The retinoblastoma homolog RBR1 mediates localization of the repair protein RAD51 to DNA lesions in Arabidopsis.

The retinoblastoma protein (Rb), which typically functions as a transcriptional repressor of E2F-regulated genes, represents a major control hub of the cell cycle. Here, we show that loss of the Arabidopsis Rb homolog RETINOBLASTOMA-RELATED 1 (RBR1) leads to cell death, especially upon exposure to genotoxic drugs such as the environmental toxin aluminum. While cell death can be suppressed by reduced cell-proliferation rates, rbr1 mutant cells exhibit elevated levels of DNA lesions, indicating a direct role of RBR1 in the DNA-damage response (DDR). Consistent with its role as a transcriptional repressor, we find that RBR1 directly binds to and represses key DDR genes such as RADIATION SENSITIVE 51 (RAD51), leaving it unclear why rbr1 mutants are hypersensitive to DNA damage. However, we find that RBR1 is also required for RAD51 localization to DNA lesions. We further show that RBR1 is itself targeted to DNA break sites in a CDKB1 activity-dependent manner and partially co-localizes with RAD51 at damage sites. Taken together, these results implicate RBR1 in the assembly of DNA-bound repair complexes, in addition to its canonical function as a transcriptional regulator.

Genome wide natural variation of H3K27me3 selectively marks genes predicted to be important for cell differentiation in Phaeodactylum tricornutum.

In multicellular organisms, Polycomb Repressive Complex2 (PRC2) is known to deposit tri-methylation of lysine 27 of histone H3 (H3K27me3) to establish and maintain gene silencing, critical for developmentally regulated processes. The PRC2 complex is absent in both widely studied model yeasts, which initially suggested that PRC2 arose with the emergence of multicellularity. However, its discovery in several unicellular species including microalgae questions its role in unicellular eukaryotes. Here, we use Phaeodactylum tricornutum enhancer of zeste E(z) knockouts and show that P. tricornutum E(z) is responsible for di- and tri-methylation of lysine 27 of histone H3. H3K27me3 depletion abolishes cell morphology in P. tricornutum providing evidence for its role in cell differentiation. Genome-wide profiling of H3K27me3 in fusiform and triradiate cells further revealed genes that may specify cell identity. These results suggest a role for PRC2 and its associated mark in cell differentiation in unicellular species, and highlight their ancestral function in a broader evolutionary context than currently is appreciated.

Genome-wide identification of RETINOBLASTOMA RELATED 1 binding sites in Arabidopsis reveals novel DNA damage regulators.

Retinoblastoma (pRb) is a multifunctional regulator, which was likely present in the last common ancestor of all eukaryotes. The Arabidopsis pRb homolog RETINOBLASTOMA RELATED 1 (RBR1), similar to its animal counterparts, controls not only cell proliferation but is also implicated in developmental decisions, stress responses and maintenance of genome integrity. Although most functions of pRb-type proteins involve chromatin association, a genome-wide understanding of RBR1 binding sites in Arabidopsis is still missing. Here, we present a plant chromatin immunoprecipitation protocol optimized for genome-wide studies of indirectly DNA-bound proteins like RBR1. Our analysis revealed binding of Arabidopsis RBR1 to approximately 1000 genes and roughly 500 transposable elements, preferentially MITES. The RBR1-decorated genes broadly overlap with previously identified targets of two major transcription factors controlling the cell cycle, i.e. E2F and MYB3R3 and represent a robust inventory of RBR1-targets in dividing cells. Consistently, enriched motifs in the RBR1-marked domains include sequences related to the E2F consensus site and the MSA-core element bound by MYB3R transcription factors. Following up a key role of RBR1 in DNA damage response, we performed a meta-analysis combining the information about the RBR1-binding sites with genome-wide expression studies under DNA stress. As a result, we present the identification and mutant characterization of three novel genes required for growth upon genotoxic stress.

Cell density and airspace patterning in the leaf can be manipulated to increase leaf photosynthetic capacity.

