Biomedical subjects

Dirk Inzé

Publications and source records attributed to Dirk Inzé.

87 records · Page 5Linked to original sources

PubMed2002

Solution structure of the single-domain prolyl cis/trans isomerase PIN1At from Arabidopsis thaliana.

The 119-amino acid residue prolyl cis/trans isomerase from Arabidopsis thaliana (PIN1At) is similar to the catalytic domain of the human hPIN1. However, PIN1At lacks the N-terminal WW domain that appears to be essential for the hPIN1 function. Here, the solution structure of PIN1At was determined by three-dimensional nuclear magnetic resonance spectroscopy. The PIN1At fold could be superimposed on that of the catalytic domain of hPIN1 and had a 19 residue flexible loop located between strand beta1 and helix alpha1. The dynamical features of this beta1/alpha1-loop, which are characteristic for a region involved in protein-protein interactions, led to exchange broadening in the NMR spectra. When sodium sulfate salt was added to the protein sample, the beta1/alpha1 loop was stabilized and, hence, a complete backbone resonance assignment was obtained. Previously, with a phospho-Cdc25 peptide as substrate, PIN1At had been shown to catalyze the phosphoserine/phosphothreonine prolyl cis/trans isomerization specifically. To map the catalytic site of PIN1At, the phospho-Cdc25 peptide or sodium sulfate salt was added in excess to the protein and chemical shift changes in the backbone amide protons were monitored in the (1)H(N)-(15)N heteronuclear single quantum coherence spectrum. The peptide caused perturbations in the loops between helix alpha4 and strand beta3, between strands beta3 and beta4, in the alpha3 helix, and in the beta1/alpha1 loop. The amide groups of the residues Arg21 and Arg22 showed large chemical shift perturbations upon phospho-Cdc25 peptide or sulfate addition. We conclude that this basic cluster formed by Arg21 and Arg22, both located in the beta1/alpha1 loop, is homologous to that found in the hPIN1 crystal structure (Arg68 and Arg69), which also is involved in sulfate ion binding. We showed that the sulfate group competed for the interaction between PIN1At and the phospho-Cdc25 peptide. In the absence of the WW domain, three hydrophobic residues (Ile33, Ile34, and Leu35) located in the long flexible loop and specific for the plant PIN-type peptidyl prolyl cis/trans isomerases (PPIases) could be an additional interaction site in PIN1At. However, phospho-peptide addition did not affect the resonances of these residues significantly. Electrostatic potential calculations revealed a negatively charged area not found in hPIN1 on the PIN1At molecular surface, which corresponds to the surface shielded by the WW domain in hPIN1. Based on our experimental results and the molecular specificities of the PIN1At enzyme, functional implications of the lack of WW domains in this plant PIN-type PPIase will be discussed.

Amino Acid Sequence↗

PubMed2002

Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor.

New plant cells arise at the meristems, where they divide a few times before they leave the cell-cycle program and start to differentiate. Here we show that the E2Fa-DPa transcription factor of Arabidopsis thaliana is a key regulator determining the proliferative status of plant cells. Ectopic expression of E2Fa induced sustained cell proliferation in normally differentiated cotyledon and hypocotyl cells. The phenotype was enhanced strongly by the co-expression of E2Fa with its dimerization partner, DPa. In endoreduplicating cells, E2Fa--DPa also caused extra DNA replication that was correlated with transcriptional induction of S phase genes. Because E2Fa--DPa transgenic plants arrested early in development, we argue that controlled exit of the cell cycle is a prerequisite for normal plant development.

Arabidopsis↗

PubMed2002

GATEWAY vectors for Agrobacterium-mediated plant transformation.

Agrobacterium tumefaciens is the preferred method for transformation of a wide range of plant species. Commonly, the genes to be transferred are cloned between the left and right T-DNA borders of so-called binary T-DNA vectors that can replicate both in E. coli and Agrobacterium. Because these vectors are generally large, cloning can be time-consuming and laborious. Recently, the GATEWAY conversion technology has provided a fast and reliable alternative to the cloning of sequences into large acceptor plasmids.

Agrobacterium tumefaciens↗

PubMed2002

Characterization of cis-acting element involved in cell cycle phase-independent activation of Arath;CycB1;1 transcription and identification of putative regulatory proteins.

