Biomedical subjects

Dirk Inzé

Publications and source records attributed to Dirk Inzé.

At least 37 records · Page 2Linked to original sources

PubMed2006

Cyclin-dependent kinase (CDK) inhibitors regulate the CDK-cyclin complex activities in endoreduplicating cells of developing tomato fruit.

The jelly-like locular (gel) tissue of tomato fruit is made up of large thin-walled and highly vacuolized cells. The development of the gel tissue is characterized by the arrest of mitotic activities, the inhibition of cyclin-dependent kinase A (CDKA) activity, and numerous rounds of nuclear DNA endoreduplication. To decipher the molecular determinants controlling these developmental events, we investigated the putative involvement of CDK inhibitors (p27(Kip)-related proteins, or KRPs) during the endoreduplication process. Two cDNAs, LeKRP1 and LeKRP2, encoding tomato CDK inhibitors were isolated. The LeKRP1 and LeKRP2 transcript expression was shown to be enhanced in the differentiating cells of the gel undergoing endoreduplication. At the translational level, LeKRP1 was shown to accumulate in the gel tissue and to participate in the inhibition of the CDK-cyclin kinase activities occurring in endoreduplicating cells of the gel tissue. We here propose that LeKRP1 participates in the control of both the cell cycle and the endoreduplication cycle.

Amino Acid Sequence↗

PubMed2006

Lateral root initiation or the birth of a new meristem.

Root branching happens through the formation of new meristems out of a limited number of pericycle cells inside the parent root. As opposed to shoot branching, the study of lateral root formation has been complicated due to its internal nature, and a lot of questions remain unanswered. However, due to the availability of new molecular tools and more complete genomic data in the model species Arabidopsis, the probability to find new and crucial elements in the lateral root formation pathway has increased. Increasingly more data are supporting the idea that lateral root founder cells become specified in young root parts before differentiation is accomplished. Next, pericycle founder cells undergo anticlinal asymmetric, divisions followed by an organized cell division pattern resulting in the formation of a new organ. The whole process of cell cycle progression and stimulation of the molecular pathway towards lateral root initiation is triggered by the plant hormone auxin. In this review, we aim to give an overview on the developmental events taking place from the very early specification of founder cells in the pericycle until the first anticlinal divisions by combining the knowledge originating from classical physiology studies with new insights from genetic-molecular analyses. Based on the current knowledge derived from recent genetic and developmental studies, we propose here a hypothetical model for LRI.

Arabidopsis↗

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↗

PubMed2006

Cell cycle regulation in plant development.

Cell cycle regulation is of pivotal importance for plant growth and development. Although plant cell division shares basic mechanisms with all eukaryotes, plants have evolved novel molecules orchestrating the cell cycle. Some regulatory proteins, such as cyclins and inhibitors of cyclin-dependent kinases, are particularly numerous in plants, possibly reflecting the remarkable ability of plants to modulate their postembryonic development. Many plant cells also can continue DNA replication in the absence of mitosis, a process known as endoreduplication, causing polyploidy. Here, we review the molecular mechanisms that regulate cell division and endoreduplication and we discuss our understanding, albeit very limited, on how the cell cycle is integrated with plant development.

Arabidopsis Proteins↗

PubMed2005

Protection against photooxidative injury of tobacco leaves by 2-alkenal reductase. Detoxication of lipid peroxide-derived reactive carbonyls.