The pattern of cell division, growth and separation during leaf development determines the pattern and volume of airspace in a leaf. The resulting balance of cellular material and airspace is expected to significantly influence the primary function of the leaf, photosynthesis, and yet the manner and degree to which cell division patterns affect airspace networks and photosynthesis remains largely unexplored. In this paper we investigate the relationship of cell size and patterning, airspace and photosynthesis by promoting and repressing the expression of cell cycle genes in the leaf mesophyll. Using microCT imaging to quantify leaf cellular architecture and fluorescence/gas exchange analysis to measure leaf function, we show that increased cell density in the mesophyll of Arabidopsis can be used to increase leaf photosynthetic capacity. Our analysis suggests that this occurs both by increasing tissue density (decreasing the relative volume of airspace) and by altering the pattern of airspace distribution within the leaf. Our results indicate that cell division patterns influence the photosynthetic performance of a leaf, and that it is possible to engineer improved photosynthesis via this approach.

Integrative epigenomic mapping defines four main chromatin states in Arabidopsis.

Post-translational modification of histones and DNA methylation are important components of chromatin-level control of genome activity in eukaryotes. However, principles governing the combinatorial association of chromatin marks along the genome remain poorly understood. Here, we have generated epigenomic maps for eight histone modifications (H3K4me2 and 3, H3K27me1 and 2, H3K36me3, H3K56ac, H4K20me1 and H2Bub) in the model plant Arabidopsis and we have combined these maps with others, produced under identical conditions, for H3K9me2, H3K9me3, H3K27me3 and DNA methylation. Integrative analysis indicates that these 12 chromatin marks, which collectively cover ∼90% of the genome, are present at any given position in a very limited number of combinations. Moreover, we show that the distribution of the 12 marks along the genomic sequence defines four main chromatin states, which preferentially index active genes, repressed genes, silent repeat elements and intergenic regions. Given the compact nature of the Arabidopsis genome, these four indexing states typically translate into short chromatin domains interspersed with each other. This first combinatorial view of the Arabidopsis epigenome points to simple principles of organization as in metazoans and provides a framework for further studies of chromatin-based regulatory mechanisms in plants.

Retinoblastoma related1 regulates asymmetric cell divisions in Arabidopsis.

Formative, also called asymmetric, cell divisions produce daughter cells with different identities. Like other divisions, formative divisions rely first of all on the cell cycle machinery with centrally acting cyclin-dependent kinases (CDKs) and their cyclin partners to control progression through the cell cycle. However, it is still largely obscure how developmental cues are translated at the cellular level to promote asymmetric divisions. Here, we show that formative divisions in the shoot and root of the flowering plant Arabidopsis thaliana are controlled by a common mechanism that relies on the activity level of the Cdk1 homolog CDKA;1, with medium levels being sufficient for symmetric divisions but high levels being required for formative divisions. We reveal that the function of CDKA;1 in asymmetric cell divisions operates through a transcriptional regulation system that is mediated by the Arabidopsis Retinoblastoma homolog RBR1. RBR1 regulates not only cell cycle genes, but also, independent of the cell cycle transcription factor E2F, genes required for formative divisions and cell fate acquisition, thus directly linking cell proliferation with differentiation. This mechanism allows the implementation of spatial information, in the form of high kinase activity, with intracellular gating of developmental decisions.

A general G1/S-phase cell-cycle control module in the flowering plant Arabidopsis thaliana.

The decision to replicate its DNA is of crucial importance for every cell and, in many organisms, is decisive for the progression through the entire cell cycle. A comparison of animals versus yeast has shown that, although most of the involved cell-cycle regulators are divergent in both clades, they fulfill a similar role and the overall network topology of G1/S regulation is highly conserved. Using germline development as a model system, we identified a regulatory cascade controlling entry into S phase in the flowering plant Arabidopsis thaliana, which, as a member of the Plantae supergroup, is phylogenetically only distantly related to Opisthokonts such as yeast and animals. This module comprises the Arabidopsis homologs of the animal transcription factor E2F, the plant homolog of the animal transcriptional repressor Retinoblastoma (Rb)-related 1 (RBR1), the plant-specific F-box protein F-BOX-LIKE 17 (FBL17), the plant specific cyclin-dependent kinase (CDK) inhibitors KRPs, as well as CDKA;1, the plant homolog of the yeast and animal Cdc2⁺/Cdk1 kinases. Our data show that the principle of a double negative wiring of Rb proteins is highly conserved, likely representing a universal mechanism in eukaryotic cell-cycle control. However, this negative feedback of Rb proteins is differently implemented in plants as it is brought about through a quadruple negative regulation centered around the F-box protein FBL17 that mediates the degradation of CDK inhibitors but is itself directly repressed by Rb. Biomathematical simulations and subsequent experimental confirmation of computational predictions revealed that this regulatory circuit can give rise to hysteresis highlighting the here identified dosage sensitivity of CDK inhibitors in this network.

