Biomedical subjects

Dirk Inzé

Publications and source records attributed to Dirk Inzé.

At least 19 recordsLinked to original sources

PubMed2007

Auxin-dependent regulation of lateral root positioning in the basal meristem of Arabidopsis.

In plants, the developmental mechanisms that regulate the positioning of lateral organs along the primary root are currently unknown. We present evidence on how lateral root initiation is controlled in a spatiotemporal manner in the model plant Arabidopsis thaliana. First, lateral roots are spaced along the main axis in a regular left-right alternating pattern that correlates with gravity-induced waving and depends on AUX1, an auxin influx carrier essential for gravitropic response. Second, we found evidence that the priming of pericycle cells for lateral root initiation might take place in the basal meristem, correlating with elevated auxin sensitivity in this part of the root. This local auxin responsiveness oscillates with peaks of expression at regular intervals of 15 hours. Each peak in the auxin-reporter maximum correlates with the formation of a consecutive lateral root. Third, auxin signaling in the basal meristem triggers pericycle cells for lateral root initiation prior to the action of INDOLE-3-ACETIC ACID14 (SOLITARY ROOT).

Arabidopsis↗

PubMed2007

Cold nights impair leaf growth and cell cycle progression in maize through transcriptional changes of cell cycle genes.

Low temperature inhibits the growth of maize (Zea mays) seedlings and limits yield under field conditions. To study the mechanism of cold-induced growth retardation, we exposed maize B73 seedlings to low night temperature (25 degrees C /4 degrees C, day/night) from germination until the completion of leaf 4 expansion. This treatment resulted in a 20% reduction in final leaf size compared to control conditions (25 degrees C/18 degrees C, day/night). A kinematic analysis of leaf growth rates in control and cold-treated leaves during daytime showed that cold nights affected both cell cycle time (+65%) and cell production (-22%). In contrast, the size of mature epidermal cells was unaffected. To analyze the effect on cell cycle progression at the molecular level, we identified through a bioinformatics approach a set of 43 cell cycle genes and analyzed their expression in proliferating, expanding, and mature cells of leaves exposed to either control or cold nights. This analysis showed that: (1) the majority of cell cycle genes had a consistent proliferation-specific expression pattern; and (2) the increased cell cycle time in the basal meristem of leaves exposed to cold nights was associated with differential expression of cell cycle inhibitors and with the concomitant down-regulation of positive regulators of cell division.

Biomechanical Phenomena↗

PubMed2007

Arabidopsis WEE1 kinase controls cell cycle arrest in response to activation of the DNA integrity checkpoint.

Upon the incidence of DNA stress, the ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signaling kinases activate a transient cell cycle arrest that allows cells to repair DNA before proceeding into mitosis. Although the ATM-ATR pathway is highly conserved over species, the mechanisms by which plant cells stop their cell cycle in response to the loss of genome integrity are unclear. We demonstrate that the cell cycle regulatory WEE1 kinase gene of Arabidopsis thaliana is transcriptionally activated upon the cessation of DNA replication or DNA damage in an ATR- or ATM-dependent manner, respectively. In accordance with a role for WEE1 in DNA stress signaling, WEE1-deficient plants showed no obvious cell division or endoreduplication phenotype when grown under nonstress conditions but were hypersensitive to agents that impair DNA replication. Induced WEE1 expression inhibited plant growth by arresting dividing cells in the G2-phase of the cell cycle. We conclude that the plant WEE1 gene is not rate-limiting for cycle progression under normal growth conditions but is a critical target of the ATR-ATM signaling cascades that inhibit the cell cycle upon activation of the DNA integrity checkpoints, coupling mitosis to DNA repair in cells that suffer DNA damage.

Aphidicolin↗

PubMed2006

Somatic cytokinesis and pollen maturation in Arabidopsis depend on TPLATE, which has domains similar to coat proteins.

