Cell cycle status of male and female gametes during Arabidopsis reproduction.

Fertilization in Arabidopsis (Arabidopsis thaliana) is a highly coordinated process that begins with a pollen tube delivering the 2 sperm cells into the embryo sac. Each sperm cell can then fertilize either the egg or the central cell to initiate embryo or endosperm development, respectively. The success of this double fertilization process requires a tight cell cycle synchrony between the male and female gametes to allow karyogamy (nuclei fusion). However, the cell cycle status of the male and female gametes during fertilization remains elusive as DNA quantification and DNA replication assays have given conflicting results. Here, to reconcile these results, we quantified the DNA replication state by DNA sequencing and performed microscopic analyses of fluorescent markers covering all phases of the cell cycle. We show that male and female Arabidopsis gametes are both arrested prior to DNA replication at maturity and initiate their DNA replication only during fertilization.

TRIMETHYLGUANOSINE SYNTHASE1 mutations decanalize female germline development in Arabidopsis.

Here, we report the characterization of a plant RNA methyltransferase, orthologous to yeast trimethylguanosine synthase1 (Tgs1p) and whose downregulation was associated with apomixis in Paspalum grasses. Using phylogenetic analyses and yeast complementation, we determined that land plant genomes all encode a conserved, specific TGS1 protein. Next, we studied the role of TGS1 in female reproduction using reporter lines and loss-of-function mutants in Arabidopsis thaliana. pAtTGS1:AtTGS1 reporters showed a dynamic expression pattern. They were highly active in the placenta and ovule primordia at emergence but, subsequently, showed weak signals in the nucellus. Although expressed throughout gametophyte development, activity became restricted to the female gamete and was also detected after fertilization during embryogenesis. TGS1 depletion altered the specification of the precursor cells that give rise to the female gametophytic generation and to the sporophyte, resulting in the formation of a functional aposporous-like lineage. Our results indicate that TGS1 participates in the mechanisms restricting cell fate acquisition to a single cell at critical transitions throughout the female reproductive lineage and, thus, expand our current knowledge of the mechanisms governing female reproductive fate in plants.

Extrachromosomal circular DNA and structural variants highlight genome instability in Arabidopsis epigenetic mutants.

Abundant extrachromosomal circular DNA (eccDNA) is associated with transposable element (TE) activity. However, how the eccDNA compartment is controlled by epigenetic regulations and what is its impact on the genome is understudied. Here, using long reads, we sequence both the eccDNA compartment and the genome of Arabidopsis thaliana mutant plants affected in DNA methylation and post-transcriptional gene silencing. We detect a high load of TE-derived eccDNA with truncated and chimeric forms. On the genomic side, on top of truncated and full length TE neo-insertions, we detect complex structural variations (SVs) notably at a disease resistance cluster being a natural hotspot of SV. Finally, we serendipitously identify large tandem duplications in hypomethylated plants, suggesting that SVs could have been overlooked in epigenetic mutants. We propose that a high eccDNA load may alter DNA repair pathways leading to genome instability and the accumulation of SVs, at least in plants.

High temperature increases centromere-mediated genome elimination frequency and enhances haploid induction in Arabidopsis.

Double haploid production is the most effective way to create true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when the mutant is outcrossed to the wild-type, and the wild-type genome remains in the haploid progeny. However, factors that affect haploid induction are still poorly understood. Here, we report that a mutant of the cenH3 assembly factor Kinetochore Null2 (KNL2) can be used as a haploid inducer when pollinated by the wild-type. We discovered that short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction 10-fold. We also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines, suggesting that haploid-inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the generic modification (GM) approach at any stage. Furthermore, a cenh3-4 mutant functioned as a haploid inducer in response to short-term heat stress, even though it did not induce haploids under standard conditions. Thus, we identified KNL2 as a new target gene for the generation of haploid-inducer lines and showed that exposure of centromeric protein mutants to high temperature strongly increases their haploid induction efficiency.

Evolution of CG Methylation Maintenance Machinery in Plants.

Cytosine methylation is an epigenetic mark present in most eukaryotic genomes that contributes to the regulation of gene expression and the maintenance of genome stability. DNA methylation mostly occurs at CG sequences, where it is initially deposited by de novo DNA methyltransferases and propagated by maintenance DNA methyltransferases (DNMT) during DNA replication. In this review, we first summarize the mechanisms maintaining CG methylation in mammals that involve the DNA Methyltransferase 1 (DNMT1) enzyme and its cofactor, UHRF1 (Ubiquitin-like with PHD and RING Finger domain 1). We then discuss the evolutionary conservation and diversification of these two core factors in the plant kingdom and speculate on potential functions of novel homologues typically observed in land plants but not in mammals.

ANCHOR: A Technical Approach to Monitor Single-Copy Locus Localization in Planta.

