Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Targets and tools for optimizing lignocellulosic biomass quality of miscanthus
    Weijde, R.T. van der - \ 2016
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade; Oene Dolstra. - Wageningen : Wageningen University - ISBN 9789462578388 - 231
    miscanthus - bioethanol - biomass - biofuels - lignocellulose - fuel crops - plant breeding - cell walls - cell wall components - genetic diversity - genetic variation - biomass conversion - biobased economy - miscanthus - bioethanol - biomassa - biobrandstoffen - lignocellulose - brandstofgewassen - plantenveredeling - celwanden - celwandstoffen - genetische diversiteit - genetische variatie - biomassaconversie - biobased economy

    Miscanthus is a perennial energy grass characterized by a high productivity and resource-use efficiency, making it an ideal biomass feedstock for the production of cellulosic biofuels and a wide range of other biobased value-chains. However, the large-scale commercialization of converting biomass into cellulosic biofuel is hindered by our inability to efficiently deconstruct the plant cell wall. The plant cell wall is a complex and dynamic structure and its components are extensively cross-linked into an unyielding matrix. The production of biofuel depends on the extraction, hydrolysis and fermentation of cell wall polysaccharides, which currently requires energetically and chemically intensive processing operations that negatively affect the economic viability and sustainability of the industry. To address this challenge it is envisioned that the bioenergy feedstocks can be compositionally tailored to increase the accessibility and extractability of cell wall polysaccharides, which would allow a more efficient conversion of biomass into biofuel under milder processing conditions.

    Extensive phenotypic and genetic diversity in cell wall composition and conversion efficiency was observed in different miscanthus species, including M. sinensis, M. sacchariflorus and interspecific hybrids between these two species. In multiple experiments a twofold increase in the release of fermentable sugars was observed in ‘high quality’ accessions compared to ‘low quality’ accessions. The exhaustive characterization of eight highly diverse M. sinensis genotypes revealed novel and distinct breeding targets for different bioenergy conversion routes. The key traits that contributed favourably to the conversion efficiency of biomass into biofuel were a high content of hemicellulosic polysaccharides, extensive cross-linking of hemicellulosic polysaccharides (revealed by a high content of trans-ferulic acids and a high ratio of arabinose-to-xylose), a low lignin content and extensive incorporation of para-coumaric acid into the lignin polymer.

    Lignin is widely recognized as one of the key factors conveying recalcitrance against enzymatic deconstruction of the cell wall. The incorporation of para-coumaric acid into the lignin polymer is hypothesized to make lignin more easily degradable during alkaline pretreatment, one of the most widely applied processing methods that is used to pretreat biomass prior to enzymatic hydrolysis. Previous studies have shown that reducing lignin content is often implicated in reduced resistance of plants to lodging. We hypothesize that extensively cross-linked hemicellulosic polysaccharides may fulfil a similar function in supporting cell wall structural rigidity and increasing the content of hemicellulosic polysaccharides may be a way to reduce lignin content without adversely affecting cell wall rigidity. This strategy can be used to improve biomass quality for biobased applications, as hemicellulosic polysaccharides are more easily degradable during industrial processing than lignin. Furthermore, hemicellulosic polysaccharides adhere to cellulose, which negatively affects the level of cellulose crystallinity. Crystalline cellulose is harder to degrade than its more amorphous form. Therefore the reduction of cellulose crystallinity is another mechanism through which increasing the content of hemicellulosic polysaccharides positively contributes to cell wall degradability. These results provided new insights into the traits that may be targeted to improve the quality of lignocellulose feedstocks.

    However, evaluation of complex biochemical traits for selection purposes is hindered by the fact that their accurate quantification is a costly, lengthy and laborious procedure. To overcome these limitations an accurate and high-throughput method was developed based on near-infrared spectroscopy. Through extensive calibration we developed accurate prediction models for a wide range of biomass quality characteristics, which may be readily implemented as a phenotyping tool for selection purposes.

    Additionally, progress through breeding may substantially be improved by marker-assisted selection, which will reduce the need for the evaluation of genotype performance in multi-year field trials. To this end, a biparental M. sinensis mapping population of 186 individuals was developed and genotyped using a genotyping-by-sequencing approach. A total of 564 short-sequence markers were used to construct a new M. sinensis genetic map. Cell wall composition and conversion efficiency were observed to be highly heritable and quantitatively inherited properties. This is the first genetic study in miscanthus to map quantitative trait loci (QTLs) for biomass quality properties and is a first step towards the application of marker-assisted selection for biomass quality properties.

    Through the evaluation of a diverse set of miscanthus genotypes in multiple locations we demonstrated that in addition to genotypic variation, growing conditions may have a substantial influence on cell wall composition and conversion efficiency. While further research is needed to identify which specific environmental parameters are responsible for the observed effects, these results clearly indicate that the environmental influence on biomass quality needs to be taken into account in order to match genotype, location and end-use of miscanthus as a lignocellulose feedstock. Moreover, significant genotype-by-environment interaction effects were observed for cell wall composition and conversion efficiency, indicating variation in environmental sensitivity across genotypes. Although the magnitude of the genotypic differences was small in comparison to genotype and environmental main effects, this affected the ranking of accession across environments. Stability analysis indicated some stable accessions performed relatively across diverse locations.

    In addition to trialing miscanthus in diverse locations, we also evaluated miscanthus biomass quality under drought conditions for a number of reasons: 1) drought stress is linked to a differential expression of cell wall biosynthesis genes, 2) incidence of drought events is increasing due to climate change, 3) irrigation is likely to be uneconomical during the cultivation of miscanthus and 4) miscanthus has many characteristics that make it a crop with a good potential for cultivation on marginal soils, where abiotic stresses such as drought may prevail. Drought stress was shown to result in a large reduction in cell wall and cellulose content and a substantial increase in hemicellulosic polysaccharides and cellulose conversion rates. We hypothesized that the reduction in cellulose content was due to an increase in the production of osmolytes, which are well-known for their role in plant protection against drought. The results indicated that drought stress had a positive effect on the cell wall degradability of miscanthus biomass.

    Overall the compendium of knowledge generated within the framework of this thesis provided insights into the variation in biomass quality properties in miscanthus, increased our understanding of the molecular, genetic and environmental factors influencing its conversion efficiency into biofuel and provided tools to exploit these factors to expand the use of miscanthus as a lignocellulose feedstock.

    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.

    Identification of novel isomeric pectic oligosaccharides using hydrophilic interaction chromatography coupled to traveling-wave ion mobility mass spectrometry
    Leijdekkers, A.G.M. ; Huang, J.H. ; Bakx, E.J. ; Gruppen, H. ; Schols, H.A. - \ 2015
    Carbohydrate Research : an international journal 404 (2015). - ISSN 0008-6215 - p. 1 - 8.
    interaction liquid-chromatography - fluorescent labels - galacturonic acid - cell walls - separation
    Separation and characterization of complex mixtures of pectic oligosaccharides still remains challenging and often requires the use of multiple analytical techniques, especially when isomeric structures are present. In this work, it is demonstrated that the coupling of hydrophilic interaction chromatography (HILIC) to traveling-wave ion mobility mass spectrometry (TWIMMS) enabled the simultaneous separation and characterization of complex mixtures of various isomeric pectic oligosaccharides. Labeling of oligosaccharides with 3-aminoquinoline (3-AQ) improved MS-ionization efficiency of the oligosaccharides and reduced the complexity of the product ion mass spectra, without losing resolution of the HILIC separation. In addition, labeling enabled quantification of oligosaccharides on molar basis using in-line fluorescence detection. Isomeric structures were distinguished using TWIMMS. The 3-AQ-HILIC–TWIMMS method was used to characterize a series of isomeric sugar beet rhamnogalacturonan I derived oligosaccharides carrying a glucuronic acid substituent. Thereby, some novel structural features were identified for the first time: glucuronic acid was attached to O-3 or to O-2 of galacturonic acid residues and a single galacturonic acid residue within an oligomer could contain both an acetyl group and a glucuronic acid substituent.
    Relatie eigenschappen maïscelwanden en fermentatiekarakteristieken in de pens van herkauwers
    Cone, J.W. - \ 2014
    Wageningen : Animal Nutrition Group (ANU rapport Oktober 2014) - 52
    maïs - celwanden - fermentatie - pens - herkauwers - diervoeding - penssap - ruwvoer (forage) - rundveehouderij - melkveehouderij - dierenwelzijn - maize - cell walls - fermentation - rumen - ruminants - animal nutrition - rumen fluid - forage - cattle husbandry - dairy farming - animal welfare
    Dit rapport beschrijft onderzoek naar de relatie tussen de eigenschappen van maïscelwanden en fermentatiekarakteristieken van deze celwanden in de pens van herkauwers. Het betreft hier zowel chemische onderzoek als anatomisch en histologisch onderzoek en in-vitro-onderzoek naar de afbreekbaarheid van maïscelwanden in pensvloeistof. Het doel is om inzicht te krijgen in de achtergrond van verschillen in celwandstructuur en samenstelling tussen verschillende monsters, ook op moleculair niveau.
    Genetics and bioenergy potential of forage maize: deconstructing the cell wall
    Torres, A.F. - \ 2014
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade; Oene Dolstra. - Wageningen : Wageningen University - ISBN 9789462570375 - 202
    zea mays - maïs - voedergewassen - plantengenetica - bio-energie - celwanden - bioethanol - bioconversie - industriële grondstoffen - brandstofgewassen - zea mays - maize - fodder crops - plant genetics - bioenergy - cell walls - bioethanol - bioconversion - feedstocks - fuel crops

    Despite gaining prominence in scientific spheres and political agendas worldwide, the production of biofuels from plant biomass is yet to achieve an economic stronghold in the renewable-energy sector. Plant lignocellulose has evolved to resist chemical and enzymatic deconstruction, and its conversion into liquid fuels requires energetically stringent processes that currently render the industry economically and environmentally unviable.

    To address this challenge, experts have envisioned the development of advanced bioenergy crops which require lower energetic and chemical inputs for their effective fractionation. At its core, this approach requires an in-depth understanding of the composition, synthesis and breeding amenability of the plant cell wall; the principal constituent of total plant dry biomass and the most recalcitrant fraction of the crop at physiological maturity to deconstruction. To this end, the primary aim of this thesis was to dissect and elucidate the biochemical and genetic factors controlling cell wall characteristics relevant to the development of bioenergy grasses with improved processing quality for cellulosic based fuel production. A focus on maize was warranted as it currently represents the de facto model system for bioenergy crop research; offering an unrivalled platform to underpin the complex genetic architecture of cell wall biosynthesis, develop advanced bioenergy-crop breeding strategies and translate cell wall research into innovations and commercial products.

    This thesis exposed that the biomass-to-fuel conversion of crops is a highly complex trait dependent on both, the balance and synergy between multiple cell wall components, and the inherent effectiveness of the conversion technology. Concerning the production of cellulosic ethanol via the combined operations of dilute-acid pretreatment and enzymatic saccharification, our results revealed that the chemical mechanisms affecting biomass conversion efficiency depend on pretreatment severity. Whereas at harsh pretreatments biomass conversion efficiency was primarily influenced by the inherent efficacy of thermochemical cell wall deconstruction, at milder pretreatments, maximum fermentable glucose release was observed for maize genotypes exhibiting systematic cell wall changes leading to higher ruminal cell wall digestibility. These results confirmed that the selection and use of cellulosic feedstocks that best match the processing conditions used in the industry can aid in reaching industrial goals aimed at improving the commercial and environmental performance of cellulosic fuels.

    In turn, the exhaustive characterization of a forage maize doubled haploid (DH) population demonstrated the vast degree of genetic diversity in maize cell wall composition and bioconversion potential amenable to breeding. Principally, these findings suggest that natural diversity in the biochemical composition of the maize cell wall and its physical properties is primarily ascribed to variation in the balance, monomeric make-up, and extent of cross-linking of non-cellulosic cell wall polymers (i.e. lignin and hemicellulose). Indeed, correlation analyses confirmed that the extent of enzymatic depolymerization of maize biomass was strongly and negatively associated to the concentration of cell wall phenolics, but positively impacted by the degree of glucuronoarabinoxylan (GAX) glycosylation and extent of hemicellulose-to-hemicellulose cross-linking. Our results also showed that natural variation in cell wall content and composition is quantitatively inherited and putatively ascribed to the segregation of multiple genetic loci with minor additive effects. In our population, genotypic diversity for cell wall composition and quality was found to be controlled by 52 quantitative trait loci (QTLs). From eight QTLs regulating bioconversion properties, five were previously unidentified and warrant further investigation.

    Despite the apparent complexity of cell wall genetics, however, the high heritability and environmentally stability of cell wall compositional and degradability properties guarantee high selection efficacy during the development of superior DH/inbred material, and predispose that multi-environment testing will only be necessary at advanced stages of bioenergy-maize breeding programs. Moreover, because genetic variation for complex cell wall characteristics appears to be predominantly additive, preliminary selection at the inbred level will expectedly lead to successful hybrid selection; thereby minimizing the need for recurrent test-crossing procedures and evaluations. In this regard, maize cell wall bioconversion efficiency constitutes an excellent selection criterion for immediate application in modern maize breeding programs.

    Ultimately, the convergence of classical selection schemes with inexpensive genotyping, advanced biometric models, high-throughput cell wall phenotyping and doubled haploid (DH) production technologies can accelerate development and commercial release of maize cultivars for bioenergy applications. To play a determinant role in the development and realization of sustainable and cost-effective cellulosic fuel processing technologies, however, novel dual-purpose maize cultivars (i.e. delivering both, grain for feed or food and fiber materials for bioconversion) will have to surpass the performance in lignocellulose processing quality and biomass yields of the best elite germplasm. These prospects seem realistic as the parallel advance of grain yield and stover productivity and quality characteristics is a feasible undertaking. Conceptually, the advance of superior bioenergy cultivars (surpassing the performance of modern elite material) would allow us to make the currently available biomass-to-fuel conversion systems more cost-effective and sustainable, and may also have favorable consequences for the ideal size and geographical distribution of biofuel refineries.

    Functional analysis of LysM effectors secreted by fungal plant pathogens
    Kombrink, A. - \ 2014
    Wageningen University. Promotor(en): Bart Thomma; P.J.G.M. de Wit. - Wageningen : Wageningen University - ISBN 9789461738578 - 119
    plantenziekteverwekkende schimmels - secretie - celwanden - chitine - bindende eiwitten - virulentie - pathogeniteit - hyfen - ziekteresistentie - verdedigingsmechanismen - plant pathogenic fungi - secretion - cell walls - chitin - binding proteins - virulence - pathogenicity - hyphae - disease resistance - defence mechanisms

    Chitin is a homopolymer of N-acetyl-d-glucosamine (GlcNAc)that is abundantly present in nature and found as a major structural component in the fungal cell wall. In Chapter 1,the role of chitin as an important factor in the interaction between fungal pathogens and their plant hosts is discussed. As plants do not produce chitin, they evolved to recognize fungal chitin as a non-self molecule by plasma membrane receptors that can activate host immune responses to stop fungal growth.To overcome those host immune responses, fungal pathogens secrete effector molecules that manipulate host physiology, including immune responses, to support colonization. The chitin-binding Lysin motif (LysM) effector Ecp6 from the fungal tomato pathogen Cladosporium fulvumwas previously demonstrated to contribute to virulence through interfering with the activation of chitin-induced host immune responses. Subsequently, LysM effector genes were found in the genomes of many fungal species.

    In Chapter 2 we describe the functional characterization of LysM effectors of the plant pathogenic fungi Mycosphaerella graminicola, Magnaporthe oryzae and Colletotrichum higginsianum, which cause leaf blotch disease of wheat, rice blast disease and anthracnose disease on Brassicaceae, respectively. This functional analysis revealed that the ability to perturb chitin-induced immunity is conserved among LysM effectors of these fungal plant pathogens. In addition, two LysM effectors that are secreted by M. graminicolawere found to protect fungal hyphae against cell wall hydrolytic enzymes from plants, demonstrating that LysM effectors can contribute to virulence of fungal plant pathogens in multiple ways.

    The M. graminicola LysM effector Mg3LysM and C. fulvum Ecp6 both contain three LysM domains and show a high overall similarity. However, whereas Mg3LysM can protect fungal hyphae against plant-derived cell wall hydrolytic enzymes, Ecp6 does not have this capacity. Chapter 3describes a functional analysis of the contribution of LysM domains of Mg3LysM to its protection ability. To this end a series of chimeric proteins were produced in whichLysM domains of Mg3LysM were swapped with the corresponding LysM domain of Ecp6.Analysis of these chimeras indicated that protection against the hydrolytic activity of plant enzymes is mediated by the concerted activity of LysM1 and LysM3 in Mg3LysM.

    LysM effectors do not only occur in foliar fungal plant pathogens, but also in soil-borne pathogens that infect their host through the roots. In Chapter 4, LysM effectors of the fungal soil-borne vascular wilt pathogen Verticillium dahliaeare described. Comparative genomics of eleven V. dahliae strains revealed that four LysM effectors are found in the core genome, which are referred to as core VdLysM effectors. Intriguingly, for none of the core LysM effector genes expression could be monitored during host colonization, and targeted deletion could not reveal a role in virulence, suggesting that the core LysM effectors do not act as virulence factors during host colonization. In addition to the core genome, V. dahliaestrains generally carry lineage-specific (LS) genomic regions. Interestingly, an additional LysM effector gene (Vd2LysM) was found in an LS region of V. dahliaestrain VdLs17 that is absent in all other sequenced V. dahliaestrains. Remarkably, the LS effector Vd2LysM was found to contribute to virulence of strain VdLs17. Like the previously characterized plant pathogen LysM effectors, also Vd2LysM was found to bind chitin and suppress chitin-induced immune responses. These results indicate that Vd2LysM interferes with chitin-induced immunity during host colonization by V. dahliaestrain VdLs17.

    Thus far, LysM effectors were demonstrated to contribute to virulence of various fungal plant pathogens through their ability to interfere with host immune responses. However, the presence of LysM effector genes in the genomes of non-pathogenic fungi and fungi with a saprophytic lifestyle suggests that LysM effectors contribute to fungal physiology in other manners as well. In Chapter 5we investigated the hypothesis that LysM effectors play a role in the interaction of fungi with other microbes in the environment, which could even be relevant for plant pathogenic fungi that encounter other microbes at the site of host infection. To investigate this hypothesis, assays were developed that allow to assess the attachment and antagonistic effects of particular bacterial species on fungi by employing the fungus Trichoderma viride, as this species is known to have accessible cell wall chitin upon growth in vitro. Assays to assess bacterial attachment and antagonistic activity in the absence or presence of LysM effectors indicate that LysM effectors play a role in the protection of fungi against bacterial competitors.

    In Chapter6, the major results described in this thesis are discussed and a perspective on the (potential) roles of LysM effectors in fungi with different lifestyles, including pathogenic as well as non-pathogenic fungi, is presented.