TPLATE was previously identified as a potential cytokinesis protein targeted to the cell plate. Disruption of TPLATE in Arabidopsis thaliana leads to the production of shriveled pollen unable to germinate. Vesicular compartmentalization of the mature pollen is dramatically altered, and large callose deposits accumulate near the intine cell wall layer. Green fluorescent protein (GFP)-tagged TPLATE expression under the control of the pollen promoter Lat52 complements the phenotype. Downregulation of TPLATE in Arabidopsis seedlings and tobacco (Nicotiana tabacum) BY-2 suspension cells results in crooked cell walls and cell plates that fail to insert into the mother wall. Besides accumulating at the cell plate, GFP-fused TPLATE is temporally targeted to a narrow zone at the cell cortex where the cell plate connects to the mother wall. TPLATE-GFP also localizes to subcellular structures that accumulate at the pollen tube exit site in germinating pollen. Ectopic callose depositions observed in mutant pollen also occur in RNA interference plants, suggesting that TPLATE is implicated in cell wall modification. TPLATE contains domains similar to adaptin and beta-COP coat proteins. These data suggest that TPLATE functions in vesicle-trafficking events required for site-specific cell wall modifications during pollen germination and for anchoring of the cell plate to the mother wall at the correct cortical position.

Amino Acid Sequence↗

PubMed2006

AtGRP2, a cold-induced nucleo-cytoplasmic RNA-binding protein, has a role in flower and seed development.

The glycine-rich protein AtGRP2 is one of the four members of the cold-shock domain (CSD) protein family in Arabidopsis. It is characterized by the presence of a nucleic acid-binding CSD domain, two glycine-rich domains and two CCHC zinc-fingers present in nucleic acid-binding proteins. In an attempt to further understand the role of CSD/GRP proteins in plants, we have proceeded to the functional characterization of the AtGRP2 gene. Here, we demonstrate that AtGRP2 is a nucleo-cytoplasmic protein involved in Arabidopsis development with a possible function in cold-response. Expression analysis revealed that the AtGRP2 gene is active in meristematic tissues, being modulated during flower development. Down-regulation of AtGRP2 gene, using gene-silencing techniques resulted in early flowering, altered stamen number and affected seed development. A possible role of AtGRP2 as an RNA chaperone is discussed.

Arabidopsis↗

PubMed2006

Metacaspase activity of Arabidopsis thaliana is regulated by S-nitrosylation of a critical cysteine residue.

Nitric oxide (NO) regulates a number of signaling functions in both animals and plants under several physiological and pathophysiological conditions. S-Nitrosylation linking a nitrosothiol on cysteine residues mediates NO signaling functions of a broad spectrum of mammalian proteins, including caspases, the main effectors of apoptosis. Metacaspases are suggested to be the ancestors of metazoan caspases, and plant metacaspases have previously been shown to be genuine cysteine proteases that autoprocess in a manner similar to that of caspases. We show that S-nitrosylation plays a central role in the regulation of the proteolytic activity of Arabidopsis thaliana metacaspase 9 (AtMC9) and hypothesize that this S-nitrosylation affects the cellular processes in which metacaspases are involved. We found that AtMC9 zymogens are S-nitrosylated at their active site cysteines in vivo and that this posttranslational modification suppresses both AtMC9 autoprocessing and proteolytic activity. However, the mature processed form is not prone to NO inhibition due to the presence of a second S-nitrosylation-insensitive cysteine that can replace the S-nitrosylated cysteine residue within the catalytic center of the processed AtMC9. This cysteine is absent in caspases and paracaspases but is conserved in all reported metacaspases.

Amino Acid Sequence↗

PubMed2006

SIAMESE, a plant-specific cell cycle regulator, controls endoreplication onset in Arabidopsis thaliana.

Recessive mutations in the SIAMESE (SIM) gene of Arabidopsis thaliana result in multicellular trichomes harboring individual nuclei with a low ploidy level, a phenotype strikingly different from that of wild-type trichomes, which are single cells with a nuclear DNA content of approximately 16C to 32C. These observations suggested that SIM is required to suppress mitosis as part of the switch to endoreplication in trichomes. Here, we demonstrate that SIM encodes a nuclear-localized 14-kD protein containing a cyclin binding motif and a motif found in ICK/KRP (for Interactors of Cdc2 kinase/Kip-related protein) cell cycle inhibitor proteins. Accordingly, SIM was found to associate with D-type cyclins and CDKA;1. Homologs of SIM were detected in other dicots and in monocots but not in mammals or fungi. SIM proteins are expressed throughout the shoot apical meristem, in leaf primordia, and in the elongation zone of the root and are localized to the nucleus. Plants overexpressing SIM are slow-growing and have narrow leaves and enlarged epidermal cells with an increased DNA content resulting from additional endocycles. We hypothesize that SIM encodes a plant-specific CDK inhibitor with a key function in the mitosis-to-endoreplication transition.

