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Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
Lindhoud, S. ; Westphal, A.H. ; Mierlo, C.P.M. van; Visser, A.J.W.G. ; Borst, J.W. - \ 2014
International Journal of Molecular Sciences 15 (2014)12. - ISSN 1661-6596 - p. 23836 - 23850.
resonance energy-transfer - beta parallel protein - single-molecule fluorescence - azotobacter-vinelandii - spectroscopic ruler - refractive-index - tryptophan residue - molten globule - wild-type - pathway
Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxin's complex folding pathway.
Disturbed excitation energy transfer in Arabidopsis thaliana mutants lacking minor antenna complexes of photosystem II
Dall'Osto, L. ; Ünlü, C. ; Cazzaniga, S. ; Amerongen, H. van - \ 2014
Biochimica et Biophysica Acta. B, Bioenergetics 1837 (2014)12. - ISSN 0005-2728 - p. 1981 - 1988.
light-harvesting-complex - primary charge separation - chlamydomonas-reinhardtii - thylakoid membrane - electron-transport - crystal-structure - green plants - redox state - wild-type - proteins
Minor light-harvesting complexes (Lhcs) CP24, CP26 and CP29 occupy a position in photosystem II (PSII) of plants between the major light-harvesting complexes LHCII and the PSII core subunits. Lack of minor Lhcs in vivo causes impairment of PSII organization, and negatively affects electron transport rates and photoprotection capacity. Here we used picosecond-fluorescence spectroscopy to study excitation-energy transfer (EET) in thylakoid membranes isolated from Arabidopsis thaliana wild-type plants and knockout lines depleted of either two (koCP26/24 and koCP29/24) or all minor Lhcs (NoM). In the absence of all minor Lhcs, the functional connection of LHCII to the PSII cores appears to be seriously impairedwhereas the “disconnected” LHCII is substantially quenched. For both double knock-out mutants, excitation trapping in PSII is faster than in NoM thylakoids but slower than in WT thylakoids. In NoM thylakoids, the loss of all minor Lhcs is accompanied by an over-accumulation of LHCII, suggesting a compensating response to the reduced trapping efficiency in limiting light,which leads to a photosynthetic phenotype resembling that of low-light-acclimated plants. Finally, fluorescence kinetics and biochemical results show that the missing minor complexes are not replaced by other Lhcs, implying that they are unique among the antenna subunits and crucial for the functioning and macroorganization of PSII.
Field vaccinated chickens with low antibody titres show equally insufficient protection against matching and non-matching genotypesof virulent Newcastle disease virus
Dortmans, J.C.F.M. ; Venema-Kemper, S. ; Peeters, B.P.H. ; Koch, G. - \ 2014
Veterinary Microbiology 172 (2014)1-2. - ISSN 0378-1135 - p. 100 - 107.
shedding following vaccination - wild-type - outbreaks - strains - sequence - china - pathogenesis - challenge - genetics - poultry
Newcastle disease (ND) is a severe threat to the poultry industry and is caused by virulent strains of Newcastle disease virus (NDV). Many countries maintain a vaccination policy, but NDV is rapidly evolving as shown by the discovery of several new genotypes in the last decades. We tested the efficacy of the currently used classical commercial ND vaccine based on the genotype II strain VG/GA, applied under standard field conditions, against outbreak strains. Field vaccinated broilers were challenged with four different viruses belonging to genotype II, V or VII. A large proportion of field vaccinated broilers showed suboptimal immunity and the protection level against early and recent NDV isolates was dramatically low. Furthermore, there were no significant differences in protection afforded by a genotype II vaccine against a genotype II virus challenge compared to a challenge with viruses belonging to the other genotypes. This study suggests that the susceptibility of vaccinated poultry to NDV infection is not the result of vaccine mismatch, but rather of poor vaccination practices
Transfer of knowledge about flowering and vegetative propagation from model species to bulbous plants
Leeggangers, H.A.C.F. ; Moreno Pachón, N.M. ; Gude, H. ; Immink, G.H. - \ 2013
International Journal of Developmental Biology 57 (2013)6-8. - ISSN 0214-6282 - p. 611 - 620.