    Nieuwe verwaarding algenbiomassa : literatuurstudie en praktische screening van enkele ontsluitingsmethoden
    Kootstra, A.M.J. ; Schipperus, R. ; Berg, W. van den; Grobben-Gaastra, S.A. ; Weide, R.Y. van der - \ 2013
    Lelystad : PPO AGV (PPO rapport 555) - 26
    algen - bioraffinage - biomassaconversie - celwanden - chemische behandeling - enzymen - biobased economy - algae - biorefinery - biomass conversion - cell walls - chemical treatment - enzymes - biobased economy
    Om de eiwitten, oliën, koolhydraten, carotenoïden en andere nuttige stoffen aanwezig in algencellen los van elkaar te kunnen benutten, is het nodig om de celwanden van de algen open te breken, ofwel om de algen te ontsluiten. Uiteraard is het van belang om dit te doen op een manier die de te winnen componenten zo min mogelijk beschadigt. Dit rapport bestaat uit twee delen. Het eerste is een literatuurstudie waarin een aantal algensoorten wordt omschreven, met waar mogelijk nadruk op de celwandstructuur. Verder wordt een aantal methoden beschreven om de celwanden te openen of te verzwakken. In het tweede deel van het rapport worden een aantal uitgevoerde proeven beschreven waarbij algen zijn behandeld met enzymen, schoonmaakazijn, of ethanol.
    Characterisation of 3-aminoquinoline-derivatised isomeric oligogalacturonic acid by travelling-wave ion mobility mass spectrometry
    Huang, J.H. ; Bakx, E.J. ; Gruppen, H. ; Schols, H.A. - \ 2013
    Rapid Communications in Mass Spectrometry 27 (2013)20. - ISSN 0951-4198 - p. 2279 - 2285.
    cell walls - oligosaccharides - matrix - chromatography - separation - pectin - quantification - resolution
    RATIONALE Mass spectrometry has become a useful technique for elucidating the chemical structures of oligosaccharides. The combined use of chromatography and mass spectrometry for the separation and identification of oligosaccharides has shown much progress in recent years. However, no powerful method has yet been developed to quickly identify isomeric oligosaccharides in complex mixtures. METHODS A rapid travelling-wave ion mobility mass spectrometry (TWIMS-MS) method was developed for the identification of various isomeric oligogalacturonic acids in mixtures and determined their structures, using 3-aminoquinoline (3-AQ) as a labelling agent. RESULTS TWIMS successfully distinguished isomeric oligogalacturonic acids of various degrees of polymerisation (DPs) and levels of methyl-esterification. After derivatisation by 3-AQ, isomeric oligosaccharides of galacturonic acid, with the DP ranging from 2 to 9 and the number of methyl esters ranging from 1 to 5, were identified by 3-AQ-TWIMS-MS. The isomeric oligosaccharides with varying sites of methyl ester substitution were identified by the post-fragmentation mode of TWIMS using 3-AQ labelling to obtain simplified mass spectra. CONCLUSIONS Using the 3-AQ-TWIMS-MS method, the precise distribution of methyl esters within the pectin molecule and isomeric oligogalacturonic acids after enzyme degradation was determined. Simplified product ion mass spectra and precise analysis of the isomers were achieved by labelling 3-AQ at the reducing end of the oligosaccharides. Series of methyl-esterified galacturonic acid oligomers have predictable drift times, depending on the precise position of the methyl ester.
    Functional analysis of tomato immune receptor Ve1 and recognition of Verticillium effector Ave1
    Zhang, Z. - \ 2013
    Wageningen University. Promotor(en): Bart Thomma; Pierre de Wit, co-promotor(en): C.M. Liu. - S.l. : s.n. - ISBN 9789461735461 - 191
    solanum lycopersicum - tomaten - celwanden - receptoren - immuunsysteem - liganden - plantenziekteverwekkende schimmels - verticillium - infectiviteit - modellen - plant-microbe interacties - solanum lycopersicum - tomatoes - cell walls - receptors - immune system - ligands - plant pathogenic fungi - verticillium - infectivity - models - plant-microbe interactions

    Similar to the animal innate immune system, plants employ extracellular leucine rich repeat (eLRR)-containing cell surface receptors to recognize conserved molecular structures that are derived from microbial pathogens. A number of these immune receptors, as well as the corresponding pathogen ligands, have been characterized. The interaction between the tomato Ve1 immune receptor and the Ave1 effector from the pathogenic fungus Verticillium serves as a model system for the study of plant innate immunity. The research described in this thesis was aimed at a further understanding of how the eLRR-containing cell surface receptor Ve1 confers recognition of the Ave1 ligand and how it activates downstream immune signaling.

    It has been shown that eLRR-containing cell surface receptors play important roles in development and innate immunity in various plant species.Chapter 1 gives an overview on the current status of research on eLRR-containing cell surface receptors, their co-receptors and corresponding ligands, with emphasis on structural aspects. The functions of distinct eLRR receptor domains, their role in structural conformation, ligand perception, signal transduction and receptor complex formation are extensively discussed.

    To facilitate studies on the Ve1-Ave1 model system, we describe the establishment of protocols to investigate Ve1-mediated recognition of Ave1 and immune signaling in tobacco in Chapter 2. We optimized an Agrobacterium tumefaciens transient expression assay (agroinfiltration) by testing various over-expression vectors, and found that co-expression of Ve1 and Ave1 leads to hypersensitive response (HR) only in particular tobacco species. We further report on virus-induced gene silencing (VIGS) in Nicotiana tabacum cv. Samsun that allows investigating signaling components involved in Ve1-mediated resistance. Collectively, we established N. tabacum as a model plant to study Ve1-mediated immunity.

    In Chapter 3, we further investigated whether co-expression of Ve1 and Ave1 in Arabidopsis leads to an HR, which may potentially be used as a straightforward screening method upon a random mutagenesis. However, although Ave1 is able to trigger an HR in resistant tomato and tobacco plants, co-expression of Ve1 and Ave1 did not activate an HR in Arabidopsis. These results suggest that the HR occurs as a consequence of Ve1-mediated resistance signaling, and it is not absolutely required for Verticillium resistance.

    In Chapter 4 we investigated the contribution of particular regions of Ve1 to the activation of immune signaling through domain swaps between Ve1 with its non-functional homolog Ve2. Agroinfiltration, as well as stable Arabidopsis transformation, revealed that chimeras in which the first thirty eLRRs of Ve1 were replaced with those of Ve2 remain able to induce HR and activate Verticillium resistance. However, a truncated Ve1 protein that lacks the first 30 eLRRs is no longer functional. We speculate that the non-functional Ve2 receptor may still interact with the Ave1 effector in the eLRR domain, but fails to activate immune signaling due to a non-functional C-terminus.

    In Chapter 5, site-directed mutagenesis was employed to further investigate the eLRR domain of Ve1. We designed alanine scanning mutants in the solvent-exposed residues across the convex surface of the eLRR domain. In each mutant, two of the five solvent-exposed residues in β-sheet of a single eLRR were substituted into alanines. Functionality of the mutants through agroinfiltration and stable transformation of Arabidopsis revealed three eLRR regions that are potentially required for ligand specificity and for co-receptor interaction. In addition, alanine substitution was employed to evaluate role of putative protein-protein interaction and endocytosis motifs in the transmembrane domain and the cytoplasmic tail of the Ve1 protein. However, no requirement of these domains for Ve1 functionality could be demonstrated.

    It has been demonstrated that eLRR-containing cell-surface immune receptors often recognize short peptide sequence stretches as epitopes of their ligands. In Chapter 6, we aimed to identify the surface epitope of the Verticilliumeffector Ave1 that is recognized by Ve1. Firstly, we assessed whether various Ave1 homologs are recognized by Ve1. Since we found that C-terminal fusion of a GFP tag to Ave1 compromised its recognition, we hypothesized that accessibility of the Ave1 C-terminus is essential for Ve1-mediated recognition. Ave1 truncations and domain swaps with Ave1 homologs that are not recognized by Ve1 showed that a nine amino acid sequence derived from the C-terminus of Ave1 is essential for recognition by Ve1. This nine amino acid epitope is sufficient to activate Ve1-mediated immunity.

    In Chapter 7 the highlights of the thesis are discussed and placed in a broader perspective. The current understanding of eLRR-containing cell surface receptors is discussed, taking the findings of this thesis into account, with specific emphasis on ligand perception and receptor complex formation. In addition, future perspectives on the future are sketched, and novel research questions are posed aimed to obtain further insights into how Ve1 may form complexes with various co-receptors and how Ave1 contributes to Verticillium pathogenicity.

    Pectin degradation by Botrytis cinerea: recognition of endopolygalacturonases by an Arabidopsis receptor and utilization of Dgalacturonic acid
    Lisha Zhang, Lisha - \ 2013
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jan van Kan. - S.l. : s.n. - ISBN 9789461735409 - 188
    botrytis cinerea - plantenziekteverwekkende schimmels - pectinen - degradatie - celwanden - arabidopsis - receptoren - polygalacturonase - galacturonzuur - botrytis cinerea - plant pathogenic fungi - pectins - degradation - cell walls - arabidopsis - receptors - polygalacturonase - galacturonic acid

    The necrotrophic fungal plant pathogenBotrytis cinerea is able to infect over 200 host plants and cause severe damage to crops, both pre- and post-harvest. B. cinerea often penetrates host leaf tissue at the anticlinal cell wall and subsequently grows into and through the middle lamella, which consists mostly of low-methylesterified pectin. Effective pectin degradation thus is important for virulence of B. cinerea. Chapter 1 describes the chemical structures of plant cell wall polysaccharides, the cell wall-associated mechanisms that confer resistance against pathogens, and the microbial enzymes involved in cell wall decomposition. It then discusses the plant cell wall degrading enzymes of pathogenic fungi and illustrates with case studies the process of pectin decomposition by B. cinerea.

    Chapter 2describes the molecular identification and functional characterization of a novel MAMP receptor RBPG1, a Leucine-Rich Repeat Receptor-Like Protein (LRR-RLP), that recognizes fungal endo-polygalacturonases (endo-PGs), in particular the B. cinerea protein BcPG3. Infiltration of the BcPG3 protein into Arabidopsis thaliana accession Col-0 induced a necrotic response. Heat-inactivated protein and a catalytically inactive mutant protein retained the ability to induce necrosis. An 11-amino acid peptide stretch was identified that is conserved among many fungal but not plant endo-PGs. A synthetic peptide comprising this sequence was unable to induce necrosis. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the RBPG1 gene, which is required for responsiveness of A. thaliana to the BcPG3 protein. Co-immunoprecipitation experiments demonstrated that RBPG1 and BcPG3 form a complex inNicotiana benthamiana, which also involves the A. thaliana LRR-RLK SOBIR1. The sobir1 mutant plants no longer respond to BcPG3. Furthermore, overexpression of RBPG1 in the BcPG3-non-responsive accession Br-0 did not enhance resistance to a number of microbial pathogens.

    Chapter 3describes the functional, biochemical and genetic characterization of the D-galacturonic acid catabolic pathway in B. cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-L-galactonate aldolase gene (Bclga1). Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on D-galacturonic acid, pectate, or pectin as the sole carbon source. The extent of growth reduction of the mutants on pectic substrates was positively correlated to the proportion of D-galacturonic acid present in the pectic substrate. The virulence of these mutants on different host plants is discussed in Chapter 4. These mutants showed reduced virulence on N. benthamiana and A. thaliana leaves, but not on tomato leaves. The cell walls of N. benthamiana and A. thaliana leaves have a higher D-galacturonic acid content as compared to tomato. Additional in vitro growth assays with the knockout mutants suggested that the reduced virulence of D-galacturonic acid catabolism-deficient mutants on N. benthamiana and A. thaliana is not only due to the inability of the mutants to utilize an abundant carbon source as nutrient, but also due to the growth inhibition by catabolic intermediates.

    In Chapter 5, the functional characterization of two putative D-galacturonic acid transporters is presented. Bchxt15 is highly and specifically induced by D-galacturonic acid, whereas Bchxt13 is highly expressed in the presence of all carbon sources tested except for glucose. Subcellular localization of BcHXT13-GFP and BcHXT15-GFP fusion proteins expressed under their native promoter suggests that the fusion proteins are localized in plasma membranes and intracellular vesicles. Knockout mutants in the Bchxt13 and Bchxt15 genes, respectively, were neither affected in their growth on D-galacturonic acid as the sole carbon source, nor in their virulence on tomato and N. benthamiana leaves.

    Chapter 6describes the genome-wide transcriptome analysis in B. cinerea grown in media containing glucose and pectate as sole carbon sources. Genes were identified that are significantly altered in their expression during growth on these two carbon sources. Conserved sequence motifs were identified in the promoters of genes involved in pectate decomposition and D-galacturonic acid utilization. The role of these motifs in regulating D-galacturonic acid-induced expression was functionally analysed in thepromoter of the Bclga1 gene, which encodes one of the key enzymes in the D-galacturonic acid catabolic pathway. The regulation by D-galacturonic acid required the presence of sequences encompassing the GAE1 motif and a binding motif for the pH-dependent transcriptional regulator PacC.

    Chapter 7 provides a general discussion of the results presented in this thesis. A model of the concerted action of pectin degradation and subsequent monosaccharide consumption and co-regulation of gene expression is proposed.

    Exocytosis and polarity in plant cells: insights by studying cellulose synthase complexes and the exocyst
    Ying Zhang, Ying - \ 2012
    Wageningen University. Promotor(en): Anne Mie Emons, co-promotor(en): Tijs Ketelaar; C.M. Liu. - S.l. : s.n. - ISBN 9789461734075 - 132
    plantencelbiologie - cellen - exocytose - cellulose - polariteit - microtubuli - celwanden - celwandstoffen - plant cell biology - cells - exocytosis - cellulose - polarity - microtubules - cell walls - cell wall components

    The work presented in this thesis covers aspects of exocytosis, plant cell growth and cell wall formation. These processes are strongly linked as cell growth and cell wall formation occur simultaneously and exocytosis is the process that delivers cell wall components to the existing cell wall and integral membrane proteins to the plasma membrane. The chapters in this thesis describe work on the exocyst, a group of proteins thought to be involved in polarized secretion, the regulation of CESA complex mediated cellulose microfibril deposition by cortical microtubules, and the organization of cortical microtubules. Chapter 1 is a review in which research on the plant exocyst is discussed. We compare the literature about the plant exocyst with knowledge about well-studied yeast and mammalian exocysts and explore which aspects of exocyst functioning are conserved in plants and which aspects are not. We propose that the plant exocyst has acquired distinct functions and mechanisms in exocytosis for plant cell growth, based on the fact that each subunit of the exocyst in yeast and mammals is encoded by one gene, whereas some exocyst subunits in plants, particularly EXO70, are encoded by multiple genes. In Chapter 2, we presented experimental data on the exocyst. Using a yeast two hybrid based approach we present novel interactions between different exocyst subunits. We continue by focusing on the exocyst subunit SEC3, which functions as a landmark protein in yeast and mammalian cells. We show that both SEC3 genes in Arabidopsis are essential for plant development; A T-DNA insertion in the SEC3A gene causes embryo development to arrest at the globular stage and a T-DNA insertion in the SEC3B gene causes gametophytic lethality. We were able to complement the sec3a mutant by introducing a pSEC3A::SEC3A:GFP construct and used the resulting lines to study the subcellular localization of SEC3A. The fusion protein shows a similar localization to cytokinetic cell plates as has been shown for other exocyst subunits. In interphase cells SEC3A:GFP localizes to the cytoplasm and to the plasma membrane, where it forms immobile, punctuate structures with discrete average lifetimes of 6-12 seconds. These puncta are equally distributed over the cell surface of root epidermal cells and tip growing root hairs and the density of puncta does not decrease after growth termination of these cells. Either SEC3a puncta may not participate in exocytosis for polarized cell expansion, or the plasma membrane recruitment of SEC3 is a default process that requires other, polarly localized factors to mediate exocytotic events. Chapter 3 focused on the role of cortical microtubules in the insertion, guidance and occurrence in the plasma membrane of cellulose microfibril producing CESA complexes. We characterized a wide range of parameters that give insight in CESA complex behavior, such as velocity, density and movement angles in the expanding tip and non-expanding tube of growing root hairs and the same areas in fully-grown root hairs. Then we performed co-localization studies of CESA complexes with cortical microtubules. In tubes of both growing and fully-grown root hair cells CESA complex insertion occurred preferentially on cortical microtubules. Part of the population of CESA complexes that were moving in the plasma membrane was tracking cortical microtubules, whereas others were moving in between cortical microtubules. CESA complexes tracking cortical microtubules had a slightly different movement direction, but also a much lower variation in movement direction than the CESA complexes that were moving in between cortical microtubules. When microtubules are absent, all CESA complexes move in the same direction as those that do not track cortical microtubules in the presence of microtubules, and the variation in the movement direction is similar to that of CESA complexes moving in between cortical microtubules. This shows that CMTs in root hairs focus CESA complex movement, by which they order cellulose microfibrils into a tighter helix. In the absence of microtubules, the average lifetime of CESA complexes increases from 12.8 minutes to 22.3 minutes, showing that there is a feedback mechanism between CESA complex insertion and CESA complex lifetime. Since their velocity of movement in the plasma membrane does not change, they produce longer cellulose microfibrils in the absence of cortical microtubules. In Chapter 4, we addressed the question how CESA complexes that are guided by widely spaced cortical microtubules can produce a uniform layer of cellulose microfibrils with a much smaller spacing in axially growing root epidermal cells. We studied the orientation, density, alignment and movement of cortical microtubules and CESA complexes using immunocytochemistry and live cell imaging of root epidermal cells. The CMTs, the rows of CESA complexes and the innermost CMFs lay in the same orientation, approximately transverse to the elongation axis in both the inner and outer periclinal cell face in the elongation zone and root hair zone. CESA complexes predominantly move in rows along CMTs in both directions. Analysis of timelapse movies of CMTs revealed that the position shifting of cortical microtubules accounts for how the uniform layer of cellulose microfibrils can be formed. Chapter 5 is the general discussion of the thesis, where we provide a framework in which the results presented in the previous chapters fall.

    From Golgi body movement to cellulose microfibril alignment
    Akkerman, M. - \ 2012
    Wageningen University. Promotor(en): Anne Mie Emons, co-promotor(en): Tijs Ketelaar. - S.l. : s.n. - ISBN 9789461733030 - 122
    plantencelbiologie - golgiapparaat - organellen - cellulose - cellen - celwanden - microtubuli - arabidopsis thaliana - plant cell biology - golgi apparatus - organelles - cellulose - cells - cell walls - microtubules - arabidopsis thaliana

    The shape and strength of plant cells is determined by a combination of turgor pressure and constraining cell wall. The main load bearing structures in the cell wall, cellulose microfibrils (CMFs), are deposited in highly organized textures. For more than 50 years scientists have tried to elucidate how the organized CMF textures are being generated and what role cortical microtubules (CMTs) play in CMF deposition. In 2006 Paredez et al. caused a breakthrough by live imaging of cellulose synthase (CESA) complexes that move along CMTs. However, the mechanism by which CMTs guide CESA complexes is still unknown at the moment of writing this thesis and many questions related to the CMF organization still are unanswered.
    This thesis illuminates the mechanism behind the highly organized CMF textures. To analyze the positioning and patterning of CESA complexes in the cell we studied the following three aspects: (1) the distribution and delivery (close) to the plasma membrane of CESA complexes via Golgi bodies, (2) the distribution and movement of CESA complexes inside the plasma membrane while producing CMFs and finally (3) their product, the CMF texture of the cell wall. We chose epidermal root cells of Arabidopsis thaliana and compared cells of different growth stages.

