than wild-type tubers and tubers with an increased length of pectin side chains. The modification of both targeted as well as non-targeted structures have now been shown to occur in many different potato transgenic lines, but precise mechanisms and consequences for the cell wall architecture remain unclear. Research performed so far, as well as research needed for getting a better understanding of plant cell wall architecture, is discussed.

    Galactosyl hydrolases from Bifidobacterium adolescentis and Bifidobacterium longum
    Hinz, S.W.A. - \ 2005
    Wageningen University. Promotor(en): Fons Voragen, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789085041832 - 133
    galactosidasen - bifidobacterium adolescentis - bifidobacterium longum - galactanen - oligosacchariden - galactosidases - bifidobacterium adolescentis - bifidobacterium longum - galactans - oligosaccharides
    The human intestine contains many bacteria, among which bifidobacteria. These can have a positive effect on human health. By consuming products containing dietary fibres (prebiotics), the amount of these intestinal bacteria can be stimulated, because they contain enzymes, which are able to degrade the fibres. Knowing which enzymes are present in the bacteria, will help to determine which kind of dietary fibres are suitable for use as a prebiotic. In this research, enzymes present in bifidobacteria, which were able to degrade the fibres galactan and galacto-oligosaccharides, were investigated. Three different enzymes were examined: a beta-galactosidase, an endo-galactanase, and an alpha-galactosidase. The results of this thesis gave more insight in how galactans and galacto-oligosaccharides can be degraded by bifidobacteria.
    Characterization of carrot arabinogalactan proteins
    Immerzeel, P. - \ 2005
    Wageningen University. Promotor(en): Sacco de Vries; Fons Voragen, co-promotor(en): Henk Schols. - Wageningen : WUR - ISBN 9789085041504 - 128
    galactanen - eiwitten - penen - karakterisering - celwanden - galactans - proteins - carrots - characterization - cell walls
    Arabinogalactan proteins (AGPs) are highly glycosylated proteins. Besides galactose and arabinose the carbohydrate part of AGPs contains other neutral sugars and uronic acids. AGPs are widely distributed in the plant kingdom, probably occurring in all tissues of every plant. Yariv phenylglycoside is a synthetic molecule and can form a complex with AGPs and this property of Yariv is used to isolate AGPs. Exposure of cell cultures or seedlings to Yariv phenylglycoside indirectly showed that AGPs have a biological function. For the study of embryogenesis cell cultures have been used.

    The cells of a cell culture can differentiate, form embryos and finally develop into plants. The growth conditions can be changed and the effect on the development of the embryos can be investigated. By adding AGPs to cell cultures the amount of embryos formed can be manipulated.

    This research showed that especially AGPs that were derived from carrot seeds were able to increase the number of embryos formed. Pre-treatment of the seed AGPs with chitinase increased the number of embryos formed when compared to the untreated AGPs. Chitinase is an enzyme able to hydrolyse the glycosidic bounds between acetylglucosamine and glucosamine. For the determination of an AGP that showed sensitivity to chitinase and the formed products, AGPs have been isolated from carrot cell culture medium and carrot seeds. In the AGPs that were isolated from the cell culture no glucosamine could be detected. The seed AGP extract contained a very low concentration of glucosamine. After fractionation of the seed AGP extract it was not possible to detect glucosamine in the obtained fractions. It could be possible that the glucosamine containing compound present in crude AGP extracts was coprecipitated with Yariv phenylglycoside during the isolation of AGPs.

    AGPs that were isolated from carrot cell cultures and the cell walls of different carrot tissues show in general different molecular weight fractions. Linkage analysis of the carbohydrates of two main fractions and additional protein analysis showed that bothfractionsposses the chemical characteristics of AGPs. Analysis of AGPs that were isolated with Yariv shows a large variation in sugar composition, depending on the purification procedure used. When AGPs from carrot cell culture medium and cell wall fractions were further purified with copper ions, galacturonic acid rich fractions were identified. Homogalacturonan hydrolysing enzymes were used to test whether the galacturonic acid was organised as homogalacturonan or present in the side chains of the AGPs. Homogalacturonan is a characteristic structural element of pectin. Oligogalacturonans were found after incubation of the galacturonic acid rich AGP fractions with polygalacturonase and pectin methylesterase. This result indicates that the galacturonic acid present in AGPs is organised as homogalacturonan. AGPs that were isolated from carrot tap root cell walls also showed a galacturonic acid rich fraction that was sensitive to homogalacturonan hydrolysing enzymes.

