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.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    No to Neocosmospora: Phylogenomic and Practical Reasons for Continued Inclusion of the Fusarium solani Species Complex in the Genus Fusarium
    O'Donnell, Kerry ; Al-Hatmi, Abdullah M.S. ; Aoki, Takayuki ; Brankovics, Balázs ; Cano-Lira, Jose F. ; Coleman, Jeffrey J. ; Hoog, G.S. de; Pietro, A. Di; Frandsen, R.J.N. ; Geiser, D.M. ; Lee, T.A.J. van der; Diepeningen, A.D. van; Waalwijk, C. - \ 2020
    mSphere 5 (2020)5. - ISSN 2379-5042
    clinical mycology - evolution - fungi - phylogenetics - taxonomy
    This article is to alert medical mycologists and infectious disease specialists of recent name changes of medically important species of the filamentous mold Fusarium. Fusarium species can cause localized and life-threating infections in humans. Of the 70 Fusarium species that have been reported to cause infections, close to one-third are members of the Fusarium solani species complex (FSSC), and they collectively account for approximately two-thirds of all reported Fusarium infections. Many of these species were recently given scientific names for the first time by a research group in the Netherlands, but they were misplaced in the genus Neocosmospora. In this paper, we present genetic arguments that strongly support inclusion of the FSSC in Fusarium. There are potentially serious consequences associated with using the name Neocosmospora for Fusarium species because clinicians need to be aware that fusaria are broadly resistant to the spectrum of antifungals that are currently available.
    Data from: DISCOMARK: nuclear marker discovery from orthologous sequences using draft genome data
    Rutschmann, Sereina ; Detering, Harald ; Simon, S. ; Fredslund, Jakob ; Monaghan, Michael T. - \ 2016
    Wageningen University & Research
    marker discovery - mayfly - non-model organism - phylogenetics - primer design - Cloeon dipterum - Cloeon peregrinator
    High-throughput sequencing has laid the foundation for fast and cost-effective development of phylogenetic markers. Here we present the program discomark, which streamlines the development of nuclear DNA (nDNA) markers from whole-genome (or whole-transcriptome) sequencing data, combining local alignment, alignment trimming, reference mapping and primer design based on multiple sequence alignments to design primer pairs from input orthologous sequences. To demonstrate the suitability of discomark, we designed markers for two groups of species, one consisting of closely related species and one group of distantly related species. For the closely related members of the species complex of Cloeon dipterum s.l. (Insecta, Ephemeroptera), the program discovered a total of 78 markers. Among these, we selected eight markers for amplification and Sanger sequencing. The exon sequence alignments (2526 base pairs) were used to reconstruct a well-supported phylogeny and to infer clearly structured haplotype networks. For the distantly related species, we designed primers for the insect order Ephemeroptera, using available genomic data from four sequenced species. We developed primer pairs for 23 markers that are designed to amplify across several families. The discomark program will enhance the development of new nDNA markers by providing a streamlined, automated approach to perform genome-scale scans for phylogenetic markers. The program is written in Python, released under a public licence (GNU GPL version 2), and together with a manual and example data set available at:
    Utilization of complete chloroplast genomes for phylogenetic studies
    Ramlee, Shairul Izan Binti - \ 2016
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Rene Smulders; Theo Borm. - Wageningen : Wageningen University - ISBN 9789462579354 - 186
    phylogenetics - genomes - chloroplasts - models - solanum - orchidaceae - phylogenomics - dna sequencing - fylogenetica - genomen - chloroplasten - modellen - solanum - orchidaceae - phylogenomica - dna-sequencing

    Chloroplast DNA sequence polymorphisms are a primary source of data in many plant phylogenetic studies. The chloroplast genome is relatively conserved in its evolution making it an ideal molecule to retain phylogenetic signals. The chloroplast genome is also largely, but not completely, free from other evolutionary processes such as gene duplication, concerted evolution, pseudogene formation and genome rearrangements. The conservation of the chloroplast genome sequence allows designing primers targeting regions conserved well beyond species boundaries, and amplification of these targets.

    The small size together with their high copy number in leaf cells greatly facilitates chloroplast genome sequencing. In this thesis, chloroplast phylogenomics was conducted using complete chloroplast DNA genomes obtained by a newly developed method of de novo assembly. The method was not only cost-effective but also has the potential to extract a wealth of useful information of thousands of chloroplast genomes from Whole Genome Shotgun (WGS) data. We used k-mer frequency tables to identify and extract the chloroplast reads from the WGS reads and assemble these using a highly integrated and automated custom pipeline. This pipeline includes steps aimed at optimizing assemblies and filling gaps that are left due to coverage variation in the WGS dataset. The pipeline enabled successful de novo assembly across a range of nuclear genome sizes, from Solanum lycopersicon (tomato, 0.9 Gb) to Paphiopedilum heryanum (slipper orchid, 35 Gb).

    The pipeline is suitable for studying structural variation in the chloroplast genome, as opposed to the common procedure of read mapping against a reference genome. To support the putative rearrangements, a flexible assembly quality comparison tool was created that combines and visualizes read mapping and alignment results in a two-dimensional plot. We have evaluated the ability of this tool using the de novo assemblies of S. lycopersicon and P. henryanum chloroplasts. The results show that not only we can immediately select the best of two options, but also determine the location of specific artefacts.

    In order to explore and evaluate the utility of complete chloroplast phylogenomics, tomato and Paphiopedilum spp were used to conduct phylogenetic inferences based on the complete chloroplast genome. In total 84 tomato chloroplast genomes within the section Lycopersicon were assembled and phylogenetic trees produced. The analyses revealed that next to the chloroplast regions and spacers traditionally used for phylogenetics, additional regions of protein coding and non-coding DNA may be exploited for intraspecific phylogenetic studies. In particular, more than 50% of all phylogenetically relevant information could be included by just using four genes (ycf1, ndhF, ndhA, and ndhH), of which 34% in ycf1 alone. The topology of the phylogenetic tree inferred from ycf1 was the same as that of trees based on all other protein coding genes, although with lower bootstrap values. The phylogenetic analyses based on 32 complete Paphiopedilum spp. chloroplast genomes confirmed the division of the genus into three subgenera Parvisepalum, Brachypetalum and Paphiopedilum. The division of five sections of subgenus Paphiopedilum was also recovered. The de novo assemblies revealed several structural rearrangements including gene loss and inversion. In addition, the chloroplast genome of Paphiopedilum has experienced extreme IR expansion that has included part of or the entire SSC region, resulting in larger IR regions than commonly observed among monocots.

    In conclusion, WGS data offer opportunities to generate partial or entire chloroplast genomes for phylogenetic studies. Species discrimination can be achieved already with partial data (subsets of genes), but evolutionarily young lineages may require more informative characters. Therefore, it is expected that many complete chloroplast genomes will be produced in the years to come. While generating these genomes, the urge for de novo assembly of chloroplast genomes rather than mapping against reference genomes is adamant in order to also uncover structural rearrangements in chloroplast genome.