mads-box genes - axillary meristem formation - tulip tulipa-gesneriana - crocus-sativus l. - agrobacterium-mediated transformation - lily lilium-longiflorum - wild-type - heterotopic expression - arabidopsis-thaliana - stalk elongation
The extensive characterization of plant genes and genome sequences summed to the continuous development of biotechnology tools, has played a major role in understanding biological processes in plant model species. The challenge for the near future is to generate methods and pipelines for an efficient transfer of this knowledge to economically important crops and other plant species. In the case of flower bulbs, which are economically very important for the ornamental industry, flowering time control and vegetative propagation constitute the most relevant processes for agronomical improvements. Those processes have been reasonably studied in reference species, making them excellent candidates for translational investigations in bulbous plant species. The approaches that can be taken for the transfer of biological knowledge from model to non-model species can be roughly categorized as "bottom-up" or "top-down". The former approach usually goes from individual genes to systems, also known as a "gene-by-gene" approach. It assumes conservation of molecular pathways and therefore makes use of sequence homology searches to identify candidate genes. "Top-down" methodologies go from systems to genes, and are e.g. based on large scale transcriptome profiling via heterologous microarrays or RNA sequencing, followed by the identification of associations between phenotypes, genes, and gene expression patterns and levels. In this review, examples of the various knowledge-transfer approaches are provided and pros and cons are discussed. Due to the latest developments in transgenic research and next generation sequencing and the emerging of systems biology as a matured research field, transfer of knowledge concerning flowering time and vegetative propagation capacity in bulbous species are now within sight
FAD C(4a)-hydroxide stabilized in a naturally fused styrene monooxygenase
Tischler, D. ; Schlömann, M. ; Berkel, W.J.H. van; Gassner, G.T. - \ 2013
FEBS Letters 587 (2013)23. - ISSN 0014-5793 - p. 3848 - 3852.
rhodococcus-opacus 1cp - phenol hydroxylase - wild-type - mechanism
StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems. Our studies identify substrate epoxidation and elimination of water from the FAD C(4a)-hydroxide as rate-limiting steps in the styrene epoxidation reaction. Efforts directed at accelerating these reaction steps are expected to greatly increase catalytic efficiency and the value of StyA2B as biocatalyst.
Light harvesting and Blue-Green light induced non-photochemical quenching in two different C-phycocyanin mutants of synechocytis PCC 6803
Tian, L. ; Stokkum, I.H.M. van; Koehorst, R.B.M. ; Amerongen, H. van - \ 2013
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 117 (2013)38. - ISSN 1520-6106 - p. 11000 - 11006.
orange carotenoid protein - excitation-energy transfer - photosystem-ii - sp pcc-6803 - phycobilisome fluorescence - photoprotective mechanism - picosecond kinetics - wild-type - cyanobacterium - photosynthesis
Cyanobacteria are oxygen-evolving photosynthetic organisms that harvest sunlight and convert excitation energy into chemical energy. Most of the light is absorbed by large light harvesting complexes called phycobilisomes (PBs). In high-light conditions, cyanobacteria switch on a photoprotective mechanism called non-photochemical quenching (NPQ): During this process, absorption of blue-green light transforms the inactive orange form of the orange carotenoid protein OCP (OCPo) into the red active form OCPr that subsequently binds to the PB, resulting in a substantial loss of excitation energy and corresponding decrease of the fluorescence. In wild-type cells, the quenching site is a bilin chomophore that fluoresces at 660 nm and which is called APCQ660. In the present work, we studied NPQ in two different types of mutant cells (CB and CK) that possess significantly truncated PBs, using spectrally resolved picosecond fluorescence spectroscopy. The results are in very good agreement with earlier in vitro experiments on quenched and unquenched PBs, although the fraction of quenched PBs is far lower in vivo. It is also lower than the fraction of PBs that is quenched in wild-type cells, but the site, rate, and location of quenching appear to be very similar.
