Biomedical subjects

Harry Van Onckelen

Publications and source records attributed to Harry Van Onckelen.

At least 19 recordsLinked to original sources

PubMed2007

Banana (Musa spp.) as a model to study the meristem proteome: acclimation to osmotic stress.

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.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Integration of gel-based proteome data with pProRep.

UNLABELLED: 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.

Computational Biology↗

PubMed2006

A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells.

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.

Amino Acid Sequence↗

PubMed2006

Zeatin accumulation and misexpression of a class I knox gene are intimately linked in the epiphyllous response of the interspecific hybrid EMB-2 (Helianthus annuus x H. tuberosus).

Epiphylly, occurring in a somaclonal variant (EMB-2) of the interspecific hybrid Helianthus annuus x H. tuberosus, was used to investigate molecular and cyto-physiological mechanisms that underlie cellular fate change. EMB-2 plants are characterized by profuse proliferation of shoot- and embryo-like structures on some leaves. We addressed the putative relationship between cytokinins and knox genes in EMB-2 plants. A class I knox gene, HtKNOT1, was isolated from H. tuberosus. A high level of HtKNOT1 transcripts was detected in EMB-2 epiphyllous leaves compared to non-epiphyllous (NEP) ones. In addition, epiphylly was related to a localized increases in zeatin and N-glycosylated cytokinins. As ectopic morphogenesis proceeded, HtKNOT1 transcripts and zeatin co-localized and showed different patterns in ectopic shoot compared with embryo-like structures, consistent with the differential role of both cytokinin and knox genes in the two morphogenetic events. Notably, a massive shoot/embryo regeneration was induced in EMB-2 NEP leaves by in vitro zeatin treatment. These results clearly indicate that localized cytokinin accumulation and ectopic expression of HtKNOT1 are closely linked in the epiphylly of EMB-2 plants.

Amino Acid Sequence↗

PubMed2006

A hormone and proteome approach to picturing the initial metabolic events during Plasmodiophora brassicae infection on Arabidopsis.

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.

Arabidopsis↗

PubMed2005

A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis.

The changes in root system architecture (RSA) triggered by phosphate (P) deprivation were studied in Arabidopsis (Arabidopsis thaliana) plants grown for 14 d on 1 mM or 3 microM P. Two different temporal phases were observed in the response of RSA to low P. First, lateral root (LR) development was promoted between days 7 and 11 after germination, but, after day 11, all root growth parameters were negatively affected, leading to a general reduction of primary root (PR) and LR lengths and of LR density. Low P availability had contrasting effects on various stages of LR development, with a marked inhibition of primordia initiation but a strong stimulation of activation of the initiated primordia. The involvement of auxin signaling in these morphological changes was investigated in wild-type plants treated with indole-3-acetic acid or 2,3,5-triiodobenzoic acid and in axr4-1, aux1-7, and eir1-1 mutants. Most effects of low P on RSA were dramatically modified in the mutants or hormone-treated wild-type plants. This shows that auxin plays a major role in the P starvation-induced changes of root development. From these data, we hypothesize that several aspects of the RSA response to low P are triggered by local modifications of auxin concentration. A model is proposed that postulates that P starvation results in (1) an overaccumulation of auxin in the apex of the PR and in young LRs, (2) an overaccumulation of auxin or a change in sensitivity to auxin in the lateral primordia, and (3) a decrease in auxin concentration in the lateral primordia initiation zone of the PR and in old laterals. Measurements of local changes in auxin concentrations induced by low P, either by direct quantification or by biosensor expression pattern (DR5::beta-glucuronidase reporter gene), are in line with these hypotheses. Furthermore, the observation that low P availability mimicked the action of auxin in promoting LR development in the alf3 mutant confirmed that P starvation stimulates primordia emergence through increased accumulation of auxin or change in sensitivity to auxin in the primordia. Both the strong effect of 2,3,5-triiodobenzoic acid and the phenotype of the auxin-transport mutants (aux1, eir1) suggest that low P availability modifies local auxin concentrations within the root system through changes in auxin transport rather than auxin synthesis.

Arabidopsis↗

PubMed2005

Identification of four adenosine kinase isoforms in tobacco By-2 cells and their putative role in the cell cycle-regulated cytokinin metabolism.

Adenosine kinase (ADK), a key enzyme in the regulation of the intracellular level of adenosine is also speculated to be responsible for the conversion of cytokinin ribosides to their respective nucleotides. To elucidate the role of ADK in the cytokinin metabolism of tobacco BY-2 cells (Nicotiana tabacum cv. "Bright Yellow-2"; TBY-2), we have identified and characterized the full-length cDNAs encoding four ADK isoforms of N. tabacum and determined their catalytic properties. The four TBY-2 ADK isoforms (designated 1S, 2S, 1T, and 2T) display a high affinity for both adenosine (Km 1.88-7.30 microm) and three distinct types of cytokinin ribosides: isopentenyladenosine; zeatin riboside; and dihydrozeatin riboside (Km 0.30-8.71 microm). The Vmax/Km values suggest that ADK2S exhibits in vitro an overall higher efficiency in the metabolism of cytokinin ribosides than the other three isoforms. The expression pattern of NtADK genes is modulated significantly during the cell cycle. We suggest that the increased transcript accumulation of NtADK coupled to an increased ADK activity just prior to mitosis is associated with a very active cytokinin metabolism at that phase of the cell cycle of synchronized TBY-2 cells.