    Chapter 1 is an introduction into cellulose deposition and an outline of this thesis.

    In chapter 2 the movement and distribution of Golgi bodies is studied in the cortex of cells of different growth stages, early elongation zone compared to late elongation zone, in relation to the configuration of the actin cytoskeleton. Golgi bodies in the cortex of cells in the early elongation zone, where growth accelerates to rapid growth, show slow random oriented movement, called wiggling. In the cortex of cells in the late elongation zone, where cell elongation ceases, they also show a second kind of motility, fast directed movement with velocities of up to 7 µm.s-1, like in cytoplasmic strands in the same cells. The cortical areas where Golgi body movement is slow and random co-localize with fine F-actin, a configuration of single or thin bundles of filaments. On the other hand, areas where Golgi body movement is fast and directed co-localize with thick actin filament bundles. When Golgi bodies enter an area with a different actin cytoskeleton configuration they change their type of motility concomitantly. We conclude that Golgi body dynamics correlate with the actin cytoskeleton organization.

    CESA complexes are known to run in rows along CMTs in Arabidopsis hypocotyl cells. In chapter 3 we studied the orientation, density, alignment and movement of CMTs and CESA complexes using immunocytochemistry and live cell imaging. Furthermore we studied the orientation and density of the product of the CESA complexes, the CMFs, in the innermost wall layer with Field Emission Scanning Electron Microscopy (FESEM). The CMTs, the tracks of CESA complexes and the innermost CMFs lay in the same orientation, approximately transverse to the elongation axis in both the inner and outer periclinal cell face in the elongation zone and root hair zone, where cell elongation ceases. CESA complexes predominantly move in rows along CMTs in both directions. While the CMFs form a uniform cell wall layer, CESA complexes run one after the other along CMTs that are wider spread from each other than the CMFs and only few CESA complexes move in between the CMTs. To understand how CESA complexes can produce a uniform layer of CMFs, instead of local CMF thickenings, we studied whether the CMTs change position during CMF production. Time lapse movies of CMTs show that CMTs reposition over time, so that CESA complexes produce an even CMF layer. In this way we can understand how the density of CMFs in the nascent cell wall can be higher than that of the CMTs and the moving rows of CMFs in the plasma membrane. CMFs are deposited consecutively next to earlier deposited ones in the same orientation.

    In chapter 4 we used several different electron microscopy techniques to visualize CMF texture: transmission Electron Microscopy (TEM) of ultrathin sections after mild or complete matrix extraction, TEM of surface preparations and FESEM of surface preparations. We used root hairs of three different species; Arabidopsis thaliana, Medicago truncatula and Vicia sativa. We compare and discuss the results of the techniques for the capacity to measure orientation, density, length and width of the CMFs. In ultrathin sections and surface preparations we observed that the three species studied have root hairs with an axial/helical wall texture. Surface preparations are best suitable for density and orientation measurements of CMFs within the most inner cell wall layer. Ultrathin sections showed that the thickness of CMFs in Arabidopsis is approximately 3 nm. which indicates that these CMFs are produced by single CESA complexes.

    Chapter 5 is a general discussion of our work in relation to the field. It describes the role of the actin cytoskeleton , Golgi body motility and CMTs in the deposition of an organized texture of CMFs.

    Characterising the cellulose synthase complexes of cell walls
    Mansoori Zangir, N. - \ 2012
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade. - S.l. : s.n. - ISBN 9789461732958 - 162
    planten - celwanden - cellulose - biosynthese - enzymen - enzymactiviteit - eiwitten - katalyse - genetische kartering - genomica - plants - cell walls - cellulose - biosynthesis - enzymes - enzyme activity - proteins - catalysis - genetic mapping - genomics

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the catalytic subunits of the complex. The objective of the research presented in this thesis was to generate more in-depth knowledge in cellulose biosynthesis and to this aim better characterize and understand the cellulose synthase complex and its components by notably investigating the similarities and differences between the CESAs in the primary and secondary cellulose complex and identifying the various interacting proteins forming the complex in the plant cell wall. KORRIGAN and specific isoforms of sucrose synthase were shown to be co-localized and physically interact with the CESAs in the Cellulose Synthase Complex at the plasma membrane supporting their participation in cellulose biosynthesis in Arabidopsis.

    Proceedings international symposium role of plant cell walls in dairy cow nutrition, 22nd and 23rd March 2010, Wageningen, The Netherlands
    Duinkerken, G. van - \ 2010
    [S.l.] : Centre for Animal Nutrition - 90
    rundveehouderij - rundvee - rundveevoeding - celwanden - cattle husbandry - cattle - cattle feeding - cell walls
    Het gebruik van plantcelwanden in rundveevoeding moet er toe bijdragen dat de CO2 uitstoot in de rundveehouderij verder omlaag wordt gebracht. Verslag van een symposium over plantcelwanden in de rundveevoeding. Het symposium werd gehouden op 22 en 23 maart 2010 in Wageningen.
    Steeds meer inzicht in de celwand. Oogsttijdstip, groeiomstandigheden en ras bepalen celwandverteerbaarheid maïs
    Cone, J.W. - \ 2009
    Veeteelt 26 (2009)3. - ISSN 0168-7565 - p. 12 - 14.
    melkveehouderij - zea mays - plantenveredeling - voedering - celwanden - verteerbaarheid - dairy farming - zea mays - plant breeding - feeding - cell walls - digestibility
    Na jaren van veredeling op het kolfaandeel in de maisplant krijgt nu ook de celwandverteerbaarheid van de restplant meer aandacht. Waarom is de ene celwand wel verteerbaar en de andere niet? Onderzoek door ASG en Wageningen UR moet nieuwe inzichten geven voor verdere veredeling
    Analyzing the complex machinery of cell wall biosynthesis
    Timmers, J.F.P. - \ 2009
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade; Jean-Paul Vincken. - [S.l. : S.n. - ISBN 9789085855163 - 120
    celwanden - biosynthese - celwandstoffen - cellulose - xyloglucanen - planten - cell walls - biosynthesis - cell wall components - cellulose - xyloglucans - plants
    The plant cell wall polymers make up most of the plant biomass and provide the raw material for many economically important products including food, feed, bio-materials, chemicals, textiles, and biofuel. This broad range of functions and applications make the biosynthesis of these polysaccharides a highly interesting target of scientific research.
    In this thesis a protein-protein interaction strategy was used to gain insight in the cell wall biosynthesis of Arabidopsis thaliana and to identify additional genes involved in this process. Using the membrane based yeast two hybrid system several distinct goals have been reached in this thesis, i) the characterization of the rosette structure by resolving the organization of the different cellulose synthase proteins in the complex, ii) the identification of unknown components of the cellulose synthezing machinery, iii) the confirmation of a xyloglucan synthesizing complex and the identification of several of its components.
    On the whole, this work has generated an effective tool in cell wall research and identified new players in the biosynthesis of both cellulose and xyloglucan.

    De fysiologie van bewegen : door beweging en aanraking blijven planten korter
    Kierkels, T. ; Heuvelink, E. - \ 2008
    Onder Glas 5 (2008)5. - p. 38 - 39.
    kassen - teelt onder bescherming - sierplanten - beweging - oscillatie - celwanden - verwringing - cultivars - remming - glastuinbouw - groenten - potplanten - greenhouses - protected cultivation - ornamental plants - movement - oscillation - cell walls - distortion - cultivars - inhibition - greenhouse horticulture - vegetables - pot plants
    Door het bewegen of aanraken van planten vervormt de celwand. Dat zet een aantal reacties in werking waardoor uiteindelijk nieuwe cellen korter blijven met dikkere celwanden. Al met al kan trillen, aanraken en borstelen wel perspectief hebben als alternatief voor chemische remming, maar het vergt heel veel uitproberen omdat met de huidige kennis niet te voorspellen valt welk soort of cultivar effectief te remmen valt
    Crop Platforms for cell wall biorefining : lignocellulose feedstocks
    Möller, R. ; Toonen, M.A.J. ; Beilen, J.B. ; Salentijn, E.M.J. ; Clayton, D. - \ 2007
    Newbury, Berks, UK : CPL Press - ISBN 9781872691138 - 161 p.
    celwanden - ruwe grondstoffen - lignocellulose - voer - biomassa - bio-energie - bioraffinage - cell walls - raw materials - lignocellulose - feeds - biomass - bioenergy - biorefinery
    The physics of cellulose biosynthesis : polymerization and self-organization, from plants to bacteria
    Diotallevi, F. - \ 2007
    Wageningen University. Promotor(en): Bela Mulder; Anne Mie Emons. - [S.l.] : S.n. - ISBN 9789085047193 - 118
    cellulose - biosynthese - planten - bacteriën - polymerisatie - kristallisatie - celwanden - acetobacter - cellulose - biosynthesis - plants - bacteria - polymerization - crystallization - cell walls - acetobacter
    This thesis deals with many different biological problems concerning cellulose biosynthesis. Cellulose is made by all plants, and therefore it is probably the most abundant organic compound on Earth. Aside from being the primary building material for plants, this biopolymer is of great economic importance globally because it is the major constituent of cotton (over 94%) and wood (over 50%). Moreover, according to how it is treated, cellulose can be used to make paper, film, explosives, and plastics, in addition to having many other industrial uses. The paper in this book, for example, contains cellulose, as do some of the clothes we are wearing.
    In addition to higher plants, cellulose is synthesized by a number of bacterial species, algae, lower eukaryotes (tunicates), and the slime mold Dictyostelium. The function of cellulose in these different groups of organisms reflects the diverse role associated with this simple structural polysaccharide. Whereas it is possible for some organisms, specifically bacteria, to survive in absence of cellulose synthesis, it may not be true for most vascular plant cells. As such, the importance of cellulose in the life of a plant cannot be overemphasized since it not only provides the necessary strength to resist the turgor pressure in plant cells, but also has a distinct role in maintaining the size, shape and differentiation of most plant cells.
    The aim of this thesis is to investigate, by mean of theoretical methods, coupled to simulation techniques, the polymerization, crystallization, and self-organization mechanism of this universal distributed polysaccharide, in different biological systems.
    We start in Chapter 2 with a general description of the chemical and mechanical features of the cellulose microfibrils (CMFs), the crystalline form of cellulose in nature. After a brief overview on the biogenesis of the CMFs in the plant cells we proceed focusing on two of the most important cellulose producer entities: the plant cells and the Acetobacter cells.
    The first part of the thesis, therefore, is concerned with all the aspects related to cellulose biosynthesis in the cell-wall of plant cells. We begin in Chapter 3 with a detailed investigation on the self-assembly mechanism of the Cellulose Synthase Complex (CSC) in higher plants, the hexagonal Rosette CSC: based on the known experimental evidences regarding the internal structure of this protein, we are able to build a theoretical scheme to characterize the interactions among the CSC subunits; then, by mean of a Monte Carlo algorithm, we implement this interaction scheme in a simulation that document step by step the formation of the hexagonal enzyme. Our model is able to explain the assembly of many types of CSCs, like the hexagonal Rosettes of plants as well as the linear CSCs present in bacteria and the clusters that form in the cell wall of some algae.
    After having clarified the formation of the Rosette CSC structure, we shift our attention to its motion in the plasma membrane of plant cell. In Chapter 4 we present a biophysical model that unravels the force generating mechanism underlying the propulsion of the Rosette CSC: the model identifies polymerization and crystallization as driving forces, and elucidates the role of polymer flexibility and membrane elasticity as force transducers. On the basis of our model and appropriate values for the relevant physical constants, we obtain a theoretical estimate for the velocity of the CSC that is in agreement with the experimental value. To have a proof a principle of the proposed mechanism, we have also developed a stochastic simulation that reproduces the movement of the Rosette CSC in the fluid membrane of the plant cell.
    The last issue related to plant cells cellulose is the formulation of a mathematical model to analyze the building of cell wall architecture (Chapter 5). The highly regular textures observed in cell walls reflect the spatial organization of the cellulose CMFs. Based on a geometrical hypothesis proposed earlier, we formulate a model that describes the space-time evolution of the density of Rosette CSCs in the plasma membrane of plant cell. The trajectories of the Rosettes are assumed to be governed by an optimal packing constraint of the CMFs that couples the direction of motion to the density of the CSCs. Our model is based on a relatively small numbers of variables that can be tuned to obtain most of the cell wall textures that have been found experimentally. Moreover, we demonstrate that it is also robust against a number of perturbations and noise effects.
    The second part of the thesis is focused on the cellulose-producing Acetobacter cells, which live at the air-liquid interface and which exhibit a peculiar motion during the cellulose polymerization process. The mechanism of formation as well as the structure of the bacterial cellulose has been studied extensively in recent decades. The cellulose product appears as a long ribbon, composed of many CMFs, which extends parallel to the longitudinal axis of the cell, and which is synthesized by a linear array of particles placed along the axis of the bacterial rod. Goal of this chapter (Chapter 6) is to correlate the peculiar motion of Acetobacter with an hydrodynamic effect caused by the interactions between the cellulose CMFs and the fluid in which they are immersed. To further assess the correctness of our model, in the last part of the thesis we implement a Brownian Dynamics simulation that is able to reproduce the main features of this particular bacterial motion.
    With this work we hope to contribute in elucidating some key questions, both regarding the cell biology of plants as well as concerning the physics of interacting filaments and complex macromolecular assemblies.
    The role of pectin degradation in pathogenesis of Botrytis cinerea
    Kars, I. - \ 2007
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jan van Kan. - - 166
    botrytis cinerea - pectinen - degradatie - pathogenese - celwanden - methodologie - botrytis cinerea - pectins - degradation - pathogenesis - cell walls - methodology
    Botrytis cinerea is a fungal plant pathogen that causes soft rot in many plant species. During the infection process, from the moment a conidium lands on the plant surface until complete host colonization, the fungus secretes numerous enzymes and metabolites that may contribute to virulence. Among the extracellular enzymes that are produced are pectin-degrading enzymes (pectinases) that facilitate the penetration of the plant surface and growth into the middle lamella, and contribute to decomposition of plant tissue and its conversion into fungal biomass. The main objective of the work was to elucidate the process of cell wall-degradation by B. cinerea, by studying when the genes were expressed during infection and determining which of the genes played an important role in the infection (functional analysis). Therefore, a PCR-based targeted mutagenesis method was developed. Using this method, many B. cinerea mutants were created in which endopolygalacturonase (Bcpg) or pectin methylesterase (Bcpme) genes were lacking. We showed that Bcpg2 played an important role in the infection from the moment that the fungus is penetrating the plant tissue. Other genes tested played no important role during the infection. Furthermore, we showed that BcPG enzymes each have specific biochemical characteristics and can degrade pectin in distinct ways. We tested whether each of the enzymes was capable of causing damage to healthy plant tissue. Especially BcPG1 and BcPG2 caused major damage resulting in rapid tissue collapse. This visible damage was caused by the enzymatic activity of BcPG2, not due to the response to protein recognition. The natural variation among Arabidopsis thaliana (thale cress) in their responses to infiltration with purified BcPGs was genetically analyzed in segregating progenies from crosses between parents that strongly differed in sensitivity to BcPGs. A genetic region that controls the response to BcPG2, BcPG3, BcPG4 and BcPG6 was identified. The identified genetic region is a starting point to identify and isolate the gene involved in response to BcPGs and study its role in resistance to B. cinerea.
    Efficiënt gebruik van snijmaïs. Deel 4: invloed rastype en oogststadium op afbraakkaraketeristieken van zetmeel en celwanden = Efficient use of silage maize. Part 4: effect of genotype and harvest stage on degradation characteristics of starch and cell walls
    Cone, J.W. ; Costa Ramos, I. da; Gelder, A.H. van - \ 2006
    Lelystad : Animal Sciences Group / Praktijkonderzoek (PraktijkRapport / Animal Sciences Group : Rundvee ) - 34
    maïs - zea mays - rassen (planten) - cultivars - maïskuilvoer - gewasopbrengst - zetmeelvertering - celwanden - microbiële afbraak - melkveehouderij - rundveevoeding - maize - zea mays - varieties - cultivars - maize silage - crop yield - starch digestion - cell walls - microbial degradation - dairy farming - cattle feeding
    In the period 2003-2005 effects of genotypes and maturity stage on yield, quality, conservation and nutrition were investigated. This research was focusing on degradability of starch and cell walls. Harvest stage had significant effects on degradability parameters. No clear systematic differences between the genotypes emerged from the research
    The role of cell wall-modifying proteins in plant penetration and feeding site proliferation by the potato cyst nematode Globodera rostochiensis
    Kudla, U. - \ 2006
    Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Hans Helder; Geert Smant. - Wageningen : Wageningen Universiteit - ISBN 9789085044147 - 121
    globodera rostochiensis - plantenparasitaire nematoden - celwanden - eiwitten - pectaat lyase - globodera rostochiensis - plant parasitic nematodes - cell walls - proteins - pectate lyase
    The main objective of this thesis was to investigate two distinct groups of proteins involved in plant cell walls modifications in the parasitism of the potato cyst nematode, Globodera rostochiensis , namely pectate lyases and expansins. Plant parasitism of potato cyst nematode proceeds through two main stages i.e. mobile and sessile. During the migratory phase, potato cyst nematode uses cell wall degrading enzymes and mechanical force of the stylet protruding from nematode's buccal cavity to disrupt cell walls in host plant tissue. In order to degrade plant cell wall components, nematodes produce a range of cell wall degrading and modifying proteins. Plant parasitic nematodes were the first animals for which it was shown that they are able to degrade cell wall polymers (i.e. cellulose microfibrils) without the help of symbiotic microorganisms. This first finding of an endoglucanase produced by G. rostochiensis turned out to be only a tip of an iceberg. To date, similar evidence is found in plant parasitic nematodes for the presence of enzymes capable of depolymerising the pectin matrix in plant cell walls (i.e. pectate lyase and exopolygalacturonase). In Chapter II, a novel putative pectate lyase gene from G. rostochiensis is described including a functional assay using Agrobacterium mediated leaf infiltration and site directed mutagenesis.

    In Chapters III and IV, we showed that potato cyst nematode is producing and secreting expansins, which are likely to aid the activity of cell wall degrading enzymes. Contrary to pectinolytic enzymes expansins are a relatively newly described group of proteins and there are still a lot of unresolved issues that need to be addressed with regard to their mode of action, biological functions and classification. Since their discovery in 1992, expansins were thought to be specific for land plants. In this thesis, we present the first evidence of a functional expansin produced outside of the plant kingdom. We demonstrated that invasive juveniles of G. rostochiensis secrete b-expansin, which most likely facilitates the disruption of cell walls in the root tissues of a host by making the structural cell wall polymers more accessible to cell wall degrading enzymes.