Degradation of lipid peroxides leads to the formation of cytotoxic 2-alkenals and oxenes (collectively designated reactive carbonyls). The novel NADPH-dependent oxidoreductase 2-alkenal reductase (AER; EC 1.3.1.74) from Arabidopsis (Arabidopsis thaliana), which is encoded by the gene At5g16970, catalyzes the reduction of the alpha,beta-unsaturated bond of reactive carbonyls, and hence is presumed to function in antioxidative defense in plants. Here we show that Arabidopsis AER (At-AER) has a broad substrate spectrum to biologically relevant reactive carbonyls. Besides 2-alkenals, the enzyme recognized as substrates the lipid peroxide-derived oxenes 9-oxo-octadeca-(10E),(12Z)-dienoic acid and 13-oxo-octadeca-(9E),(11Z)-dienoic acid, as well as the potent genotoxin 4-oxo-(2E)-nonenal, altogether suggesting AER has a key role in the detoxification of reactive carbonyls. To validate this conclusion by in vivo studies, transgenic tobacco (Nicotiana tabacum) plants that had 100- to 250-fold higher AER activity levels than control plants were generated. The engineered plants exhibited significantly less damage from either (1) the exogenously administered 4-hydroxy-(2E)-nonenal, (2) treatment with methyl viologen plus light, or (3) intense light. We further show that the At-AER protein fused with the Aequorea victoria green fluorescent protein localizes in cytosol and the nucleus in Bright-Yellow 2 cells. These results indicate that reactive carbonyls mediate photooxidative injury in leaf cells, and At-AER in the cytosol protects the cells by reducing the alpha,beta-unsaturated bond of the photoproduced reactive carbonyls.

Base Sequence↗

PubMed2005

The Arabidopsis leaf as a model system for investigating the role of cell cycle regulation in organ growth.

The role of cell cycle regulation and cell division in plant growth and organ development is controversial. Some experimental data are most easily interpreted from the 'cellular perspective' that cell division drives growth, whereas other observations are more consistent with the 'organismal perspective' that cell division is merely a consequence of growth, and to a large extent facultative. Here we develop a model of cell cycle regulation in the context of leaf development based on literature, published kinematic analysis, flow-cytometric and transcriptomic data obtained from growing Arabidopsis leaves. We tested this model by comparing the in silico inhibition of the cell cycle progression with the experimental observations of transgenic plants overexpressing the cell cycle inhibitor Arath;KRP2. The model simulates the behaviour of proliferating cells quite well, but is inadequate in describing the effects on expanding cells. This may point to a difference in the nature of the expansion process during the proliferating and non-dividing phase of leaf development.

Arabidopsis↗

PubMed2005

Cell cycle progression in the pericycle is not sufficient for SOLITARY ROOT/IAA14-mediated lateral root initiation in Arabidopsis thaliana.

To study the mechanisms behind auxin-induced cell division, lateral root initiation was used as a model system. By means of microarray analysis, genome-wide transcriptional changes were monitored during the early steps of lateral root initiation. Inclusion of the dominant auxin signaling mutant solitary root1 (slr1) identified genes involved in lateral root initiation that act downstream of the auxin/indole-3-acetic acid (AUX/IAA) signaling pathway. Interestingly, key components of the cell cycle machinery were strongly defective in slr1, suggesting a direct link between AUX/IAA signaling and core cell cycle regulation. However, induction of the cell cycle in the mutant background by overexpression of the D-type cyclin (CYCD3;1) was able to trigger complete rounds of cell division in the pericycle that did not result in lateral root formation. Therefore, lateral root initiation can only take place when cell cycle activation is accompanied by cell fate respecification of pericycle cells. The microarray data also yielded evidence for the existence of both negative and positive feedback mechanisms that regulate auxin homeostasis and signal transduction in the pericycle, thereby fine-tuning the process of lateral root initiation.

Arabidopsis↗

PubMed2005

Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis.

