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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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The biology of plant metabolomics
Hall, R.D. - \ 2011
Oxford : Blackwell/Wiley (Annual plant reviews vol. 43) - ISBN 9781405199544 - 420
planten - metabolomica - genexpressieanalyse - plantenfysiologie - genetica - statistiek - bio-informatica - plants - metabolomics - genomics - plant physiology - genetics - statistics - bioinformatics
Following a general introduction, this book includes details of metabolomics of model species including Arabidopsis and tomato. Further chapters provide in-depth coverage of abiotic stress, data integration, systems biology, genetics, genomics, chemometrics and biostatisitcs. Applications of plant metabolomics in food science, plant ecology and physiology are also comprehensively covered.
Metabolomics of carotenoid accumulation in Dunaliella salina
Lamers, P.P. - \ 2011
Wageningen University. Promotor(en): Rene Wijffels; Raoul Bino, co-promotor(en): Marcel Janssen; Ric de Vos. - [S.l. : S.n. - ISBN 9789085858522 - 173
dunaliella - carotenoïden - metabolomica - industriële microbiologie - dunaliella - carotenoids - metabolomics - industrial microbiology
Innovative production processes based on renewable resources are required to stop the exhaustion of our natural resources. Microalgae are one of the most promising feedstocks for such sustainable processes, since they can produce valuable biochemicals at high productivity using sunlight, water, carbon dioxide and a few other nutrients, without the need for arable land. This high productivity is attained in (semi)controlled photobioreactors designed to prevent any limitation but light.
Currently, the high costs for investment and operation of such systems only allow for economic production of high-value molecules such as carotenoids and omega-3 fatty acids. Though economically sound, these production processes typically require large amounts of energy and thus cannot be termed sustainable. Nevertheless, they do provide the essential know-how to develop sustainable and cost-effective production of microalgal low-value bulk chemicals and biofuels. To this end, breakthroughs are needed in all areas of microalgal biotechnology, including cell physiology.
One of the most important objectives of physiological studies is the improved understanding of how microalgal metabolism is affected by cultivation conditions. Design of optimal production process conditions can be enabled if the metabolism is fully understood and controlled.
A good example of the interaction of cultivation conditions on microalgal metabolism is the stress-induced accumulation of β-carotene in the unicellular green microalga Dunaliella salina. β-Carotene is a lipid-soluble orange pigment and an antioxidant, that is used in cosmetics and as a colorant for feed and food. This metabolite is accumulated by D. salina up to 10% of the cellular dry weight when the alga is exposed to extreme environmental conditions. However, the knowledge about the regulatory mechanisms that underlie β-carotene accumulation in D. salina is limited.
Therefore, the aim of this thesis was to gain more insight in how D. salina senses and responds to changes in process conditions with the ultimate goal of defining optimal strategies for β-carotene production, and possibly also for other stress-inducible metabolites in other microalgae.

In Chapter 2, the cell-factory potential of D. salina was reviewed and the general ideas concerning mechanisms related to β-carotene accumulation were discussed. D. salina accumulates β-carotene when exposed to a high light intensity or to conditions that lead to a reduced growth rate, such as high salinity, nutrient deprivation or extreme temperatures. In addition, the levels of β-carotene in D. salina correlate positively with the overall amount of irradiation perceived during the cell division cycle. This positive correlation suggests the involvement of a sensing mechanism that is responsive to the imbalance between energy input, i.e. light harvesting in the chloroplasts, and energy demand, i.e. requirement for cell growth.
Singlet oxygen, an excited oxygen species that can be produced upon excess of electron transfer in the chloroplast, seems a likely candidate, as it can induce β-carotene accumulation and is known to be released under the conditions that favor β-carotene accumulation. Another potential sensing mechanism involves the redox state of the plastoquinone pool of the photosynthetic electron transport chain. Since oxidation of the reduced form of plastoquinone is generally assumed to be the rate-limiting step in this electron transport chain, an imbalance between the supply and demand of photosynthetic products may be reflected in the redox-state of the plastoquinone pool. Upon excess of electron production by the photosynthetic machinery, the plastoquinone redox state will shift towards a more reduced form.