    The cell wall modifications occurring during feeding site formation by potato cyst nematode, described in the fourth chapter of this thesis, are part of the elaborate changes in the plant cell wall architecture during syncytium induction and maintenance. In contrast to the rigorous breakdown of cell walls and subsequent collapse of the protoplast during nematode migration, the modifications during feeding site formation aim at a subtle remodeling of the cell wall, including both cell wall dissolution and synthesis. The uniform structure of nematode induced syncytia suggests that these cell wall modifications are precisely controlled. A key role for the recruitment of plant enzymes (e.g. cellulases and pectin methyl esterases), by nematodes to modify plant cell wall structure to their advantage has been pointed at in several papers. The data presented in Chapter V shows that these cell wall modifications in a nematode-induced syncytium are mediated by enzymes and -expansins and expansin-like proteins of the host plant. This is the first report that links morphological changes in the cell walls of dicotes plants to the expression ofb- expansin genes.
    Bioengineering cellulose-hemicellulose networks in plants
    Obembe, O. - \ 2006
    Wageningen University. Promotor(en): Evert Jacobsen; Richard Visser, co-promotor(en): Jean-Paul Vincken. - [S.l.] : S.n. - ISBN 9789085043522 - 127
    cellulose - hemicellulosen - celwanden - vezelkwaliteit - plantenveredeling - solanum tuberosum - nicotiana tabacum - kruisingen - genexpressie - aardappelen - tabak - interacties - plantensamenstelling - bindende eiwitten - solanum tuberosum - potatoes - nicotiana tabacum - tobacco - cellulose - hemicelluloses - interactions - plant composition - cell walls - fibre quality - plant breeding - crosses - gene expression - binding proteins
    The interactions between cellulose and hemicellulose in the cell walls are important in the industrial application of the cellulose (natural) fibres. We strive to modify these interactions (i) by interfering with cellulose biosynthesis and (ii) by direct interference of the interactions through the use of carbohydrate binding modules (CBMs). Additionally, we strive to establish a link between the putative expansin CBM and cell wall/plant modification. The investigation generates structural changes in the cell walls leading to altered cellular, organ and whole plant morphology. The results demonstrate that a gain of control over the inherent cellulose fibre characteristics for paper and textile applications is possible in the future. This can complement the current use of enzymes and chemicals for surface modification of cellulose fibre. The approaches described in the thesis hold great promise for modifying the fibre properties inside the plant. The technology can be transferred to economically important fibre species, e.g. flax, hemp and poplar.
    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
    Hemicellulose biosynthesis and degradation in tobacco cell walls
    Compier, M.G.M. - \ 2005
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Jean-Paul Vincken. - Wageningen : s.n. - ISBN 9789085042785 - 120
    nicotiana tabacum - tabak - hemicellulosen - biosynthese - degradatie - celwanden - xylaan - transgene planten - plantenveredeling - vezelgewassen - nicotiana tabacum - tobacco - fibre plants - xylan - hemicelluloses - cell walls - biosynthesis - degradation - transgenic plants - plant breeding
    Natural fibres have a wide range of technological applications, such as in paper and textile industries. The basic properties and the quality of plant fibres are determined by the composition of the plant cell wall. Characteristic for fibres are thick secondary cell walls, which consist of cellulose microfibrils in a matrix of lignin and hemicelluloses. The major hemicellulose component of the dicot secondary wall is xylan. This component can establish tight interactions with cellulose and covalent bonds with lignin and is thereby involved in strengthening of the wall. Since high amounts of hemicelluloses as well as lignins have a negative effect on the industrial processing of fibres, the development of plants with altered cell wall composition is of great value. This thesis focuses on modification of the hemicellulose (in particular xylan) content and thereby also the attachment of lignin in the cell wall.Different approaches to generate transgenically modified plants with altered xylan composition have been examined. (i) Decreasing the expression levels of putative xylan synthases, responsible for the polymerisation of the xylan backbone. (ii) Degradation of already deposited xylan polymers by the introduction of xylan degrading enzymes (xylanases). In our research, tobacco ( Nicotiana tabacum ) is used as a model species, since it is widely used in fundamental cell wall research. Once interesting results are obtained in tobacco, technologies can be transferred into economically more important species, such as flax ( Linum usitatissimum ) and poplar ( Populus spp). 

    At the start of the work described in this thesis, genes involved in hemicellulose backbone biosynthesis were not identified yet. This favoured the use of a set of candidate genes, the cellulose synthase-like genes ( Csl s), which are generally suggested to be involved in the biosynthesis of the non-cellulosic cell wall polysaccharides. Plants contain at least eight families of Csl s ( CslA though H ), which all show the conserved motifs common to polymerizing β-glycosyltransferases. In order to investigate the function of six Csl families ( CslA , B , C , D , E and G )we introduced Inverted Repeat (IR) constructs, based on potato ( Solanum tuberosum ) Csl cDNA sequences into tobacco. If one of the Csl family members is coding for a xylan synthase, this strategy consequently provides us with transgenic tobacco plants with altered xylan levels.Sugar compositional analysis of cell wall material isolated from in vitro grown IR transformants revealed a reduction of xylose exclusively in the CslG transformants. These data led us to speculate about the involvement of CslG in xylan biosynthesis. Microscopic analysis of stem samples from mature CslG transformants revealed some very local effects like the multiplication of ray cells, enlargement of the size of vessels and the occurrence of abnormal fibres with thin walls and unusually high starch content. This result could indicate the tissue- or cell-type specificity of the CslG gene. Sugar compositional analyses of mature CslG plants were rather ambiguous and a second series of in vitro grown CslG transformants did not confirm the decrease of xylose that was observed during the initial screening. Additionally, the absence of tobacco CslG sequence information did not allow us to link the reduced xylose levels with altered CslG mRNA expression levels. Therefore, it required searching for tobacco CslG genes.We report the isolation of full length tobacco CslE and CslG cDNA sequences from a cDNA library, constructed from mRNAs isolated from a xylogenic suspension cell culture of tobacco. The proteins encoded by the NtCslE and NtCslG cDNA clones, contain eight putative transmembrane domains, alternating conserved and variable domains, and the processive glycosyltransferase signature. Southern blot analysis revealed that the tobacco CslEand CslG gene families consist of two to four and at least three genes, respectively. Gene expression studies in wild type tobacco tissues showed that CslE expression was highest in tissues associated with secondary wall biosynthesis, whereas CslG mRNA levels were highest in tissues undergoing primary wall formation. An involvement for CslG in xylan biosynthesis in tobacco, as indicated by the heterologous IR approach, is not supported by the gene expression analysis.

    Apart from modifying the xylan content by down regulation of putative xylan backbone synthesizing enzymes, a second strategy to generate transformants with altered xylan composition was applied. This strategy involved the specific degradation of the xylan polymers, which are already deposited in the cell wall, by the introduction of a xylanase. We describe the heterologous expression of AtXyn2 , a modular enzyme from Arabidopsis thaliana , consisting of four contiguous CBM22 modules at the N-terminus, joined to a glycoside hydrolase Family 10 (GH10) catalytic domain, into tobacco. The full length AtXyn2 as well as a derivative, comprising the catalytic GH10 domain lacking the four CBMs, was ectopically expressed in tobacco.An N-terminal signal peptide and RGS×(HIS) 6 -tag mediatedtranslocation into the cell wall and detection of the protein. Although both transgenes were successfully expressed in tobacco, presence of the corresponding proteins could not be demonstrated. We speculate that this could be caused by removal of the RGS×(HIS) 6 -tag due to either post-translational processing of AtXyn2 in tobacco or due to cleavage of the epitope-tag by a tobacco protease. Our data showed that tobacco transformants, expressing the complete AtXyn2 protein, possessed a significantly higher wheat xylan and HE-cellulose degrading activity than wild type tobacco. This result indicated that AtXyn2 displays a dual hydrolytic activity towards specific β-1,4-linked xylans and glucans. Since the catalytic activity was inversely proportional to the AtXyn2 transcript level, we speculate that high amounts of AtXyn2, or released oligosaccharides, could result in a negative feedback mechanism and subsequent inactivation or degradation of the enzyme. The cellulose degrading activity of AtXyn2 appeared to be even higher than the xylan degrading activity. This raises the question whether AtXyn2 should be considered as a cellulase instead of a xylanase. Transgenic tobacco plants expressing the truncated AtXyn2 protein did not show an altered hydrolytic activity against any of the substrates tested. It can therefore be concluded that the presence of the CBM22 domains adjacent to the catalytic module seems to play an essential role in the enzyme activity.Based on our results and additional information found in literature, we propose a model, which might explain the regulation of AtXyn2 activity in the plant cell wall. This model suggests that the CBM22 domains are responsible for immobilization of the enzyme in proximity of the substrate and for correct folding and stability of the protein. The enzyme activity is in turn suggested to be regulated by post-translational cleavage by an endoprotease.   

    The thesis is concluded with a general discussion on the outcome of the different approaches to generate tobacco transformants with reduced xylan content. Although both approaches initially seemed to be very promising, we were not successful in altering the xylan level in the cell wall. The drawbacks related to the down regulation of Csl candidate genes are discussed. Alternative approaches are therefore essential to identify xylan synthases and to modulate the xylan content in the cell wall. Various strategies to identify and characterise genes involved in hemicellulose biosynthesis are discussed. The heterologous expression of AtXyn2 was expected to result in a reduction of the xylan level in the cell wall. However, the dual substrate activity and the regulation of AtXyn2 activity, make this enzyme less suitable for reduction of the xylan content. Heterologous expression of well-studied microbial xylanases could be a suitable alternative to degrade xylan polymers in planta
    Wood fibre cell walls: methods to study their formation structure and properties
    Schmitt, U. ; Ander, P. ; Barnett, J.R. ; Emons, A.M.C. ; Jeronimidis, G. ; Saranpaä, P. ; Tschegg, S. - \ 2004
    Uppsala, Sweden : Swedish University of Agr. Sciences, Dep. of Wood Science - ISBN 9789157668035 - 308
    celwanden - plantenvezels - hout - houtchemie - celultrastructuur - cell walls - plant fibres - wood - wood chemistry - cell ultrastructure
    Sturing calcium gecompliceerde zaak : sturing calcium gecompliceerde zaak
    Heuvelink, E. ; Kierhels, T. - \ 2004
    Onder Glas 1 (2004)8. - p. 12 - 13.
    plantenvoeding - tomaten - solanum lycopersicum - capsicum - calcium - celwanden - voedingsstoffentekorten - afwijkingen - paprika - glastuinbouw - groenten - plant nutrition - tomatoes - solanum lycopersicum - capsicum - calcium - cell walls - nutrient deficiencies - abnormalities - sweet peppers - greenhouse horticulture - vegetables
    Calcium zorgt voor stevige cellen en heeft ook nog een functie als boodschapper in de cellen. De verdeling van calcium is van groot belang. Die verdeling is te sturen, maar vergt wel veel aandacht. Een tekort aan calcium veroorzaakt neusrot bij tomaat en paprika en bladpuntverbranding bij siergewassen. Een overschot veroorzaakt goudspikkels bij tomaat en stip bij paprika
    Editorial: Plant degradation: a nematode expansin acting on plants
    Qin Ling, ; Kudla, U. ; Roze, E.H.A. ; Goverse, A. ; Popeijus, H. ; Nieuwland, J. ; Overmars, H.A. ; Jones, J.T. ; Schots, A. ; Smant, G. ; Bakker, J. ; Helder, J. - \ 2004
    Nature 427 (2004). - ISSN 0028-0836 - p. 30 - 30.
    cell walls
    Expansin proteins, which have so far been identified only in plants, rapidly induce extension of plant cell walls by weakening the non-covalent interactions that help to maintain their integrity1. Here we show that an animal, the plant-parasitic roundworm Globodera rostochiensis, can also produce a functional expansin, which it uses to loosen cell walls when invading its host plant. As this nematode is known to be able to disrupt covalent bonds in plant cell walls2, 3, its accompanying ability to loosen non-covalent bonds challenges the prevailing view that animals are genetically poorly equipped to degrade plant cell walls.
    Al toxicity and plant nutrient uptake: a role for root cell walls, pH and organic chelators
    Postma, J. - \ 2003
    Wageningen University. Promotor(en): Willem van Riemsdijk, co-promotor(en): W.G. Keltjens. - Wageningen : S.n. - ISBN 9789058089298 - 169
    planten - aluminium - voedingsstoffenopname (planten) - plantenvoeding - fytotoxiciteit - wortels - celwanden - chelaatvormers - plants - aluminium - nutrient uptake - plant nutrition - phytotoxicity - roots - cell walls - chelating agents
    Enzymes with activity toward Xyloglucan
    Vincken, J.P. - \ 2003
    In: Handbook of food enzymology / Whitaker, J.R., Voragen, A.G.J., Wong, D.W.S., New York : - ISBN 9780824706869 - p. 917 - 930.
    enzymen - xyloglucanen - celwanden - enzymes - xyloglucans - cell walls
    Xyloglucans are plant cell wall polysaccharides, which belong to the hemicellulose class. Here the structural variations of xyloglucans will be reviewed. Subsequently, the anchoring of xyloglucan in the plant cell wall will be discussed. Enzymes involved in degradation or modification of xyloglucan are dealt with, especially with the determination of transferase activity. Fially, a few applications in the food industry will be described
    Pectic Polysaccharides
    Schols, H.A. ; Voragen, A.G.J. - \ 2003
    In: Handbook of food enzymology / Whitaker, J.R., Voragen, A.G.J., Wong, D.W.S., New York : Dekker - ISBN 9780824706869 - p. 829 - 843.
    enzymen - polysacchariden - pectinen - celwanden - enzymes - polysaccharides - pectins - cell walls
    Pectins or pectic substances are names of a group of closely associated polysaccharides present in plant cell walls. Due to their anionic nature, pectic polysaccharides are considered to be involved in the regulation of ion transport and may control the permeability of the walls for enzymes. Also they determine the water-holding capacity of the wall
    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
    Cloning, characterisation and assessment of the cell wall modifying ability of a fungal cellulose-binding domain
    Quentin, M.G.E. - \ 2003
    Radboud University Nijmegen. Promotor(en): C. Mariani, co-promotor(en): Johan Derksen; H.C.P.M. van der Valk. - Nijmegen : Katholieke Universiteit Nijmegen - ISBN 9789064642586 - 149
    plantenvezels - celwanden - enzymactiviteit - bindende eiwitten - chemische samenstelling - genexpressie - arabidopsis thaliana - schimmels - cellulose - genetische modificatie - bacteriën - dierkloneren - arabidopsis thaliana - fungi - bacteria - plant fibres - cell walls - enzyme activity - cellulose - binding proteins - chemical composition - gene expression - genetic engineering - animal cloning
    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
    In planta modification of the potato tuber cell wall
    Oomen, R.J.F.J. - \ 2003
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Jean-Paul Vincken. - [S.I.] : S.n. - ISBN 9789058087843 - 131
    solanum tuberosum - aardappelen - genetische modificatie - celwanden - polygalacturonase - celwandstoffen - cellulose - enzymactiviteit - genexpressie - pectinen - plantenveredeling - solanum tuberosum - potatoes - cell walls - cell wall components - genetic engineering - pectins - polygalacturonase - cellulose - enzyme activity - gene expression - plant breeding

    Apart from its well known uses in the human diet a large amount of the grown potatoes (about one third in the Netherlands) is used for the isolation of starch which is used in several food and non-food applications. The cell wall fibres comprise a large portion of the waste material remaining after the starch isolation process. While cell wall fibres from some other plant species are used in food and non-food industry, a structural alteration of the potato fibres is necessary before similar applications are possible. However, before it is possible to generate plants with a tailor-made cell wall composition, questions concerning the best approach have to be answered and the necessary tools will have to be identified and made available. This thesis describes the results of a study investigating the possibilities to generate transgenically modified potato plants with an altered cell wall composition. These experiments were mostly focussed on altering pectin composition. This is particularly interesting because several studies already showed that many different pectin structures occur in specific plants, plant tissues and developmental stages. Plants with a specific alteration in pectin structure may aid in revealing the biological significance of these different structures. Additionally, the possibility to produce a particular pectin structure may be useful for the food industry, in which pectins from other plant species are already used as a gelling agent.

    At the start of the work described in this thesis only few genes involved in cell wall biosynthesis were identified which favoured the heterologous expression of fungal pectin degrading enzymes. A rhamnogalacturonan lyase ( e RGL) from Aspergillus aculeatus , which is able to cleave the rhamnogalacturonan I (RG I) at specific sites, was introduced. The e RGL was successfully expressed (under control of the granule-bound starch synthase promoter) and translated into an active protein, demonstrated by e RGL activity in the tuber extracts. These tubers showed clear morphological alterations, including radial swelling of the periderm cells and development of intercellular spaces in the cortex. Sugar compositional analysis and antibody labelling studies showed a large reduction in galactan and arabinan side-chains of RG I. These data show the possibility of specifically modifying cell wall polysaccharide structures by the introduction of such a pectin degrading enzyme. Additionally, the results suggest that RG I has an important role in anchoring galactans and arabinans at particular regions in the wall and in normal development of the periderm. The utility of these transgenic plants in answering questions concerning the biological importance of cell wall polysaccharides is evident.

    Apart from modifying the cell wall composition by the introduction of pectin degrading enzymes two experiments were performed focussed on interference with the biosynthetic machinery of the plant cell wall at different levels. The first study concerns the modulation of cellulose synthase ( CesA ) gene expression. Since this enzyme is polymerising theb-1,4 glucan chains forming cellulose, its altered expression is likely to directly affect the level of cellulose in the wall. In the second study the expression of the UDP-Glc-4-epimerase ( UGE ) was modulated. The UGE is responsible for the conversion of UDP-glucose to UDP-galactose and vice-versa. Its altered expression is likely to affect the amount of cell wall bound galactan.

    Four CesA genes were isolated from potato and one full length cDNA clone was used for up- and down-regulation of the corresponding RNA expression levels controlled by the granule-bound starch synthase promoter. Fourier Transform Infra-Red microspectroscopy (FTIR) was used for the identification of transformants with altered levels of cellulose in their tuber cell walls in comparison to WT plants. A further quantification of these results, by measuring the cellulose content in the cell wall material, showed that by modulating the CesA expression levels, tubers with levels of cellulose ranging from 50 to 200% of the WT amount were obtained. Especially the increase in cellulose is quite remarkable and in contradiction with the general believe that expression of more than one CesA gene (and possibly even more genes) is necessary to achieve such a modification. By using a specific region of the other three CesA genes in antisense experiments we managed to individually down regulate these genes and concomitantly the cellulose levels in the tubers of these plants. The use of this so-called class specific region (CSR), which is only present in plant cellulose synthases and is believed to determine the genetic difference between the different CesA genes in one plant, showed to be sufficient to down regulate the corresponding gene. In contrast to many other plants and plant systems, depletion of cellulose (to 50% of WT level) in potato tubers did not result in any phenotypic alterations. However, our potato plants were grown at normal conditions while some of the cellulose synthase mutants only revealed a phenotype when grown at restrictive conditions. Another important result is the fact that not all potato transformants with decreased cellulose levels show modifications in their pectin composition. This indicates a delicate balance between cellulose and pectin levels and that an altered pectin composition in plants with depleted cellulose is not necessarily a response upon reduced strength of the cell wall.