Nuclear trapping by GL3 controls intercellular transport and redistribution of TTG1 protein in Arabidopsis.

Trichome patterning on Arabidopsis leaves is one of the best-studied model systems for two-dimensional de novo patterning. In addition to an activator-inhibitor-related mechanism, we previously proposed a depletion mechanism to operate during this process such that GLABRA3 (GL3) traps the trichome-promoting factor TRANSPARENT TESTA GLABRA1 (TTG1) in trichomes that, in turn, results in a depletion of TTG1 in trichome neighbouring cells. In this manuscript we analyze the molecular basis underlying this trapping mechanism. We demonstrate the ability of GL3 to regulate TTG1 mobility by expressing TTG1 and GL3 in different tissue layers in different combinations. We further show that TTG1 trapping by GL3 is based on direct interaction between both proteins and recruitment in the nucleus.

Polycomb repressive complex 2 controls the embryo-to-seedling phase transition.

Polycomb repressive complex 2 (PRC2) is a key regulator of epigenetic states catalyzing histone H3 lysine 27 trimethylation (H3K27me3), a repressive chromatin mark. PRC2 composition is conserved from humans to plants, but the function of PRC2 during the early stage of plant life is unclear beyond the fact that it is required for the development of endosperm, a nutritive tissue that supports embryo growth. Circumventing the requirement of PRC2 in endosperm allowed us to generate viable homozygous null mutants for FERTILIZATION INDEPENDENT ENDOSPERM (FIE), which is the single Arabidopsis homolog of Extra Sex Combs, an indispensable component of Drosophila and mammalian PRC2. Here we show that H3K27me3 deposition is abolished genome-wide in fie mutants demonstrating the essential function of PRC2 in placing this mark in plants as in animals. In contrast to animals, we find that PRC2 function is not required for initial body plan formation in Arabidopsis. Rather, our results show that fie mutant seeds exhibit enhanced dormancy and germination defects, indicating a deficiency in terminating the embryonic phase. After germination, fie mutant seedlings switch to generative development that is not sustained, giving rise to neoplastic, callus-like structures. Further genome-wide studies showed that only a fraction of PRC2 targets are transcriptionally activated in fie seedlings and that this activation is accompanied in only a few cases with deposition of H3K4me3, a mark associated with gene activity and considered to act antagonistically to H3K27me3. Up-regulated PRC2 target genes were found to act at different hierarchical levels from transcriptional master regulators to a wide range of downstream targets. Collectively, our findings demonstrate that PRC2-mediated regulation represents a robust system controlling developmental phase transitions, not only from vegetative phase to flowering but also especially from embryonic phase to the seedling stage.

Two-dimensional patterning by a trapping/depletion mechanism: the role of TTG1 and GL3 in Arabidopsis trichome formation.

Trichome patterning in Arabidopsis serves as a model system to study how single cells are selected within a field of initially equivalent cells. Current models explain this pattern by an activator-inhibitor feedback loop. Here, we report that also a newly discovered mechanism is involved by which patterning is governed by the removal of the trichome-promoting factor TRANSPARENT TESTA GLABRA1 (TTG1) from non-trichome cells. We demonstrate by clonal analysis and misexpression studies that Arabidopsis TTG1 can act non-cell-autonomously and by microinjection experiments that TTG1 protein moves between cells. While TTG1 is expressed ubiquitously, TTG1-YFP protein accumulates in trichomes and is depleted in the surrounding cells. TTG1-YFP depletion depends on GLABRA3 (GL3), suggesting that the depletion is governed by a trapping mechanism. To study the potential of the observed trapping/depletion mechanism, we formulated a mathematical model enabling us to evaluate the relevance of each parameter and to identify parameters explaining the paradoxical genetic finding that strong ttg1 alleles are glabrous, while weak alleles exhibit trichome clusters.

Male fertility in Arabidopsis requires active DNA demethylation of genes that control pollen tube function.

Active DNA demethylation is required for sexual reproduction in plants but the molecular determinants underlying this epigenetic control are not known. Here, we show in Arabidopsis thaliana that the DNA glycosylases DEMETER (DME) and REPRESSOR OF SILENCING 1 (ROS1) act semi-redundantly in the vegetative cell of pollen to demethylate DNA and ensure proper pollen tube progression. Moreover, we identify six pollen-specific genes with increased DNA methylation as well as reduced expression in dme and dme;ros1. We further show that for four of these genes, reinstalling their expression individually in mutant pollen is sufficient to improve male fertility. Our findings demonstrate an essential role of active DNA demethylation in regulating genes involved in pollen function.