    The chemical analysis of carrot cell wall AGPs from two different tissues showed that a small fraction of AGPs is present that contains galacturonic acid in the form of homogalacturonan. The interaction of AGPs and pectin has been suggested earlier and could be due to non-covalent interactions or a covalent linkage. The galacturonic acid rich AGP fractions were isolated from the cell walls with EDTA buffer and it is very unlikely that the interaction between AGPs and pectin found in this study is accomplished by ionic interactions. This research has shown that AGP-pectin complexes exist in carrot tissues and this finding could be a starting point for a more precise determination of the linkage
    Pectic substances from soybean cell walls distinguish themselves from other plant cell wall pectins
    Huisman, M.M.H. ; Schols, H.A. ; Voragen, A.G.J. - \ 2003
    In: Advances in pectin and pectinases / Voragen, A.G.J., Schols, H.A., Visser, R.G.F., Dordrecht : Kluwer - ISBN 9781402011443 - p. 159 - 168.
    pectinen - celwanden - galactanen - sojabonen - glucanen - xyloglucanen - enzymen - pectins - cell walls - galactans - soyabeans - glucans - xyloglucans - enzymes
    The uncommon structural features of soybean cell wall pectic substances explain their resistance to degradation by enzymes generally used to degrade this kind of polymers, and indicates that a search for new enzymes is required to enable enzymatic modification of these polysaccharides
    Pectin : the hairy thing : evidence that homogalacturonan is a side chain of rhamnogalacturonan I
    Vincken, J.P. ; Schols, H.A. ; Oomen, R.J.F.J. ; Beldman, G. ; Visser, R.G.F. ; Voragen, A.G.J. - \ 2003
    In: Advances in Pectin and Pectinase Research : 2nd International Symposium on Pectins and Pectinases, Rotterdam, 2001 / F. Voragen, H. Schols & R. Visser Dordrecht : Kluwer Academic Publishers - ISBN 9781402011443 - p. 47 - 61.
    pectinen - galactanen - celwanden - pectins - galactans - cell walls
    In vitro degradation studies of pectic polysaccharides with novel fungal pectinases, investigations in which these polymers were treated with dilute acid, and microscopic analysis of extracted pectins have provided clues on how these polysaccharides are linked. Therefore it is believed that pectin is not an extended backbone consisting of homogalacturonan and rhamnogalacturonan regions, but rather a rhamnogalacturonan with neutral sugar and homogalacturonan side chains
    Towards unravelling the biological significance of the individual components of pectic hairy regions in plants
    Oomen, R.J.F.J. ; Vincken, J.P. ; Bush, M.S. ; Skjot, M. ; Voragen, C.H.L. ; Ulvskov, P. ; Voragen, A.G.J. ; Mccann, M.C. ; Visser, R.G.F. - \ 2003
    In: Advances in Pectin and Pectinase Research : 2nd International Symposium on Pectins and Pectinases, Rotterdam, 2001 / F. Voragen, H. Schols & R. Visser Dordrecht : Kluwer Academic Publishers - ISBN 9781402011443 - p. 15 - 34.
    pectinen - galactanen - celwanden - poriëngrootte - plantenweefsels - pectins - galactans - cell walls - pore size - plant tissues
    This review compares the results from the developmental studies together with those from mutagenized and genetically modified plants with compositional alterations to the hairy region
    Elucidation of the chemical fine structure of polysaccharides from soybean and maize kernel cell walls
    Huisman, M.M.H. - \ 2000
    Agricultural University. Promotor(en): A.G.J. Voragen; H.A. Schols. - S.l. : S.n. - ISBN 9789058081872 - 159
    celwanden - polysacchariden - pectinen - galactanen - galacturonzuur - zea mays - glycine max - cell walls - polysaccharides - pectins - galactans - galacturonic acid - zea mays - glycine max

    The subject of this thesis was the elucidation of the chemical fine structure of polysaccharides from cell walls of soybean and maize kernel. The two species investigated represent different taxonomic groups, soybean belonging to the dicotyledonous and maize to the monocotyledonous plants. Besides representing the most important structures present in cell wall material, these raw materials are of great importance in food and feed industry.

    The characterisation of the soybean cell wall polysaccharides started with the isolation of the cell wall material as Water-Unextractable Solids (WUS) from soybean meal (chapter 2). The isolation procedure yielded a WUS fraction containing almost all polysaccharides present in the meal and only few other components. WUS was sequentially extracted with chelating agent (Chelating agent Soluble Solids, ChSS), dilute alkali (Dilute Alkali Soluble Solids, DASS), 1 m alkali (1 m Alkali Soluble Solids, 1 MASS) and 4 m alkali (4 m Alkali Soluble Solids, 4 MASS) to leave a cellulose-rich residue (RES). The pectin-rich extracts (ChSS and DASS) were found to have identical sugar compositions and contained predominantly galactose, arabinose, and uronic acid residues. The 1 MASS fraction contained xylose in addition to the former three sugars. The hemicellulose-rich fraction (4 MASS) contained mainly xylose and glucose. No indications were found that ChSS and DASS were structurally different, although it is obvious that their arrangement in the cell wall was not identical.

    The intact cell wall polysaccharides in the meal and WUS were hardly degradable by enzymes. Once extracted, the polysaccharides from WUS were degraded more easily (chapter 3). The arabinogalactan side chains in the pectin-rich ChSS fraction could to a large extent be removed by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase, and arabinofuranosidase B. The remaining polymer (fraction P) was isolated and represented 30% of the polysaccharides in the ChSS fraction (12% of the polysaccharides in the WUS). This polymer still contained some remaining arabinose and galactose residues, which could not be removed by the enzyme mixture used.