However, the way these stress-induced changes in singlet oxygen or plastoquinone redox state are transduced and finally stimulate β-carotene accumulation is still largely unknown. Transcriptional and translational activity have been found essential for β-carotene accumulation, although a clear understanding of the involvement of structural genes that encode enzymes of the carotenoid biosynthetic pathway is still lacking.
Finally, β-carotene accumulation may also be driven by the formation of β-carotene-sequestering lipid globules. In this case, β-carotene accumulation does not require upregulation of genes encoding enzymes of the carotenoid biosynthetic pathway, but relies on activity-enhancement of these enzymes through removal of their end-product (i.e. β-carotene) by newly formed oil globules.

In Chapters 3 and 4, the effects of either a sudden light increase or nitrogen starvation on the carotenoid metabolism of D. salina were described. Since high-light is one of the most potent inducers of β-carotene accumulation, the experiments were done in flat panel photobioreactors that were run in turbidostat mode to ensure a constant light regime throughout the entire duration of the experiments.
A 7-fold increase in light intensity proved more powerful in inducing β-carotene accumulation than complete nitrogen starvation, as was demonstrated by a 2 times higher maximum productivity (37 mg per liter reactor volume per day as compared to 18.5 mg per liter reactor volume per day). However, nitrogen depletion appears more efficient with regard to energy usage, since 7 times more light energy was used in the high-light experiment.
Interestingly, in our experimental turbidostat systems the maximal productivities found for both stresses were about an order of magnitude larger than the average productivity reported for a commercial β-carotene production facility, which indicates a vast potential for improvement in the latter.
Apart from β-carotene accumulation, the growth characteristics of the D. salina cells were also influenced by the stress treatments. Induction by either type of stress resulted in cell swelling and an increase in the cell-specific density. The increased amount of β-carotene could only partially explain the increase in cell-specific density, which suggests that other metabolites accumulated as well.
The initial cell division rate was differentially affected, with high-light causing a temporary cell cycle arrest and nitrogen starvation leading to a transient increase in the cell division rate. Nevertheless, the volumetric biomass productivity increased temporarily for both nitrogen starvation (2-fold) and high-light stress (6-fold).

These results implied that the 7-fold increase in incident light caused a decrease in the biomass yield on absorbed light energy, whereas nitrogen depletion led to a transient increase in the yield caused by the accumulation of non-nitrogenous biomass. This finding points towards the potential of nitrogen-limitation strategies for permanent improvement of lipid or carbohydrate yield on light in large scale microalgal production systems.
Since D. salina accumulates β-carotene in lipid globules and it has been suggested that fatty acid biosynthesis determines the amount of accumulating β-carotene, we determined the fatty acid content and composition during both stress treatments and correlated the exact time course of fatty acid levels to that of β-carotene production. The intracellular concentration of the total fatty acid pool did not change significantly during nitrogen starvation and decreased following treatment with high-light.
These results indicated that β-carotene and total fatty acid accumulation were unrelated, at least in our turbidostat experiments. Nevertheless, carotenoid overproduction was associated with oil globule formation and a decrease in the degree of fatty acid unsaturation. The accumulation of β-carotene appeared to correlate positively with oleic acid production, suggesting that oleic acid may be a key component of the lipid globule-localized triacylglycerols and thereby in β-carotene accumulation.

In Chapter 5, the cellular mechanisms that could be related with stress-induced β-carotene accumulation in D. salina were investigated. Samples were taken from the aforementioned high-light and nitrogen-starvation experiments, both before and during stress induction. Subsequent untargeted GC-TOF-MS-based analysis of derivatized polar extracts, mainly containing primary metabolites, in combination with unbiased peak picking and clustering of signals into metabolite mass spectra, yielded 87 unique polar metabolites of which 31 were in common between both stress experiments.