    Ecophysiology and environmental distribution of organohalide-respiring bacteria
    Lu, Y. - \ 2016
    Wageningen University. Promotor(en): Hauke Smidt, co-promotor(en): Siavash Atashgahi. - Wageningen : Wageningen University - ISBN 9789462578418 - 239
    bacteria - halides - ecophysiology - phylogenetics - genomics - lakes - halogens - pollutants - bacteriën - haliden - ecofysiologie - fylogenetica - genomica - meren - halogenen - verontreinigende stoffen

    Organohalide-respiring bacteria (OHRB) are able to breathe natural and anthropogenically produced organohalides persistent in a broad range of oxygen-depleted environments. Therefore, these microorganisms are of high interest for organohalide-contaminated site bioremediation and natural halogen and carbon cycle. Nevertheless, to assess and adjust in situ bioremediation strategies and to enhance current understanding about the role of OHRB in natural habitats, thorough understanding of their ecophysiology and interaction with surrounding biotic and abiotic forces is necessary. To this end, this thesis focused on exploring ecophysiology and environmental distribution of OHRB in pristine and contaminated sites and unraveling their interactions with the co-existing microbial guilds in the community and geochemical parameters by application of a suite of physiological, molecular and geochemical analyses.

    Based on a comprehensive overview of currently known organohalide-respiring isolates and their environmental distribution, the presence of yet unknown OHRB in extreme environments was proposed as the known organohalide-respiring isolates survive/thrive at a moderate range of pH and salinity in laboratory culture. Therefore, the OHRB were surveyed in alkaline and hypersaline sediments collected from Lake Strawbridge, Western Australia, that was known to emit organohalides. As a result, for the first time, the dechlorination of chloroform and perchloroethene (PCE) to dichloromethane and trichloroethene, respectively, was documented from an alkaline hypersaline pristine environment.

    Corrinoids are essential cofactors for the activity of reductive dehalogenase enzymes. Ironically, some OHRB are reported to be corrinoid auxotrophs. Using transcriptional analysis and shotgun proteomics, here we show corrinoid auxotrophy in Dehalobacter restrictus PER-K23T. This detrimental deficiency seems to be compensated by up-regulation of relevant cobalamin salvaging and transport pathways to ensure sufficient corrinoid supply under partial corrinoid starvation. Hence, such OHRB incapable of de novo corrinoid synthesis will be dependent on non-dechlorinating community members to fulfill their nutritional needs indicating paramount importance of syntrophic interactions in supporting robust growth and activity of OHRB.

    Bacterial community analysis of chlorinated benzene dechlorinating consortia derived from contaminated harbour sludge suggested members of the Bacteroidetes phylum and Clostridiales order as well as sulfate-reducing Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to OHRB i.e. D. mccartyi and Dehalobacter. However, despite well-controlled lab condition, syntrophic interactions could be influenced by geochemical parameters under field settings. Accordingly, analysis of geochemical and microbial determinants of OHR at a site biostimulated by glycerol injection further verified supportive role of fermenters and sulfate reducers under highly reduced condition following biostimulation. However, towards the end of field experiment, reducing condition faded and sulfate increased concurrent with the appearance of Epsilonproteobacteria and Deferribacteres as putative oxidizers of reduced sulfur compounds. The latter guilds might serve as detoxifiers of sulfide and thereby stimulate D. mccartyi, but could also be inhibitory as successors of the more important syntrophic fermenting and sulfate reducing bacteria.

    In conclusion, this thesis expands our understanding of ecophysiology and environmental distribution of OHRB, addressing their presence in pristine environments as well as providing further evidence for their dependencies on other microbial community members in order to meet their nutritional requirements. Hence, research described here strengthens the scientific foundation for evaluating and optimizing strategies for the bioremediation of organohalide-contaminated sites and expands the natural niche of OHRB to extreme pristine environments.

    Induction of prenylated isoflavonoids and stilbenoids in legumes
    Aisyah, S. - \ 2015
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789462574816 - 154
    flavonoïden - stilbenoïden - isoflavonoïden - peulgewassen - glycine max - sojabonen - arachis hypogaea - fylogenetica - phaseolus - lupinus - rhizopus - aspergillus - kwantitatieve analyse - flavonoids - stilbenoids - isoflavonoids - legumes - glycine max - soyabeans - arachis hypogaea - phylogenetics - phaseolus - lupinus - rhizopus - aspergillus - quantitative analysis

    The germination of legume seeds in the presence or absence of stress factors was studied with respect to compositional changes in prenylated isoflavonoids and stilbenoids. Different strategies were applied using (i) different types of legume seed, (ii) different stress factors i.e. biotic, abiotic and their combination, and (iii) different time point of application of the fungus. Mass spectrometric tools to better characterize the position of prenyl groups in the molecules were optimized. Isoflavonoids and stilbenoids appeared more inducible than flavonoids. Fungus was a more effective stress factor than light and wounding. The impact of fungus might be enhanced by combining it with other stress factors; the combination of fungus and light was more promising than that of fungus and wounding. The seeds of various legume species appeared to respond differently towards elicitation by Rhizopus during germination. The kind of molecules induced followed the phylogenetic relationship of the various species, but their amounts induced during germination, alone or combined with elicitation, did not. In terms of quantities of compounds induced, some species such as Glycine max, Phaseolus spp., Lupinus spp. and Arachis hypogaea were more promising than Vigna spp., Lablab purpureus and Psophocarpus tetragonolobus. Moreover, the fact that Rhizopus and Aspergillus could metabolize the stilbenoids induced during the process of simultaneous germination and elicitation of peanut seedlings showed that the type of fungus was a crucial parameter for optimizing accumulation of potentially bioactive compounds.

    Systematics, evolution and historical biogeography of the family Ochnaceae with emphasis on the genus Campylospermum
    Bissiengou, P. - \ 2014
    Wageningen University. Promotor(en): Marc Sosef, co-promotor(en): Lars Chatrou; L. Ngok Banak. - Wageningen : Wageningen University - ISBN 9789462572225 - 357
    ochnaceae - biosystematiek - taxonomie - evolutie - biogeografie - plantengeografie - fylogenie - taxonomische revisies - fylogenetica - ochnaceae - biosystematics - taxonomy - evolution - biogeography - phytogeography - phylogeny - taxonomic revisions - phylogenetics


    Ochnaceae s.l. is a family of trees, shrubs or rarely herbs widely distributed in tropical and subtropical forests and savannas of the Old and New World, and has about 500 species in 32 genera. The family is divided into three subfamilies: Medusagynoideae, Quiinoideae and Ochnoideae. We have provided, for the first time, a nearly complete molecular phylogenetic analysis of Ochnaceae s.l. resolving most of the phylogeny backbone of the family using five DNA regions. Based on this, dating analyses were performed using a secondary calibration, and relaxed molecular clock models. The historical biogeography of Ochnaceae s.l. was reconstructed using Dispersal-Vicariance Analysis and Bayesian Binary MCMC. The Neotropics were inferred as being the geographical origin of the family and the Old World was most likely colonized via the North Atlantic Land Bridge during a period when climatic conditions allowed establishment of a boreotropical flora. A full taxonomic revision of the continental African species of the genus Campylospermum has been prepared and additional historical biogeographic analyses were performed with a focus on the genus Campylospermum.

