Harry Van Onckelen

University of Antwerp, Antwerpen, Flanders, Belgium

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Publications (74)286.55 Total impact

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    ABSTRACT: The physiological state of dormancy is one of the important stages in the plant life cycle. It is accepted that ABA and GAs are among the ruling regularities of the process. Here we report for the first time of endogenous ABA levels determination in Lilium bulblets, regenerated at various conditions. An indirect ELISA was applied using monoclonal antibody validated by LC-MS. The mother bulbls tissues were with low ABA levels—a possible consiquence of the stock storing conditions. No correlation was found between development, maintainence and release of dormancy and the ABA contents of the explants as well as of the regenerated at various temperature conditions, bulblets. Dormancy of Lilium bulblets regenerated in vitro depends not only on their endogenous ABA levels butq in great extent on their ABA—and GA—sensitivity.
    Biotechnology & Biotechnological Equipment 04/2014; 8(1):30-35. DOI:10.1080/13102818.1994.10818748 · 0.38 Impact Factor
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    Biotechnology & Biotechnological Equipment 04/2014; 8(2):3-6. DOI:10.1080/13102818.1994.10818761 · 0.38 Impact Factor
  • A. Ivanova · M. Eshkenazy · H. van Onckelen
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    ABSTRACT: Direct enzyme immunoassays (ELISA) for determination of zeatin- and isopentenyladenine-cytokinins were developed. The assays are sensitive and measuring ranges extend from 0.5 to 75 pmol for zeatinriboside and from 0.5 to 50 pmol for isopentenyladenosine. The accuracy and specificity of ELISA were checked by LC-MS. Direct enzyme immunoassay (ELISA) for indole-3-acetic acid determination was developed. The method was verified by HPLC with fluorometric detection.
    Biotechnology & Biotechnological Equipment 04/2014; 8(2):7-11. DOI:10.1080/13102818.1994.10818762 · 0.38 Impact Factor
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    ABSTRACT: When transferred to the plant genome, the rolA gene of Agro- bacterium rhizogenes induces numerous changes in plant phenotype, including prominent leaf wrinkling and dwarfism. Cytological, physiological and molecular studies were carried out on tobacco plants expressing rolA, with the goal of explaining these developmental modifications.
    Acta botanica Gallica: bulletin de la Société botanique de France 04/2013; 140(6):685-691. DOI:10.1080/12538078.1993.10515646 · 0.48 Impact Factor
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    ABSTRACT: Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)-cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK-cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.
    Molecular Systems Biology 08/2010; 6:397. DOI:10.1038/msb.2010.53 · 14.10 Impact Factor
  • Luc Roef · Harry Van Onckelen
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    ABSTRACT: Contrary to the situation in animal systems, plant shape and size are predominantly determined by developmental programs that govern the timely initiation and outgrowth of meristems. In plants, meristematic zones almost exclusively constitute the regions of dividing cells. The role of mitotic cell division in morphogenetic and developmental processes has been the subject of intense debate, but the present view seems to acknowledge the importance of proper cell division regulation for the correct elaboration and execution of developmental programs (29)
    12/2009: pages 241-261;
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    ABSTRACT: While protein interaction studies and protein network modeling come to the forefront, the isolation and identification of protein complexes in a cellular context remains a major challenge for plant science. To this end, a nondenaturing extraction procedure was optimized for plant whole cell matrices and the combined use of gel filtration and BN-PAGE for the separation of protein complexes was studied. Hyphenation to denaturing electrophoresis and mass spectrometric analysis allows for the simultaneous identification of multiple (previously unidentified) protein interactions in single samples. The reliability and efficacy of the technique was confirmed (i) by the identification of well-studied plant protein complexes, (ii) by the presence of nonplant interologs for several of the novel complexes (iii) by presenting physical evidence of previously hypothetical plant protein interactions and (iv) by the confirmation of found interactions using co-IP. Furthermore practical issues concerning the use of this 2-D BN/SDS-PAGE display method for the analysis of protein–protein interactions are discussed.
    Proteomics 03/2009; 9(5):1416. DOI:10.1002/pmic.200990016 · 3.97 Impact Factor
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    ABSTRACT: The effect of ecto-nucleotide pyrophosphatase (ecto-NPPase; EC 3.6.1.9) on the ATP- and ADP-mediated receptor activation was studied in rat C6 glioma cells. The P2-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC50=12±3 μM) of the latter enzyme. 4,4′-diisothiocyanatostilbene-2,2′ disulfonic acid (DIDS), 5′-phosphoadenosine 3′-phosphate (PAP) and suramin were less potent inhibitors with an IC50 of 22±4, 36±7 and 72±11 μM respectively.P1-purinoceptor antagonists CGS 15943, cyclo-pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto-NPPase.ATP- and ADP-mediated P2Y1-like receptor activation inhibited the (−)-isoproterenol-induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y-antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto-NPPase.We conclude that ecto-NPPase has a modulatory effect on purinoceptor-mediated signalling in C6 glioma cell cultures.British Journal of Pharmacology (2000) 130, 139–145; doi:10.1038/sj.bjp.0703289