    For the UDP-Glc-4-epimerase two potato cDNA clones ( StUGE45 and StUGE51 ) were identified and used for overexpression in potato tubers. The increased levels of cell wall bound galactan in these tubers indicates the importance of UDP-galactose levels for galactan deposition in the cell wall. Additionally these plants showed a small decrease in the amount of galacturonan. This suggests that alterations in the UDP-galactose pool size can influence the levels of nucleotide sugars which are used for the synthesis of other polysaccharides. Additionally, the elevated expression levels of the two UGE s showed to have different effects, which suggests that they have a different function in plant development. Further research has to show whether other polysaccharides than cell wall galactan are affected by this decrease in galactose. Xyloglucan and galactomannan also contain galactosyl residues and the decrease does not necessarily affect the levels of RG I bound galactan.

    Both these studies show the possibility to induce alterations in the cell wall composition by interfering with the biosynthetic machinery. Identification of more genes involved in cell wall biosynthesis is necessary to enable new studies in the future. An RNA fingerprinting experiment was performed to investigate the possibility of identifying new genes involved in primary cell wall biosynthesis. Potato leaf protoplasts showed to regenerate a new cell wall in the first 18h after transfer to a culture medium. At 5 distinct time-points RNA was isolated and the expressed genes were visualised using cDNA-AFLP. Around 8500 transcript derived fragments (TDFs) were visualised from which 156 were isolated and sequenced. However, no cell wall related TDFs were identified. This indicates that even though the protoplasts actively regenerate a new cell wall, this did not result in highly increased expression of genes involved in cell wall biosynthesis or modification.

    In summary the experiments described in this thesis showed that different approaches can be used to generate a modified cell wall composition in potato tubers. These genetically modified plants have shown to be an interesting study material for unravelling the biological function of different cell wall polysaccharide structures. Additionally these transformants obviously showed that the potato tuber cell wall is amenable to genetic modification. There is a wide range of wall modifications which is tolerated by the tubers, which may hold a promise for the future in valorising the fibre fraction of potato after starch isolation.

    The identification of cell wall degrading enzymes in Globodera rostochiensis
    Popeijus, H.E. - \ 2002
    Wageningen University. Promotor(en): J. Bakker; A. Schots; G. Smant. - S.l. : S.n. - ISBN 9789058086914 - 86
    globodera rostochiensis - plantenparasitaire nematoden - pectaat lyase - genen - genexpressie - cellulase - celwanden - complementair dna - globodera rostochiensis - plant parasitic nematodes - pectate lyase - cellulase - genes - gene expression - cell walls - complementary dna

    This thesis describes the identification of cell wall degrading enzymes of the potato cyst nematode Globodera rostochiensis . A robust method using expressed sequence tags (ESTs) was applied to identify new parasitism related enzymes. One of the ESTs revealed the first pectate lyase from a metazoan origin. Another tag shared a strong identity towards a previously determined N-terminal amino acid sequence. Further analysis of corresponding cDNA sequence and the gene revealed two closely related beta-1,4-endoglucanases. Heterologous expression of the pectate lyase and both beta-1,4-endoglucanases showed that they are active enzymes towards their appropriate substrates ( e.g. polygalacturonic acid for the pectate lyase and carboxy methyl cellulose for both beta-1,4-endoglucanases respectively). The application of in situ hybridisation predict that these cell wall degrading enzymes are produced in the subventral oesophageal gland cells. Evidence is provided that nematodes use mixtures of cell wall degrading enzymes in order to penetrate and migrate in the plant root.

    The Botrytis cinerea endopolygalacturonase gene family
    Have, A. ten - \ 2000
    Agricultural University. Promotor(en): P.J.G.M. de Wit; J.A.L. van Kan. - S.l. : S.n. - ISBN 9789058082275 - 119
    tomaten - solanum lycopersicum - plantenziekteverwekkende schimmels - botrytis cinerea - polygalacturonase - pectinen - celwanden - degradatie - genetische analyse - genen - tomatoes - solanum lycopersicum - plant pathogenic fungi - botrytis cinerea - pectins - cell walls - degradation - genetic analysis - genes - ?

    C ell w all d egrading e nzyme s (CWDEs) secreted by microbial plant pathogens have been suggested to function as virulence factors. Evidence that particular bacterial CWDEs contribute to virulence has emerged in the last two decades. Targeted gene replacement of different genes encoding CWDEs resulted in mutants with reduced virulence on a number of host plants. Similar molecular genetic approaches in plant pathogenic fungi have, until recently, been unsuccessful in elucidating a role for fungal CWDEs in pathogenesis. This thesis describes molecular genetic analyses of CWDEs secreted by the necrotrophic plant pathogenic fungus Botrytis cinerea , the causal agent of gray mould.

    From literature it was known that B. cinerea secretes many CWDEs when grown in liquid culture. The number of CWDE encoding genes present in the B. cinerea genome was unknown and detailed expression studies were lacking. In order to fill this knowledge gap we used the following strategy:

    1. Cloning of genes encoding CWDEs
    2. Study of the expression of CWDE genes both in liquid cultures and in planta
    3. Targeted deletion of CWDE genes that have expression patterns that indicate a function in the infection process

    Chapter 1 introduces the research area and gives an outline of the thesis. It describes a model of the chemical and structural composition of the plant cell wall and reviews various classes of microbial CWDEs. It summarises previously published data on the role of bacterial and fungal CWDEs in pathogenesis in general and on the CWDEs secreted by B. cinerea in particular. B. cinerea has a wide host range but prefers hosts that contain high amounts of pectin. Therefore the focus was on endo p oly g alacturonases (endoPGs), enzymes that cleave homogalacturonan, a major constituent of pectin.

    In order to study gene expression of B. cinereain planta , it was essential to develop a standardised inoculation procedure that enables reproducible infections both in time and space. The development of this inoculation procedure for tomato leaves is described in Chapter 2. The expression of two fungal genes and a number of plant PR-protein genes was investigated in time course experiments performed at two different incubation temperatures.

    Subsequently, we set out to clone the genes of interest, analysed their expression and studied the effect in pathogenesis by targeted gene replacement. The genes were isolated by hybridisation with heterologous probes. The first gene that was cloned and characterised, Bcpg 1, is constitutively expressed. Targeted replacement of this gene resulted in a mutant with reduced virulence on apple fruits and tomato (Chapter 3). Subsequently, five additional endoPG genes were isolated (Chapter 4). The gene products were compared with other fungal endoPGs and it was shown that the members of the B. cinereaBcpg gene family fall into at least three distinct monophyletic groups (Chapter 4).

    The members of the endoPG gene family, denoted as Bcpg 1-6, are differentially expressed in liquid cultures that differed in carbon source or pH (Chapters 4). The constitutive expression pattern of Bcpg 1, as found in Chapter 3, was further confirmed. Bcpg 2 is expressed under all circumstances tested except when B. cinerea is grown in glucose-containing medium at low pH. Bcpg 3 is expressed at low ambient pH. Bcpg 4 is induced by the pectin breakdown end-product galacturonic acid, and is repressed by glucose. Bcpg 5 expression can be induced by a yet unknown factor present in apple pectin. Bcpg 6 is, like Bcpg 4, induced by galacturonic acid but is, unlike Bcpg 4, not repressed by glucose. The expression of the endoPG gene family enables the fungus to degrade pectate in a flexible manner. It enables the fungus to respond to environmental signals like nutrient availability and pH.

    The expression of the endoPG gene family during infection of tomato leaf, broad bean leaf, apple fruit and courgette fruit was studied (Chapter 5). Expression of the genes in planta is differential and most expression patterns can be explained by the results of expression studies in liquid cultures. Bcpg 1 is expressed in all host tissues tested, whereas expression of Bcpg 2 is evident in tomato, broad bean and courgette. Bcpg 3 and Bcpg 5 are expressed in apple fruit. Bcpg 4 and Bcpg 6 are expressed in all host tissues tested.

    Chapter 6 discusses the results in a broader context. It is hypothesised that, besides Bcpg 1, additional members of the Bcpg gene family contribute to virulence, albeit likely under specific circumstances. It is suggested that fungal CWDEs can play a role in plant pathogenesis but that this role also strongly depends on the lifestyle of the fungus. It is postulated that B. cinerea depends strongly on endoPGs for successful infection. The research described in this thesis may lead to novel disease control strategies that rely on P oly G alacturonase I nhibiting P rotein (PGIP) expression in transgenic host plants.

    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.

    Morphology of pits in hardwood fibres
    Magendans, J.F.C. - \ 1999
    Wageningen : Wageningen Agricultural University (Wageningen Agricultural University papers 99-2) - ISBN 9789057820366 - 100
    houtanatomie - morfologie - xyleem - putjes - celwanden - celstructuur - hardhout - vezels - wood anatomy - morphology - xylem - pits - cell walls - cell structure - hardwoods - fibres
    Structure, morphogenesis and function of tubular structures induced by cowpea mosaic virus
    Kasteel, D.T.J. - \ 1999
    Agricultural University. Promotor(en): R.W. Goldbach; J.W.M. van Lent. - S.l. : Kasteel - ISBN 9789058081094 - 71
    koebonenmozaïekvirus - plantenvirussen - plantenziekteverwekkers - pathogenese - plasmodesmata - celwanden - celstructuur - morfogenese - cowpea mosaic virus - plant viruses - plant pathogens - pathogenesis - plasmodesmata - cell walls - cell structure - morphogenesis

    During systemic plant infection, viruses move from the initially infected cells through plasmodesmata to neighbouring cells. Different mechanisms have been proposed for this cell-to-cell movement. Cowpea mosaic virus (CPMV) employs one of the major movement mechanisms, i.e. tubule-guided transport of virions, and this mechanism has been the subject of this thesis. The tubule-guided movement mechanism involves the assembly of movement protein (MP) into tubular structures within the plasmodesmal channel of infected cells, which pave the way for translocation of mature virus particles. These transport tubules are also induced on isolated plant cells (protoplasts) in the absence of cell walls and plasmodesmata. Employing this protoplast system, the structure, morphogenesis and function of CPMV tubules was studied.

    By mutational analysis of RNA-2 of CPMV (Chapter 2) it was established that mutations in the coding region of the overlapping 48 kDa/58 kDa proteins, but not the capsid proteins, resulted in abolishment of tubule formation in protoplasts. Deletion of the capsid proteins resulted in the formation of tubules without virions. As the 58 kDa protein contains the entire 48 kDa sequence, mutations made in the overlapping coding region affect the function of both proteins. To establish the involvement of each protein in tubule formation, antisera specific to the unique 10 kDa N-terminus of the 58 kDa protein were made by using synthetic peptides. Although the antisera reacted to purified 58 kDa protein in immunoblots, they failed to react in immunocytochemical experiments. Alternatively, the 58 kDa gene alone was transiently expressed in protoplasts. In these protoplasts no tubular structures were formed and the 58 kDa protein apparently localized to the nucleus. As production of the 48 kDa protein in protoplasts was previously shown to result in tubule formation it was concluded that this protein constitutes the actual viral movement protein (MP). The possible function and significance of the 58 kDa protein are discussed in Chapter 3.

    Having established that the 48 kDa protein is the viral MP responsible for tubule induction, the possible role of host proteins in this process was investigated following two different approaches. First, the expression of the MP gene in a heterologous (insect) cell system was studied (Chapter 4). As movement of plant viruses occurs through plasmodesmata, intercellular channels unique to plant cells, it was speculated that plant specific host factors could play a role in targetting and assembly of the tubules.

    However, upon production of the MP in insect cells, tubule formation occurred in a fashion similar to that in plant protoplasts. This led to the conclusion that if host proteins were involved in this mechanism, these should be of a conserved (among plant and animal) nature (Chapter 4). The second approach involved the isolation and subsequent biochemical analysis of tubules from infected protoplasts. This showed that the MP was (apart from the coat proteins) the sole major component of the movement tubules and host proteins were not obviously present in these structures (Chapter 5).

    Tubule-guided movement of virions appears to be an important mechanism used by a large variety of plant viruses. For two other viruses which are genetically unrelated to CPMV, i.e. brome mosaic virus and alfalfa mosaic virus, evidence for this mechanism was obtained during the course of this PhD research. These two representatives of the Bromoviridae are genetically more closely related to tobacco mosaic virus (TMV) which supposedly does not move using a tubule-guided mechanism, but moves as an RNA-MP complex through plasmodesmata. The fact that the MP of these viruses also forms tubules in protoplasts indicates that tubule formation may be a more general ability of plant virus MPs (Chapter 6). This hypothesis is discussed and corroborated by literature data and additional experimental evidence in Chapter 7.

    Effects of thermal processing on cell walls of green beans: a chemical and ultrastructural study.
    Stolle-Smits, T. - \ 1998
    Stolle-Smits - ISBN 9789090111919 - 145
    bonen - voedselverwerking - celwanden - beans - food processing - cell walls
    Characterization of changes in potato tissue during cooking in relation to texture development
    Marle, N. van - \ 1997
    Agricultural University. Promotor(en): A.G.J. Voragen. - S.l. : Van Marle - ISBN 9789054856610 - 141
    solanum tuberosum - aardappelen - voedingsmiddelen - chemische samenstelling - voedselbereiding - eten koken - celwanden - kookkunst - celinteracties - solanum tuberosum - potatoes - foods - chemical composition - food preparation - cooking - cell walls - cookery - cell interactions

    Texture of cooked potatoes is an important quality aspect. The diversity in texture types was sensory evaluated. Most of the differences between texture types could be explained by differences between mealy and non-mealy characteristics. Furthermore, cultivars with similar mealy/non-mealy characteristics could be discriminated on basis of firmness of cooked potato tissue.

    Cryo-scanning electron microscopy showed differences in intercellular contact and appearance of cell walls between fracture planes of cooked tissue from mealy and non- mealy cooking potato cultivars. Therefore, further research was focused on structure and composition of cell walls from the mealy cooking cv. Irene and the nonmealy cooking cv. Nicola.

    The degradation of middle lamellae during cooking was determined by recording the release of pectic material in cooking media. It was found that a given percentage release of pectic material results in more cell sloughing for cv. Irene than for cv. Nicola.

    Firstly, the effect of differences in ionic conditions in cell walls and middle lamellae on the degradation of pectic material was studied by recording the transfer of calcium, potassium and citrate during cooking of potato tissue. The transfer rates of potassium and citrate for cv. Irene are lower than expected in comparison with cv. Nicola. Calcium showed a deviant behaviour, since 80% of the calium initially present remains in tissue during cooking.

    Furthermore, composition and structure of the pectic polysaccharides in the cell walls and middle lamellae of both cultivars were studied. Although, isolated cell wall material has a comparable molar composition for both cultivars, different types of pectic polysaccharides are solubilized during cooking.

    The structure of pectic polysaccharides; was further elucidated using chemical fractionation and enzymic degradation. It is proposed that the pectin matrix in the primary cell wall of cv. Irene had a thicker and/or less porous structure than the matrix in the cell wall of cv. Nicola. Additionally, the primary cell wall of cv. Irene has a more dense and/or thicker cellulose-xyloglucan network than the primary cell wall of cv. Nicola.

    Electrochemical characterization of the bacterial cell surface
    Wal, A. van der - \ 1996
    Agricultural University. Promotor(en): J. Lyklema; A.J.B. Zehnder; W. Norde. - S.l. : Van der Wal - ISBN 9789054854920 - 101
    colloïden - bacteriën - celwanden - elektrokinetische potentiaal - elektrochemie - colloids - bacteria - cell walls - electrokinetic potential - electrochemistry

    Bacterial cells are ubiquitous in natural environments and also play important roles in domestic and industrial processes. They are found either suspended in the aqueous phase or attached to solid particles. The adhesion behaviour of bacteria is influenced by the physico-chemical properties of their cell surfaces, such as hydrophobicity and cell wall charge. The charge in the bacterial wall originates from carboxyl, phosphate and amino groups. The degree of dissociation of these anionic and cationic groups is determined by the pH and the activity of the surrounding electrolyte solution. Almost all bacterial cells are negatively charged at neutral pH, because the number of carboxyl and phosphate groups is generally higher than that of the amino groups. The presence of the charged cell wall groups leads to the spontaneous formation of an electrical double layer. The purpose of the present investigation is to elucidate the structure of the electrical double layer of bacterial cell surface. Such a study serves at least two goals. It allows the quantification of electrostatic interactions in the adhesion process and it contributes to gain better insight into the availability of (in)organic compounds for bacterial cells.

    The characteristics of the electrical double layer of bacterial cell surfaces have been revealed by applying a combination of experimental techniques, which include: chemical cell wall analysis, potentiometric proton titration and electrokinetic studies such as micro-electrophoresis, static conductivity and dielectric dispersion measurements.

    For the present study five Gram-positive bacterial strains, including four coryneforms and a Bacillus brevis, have been selected. Cell walls of these bacterial strains have been isolated and were subsequently subjected to chemical analyses and proton titration studies. Both methods provide information on the number of carboxyl, phosphate and amino groups.

    The chemical analysis of isolated cell walls involves the quantitative determination of both peptidoglycan and protein content. These analyses indicate that the chemical composition of the walls of the coryneforms are very similar, but considerably different from that of Bacillus brevis. Peptidoglycan is an important cell wall constituent of the coryneform bacteria and determines about 23 to 31 % of the cell wall dry weight. The protein fractions are somewhat lower, between 7 to 14%. The cell wall structure of the Bacillus brevis strain is more complex and multi-layered. It contains a thin peptidoglycan layer, which only determines 5 % of the cell wall dry weight. On the other hand, the protein content of these walls is higher than 56%. These proteins most likely can be attributed to a so-called S(urface)-layer, which is the outermost cell wall layer.

    The surface charge density of the bacterial cells is assessed by proton titrations of isolated cell walls at different electrolyte concentrations. Rather high values, i.e. between 0.5 and 1.0 C/m 2are found at neutral pH. The absence of hysteresis in the titration curves leads to the conclusion that the charging process can be considered as reversible. It also implies that the cell wall charge is continuously in equilibrium with the surrounding electrolyte solution, at any pH and salt concentration. This observation considerably facilitates the interpretation of the titration curves, because it allows a rigorous (thermodynamic) analysis. The anionic and cationic groups in the bacterial wall could be identified and their numbers determined by representing the differential titration curves as functions of pH and cell wall charge. The carboxyl and phosphate groups are almost entirely titrated in the pH range accessible by proton titration, allowing precise estimation of their numbers. These numbers compare very well with those based on a chemical analysis of the isolated cell walls. Estimates for the number of amino groups were less accurate, because these groups are only partly titrated in the pH range were precise titration measurements are feasible. Nevertheless, it could be concluded that the number of amino groups in the bacterial wall are lower than those of the carboxyl groups.