Probing the picosecond kinetics of the photosystem II core complex in vivo
Tian, L. ; Farooq, S. ; Amerongen, H. van - \ 2013
Physical Chemistry Chemical Physics 15 (2013)9. - ISSN 1463-9076 - p. 3146 - 3154.
excitation-energy transfer - sp pcc 6803 - a antenna states - charge separation - synechococcus-elongatus - crystal-structure - 3-dimensional structure - decay kinetics - higher-plants - wild-type
Photosystems I (PSI) and II (PSII) are two major pigment–protein complexes of photosynthetic organisms that function in series to convert sunlight energy into chemical energy. We have studied the picosecond fluorescence behaviour of the core of both photosystems in vivo by using two Synechocystis PCC 6803 mutants: BE cells contain PSI but are lacking both PSII and the light-harvesting complexes called phycobilisomes (PBs) whereas PAL cells contain both PSI and PSII but lack the PBs. Measurements were performed at room temperature and at 77 K. The fluorescence kinetics of PSI and PSII can nicely be separated, en passant providing the PSI/PSII ratio. At room temperature, the PSI kinetics are identical to those of isolated PSI whereas the PSII kinetics can equally well be described by the in vitro trap-limited model of Y. Miloslavina, M. Szczepaniak, M. G. Muller, J. Sander, M. Nowaczyk, M. Rogner and A. R. Holzwarth, Biophys J., 2009, 96(2), 621–631, and by the transfer-to-the-trap-limited model of C. D. van der Weij-de Wit, J. P. Dekker, R. van Grondelle and I. H. M. van Stokkum, J. Phys. Chem. A, 2011, 115(16), 3947–3956, albeit that the in vivo kinetics turn out to be somewhat slower. At 77 K several low-energy pigments are observed in both photosystems which complicate the overall dynamics but the PSII kinetics can still be described by both a trap-limited and a transfer-to-the-trap-limited model.
POPCORN Functions in the Auxin Pathway to Regulate Embryonic Body Plan and Meristem Organization in Arabidopsis
Xiang, D.Q. ; Yang, H. ; Venglat, P. ; Cao, Y.G. ; Wen, R. ; Ren, M.Z. ; Stone, S. ; Wang, E. ; Wang, H. ; Xiao, W. ; Weijers, D. ; Berleth, T. ; Laux, T. ; Selvaraj, G. ; Datla, R. - \ 2011
The Plant Cell 23 (2011)12. - ISSN 1040-4651 - p. 4348 - 4367.
stem-cell niche - cup-shaped-cotyledon - shoot meristem - wild-type - gene - embryogenesis - wuschel - fate - monopteros - thaliana
The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are temperature sensitive, showing defective embryo development when grown at 22 degrees C that is rescued when grown at 29 degrees C. In pcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link PCN to auxin signaling and meristem function: ectopic expression of DR5(rev):green fluorescent protein (GFP), pBDL:BDL-GFP, and pMP:MP-beta-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn, suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM.
Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum)
Andreote, F.D. ; Rocha, U.N. da; Araujo, W.L. ; Azevedo, J.L. ; Overbeek, L.S. van - \ 2010
Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 97 (2010)4. - ISSN 0003-6072 - p. 389 - 399.
gradient gel-electrophoresis - 16s ribosomal-rna - land-use history - transgenic eucalyptus - multivariate analyses - xylella-fastidiosa - wild-type - rhizosphere - diversity - soil
Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.