Progression through the cell cycle is driven by cyclin-dependent kinases (CDKs) whose activity is controlled by regulatory subunits called cyclins. The expression of cyclins is subject to numerous controls at multiple levels, not least at the level of transcription. As a first step to unravel the mechanisms that regulate expression of B-cyclins in plants, we undertook the identification of the required promoter elements of the Arath;CycB1;1 gene. A detailed analysis of different promoter fragments consisted in analysing their ability to mediate cell cycle-dependent transcriptional oscillations of the gus reporter gene in transformed BY-2 cell lines. We showed that different promoter regions took part in transcriptional activation. Furthermore, 202 bp upstream of the ATG were sufficient to induce M-phase-specific expression. This region contains an 18 bp sequence including a Myb-binding core (AACGG) which is able to activate reporter gene without leading to M-phase-specific expression. Electrophoretic mobility shift assays showed that this 18 bp sequence specifically binds protein complexes from Arabidopsis cell suspension enriched either in G1 or G2 phase. Furthermore, the Myb core, AACGG, was characterized as necessary for the binding of proteins. DNA affinity purification of the complexes bound to the 18 bp sequence allowed the isolation of three different complexes and two proteins from these complexes were identified by mass spectrometry analyses. A new putative Myb transcription factor and a hypothetical protein, HYP containing with a leucine zipper and Myc-type dimerization domains were identified. When over-expressed in plants, HYP factor is able to trans-activate the expression of gus reporter gene downstream from the -202 promoter fragment as well as the endogenous CycB1;1 gene.

Arabidopsis↗

PubMed2002

Double antisense plants lacking ascorbate peroxidase and catalase are less sensitive to oxidative stress than single antisense plants lacking ascorbate peroxidase or catalase.

The plant genome is a highly redundant and dynamic genome. Here, we show that double antisense plants lacking the two major hydrogen peroxide-detoxifying enzymes, ascorbate peroxidase (APX) and catalase (CAT), activate an alternative/redundant defense mechanism that compensates for the lack of APX and CAT. A similar mechanism was not activated in single antisense plants that lacked APX or CAT, paradoxically rendering these plants more sensitive to oxidative stress compared to double antisense plants. The reduced susceptibility of double antisense plants to oxidative stress correlated with suppressed photosynthetic activity, the induction of metabolic genes belonging to the pentose phosphate pathway, the induction of monodehydroascorbate reductase, and the induction of IMMUTANS, a chloroplastic homologue of mitochondrial alternative oxidase. Our results suggest that a co-ordinated induction of metabolic and defense genes, coupled with the suppression of photosynthetic activity, can compensate for the lack of APX and CAT. In addition, our findings demonstrate that the plant genome has a high degree of plasticity and will respond differently to different stressful conditions, namely, lack of APX, lack of CAT, or lack of both APX and CAT.

Antisense Elements (Genetics)↗

PubMed2002

The NADPH:quinone oxidoreductase P1-zeta-crystallin in Arabidopsis catalyzes the alpha,beta-hydrogenation of 2-alkenals: detoxication of the lipid peroxide-derived reactive aldehydes.

P1-zeta-crystallin (P1-ZCr) is an oxidative stress-induced NADPH:quinone oxidoreductase in Arabidopsis thaliana, but its physiological electron acceptors have not been identified. We found that recombinant P1-ZCr catalyzed the reduction of 2-alkenals of carbon chain C(3)-C(9) with NADPH. Among these 2-alkenals, the highest specificity was observed for 4-hydroxy-(2E)-nonenal (HNE), one of the major toxic products generated from lipid peroxides. (3Z)-Hexenal and aldehydes without alpha,beta-unsaturated bonds did not serve as electron acceptors. In the 2-alkenal molecules, P1-ZCr catalyzed the hydrogenation of alpha,beta-unsaturated bonds, but not the reduction of the aldehyde moiety, to produce saturated aldehydes, as determined by gas chromatography/mass spectrometry. We propose the enzyme name NADPH:2-alkenal alpha,beta-hydrogenase (ALH). A major portion of the NADPH-dependent HNE-reducing activity in A. thaliana leaves was inhibited by the specific antiserum against P1-ZCr, indicating that the endogenous P1-ZCr protein has ALH activity. Because expression of the P1-ZCr gene in A. thaliana is induced by oxidative stress treatments, we conclude that P1-ZCr functions as a defense against oxidative stress by scavenging the highly toxic, lipid peroxide-derived alpha,beta-unsaturated aldehydes.

Alcohol Oxidoreductases↗

PubMed2002

Variation in growth rate between Arabidopsis ecotypes is correlated with cell division and A-type cyclin-dependent kinase activity.