    Information about the ionic composition of the countercharge has been obtained from Esin-Markov analysis of the titration curves and from estimates of the cell wall potential based on a Donnan-type model. The Esin-Markov analysis is purely thermodynamic and based on first principles, whereas the Donnan model requires several assumptions about the structure of the bacterial wall. Both approaches lead to the same conclusion that at salt concentrations below 0.01 M the cell wall charge is predominantly compensated by counterions, with the excluded co-ions hardly contributing to the countercharge. This observation has considerably facilitated the interpretation of the electrokinetic properties of bacterial cell suspensions.

    Electrophoresis, static conductivity and dielectric response are related (electrokinetic) techniques and therefore share common physical bases. This also implies that the physical and mathematical problems that have to be solved in order to interpret the experimental data are very similar. Analytical solutions only exist for colloidal particles for which the electrical double layer is very thin compared to the particle dimensions. Most bacterial cells are relatively large colloidal particles and therefore the largeKa theory may be of help in the evaluation of their electrokinetic properties. However, the original theories do not include surface conductance in the hydrodynamically stagnant layer. Therefore, they had to be extended to account for the finite conductivity of ions in the bacterial wall.

    Static conductivity and dielectric dispersion both show that the counterions in the bacterial wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the counterions in the bacterial wall are of the same order but somewhat lower than those in the electrolyte solution.

    Due to surface conductance the electrophoretic mobility may be strongly retarded compared to the classical Helmholtz-Smoluchowski theory, especially at low electrolyte concentrations. In 1 mM and 10 mM electrolyte solution, the Helmholtz-Smoluchowski equation underestimates the ζ-potential by approximately a factor of 2 and 1.3, respectively.

    Resolving the fundamentals of the electrochemical characteristics of bacterial cell surfaces is a key step towards a quantitative understanding of the electrostatic interactions of bacterial cells with their surroundings. The success of such an investigation depends on the state of the art of the disciplines involved. Both microbiology and colloid chemistry have the microscopically small particle as object of study. Until recently there has hardly been any exchange of scientific knowledge between these two disciplines, despite their common interest. Colloid chemists prefered to study relatively simple particles to test their basic theories and bacterial cells were considered far too complex to serve as model colloids. However, the progress that has been made during the last decades in both colloid chemistry and microbiology provide the right tools for a successful cooporation. The present study is born from such a symbiosis and shows that many physicochemical characteristics of bacterial cell surfaces are accessible with (classical) colloid chemical techniques. In fact, for testing more advanced colloid chemical theories bacteria may even be better model particles than the generally used ionorganic colloids, because of their ability to produce a homogeneous population of identical cells.

    For the time being only Gram-positive strains have been considered, because of their relatively less complex cell wall structures. Nevertheless, the techniques used may mutatis mutandis also be applied to Gram-negative cells. In fact, such a study would be highly interesting, because it would contribute to a more complete description of the composition of the electrical double layer of bacterial cell surfaces.

    Physico - chemical stability of tomato products
    Ouden, F.W.C. den - \ 1995
    Agricultural University. Promotor(en): A.G.J. Voragen; T. van Vliet. - S.l. : Den Ouden - ISBN 9789054853985 - 113
    vloeistofmechanica - reologie - visco-elasticiteit - celwanden - enzymen - enzymologie - fermentatie - voedselindustrie - voedseltechnologie - solanum lycopersicum - tomaten - fluid mechanics - rheology - viscoelasticity - cell walls - enzymes - enzymology - fermentation - food industry - food technology - solanum lycopersicum - tomatoes

    The effect of some physical processes and enzymatic hydrolysis on the physicochemical properties of tomato suspensions was studied.

    Concentration degree has a large effect on the apparent viscosity and the storage modulus of suspensions after being diluted to a standardized water insoluble solids level. Besides decrease in average particle size, microscopic fracture of the cellulosic microfibrillar network during concentration are thought to be responsible for this phenomenon. By wet sieving it was shown that the bulk of the particles has a size between 45-180 μm. The tomato cell wall seems to be highly deformable. The 90- 180 μm wet sieve fraction had highest apparent viscosity and yield stress as well before as after homogenization.

    Homogenization of tomato suspensions as well as of strawberry sauce led to an increase in the apparent viscosity and storage modulus, whereas that of apple sauce led to a decrease in the apparent viscosity. The difference in behaviour upon homogenization is due to a difference in fracture behaviour of the plant cells, which is probably a result of the cell wall structure, especially the microfibrillar cellulose structure.

    Incubation of tomato suspensions with highly purified well specified polysaccharide degrading enzymes resulted in a decrease in rheological parameters. By homogenization the apparent viscosity increased to higher values compared to that of the non enzyme treated tomato suspension. The enzyme preparations gave rise to more serum separation. Hydrolysis of diluted hot break paste by pectin esterase from oranges or fungi resulted in a much higher yield stress, which is probably due to the formation of a calcium pectinate network. The apparent viscosity became only slightly higher. Serum viscosity increased initially, after which it decreased to about 50% of the original value.

    The main physical problem of tomato suspensions is the formation of a serum layer on top of it. Several mechanisms are responsible: uniaxial compression of the weak particle network due the gravitational force and drainage of serum as a result of unevenness in the surface. The physical behaviour of tomato suspensions can be better understood by considering the tomato cell wall as a concentrated, composite gel consisting of cellulose microfibrils embedded in a "jelly" matrix of pectic and hemicellulosic substances.

    A molecular analysis of L-arabinan degradation in Aspergillus niger and Aspergillus nidulans
    Flipphi, M.J.A. - \ 1995
    Agricultural University. Promotor(en): A.J.J. van Ooyen; J. Visser. - S.l. : Flipphi - ISBN 9789054853923 - 165
    aspergillus - celwanden - koolhydraten - cellulose - celmembranen - fermentatie - voedselbiotechnologie - glycosidasen - polysacchariden - genexpressie - pleiotropie - moleculaire genetica - aspergillus - cell walls - carbohydrates - cellulose - cell membranes - fermentation - food biotechnology - glycosidases - polysaccharides - gene expression - pleiotropy - molecular genetics

    This thesis describes a molecular study of the genetics ofL-arabinan degradation in Aspergillus niger and Aspergillus nidulans. These saprophytic hyphal fungi produce an extracellular hydrolytic enzyme system to depolymerize the plant cell wall polysaccharideL-arabinan. Chapter 1 surveys the occurrence, properties and applications ofL-arabinanolytic enzymes (arabinases). The A.niger system, which constitutes an endolytic endo-1,5-α-L-arabinase (ABN A) and two distinct α-L-arabinofuranosidases (ABF A and ABF B), has been a frequent subject of investigation in the past and represents the best characterizedL-arabinanolytic system to date. These three enzymes are all glycosylated. Current knowledge on the induction of fungal arabinase expression is summarized in this Chapter. Furthermore, the structure of the polysaccharide substrate and its function in the plant cell wall matrix are introduced.

    In Chapters 2 to 5, the cloning and characterization of the structural genes coding for the three glycosyl hydrolases from the A. nigerL-arabinan-degrading complex are described. A. niger abf A and abf B ar e the first eukaryotic ABF-encoding genes to be isolated and sequenced, abn A is the first ABN-encoding gene published. Chapter 2 reports on the isolation of the abf A gene encoding ABF A, the minor extracellular ABF. This gene could be cloned by utilizing ABF Aspecific cDNA as the probe. This cDNA was immunochemically identified from a cDNA library generated fromL-arabitol-induced myceliurn of an A. nigerD-xylulose kinase mutant. This mutant is unable to grow onL-arabitol and features enhanced expression of all three arabinases when transferred to medium containing this pentitol as sole carbon source. In Chapter 3 , the cloning of the ABN A-encoding gene (abn A) is described. This gene was isolated following the same strategy as with abf A, although a second cDNA library had to be generated first. The induction process was immunochemically monitored in order to establish the proper induction conditions for the new library. The abn A gene and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and the cDN A. The N-terminal amino acid sequences of ABN A and a CNBr-derived peptide were determined. Several transcription initiation sites and one polyadenylation site could be identified. The structural region codes for a protein of 321 amino acids and is interrupted by three introns. Extracellular ABN A consists of 302 amino acid residues with a deduced molecular weight of 32.5 kDa and a theoretical pl of 3.5. For the protein, an apparent pl of 3.0 and an apparent molecular weight of 43 kDa, determined upon SDS-PAGE, were previously reported. Chapter 4 documents the isolation and characterization of the abf B gene, coding for the major extracellular ABF. The determination of N-terminal amino acid sequences from ABF B and CNBr-generated peptides allowed the design of deoxyoligonucleotide mixtures which enabled the cloning of abf B. When utilized as primers in a polymerase chain reaction (PCR), ABF B-specific amplification products emerged, one of which was used to probe the gene. The abf B gene and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and of ABF B- specific cDNA isolated from the library described in Chapter 3. Several transcription initiation sites and one polyadenylation site could be identified. The structural region is a single open reading frame and codes for a protein of 499 amino acids. The mature enzyme consists of 481 amino acid residues with a deduced molecular weight of 50.7 kDa and a theoretical pl of 3.8. An apparent pl of 3.5 and an apparent molecular weight of 67 kDa, determined upon SDS-PAGE, were previously reported. The abf B gene product was suggested to be identical to the ABF purified and characterized by Kaji and Tagawa (Biochim Biophys Acta 207 : 456-464 (1970)). Considering the non-amino acid content of the latter protein, a molecular weight of 64 kDa could be deduced for ABF B. In Chapter 5 , the abf A gene and its gene product were characterized by DNA sequence analyses of the genomic DNA and of the cDNA for which the isolation was described in Chapter 2. The N-terminal amino acid sequences of ABF A and a CNBr-derived peptide were determined. One transcription initiation site and two polyadenylation sites could be identified. The structural region is interrupted by seven introns and codes for a protein of 628 amino acids. Mature ABF A consists of 603 amino acid residues with a deduced molecular weight of 65.4 kDa and a theoretical pl of 3.7. For this ABF, an apparent pi of 3.3 and an apparent molecular weight of 83 kDa, determined upon SDS-PAGE, were previously documented.

    Although the three enzymes are all active against (1->5)-α-glycosidic bonds betweenL-arabinofuranosides, ABF A, ABF B and ABN A are genetically unrelated. ABF A was found to be N -glycosylated whereas ABF B and ABN A were not - these enzymes are only O -glycosylated. For each gene, arabinaseoverproducing strains were generated by introducing multiple gene copies in A.niger or in A.nidulans uridine auxotrophic strains through co-transformation. Transformants were isolated upon primary selection for uridine prototrophy. Subsequent overproduction of the genes introduced was demonstrated in these recombinant strains upon growth on sugar beet pulp, both immunochemically and by assaying enzyme activity. abf A was shown to be expressed in the heterologous host A.nidulans, despite the absence of an abf A gene equivalent in this organism. High-copy number A.niger abf B transformants featured impaired secretion of other extracellular proteins upon growth on sugar beet pulp. ABN A overproduction was found to be limited to approximately five times the wild-type level in A.niger abn A transformants, but not in A.nidulans transformants. Such a limitation was not observed in case of the ABFs.

    In Chapters 5 and 6, the regulation ofL-arabinan degradation is addressed. The structural genes seem to be regulated mainly at the transcriptional level. Additional copies of either A13F-encoding gene in A.niger were shown to result in a reduction, but not in total silencing of the expression of the wild-type ABN Aencoding gene upon induction with either sugar beet pulp orL-arabitol ( Chapter 5 ). The reduction of the expression level of abn A correlated with the abf gene dosage. The repression effected by extra abf B gene copies was more stringent and more persistent than that elicited by additional abf A copies. Although observed with both inducers, these phenomena were more outspoken and more persistent on sugar beet pulp. Similar, but more moderate effects were observed towards the expression of the other abf gene in multiple copy abf A- and abf B-transformants. It was proposed that the abf genes titrate two distinct gene activators both involved in coordination of arabinase gene expression. However, the three genes were shown to respond differently upon a mycelial transfer toL-arabitol-containing medium, indicating that gene-specific factors are also involved. Four distinct sequence motifs were found in common in the promoter regions of the three genes. One of these elements is identical to the A.nidulans CREA-motif, which has been shown to mediate carbon catabolite repression on several A.nidulans enzyme systems. Arabinase expression in A.niger is known to be repressed in the presence ofD-glucose. Two other motifs are highly homologous to cAMP-responsive elements described in other organisms. For the fourth motif no functional analogues could be found, but the element was found to be present in several other fungal genes which are not involved inL-arabinan degradation at all. It is therefore likely that none of these common elements confer system-specific regulation.

    The presumed involvement ofL-arabitol in the induction process of fungal arabinases was further emphasized by the induction characteristics of an A. nidulans mutant unable to grow on the end-product ofL-arabinan degradation,L-arabinose, nor onL-arabitol ( Chapter 6).L-Arabitol is an intermediate ofL-arabinose catabolism in Aspergilli. This mutant was shown to lack NAD +-dependentL-arabitol dehydrogenase activity resulting inL-arabitol accumulation, both intracellularly and in the culture medium, wheneverL-arabinose is present. Upon submerged growth on various carbon sources in the presence ofL-arabinose, the mutant featured enhanced expression of the enzymes involved in extracellularL-arabinan degradation, and of those of the intracellularL-arabinose catabolism. The co-substrates on which the mutant secreted large amounts of arabitol simultaneously exhibited high arabinase expression and featured reduced growth.L-Arabitol secretion and enzyme production were also observed on a mixed carbon source ofD-glucose andL-arabinose, resulting in normal growth. Hence, in the presence ofL- arabinose, the carbon catabolite repression conferred byD-glucose in the wild-type, is overruled in the mutant.

    In Chapter 7 , ABN A is shown to have remote sequence similarity with four bacterial xylanolytic glycosyl hydrolases (three β-D-xylosidases and an endo-1,4-β-D-xylanase), three of which feature activity against para -nitrophenyl-α-L-arabinofuranoside. This synthetic compound is commonly utilized to assay potential ABF activity, whereas it is known to be an inhibitor of the fourth enzyme. The homology became evident only after multi pie-sequence alignments and hydrophobic cluster analysis. It was proposed that these enzymes share a binding site for a terminal non-reducing α-linkedL-arabinofuranosyl residue and that they all belong to glycosyl hydrolase family 43. Implications from these suggestions were discussed. The ABFs could not be assigned to an established glycosyl hydrolase family.

    Based on theL-arabinolytic system of the brown-rot fungus Monilinia fructigena, the sequence similarity found amongst ABF A and bacterial pullulan-degrading enzymes, and ABF expression levels under carbon starvation conditions and onD-glucose as the carbon source, distinct functions inL-arabinan and plant cell-wall degradation were proposed for ABF A and ABF B. ABF A would be essentially cell-wall associated and act to degradeL-arabinan fragments generated by ABN A. ABF B activity would be important for the primary release of small amounts ofL-arabinose which initiate induction of various endolytic systems to degrade plant cell walls, and thus function in substrate sensing. In line with these considerations, the involvement of other, not yet identified glycosyl hydrolases inL-arabinan degradation by A.niger was suggested.

    Induction and repression of arabinase gene expression are further discussed in Chapter 7 . The results of the studies in A.niger (Chapter 5) and A.nidulans (Chapter 6) were interpreted in a mutual context. The identity of the lowmolecular-weight compound directly responsible for induction of arabinase gene expression, was addressed. BothL-arabinose andL-arabitol are likely candidates to fulfil such a role. However, it was not possible to weigh the actual inductive capacities ofL-arabinose andL-arabitol due to their in vivo convertibility and the carbon catabolite repression elicited by the pentose. Competition for such a compound provides an alternative explanation for the phenomena observed in Chapter 5. The involvement of the transcriptional repressor CREA in arabinase gene expression is not limited to the direct repression of structural and regulatory genes of theL-arabinan-degrading system. It also plays a role in inducer exclusion and end-product repression, two processes shown to be eminently involved in the regulation ofL-arabinan degradation in wild-type A.nidulans. Fungal growth rate was suggested to be related to derepression of theL-arabinan-degrading system. The possible involvement of cAMP in arabinase gene expression, as suggested by the presence of potential cis -acting cAMP-responsive elements in the structural genes, was considered. Various ways by which cAMP might modulate arabinase synthesis were surveyed.

    Xylose metabolism in Bacteroides xylanolyticus X5-1
    Biesterveld, S. - \ 1994
    Agricultural University. Promotor(en): A.J.B. Zehnder; A.J.M. Stams. - S.l. : Biesterveld - ISBN 9789054852308 - 119
    bacteriën - anaërobe micro-organismen - micro-organismen - biochemie - metabolisme - synthese - koolhydraten - cellulose - celmembranen - celwanden - bacteria - anaerobes - microorganisms - biochemistry - metabolism - synthesis - carbohydrates - cellulose - cell membranes - cell walls

    Plant cell walls represent a major part of the available biomass on earth. They are mainly composed of the energy-rich polymers lignin, cellulose, and hemicellulose. For many decades, research is done to exploit agricultural and forestry wastes as renewable resources. Much research was focused on the degradation of cellulose. In contrast, hemicellulose. has got less attention, though it can account for up to 40% of the total dry weight of plant cell walls. Fermentation by anaerobic bacteria offers the possibility to conserve most energy fixed in the energy-rich polymeric and monomeric sugars in the form of organic acids and solvents (e.g. acetic acid, butanol and acetone).

    A project in which the anaerobic conversion of hemicellulose to potentially biotechnological interesting products was investigated, was divided into two parts. One part, performed by Philippe Schyns, concerned the microbial degradation of xylan, which was used as a model substrate for hemicellulose. Several xylanolytic enzymes (endo-xylanase, β-xylosidase, acetylesterase, α-L-arabinofuranosidase) were purified and characterized. The mode of action of some of these enzymes was investigated. Furthermore, the induction mechanism of xylanase and β-xylosidase was studied. The results of this research will be presented in a separate thesis. The other part of the project, of which the outcomes are given in this thesis, was focused mainly on the fermentation of xylose, a major constituent of hemicelluloses.

    Bacteroides xylanolyticus X5-1 was used as a model organism. This organism had been isolated from fermenting cattle manure. B. xylanolyticus X5-1 can only grow on one specific hemicellulose, xylan. Cellulose and other hemicelluloses could not be utilized for growth. This fact made the organism interesting for studying the (regulation of the) xylanolytic enzyme synthesis, since interferences from other (hemi)cellulolytic enzymes could be excluded. In addition, the organism could ferment a wide variety of monomeric sugars, produced a mixture of end products, and showed a relatively high growth rate. These latter features made B. xylanolyticus X5-1 a suitable microorganism for studying the regulation of the anaerobic xylose fermentation.