    The pectic backbone (fraction P) appeared to be resistant to enzymatic degradation by both established (like polygalacturonase) and novel pectic enzymes (like RG-hydrolase). After partial acid hydrolysis of the isolated pectic backbone, one oligomeric and two polymeric populations were obtained by size-exclusion chromatography. Monosaccharide and linkage analyses, enzymatic degradation, and NMR spectroscopy of these two polymeric populations showed that the pectic substances in the original extract (ChSS) contained both rhamnogalacturonan and xylogalacturonan regions, while homogalacturonan was absent (chapter 4). The absence of homogalacturonan distinguishes the pectic substances from soybean from pectic polysaccharides extracted from other sources, which contain homogalacturonan and rhamnogalacturonan regions and can be degraded with polygalacturonase and RG-hydrolase, respectively. Acid hydrolysis of fraction P improves the susceptibility of the remaining polymers for RG hydrolase and exo-galacturonase.

    The xylogalacturonan present in the ChSS fraction distinguishes itself from xylogalacturonan from other sources known so far. A part of the xylose residues in the xylogalacturonan is substituted with fucose and the xylogalacturonan is resistant to degradation with XGH.

    The arabinogalactan side chains, which were removed from the ChSS fraction to obtain fraction P, were the subjects of investigation in chapter 5. Fractionation, monosaccharide and linkage analyses, enzymatic degradation, and mass spectrometry of the oligosaccharides in the digest of ChSS after enzymatic digestion with arabinogalactan degrading enzymes indicated the presence of common linear (1,4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1,4)-linked galactose residues bearing an arabino pyranose residue at the non-reducing terminus, and a mixture of linear oligosaccharides constructed of (1,4)-linked galactose residues interspersed with one internal (1,5)-linked arabinofuranose residue. The presence of an internal arabinofuranose residue in a pectic arabinogalactan chain in cell wall polysacchairdes has not been reported previously, not in soybean, nor in other fruit or vegetable cell walls. Another uncommon feature is the presence of arabinopyranose residues in pectic arabinogalactan.

    The pectic substances form only one network of the plant cell wall, the other is the cellulose/hemicellulose network. The hemicelluloses were solubilised from the residue with 1 and 4 m KOH solutions (chapter 6). The polysaccharides extracted with 1 m KOH were fractionated by ion-exchange chromatography, yielding a neutral and a pectic population. The sugar composition of the neutral population indicated the presence of xyloglucans and possibly xylans. Enzymatic degradation with endo-xylanases and endo-glucanases showed the presence of xyloglucan fragments only. Analysis of the digest formed after incubation of the neutral population with endo-glucanase V showed the formation of the characteristic poly-XXXG xyloglucan oligomers (XXG, XXXG, XXFG, XLXG, and XLFG), so three out of four glucose residues carry a side chain.

    In chapter 7, the structural features of glucuronoarabinoxylans from maize kernels are described. First of all, maize kernel cell wall material was isolated as Water-Unextractable Solids (WUS). As expected the non-starch polysaccharides (NSP) had concentrated in the WUS (57%). These NSP were composed mainly of glucose, xylose, arabinose, and glucuronic acid. Sequential extractions with a saturated Ba(OH) 2 -solution (BE1 extract), and distilled water (BE2 extract) were used to solubilise glucuronoarabinoxylans from maize WUS. The glycosidic linkage composition of the extracts and their resistance to endo-xylanase treatment indicated that the extracted glucuronoarabinoxylans were highly substituted. In the maize BE1 extract 25% of the xylose was unsubstituted, 38% was monosubstituted and 15% was disubstituted. The glucuronoarabinoxylans in maize BE1 appeared to be resistant to endo-xylanase treatment, but could be degraded by a sub-fraction of Ultraflo, a commercial enzyme preparation from Humicola insolens . The digest contained a number of series of oligomers: pentose n , pentose n GlcA, pentose n hexose, and pentose n GlcA 2 . The presence of these glucuronic acid-containing series of oligomers showed that the glucuronic acids in the glucuronoarabinoxylancan can be very close to each other, but are not distributed blockwise. Finally, a new measure for the degree of substitution of glucuronoarabinoxylans was defined. It turned out that the degree of substitution in maize BE1 is much higher (87%) than in sorghum (70%) and wheat flour BE1 (56%). This indicates that the glucuronoarabinoxylans in maize BE1 are more complex than those in sorghum BE1 and explains their resistance to endo-xylanase treatment.

    From this research, it can be concluded that both soybean and maize kernel cell wall polysaccharides distinguish themselves in a number of respects from other plant cell walls polysaccharides. The absence of homogalacturonan, but also the presence of internal (1,5)-linked arabinofuranose and terminal arabinopyranose in the pectic arabinogalactan side chains from soybean cell walls and the complexity of the glucuronoarabinoxylan from maize kernel cell walls are discussed in chapter 8. In addition, it was shown that techniques like mass spectrometry and NMR spectroscopy are powerfull techniques to be used after (enzymatic) fragmentation, for chemical characterisation of the original polysaccharides.

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