    Conservation genetics of local and wild pig populations : insight in genetic diversity and demographic history
    Herrero Medrano, J. - \ 2013
    Wageningen University. Promotor(en): Martien Groenen, co-promotor(en): Richard Crooijmans; Hendrik-Jan Megens. - S.l. : s.n. - ISBN 9789461737519 - 160
    wilde varkens - sus scrofa - varkens - dna - genetische diversiteit - genetische bronnen van diersoorten - genomica - fylogenetica - zoögeografie - populatiedynamica - single nucleotide polymorphism - wildbescherming - wild pigs - sus scrofa - pigs - dna - genetic diversity - animal genetic resources - genomics - phylogenetics - zoogeography - population dynamics - single nucleotide polymorphism - wildlife conservation
    Het doel van het onderzoek zoals beschreven in dit proefschrift was om de genetische diversiteit en demografische geschiedenis van lokale varkenspopulaties te verkennen middels het integreren van verschillende genetische merker systemen.
    Phylogenetic relationships within major nematode clades based on multiple molecular markers
    Rybarczyk-Mydlowska, K. - \ 2013
    Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Hans Helder; Geert Smant. - [S.l. : s.n. - ISBN 9789461736529 - 125
    vrijlevende nematoden - dorylaimidae - mononchidae - aphelenchidae - fylogenetica - moleculaire merkers - moleculaire genetica - free living nematodes - dorylaimidae - mononchidae - aphelenchidae - phylogenetics - molecular markers - molecular genetics

    Nematodes are probably the most abundant Metazoans on our planet. They are present in densities of millions of individuals per square meter in soil and sediments. However, these inconspicuous animals are hardly known to the general public as most individuals are colorless and smaller than 1 mm. If people are aware of nematodes, it is because of the damage they inflict on humans and animals such as elephantiasis (Wucheria bancrofti) and ascariasis (in humans Ascaris lubricoides; in pigs Ascaris suum), or on plants such as potato (potato cyst nematode) and tomato (root-knot nematodes). To a far lesser extent it is known that majority of nematodes are key players in the soil food web, and as such they can be used as indicators for the biological condition of the environment they live in.

    This PhD thesis focuses on terrestrial nematodes belonging to four orders: Dorylaimida, Mononchida, Aphelenchida and Tylenchida, which represent animals of ecological and economical relevance. Members of families belonging to the first two are highly sensitive to the environmental disturbances, and are informative as biological indicators. The order Aphelenchida harbors numerous facultative plant-parasitic species. In the absence of a host plant, most of them are able to feed on fungi as an alternative food source. This is in contrast to the distal representatives of the order Tylenchida that are invariably obligate parasites of higher plants.

    Stress-sensitive nematode orders Dorylaimida and Mononchida have a high potential for soil health assessment. The SSU rDNA-based analysis of these two orders resulted in two highly distinct phylogenies. Relationships among the Mononchida, an order dominated by carnivorous nematodes, were to some extent in accordance with the classical nematode systematics. It is noted that the families Mylonchulidae, Mononchidae and Anatonchidae are not monophyletic. Nevertheless, it was possible to design family-specific primers for rDNA-based molecular detection. On the contrary, resolution of the SSU rDNA tree of the Dorylaimida was extremely poor, except for the plant-parasitic family Longidoridae and the mainly predaceous family Nygolaimidae. Analysis of a 1,000 bp fragment of the 5’ region of large subunit (LSU) rDNA resulted in an improved resolution. Twelve subclades were distinguished and this topology was only in slight agreement with the classical systematics of the suborder Dorylaimina. The poor resolution generated by SSU rDNA sequence analysis within this species-rich suborder is remarkable; it has not been observed in any other suborder in the phylum Nematoda. Possibly, Dorylaimina diversification is the result of rapid speciation events.

    A plant-parasitic lifestyle apparently accelerates the rate of change of rDNA genes. This was not only true for the obligate plant-parasitic Longidoridae, but also for the facultative plant-parasitic Aphelenchoididae. Most members of the genus Aphelenchoides are fungivores, but a few of them feed on higher plants as well. As they feed on aboveground parts of higher plants they are usually called ‘foliar nematodes’. Species such as Aphelenchoides besseyi, A. fragariae and A. ritzemabosi parasitize on ornamental plants in greenhouses and nurseries, and some field crops such as rice or strawberry. Moreover, A. subtenuis causes serious damage infecting flower bulbs. Identification of foliar nematode species, and the distinction between plant- parasitic species and other, mostly harmless, fungal feeding representatives of the genus Aphelenchoides is hampered by the scarcity of informative morphological characters and lack of well-established systematics. Based on nearly full-length SSU rDNA sequences, a phylogenetic tree was generated, where the four target species appeared as distinct, well-supported, monophyletic groups. The presence of species-specific DNA motifs made it possible to design PCR primers for the detection and quantification of the foliar nematode species in complex DNA backgrounds such as plant material and soil samples.

    The order Tylenchida is dominated by obligatory plant-parasitic nematode taxa and includes economically high-impact species such as the lesion (Pratylenchus spp.), root-knot (Meloidgyne spp.) and cyst (Heterodera, Globodera) nematodes. The sequence diversification of cellulases, a non-neutral, plant pathogenicity-related genes, was investigated. Unlike by far most other animals, nematodes do not depend on endosymbionts for the production of cell wall-degrading enzymes. Among a repertoire of these proteins, glycoside hydrolase family 5 (GHF5) cellulases are best studied. It is hypothesized that they were acquired by one or multiple horizontal gene transfer (HGT) events. Moreover, the nature of the donor - hypothesized to be a plant-parasitic soil bacterium - and the recipient, possibly a bacterivorous nematode is fully unclear. Using a range of primers, partial GHF5 cellulase sequences spanning the core catalytic domain were amplified and sequenced from basal Meloidogyne, and a range of Pratylenchus and Hirschmanniella species. Phylogenetic analysis of more than 100 partial GHF5 cellulase sequences resulted in a division of the enzymes’ catalytic domains into three types (A, B, C). Type B was numerically dominant, and notably the P. thornei cellulase was positioned sister to all type B root-knot nematode cellulases. Moreover, the overall topology of the catalytic domain B-type showed remarkable resemblance with trees based on rDNA sequences. This analysis suggests that most likely the cellulases were passed on by ancestors of a family nowadays known as the Pratylenchidae, and root-knot and cyst nematodes did not acquire these genes directly by lateral genes transfer.

