Mieke Van Lijsebettens

The Arabidopsis thaliana Homolog of Yeast BRE1 Has a Function in Cell Cycle Regulation during Early Leaf and Root Growth

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.

Leaf development in Arabidopsis

In this review, we summarize recent research on leaf development in Arabidopsis. Topics include leaf initiation, ontogeny, the acquisition of leaf shape, heteroblasty, and the differentiation of the tissue types that make up a leaf. New data obtained by a molecular genetic approach and advanced microscopy are discussed in relation to existing hypotheses derived from earlier surgical experiments, morphological and anatomical descriptions, and mutational analyses. Molecular markers are providing new tools with which to investigate cell identity and growth patterns. An example is shown in which cell division is monitored during leaf growth. The cloning of the first set of genes critical for leaf development has shown that transcription factors control a number of important switches in developmental programs, such as the transition from indeterminate to determinate growth during leaf initiation, dorsiventral patterning and cell determination during trichome formation. In short, the study of leaf development seems to be about to come of age.

A local maximum in gibberellin levels regulates maize leaf growth by spatial control of cell division

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.

Plant Elongator regulates auxin-related genes during RNA polymerase II transcription elongation

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.

The transcript elongation factor FACT affects Arabidopsis vegetative and reproductive development and genetically interacts with HUB1/2

The facilitates chromatin transcription (FACT) complex, consisting of the SSRP1 and SPT16 proteins, is a histone chaperone that assists the progression of transcribing RNA polymerase on chromatin templates by destabilizing nucleosomes. Here, we examined plants that harbour mutations in the genes encoding the subunits of Arabidopsis FACT. These experiments revealed that (i) SSRP1 is critical for plant viability, and (ii) plants with reduced amounts of SSRP1 and SPT16 display various defects in vegetative and reproductive development. Thus, mutant plants display an increased number of leaves and inflorescences, show early bolting, have abnormal flower and leaf architecture, and their seed production is severely affected. The early flowering of the mutant plants is associated with reduced expression of the floral repressor FLC in ssrp1 and spt16 plants. Compared to control plants, reduced amounts of FACT in mutant plants are detected at the FLC locus as well as at the locations of housekeeping genes (whose expression is not affected in the mutants), suggesting that expression of FLC is particularly sensitive to reduced FACT activity. Analysis of double mutants that are affected in the expression of both FACT subunits and factors catalysing the mono-ubiquitination of histone H2B (HUB1/2) demonstrates that they genetically interact to regulate various developmental processes (i.e. branching, leaf venation pattern, silique development) but independently regulate the growth of leaves and the induction of flowering.

DRL1, a Homolog of the Yeast TOT4/KTI12 Protein, Has a Function in Meristem Activity and Organ Growth in Plants

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 TORNADO1 and TORNADO2 Genes Function in Several Patterning Processes during Early Leaf Development in Arabidopsis thaliana

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.

Ribosomes and translation in plant developmental control

Ribosomes play a basic housekeeping role in global translation. However, a number of ribosomal-protein-defective mutants show common and rare developmental phenotypes including growth defects, changes in leaf development, and auxin-related phenotypes. This suggests that translational regulation may be occurring during development. In addition, proteomic and bioinformatic analyses have demonstrated a high heterogeneity in ribosome composition. Although this might be a sign of unequal roles of individual ribosomal proteins, it does not explain every ribosomal-protein-defective phenotype. Moreover, comprehensive interpretations concerning the relationship between ribosomal-protein-defective phenotypes and molecular changes in ribosome status are lacking. In this review, we address these phenotypes based on three models, ribosome insufficiency, heterogeneity, and aberrancy, to consider how ribosomes play developmental roles. We propose that the three models are not mutually exclusive, and ribosomal-protein-defective phenotypes can be explained with one or more of these models. The three models with reference to genetic, biochemical, and bioinformatic knowledge will serve as a foundation for future studies of translational regulation.

The RON1/FRY1/SAL1 gene is required for leaf morphogenesis and venation patterning in Arabidopsis

To identify genes involved in vascular patterning in Arabidopsis (Arabidopsis thaliana), we screened for abnormal venation patterns in a large collection of leaf shape mutants isolated in our laboratory. The rotunda1-1 (ron1-1) mutant, initially isolated because of its rounded leaves, exhibited an open venation pattern, which resulted from an increased number of free-ending veins. We positionally cloned the RON1 gene and found it to be identical to FRY1/SAL1, which encodes an enzyme with inositol polyphosphate 1-phosphatase and 3′ (2′),5′-bisphosphate nucleotidase activities and has not, to our knowledge, previously been related to venation patterning. The ron1-1 mutant and mutants affected in auxin homeostasis share perturbations in venation patterning, lateral root formation, root hair length, shoot branching, and apical dominance. These similarities prompted us to monitor the auxin response using a DR5-GUS auxin-responsive reporter transgene, the expression levels of which were increased in roots and reduced in leaves in the ron1-1 background. To gain insight into the function of RON1/FRY1/SAL1 during vascular development, we generated double mutants for genes involved in vein patterning and found that ron1 synergistically interacts with auxin resistant1 and hemivenata-1 but not with cotyledon vascular pattern1 (cvp1) and cvp2. These results suggest a role for inositol metabolism in the regulation of auxin responses. Microarray analysis of gene expression revealed that several hundred genes are misexpressed in ron1-1, which may explain the pleiotropic phenotype of this mutant. Metabolomic profiling of the ron1-1 mutant revealed changes in the levels of 38 metabolites, including myoinositol and indole-3-acetonitrile, a precursor of auxin.

ASYMMETRIC LEAVES2 and Elongator, a Histone Acetyltransferase Complex, Mediate the Establishment of Polarity in Leaves of Arabidopsis thaliana

Leaf primordia are generated around the shoot apical meristem. Mutation of the ASYMMETRIC LEAVES2 (AS2) gene of Arabidopsis thaliana results in defects in repression of the meristematic and indeterminate state, establishment of adaxial-abaxial polarity and left-right symmetry in leaves. AS2 represses transcription of meristem-specific class 1 KNOX homeobox genes and of the abaxial-determinant genes ETTIN/ARF3, KANADI2 and YABBY5. To clarify the role of AS2 in the establishment of leaf polarity, we isolated mutations that enhanced the polarity defects associated with as2. We describe here the enhancer-of-asymmetric-leaves-two1 (east1) mutation, which caused the formation of filamentous leaves with abaxialized epidermis on the as2-1 background. Levels of transcripts of class 1 KNOX and abaxial-determinant genes were markedly higher in as2-1 east1-1 mutant plants than in the wild-type and corresponding single-mutant plants. EAST1 encodes the histone acetyltransferase ELONGATA3 (ELO3), a component of the Elongator complex. Genetic analysis, using mutations in genes involved in the biogenesis of a trans-acting small interfering RNA (ta-siRNA), revealed that ELO3 mediated establishment of leaf polarity independently of AS2 and the ta-siRNA-related pathway. Treatment with an inhibitor of histone deacetylases (HDACs) caused additive polarity defects in as2-1 east1-1 mutant plants, suggesting the operation of an ELO3 pathway, independent of the HDAC pathway, in the determination of polarity. We propose that multiple pathways play important roles in repression of the expression of class 1 KNOX and abaxial-determinant genes in the development of the adaxial domain of leaves and, thus, in the establishment of leaf polarity.