By combining these polar metabolite profiles with the previously determined levels of carotenoids, chlorophylls and fatty acids, it was found that D. salina cells exhibit essentially similar overall responses towards both types of stress, with the principal stress-specific variation being caused by only 3 out of the 44 polar and apolar metabolites.
Furthermore, we observed accumulation of various metabolites that are usually linked with energy storage, both during high-light stress and during nitrogen depletion. Because both stress treatments are known to cause an imbalance in absorbed and required light energy, it was suggested that β-carotene accumulation and an increased production of energy storage molecules reflect a uniform and concerted effort of D. salina cells to cope with such damaging imbalances.

In Chapter 6, data of preliminary experiments towards elucidating the mechanisms potentially involved in the sensing of imbalances in the ratio between energy supply and energy consumption in D. salina are presented. In these experiments we used specific inhibitors of the reduction (DCMU) and the oxidation (DBMIB) of the plastoquinone pool in the photosynthetic electron transport chain, under both inducing and non-inducing conditions. It was found that DCMU inhibited both growth and β-carotene accumulation under otherwise carotenogenic conditions, whereas DBMIB inhibited only the growth.
These results pointed towards a possible role of the plastoquinone redox state in the regulation of β-carotene accumulation in D. salina. In addition, these findings suggested that singlet oxygen did not serve as a signaling agent for stress-induced β-carotene accumulation, since this accumulation was inhibited in the presence of DCMU, despite this inhibitor being a well-known stimulator of singlet oxygen release.
Next to performing these photosynthesis inhibitor experiments, we also showed the importance of metabolic profiling techniques for a better understanding of microalgal metabolism. We discussed the relations between β-carotene accumulation and the observed temporal changes in polar and apolar metabolites. This enabled evaluation of several hypotheses concerning the mechanisms involved in β-carotene accumulation, with upregulation of the entire β-carotene biosynthetic pathway proving a likely element of such a mechanism.
Furthermore, because light intensity and growth-reducing stress conditions act together upon the accumulation of secondary metabolites in microalgae, proper physiological studies, as well as optimal commercial production strategies, require cultivation systems that enable tight control of both light and nutrient supply. We therefore advocated the use of turbidostats, both for research on and for commercial production of stress-induced accumulation of microalgal metabolites.

In conclusion, this thesis illustrates the potential of combining sophisticated cultivation techniques with broad-scale metabolite profiling approaches for the detailed study of microalgal metabolism. Hence, similar studies should be applied for gaining the understanding that is needed for the development of optimal and sustainable production processes that will ultimately put a stop to the exhaustion of our natural resources.
Hormonal effects of prohormones : novel approaches towards effect based screening in veterinary growth promoter control
Rijk, J.C.W. - \ 2010
Wageningen University. Promotor(en): Michel Nielen; Ivonne Rietjens, co-promotor(en): Maria Groot; Ad Peijnenburg. - [S.l. : S.n. - ISBN 9789085858195 - 207
groeibevorderaars - hormonen - biotesten - metabolisme - metabolomica - growth promoters - hormones - bioassays - metabolism - metabolomics
Within the European Union the use of growth promoting agents in cattle fattening is prohibited according to Council Directive 96/22/EC. Interestingly, there is not a black list of substances, but 96/22/EC states that all substances having thyrostatic, estrogenic, androgenic or gestagenic activity are prohibited. Besides abuse of the “classical” synthetic steroids there is a tendency towards misuse of natural steroids and prohormones. Prohormones are compounds that exhibit limited or no hormonal activity but are direct precursors of bioactive hormones and are intended to be converted to full active hormones via enzymatic processes in the body. However, knowledge about metabolism, the mode of action and excretion profiles in cattle is often unclear, and methods to detect abuse of prohormones in livestock production are lacking. Therefore, the aim of this thesis was to get insight into the hormonal action of prohormones and to develop novel in vitro and in vivo screening methods allowing effective surveillance on the illegal use of prohormones in livestock production. Hereby the emphasis was on developing effect based approaches to better meet Council Directive 96/22/EC.