    To further elucidate the relationship between a part of the family Pratylenchidae (namely the subfamilies Pratylenchinae and Hirschmanniellinae) and the Meloidogynidae, two neutral (= pathogenicity-unrelated) genes were taken into consideration: SSU rDNA and a part of the largest subunit of the RNA polymerase II gene (rpb1). Both morphological and molecular data seem to point at root-knot nematodes being a subclade branching from the migratory endoparasites Pratylenchidae. Extension of the SSU rDNA data set - more sequences from a broader range of species – did not result in a well- resolved relationship between the Pratylenchidae and the Meloidogynidae. A switch to another gene that was previously exploited to investigate relationships within the genus Meloidogyne, a fragment of the largest subunit of RNA polymerase II sequences, did not provide us with more robust information about the evolutionary transition between lesion and root-knot nematodes. The genus Pratylenchus comprises more than 100 species. It is referred to as a stenomorphic genus since only a few subtle characteristics are used for species identification. In the current study, only a subset of these species was taken to consideration; predominantly species that are relatively well-characterized as pathogens in agro-ecosystems. It was postulated that the Pratylenchus species closest to the basal root-knot nematodes should be sought among the less well-known and agronomically less relevant lesion nematode species.

    A concise bibliographic overview of Annonaceae
    Erkens, R.H.J. ; Mennega, E.A. ; Westra, L.I.T. - \ 2012
    Botanical Journal of the Linnean Society 169 (2012)1. - ISSN 0024-4074 - p. 41 - 73.
    for-plant-conservation - taxonomists - science - classification - phylogenetics - collections - family - policy
    It is important that taxonomic knowledge is disseminated by those who possess it. This can be carried out via online databases, printed publications or training in taxonomic practice. For Annonaceae, the online database AnnonBase includes all published names of Annonaceae at any rank worldwide. Recently, an updated index to genera, species and infraspecific taxa of Neotropical Annonaceae was published in hard copy. This manuscript contains a concise update to the Bibliography of Annonaceae published by the late E. A. Mennega in 1993. Since his publication, much work has been carried out on systematics and evolution of Annonaceae, with the advent of molecular phylogenetics providing an additional stimulus. Here, however, we concentrate only on taxonomic papers focusing specifically on the description and circumscription of taxa (e.g. revisions, monographs, floras, checklists, descriptions of new species or phylogenetic studies that show monophyly/polyphyly/paraphyly of genera). The index should provide an easy entrance into the most important taxonomic literature on Annonaceae from 1900 onwards for the trained and untrained taxonomist with an interest in Annonaceae.
    Co-option of pre-existing pathways during Rhizobium-legume symbiosis evolution
    Lillo, A. - \ 2012
    Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - S.l. : s.n. - ISBN 9789461733443 - 151
    rhizobium - fabaceae - symbiose - evolutie - stikstoffixatie - wortels - fylogenetica - genomen - medicago - eerste wortels - rhizobium - fabaceae - symbiosis - evolution - nitrogen fixation - roots - phylogenetics - genomes - medicago - root primordia

    Fixed nitrogen is one of the most limiting factors for plant growth. One of the most important nitrogen-fixing systems is the rhizobium root nodule symbiosis. In this Thesis I have studied the legume-rhizobium symbiosis, starting from the idea that part of pre-existing signalling pathways have been co-opted during evolution of this mutualistic interaction. Gene duplications -of which a whole genome duplication (WGD) is the most dramatic variant- are known as important driving forces in evolution of new traits. 56 to 65 million years ago an ancestral legume species within the Papilionoidae subfamily (Papilionoids) experienced a WGD event and subsequently gave rise to several major phylogenetic crowns. I hypothesize that among the orthologous gene pairs maintained are genes that are essential for nodulation. I adopted a phylogenetic strategy to identify new candidate genes involved in the legume-Rhizobium symbiosis
    In a targeted approach, we focussed on the cytokinin phosphorelay pathway. This resulted in the identification of one gene pair encoding type-A Response Regulators (RRs) with a positive regulatory role for these proteins in root nodule formation. Yet the illustrated role for MtRR9 and MtRR11 in rhizobial symbiosis provides a proof of principle of this method to identify gene pairs involved in legume specific characters. An unbiased search for paralogous gene pairs revealed two conserved gene duplications in the NADPH oxidases gene family. NADPH oxidases are reactive oxygen species (ROS) producing enzymes. We identified two sets of duplicated genes that have been maintained after the Papilionoid specific WGD and we show that MtRBOHA and MtRBOHG are redundant, yet essential during symbiosis.
    Moreover, although it is commonly believed that exclusively pericycle cells give rise to the lateral root primordium, similar as seen in Arabidopsis thaliana, we provide morphological evidence that in the studied legume species this is not the case. In both, Lotus and Medicago, also root cortical cell divisions occur during lateral root formation. Furthermore, we found a striking correlation in the cell layers that are recruited during lateral root and nodule primordium formation. This supports the hypothesis that at least parts of the lateral root developmental program have been recruited during evolution of symbiotic root nodules.

    Functional aspects of baculovirus DNA photolyases
    Xu, F. - \ 2010
    Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Monique van Oers. - [S.l. : S.n. - ISBN 9789085857730 - 112
    baculoviridae - organismen ingezet bij biologische bestrijding - insectenplagen - fotolyse - chrysodeixis chalcites - kernpolyedervirussen - lyasen - genen - genexpressie - fylogenetica - lokalisatie - ultraviolette straling - eiwitexpressieanalyse - gevoeligheid - baculoviridae - biological control agents - insect pests - photolysis - chrysodeixis chalcites - nuclear polyhedrosis viruses - lyases - genes - gene expression - phylogenetics - localization - ultraviolet radiation - proteomics - sensitivity
    Keywords: baculovirus, ChchNPV, CPD photolyase, phylogeny, UV resistance, DNA binding, localization, proteomics