In plants, reactive oxygen species and, more particularly, hydrogen peroxide (H(2)O(2)) play a dual role as toxic by-products of normal cell metabolism and as regulatory molecules in stress perception and signal transduction. Peroxisomal catalases are an important sink for photorespiratory H(2)O(2). Using ATH1 Affymetrix microarrays, expression profiles were compared between control and catalase-deficient Arabidopsis (Arabidopsis thaliana) plants. Reduced catalase levels already provoked differences in nuclear gene expression under ambient growth conditions, and these effects were amplified by high light exposure in a sun simulator for 3 and 8 h. This genome-wide expression analysis allowed us to reveal the expression characteristics of complete pathways and functional categories during H(2)O(2) stress. In total, 349 transcripts were significantly up-regulated by high light in catalase-deficient plants and 88 were down-regulated. From this data set, H(2)O(2) was inferred to play a key role in the transcriptional up-regulation of small heat shock proteins during high light stress. In addition, several transcription factors and candidate regulatory genes involved in H(2)O(2) transcriptional gene networks were identified. Comparisons with other publicly available transcriptome data sets of abiotically stressed Arabidopsis revealed an important intersection with H(2)O(2)-deregulated genes, positioning elevated H(2)O(2) levels as an important signal within abiotic stress-induced gene expression. Finally, analysis of transcriptional changes in a combination of a genetic (catalase deficiency) and an environmental (high light) perturbation identified a transcriptional cluster that was strongly and rapidly induced by high light in control plants, but impaired in catalase-deficient plants. This cluster comprises the complete known anthocyanin regulatory and biosynthetic pathway, together with genes encoding unknown proteins.

Anthocyanins↗

PubMed2005

Genome-wide identification of potential plant E2F target genes.

Entry into the S phase of the cell cycle is controlled by E2F transcription factors that induce the transcription of genes required for cell cycle progression and DNA replication. Although the E2F pathway is highly conserved in higher eukaryotes, only a few E2F target genes have been experimentally validated in plants. We have combined microarray analysis and bioinformatics tools to identify plant E2F-responsive genes. Promoter regions of genes that were induced at the transcriptional level in Arabidopsis (Arabidopsis thaliana) seedlings ectopically expressing genes for the E2Fa and DPa transcription factors were searched for the presence of E2F-binding sites, resulting in the identification of 181 putative E2F target genes. In most cases, the E2F-binding element was located close to the transcription start site, but occasionally could also be localized in the 5' untranslated region. Comparison of our results with available microarray data sets from synchronized cell suspensions revealed that the E2F target genes were expressed almost exclusively during G1 and S phases and activated upon reentry of quiescent cells into the cell cycle. To test the robustness of the data for the Arabidopsis E2F target genes, we also searched for the presence of E2F-cis-acting elements in the promoters of the putative orthologous rice (Oryza sativa) genes. Using this approach, we identified 70 potential conserved plant E2F target genes. These genes encode proteins involved in cell cycle regulation, DNA replication, and chromatin dynamics. In addition, we identified several genes for potentially novel S phase regulatory proteins.

Arabidopsis↗

PubMed2005

Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions.

Vitamin C (L-ascorbic acid) is an important primary metabolite of plants that functions as an antioxidant, an enzyme cofactor, and a cell-signalling modulator in a wide array of crucial physiological processes, including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, and growth. Plants synthesize ascorbic acid via de novo and salvage pathways, but the regulation of its biosynthesis and the mechanisms behind ascorbate homeostasis are largely unknown. Jasmonic acid and its methyl ester (jasmonates) mediate plant responses to many biotic and abiotic stresses by triggering a transcriptional reprogramming that allows cells to cope with pathogens and stress. By using 14C-mannose radiolabelling combined with HPLC and transcript profiling analysis, it is shown that methyl jasmonate treatment increases the de novo synthesis of ascorbic acid in Arabidopsis and tobacco Bright Yellow-2 (BY-2) suspension cells. In BY-2 cells, this stimulation coincides with enhanced transcription of at least two late methyl jasmonate-responsive genes encoding enzymes for vitamin C biosynthesis: the GDP-mannose 3'',5''-epimerase and a putative L-gulono-1,4-lactone dehydrogenase/oxidase. As far as is known, this is the first report of a hormonal regulation of vitamin C biosynthesis in plants. Finally, the role of ascorbic acid in jasmonate-regulated stress responses is reviewed.

Acetates↗

PubMed2005

The role of the Arabidopsis E2FB transcription factor in regulating auxin-dependent cell division.