We used a kinematic analysis to investigate the growth processes responsible for variation in primary root growth between 18 ecotypes of Arabidopsis. Root elongation rate differed 4-fold between the slowest (Landsberg erecta, 71 microm h(-1)) and fastest growing line (Wassilewskija [Ws]; 338 microm h(-1)). This difference was contributed almost equally by variations in mature cortical cell length (84 microm [Landsberg erecta] to 237 microm [Ws]) and rate of cell production (0.63 cell h(-1) [NW108] to 1.83 cell h(-1) [Ws]). Cell production, in turn, was determined by variation in cell cycle duration (19 h [Tsu] to 48 h [NW108]) and, to a lesser extent, by differences in the number of dividing cells (32 [Weiningen] to 61 [Ws]). We found no correlation between mature cell size and endoreduplication, refuting the hypothesis that the two are linked. However, there was a strong correlation between cell production rates and the activity of the cyclin-dependent kinase (CDKA). The level of the protein could explain 32% of the variation in CDKA. Therefore, it is likely that regulators of CDKA, such as cyclins and inhibitors, are also involved. These data provide a functional link between cell cycle regulation and whole-plant growth rate as affected by genetic differences.

Algorithms↗

PubMed2002

Gamma-glutamyl transpeptidase in transgenic tobacco plants. Cellular localization, processing, and biochemical properties.

gamma-Glutamyl transpeptidase (gamma-GT) is a ubiquitous enzyme that catalyzes the first step of glutathione (GSH) degradation in the gamma-glutamyl cycle in mammals. A cDNA encoding an Arabidopsis homolog for gamma-GT was overexpressed in tobacco (Nicotiana tabacum) plants. A high level of the membrane-bound gamma-GT activity was localized outside the cell in transgenic plants. The overproduced enzyme was characterized by a high affinity to GSH and was cleaved post-translationally in two unequal subunits. Thus, Arabidopsis gamma-GT is similar to the mammalian enzymes in enzymatic properties, post-translational processing, and cellular localization, suggesting analogous biological functions as a key enzyme in the catabolism of GSH.

Amino Acid Sequence↗

PubMed2002

Molecular characterization of plant ubiquitin-conjugating enzymes belonging to the UbcP4/E2-C/UBCx/UbcH10 gene family.

The anaphase promoting complex or cyclosome is the ubiquitin-ligase that targets destruction box-containing proteins for proteolysis during the cell cycle. Anaphase promoting complex or cyclosome and its activator (the fizzy and fizzy-related) proteins work together with ubiquitin-conjugating enzymes (UBCs) (E2s). One class of E2s (called E2-C) seems specifically involved in cyclin B1 degradation. Although it has recently been shown that mammalian E2-C is regulated at the protein level during the cell cycle, not much is known concerning the expression of these genes. Arabidopsis encodes two genes belonging to the E2-C gene family (called UBC19 and UBC20). We found that UBC19 is able to complement fission yeast (Schizosaccharomyces pombe) UbcP4-140 mutant, indicating that the plant protein can functionally replace its yeast ortholog for protein degradation during mitosis. In situ hybridization experiments were performed to study the expression of the E2-C genes in various tissues of plants. Their transcripts were always, but not exclusively, found in tissues active for cell division. Thus, the UBC19/20 E2s may have a key function during cell cycle, but may also be involved in ubiquitylation reactions occurring during differentiation and/or in differentiated cells. Finally, we showed that a translational fusion protein between UBC19 and green fluorescent protein localized both in the cytosol and the nucleus in stable transformed tobacco (Nicotiana tabacum cv Bright Yellow 2) cells.

Amino Acid Sequence↗

PubMed2002

Genetic complexity of cellulose synthase a gene function in Arabidopsis embryogenesis.

The products of the cellulose synthase A (CESA) gene family are thought to function as isoforms of the cellulose synthase catalytic subunit, but for most CESA genes, the exact role in plant growth is still unknown. Assessing the function of individual CESA genes will require the identification of the null-mutant phenotypes and of the gene expression profiles for each gene. Here, we report that only four of 10 CESA genes, CESA1, CESA2, CESA3, and CESA9 are significantly expressed in the Arabidopsis embryo. We further identified two new mutations in the RADIALLY SWOLLEN1 (RSW1/CESA1) gene of Arabidopsis that obstruct organized growth in both shoot and root and interfere with cell division and cell expansion already in embryogenesis. One mutation is expected to completely abolish the enzymatic activity of RSW1(CESA1) because it eliminated one of three conserved Asp residues, which are considered essential for beta-glycosyltransferase activity. In this presumed null mutant, primary cell walls are still being formed, but are thin, highly undulated, and frequently interrupted. From the heart-stage onward, cell elongation in the embryo axis is severely impaired, and cell width is disproportionally increased. In the embryo, CESA1, CESA2, CESA3, and CESA9 are expressed in largely overlapping domains and may act cooperatively in higher order complexes. The embryonic phenotype of the presumed rsw1 null mutant indicates that the RSW1(CESA1) product has a critical, nonredundant function, but is nevertheless not strictly required for primary cell wall formation.

Amino Acid Sequence↗

PubMed2002

Auxin-mediated cell cycle activation during early lateral root initiation.