    Baculoviruses are insect viruses that are applied as biological control agents due to adequate virulence, host specificity and safety for the environment. Solar light negatively affects field performance of baculoviruses by reducing their infectivity, most likely as a consequence of the formation of cyclobutane pyrimidine dimers (CPDs) in the viral DNA upon ultraviolet (UV) irradiation. CPDs can be repaired by CPD photolyases when exposed to blue light photons, a process called photoreactivation. From previous work it was known that the Cc-phr2 gene of the baculovirus Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) encodes a biochemically active photolyase. The research in this thesis focuses on (i) the degree of conservation of CPD photolyase (phr) genes in a subgroup of baculoviruses, (ii) the localization of baculovirus photolyase proteins in insect cells and occlusion derived virus (ODV), and (iii) the in vivo effect of phr genes on the UV sensitivity of baculoviruses. Homologues of the Cc-phr genes were found in all studied group II NPVs in the genus Alphabaculovirus that infect insects in the subfamily Plusiinae insects. Phylogenetic analysis suggested that these phr-like genes have a common ancestor. Intracellular localization of the two ChchNPV encoded PHR proteins in insect cells was studied using enhanced GFP fusion. Both PHR1 and PHR2 localized in the nucleus and associated with chromosomes, spindle, aster and midbody structures during host cell mitosis. Moreover, Cc-PHR2 co-localized with virogenic stroma, when PHR2-EGFP-transfected cells were infected with Autographa californica (Ac) MNPV. Neither of the two Cc-PHR proteins was identified by LC/MS-MS in the ODVs of ChchNPV. To evaluate the potential of the Cc-PHR2 protein to reduce the UV sensitivity of a baculovirus, the Cc-phr2 gene was incorporated in the genome of Helicoverpa armigera (Hear) NPV, which does not have a UV damage repair system. This resulted in a decreased sensitivity to UV-light compared to wild type HearNPV. A cell line was established from embryos of the insect C. chalcites. This cell line was shown to be permissive for both ChchNPV and the related Trichoplusia ni NPV (TnSNPV). This novel cell line will be a useful tool for making ChchNPV phr mutant viruses to study the impact of DNA repair mediated by photolyases on baculovirus ecology. The collected data support the hypothesis that the Cc- phr2 gene provides a baculovirus with an ecological benefit by increasing the resistance to UV.