Adenosine↗

PubMed2005

Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle.

The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants. In Spcdc25-expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in early S phase but thereafter remained lower. Spcdc25-expressing cells also bypassed a block on G2/M imposed by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels in Spcdc25-expressing cells compared with the empty vector, explaining why these cells retained mitotic competence despite the presence of LVS. In conclusion, synchronised Spcdc25-expressing BY-2 cells divided prematurely at a small cell size, and they exhibited premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable.

Aphidicolin↗

PubMed2004

Jasmonic acid prevents the accumulation of cyclin B1;1 and CDK-B in synchronized tobacco BY-2 cells.

Jasmonic acid (JA) plays a crucial role in plant fertility and defense responses. It exerts an inhibitory effect on plant growth when applied exogenously. This effect seems to be somehow related to a negative regulation of cell cycle progression in the meristematic tissues. In this report, we focus on the molecular events that occur during JA-induced G2 arrest. We demonstrate that JA prevents the accumulation of B-type cyclin-dependent kinases and the expression of cyclin B1;1, which are both essential for the initiation of mitosis. This feature suggests the existence of an early G2 checkpoint that is affected by JA.

Aphidicolin↗

PubMed2004

Metabolic fate of jasmonates in tobacco bright yellow-2 cells.

Jasmonic acid and methyl jasmonate play an essential role in plant defense responses and pollen development. Their levels are temporarily and spatially controlled in plant tissue. However, whereas jasmonate biosynthesis is well studied, metabolic pathways downstream of jasmonic acid are less understood. We studied the uptake and metabolism of jasmonic acid and methyl jasmonate in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture. We found that upon uptake, jasmonic acid was metabolized to its Glc and gentiobiose esters, and hydroxylation at C-11 or C-12 occurred. Free hydroxylated jasmonates were the preferential fraction of the culture medium. Upon hydrolysis of methyl jasmonate to jasmonic acid, a similar set of conversions occurs. In contrast to jasmonic acid, none of its derivatives interfere with the G2/M transition in synchronized tobacco Bright Yellow-2 cells.

Acetates↗

PubMed2004

Construction of a two-dimensional gel electrophoresis protein database for the Nicotiana tabacum cv. Bright Yellow-2 cell suspension culture.

Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2). The BY-2 cell suspension culture is widely used as a model system to study the growth and development of plant cells. We present a protocol describing the sample preparation and 2-DE, enabling us to separate and display more than 1000 proteins from this cell culture. A reference gel was generated, using immobilized pH gradient isoelectric focusing in a linear gradient from pH 3 to 10 and 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the tobacco genome is not sequenced yet, a range of protein spots from this reference map was identified by means of a semi-automated liquid chromatography-ESI-quadrupole time of flight-tandem MS (LC-ESI-QTOF-MS-MS) setup and cross-species matching. These data were integrated in a database, which can be accessed at http://tby2-www.uia.ac.be/tby2/. On the on-line reference map, the identified protein spots are hyperlinked to individual protein entries. Each protein entry contains all identification information, as well as links to relevant entries in other on-line databases. Comprehensive search functions are implemented. Especially for an unsequenced but widespread model organism like tobacco BY-2, such a reference database is a convenient source for protein information that brings protein identification within reach without the need for extensive MS. This publicly accessible database provides a solid basis for tobacco BY-2 proteomics in the future.

Databases as Topic↗

PubMed2004

Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum.

In view of the ongoing debate on plant cyclic nucleotide metabolism, especially the functional presence of adenylyl cyclase, a novel detection method has been worked out to quantify the reaction product. Using uniformly labelled (15)N-ATP as a substrate for adenylyl cyclase, a qualitative and quantitative liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method was developed to measure de novo formed (15)N-adenosine 3',5'-cyclic monophosphate. Adenylyl cyclase activity was observed in chloroplasts obtained from Nicotiana tabacum cv. Petit Havana and the kinetic parameters and influence of various metabolic effectors are discussed in their context.

Adenosine Triphosphate↗

PubMed2003

Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity.

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.

Arabidopsis↗

PubMed2003

Cytokinin levels in leaves, leaf exudate and shoot apical meristem of Arabidopsis thaliana during floral transition.