The molecular mechanisms by which the phytohormone auxin coordinates cell division with cell growth and differentiation are largely unknown. Here, we show that in Arabidopsis thaliana E2FB, accumulation and stability are positively regulated by auxin. Coexpression of E2FB, but not of E2FA, with its dimerization partner A, stimulated cell proliferation in the absence of auxin in tobacco (Nicotiana tabacum) Bright Yellow-2 cells. E2FB regulated the entry into both S- and M-phases, the latter corresponding to the activation of a plant-specific mitotic regulator, CDKB1;1. Increased E2FB levels led to shortened cell cycle duration, elevated cell numbers, and extremely small cell sizes. In the absence of auxin, cells elongated with concomitant increase in their ploidy level, but both were strongly inhibited by E2FB. We conclude that E2FB is one of the key targets for auxin to determine whether cells proliferate or whether they exit the cell cycle, enlarge, and endoreduplicate their DNA.

Arabidopsis↗

PubMed2005

Fatty acid hydroperoxides and H2O2 in the execution of hypersensitive cell death in tobacco leaves.

We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species (ROS)-mediated lipid peroxidation from 9- and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H(2)O(2)-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H(2)O(2) plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H(2)O(2) and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H(2)O(2) and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.

Catalase↗

PubMed2005

The elongata mutants identify a functional Elongator complex in plants with a role in cell proliferation during organ growth.

The key enzyme for transcription of protein-encoding genes in eukaryotes is RNA polymerase II (RNAPII). The recruitment of this enzyme during transcription initiation and its passage along the template during transcription elongation is regulated through the association and dissociation of several complexes. Elongator is a histone acetyl transferase complex, consisting of six subunits (ELP1-ELP6), that copurifies with the elongating RNAPII in yeast and humans. We demonstrate that point mutations in three Arabidopsis thaliana genes, encoding homologs of the yeast Elongator subunits ELP1, ELP3 (histone acetyl transferase), and ELP4 are responsible for the phenotypes of the elongata2 (elo2), elo3, and elo1 mutants, respectively. The elo mutants are characterized by narrow leaves and reduced root growth that results from a decreased cell division rate. Morphological and molecular phenotypes show that the ELONGATA (ELO) genes function in the same biological process and the epistatic interactions between the ELO genes can be explained by the model of complex formation in yeast. Furthermore, the plant Elongator complex is genetically positioned in the process of RNAPII-mediated transcription downstream of Mediator. Our data indicate that the Elongator complex is evolutionarily conserved in structure and function but reveal that the mechanism by which it stimulates cell proliferation is different in yeast and plants.

Acetyltransferases↗

PubMed2005

Genome-wide analysis of gene expression profiles associated with cell cycle transitions in growing organs of Arabidopsis.

Organ growth results from the progression of component cells through subsequent phases of proliferation and expansion before reaching maturity. We combined kinematic analysis, flowcytometry, and microarray analysis to characterize cell cycle regulation during the growth process of leaves 1 and 2 of Arabidopsis (Arabidopsis thaliana). Kinematic analysis showed that the epidermis proliferates until day 12; thereafter, cells expand until day 19 when leaves reach maturity. Flowcytometry revealed that endoreduplication occurs from the time cell division rates decline until the end of cell expansion. Analysis of 10 time points with a 6k-cDNA microarray showed that transitions between the growth stages were closely reflected in the mRNA expression data. Subsequent genome-wide microarray analysis on the three main stages allowed us to categorize known cell cycle genes into three major classes: constitutively expressed, proliferative, and inhibitory. Comparison with published expression data obtained from root zones corresponding to similar developmental stages and from synchronized cell cultures supported this categorization and enabled us to identify a high confidence set of 131 proliferation genes. Most of those had an M phase-dependent expression pattern and, in addition to many known cell cycle-related genes, there were at least 90 that were unknown or previously not associated with proliferation.