    Decoupled leaf and stem economics in rain forest trees
    Baraloto, C. ; Paine, C.E.T. ; Poorter, L. ; Beauchene, J. ; Bonal, D. ; Domenach, A.M. ; Herault, B. ; Patiño, S. ; Roggy, J.C. ; Chave, J. - \ 2010
    Ecology Letters 13 (2010)11. - ISSN 1461-023X - p. 1338 - 1347.
    functional traits - neotropical forests - wood density - strategies - diversity - spectrum - size - phylogenetics - conductivity - architecture
    Cross-species analyses of plant functional traits have shed light on factors contributing to differences in performance and distribution, but to date most studies have focused on either leaves or stems. We extend these tissue-specific analyses of functional strategy towards a whole-plant approach by integrating data on functional traits for 13 448 leaves and wood tissues from 4672 trees representing 668 species of Neotropical trees. Strong correlations amongst traits previously defined as the leaf economics spectrum reflect a tradeoff between investments in productive leaves with rapid turnover vs. costly physical leaf structure with a long revenue stream. A second axis of variation, the ‘stem economics spectrum’, defines a similar tradeoff at the stem level: dense wood vs. high wood water content and thick bark. Most importantly, these two axes are orthogonal, suggesting that tradeoffs operate independently at the leaf and at the stem levels. By simplifying the multivariate ecological strategies of tropical trees into positions along these two spectra, our results provide a basis to improve global vegetation models predicting responses of tropical forests to global chan
    Taxonomy of Atlantic Central African Orchids 1. A New Species of Angraecum set. Pectinaria (Orchidaceae) from Gabon and Equatorial Guinea.
    Stévart, T. ; Cawoy, V. ; Damen, T.H.J. ; Droissart, V. - \ 2010
    Systematic Botany 35 (2010)2. - ISSN 0363-6445 - p. 252 - 256.
    vandeae - phylogenetics
    During a recent survey of Atlantic central African orchids, we collected four orchid specimens in Rio Muni (Equatorial Guinea) that share the general morphology of Angraecum gabonense, the most frequent member of Angraecum section Pectinaria in Central Africa, but differ in leaf shape and flower size. Further inspection of specimens deposited at the Wageningen herbarium and cultivated in their greenhouse led to the discovery of additional specimens from the Monts Doudou area in Gabon. Comparison with other Angraecum specimens indicate that these collections represent a new species, which we describe here as Angraecum atlanticum, the fifth species of Angraecum section Pectinaria recorded in central Africa. The new species is restricted to submontane forests covering the mountain chain situated along the coasts of Gabon and Equatorial Guinea. The distinguishing features of the species include its leaves, which are more widely spaced than A. gabonese, and are 2.2 mm wide; its petals and sepals, which are slightly longer than the lip; its spur, which is somewhat inflated in the middle; and its larger ovary. Information on the ecology, phenology and distribution of Angraecum atlanticum is presented, along with a preliminary conservation assessment using the IUCN Red List Categories and Criteria.
    The development of FISH tools for genetic, phylogenetic and breeding studies in tomato (Solanum lycopersicum)
    Szinay, D. - \ 2010
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Hans de Jong; Yuling Bai. - [S.l. : S.n. - ISBN 9789085856351 - 147
    solanum lycopersicum - plantenveredeling - fylogenetica - fluorescentie - dna - chromosoomanalyse - methodologie - genexpressieanalyse - in situ hybridisatie - solanum lycopersicum - plant breeding - phylogenetics - fluorescence - dna - chromosome analysis - methodology - genomics - in situ hybridization
    In this thesis various fluorescence in situ hybridization (FISH) technologies are described to support genome projects, plant breeding and phylogenetic analysis on tomato (Solanum lycopersicum, 2n=24). Its genome is 980 Mb and only 30 % are single copy sequences, which are mostly found in the euchromatin regions. These regions in all 12 chromosomes were therefore focus of the International Solanaceae Genome Sequencing Project. Based on the F2.2000 linkage map bacterial artificial chromosomes (BACs) were selected from three libraries for validating their physical locations by Fluorescence in situ Hybridization (FISH). In Chapter 2 I describe a five-color high-resolution BAC FISH approach and results of the mapping of 75 seed BACs on pachytene complements of chromosome 6. We found differences between the cytogenetic map and the linkage map. Most of the discrepancies occurred in the pericentromeric heterochromatin where recombination is highly suppressed. For establishing the BAC coverage of chromosome 6 a pooled BAC FISH method was used to hybridize all seed BACs simultaneously. A few larger gaps were discovered mostly on the long arm, where our ‘BAC-by-BAC’ sequencing approach could not manage to close the gaps by extending contigs. Afterwards new candidate BACs were tested by pooled-BAC FISH. Finally we demonstrated the heterochromatin / euchromatin distribution focusing on its borders by mapping pooled repetitive sequences (Cot 100) together with border BACs. In Chapter 3 the repeat content of chromosome 7 was analyzed by combining BAC and extended fiber FISH mapping with bioinformatics of 169 BACs. Repeats are important due to their challenging interpretations in genome sequencing. Tandem arrays of Tomato Genome Repeat I (TGRI) were found in BACs close to the distal end of chromosome 7 as well as on the long arm interstitial knobs. Phylogenetic analysis by neighbor-joining approach showed clustering of the TGRI blocks that suggested their independent origin. TGRI is likely to be transposed by extrachromosomal circular DNA molecules during anaphase. The dispersed TGR repeats (TGRII, TGRIII, TGRIV) all belong to the Ty3-Gypsy LTR class of retrotransposons. All of them cover the pericentromeric heterochromatin but overlap only partly as shown by FISH and BAC sequencing. TGRII hybridized through the whole pericentromere, TGRIII overlapped with TGRII except for the distal regions of the heterochromatin on the long arm, whereas TGRIV showed coverage in the most proximal parts of the short arm heterochromatin. BAC sequences corresponded well to the FISH data except that there were solo LTRs of TGRII found in the euchromatin. In the pericentromere heterochromatin truncated and solo LTRs were present of both TGRII and TGRIII. The TGRIV repeat could not be further investigated due to too high repeat content. Further this chapter offers some clues why TGR repeats are distributed in a certain way in the pericentromere.
    In Chapter 4 a comparative mapping study was carried out between tomato and potato (Solanum tuberosum) chromosome 6 using BACs from both species. The BACs were hybridized on both species by FISH. Due to some repeat-rich BACs Cot 100 blocking was necessary as well as lowered stringent washing to achieve unique and clear signals. We detected a novel paracentric inversion on the short arm of chromosome 6. The two break points are close to the distal heterochromatin end and to the eu- heterochromatin border. The BAC order revealed colinearity on the long arm. The two investigated tomato cultivars- Heinz 1706 and Cherry VFNT- were colinear for all of the used BACs. One (RH98-856-18) out of six potato clones differed by a small rearrangement in the middle of the inversion. This study gave a first idea for evolutionary investigative studies in the Solanum genus using chromosomal rearrangements as detected by FISH and which are elaborated in Chapter 5. It is known that chromosomal rearrangements happen frequently, but rarely get fixed during evolution. The reason is that chromosomal rearrangements have often a negative influence on fertility and on the progeny. In Solanum mostly inversions were previously reported. We selected repeat poor and evenly distributed tomato and potato BACs and after labeling those by fluorescence dyes we hybridized them across related wild species, tomato breeding lines, potato, eggplant and pepper (which is a close relative outside of the genus). We could reveal synteny between these species. In this way we discovered five undescribed inversions and found discrepancies with previous literature claiming chromosomal rearrangements. Our results correspond well to published phylogeny on Solanum, suggesting that our approach would be suitable for studying unknown genomes and resolving relationships on a higher level, such as sections.
    Finally this thesis discusses the crucial points of FISH technology; such as spatial resolution, detection sensitivity and applicability. It highlights the strength, weaknesses, opportunities and threads of FISH. In conclusion FISH is an indispensible technique for sequencing large genomes and defining repeat content with support of bioinformatics. Moreover, hidden chromosomal rearrangements can be visualized in regions where recombination is suppressed, which is important for plant breeding and definitely for phylogenetic studies.
    A phylogenetic re-evaluation of Dothideomycetes
    Schoch, C.L. ; Spatafora, J.W. ; Lumbsch, H.T. ; Huhndorf, S.M. ; Hyde, K.D. ; Groenewald, J.Z. ; Crous, P.W. - \ 2009
    Utrecht, the Netherlands : CBS-KNAW (Studies in mycology 64) - ISBN 9789070351786 - 220
    pezizomycotina - fylogenetica - fylogenie - evolutie - taxonomie - moleculaire taxonomie - pezizomycotina - phylogenetics - phylogeny - evolution - taxonomy - molecular taxonomy
    This volume presents a re-evaluation of phylogenetic relationships within the class Dothideomycetes, which is by far the largest and arguably most phylogenetically diverse class within the largest fungal phylum, Ascomycota.
    Two-component signal transduction in Bacillus cereus and closely related bacteria
    Been, M.W.H.J. de - \ 2009
    Wageningen University. Promotor(en): Tjakko Abee; R.J. Siezen, co-promotor(en): C. Francke. - [S.l.] : S.n. - ISBN 9789085854357 - 176
    bacillus cereus - stressreactie - fylogenetica - signaaltransductie - genregulatie - bacillus cereus - stress response - phylogenetics - signal transduction - gene regulation
    Bacillus cereus is a Gram-positive pathogen that is recognised as an important cause of food-borne disease worldwide. Within the genus Bacillus, B. cereus and its closest relatives form a homogeneous subdivision that has been termed the B. cereus group. This group includes B. anthracis, a pathogen that can cause anthrax in mammals, and B. thuringiensis, an insect pathogen that is used as an insecticide worldwide. Members of the B. cereus group can adapt to a wide range of environmental challenges. In bacteria, these challenges are generally monitored by two-component systems (TCS), which consist of a histidine kinase (HK) and a partner response regulator (RR). Upon sensing a specific environmental stimulus, the HK activates its cognate RR, which in turn controls the expression of genes that are involved in the appropriate response. This thesis describes the functional analysis of TCSs in the B. cereus group. By using in silico techniques, 50-58 HKs and 48-52 RRs were identified in eight different B. cereus group genomes. Biological functions, including the involvement in sporulation, biofilm formation and host-microbe interactions were predicted for these TCS proteins. A phylogenetic footprinting approach was developed and used to identify specific binding sites and target genes for over 50% of the B. cereus group DNA-binding RRs. These predictions allowed relating several RRs to a minimal regulon and thereby to a characteristic transcriptional response. To further support these predictions, the transcriptomes of two B. cereus TCS deletion mutants (ΔyvrHG and ΔyufLM) were analysed and compared with the transcriptome of wild-type B. cereus. This revealed that the minimal regulon predictions were correct for the two respective TCSs. Furthermore, the predicted biological roles for these TCSs, including roles in antibiotic resistance (YvrHG) and fumarate metabolism (YufLM), were supported by phenotypic tests. Besides the many “classical” HKs and RRs detected in the B. cereus group, several a-typical TCS proteins were found. These included five RRs without a DNA-binding output domain and two hybrid HKs (HK-RR fusions). Genome analyses revealed that one of the hybrid HK-encoding genes (BC1008) is located in a conserved gene cluster that also encodes the a-typical RR RsbY. In B. cereus, RsbY is known to activate the key stress-responsive sigma factor σB. As a partner HK for RsbY was still “missing”, the role of BC1008 in the σB-mediated stress response was tested. Indeed, a bc1008 deletion strain appeared incapable of inducing σB and its associated regulon upon stress conditions and appeared impaired in its heat adaptive response. In addition, truncation of the BC1008 fused RR receiver domain indicated that this domain plays a role in fine-tuning BC1008 activity. A comparative genome analysis further indicated that BC1008-type hybrid HKs control σB-like sigma factors in at least several other Gram-positive bacteria, including Geobacillus, Paenibacillus and actinobacteria. In summary, the research described in this thesis contributes to our understanding of B. cereus adaptive responses through TCSs. This knowledge may be applied for the development of novel intervention strategies for an improved control of B. cereus in food production environments.