Understanding the complete picture of floral transition is still impaired by the fact that physiological studies mainly concern plant species whose genetics is poorly known, and vice versa. Arabidopsis thaliana has been successfully used to unravel signalling pathways by genetic and molecular approaches, but analyses are still required to determine the physiological signals involved in the control of floral transition. In this work, the putative role of cytokinins was investigated using vegetative plants of Arabidopsis (Columbia) induced to flower synchronously by a single 22 h long day. Cytokinins were analysed in leaf extracts, leaf phloem exudate and in the shoot apical meristem at different times during floral transition. It was found that, in both the leaf tissues and leaf exudate, isopentenyladenine forms of cytokinins increased from 16 h after the start of the long day. At 30 h, the shoot apical meristem of induced plants contained more isopentenyladenine and zeatin than vegetative controls. These cytokinin increases correlate well with the early events of floral transition.

Arabidopsis↗

PubMed2003

Cytokinin affinity purification and identification of a tobacco BY-2 adenosine kinase.

Adenosine kinase is one of the enzymes potentially responsible for the formation of cytokinin nucleotides in plants. Using a zeatin affinity column a 40 kDa protein was isolated from tobacco Bright Yellow 2 (TBY-2) and identified by mass spectrometry as adenosine kinase. The ligand interaction reported here can be disrupted by several other adenine- but not guanine-based purine derivatives. The observed interaction with cytokinins is discussed in view of a putative role for adenosine kinase in TBY-2 cytokinin metabolism. The presented results show for the first time a plant adenosine kinase affinity-purified to homogeneity that was identified by primary structure analysis.

Adenosine Kinase↗

PubMed2003

Fast liquid chromatography coupled to electrospray tandem mass spectrometry peptide sequencing for cross-species protein identification.

Using a parallel microcolumn switching liquid chromatography set-up coupled to a quadrupole time-of-flight mass spectrometer, a rapid liquid chromatography/mass spectrometric (LC/MS) protein identification method is presented. Without prior sample clean-up up to 300 protein digest samples a day can be processed. Using data-directed acquisition, up to 10 fragmentation analyses for each protein sample can be acquired in the same chromatographic run that can be used for database searching. Using internal peptide sequence information, protein databases and the various nucleic acid databases can both be queried for cross-species identification of the protein sample. The method was evaluated and put into force to generate data for a tobacco cell culture protein database.

Cell Line↗

PubMed2003

New cytokinin metabolites in IPT transgenic Arabidopsis thaliana plants.

Two novel cytokinin metabolites were identified in transgenic Arabidopsis thaliana (L.) Heynh. plants containing the bacterial IPT gene under the transcriptional control of a heat-regulated promoter. After cyclic heat-shock treatment, the endogenous cytokinin concentrations were elevated up to 100-fold compared to the wild-type plants. More then 20 different cytokinin metabolites were found, with zeatin-type cytokinins being the most abundant. The metabolic inactivation of these compounds occurred predominantly through N-glucosylation. No significant accumulation of isopentenyladenine-type cytokinins, and only a small increase in dihydrozeatin metabolites, was observed. Subsequent studies of the abundant, unidentified conjugates revealed the presence of zeatin and dihydrozeatin diglucoside conjugates. Structural analysis, utilizing electrospray-liquid tandem mass spectrometry, identified these as a zeatin-O-glucoside-9-glucoside and dihydrozeatin-O-glucoside-9-glucoside, respectively. A third unknown metabolite, was tentatively identified as a phosphorylated form of zeatin-9-glucoside. The biological activity of these compounds in three cytokinin bioassays was low. A comparison of the cytokinin pattern in transgenic and wild-type plants indicates that these specific metabolites accumulate as a consequence of enhanced cytokinin biosynthesis, and are probably involved in the homeostatic mechanisms that control endogenous cytokinin levels.

Journal Article↗

PubMed2003

The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin.

Ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) can stimulate hypocotyl elongation in light-grown Arabidopsis seedlings. A mutant, designated ACC-related long hypocotyl 1 (alh1), that displayed a long hypocotyl in the light in the absence of the hormone was characterized. Etiolated alh1 seedlings overproduced ethylene and had an exaggerated apical hook and a thicker hypocotyl, although no difference in hypocotyl length was observed when compared with wild type. Alh1 plants were less sensitive to ethylene, as reflected by reduction of ACC-mediated inhibition of hypocotyl growth in the dark and delay in flowering and leaf senescence. Alh1 also had an altered response to auxin, whereas auxin levels in whole alh1 seedlings remained unaffected. In contrast to wild type, alh1 seedlings showed a limited hypocotyl elongation when treated with indole-3-acetic acid. Alh1 roots had a faster response to gravity. Furthermore, the hypocotyl elongation of alh1 and of ACC-treated wild type was reverted by auxin transport inhibitors. In addition, auxin up-regulated genes were ectopically expressed in hypocotyls upon ACC treatment, suggesting that the ethylene response is mediated by auxins. Together, these data indicate that alh1 is altered in the cross talk between ethylene and auxins, probably at the level of auxin transport.

Amino Acids, Cyclic↗