    Information concerning the composition and degradation of biomass, and the (regulation of) product formation from biomass has been reviewed in a general context in chapter 1. Some biotechnological applications of biomass fermentation have been mentioned in this chapter as well.

    Using 14C-labelled xylose, the xylose uptake system of this organism was studied. It was shown that xylose transport occurs via an active uptake system, and probably a binding protein was involved. The exact mechanism of xylose uptake remains to be elucidated. Based on mass balance calculations, measuring specific enzyme activities of key enzymes of catabolic pathways, and determining label distribution patterns with 13C-NMR, the pentose phosphate pathway in conjunction with the glycolysis was shown to be operative in xylose fermentation by B. xylanolyticus X5-1. Acetate, ethanol H 2 , CO 2 and formate were the main end products formed during xylose metabolism. At higher xylose concentrations, lactate and 1,2-propanediol were produced in small amounts as additional products. Reducing equivalents formed during the oxidation of glyceraldehyde-3-PO 4 and pyruvate, were used for the production of H 2 , formate, and ethanol. According to the proposed pathway about 2.5 mol of ATP, synthesized at substrate level, were generated per mol of xylose degraded. This part of the research is presented in chapter 2.

    The degradation of mixtures of hexoses and pentoses by B. xylanolyticus X5-1 is described in chapter 3. Batch culture cells did not show diauxic growth or a substrate preference for either glucose, xylose, arabinose or rhamnose, independent of the substrate the organism was grown on. In contrast, glucoselimited continuous culture cells were not able to consume xylose, unless some glucose or pyruvate was present as additional substrate. Glucose-limited continuous culture cells exhibited low activities of xylose transport and of xylose isomerase. Xylulose kinase could not be detected at all. Upon addition of xylose as single substrate to the glucose grown cells no increase in the transport rate and the isomerase and kinase activities was observed. However, when together with the xylose some glucose was added, all activities were induced. In the presence of chloramphenicol, an inhibitor of protein synthesis, xylose isomerase and xylulose kinase were not induced. The transport activity increased in a similar fashion as in the absence of chloramphenicol, suggesting that the transport system had to be activated and not induced. These experiments showed that i) xylose isomerase and xylulose kinase were regulated at the level of protein synthesis, ii) xylose transport was constitutively present, and iii) apparently, the glucose grown cells were carbon and energy limited. When grown under non-limiting conditions, as will probably happen in hemicellulose hydrolysates, B. xylanolyticus X5-1 can use sugar mixtures. This certainly is of biotechnological relevance, as conversion of the major substrate xylose will not be negatively affected by the minor, often preferred substrate glucose.

    Chapter 4 describes the effect of a low partial hydrogen pressure on the xylose metabolism in B. xylanolyticus X5-1. When grown in pure culture in the chemostat with xylose as the growth limiting substrate, B. xylanolyticus X5-1 produced acetate, ethanol, H 2 and CO 2 as the only end products. When grown in the presence of the methanogen Methanospirillum hungatei JF-1, xylose was converted to mainly acetate and CO 2 and presumably H 2 . Due to the cocultivation an increased biomass production was observed. H 2 could hardly be detected because it was efficiently converted to CH 4 by the methanogen. Ethanol was no longer produced. This type of regulation of product formation has been observed in many anaerobic microorganisms. However, xylose fermentation in B. xylanolyticus X5-1 was not only regulated at product level, but also on enzyme level. In cell free extracts of the pure culture of B. xylanolyticus X5-1 NAD and NADP-linked acetaldehyde and ethanol dehydrogenases could be detected. When grown in mixed culture with M.hungatei JF-1 these enzymes were no longer observed. The NAD and NADP-linked dehydrogenases were induced sequentially, when the interspecies electron transfer was inhibited, unless chloramphenicol was present. These results showed that product formation at low partial hydrogen pressure in B. xylanolyticus X5-1 is regulated at the level of enzyme synthesis.

    Several environmental conditions were used to affect xylose metabolism of B. xylanolyticus X5-1 ( chapter 5 ). Growth under a hydrogen atmosphere did not affect the xylose metabolism significantly. CO inhibited H 2 production from xylose completely with formate and ethanol as major reduced products. An increased ethanol yield resulted in a reduced amount of acetate and biomass formation. Xylose metabolism could also be affected by using alternative electron acceptors such as acetol, acetone, acetoin, and dihydroxy acetone. They were reduced to their corresponding alcohols 1,2-propanediol, 2-propanol, 2,3-butanediol, and glycerol, respectively. With these electron acceptors mainly acetate and CO 2 were formed and hardly any H 2 , formate and ethanol. As a result of more acetate formation, biomass production increased. In continuous culture with xylose as growth limiting substrate and acetol as electron acceptor, product formation from xylose shifted to mainly acetate and CO 2 as well. Acetol was not only reduced to 1,2-propanediol, but also converted to acetone. In gel activity staining of the alcohol dehydrogenases revealed that i) the NADP-linked ethanol dehydrogenase was repressed in the xylose + acetol grown culture, ii) the NADP-linked ethanol dehydrogenase in the xylose grown cells exhibited a nonspecific activity for both ethanol and 1,2-propanediol, and iii) another, also NADP-linked, 1,2-propanediol dehydrogenase was induced in the xylose + acetol grown cells.

    The data presented in this thesis show that it is possible to modulate the xylose metabolism of B. xylanolyticus X5-1 by several methods and at different levels during metabolism. The outcomes of this research might be applicable for other microorganisms of biotechnological value as well. Accordingly, the results can be used for biotechnological production processes and the biotechnological formation of valuable products (e.g. microbiological reduction processes, optically active products, enzymes like (stereospecific) alcohol dehydrogenases).

    Characterization and mode of action of enzymes degrading galactan structures of arabinogalactans
    Vis, J.W. van de - \ 1994
    Agricultural University. Promotor(en): A.G.J. Voragen. - S.l. : Van de Vis - ISBN 9789054852377 - 161
    koolhydraten - cellulose - celmembranen - celwanden - fermentatie - voedselbiotechnologie - enzymen - carbohydrates - cellulose - cell membranes - cell walls - fermentation - food biotechnology - enzymes

    Agricultural biomass consisting mainly of cellulose, hemicellulose and lignin, is a renewable source of fuels and chemicals. An interesting option is enzymic conversion of biomass to readily usable material. To improve the overall economics of enzymic conversion of biomass not only cellulose but also hemicelluloses have to be degraded to monomeric sugars (saccharification). The aim of the work presented in this thesis was to study saccharification of arabinogalactans, a subgroup of the hemicelluloses.

    Arabinogalactans (AGs) have been found in numerous higher plants. In most plants these arabinogalactans occur only in small amounts, with exception of Larix and Acacia species. Their role in maintaining cell wall rigidity is discussed in chapter 1).

    Chapter 2 discusses structural features of AGs, subdivided in arabino-β(1->4)-galactans (type I) and arabino-β(1->3,1->6)galactans (type II), and gives a brief overview of enzymes degrading galactan structures of AGs.

    In chapter 3 the alkaline extraction of type I AGs from potato fibre, onion powder and citrus pomace is described. The extracts appeared to be mixtures of various polysaccharides. The presence of arabinan in all of these extracts is likely. By means of graded ethanol precipitation a major fraction enriched in type I AG could be precipitated in 40% v/v ethanol (denoted as F40). Results indicated that the 1,4-linked galactan backbone of onion F40 was substituted at C 6 with single unit galactose side-chains and for potato F40 also with 1,5-arabinans. Citrus TF40, (F40 treated with an endoglucanase for removal of contaminating xyloglucan), was suggested to contain a 1,4-linked galactan substituted at C 6 with short arabinose or highly branched arabinan side-chains and single unit galactose side-chains. A type I AG extracted from soy meal with alkali may be substituted at C 6 to a small extent with appendages of arabinose and single unit galactose side-chains.

    A type II AG from green coffee beans was indicated to consist of 1,3- linked galactan backbone substituted at C 6 with sidechains of arabinofuranose and galactose (chapter 3). The presence of terminal mannose possibly substituted on the sidechains, indicates a more complex structure for this polysaccharide. Analysis of a commercially available type II AG from larch (stractan) showed that the side-chains consisted also of arabinopyranose residues and this AG was a more heavily branched polymer than coffee AG.

    Endo-1,4-β-D-galactanases involved in the bioconversion of type I AG were purified from experimental enzyme preparations derived from Aspergillus niger and A. aculeatus (chapter 4). Their molecular weights were 42-43 kD and maximal activities were measured at pH 4.0- 4.3 and 50-55 °C on de-arabinosylated potato AG. In absence of substrate the A. aculeatus endogalactanase showed less thermal stability than the A. niqer endo-galactanase. Both endo-galactanases, which were similar in their mode of action, were suggested to degrade type I AG according to a multiple attack mechanism. It appeared that a combination of endo-galactanase and endo-1,5-α-L-arabinanase exerted synergistic effects in the initial stage of degradation of the potato AG. The action of these enzymes resulted in an increase in the downward shift of the molecular weight distribution of the digest and increased amounts of galactose, galactobiose and galactotriose (chapter 4). No synergism was observed for a combination of endo- galactanase and arabinofuranosidase B.

    Chapter 5 describes the purification of a β-D-galactopyranosidase. This β-galactosidase showed maximal activity on PNP-β-D-galactopyranose at pH 5 and 50 °C and was stable up to 50 °C and in the range of pH 3.5 to 7. It released non-reducing terminal galactose residues from type I AGs but not from type II AGs. With respect to polymeric substrates the enzyme showed highest activity towards 1,4- linkages but was also able to release 1,6-linked single unit galactopyranose side-chains.
    Chapter 6 describes that the differences in structural features of type I AGs were reflected in the combinations of enzymes which exerted synergistic effects in degradation. In the degradation of onion and potato F40 synergism occurred in the initial stage of degradation for the endo-galactanase/β-D-galactosidase combination. In the degradation of potato F40 the endo-galactanase/endo-arabinanase combination exerted also synergistic effects. The β-D-galactosidase released single unit galactose side-chains from both substrates thereby improving the affinity for endo-galactanase. These results were consistent with the structural features of these substrates reported in chapter 3.

    The activity of β-D-galactosidase on oligomeric reaction products released by endo-galactanase also enhanced degradation of potato and onion F40. This synergism occurred only in degradation of soy AG and citrus TF40. In the degradation of citrus TF40 synergistic effects were exerted also by the endo-galactanase/arabinofuranosidase B and endo-galactanase/endo-arabinanase combinations.

    An exo-1,3-β-D-galactanase purified from an experimental enzyme preparation derived from A. niger preferentially degraded 1,3-β-D-galactans (chapter 7). Mainly galactose and 1,6-galactobiose were released as reaction products from partly de-arabinosylated coffee AG. Hydrolysis of coffee AG by this exo-galactanase was accompanied by formation of small amounts of several arabinogalacto-oligomers. This indicated a limited capability of this enzyme of bypassing branching points. Optimal activity was measured at pH 5.0 and 40 °C and thermal stability was found in the pH range of 2.5 to 7.5 and up to 45 °C

    In the enzymic degradation of coffee bean AG a combination of exo-galactanase and α-L-arabinofuranosidase B exerted synergistic effects. For a combination of exo-galactanase and endo-arabinanase no enhancement in degradation of this substrate occurred. None of these combinations showed activity towards a type II arabinogalactan from larch wood (chapter 7).

    In chapter 8 discusses the isolation of type I and II AGs, the purification procedure of the endo-1,4-β-D-galactanases, the properties and mode action of the purified galactan degrading enzymes, their role in saccharification of AGs and other fields of possible application of galactan degrading enzymes.

    Characterisation and enzymic degradation of non-starch polysccharides in lignocellulosic by-products : a study on sunflower meal and palm-kernel meal
    Duesterhoeft, E.M. - \ 1993
    Agricultural University. Promotor(en): A.G.J. Voragen, co-promotor(en): W. Pilnik. - S.l. : Duesterhoeft - ISBN 9789054850762 - 134
    lignocellulose - lignine - zonnebloemolie - asteraceae - plantaardige oliën - palmpitolie - helianthus annuus - zonnebloemen - elaeis guineensis - oliepalmen - fermentatie - voedselbiotechnologie - bijproducten - koolhydraten - cellulose - celmembranen - celwanden - lignocellulose - lignin - sunflower oil - asteraceae - plant oils - palm kernel oil - helianthus annuus - sunflowers - elaeis guineensis - oil palms - fermentation - food biotechnology - byproducts - carbohydrates - cellulose - cell membranes - cell walls

    Non-starch polysaccharides (NSP) constitute a potentially valuable part of plant by- products deriving from the food and agricultural industries. Their use for various applications (fuel, feed, food) requires the degradation and modification of the complex plant materials. This can be achieved by enzymatic processes which, in comparison with chemical or physical methods, are regarded as energy-saving and non-polluting. However, a major disadvantage of enzymic processes often is their low effectivity and consequently high costs.

    The investigations described in this thesis were conducted to understand the reasons for the low susceptibility to enzymic hydrolysis of such by-products and, in particular, of their non-starch polysaccharides, and to find out whether and how the efficacy of enzymic treatment could be enhanced. The studies should provide information necessary for the development of polysaccharidase-preparations, tailored for the use in different applications.

    Sunflower ( Helianthus annuus L.) meal and palm-kernel ( Elaeis guineensis Jacq) meal, by-products from the production of edible oil and used as animal feed compounds, were chosen for our studies.

    In chapter 1 an introduction is given to the macroscopic and microscopic structure of the raw materials, to plant cell walls and their constituent polymers. The biodegradation of cell walls and its limitations are briefly reviewed and major non-starch polysaccharide degrading enzymes are summarised. Chapter 1 closes with an outline of the thesis.

    For a detailed study of type and structure of the non-starch polysaccharides, cell wall materials (CWM) were prepared from the meals by enzymatic digestion of protein and removal of small amounts of buffer-extractable material ( chapter 2 ). The resulting CWM's were enriched in NSP (55% of sunflower CWM and 75% of palm-kernel CWM) and had a high lignin content. Two different chemical methods, sequential extraction with alkali and sodium chlorite and treatment with 4-methylmorpholine-N-oxide (MMNO) were tested to extract all constituent polysaccharides. Almost complete dissolution could be achieved by a combination of both methods, but the recovery of sugars, especially during MMNO treatment, was low. From the sugar composition of polysaccharide fractions, obtained by sequential chemical extraction, a tentative identification of major polysaccharides was achieved. Their distribution in different botanical fractions of the meals could be deduced by comparison with data from literature (sunflower) or by own experiments (palm-kernel).

    The polysaccharide extracts of different purity were further fractionated by graded precipitation with ethanol, size-exclusion or adsorption-chromatography. By determination of the sugar- and glycosidic linkage composition of extracts, (partially) purified subfractions and intact cell wall materials, the identification, partial characterisation and quantification of major non-starch polysaccharides were achieved ( chapter 3 ). In sunflower meal, cellulose (42% of NSP), pectic polysaccharides (24%) and (4-O-methyl)-glucuronoxylans (24%) with about 10% glucuronosyl-substitution were major constituents. Minor amounts of (gluco)mannans (5%) and fucoxyloglucans (4.5%) were also identified. Major polysaccharides in palm-kernel meal were mannans (78% of NSP) with very low degrees of galactose-substitution and of apparently small molecular size (DP 12 to 14), and cellulose (12%). Arabinoxylans (3%) and (4- O -methyl)-glucuronoxylans (3%), deriving from the endocarp fraction of the meal, were present in low amounts in this monocotyledenous material.

    For a study of the enzymic hydrolysis of the cell wall materials ( chapter 4 ), three multi-component enzyme preparations were chosen. Solubilisation occurred as a bi-phasic process with high reaction velocities in the first stage of the incubation and only slow progress during extended incubation up to 72h. The solubilisation could markedly be improved by reduction in particle size; partial delignification or increasing enzyme concentration, however, had almost no effect. Maximally 30% of NSP in sunflower meal and 50% in palm-kernel meal could be solubilised from the finely milled CWM's. Although the composition of the enzyme preparations was found to influence the type of reaction products, the extent of their release and, as observed by transmission electron microscopy, the site of enzymic attack in different cell wall layers, our results suggested that substrate accessibility was the major factor limiting enzymic hydrolysis.

    A detailed study of the reaction products obtained by incubation with the crude enzyme preparations or fractions thereof (prepared by anion-exchange chromatography) revealed, that pectic compounds and mannose-containing polysaccharides in sunflower CWM were readily degradable ( chapter 5 ). The hydrolysis of mannans in palm-kernel CWM varied from 20% to 50%. In both CWM's, xylans and cellulose were most resistant to hydrolysis. The results indicate the preferential degradation of parenchyma and endosperm tissues and the resistance of hull and endocarp fractions to enzymic hydrolysis. The reaction products formed during all stages of the treatment were of small oligomeric and monomeric size.

    The contribution of different enzyme activities to the total solubilisation achieved by the heterogeneous enzyme preparations was studied with (partially) purified subfractions which were prepared by various chromatographic techniques from the crude preparations, and with highly purified enzymes from other microbial sources ( chapter 6 ). In general, the effect of these purified enzyme fractions was low (solubilisation of NSP: 1 % to 5 %). Supplementation of main enzyme fractions with pectolytic, cellulolytic or mannanolytic subfractions did only slightly enhance the total solubilisation. Synergistic action was observed between glucanases and mannanases in palm-kernel incubations and between arabinanases, polygalacturonases and rhamnogalacturonan-degrading enzyme fractions in the hydrolysis of pectic polysaccharides in sunflower CWM. The enzymic hydrolysis of (4- O -methyl)-glucuronoxylans was studied in-situ and with the isolated polysaccharide. The results indicated that the resistance of the xylans to enzymic degradation is not only due to their interlinkage with other polymers and location in the cell wall but also to their primary structure.

    In chapter 7 , important aspects concerning the approach and the methodology used are discussed. Implications arising for different fields of application are shown and suggestions for the formulation of enzyme preparations, which merit further research, are made.

    Cationic starches on cellulose surfaces : a study of polyelectrolyte adsorption
    Steeg, H.G.M. van de - \ 1992
    Agricultural University. Promotor(en): B.H. Bijsterbosch; A. de Keizer. - S.l. : S.n. - 142
    adsorptie - sorptie - elektrolyten - zetmeel - koolhydraten - cellulose - celmembranen - celwanden - adsorption - sorption - electrolytes - starch - carbohydrates - cellulose - cell membranes - cell walls

    Cationic starches are used on a large scale in paper industry as wet-end additives. They improve dry strength. retention of fines and fillers, and drainage. Closure of the white water systems in the paper mills hase increased the concentration of detrimental substances. This might be the reason for the poor retention of cationic starches observed in the last few years.

    The purpose of the research described in this thesis was to obtain a better understanding of the adsorption of cationic starch on cellulose and how this interaction can be disturbed. In contrast to most research in papermaking we have used a colloid-chemical approach. This means that we kept our experimental system as simple as possible and therefore far from the reality of papermaking.