Chikungunya Virus Nonstructural Protein 2 Inhibits type I/II Interferon-Stimulated JAK-STAT Signaling
Fros, J. ; Liu, W.J. ; Prow, A. ; Geertsema, C. ; Ligtenberg, M. ; Vanlandingham, D.L. ; Schnettler, E. ; Vlak, J.M. ; Suhrbier, A. ; Khromykh, A.A. ; Pijlman, G.P. - \ 2010
Journal of Virology 84 (2010)20. - ISSN 0022-538X - p. 10877 - 10887.
persistent infection - replication complex - equine encephalitis - gene-expression - wild-type - rnase-l - alphaviruses - induction - disease - cells
Chikungunya virus (CHIKV) is an emerging human pathogen transmitted by mosquitoes. Like that of other alphaviruses, CHIKV replication causes general host shutoff, leading to severe cytopathicity in mammalian cells, and inhibits the ability of infected cells to respond to interferon (IFN). Recent research, however, suggests that alphaviruses may have additional mechanisms to circumvent the host's antiviral IFN response. Here we show that CHIKV replication is resistant to inhibition by interferon once RNA replication has been established and that CHIKV actively suppresses the antiviral IFN response by preventing IFN-induced gene expression. Both CHIKV infection and CHIKV replicon RNA replication efficiently blocked STAT1 phosphorylation and/or nuclear translocation in mammalian cells induced by either type I or type II IFN. Expression of individual CHIKV nonstructural proteins (nsPs) showed that nsP2 was a potent inhibitor of IFN-induced JAK-STAT signaling. In addition, mutations in CHIKV-nsP2 (P718S) and Sindbis virus (SINV)-nsP2 (P726S) that render alphavirus replicons noncytopathic significantly reduced JAK-STAT inhibition. This host shutoff-independent inhibition of IFN signaling by CHIKV is likely to have an important role in viral pathogenesis.
Does abscisic acis affect strigolactone biosynthesis?
Lopez-Raez, J.A. ; Kohlen, W. ; Charnikhova, T. ; Mulder, P.P.J. ; Undas, A.K. ; Sergeant, J. ; Verstappen, F. ; Bugg, T.D.H. ; Thompson, A.J. ; Ruyter-Spira, C.P. ; Bouwmeester, H.J. - \ 2010
New Phytologist 187 (2010)2. - ISSN 0028-646X - p. 343 - 354.
arbuscular mycorrhizal fungi - parasitic plants - germination stimulants - arabidopsis-thaliana - phosphorus stress - mutant notabilis - orobanche spp. - tomato plants - water-stress - wild-type
Strigolactones are considered a novel class of plant hormones that, in addition to their endogenous signalling function, are exuded into the rhizosphere acting as a signal to stimulate hyphal branching of arbuscular mycorrhizal (AM) fungi and germination of root parasitic plant seeds. Considering the importance of the strigolactones and their biosynthetic origin (from carotenoids), we investigated the relationship with the plant hormone abscisic acid (ABA). Strigolactone production and ABA content in the presence of specific inhibitors of oxidative carotenoid cleavage enzymes and in several tomato ABA-deficient mutants were analysed by LC-MS/MS. In addition, the expression of two genes involved in strigolactone biosynthesis was studied. The carotenoid cleavage dioxygenase (CCD) inhibitor D2 reduced strigolactone but not ABA content of roots. However, in abamineSG-treated plants, an inhibitor of 9-cis-epoxycarotenoid dioxygenase (NCED), and the ABA mutants notabilis, sitiens and flacca, ABA and strigolactones were greatly reduced. The reduction in strigolactone production correlated with the downregulation of LeCCD7 and LeCCD8 genes in all three mutants. The results show a correlation between ABA levels and strigolactone production, and suggest a role for ABA in the regulation of strigolactone biosynthesis.