Lateral root formation can be divided into two major phases: pericycle activation and meristem establishment. In Arabidopsis, the first lateral root initiation event is spatially and temporally asynchronous and involves a limited number of cells in the xylem pericycle. To study the molecular regulation during pericycle activation, we developed a lateral root-inducible system. Successive treatments with an auxin transport inhibitor and exogenous auxin were used to prevent the first formative divisions and then to activate the entire pericycle. Our morphological and molecular data show that, in this inducible system, xylem pericycle activation was synchronized and enhanced to cover the entire length of the root. The results also indicate that the inducible system can be considered a novel in planta system for the study of synchronized cell cycle reactivation. In addition, the expression patterns of Kip-Related Protein2 (KRP2) in the pericycle and its ectopic expression data revealed that the cyclin-dependent kinase inhibitor plays a significant role in the regulation of lateral root initiation. KRP2 appears to regulate early lateral root initiation by blocking the G1-to-S transition and to be regulated transcriptionally by auxin.

Arabidopsis↗

PubMed2002

Genome-wide analysis of core cell cycle genes in Arabidopsis.

Cyclin-dependent kinases and cyclins regulate with the help of different interacting proteins the progression through the eukaryotic cell cycle. A high-quality, homology-based annotation protocol was applied to determine the core cell cycle genes in the recently completed Arabidopsis genome sequence. In total, 61 genes were identified belonging to seven selected families of cell cycle regulators, for which 30 are new or corrections of the existing annotation. A new class of putative cell cycle regulators was found that probably are competitors of E2F/DP transcription factors, which mediate the G1-to-S progression. In addition, the existing nomenclature for cell cycle genes of Arabidopsis was updated, and the physical positions of all genes were compared with segmentally duplicated blocks in the genome, showing that 22 core cell cycle genes emerged through block duplications. This genome-wide analysis illustrates the complexity of the plant cell cycle machinery and provides a tool for elucidating the function of new family members in the future.

Amino Acid Sequence↗

PubMed2002

Differential effect of jasmonic acid and abscisic acid on cell cycle progression in tobacco BY-2 cells.

Environmental stress affects plant growth and development. Several plant hormones, such as salicylic acid, abscisic acid (ABA), jasmonic acid (JA), and ethylene play a crucial role in altering plant morphology in response to stress. Developmental regulation often has the cell cycle machinery among its targets. We analyzed the effect of JA and ABA on cell cycle progression in synchronized tobacco (Nicotiana tabacum) BY-2 cells. Both compounds were found to prevent DNA replication, keeping the cells in the G1 stage, when applied just before the G1/S transition. However, ABA did not have any effect on subsequent phases of the cell cycle when applied at a later stage, whereas JA effectively prevented mitosis on application during DNA synthesis. This demonstrates that JA treatment can freeze synchronized BY-2 cells in both the G1 and G2 stages of the cell cycle. Jasmonate administered after the S-phase was less effective in decreasing the mitotic index, suggesting that cell sensitivity toward JA is dependent on the cell cycle phase. In cultures detained in the G2-phase, we observed a reduced histone H1 kinase activity of kinases associated with the p13(suc1) protein.

Abscisic Acid↗

PubMed2002

Signal transduction during oxidative stress.

As an unfortunate consequence of aerobic life, active oxygen species (AOS) are formed by partial reduction of molecular oxygen. Plants possess a complex battery of enzymatic and non-enzymatic antioxidants that can protect cells from oxidative damage by scavenging AOS. It is becoming evident that AOS, which are generated during pathogen attack and abiotic stress situations, are recognized by plants as a signal for triggering defence responses. An overview of the literature is presented on the signalling role of AOS in plant defence responses, cell death, and development. Special attention is given to AOS and redox-regulated gene expression and the role of kinases and phosphatases in redox signal transduction.

Aerobiosis↗

PubMedDate not supplied

Cyclin-dependent kinase inhibitors in yeast, animals, and plants: a functional comparison.

The cell cycle is remarkably conserved in yeast, animals, and plants and is controlled by cyclin-dependent kinases (CDKs). CDK activity can be inhibited by binding of CDK inhibitory proteins, designated CKIs. Numerous studies show that CKIs are essential in orchestrating eukaryotic cell proliferation and differentiation. In yeast, animals, and plants, CKIs act as regulators of the G1 checkpoint in response to environmental and developmental cues and assist during mitotic cell cycles by inhibiting CDK activity required to arrest mitosis. Furthermore, CKIs play an important role in regulating cell cycle exit that precedes differentiation and in promoting differentiation in cooperation with transcription factors. Moreover, CKIs are essential to control CDK activity in endocycling cells. So, in yeast, animals, and plants, CKIs share many functional similarities, but their functions are adapted toward the specific needs of the eukaryote.

Animals↗