    In chapter 2 we tried to generalize the specific problem of cationic starch adsorption on cellulose to polyelectrolyte adsorption on an oppositely charged surface. We used a recent extension of the polymer adsorption theory of Scheutjens and Fleer for polyelectrolyte adsorption to perform model calculations. It emerged that, for the adsorption of a strong polyelectrolyte on an oppositely charged surface, two regimes can be distinguished based on the effect of salt concentration on the adsorption. We call these the screening-enhanced adsorption regime and the screening-reduced adsorption regime. In the former regime the adsorption increases with increasing salt concentration because the repulsion between the segments is screened and a nonelectrostatic interaction between polyelectrolyte and surface is present. The adsorption decreases with increasing salt concentration in the latter regime, because the mainly electrostatic attraction between polyelectrolyte and surface is screened. A transition between these two regimes can take place if the balance between the electrostatic and nonelectrostatic interactions is changed. The electrostatic interactions are determined by the segment charge and the surface charge density. The strength of the nonelectrostatic interaction is described with a χs parameter, the net adsorption energy in units kT. From the model calculations it appears that the screening-reduced adsorption regime always occurs if the interaction between polyelectrolyte and surface is only electrostatic (χs=0). The polyelectrolyte can then be completely displaced from the surface with salt ions. If there is a nonelectrostatic attraction between polyelectrolyte and surface (χs>0) the screening-enhanced adsorption regime shows up in most cases. Only for very low segment charges and not too low surface charge densities, which is often the case for polyelectrolytes used in papermaking, we are dealing with the screening-reduced adsorption regime. The theory also predicts that the adsorbed amount shows a maximum as a function of the segment charge, irrespective of the value of χs. For a very low salt concentration this occurs at a segment charge of about 0.01 unit charges, or even lower.
    If the counter ions have a specific interaction with the surface, the adsorption of a polyelectrolyte can pass through a maximum as a function of the salt concentration provided χs is not too small.
    The predictions of the model calculations agree very well with experimental results reported in literature.

    The careful characterization of the materials and the experimental methods we used are described in chapter 3. We showed that the microcrystalline cellulose, which we use as a model for cellulose fibers, is level-off DP cellulose. This means that we are dealing with fibers chemically cut into pieces. The microcrystalline cellulose had to be cleaned before use. because hemicellulose came off in aqueous solutions disturbing the determination of the equilibrium concentration of starch with a carbohydrate determination. This was accomplished by washing the microcrystalline cellulose with concentrated NaOH solutions. The surface charge of the microcrystalline cellulose, originating from carboxylate groups, was determined by potentiometric titrations to be about -1 C/g at pH=7, which is a little lower than reported for cellulose fibers. The specific surface area is a somewhat problematic quantity for a porous substrate as microcrystalline cellulose. Based on the adsorption of cationic polyelectrolytes with different molecular weights on microcrystalline cellulose, we estimated the accessible surface area for cationic starch to be about 6 m 2/g, which is only 10% of the surface area accessible to small ions.

    We used two different types of cationic starch, namely cationic potato starch and cationic waxy maize starch. Potato starch consists of two components, an essentially linear polymer of α-1,4 glucose, called amylose, and a much larger branched polymer, called amylopectin. The fraction of amylose is about 21%. Waxy maize starch consists of amylopectin. only. From their sedimentation coefficients we estimated the molecular weight of cationic amylose to be about 3.5.10 5and of cationic amylopectin from potato starch between 5.10 7and 5.10 8. The molecular weight of cationic amylopectin from waxy maize was estimated to be between 1.10 7and 6.5.10 7. Both cationic starches showed a marked decrease in viscosity and hydrodynamic radius, as measured by dynamic light scattering, with increasing electrolyte concentration. This indicates that cationic amylopectin has enough flexibility to shrink, even though it has a branched structure.
    Special attention is paid to the methods with which the starch concentration can be determined, especially to the well-known iodine determination. It is shown that it is very important to specify the iodine and iodide concentrations in the final solution with the blue starch-iodine complex and the wavelength at which the absorbance is measured.
    Finally we describe how the adsorbed amounts are measured by depletion.

    In chapter 4 we investigated the adsorption of cationic amylopectin (from waxy maize, DS(Degree of Substitution) =0.035) on microcrystalline cellulose in the presence of simple electrolytes and at different pH values. The adsorption isotherms of cationic amylopectin were all of the high affinity type, as is expected for polyelectrolyte adsorption. The plateau value of the adsorbed amount showed a maximum as a function of the salt concentration. It was also found that the adsorbed amount, in the region
    where it decreases with increasing salt concentration, was very sensitive to the type of cation used. We obtained a Iyotropic series for the alkali cations, where the adsorbed amount in the presence of the cations decreased as Li +>Na +=K +>Cs +. The trend of these experimental results could be explained very well with the theory on polyelectrolyte adsorption described in chapter 2.

    The plateau value of the adsorbed amount increased with increasing pH in the same way as the surface charge. The adsorbed amount of charge was estimated to be 10% of the titratable surface charge.

    Based on the dependence of the adsorption on the pH and the salt concentration, we concluded that for cationic amylopectin charge interactions are the main driving forces for adsorption on cellulose.

    The adsorption of cationic potato starch (usually DS=0.035) on microcrystalline cellulose is investigated in chapter 5. Special attention is paid to the fact that cationic potato starch is a mixture of 21% amylose and 79% amylopectin. It was found that amylose adsorbs preferentially. This was attributed to a larger accessible surface area for amylose due to its ability to enter the pores of microcrystalline cellulose during the equilibration time (15 hours).

    The adsorption isotherms of cationic potato starch are also of the highaffinity type. There is a strong dependence on the cellulose concentration, caused by heterodispersity of the amylose and the amylopectin fractions.

    The adsorption of cationic potato starch strongly and monotonously decreased with increasing salt concentration. It was completely displaced by salt ions at concentrations larger than 0.05 M. The divalent cations Ca 2+and Mg 2+appeared to be ten times as effective as Na+ in suppressing the adsorption of cationic potato starch, which is due both to their higher charge and a specific interaction with the cellulose surface. From the small difference in effect of Ca 2+and Mg 2+we concluded that the phosphate groups in cationic Potato starch play no relevant role in the adsorption.

    Increasing the pH led to increasing adsorption. The adsorbed amount of charge was estimated to be 10% of the titratable charge.
    Finally, we investigated the effect of DS on the adsorption of cationic potato starch. At 2 mM NaCl the adsorbed amount of the starch with the lowest DS (0.017) was largest, but at 10 mM NaCl the difference between starches with DS=0.017, 0.035 and 0.047 was very small. The adsorbed amounts decreased slightly with decreasing DS. Theory on polyelectrolyte adsorption predicts that at a salt concentration of about 0.01 M the adsorbed amounts of polyelectrolytes with various segment charges can be the same indeed. The effect of segment charge is larger at lower and higher salt concentration. At a low salt concentration the starch with the lowest DS is expected to adsorb most, whereas at high salt concentration the starch with the highest DS will adsorb best.
    We concluded from the strong dependence on salt concentration and pH that the adsorption of cationic potato starch on cellulose is mainly driven by electrostatic attraction.

    In chapter 6 we conclude that the adsorption of cationic starch on cellulose is mainly determined by the presence of charges and not by certain special properties of starch and cellulose. We point out that this thesis has general relevance because of the new light it sheds on polyelectrolyte adsorption. For papermaking this thesis is particularly relevant, because it explains the adsorption behaviour of cationic starch as that of polyelectrolytes and it therefore also improves the understanding of the adsorption of other polyelectrolytes used in papermaking. The adsorption behaviour of cationic potato starch is compared with that of cationic amylopectin from waxy maize. We suggest that this may be caused by differences in size and shape. Finally we indicate which results can be of direct relevance for papermaking in practice.

    Biological, chemical and physical treatment of fibrous crop residues for use as animal feed.
    Singh, K. ; Flegel, T.W. ; Schiere, J.B. - \ 1987
    New Delhi [etc.] : Indian Council of Agricultural Research [etc.] - 240
    koolhydraten - celmembranen - celwanden - cellulose - voer - rijststro - stro - strobehandeling - vezelgewassen - carbohydrates - cell membranes - cell walls - cellulose - feeds - rice straw - straw - straw treatment - fibre plants
    The in sacco degradation of curde protein and cell wall constituents in grass, alfalfa and maize silages.
    Kwakkel, R.P. ; Bruchem, J. van; Hof, G. ; Boer, H. - \ 1986
    Netherlands Journal of Agricultural Science 34 (1986). - ISSN 0028-2928 - p. 116 - 119.
    biological treatment - carbohydrates - cell membranes - cell walls - cellulose - experiments - feeds - in vitro - microbial degradation - proteins - silage
    Isolatie en analyse van plantaardige celwanden
    Pannebakker, E.G. - \ 1983
    Wageningen : CABO (CABO-verslag no. 46) - 30
    celwanden - cytologie - histologie - plantenfysiologie - celfysiologie - cell walls - cytology - histology - plant physiology - cell physiology
    Ontsluiting van stro, hooi, ook met een loog of zwakzuur
    Anonymous, - \ 1981
    Wageningen : Pudoc (Literatuurlijst / Centrum voor Landbouwpublikaties en Landbouwdocumentatie no. 4536)
    bibliografieën - koolhydraten - celmembranen - celwanden - cellulose - voer - hooi - rijststro - stro - bibliographies - carbohydrates - cell membranes - cell walls - cellulose - feeds - hay - rice straw - straw
    Utilization of cellulose and hemicellulose of pig faeces by Trichoderma viride
    Wit, W. de - \ 1980
    Landbouwhogeschool Wageningen. Promotor(en): E.G. Mulder. - Wageningen : Veenman - 77
    deuteromycotina - koolhydraten - cellulose - celmembranen - celwanden - stalmest - microbiologie - afvalverwerking - afvalwaterbehandeling - trichoderma viride - hemicellulosen - deuteromycotina - carbohydrates - cellulose - cell membranes - cell walls - farmyard manure - microbiology - waste treatment - waste water treatment - trichoderma viride - hemicelluloses

    The purpose of this investigation was to study the microbiological degradation of the cellulose-hemicellulose-lignin complexes of the faeces of pigs. Cellulose, hemicellulose and lignin are components of the cell wall of plants and residues of plant material occur in large quantities in faeces and other organic waste material. The development of the intensive livestock farming is leading to the production of large quantities of manure that cannot always be disposed of in the usual way by lack of agricultural land. To prevent deterioration of the environment, other ways of handling the manure should be sought. Priority should be given to those procedures according to which the insoluble organic components of the faeces are decomposed by microorganisms and the energy produced utilized by man. Examples of such procedures are the aerobic breakdown of the organic residues with production of large amounts of biomass that can be utilized as feed additive (single-cell protein). Anaerobic treatment of manure may lead to the production of methane. In the present investigation attention was centred on the aerobic break-down of the undigested organic residues of pig faeces.

    Chapter 1 contains a general introduction and gives a review of literature pertaining to waste material with cellulose, hemicellulose and lignin as main components. In the Netherlands most of the organic waste material occurs as manure, sewage sludge and domestic wastes.
    The chemistry and structure of the plant cell wall were discussed. The presence of lignin strengthens the structure of plant tissue but seriously hampers the break-down of other cell wall components like cellulose and hemicellulose which are closely linked to the lignin molecules.
    Fungi play an important part in the degradation of cellulose-hemicellulose-lignin complexes of plant residues. Some fungi are able to decompose lignin, others utilize cellulose and hemicellulose, leaving lignin untouched. Cellulolytic bacteria occur in considerable numbers in nature, but break-down of lignin by bacteria is hardly reported in the literature.
    Extra-cellular, substrate-specific enzymes produced by microorganisms are involved in the break-down of plant residues. Several enzymes play a part in the degradation of the cellulose-hemicellulose-lignin complexes. Those involved in the decomposition of cellulose are known under the collective name of cellulase. Hemicellulose is a group name for several plant cell wall heteropolysaccharides which are degraded by substrate-specific enzymes. Little is known of enzymes involved in lignin break-down.

    In Chapter 2 a survey is given of materials and methods used in this investigation. A method was described for the collection of insoluble residues of plant material from pig faeces. For practical reasons, this preparation was used in the experiments to be described.
    Chapter 3 gives the results of microscopic and chemical analyses of undigested plant residues of pig faeces. These residues were derived from plant material. mainly ground seeds and fruits, supplied as mixed feed to the animals. Various types of plant tissue (parenchyma, collenchyma and sclerenchyma) were observed showing that considerable amounts of cellulose-, hemicellulose-, and lignin-containing cell walls had passed more or less undamaged the digestive track of the pigs (Figs. 3.1 - 3.11).
    The chemical analysis of pig faeces showed that 30-40 % of the dried matter consisted of insoluble plant residues containing 65 - 70 % polysaccharides (cellulose and hemicellulose) and nearly 30 % lignin. In order to use these plant residues as carbon source for fungi, pretreatment of the material is necessary as the high lignin content hampers the ready utilization of the cellulose and hemicellulose by the cellulolytic fungi.

    Chapter 4 described the isolation of cellulolytic microorganisms. Mainly fungi were isolated and a number of them together with some fungi obtained from culture collections were tested for their ability to utilize as carbon source: (a) monosaccharides that occur in the polysaccharides of plants (b) some model polysaccharides related to plant cell walls (c) lignin and (d) undigested plant residues from pig faeces. The results of these experiments are shown in Tables 4.1, 4.2 and 4.3. None of the fungi tested was able to decompose lignin. When the undigested plant residues of pig faeces had been supplied as carbon source only 30-40 % of the material was utilized. This was mainly due to the protection of the polysaccharides against fungal attack by the lignin. In order to improve the availability of the polysaccharides to the fungi, breaking or removal of the lignin matrix of the plant tissue is required.
    Of the fungi tested for growth on residues of faeces, Penicillium nigricans, Myrothecium verrucaria and Trichoderma viride QM 9419 gave the best results. As the Trichoderma strain produced the highest amounts of cellulolytic enzymes, this organism was chosen for further research.

    In Chapter 5 experiments were described concerning the growth of Trichoderma viride on (a) crystalline cellulose (Avicel), (b) undigested plant residues from pig faeces, and (c) plant residues from faeces treated with alkali to dissolve and remove lignin. Crystalline cellulose was the best carbon source. It was almost completely utilized by T.viride giving about 250 mg of protein per g of cellulose consumed. The undigested carbon compounds of pig faeces were utilized for about 49 % and the alkali-treated residues for 60 %.
    The amount of cellulolytic enzymes in the culture liquid of T.viride, calculated per unit of soluble protein, was with cellulose as carbon source three times as high, and with alkali- treated residues twice as high as with untreated plant residues from pig faeces. Spent culture solutions of T. viride supplied with pure ,cellulose or undigested residues of pig faeces contained relatively large amounts of soluble protein. An unknown proportion of this protein represented enzymes, presumably mainly cellulolytic enzymes.
    The production of biomass from the undigested residues of pig faeces by use of Trichoderma viride ('single-cell protein') to prepare mixed feed is for the following reasons no appropriate procedure: (a) T.viride only partly utilizes the polymers of the undigested residues of pig faeces, (b) the fungal protein is mixed with part of the faeces constituents which could not be metabolized by the fungus and which are also resistant to digestion by the animal owing to the protection of cellulose and hemicellulose against break-down by the presence of lignin, (c) the soluble protein of the culture solution of the fungus is difficult to collect.

    Chapter 6. A procedure was proposed (Fig 6.1) according to which the insoluble residues of pig faeces are submitted to a solution of cellulolytic enzymes. The hydrolysis of the polysaccharides (at 50°C and pH 4.8) proceeds in a relatively short time giving a solution of sugars which can be used for further purposes. A suitable enzyme solution can be obtained by growing T.viride on residues of faeces. The enzyme solution of the spent nutrient solution of the fungus can be easily concentrated by acetone precipitation or by vacuum evaporation. The substrate for the enzymic procedure, the undigested residues of faeces, are adequately degraded when a pretreatment has been applied (grinding to a particle size of 0.08 mm).
    When the proposed procedure is applied, 150 g of insoluble residues per 1 of enzyme solution can be hydrolysed; 70 % of the residues is converted-into soluble sugars within 4 h. The cellulolytic enzymes are initially adsorbed to the insoluble substrate but are released when hydrolysis proceeds; they can be separated from the undissolved material together with the dissolved sugars. Separation of sugars and enzymes can be achieved by mixing with new substrate which adsorbs the enzymes. After separation of the sugar solution the substrate with enzymes are transferred to the reactor. In this way part of the enzymes can be re-used.
    In Chapter 6 also the results are given of the purification and separation of a number of enzymes involved in the degradation of the cellulose-, hemicellulose-, lignin complex (Fig. 6.8). The mixture of enzymes, excreted by T. viride when the organism was growing on undigested plant residues of pig faeces, was separated into several protein fractions each with its own enzyme activity with respect to different substrates, i.c. endoglucanase, exoglucanase and hemicellulase activity (Figs. 6.9-6.12).
    Degradation of undigested components of pig faeces in view of recycling of the faeces is no simple procedure. Microbial (enzymic) break-down is possible if a suitable pretreatment is applied. The economical applicability of the results obtained in this investigation will depend on: (a) the occurrence of excessive amounts of manure, (b) disposal problems, particularly in connection with the environment, (c) alternative methods for processing excessive amounts of manure.

    De bepaling van ruwe celstof in veevoeders en mest
    Kemmink, A. - \ 1968
    Hoorn : [s.n.] (Intern rapport / Instituut voor veevoedingsonderzoek "Hoorn" no. 23)
    analyse - koolhydraten - celmembranen - celwanden - cellulose - chemie - kwantitatieve analyse - analytische scheikunde - analysis - carbohydrates - cell membranes - cell walls - cellulose - chemistry - quantitative analysis - analytical chemistry
    Chromatografisch onderzoek naar de samenstelling van de polysacchariden uit de celwand in verband met de analyse van ruwvoeders
    Gaillard, B.D.E. - \ 1954
    Wageningen : [s.n.] - 65
    polysacchariden - voer - chemische samenstelling - voedergewassen - zoötechniek - fysiologie - celwanden - polysaccharides - feeds - chemical composition - fodder crops - zootechny - physiology - cell walls
    nieuw apparaat voor de bepaling van ruwe celstof; Bereiding van het uranylreagens volgens Kahane door verwerking van uraanresten
    Kampen, G.B. van; Westenberg, L. - \ 1936
    's-Gravenhage : [s.n.] (Verslagen van landbouwkundige onderzoekingen no. 42,10) - 8
    analyse - koolhydraten - celmembranen - celwanden - cellulose - chemische reacties - chemie - samenstelling - uitrusting - voer - verwerking - uranium - analytische scheikunde - chemische proceskunde - chemische processen - analysis - carbohydrates - cell membranes - cell walls - cellulose - chemical reactions - chemistry - composition - equipment - feeds - processing - uranium - analytical chemistry - chemical engineering - chemical processes
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