[show abstract][hide abstract] ABSTRACT: DNA methylation is an important and widespread epigenetic modification in plant genomes, mediated by DNA methyltransferases (DMTs). DNA methylation is known to play a role in genome protection, regulation of gene expression, splicing, and was previously associated with major developmental reprogramming in plants, such as vernalization and transition to flowering. Here, we show that DNA methylation is also controlling the growth processes of cell division and cell expansion within a growing organ. The maize (Zea mays) leaf offers a great tool to study growth processes as the cells progressively move through the spatial gradient encompassing the division zone, transition zone, elongation zone, and mature zone. Opposite to de novo DMTs, the maintenance DMTs were transcriptionally regulated throughout the growth zone of the maize leaf, concomitant with differential CCGG methylation levels in the four zones. Surprisingly, the majority of differentially methylated sequences mapped on or close to gene bodies, and not to repeat-rich loci. Moreover, especially the 5' and 3' regions of genes, which show overall low methylation levels, underwent differential methylation in a developmental context. Genes involved in processes such as chromatin remodeling, cell cycle progression, and growth regulation were differentially methylated. The presence of differential methylation located upstream of the gene anti-correlated with transcript expression, while gene body differential methylation was unrelated to the expression level. These data indicate that DNA methylation is correlated with the decision to exit mitotic cell division and to enter cell expansion, which adds a new epigenetic level to the regulation of growth processes.
[show abstract][hide abstract] ABSTRACT: The cellular level processes cell division and cell expansion form a crucial level linking regulatory processes at the molecular level to whole plant growth rates and organ size and shape. With the rapid progress in molecular profiling, quantification of cellular activities becomes increasingly important to determine sampling strategies that are most informative to understand the molecular basis for organ and plant level phenotypes. Inversely, to understand phenotypes caused by genetic or environmental perturbations it is crucial to know how the cell division and expansion parameters are affected spatially and temporally. Kinematic analyses provide a powerful and rigorous mathematical framework to quantify cell division and cell expansion rates. In dicotyledonous leaves, these processes are primarily changing over time, resulting in division, expansion, and mature phases of development. Monocotyledonous leaves have a persistent spatial gradient, with an intercalary meristem where division takes place, an expansion zone, and a mature part of the leaf. Here we describe in detail how to perform kinematic analyses in leaves of the model species Arabidopsis thaliana and in the leaves of the monocotyledonous crop species Zea mays. These methods can readily be used and adapted to suit other species using relatively standard equipment present in most laboratories. Importantly, the obtained results can be used to design sampling techniques for proliferating, expanding and mature cells.
Methods in molecular biology (Clifton, N.J.) 01/2013; 959:247-64.
[show abstract][hide abstract] ABSTRACT: Previously, we identified HISTONE MONOUBIQUITINATION1 (HUB1) as an unconventional ubiquitin E3 ligase that is not involved in protein degradation but in the histone H2B modification that is implicated in transcriptional activation in plants. HUB1-mediated regulation of gene expression played a role in periodic and inducible processes such as the cell cycle, dormancy, flowering time and defense responses. Here, we determined the effects of the hub1-1 mutation on expression of a set of diurnally induced circadian clock genes identified from a comparative microarray analysis between the hub1-1 mutant and an HUB1 over-expression line. The hub1-1 mutation reduced the amplitudes of a number of induced clock gene expression peaks, as well as the HUB1-mediated histone H2BUb and H3K4Me3 marks associated with the coding regions, suggesting a role for HUB1 in facilitating transcriptional elongation in plants. Furthermore, double mutants between hub1-1 and elongata (elo) showed an embryo-lethal phenotype, indicating a synergistic genetic interaction. The double mutant embryos arrested at the torpedo stage, implying that together histone ubiquitination and acetylation marks are essential to activate expression of target genes in multiple pathways.
The Plant Journal 07/2012; 72(2):249-60. · 6.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plant growth rate is largely determined by the transition between the successive phases of cell division and expansion. A key role for hormone signaling in determining this transition was inferred from genetic approaches and transcriptome analysis in the Arabidopsis root tip. We used the developmental gradient at the maize leaf base as a model to study this transition, because it allows a direct comparison between endogenous hormone concentrations and the transitions between dividing, expanding, and mature tissue. Concentrations of auxin and cytokinins are highest in dividing tissues, whereas bioactive gibberellins (GAs) show a peak at the transition zone between the division and expansion zone. Combined metabolic and transcriptomic profiling revealed that this GA maximum is established by GA biosynthesis in the division zone (DZ) and active GA catabolism at the onset of the expansion zone. Mutants defective in GA synthesis and signaling, and transgenic plants overproducing GAs, demonstrate that altering GA levels specifically affects the size of the DZ, resulting in proportional changes in organ growth rates. This work thereby provides a novel molecular mechanism for the regulation of the transition from cell division to expansion that controls organ growth and size.