Mitochondrial recombination increases with age in Podospora anserina
Diepeningen, A.D. van; Goedbloed, D.J. ; Slakhorst, S.M. ; Koopmanschap-Memelink, A.B. ; Maas, M.F.P.M. ; Hoekstra, R.F. ; Debets, A.J.M. - \ 2010
Mechanisms of Ageing and Development 131 (2010)5. - ISSN 0047-6374 - p. 315 - 322.
group-ii introns - life-span - excision-amplification - sexual reproduction - sequence-analysis - plasmid pal2-1 - dna-sequence - wild-type - senescence - selection
With uniparental inheritance of mitochondria, there seems little reason for homologous recombination in mitochondria, but the machinery for mitochondrial recombination is quite well-conserved in many eukaryote species. In fungi and yeasts heteroplasmons may be formed when strains fuse and transfer of organelles takes place, making it possible to study mitochondrial recombination when introduced mitochondria contain different markers. A survey of wild-type isolates from a local population of the filamentous fungus Podospora anserina for the presence of seven optional mitochondrial introns indicated that mitochondrial recombination does take place in nature. Moreover the recombination frequency appeared to be correlated with age: the more rapidly ageing fraction of the population had a significantly lower linkage disequilibrium indicating more recombination. Direct confrontation experiments with heterokaryon incompatible strains with different mitochondrial markers at different (relative) age confirmed that mitochondrial recombination increases with age. We propose that with increasing mitochondrial damage over time, mitochondrial recombination – even within a homoplasmic population of mitochondria – is a mechanism that may restore mitochondrial function
Mixed-genotype infections of Trichoplusia ni larvae with Autographa californica multicapsid nucleopolyhedrovirus: Speed of action and persistence of a recombinant in serial passage
Zwart, M.P. ; Werf, W. van der; Georgievska, L. ; Oers, M.M. van; Vlak, J.M. ; Cory, J.S. - \ 2010
Biological Control 52 (2010)1. - ISSN 1049-9644 - p. 77 - 83.
single-nucleocapsid nucleopolyhedrovirus - improved baculovirus insecticide - wild-type - spodoptera-exigua - beet armyworm - preoccluded virions - biological-activity - egt gene - lepidoptera - transmission
Fast-acting recombinant baculoviruses have potential for improved insect pest suppression. However, the ecological impact of using such viruses must be given careful consideration. One strategy for mitigating risks might be simultaneous release of a wild-type baculovirus, so as to facilitate rapid displacement of the recombinant baculovirus by a wild-type. However, at what ratio must the two baculoviruses be released? An optimum release ratio must ensure both fast action, and the eventual competitive displacement of the recombinant virus and fixation of the wild-type baculovirus in the insect population. Here we challenged Trichoplusia ni larvae with different ratios of wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and a derived recombinant, vEGTDEL, which has the endogenous egt gene (coding for ecdysteroid UDP-glucosyltransferase) deleted. Time to death increased with the proportion wild-type virus in the inoculum mixture, although a 1:10 ratio (wild-type: recombinant) resulted in equally rapid insecticidal action as vEGTDEL alone. Five serial passages of three different occlusion body (OB) mixtures of the two viruses were also performed. OBs from 10 larval cadavers were pooled and used to initiate the following passage. Although the wild-type baculovirus was maintained over five passages, it did not go to fixation in most replicates of the serial passage experiment (SPE), and there was no good evidence for selection against the recombinant. Long-term maintenance of a recombinant in serial passage suggests an ecosystem safety risk. We conclude that for assessing ecological impact of recombinant viruses, SPEs in single and multiple larvae are relevant because of potential modulating effects at the between-host level.