Arabidopsis↗

PubMed2005

The cyclin-dependent kinase inhibitor KRP2 controls the onset of the endoreduplication cycle during Arabidopsis leaf development through inhibition of mitotic CDKA;1 kinase complexes.

Exit from the mitotic cell cycle and initiation of cell differentiation frequently coincides with the onset of endoreduplication, a modified cell cycle during which DNA continues to be duplicated in the absence of mitosis. Although the mitotic cell cycle and the endoreduplication cycle share much of the same machinery, the regulatory mechanisms controlling the transition between both cycles remain poorly understood. We show that the A-type cyclin-dependent kinase CDKA;1 and its specific inhibitor, the Kip-related protein, KRP2 regulate the mitosis-to-endocycle transition during Arabidopsis thaliana leaf development. Constitutive overexpression of KRP2 slightly above its endogenous level only inhibited the mitotic cell cycle-specific CDKA;1 kinase complexes, whereas the endoreduplication cycle-specific CDKA;1 complexes were unaffected, resulting in an increase in the DNA ploidy level. An identical effect on the endoreduplication cycle could be observed by overexpressing KRP2 exclusively in mitotically dividing cells. In agreement with a role for KRP2 as activator of the mitosis-to-endocycle transition, KRP2 protein levels were more abundant in endoreduplicating than in mitotically dividing tissues. We illustrate that KRP2 protein abundance is regulated posttranscriptionally through CDK phosphorylation and proteasomal degradation. KRP2 phosphorylation by the mitotic cell cycle-specific CDKB1;1 kinase suggests a mechanism in which CDKB1;1 controls the level of CDKA;1 activity through regulating KRP2 protein abundance. In accordance with this model, KRP2 protein levels increased in plants with reduced CDKB1;1 activity. Moreover, the proposed model allowed a dynamical simulation of the in vivo observations, validating the sufficiency of the regulatory interactions between CDKA;1, KRP2, and CDKB1;1 in fine-tuning the mitosis-to-endocycle transition.

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

Green light for the cell cycle.

In recent years, considerable progress has been made in unraveling the control mechanisms operating on the plant cell cycle and most of the key regulators have now been identified, including cyclin-dependent kinases (CDKs), cyclins, CDK-inhibitory proteins, the WEE kinase and proteins of the retinoblastoma-related protein (RBR)/E2F/DP pathway. The review discusses recent developments in our understanding of the plant cell cycle machinery and highlights the role of the cell cycle in plant development.

Animals↗

PubMed2005

The DP-E2F-like gene DEL1 controls the endocycle in Arabidopsis thaliana.

Endoreduplication or DNA replication without mitosis is widespread in nature. Well-known examples are fruit fly polytene chromosomes and cereal endosperm. Although endocycles are thought to be driven by the same regulators as those that control the G1-S transition of the mitotic cell cycle, the molecular mechanisms that differentiate mitotically dividing cells from endoreduplicating ones are largely unknown. A novel class of atypical E2F-like proteins has recently been identified and is designated E2F7 in mammals and DP-E2F-like (DEL) in Arabidopsis thaliana . We demonstrate that loss of DEL1 function resulted in increased ploidy levels, whereas ectopic expression of DEL1 reduced endoreduplication. Ploidy changes were correlated with altered expression of a subset of E2F target genes encoding proteins necessary for DNA replication. Because DEL1 proteins were postulated to antagonize the E2F pathway, we generated DEL1-E2Fa-DPa triple transgenics. DEL1 inhibited the endoreduplication phenotype, but not the ectopic cell divisions that resulted from the overexpression of both E2Fa and DPa, illustrating that DEL1 specifically represses the endocycle. Because DEL1 transcripts were detected exclusively in mitotically dividing cells, we conclude that DEL1 is an important novel inhibitor of the endocycle and preserves the mitotic state of proliferating cells by suppressing transcription of genes that are required for cells to enter the DNA endoreduplication cycle.

Arabidopsis↗