Current biology: CB 06/2012; 22(13):1183-7. · 10.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: To enable easy access and interpretation of heterogeneous and scattered data, we have developed a user-friendly tool for data mining and integration in Arabidopsis, named CORNET. This tool allows the browsing of microarray data, the construction of coexpression and protein-protein interaction (PPI) networks and the exploration of diverse functional annotations. Here, we present the new functionalities of CORNET 2.0 for data integration in plants. First of all, CORNET allows the integration of regulatory interaction datasets accessible through the new transcription factor (TF) tool that can be used in combination with the coexpression tool or the PPI tool. In addition, we have extended the PPI tool to enable the analysis of gene-gene associations from AraNet as well as newly identified PPIs. Different search options are implemented to enable the construction of networks centered around multiple input genes or proteins. New functional annotation resources are included to retrieve relevant literature, phenotypes, plant ontology and biological pathways. We have also extended CORNET to attain the construction of coexpression and PPI networks in the crop species maize. Networks and associated evidence of the majority of currently available data types are visualized in Cytoscape. CORNET is available at https://bioinformatics.psb.ugent.be/cornet.
New Phytologist 05/2012; 195(3):707-20. · 6.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: In eukaryotes, transcription of protein-encoding genes is strongly regulated by posttranslational modifications of histones that affect the accessibility of the DNA by RNA polymerase II (RNAPII). The Elongator complex was originally identified in yeast as a histone acetyltransferase (HAT) complex that activates RNAPII-mediated transcription. In Arabidopsis thaliana, the Elongator mutants elo1, elo2, and elo3 with decreased leaf and primary root growth due to reduced cell proliferation identified homologs of components of the yeast Elongator complex, Elp4, Elp1, and Elp3, respectively. Here we show that the Elongator complex was purified from plant cell cultures as a six-component complex. The role of plant Elongator in transcription elongation was supported by colocalization of the HAT enzyme, ELO3, with euchromatin and the phosphorylated form of RNAPII, and reduced histone H3 lysine 14 acetylation at the coding region of the SHORT HYPOCOTYL 2 auxin repressor and the LAX2 auxin influx carrier gene with reduced expression levels in the elo3 mutant. Additional auxin-related genes were down-regulated in the transcriptome of elo mutants but not targeted by the Elongator HAT activity showing specificity in target gene selection. Biological relevance was apparent by auxin-related phenotypes and marker gene analysis. Ethylene and jasmonic acid signaling and abiotic stress responses were up-regulated in the elo transcriptome and might contribute to the pleiotropic elo phenotype. Thus, although the structure of Elongator and its substrate are conserved, target gene selection has diverged, showing that auxin signaling and influx are under chromatin control.
Proceedings of the National Academy of Sciences 01/2010; 107(4):1678-83. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Leaf growth is a complex developmental process controlled by genetic and environmental factors and is determined by a proliferation, expansion and maturation phase. Mutational analysis in Arabidopsis thaliana showed that leaf size and shape is dependent on cell division and cell expansion activity. An investigation was made at the cytophysiological and ultrastructural level of the elo1 mutant of Arabidopsis thaliana, which is defective in one of the components of the histone acetyl transferase Elongator complex and displays a distinct 'narrow leaves' phenotype, owing to a reduced cell number and no transition between petiole and lamina. Relative expression levels of three sucrose metabolism/transport-related genes were also investigated. The aim was to determine the physiological basis of leaf morphology in this mutant, by investigating the modulatory role of sucrose.
The elo1 mutant was taken as representative of all the elo mutations and investigated at cytophysiological level. A germination test and growth assays were performed on seedlings grown for 21 d at different sucrose concentrations. Leaf morphometric and ultrastructural features were also investigated by image analysis and electron microscopy, respectively. Finally, a quantitative PCR (qPCR) analysis was performed with three sucrose metabolism/transport-related genes that were investigated under different sucrose concentrations.
elo1 plants at high sucrose concentrations exhibited an enhancement of germination and inhibition of leaf growth as compared with wild-type plants. qPCR experiments with three sucrose metabolism/transport-related genes showed an interaction between sucrose availability and the elo1 mutation. Furthermore, electron microscopy analysis provided the first ultrastructural description of an elo mutant, which showed a hypotonic vacuole, alterations in the size of grana and starch grains in the chloroplasts, and the massive presence of Golgi vesicles in the cytoplasm.
Based on the results obtained it is proposed that mechanisms producing carbon assimilates or importing sucrose could be affected in elo1 plants and could account for the observed differences, implying a role for Elongator in the regulation of these processes.