The bioactivity of a wide variety of supplements which contained prohormones were tested using a yeast androgen bioassay. For supplements containing solely prohormones the value of this bioactivity based screening appeared to be limited as they require metabolism to become active. Therefore, screening methods for animal feed, supplements and preparations were set-up by using the same yeast androgen bioassay in combination with bovine liver models as well as enzymatic and chemical deconjugation procedures to mimic in vivo metabolic bioactivation. The use of either bovine liver S9, liver slices, pure enzymes or alkaline hydrolysis showed that prohormones could be activated, resulting in a significant increase in bioactivity as determined by the androgen yeast bioassay.
For the detection of prohormone abuse at the farm and/or slaughterhouse the usefulness of ‘omics’ based profiling techniques was investigated. Within this scope a comprehensive metabolomics based screening strategy for steroid urine profiling was developed. Comparison of urinary profiles revealed large differences between the profiles of controls and dehydroepiandrosterone (DHEA) as well as pregnenolone treated animals. Moreover this steroid urine profiling approach allowed identification of biomarkers for treatment by specific prohormones. This resulted in respectively 7 and 12 specific mass peak loadings which could potentially be used as biomarkers for pregnenolone and DHEA treatment.
In addition, the feasibility of a liver gene expression profiling approach was investigated to monitor the effects of DHEA treatment at the transciptome level. It was shown that identification and application of genomic biomarkers for screening of DHEA abuse in cattle is substantially hampered by biological variation. On the other hand, it was demonstrated that comparison of pre-defined gene sets versus the whole genome expression profile of an animal allows to distinguish DHEA treatment effects from variations in gene expression due to inherent biological variation.
Altogether the results of this thesis increase the knowledge about the metabolism and bioactivation of prohormones in vitro as well as in vivo. Based on this knowledge, a panel of new effect based concepts and screening methods was developed that complement and improve the current testing programs. These new concepts will facilitate better implementation of the European ban on growth promoters in livestock production as described in Council Directive 96/22/EC.


Marine Biotechnology: A New Vision and Strategy for Europe
Querellou, J. ; Børresen, T. ; Boyen, C. ; Dobson, A. ; Höfle, M. ; Ianora, A. ; Jaspars, M. ; Kijjoa, A. ; Olafsen, J. ; Rigos, G. ; Wijffels, R.H. - \ 2010
Beernem, Belgium : Drukkerij De Windroos NV (Position paper / European Science Foundation, Marine Board 15) - ISBN 9782918428268 - 93
biotechnologie - marien milieu - metabolomica - overheidsbeleid - europa - strategisch management - bio-energie - biotechnology - marine environment - metabolomics - government policy - europe - strategic management - bioenergy
Marine Board-ESF The Marine Board provides a pan-European platform for its member organisations to develop common priorities, to advance marine research, and to bridge the gap between science and policy in order to meet future marine science challenges and opportunities. The Marine Board was established in 1995 to facilitate enhanced cooperation between European marine science organisations (both research institutes and research funding agencies) towards the development of a common vision on the research priorities and strategies for marine science in Europe. In 2010, the Marine Board represents 30 Member Organisations from 19 countries. The Marine Board provides the essential components for transferring knowledge for leadership in marine research in Europe. Adopting a strategic role, the Marine Board serves its Member Organisations by providing a forum within which marine research policy advice to national agencies and to the European Commission is developed, with the objective of promoting the establishment of the European Marine Research Area.