Phytochrome A requires jasmonate for photodestruction.

The plant photoreceptor phytochrome is organised in a small gene family with phytochrome A (phyA) being unique, because it is specifically degraded upon activation by light. This so called photodestruction is thought to be important for dynamic aspects of sensing such as measuring day length or shading by competitors. Signal-triggered proteolytic degradation has emerged as central element of signal crosstalk in plants during recent years, but many of the molecular players are still unknown. We therefore analyzed a jasmonate (JA)-deficient rice mutant, hebiba, that in several aspects resembles a mutant affected in photomorphogenesis. In this mutant, the photodestruction of phyA is delayed as shown by in vivo spectroscopy and Western blot analysis. Application of methyl-JA (MeJA) can rescue the delayed phyA photodestruction in the mutant in a time- and dose-dependent manner. Light regulation of phyA transcripts thought to be under control of stable phytochrome B (phyB) is still functional. The delayed photodestruction is accompanied by an elevated sensitivity of phytochrome-dependent growth responses to red and far-red light.

DNA methylation dynamics during early plant life.

BACKGROUND: Cytosine methylation is crucial for gene regulation and silencing of transposable elements in mammals and plants. While this epigenetic mark is extensively reprogrammed in the germline and early embryos of mammals, the extent to which DNA methylation is reset between generations in plants remains largely unknown. RESULTS: Using Arabidopsis as a model, we uncovered distinct DNA methylation dynamics over transposable element sequences during the early stages of plant development. Specifically, transposable elements and their relics show invariably high methylation at CG sites but increasing methylation at CHG and CHH sites. This non-CG methylation culminates in mature embryos, where it reaches saturation for a large fraction of methylated CHH sites, compared to the typical 10-20% methylation level observed in seedlings or adult plants. Moreover, the increase in CHH methylation during embryogenesis matches the hypomethylated state in the early endosperm. Finally, we show that interfering with the embryo-to-seedling transition results in the persistence of high CHH methylation levels after germination, specifically over sequences that are targeted by the RNA-directed DNA methylation (RdDM) machinery. CONCLUSION: Our findings indicate the absence of extensive resetting of DNA methylation patterns during early plant life and point instead to an important role of RdDM in reinforcing DNA methylation of transposable element sequences in every cell of the mature embryo. Furthermore, we provide evidence that this elevated RdDM activity is a specific property of embryogenesis.

Emerging concepts in chromatin-level regulation of plant cell differentiation: timing, counting, sensing and maintaining.

Plants are characterized by a remarkable phenotypic plasticity that meets the constraints of a sessile lifestyle and the need to adjust constantly to the environment. Recent studies have begun to reveal how chromatin dynamics participate in coordinating cell proliferation and differentiation in response to developmental cues as well as environmental fluctuations. In this review, we discuss the pivotal function of chromatin-based mechanisms in cell fate acquisition and maintenance, within as well as outside meristems. In particular, we highlight the emerging role of specific epigenomic factors and chromatin pathways in timing the activity of stem cells, counting cell divisions and positioning cell fate transitions by sensing phytohormone gradients.

Deciphering and modifying LAFL transcriptional regulatory network in seed for improving yield and quality of storage compounds.

Increasing yield and quality of seed storage compounds in a sustainable way is a key challenge for our societies. Genome-wide analyses conducted in both monocot and dicot angiosperms emphasized drastic transcriptional switches that occur during seed development. In Arabidopsis thaliana, a reference species, genetic and molecular analyses have demonstrated the key role of LAFL (LEC1, ABI3, FUS3, and LEC2) transcription factors (TFs), in controlling gene expression programs essential to accomplish seed maturation and the accumulation of storage compounds. Here, we summarize recent progress obtained in the characterization of these LAFL proteins, their regulation, partners and target genes. Moreover, we illustrate how these evolutionary conserved TFs can be used to engineer new crops with altered seed compositions and point out the current limitations. Last, we discuss about the interest of investigating further the environmental and epigenetic regulation of this network for the coming years.

Evolutionarily conserved histone methylation dynamics during seed life-cycle transitions.