Annals of Botany 09/2007; 100(2):261-70. · 3.45 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chromatin modification and transcriptional activation are novel roles for E3 ubiquitin ligase proteins that have been mainly associated with ubiquitin-dependent proteolysis. We identified HISTONE MONOUBIQUITINATION1 (HUB1) (and its homolog HUB2) in Arabidopsis thaliana as RING E3 ligase proteins with a function in organ growth. We show that HUB1 is a functional homolog of the human and yeast BRE1 proteins because it monoubiquitinated histone H2B in an in vitro assay. Hub knockdown mutants had pale leaf coloration, modified leaf shape, reduced rosette biomass, and inhibited primary root growth. One of the alleles had been designated previously as ang4-1. Kinematic analysis of leaf and root growth together with flow cytometry revealed defects in cell cycle activities. The hub1-1 (ang4-1) mutation increased cell cycle duration in young leaves and caused an early entry into the endocycles. Transcript profiling of shoot apical tissues of hub1-1 (ang4-1) indicated that key regulators of the G2-to-M transition were misexpressed. Based on the mutant characterization, we postulate that HUB1 mediates gene activation and cell cycle regulation probably through chromatin modifications.
The Plant Cell 03/2007; 19(2):417-32. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: In eukaryotic organisms a wide range of regulatory mechanisms are required to complete the developmental program. Recently, the information contained in the histone code has been recognized to offer an additional mode of regulation of many processes in development. The histone code consists of covalent modifications of the core histone tails by ubiquitination, acetylation, methylation, ribosylation, sumoylation, phosphorylation, carbonylation, and glycation and the cross-talk between these modifications. These modifications reversibly alter the accessibility of the genome by the transition from heterochromatin to euchromatin and vice versa and play a role in almost all aspects of DNA metabolism: transcription, DNA repair, DNA recombination, and DNA replication. This review highlights the mechanisms that regulate the transcriptional activation or repression through modification of core histone tails and how these processes affect plant development. Chromatin control of leaf and root growth and the developmental transition from vegetative to reproductive phase is emphasized. In addition, the environmental impact on histone modifications will be discussed to support the view that chromatin acts as an interface to sense external signals and to regulate RNAPII transcription activity to adjust growth and developmental transitions.
[show abstract][hide abstract] ABSTRACT: In multicellular organisms, patterning is a process that generates axes in the primary body plan, creates domains upon organ formation, and finally leads to differentiation into tissues and cell types. We identified the Arabidopsis thaliana TORNADO1 (TRN1) and TRN2 genes and their role in leaf patterning processes such as lamina venation, symmetry, and lateral growth. In trn mutants, the leaf venation network had a severely reduced complexity: incomplete loops, no tertiary or quaternary veins, and vascular islands. The leaf laminas were asymmetric and narrow because of a severely reduced cell number. We postulate that the imbalance between cell proliferation and cell differentiation and the altered auxin distribution in both trn mutants cause asymmetric leaf growth and aberrant venation patterning. TRN1 and TRN2 were epistatic to ASYMMETRIC LEAVES1 with respect to leaf asymmetry, consistent with their expression in the shoot apical meristem and leaf primordia. TRN1 codes for a large plant-specific protein with conserved domains also found in a variety of signaling proteins, whereas TRN2 encodes a transmembrane protein of the tetraspanin family whose phylogenetic tree is presented. Double mutant analysis showed that TRN1 and TRN2 act in the same pathway.
The Plant Cell 05/2006; 18(4):852-66. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 06/2005; 102(21):7754-9. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The DEFORMED ROOTS AND LEAVES1 (DRL1) gene is single copy in the Arabidopsis genome, and based on overall amino acid similarity and conservation of functional domains, the DRL1 protein is homologous with yeast TOT4/KTI12. TOT4/KTI12 associates with Elongator, a multisubunit complex that binds the RNA polymerase II transcription elongation complex. Recessive mutations at the DRL1 locus caused defective organ formation indicative of disorganized shoot, inflorescence, flower, and root meristems. DRL1 is a putative ATP/GTP binding protein; in addition, calmodulin binding activity was demonstrated in vitro for the C terminus of the DRL1 protein. Phenotypic and genetic data position DRL1 relative to regulatory loci for leaf development, in which it acts early. We identified Arabidopsis homologs for the six Elongator components and hypothesize that DRL1 regulates transcription elongation through a putative plant Elongator. Upregulation of the ANGUSTIFOLIA transcript in the strong drl1-2 allele supports this model.
The Plant Cell 04/2003; 15(3):639-54. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Control of leaf shape and size was studied at the molecular level by a mutational approach in Arabidopsis thaliana. Mutations that affected leaf growth allowed the identification of genes that are important in that morphological process. The Activator/Dissociation (Ac/Ds) transposable element system of maize was modified to be used as mutagen of the Arabidopsis genome. We isolated the drl 1-2 mutation that is characterized by narrow leaf blades. We describe the mutant phenotype and have cloned and analyzed the gene. These combined analyses will give insight into the molecular mechanisms that control leaf growth.
Mededelingen (Rijksuniversiteit te Gent. Fakulteit van de Landbouwkundige en Toegepaste Biologische Wetenschappen) 02/2001; 66(3b):409-15.