Together with local chromatin structure, gene accessibility, and the presence of transcription factors, gene positioning is implicated in gene expression regulation. Although the basic mechanisms are expected to be conserved in eukaryotes, less is known about the role of gene positioning in plant cells, mainly due to the lack of a highly resolutive approach. In this study, we adapted the use of the ANCHOR system to perform real-time single locus detection in planta. ANCHOR is a DNA-labeling tool derived from the chromosome partitioning system found in many bacterial species. We demonstrated its suitability to monitor a single locus in planta and used this approach to track chromatin mobility during cell differentiation in Arabidopsis thaliana root epidermal cells. Finally, we discussed the potential of this approach to investigate the role of gene positioning during transcription and DNA repair in plants.

DNA Methylation Readers in Plants.

In plants, DNA methylation occurs in distinct sequence contexts, including CG, CHG, and CHH. Thus, plants have developed a surprisingly diverse set of DNA methylation readers to cope with an extended repertoire of methylated sites. The Arabidopsis genome contains twelve Methyl-Binding Domain proteins (MBD), and nine SET and RING finger-associated (SRA) domain containing proteins belonging to the SUVH clade, in addition to three homologs of UHRF1, namely VIM1-3, all containing SRA domains. In this review, we will highlight several research questions that remain unresolved with respect to the function of plant DNA methylation readers, which can have both de novo demethylase and maintenance activity. We argue that maintenance of CG methylation in plants likely involved actors not found in their mammalian counterparts, and that new evidence suggests significant reprogramming of DNA methylation during plant reproduction as an important new development in the field.

A protocol combining multiphoton microscopy and propidium iodide for deep 3D root meristem imaging in rice: application for the screening and identification of tissue-specific enhancer trap lines.

BACKGROUND: The clear visualization of 3D organization at the cellular level in plant tissues is needed to fully understand plant development processes. Imaging tools allow the visualization of the main fluorophores and in vivo growth monitoring. Confocal microscopy coupled with the use of propidium iodide (PI) counter-staining is one of the most popular tools used to characterize the structure of root meristems in A. thaliana. However, such an approach is relatively ineffective in species with more complex and thicker root systems. RESULTS: We adapted a PI counter-staining protocol to visualize the internal 3D architecture of rice root meristems using multiphoton microscopy. This protocol is simple and compatible with the main fluorophores (CFP, GFP and mCherry). The efficiency and applicability of this protocol were demonstrated by screening a population of 57 enhancer trap lines. We successfully characterized GFP expression in all of the lines and identified 5 lines with tissue-specific expression. CONCLUSIONS: All of these resources are now available for the rice community and represent critical tools for future studies of root development.

Sub-cellular markers highlight intracellular dynamics of membrane proteins in response to abiotic treatments in rice.

BACKGROUND: Cell biology approach using membrane protein markers tagged with fluorescent proteins highlights the dynamic behaviour of plant cell membranes, not only in the standard but also in changing environmental conditions. In the past, this strategy has been extensively developed in plant models such as Arabidopsis. RESULTS: Here, we generated a set of transgenic lines expressing membrane protein markers to extend this approach to rice, one of the most cultivated crop in the world and an emerging plant model. Lines expressing individually eight membrane protein markers including five aquaporins (OsPIP1;1, OsPIP2;4, OsPIP2;5, OsTIP1;1, OsTIP2;2) and three endosomal trafficking proteins (OsRab5a, OsGAP1, OsSCAMP1) were obtained. Importantly, we challenged in roots the aquaporin-expressing transgenic lines upon salt and osmotic stress and uncovered a highly dynamic behaviour of cell membrane. CONCLUSION: We have uncovered the relocalization and dynamics of plasma membrane aquaporins upon salt and osmotic stresses in rice. Importantly, our data support a model where relocalization of OsPIPs is concomitant with their high cycling dynamics.

Arabidopsis pollen tube germination and growth depend on the mitochondrial calcium uniporter complex.

The mitochondrial calcium uniporter complex (MCUc) was recently characterized in details in metazoans and consists of pore-forming units (MCUs) and regulatory factors that channel calcium (Ca2+ ) ion into the mitochondria. MCUs participate in many stress and developmentally related processes involving Ca2+ . Although multiple homologues of MCUs and one regulatory subunit are usually present in plants, the first functional characterization and contribution to Ca2+ related processes of these proteins have been reported recently. Here, we focused on two predicted Arabidopsis MCUs and studied their role in the germination and the growth of pollen tube, a tip-growing cell type highly dependent on Ca2+ homeostasis. Heterologous expression of MCU1 or MCU2 in yeast is sufficient to generate a mitochondrial Ca2+ influx. MCU1 and MCU2 fluorescent reporters are co-expressed in the vegetative cell mitochondria of the pollen grain but are undetectable in the embryo sac. We demonstrate that MCU1 and MCU2 can form a heterotypic complex. Phenotypic analyses revealed an impaired pollen tube germination and growth in vitro only for the mcu2 mutants suggesting a predominant role of MCU2. Our results show that mitochondrial Ca2+ controlled by MCUs is an additional player in Arabidopsis pollen tube germination and growth.

Dynamic Regulation of Auxin Response during Rice Development Revealed by Newly Established Hormone Biosensor Markers.