Lack of transparency on environmental risks of genetically modified micro-organisms in industrial biotechnology
Tamis, W.L.M. ; Dommelen, A. van; Snoo, G.R. de - \ 2009
Journal of Cleaner Production 17 (2009)6. - ISSN 0959-6526 - p. 581 - 592.
horizontal gene-transfer - modified organisms - engineered microorganisms - transgenic plants - escherichia-coli - ecological risks - wild-type - soil - survival - chemicals
For the sustainable development of technological innovations the involvement of non-specialist stakeholders is crucial, which requires transparency of the knowledge base of the risks and benefits concerned. This paper evaluates the basic assumptions of the Organisation for Economic Cooperation and Development regarding the environmental risks of genetically modified micro-organisms, in industrial biotechnology under Good Industrial Large-scale Practice. A clear factual basis for these assumptions could be only partially traced, if at all, and when facts were found they proved to provide only limited support, if any. In addition, several assumptions appeared to be questionable when contrasted with knowledge from chemical and ecological sciences. There remain a number of open questions with respect to risks, most of them relating to improvement and extension of procedures, through greater attention to baselines, safety limits and further standardisation. The necessity of transparency in stakeholder communication is discussed and possible ways of improving such communication are presented.
The MADS domain protein DIANA together with AGAMOUS-LIKE80 to specify the central cell in Arabidopsis ovules
Bemer, M. ; Wolters-Arts, M. ; Grossniklaus, U. ; Angenent, G.C. - \ 2008
The Plant Cell 20 (2008). - ISSN 1040-4651 - p. 2088 - 2101.
female gametophyte development - polycomb group gene - seed development - box gene - endosperm development - wild-type - thaliana - identification - fertilization - expression
MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein¿-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt.
Development of a quantitative real-time PCR for determination of genotype frequencies for studies in baculovirus population biology
Zwart, M.P. ; Oers, M.M. van; Cory, J.S. ; Lent, J.W.M. van; Werf, W. van der; Vlak, J.M. - \ 2008
Journal of Virological Methods 148 (2008)1-2. - ISSN 0166-0934 - p. 146 - 154.
nuclear polyhedrosis-virus - polymerase-chain-reaction - trichoplusia-ni - sybr-green - mosaic-virus - wild-type - rt-pcr - nucleopolyhedrovirus - mutants - larvae
Two bacmid-derived Autographa californica Multiple-capsid Nucleopolyhedrovirus genotypes ¿ that differ only in a short tag sequence for differential PCR recognition ¿ were generated. By electron microscopy, these genotypes were found to have identical polyhedra morphology. Mixtures of quantified polyhedra were made and used to validate a SYBR Green I-based quantitative real-time PCR (qPCR) to determine genotype frequencies in mixed genotype populations. The PCR could accurately quantify genotype ratios over a range of 8 orders of magnitude. Only a small correction of the genotype ratio was necessary to obtain a valid result. Low levels of aspecific background (a fluorescent signal when the template corresponding with the primer set used is not present) were measured in these validation experiments and in a typical laboratory setup. A small fitness difference between the genotypes generated was observed in a median lethal dose bioassay. The bacmid-derived virus genotypes generated and the qPCR assays are valuable tools for studying the population biology of baculoviruses.
Monomer formation and functioning of p hydroxybenzoate hydroxylase in reverse micelles and in dimethylsulfoxide-water mixtures
Kudryashova, E.V. ; Visser, A.J.W.G. ; Berkel, W.J.H. van - \ 2008
ChemBioChem 9 (2008)3. - ISSN 1439-4227 - p. 413 - 419.
time-resolved fluorescence - flavin adenine-dinucleotide - pseudomonas-fluorescens - alpha-chymotrypsin - catalytic activity - crystal-structure - escherichia-coli - mobile flavin - aerosol ot - wild-type
It has previously been postulated that the dimeric form of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH) is important for catalysis. Here it is demonstrated that the monomeric form of PHBH is active. In a water/AOT/isooctane reverse micellar system, the function of the monomeric and dimeric forms of PHBH could be observed separately by varying the size of the micelles. A considerable decrease in the KM value for p-hydroxybenzoate (POHB) was found for monomeric PHBH, accompanied by a 1.5-fold decrease in enzymatic activity. The same tendency was observed when monomers of PHBH were formed by adding DMSO to the buffer. The FAD in PHBH and PHBH labeled with the fluorescence dye Alexa488 was investigated by time-resolved fluorescence anisotropy to observe monomer formation in water/DMSO mixtures. Monomer formation of PHBH occurred gradually with increasing DMSO content in the mixture. Pure PHBH monomers were detected at DMSO concentrations of 30 % (v/v) and higher.
Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase.
Berg, P.A.W. van den; Grever, K. ; Hoek, A. van; Berkel, W.J.H. van; Visser, A.J.W.G. - \ 2007
Journal of Chemical Sciences 119 (2007)2. - ISSN 0974-3626 - p. 123 - 133.
maximum-entropy method - pseudomonas-fluorescens - crystal-structure - 4-hydroxybenzoate hydroxylase - glutathione-reductase - effector specificity - adenine-dinucleotide - protein nanospace - nadph binding - wild-type
Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the ‘in’ conformation with the isoalloxazine ring located in the active site, and the ‘out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the ‘in’ and ‘out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes
Environmental influence on the genetic correlations between life-history traits in Caenorhabditis elegans
Gutteling, E.W. ; Doroszuk, A. ; Riksen, J.A.G. ; Prokop, Z. ; Reszka, J. ; Kammenga, J.E. - \ 2007
Heredity 98 (2007)4. - ISSN 0018-067X - p. 206 - 213.
phenotypic plasticity - offspring size - trade-offs - egg-size - arabidopsis-thaliana - seed beetle - c-elegans - wild-type - loci - pleiotropy
Empirical evidence is mounting to suggesting that genetic correlations between life-history traits are environment specific. However, detailed knowledge about the loci underlying genetic correlations in different environments is scant. Here, we studied the influence of temperature (12°C and 24°C) on the genetic correlations between egg size, egg number and body mass in the nematode Caenorhabditis elegans. We used a quantitative trait loci (QTL) approach based on a genetic map with evenly spaced single nucleotide polymorphism markers in an N2 CB4856 recombinant inbred panel. Significant genetic correlations between various traits were found at both temperatures. We detected pleiotropic or closely linked QTL, which supported the negative correlation between egg size and egg number at 12°C, the positive correlation across temperatures for body mass, and the positive correlation between body mass and egg size at 12°C. The results indicate that specific loci control the covariation in these life-history traits and the locus control is prone to environmental conditions
A Bsister MADS-box gene involved in ovule and seed development in petunia and Arabidopsis.
Folter, S. de; Shchennikova, A.V. ; Franken, J. ; Busscher, M. ; Baskar, R. ; Grossniklaus, U. ; Angenent, G.C. ; Immink, G.H. - \ 2006
The Plant Journal 47 (2006)6. - ISSN 0960-7412 - p. 934 - 946.
protein-protein interactions - floral organ identity - transcription factor family - flower development - wild-type - plant transformation - coat development - homeotic genes - thaliana - encodes
MADS-domain transcription factors are essential for proper flower and seed development in angiosperms and their role in determination of floral organ identity can be described by the 'ABC model' of flower development. Recently, close relatives of the B-type genes were identified by phylogenetic studies, which are referred to as Bsister (Bs) genes. Here, we report the isolation and characterization of a MADS-box Bs member from petunia, designated FBP24. An fbp24 knock-down line appeared to closely resemble the Arabidopsis Bs mutant abs and a detailed and comparative analysis led to the conclusion that both FBP24 and ABS are necessary to determine the identity of the endothelial layer within the ovule. Protein interaction studies revealed the formation of higher-order complexes between Bs¿C¿E and Bs¿D¿E type MADS-box proteins, suggesting involvement of these specific complexes in determination of endothelium identity. However, although there are many similarities between the two genes and their products and functions, interestingly FBP24 cannot replace ABS in Arabidopsis. The results presented here demonstrate the importance of the comparative analysis of key regulatory genes in various model systems to fully understand all aspects of plant development