    British Journal of Pharmacology 01/2009; 130(1):139 - 145. DOI:10.1038/sj.bjp.0703289 · 4.99 Impact Factor
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    ABSTRACT: The Arabidopsis thaliana (L.) Heynh. 1-aminocyclopropane-1-carboxylate synthase gene 1 (AT-ACS1) was found to be developmentally regulated with pronounced activity in immature tissues. The effect of different hormones on this expression pattern was studied by using a transgenic line carrying the β-glucuronidase (GUS) gene under control of the AT-ACS1 promoter. In addition, the level of mRNA for AT-ACS1 was assayed in different ethylene and auxin-insensitive backgrounds, as well as in an auxin homeostasis mutant by using competitive reverse transcriptase polymerase chain reaction (RT-PCR). The results support the existence of a hormonal cross-talk involving auxin, cytokinin, and ethylene in the regulation of AT-ACS1. These hormonal controls act in a developmental and tissue-dependent manner.
    Physiologia Plantarum 09/2008; 105(2):312 - 320. DOI:10.1034/j.1399-3054.1999.105217.x · 3.26 Impact Factor
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    Phytochemistry 07/2008; 69(10):2095-2095. DOI:10.1016/j.phytochem.2008.05.001 · 3.35 Impact Factor
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    ABSTRACT: Although secondary metabolism in Nicotiana tabacum (L.) (tobacco) is rather well studied, many molecular aspects of the biosynthetic pathways and their regulation remain to be disclosed, even for prominent compounds such as nicotine and other pyridine alkaloids. To identify players in tobacco pyridine alkaloid biosynthesis a functional screen was performed, starting from a tobacco gene collection established previously by means of combined transcript profiling and metabolite analysis. First, full-length cDNA clones were isolated for 34 genes, corresponding to tobacco transcript tag sequences putatively associated with pyridine alkaloid metabolism. Full-length open reading frames were transferred to pCaMV35S-steered overexpression vectors. The effects of plant transformation with these expression cassettes on the accumulation of nicotine and other pyridine alkaloids were assessed in transgenic tobacco Bright-Yellow 2 (BY-2) cell suspensions and hairy root cultures. This screen identified potential catalysers of tobacco pyridine metabolism, amongst which a lysine decarboxylase-like gene and a GH3-like enzyme. Overexpression of the GH3-like enzyme, presumably involved in auxin homeostasis and designated NtNEG1 (Nicotiana tabacum Nicotine-Enhancing GH3 enzyme 1), increased nicotine levels in BY-2 hairy roots significantly. This study shows how functional genomics-based identification of genes potentially involved in biosynthetic pathways followed by systematic functional assays in plant cells can be used at large-scale to decipher plant metabolic networks at the molecular level.
    Phytochemistry 11/2007; 68(22-24):2773-85. DOI:10.1016/j.phytochem.2007.09.010 · 3.35 Impact Factor
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    ABSTRACT: Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.
    Molecular &amp Cellular Proteomics 07/2007; 6(7):1226-38. DOI:10.1074/mcp.M700078-MCP200 · 7.25 Impact Factor
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    ABSTRACT: Introduction of the Agrobacterium ipt gene, coding for isopentenyl transferase, under control of a tetracycline (Tc)-inducible promoter results in a very specific system in which cytokinin levels can be changed. Because Tc belongs to the group of antibiotics that affect 70S ribosomes, it is important to study the effects of Tc on untransformed plants. Although 1 mg l−1 Tc was previously reported to have no physiological effects, this study revealed several changes in hydroponically grown wild-type Nicotiana tabacum L. cv. Wisconsin. Therefore, lower Tc concentrations (0.1 and 0.2 mg l−1 Tc) were used to induce ipt-transgenic (tr) plants. Upon induction, real-time PCR analysis showed that the ipt gene was expressed several times higher in roots of tr plants, but not in leaves. Consequently, cytokinin levels were also elevated to a large extent in roots. This resulted in a disturbance of the cytokinin to auxin ratio, leading to an obstructed root growth. In leaves, no significant increase in cytokinins was observed. However, phenotypic and physiological effects, which could be attributed to cytokinin, were apparent in leaves of ipt-induced trs: chlorophyll and carotenoid content were elevated and grana stacking increased. Our study demonstrates that caution has to be taken to determine the ‘safe’ concentration of inducers when using inducible gene-expression systems.
    Physiologia Plantarum 05/2007; 130(2):290 - 300. DOI:10.1111/j.1399-3054.2007.00888.x · 3.26 Impact Factor
  • Sasha Daskalova · Alex McCormac · Nigel Scott · Harry Van Onckelen · Malcolm Elliott
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    ABSTRACT: A transgenic approach to manipulation of endosperm development has been investigated. Nicotiana tabacum cv. Xanthi, an endosperm-containing dicotyledon, has been used as a model plant and the 2.6kb wheat high molecular weight (HMW) glutenin subunit 12 promoter has been used fused either to the gus reporter gene (HMWgus construct)—to study promoter characteristics—or to the Agrobacterium ipt gene—to study the effect of cytokinin (CK) over-expression on assimilate accumulation in the seed. In transgenic tobacco the promoter:gus fusion showed that HMW is an endosperm-specific promoter with maximum expression 20days after anthesis (DAA), corresponding to the mid to late stages of seed development. Transgenic plants containing the HMWipt construct showed no morphological abnormalities but they had an average increase in seed weight and total ethanol-insoluble carbohydrates and protein content of 8.1%, 7.0% and 8.3%, respectively. SDS PAGE analysis demonstrated that the effect on protein accumulation was non-specific. The highest values of the parameters analysed correlated with moderate increases in the levels of biologically active CKs. These results suggest that ectopic expression of small amounts of CKs can be used to increase storage assimilate accumulation without a detrimental effect on development.