    Searching for species, relationships and resistance in Solanum section Petota
    Jacobs, M.M.J. - \ 2008
    Wageningen University. Promotor(en): Marc Sosef; Richard Visser, co-promotor(en): Ben Vosman; Ronald van den Berg. - [S.l.] : S.n. - ISBN 9789085852049 - 187
    solanum - taxonomie - plantkunde - taxa - soorten - moleculaire taxonomie - biosystematiek - fylogenetica - ziekteresistentie - phytophthora infestans - genexpressieanalyse - solanum - taxonomy - botany - taxa - species - molecular taxonomy - biosystematics - phylogenetics - disease resistance - phytophthora infestans - genomics
    An integrative algorithmic approach towards knowledge discovery by bioinformatics
    Alako Tadontsop, F.B. - \ 2008
    Wageningen University. Promotor(en): Jack Leunissen. - [S.l. : S.n. - ISBN 9789085048190 - 124
    bio-informatica - nomenclatuur - computeranalyse - nucleotidenvolgordes - algoritmen - fylogenetica - moleculaire biologie - fylogenie - classificatie - genexpressieanalyse - datamining - microarrays - ontologieën - bioinformatics - nomenclature - computer analysis - nucleotide sequences - algorithms - phylogenetics - molecular biology - phylogeny - classification - genomics - data mining - microarrays - ontologies
    In this thesis we describe different approaches aiding in the utilization of the exponentially growing amount of information available in the life sciences. Briefly, we address two issues in molecular biology, on sequence analysis, and on text mining. The former issue addresses the problem how to determine remote sequence homology especially when the sequence similarity is very low. For this a visualisation tool is introduced that combines sequence alignment, domain prediction and phylogeny. The second topic on text mining centres on the question how to unambiguously formulate queries for efficient information retrieval. It tackles the problem of gene nomenclature — one in two gene symbols being ambiguous - by introducing a new text-clustering- and taxonomy-based disambiguation methodology.
    Phylogenetic relationships within the phylum Nematoda as revealed by ribosomal DNA, and their biological implications
    Holterman, M.H.M. - \ 2007
    Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Hans Helder. - [S.l.] : S.n. - ISBN 9789085048800 - 208
    nematoda - ribosomaal dna - fylogenetica - klassering volgens erfelijke eigenschappen - fylogenie - plantenparasitaire nematoden - vrijlevende nematoden - dorylaimidae - chromadoridae - tylenchidae - zeenematoden - single nucleotide polymorphism - nematoda - ribosomal dna - phylogenetics - cladistics - phylogeny - plant parasitic nematodes - free living nematodes - dorylaimidae - chromadoridae - tylenchidae - marine nematodes - single nucleotide polymorphism
    Nematodes – “eel worms”; members of the phylum Nematoda – can be considered as a success story within the Metazoa (multicellular, heterotrophic eukaryotes in which cells lack cell walls): they are speciose and – probably - the most numerous group of multicellular animals on our planet. Nematodes are present in virtually all terrestrial, freshwater and marine habitats. Nematodes are trophically diverse; they may feed on bacteria, fungi/oomycetes, algae and protozoa, other nematodes or on a combination of these (omnivores), or live as facultative or obligatory parasites of plants or animals. As they are abundant, ubiquitous and occupy several trophic levels, they play an important role in the soil food web. Nematode parasites of animals affect billions of humans and livestock, while plant parasites such as cyst, root knot and lesion nematodes cause large agricultural losses worldwide.
    Despite their undisputed ecological and economical relevance, the systematics of the phylum Nematoda is far from established. One of the aims of this research was to further elucidate nematode phylogeny using molecular data. First a phylogenetic tree was constructed of 349 taxa, spanning the entire phylum Nematoda, on the basis of full length small subunit ribosomal DNA (SSU rDNA) sequences. A series of mostly well-supported bifurcations defined twelve major clades, whereas the most basal clade was defined by representatives of the Enoplida and Triplonchida. Our analysis confirmed the paraphyly of the Adenophorea. Furthermore it was found that the SSU rDNA from representatives of the distal clades evolved at a higher rate than the SSU rDNA from the basal clades. In the meantime, a substantial number of sequences was added to our overall SSU rDNA nematode alignment - both public data (GenBank) and data generated by ourselves (≈ 1,500 sequences in total; February 2008). It is noted that the clade division as proposed in 2006 on the basis of “only” 349 taxa still seems to be valid.
    Subsequent research focused on three specific groups; Dorylaimia, Chromadoria and Tylenchomorpha. Within the suborder Dorylaimina, the SSU rDNA provided an exceptionally low phylogenetic signal, and - therefore – a part (≈ 1,000 bp) of the more variable large subunit ribosomal DNA (LSU rDNA) was analyzed. In most cases nematode relationships could be elucidated with good support, although some areas in the trees remained unresolved. Generally speaking the results of molecular phylogenetics corresponded fairly well with classical nematode taxonomy. The main exception was the order Dorylaimida where twelve subclades could be distinguished which bore little resemblance to classical taxonomy. Furthermore the suitability of ribosomal DNA for a (semi-) quantative molecular identification method was demonstrated using quantitative PCR (q-PCR) and primers designed to specifically amplify members of the order Mononchida and the potato cyst nematodes Globodera pallida and G. rostochiensis.
    Plant parasitism has arisen several times within the phylum Nematoda (once in the Triplonchida, at least three times in the Dorylaimida and at least twice in the Tylenchomorpha). The long-standing and generally accepted hypothesis states that plant parasites evolved from fungal feeding ancestors. However, while in most cases plant parasites were associated with fungal feeding nematodes, this hypothesis could neither be confirmed nor denied with the results of our phylogenetic analyses. In the case of two Dorylaimida (Pungentus and Longidorella), however, the ancestor was probably an omnivore. The analysis of this problem was substantially hampered by the lack of knowledge on feeding behavior of basal Tylenchomorpha.
    Presumably, the common ancestor of the nematodes lived in a marine environment and - if this assumption is correct - the transition to a limnoterrestrial environment must have taken place at least once. Surprisingly, analysis of the Chromadoria (minus the Rhabditida) revealed that transitions from a thalassic to a limnoterrestrial habitat (and vice versa) have taken place at least 11 times in the Chromadoria. Given their frequency these transitions are apparently fairly easy to achieve for nematodes and the possible adaptations involved were discussed.
    Nematodes vary widely in their responses to environmental disturbance, making them good bio-indicators of soil health. Yet it is not known with certainty which traits are responsible for tolerance to stress in nematodes. A framework was laid out to study correlations between nematode traits and stress tolerance. Furthermore the importance of accounting for the confounding effects of phylogeny was demonstrated. This is a first step towards a transparent, ecological grouping of free-living nematodes.
    It is worthwhile mentioning that - on the basis of the rDNA-based molecular framework described in this PhD thesis - DNA sequences signatures were identified for nearly all North-West European terrestrial and freshwater nematodes families. The relationship between quantitative PCR signal and numbers of individuals has been established for nearly all families and a first testing of DNA barcode-based community analysis is planned for spring 2008.