The genetics of the metabolome in Brassica rapa
Pino del Carpio, D. - \ 2010
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Guusje Bonnema. - [S.l. : S.n. - ISBN 9789085857211 - 168
brassica campestris - koolsoorten - metabolieten - genexpressie - genetische regulatie - genotypische variatie - genetische diversiteit - genetische merkers - selectiemethoden - metabolomica - brassica campestris - cabbages - metabolites - gene expression - genetic regulation - genetic variance - genetic diversity - genetic markers - selection methods - metabolomics
In this thesis the metabolic variation in Brassica rapa is described based on results of metabolic profiling of a core collection of 168 accessions representing the different crop types and geographical origin and a Doubled Haploid population. In Chapter 2 we describe the genetic and phenotypic variation of this core collection to explore the possibility of following association mapping methods to identify genes involved in metabolic regulation. We explored through a genome wide and candidate gene approach different association mapping methods in a core collection in Chapters 3 and 4 respectively and in Chapter 5 we combined the QTL analysis of targeted and untargeted metabolites profiled through LC-MS with expression QTLs following a genetical genomics approach aiming to detect genes underlying the metabolite QTL. The genetic diversity evaluated through the screening of AFLP and SSR markers was correlated with classification of accessions using morphological and metabolic trait values. The relationship between accessions in groups was compared using hierarchical clustering and the STRUCTURE program. Using Random Forests classification a set of metabolites was selected that differentiated the different sub groups as determined by STRUCTURE (Chapter 2). Based on the classification into subpopulations using the STRUCTURE program we included the subpopulations as a correction term in our statistical model for association studies (Chapter 3). Additionally, because of the increasing amount of data that will be soon available through sequencing technology we tested the use of Random Forests in the search for marker-trait association for the isoprenoids pathway. Using the results obtained with the linear models as implemented in TASSEL and the results obtained in Random Forests we found a set of 16 significant markers with potential use for marker assisted selection in breeding for several isoprenoidsThe determination of map positions through synteny prediction and genetic mapping of a group of genes from the glucosinolate pathway lead us to identify Myb28 and MAM as candidate genes mapping under a previously detected major QTL for glucosinolates We followed an association mapping approach to investigate their role in the variation in glucosinolates in the core collection by profiling 37 SSR markers, which included markers linked to these candidate genes and markers distributed along different positions in linkage group A03 (Chapter 4). Interestingly, not only MAM and Myb28, but the AOP and GS-OH genes involved in side chain modification and Myb29 in transcriptional regulation were also associated with glucosinolate levels. A genetical genomics approach was followed to identify candidate genes for variation inmetabolites of six biosynthetic pathways: carotenoids, tocopherols, folates, glucosinolates, flavonoids and phenylpropanoids, based on the co-localization analysis and comparison between metabolic (m)QTLs and expression (e)QTLs (Chapter 5). A Doubled Haploid (DH) population was profiled for metabolite content and variation through targeted and LC-MS untargeted approaches. Additionally, the same population was profiled for transcript variation with a newly developed 105K Cogenics array assembled using mainly EST sequences from three species: B. napus, B. rapa and B. oleracea. Co-localization of eQTLs and mQTLs for several isoprenoids (tocopherols and carotenoids) and glucosinolates lead us to the identification of candidate genes for these pathways. However, further work is needed to identify the gene or genes underlying a major cluster of QTLs for 112 centrotypes derived from the LC-MS untargeted data. The results obtained through this combined approach and considerations that need to be taken into account when performing these types of studies with regard to identification of paralogues and the use of a multi Brassica species microarray for transcript profiling in Brassica rapa are discussed.In the final Chapter, the combined use of core collections encompassing the genetic diversity within B. rapa and biparental DH populations to unravel the genetic regulation of the metabolome are discussed.