Amino Acid Sequence↗

PubMed2006

The cyclin-dependent kinase inhibitor Orysa;KRP1 plays an important role in seed development of rice.

Kip-related proteins (KRPs) play a major role in the regulation of the plant cell cycle. We report the identification of five putative rice (Oryza sativa) proteins that share characteristic motifs with previously described plant KRPs. To investigate the function of KRPs in rice development, we generated transgenic plants overexpressing the Orysa;KRP1 gene. Phenotypic analysis revealed that overexpressed KRP1 reduced cell production during leaf development. The reduced cell production in the leaf meristem was partly compensated by an increased cell size, demonstrating the existence of a compensatory mechanism in monocot species by which growth rate is less reduced than cell production, through cell expansion. Furthermore, Orysa;KRP1 overexpression dramatically reduced seed filling. Sectioning through the overexpressed KRP1 seeds showed that KRP overproduction disturbed the production of endosperm cells. The decrease in the number of fully formed seeds was accompanied by a drop in the endoreduplication of endosperm cells, pointing toward a role of KRP1 in connecting endocycle with endosperm development. Also, spatial and temporal transcript detection in developing seeds suggests that Orysa;KRP1 plays an important role in the exit from the mitotic cell cycle during rice grain formation.

Amino Acid Sequence↗

PubMed2006

Serpin1 of Arabidopsis thaliana is a suicide inhibitor for metacaspase 9.

Metacaspases are distant relatives of animal caspases found in plants, fungi and protozoa. We demonstrated previously that two type II metacaspases of Arabidopsis thaliana, AtMC4 and AtMC9 are Arg/Lys-specific cysteine-dependent proteases. We screened a combinatorial tetrapeptide library of 130,321 substrates with AtMC9. Here, we show that AtMC9 is a strict Arg/Lys-specific protease. Based on the position-specific scoring matrix derived from the substrate library results, the tetrapeptide Val-Arg-Pro-Arg was identified as an optimized substrate. AtMC9 had a kcat/KM of 4.6x10(5) M-1 s-1 for Ac-Val-Arg-Pro-Arg-amido-4-methyl-coumarin, representing a more than 10-fold improvement over existing fluorogenic substrates. A yeast two-hybrid screen with catalytically inactive AtMC9 as bait identified a serine protease inhibitor, designated AtSerpin1, which was found to be a potent inhibitor of AtMC9 activity in vitro through cleavage of its reactive center loop and covalent binding to AtMC9. On the basis of the substrate profiling of AtMC9 and confirmation through site-directed mutagenesis, the inhibitory P4-P1 cleavage site of AtSerpin1 was determined to be Ile-Lys-Leu-Arg351. Further mutagenesis of the AtSerpin1 inhibitory cleavage site modulated AtMC9 inhibition positively or negatively. Both AtMC9 and AtSerpin1 were localized in the extracellular space, suggesting an in vivo interaction as well. To our knowledge, this is the first report of plant protease inhibition by a plant serpin.

Arabidopsis Proteins↗

PubMed2006

What if higher plants lack a CDC25 phosphatase?

Progression through the cell cycle is regulated by cyclin-dependent kinases (CDKs). Plants possess a unique class of CDKs, designated B-type CDKs, but seem to lack a functional CDC25 phosphatase, which is a crucial activator of the onset of mitosis in non-plant species. Based on a striking number of functional parallels between the Arabidopsis thaliana CDKB1;1 and the Drosophila melanogaster CDC25 (string), we hypothesize that the acquisition of B-type CDKs and the disappearance of CDC25 in plants might have been associated; in these coupled events, the CDC25-controlled onset of mitosis might have been evolutionarily replaced by a B-type CDK-dominated pathway, eventually resulting in the loss of the CDC25 gene.

Cell Cycle↗

PubMed2006

A novel role for abscisic acid emerges from underground.

The continuous formation of lateral roots is a vital part of establishing a root system and enables plants to react with developmental plasticity to changing soil conditions. Evidence is accumulating that abscisic acid (ABA), which is known to be involved in stress responses, has an important role in lateral root formation. An ABA receptor mutant, fca-1, shows an altered response to ABA during lateral root formation. Interestingly, ABA seems to have distinct roles at different stages in lateral root development. The emerging role of ABA in lateral root development fits well with its general functional properties as a stress hormone, including its role in dormancy.

Abscisic Acid↗

PubMed2006

Arabidopsis PASTICCINO2 is an antiphosphatase involved in regulation of cyclin-dependent kinase A.

