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.
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