Metabolomics technologies applied to the identification of compounds in plants : a liquid chromatography-mass spectrometry - nuclear magnetic resonance perspective over the tomato fruit
Moco, S.I.A. - \ 2007
Wageningen University. Promotor(en): Raoul Bino; Sacco de Vries, co-promotor(en): Jacques Vervoort; Ric de Vos. - [S.l.] : S.n. - ISBN 9789085047421 - 222
solanum lycopersicum - tomaten - metabolieten - fytochemicaliën - kernmagnetische resonantiespectroscopie - metabolomica - lc-ms - solanum lycopersicum - tomatoes - metabolites - phytochemicals - nuclear magnetic resonance spectroscopy - metabolomics - liquid chromatography-mass spectrometry
A new era of plant biochemistry at the systems level is emerging in which the detailed description of biochemical phenomena, at the cellular level, is important for a better understanding of physiological, developmental, and biomolecular processes in plants. This emerging field is oriented towards the characterisation of small molecules (metabolites) that act as substrates, products, ligands or signalling entities in cells. This thesis concerns the development and establishment of such metabolomics strategies for screening and identifying metabolites in biological systems. Most technological strategies were applied to the assignment of metabolites from tomato (Solanum lycopersicum) fruit. Tomato was chosen for being a widely consumed crop with nutritional attributes, representing a model for the Solanaceae family. In order to achieve both high coverage of detected metabolites and valuable information for identification purposes, liquid chromatography coupled to mass spectrometry (LC-MS) and nuclear magnetic resonances (NMR) technologies were used. In addition, metabolite databases, based on experimental data (mass-based, in the case of LC-MS and chemical shift-based, in the case of NMR) were initiated, in order to systemize the extensive metabolite information. The chapters in this thesis describe method developments and their applications in plant metabolomics that are also feasible to be implemented on other biological systems. A review on the technologies used for metabolomics with a perspective on compound identification is presented in Chapter 1. In Chapter 2, a robust large scale LC-MS method for the analysis of metabolites in plants is described in detail. It presents a step-by-step protocol with thorough information about the reagents used, sample preparation, instrument set­up, methods of analysis and data processing strategies. The described analytical method combines LC with photo diode array (PDA) and MS detection, and allows the analysis of mostly semi-polar secondary metabolites present in plants, such as phenolic acids, flavonoids, glucosinolates, saponins, alkaloids and derivatives thereof. Chapter 3 presents an application of the LC-PDA-MS method for the profiling of metabolites present in tomato fruit. The metabolites putatively identified in this fruit were included in a tomato dedicated-database (the MoTo DB) that is available for public search on the web (see: http://appliedbioinformatics.wur.nl). A comparison between two tomato fruit tissues, peel and flesh, for their metabolite content was made using this MoTo DB. Using the same LC-PDA-MS setup, several different tomato fruit tissues were compared in more detail, along the fruit ripening timeline, in Chapter 4. The presence of tissue-specific metabolites, at determined ripening stages, suggests developmental control of metabolite biosynthesis. Such tissue-specific metabolomics approach may give rise to a biological view over metabolite compartmentalisation. Chapters 5 and 6 describe the implementation of a NMR database for secondary metabolites, mostly including flavonoids, the Flavonoid Database (see: Flavonoid Database under http://www.wnmrc.nl). The acquisition of a large data set of related standard compounds allowed the analysis of shifts in NMR characteristics by the presence of certain functional groups or substituents in the flavonoid backbone. In addition, a 1H NMR-based prediction model was iteratively trained from the acquired experimental data and can be used for the prediction of unknown related molecules. This approach greatly increases the efficiency in the identification of (flavonoid) metabolites. Chapter 7 describes correlations of metabolomics data derived from LC-MS and NMR analyses of a large number of different tomato cultivars. The identification of metabolites is obtained among other available sources, the MoTo DB and the Flavonoid Database. This approach illustrates the complementariness and coincidence of NMR and MS as analytical techniques, applied to the detection of metabolites in tomato fruit. The summarizing discussion and conclusions, sets the work presented in this thesis into a biochemical perspective, and prospects suggestions for the future.
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