PASTICCINO2 (PAS2), a member of the protein Tyr phosphatase-like family, is conserved among all eukaryotes and is characterized by a mutated catalytic site. The cellular functions of the Tyr phosphatase-like proteins are still unknown, even if they are essential in yeast and mammals. Here, we demonstrate that PAS2 interacts with a cyclin-dependent kinase (CDK) that is phosphorylated on Tyr and not with its unphosphorylated isoform. Phosphorylation of the conserved regulatory Tyr-15 is involved in the binding of CDK to PAS2. Loss of the PAS2 function dephosphorylated Arabidopsis thaliana CDKA;1 and upregulated its kinase activity. In accordance with its role as a negative regulator of the cell cycle, overexpression of PAS2 slowed down cell division in suspension cell cultures at the G2-to-M transition and early mitosis and inhibited Arabidopsis seedling growth. The latter was accompanied by altered leaf development and accelerated cotyledon senescence. PAS2 was localized in the cytoplasm of dividing cells but moved into the nucleus upon cell differentiation, suggesting that the balance between cell division and differentiation is regulated through the interaction between CDKA;1 and the antiphosphatase PAS2.

Arabidopsis↗

PubMed2006

Quantitation of artemisinin and its biosynthetic precursors in Artemisia annua L. by high performance liquid chromatography-electrospray quadrupole time-of-flight tandem mass spectrometry.

This study reports the development and validation of a rapid, sensitive and selective assay for the quantitation of artemisinin, arteannuin B, artemisitene and artemisinic acid in Artemisia annua L. by reversed phase high performance liquid chromatography (HPLC) electrospray (ESI) quadrupole time of flight (Q-TOF) tandem mass spectrometry (MS/MS). A recovery of >97% for all analytes was achieved by immersing one gram of fresh plant material in chloroform for 1 min. This result supports the hypothesis that artemisinin and some of its structural analogs present in the leaves A. annua L. are localized entirely in the subcuticular space of the glands on the surface of the leaves. We validated the use of this chloroform extract, without additional sample preparation steps, for quantitative Q-TOF MS/MS. No ion suppression (matrix effect) resulting from interference with other compounds was detected. For every concentration within the range of the standard curve (0.1 to 3.00 microg/ml), accuracy was between 85% and 115%. Within- and between-day variations for the analysis of A. annua L. samples were <20%.

Artemisia↗

PubMed2006

Nitric oxide- and hydrogen peroxide-responsive gene regulation during cell death induction in tobacco.

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are regulatory molecules in various developmental processes and stress responses. Tobacco (Nicotiana tabacum) leaves exposed to moderate high light dramatically potentiated NO-mediated cell death in catalase-deficient (CAT1AS) but not in wild-type plants, providing genetic evidence for a partnership between NO and H(2)O(2) during the induction of programmed cell death. With this experimental model system, the specific impact on gene expression was characterized by either NO or H(2)O(2) alone or both molecules combined. By means of genome-wide cDNA-amplified fragment length polymorphism analysis, transcriptional changes were compared in high light-treated CAT1AS and wild-type leaves treated with or without the NO donor sodium nitroprusside. Differential gene expression was detected for 214 of the approximately 8,000 transcript fragments examined. For 108 fragments, sequence analysis revealed homology to genes with a role in signal transduction, defense response, hormone interplay, proteolysis, transport, and metabolism. Surprisingly, only 16 genes were specifically induced by the combined action of NO and H(2)O(2), whereas the majority were regulated by either of them alone. At least seven transcription factors were mutually up-regulated, indicating significant overlap between NO and H(2)O(2) signaling pathways. These results consolidate significant cross-talk between NO and H(2)O(2), provide new insight into the early transcriptional response of plants to increased NO and H(2)O(2) levels, and identify target genes of the combined action of NO and H(2)O(2) during the induction of plant cell death.

Base Sequence↗

PubMed2006

Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis.