    Plant Growth Regulation 03/2007; 51(3):217-229. DOI:10.1007/s10725-006-9162-y · 1.63 Impact Factor
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    ABSTRACT: We report on the early response of Arabidopsis thaliana to the obligate biotrophic pathogen Plasmodiophora brassicae at the hormone and proteome level. Using a CYCB1;1::GUS construct, the re-initiation of infection-related cell division is shown from 4 days after inoculation on. Sensitivity to cytokinins and auxins as well as the endogenous hormone levels are evaluated. Both an enhanced cytokinin gene response and an accumulation of isopentenyl adenine and adenosine precede this re-initiation of cell division, whereas an enhanced auxin gene response is observed from 6 days after inoculation on. The alhl mutant, impaired in the cross talk between ethylene and auxins, is resistant to P. brassicae. A differential protein analysis of infected versus noninfected roots and hypocotyls was performed using two-dimensional gel electrophoresis and quantitative image analysis, coupled to matrix-assisted laser desorption ionization time of flight-time of flight mass spectrometry-based protein identification. Of the visualized proteins, 12% show altered abundance compared with the noninfected plants, including proteins involved in metabolism, cell defense, cell differentiation, and detoxification. Combining the hormone and proteome data, we postulate that, at the very first stages of Plasmodiophora infection, plasmodial-produced cytokinins trigger a local re-initiation of cell division in the root cortex. Consequently, a de novo meristematic area is established that acts as a sink for host-derived indole-3-acetic acid, carbohydrates, nitrogen, and energy to maintain the pathogen and to trigger gall development.
    Molecular Plant-Microbe Interactions 01/2007; 19(12):1431-43. DOI:10.1094/MPMI-19-1431 · 4.46 Impact Factor
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    ABSTRACT: Banana (Musa spp.) multiple shoot meristems are an excellent model to study the meristem proteome. Using a 2-DE protocol developed for small amounts of tissue and MS-based cross species polypeptide identification, we have revealed the meristem proteome and investigated the influence of sucrose-mediated osmotic stress in a dehydration-tolerant variety. Proteins that were significantly up- or down-regulated due to the high-sucrose treatment were classified using non-parametric univariate statistics. Our results suggest that the maintenance of an osmoprotective intracellular sucrose concentration, the enhanced expression of particular genes of the energy-conserving glycolysis and the conservation of the cell wall integrity are essential to maintain homeostasis, to acclimate and to survive dehydration. By comparing the dehydration-tolerant variety with a dehydration-sensitive variety, we were able to distinguish several genotype-specific proteins (isoforms), and could associate the dehydration-tolerant variety with proteins involved in energy metabolism (e.g., phosphoglycerate kinase, phosphoglucomutase, UDP-glucose pyrophosphorylase) and proteins that are associated with stress adaptation (e.g., OSR40-like protein, abscisic stress ripening protein-like protein). This work shows that proteome analysis can be used successfully to perform quantitative difference analysis and to characterize genetic variations in a recalcitrant crop.
    PROTEOMICS 01/2007; 7(1):92-105. DOI:10.1002/pmic.200600533 · 3.97 Impact Factor
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    ABSTRACT: pProRep is a web application integrating electrophoretic and mass spectral data from proteome analyses into a relational database. The graphical web-interface allows users to upload, analyse and share experimental proteome data. It offers researchers the possibility to query all previously analysed datasets and can visualize selected features, such as the presence of a certain set of ions in a peptide mass spectrum, on the level of the two-dimensional gel. AVAILABILITY: The pProRep package and instructions for its use can be downloaded from http://www.ptools.ua.ac.be/pProRep. The application requires a web server that runs PHP 5 (http://www.php.net) and MySQL. Some (non-essential) extensions need additional freely available libraries: details are described in the installation instructions.
    Bioinformatics 12/2006; 22(22):2838-40. DOI:10.1093/bioinformatics/btl487 · 4.62 Impact Factor
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    ABSTRACT: Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.
    Naturwissenschaften 07/2006; 93(6):278-85. DOI:10.1007/s00114-006-0098-x · 1.97 Impact Factor

Publication Stats

3k Citations
286.55 Total Impact Points

Institutions

  • 1994–2013
    • University of Antwerp
      • Department of Biology
      Antwerpen, Flanders, Belgium
  • 2004
    • University of New Hampshire
      • Department of Plant Biology
      Durham, NH, United States
  • 2002
    • Swansea University
      • National Mass Spectrometry Service Centre "EPSRC"
      Swansea, Wales, United Kingdom