Hilde Nelissen

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.

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.

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.

CORNET 2.0: integrating plant coexpression, protein-protein interactions, regulatory interactions, gene associations and functional annotations.

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.

Dynamic Changes in ANGUSTIFOLIA3 Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf

TAP analysis to unravel protein complex composition in dividing and expanding tissue in a growing maize leaf reveals ANGUSTIFOLIA3 association with distinct GROWTH-REGULATING FACTORs to fine-tune growth. Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated.

Genetic properties of the MAGIC maize population: a new platform for high definition QTL mapping in Zea mays.

BackgroundMaize (Zea mays) is a globally produced crop with broad genetic and phenotypic variation. New tools that improve our understanding of the genetic basis of quantitative traits are needed to guide predictive crop breeding. We have produced the first balanced multi-parental population in maize, a tool that provides high diversity and dense recombination events to allow routine quantitative trait loci (QTL) mapping in maize.ResultsWe produced 1,636 MAGIC maize recombinant inbred lines derived from eight genetically diverse founder lines. The characterization of 529 MAGIC maize lines shows that the population is a balanced, evenly differentiated mosaic of the eight founders, with mapping power and resolution strengthened by high minor allele frequencies and a fast decay of linkage disequilibrium. We show how MAGIC maize may find strong candidate genes by incorporating genome sequencing and transcriptomics data. We discuss three QTL for grain yield and three for flowering time, reporting candidate genes. Power simulations show that subsets of MAGIC maize might achieve high-power and high-definition QTL mapping.ConclusionsWe demonstrate MAGIC maize’s value in identifying the genetic bases of complex traits of agronomic relevance. The design of MAGIC maize allows the accumulation of sequencing and transcriptomics layers to guide the identification of candidate genes for a number of maize traits at different developmental stages. The characterization of the full MAGIC maize population will lead to higher power and definition in QTL mapping, and lay the basis for improved understanding of maize phenotypes, heterosis included. MAGIC maize is available to researchers.

Impact of Core Histone Modifications on Transcriptional Regulation and Plant Growth

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.

Histone H2B monoubiquitination is required to reach maximal transcript levels of circadian clock genes in Arabidopsis

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.