Reactive oxygen species (ROS) are key players in the regulation of plant development, stress responses, and programmed cell death. Previous studies indicated that depending on the type of ROS (hydrogen peroxide, superoxide, or singlet oxygen) or its subcellular production site (plastidic, cytosolic, peroxisomal, or apoplastic), a different physiological, biochemical, and molecular response is provoked. We used transcriptome data generated from ROS-related microarray experiments to assess the specificity of ROS-driven transcript expression. Data sets obtained by exogenous application of oxidative stress-causing agents (methyl viologen, Alternaria alternata toxin, 3-aminotriazole, and ozone) and from a mutant (fluorescent) and transgenic plants, in which the activity of an individual antioxidant enzyme was perturbed (catalase, cytosolic ascorbate peroxidase, and copper/zinc superoxide dismutase), were compared. In total, the abundance of nearly 26,000 transcripts of Arabidopsis (Arabidopsis thaliana) was monitored in response to different ROS. Overall, 8,056, 5,312, and 3,925 transcripts showed at least a 3-, 4-, or 5-fold change in expression, respectively. In addition to marker transcripts that were specifically regulated by hydrogen peroxide, superoxide, or singlet oxygen, several transcripts were identified as general oxidative stress response markers because their steady-state levels were at least 5-fold elevated in most experiments. We also assessed the expression characteristics of all annotated transcription factors and inferred new candidate regulatory transcripts that could be responsible for orchestrating the specific transcriptomic signatures triggered by different ROS. Our analysis provides a framework that will assist future efforts to address the impact of ROS signals within environmental stress conditions and elucidate the molecular mechanisms of the oxidative stress response in plants.

Arabidopsis↗

PubMed2006

Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells.

Rational engineering of complicated metabolic networks involved in the production of biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Whereas comprehensive genome-wide functional genomics approaches can be successfully applied to analyze a select number of model plants, these holistic approaches are not yet available for the study of nonmodel plants that include most, if not all, medicinal plants. We report here a comprehensive profiling analysis of the Madagascar periwinkle (Catharanthus roseus), a source of the anticancer drugs vinblastine and vincristine. Genome-wide transcript profiling by cDNA-amplified fragment-length polymorphism combined with metabolic profiling of elicited C. roseus cell cultures yielded a collection of known and previously undescribed transcript tags and metabolites associated with terpenoid indole alkaloids. Previously undescribed gene-to-gene and gene-to-metabolite networks were drawn up by searching for correlations between the expression profiles of 417 gene tags and the accumulation profiles of 178 metabolite peaks. These networks revealed that the different branches of terpenoid indole alkaloid biosynthesis and various other metabolic pathways are subject to differing hormonal regulation. These networks also served to identify a select number of genes and metabolites likely to be involved in the biosynthesis of terpenoid indole alkaloids. This study provides the basis for a better understanding of periwinkle secondary metabolism and increases the practical potential of metabolic engineering of this important medicinal plant.

Catharanthus↗

PubMed2006

Mitosis-specific kinesins in Arabidopsis.

Kinesins are a class of microtubule-associated proteins that possess a motor domain for binding to microtubules and, in general, allows movement along microtubules. In animal mitosis, they function in spindle formation, chromosome movement and in cytokinesis. In addition to the spindle, plants develop a preprophase band and a phragmoplast that might require multiple kinesins for construction and functioning. Indeed, several kinesins play a role in phragmoplast and cell plate dynamics. Surprisingly few kinesins have been associated with the spindle and the preprophase band. Analysis of expression datasets from synchronized cell cultures indicate that at least 23 kinesins are in some way implicated in mitosis-related processes. In this review, the function of kinesins in animal and plant mitoses are compared, and the divergence that originates from plant-specific aspects is highlighted.

Animals↗

PubMed2006

Cell cycle-dependent targeting of a kinesin at the plasma membrane demarcates the division site in plant cells.

Eukaryotic cells have developed different mechanisms to establish the division plane. In plants, the position is determined before the onset of mitosis by the preprophase band (PPB). This ring of microtubules surrounds the nucleus and disappears completely by prometaphase. An unknown marker is left behind by the PPB, providing the necessary spatial cues during cytokinesis. At the position of the PPB, cortical actin is removed or modified to generate an actin-depleted zone that was proposed to provide the structural means for phragmoplast guidance. Here, we identify a plasma membrane domain that emerges at the onset of mitosis and persists until the end of cytokinesis. The narrow band in the plasma membrane corresponds to the position of the PPB and is prevented from accumulation of a GFP-tagged kinesin GFP-KCA1; hence, it is called the KCA-depleted zone (KDZ). The KDZ demarcates the cortical division site independent from the mitotic cytoskeleton. Cell divisions in the absence of a KDZ resulted in misplaced cell plates, suggesting that the PPB transmits a signal to the plasma membrane required for correct cell plate guidance and vesicular targeting to the cortical division site.

Arabidopsis↗