    Phylogenetworks : exploring reticulate evolution and its consequences for phylogenetic reconstruction
    Vriesendorp, B. - \ 2007
    Wageningen University. Promotor(en): Marc Sosef, co-promotor(en): Freek Bakker; Ronald van den Berg. - [S.l.] : S.n. - ISBN 9789085047032 - 180
    fylogenetica - evolutie - taxonomie - hybridisatie - bedektzadigen - terminologie - phylogenetics - evolution - taxonomy - hybridization - angiosperms - terminology
    Minerals are essential for humans, plants and animals and have an important micronutrient role in physiological and metabolic processes of plants. Next to this essential role of minerals, they can also be very toxic when available to the plant in elevated amounts. Plants therefore need to keep very tight control over the intracellular mineral concentrations in a process called metal homeostasis. Although the metal homeostasis mechanisms are supposed to be universal within plants, there are plant species that can tolerate and even accumulate large amounts of metals without any sign of toxicity. Thlaspi caerulescens J. & C. Presl (Brassicaceae), a close relative of the plant reference species Arabidopsis thaliana (Arabidopsis), is one of these natural metal hyperaccumulator species. The overall aim of this project is to unravel the molecular genetic mechanism of heavy metal tolerance and hyperaccumulation of the metal hyperaccumulating plant species T. caerulescens. To achieve this goal heterologous transcript profiling experiments were performed, which involved comparative microarray hybridization experiments of the hyperaccumulator T. caerulescens and Arabidopsis. Arabidopsis is used as the reference species for heterologous transcript profiling because of the availability of genetic resources and the complete genome sequence. The micronutrient zinc has an essential role in physiological and metabolic processes in plants as a cofactor or structural element. Thlaspi caerulescens can accumulate up to 3% of zinc on a dry weight basis without any sign of toxicity. The question postulated here is if this has drastic effects on the zinc homeostasis mechanism. We examined in detail the transcription profiles of roots of Arabidopsis and T. caerulescens plants grown under deficient, sufficient and excess supply of zinc (Chapter 2). A total of 608 genes were detected in Arabidopsis and 352 in T. caerulescens that responded transcriptionally to changes in zinc supply. Only 14% of these genes were also zinc-responsive in Arabidopsis. When comparing Arabidopsis and T. caerulescens at comparable zinc exposures, over 2200 genes were significantly differentially expressed. While a large fraction of these genes are of yet unknown function, many genes with a different expression between Arabidopsis and T. caerulescens appear to function in metal homeostasis, in abiotic stress response and in lignin biosynthesis. The high expression of lignin biosynthesis genes corresponds to the deposition of lignin in the endodermis. Contrary to Arabidopsis roots, which have one endodermal cell layer, we found there are two endodermal layers in T. caerulescens roots. This extra physical barrier could enhance the control of metal fluxes in the plant, in addition to the higher expression of metal transporters in the root. Cadmium is a widespread, naturally occurring non-essential element that is toxic for plants in higher concentrations. In chapter 3 we compare between and within species transcript profiles of Arabidopsis and T. caerulescens roots exposed to cadmium, with the aim to establish which genes are most likely to be relevant for the tolerance to cadmium exposure of T. caerulescens. The comparative transcriptional analysis of the cadmium response of roots of the T. caerulescens and Arabidopsis emphasizes the role of genes involved in lignin-, glutathione- and sulfate metabolism. Furthermore two transcription factors, MYB72 and bHLH100, with an altered expression after exposure to cadmium, are studied for their involvement in metal homeostasis. Analysis of a myb72 knock-out mutant showed enhanced sensitivity to excess zinc or iron deficiency. Rather than controlling Cd tolerance, this gene appears to be involved in iron homeostasis, affecting the response to Cd indirectly. Arabidopsis transformants overexpressing the transcription factor bHLH100 showed enhanced zinc and nickel tolerance, and although the exact role of this gene still needs to be resolved, the genes appears to have a role in metal homeostasis in Arabidopsis. T. caerulescens accessions exhibit distinct metal accumulation, translocation and tolerance characteristics. T. caerulescens accession Ganges can accumulate high amounts of cadmium and is extremely tolerant to cadmium, whereas the La Calamine accession is also tolerant to cadmium but accumulates much less cadmium compared to Ganges. The transcription profiles of leaves and roots of T. caerulescens accessions Ganges and La Calamine plants grown with and without cadmium were examined using the Qiagen-Operon Arabidopsis Genome Array and results are described in chapter 4. A total of 161 genes were differentially expressed between the two T. caerulescens accessions in response to changes in cadmium supply and 38 genes were differentially expressed in T. caerulescens accession Ganges leaves in response to cadmium. The comparative transcriptional analysis emphasizes that there are just minor differences between the two accessions but the genes which are differentially expressed could play an important role in the hyperaccumulation of cadmium in Ganges. The microarray data suggest that especially genes involved in cell wall modification and stress response relate to the major difference between the two accessions in cadmium hyperaccumulation. Plants have evolved a complex network of homeostatic mechanisms that serve to control the uptake, accumulation, trafficking and detoxification of metals. One potential mechanism for heavy metal detoxification in plants is the chelation of metal ions to ligands like organic acids, amino acids, peptides and polypeptides. This mechanism is important for the distribution of metal ions by keeping metal ions mobile within the plant. In plants metals are often found to be chelated to nicotianamine. Nicotianamine is formed by trimerization of S-adenosylmethionine, which is catalyzed by the enzyme nicotianamine synthase. Arabidopsis contains four nicotianamine synthase (NAS) genes. Also in T. caerulescens four full-length cDNAs encoding nicotianamine synthase members were identified (Chapter 5). The four genes were named TcNAS1-TcNAS4, analogous to the corresponding closest homologue in Arabidopsis. Arabidopsis plants over-expressing TcNAS1, TcNAS2, TcNAS3 or TcNAS4 that were tested for their response to growth on media with different zinc, iron, nickel or cadmium supply, provided evidence that the Thlaspi genes all have a genuine NAS function because they complement the NAS deficiency in specific triple knock-out Arabidopsis mutants. Evidence for a functional role in metal homeostasis was sought in studying the Arabidopsis single, double, triple and a quadruple nicotianamine synthase T-DNA insertion mutants. The combination of null mutations in three or four AtNAS genes, results in a severe phenotype that includes interveinal chlorosis and altered metal concentrations in leaves, roots and seeds. Arabidopsis transformants overexpressing TcNAS3 or TcNAS4 showed enhanced zinc and nickel tolerance compared to wild type plants. The research described in this thesis does contribute to a better understanding of heavy metal hyperaccumulation in T. caerulescens and it can be concluded that it seems unlikely that altered regulation and overexpression of single genes will be sufficient to convert metal nonaccumulators into hyperaccumulators. However, the possibility that overexpression of one or two key regulatory loci have this effect remains.
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