Plants have a remarkable ability to react to seasonal changes by synchronizing life-cycle transitions with environmental conditions. We addressed the question of how transcriptional re-programming occurs in response to an environmental cue that triggers the major life cycle transition from seed dormancy to germination and seedling growth. We elucidated an important mechanistic aspect of this process by following the chromatin dynamics of key regulatory genes with a focus on the two antagonistic marks, H3K4me3 and H3K27me3. Histone methylation patterns of major dormancy regulators changed during the transition to germination and seedling growth. We observed a switch from H3K4me3 and high transcription levels to silencing by the repressive H3K27me3 mark when dormancy was broken through exposure to moist chilling, underscoring that a functional PRC2 complex is necessary for this transition. Moreover, this reciprocal regulation by H3K4me3 and H3K27me3 is evolutionarily conserved from gymnosperms to angiosperms.

Genetic framework of cyclin-dependent kinase function in Arabidopsis.

Cyclin-dependent kinases (CDKs) are at the heart of eukaryotic cell-cycle control. The yeast Cdc2/CDC28 PSTAIRE kinase and its orthologs such as the mammalian Cdk1 have been found to be indispensable for cell-cycle progression in all eukaryotes investigated so far. CDKA;1 is the only PSTAIRE kinase in the flowering plant Arabidopsis and can rescue Cdc2/CDC28 mutants. Here, we show that cdka;1 null mutants are viable but display specific cell-cycle and developmental defects, e.g., in S phase entry and stem cell maintenance. We unravel that the crucial function of CDKA;1 is the control of the plant Retinoblastoma homolog RBR1 and that codepletion of RBR1 and CDKA;1 rescued most defects of cdka;1 mutants. Our work further revealed a basic cell-cycle control system relying on two plant-specific B1-type CDKs, and the triple cdk mutants displayed an early germline arrest. Taken together, our data indicate divergent functional differentiation of Cdc2-type kinases during eukaryote evolution.

Ectopic D-type cyclin expression induces not only DNA replication but also cell division in Arabidopsis trichomes.

Although the mechanisms controlling the two cell-cycle checkpoints G(1)-S and G(2)-M are well studied, it remains elusive how they are linked in higher eukaryotes. In animals, D-type cyclins have been implicated in the control of cell-cycle progression in mitotic as well as in endoreduplicating cells. By contrast, we show that the expression of the D-type cyclin CYCD3;1 in endoreduplicating Arabidopsis trichome cells not only induced DNA replication but also cell divisions.

Misexpression of the cyclin-dependent kinase inhibitor ICK1/KRP1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death.

A positive correlation between cell size and DNA content has been recognized in many plant cell types. Conversely, misexpression of a dominant-negative cyclin-dependent kinase (CDK) or CDK inhibitor proteins (ICK/KRPs) in Arabidopsis and tobacco leaves has revealed that cell growth can be uncoupled from cell cycle progression and DNA content. However, cell growth also appears to be controlled in a non-cell-autonomous manner by organ size, making it difficult in a ubiquitous expression assay to judge the cell-autonomous function of putative cell growth regulators. Here, we investigated the function of the CDK inhibitor ICK1/KRP1 on cell growth and differentiation independent of any compensatory influence of an organ context using Arabidopsis trichomes as a model system. By analyzing cell size with respect to DNA content, we dissected cell growth in a DNA-dependent and a DNA-independent process. We further found that ICK1/KRP1 misexpression interfered with differentiation and induced cell death, linking cell cycle progression, differentiation, and cell death in plants. The function of ICK1/KRP1 in planta was found to be dependent on a C-terminal domain and regulated negatively by an N-terminal domain. Finally, we identified CDKA;1 and a D-type cyclin as possible targets of ICK1/KRP1 expression in vivo.

The Arabidopsis STICHEL gene is a regulator of trichome branch number and encodes a novel protein.

Here, we analyze the STICHEL (STI) gene, which plays an important role in the regulation of branch number of the unicellular trichomes in Arabidopsis. We have isolated the STI locus by positional cloning and confirmed the identity by sequencing seven independent sti alleles. The STI gene encodes a protein of 1,218 amino acid residues containing a domain with sequence similarity to the ATP-binding eubacterial DNA-polymerase III gamma-subunits. Because endoreduplication was found to be normal in sti mutants the molecular function of STI in cell morphogenesis is not linked to DNA replication and, therefore, postulated to represent a novel pathway. Northern-blot analysis shows that STI is expressed in all organs suggesting that STI function is not trichome specific. The analysis of sti alleles and transgenic lines overexpressing STI suggests that STI regulates branching in a dosage-dependent manner.