The hormone auxin is critical for many plant developmental processes. Unlike the model eudicot plant Arabidopsis (Arabidopsis thaliana), auxin distribution and signaling in rice tissues has not been systematically investigated due to the absence of suitable auxin response reporters. In this study we observed the conservation of auxin signaling components between Arabidopsis and model monocot crop rice (Oryza sativa), and generated complementary types of auxin biosensor constructs, one derived from the Aux/IAA-based biosensor DII-VENUS but constitutively driven by maize ubiquitin-1 promoter, and the other termed DR5-VENUS in which a synthetic auxin-responsive promoter (DR5rev ) was used to drive expression of the yellow fluorescent protein (YFP). Using the obtained transgenic lines, we observed that during the vegetative development, accumulation of DR5-VENUS signal was at young and mature leaves, tiller buds and stem base. Notably, abundant DR5-VENUS signals were observed in the cytoplasm of cortex cells surrounding lateral root primordia (LRP) in rice. In addition, auxin maxima and dynamic re-localization were seen at the initiation sites of inflorescence and spikelet primordia including branch meristems (BMs), female and male organs. The comparison of these observations among Arabidopsis, rice and maize suggests the unique role of auxin in regulating rice lateral root emergence and reproduction. Moreover, protein localization of auxin transporters PIN1 homologs and GFP tagged OsAUX1 overlapped with DR5-VENUS during spikelet development, helping validate these auxin response reporters are reliable markers in rice. This work firstly reveals the direct correspondence between auxin distribution and rice reproductive and root development at tissue and cellular level, and provides high-resolution auxin tools to probe fundamental developmental processes in rice and to establish links between auxin, development and agronomical traits like yield or root architecture.

Live-cell analysis of DNA methylation during sexual reproduction in Arabidopsis reveals context and sex-specific dynamics controlled by noncanonical RdDM.

Cytosine methylation is a key epigenetic mark in many organisms, important for both transcriptional control and genome integrity. While relatively stable during somatic growth, DNA methylation is reprogrammed genome-wide during mammalian reproduction. Reprogramming is essential for zygotic totipotency and to prevent transgenerational inheritance of epimutations. However, the extent of DNA methylation reprogramming in plants remains unclear. Here, we developed sensors reporting with single-cell resolution CG and non-CG methylation in Arabidopsis. Live imaging during reproduction revealed distinct and sex-specific dynamics for both contexts. We found that CHH methylation in the egg cell depends on DOMAINS REARRANGED METHYLASE 2 (DRM2) and RNA polymerase V (Pol V), two main actors of RNA-directed DNA methylation, but does not depend on Pol IV. Our sensors provide insight into global DNA methylation dynamics at the single-cell level with high temporal resolution and offer a powerful tool to track CG and non-CG methylation both during development and in response to environmental cues in all organisms with methylated DNA, as we illustrate in mouse embryonic stem cells.

Imaging sexual reproduction in Arabidopsis using fluorescent markers.

Sexual reproduction in higher plants is a stealth process as most events occur within tissues protected by multiple surrounding cell layers. Female gametes are produced inside the embryo sac surrounded by layers of ovule integument cells. Upon double fertilization, two male gametes are released at one end of the embryo sac and migrate towards their respective female partner to generate the embryo and its feeding tissue, the endosperm, within a seed. Since the early discovery of plant reproduction, advances in microscopy have contributed enormously to our understanding of this process (Faure and Dumas, Plant Physiol 125:102-104, 2001). Recently, live imaging of double fertilization has been possible using a set of fluorescent markers for gametes in Arabidopsis. The following chapter will detail protocols to study male and female gametogenesis and double fertilization in living tissues using fluorescent markers.

Zygotic resetting of the HISTONE 3 variant repertoire participates in epigenetic reprogramming in Arabidopsis.

In most eukaryotes, the HISTONE 3 family comprises several variants distinguished by their amino acid sequence, localization, and correlation with transcriptional activity. Transgenerational inheritance of epigenetic information carried by histones is still unclear. In addition to covalent histone modifications, the mosaic distribution of H3 variants onto chromatin has been proposed to provide a new level of epigenetic information. To study the transmission of patterns of H3 variants through generations, we combined transcriptional profiling and live imaging of the 13 H3 variants encoded by the Arabidopsis plant genome. In comparison with somatic cells, only a restricted number of H3 variants are present in male and female gametes. Upon fertilization, H3 variants contributed by both gametes are actively removed from the zygote chromatin. The somatic H3 composition is restored in the embryo by de novo synthesis of H3 variants. A survey of Arabidopsis homologs of animal H3 chaperones suggests that removal of parental H3 from the zygote nucleus relies on a new mechanism. Our results suggest that reprogramming of parental genomes in the zygote limits the inheritance of epigenetic information carried by H3 variants across generations.

Histone3 variants in plants.

Regulation of chromatin activity by covalent histone modifications has been long recognized. Histones that constitute the nucleosome are encoded by large families of genes and display a strong degree of conservation. However, histone variants exist and it is becoming clear that they play important roles in genome regulation. While most studies of the role of histone3 (H3) variants in transcriptional control comes from animal models, emerging data in plants suggest functional conservation, although plant-specific roles are likely. We review these data and speculate on the biological significance of H3 variants in plants.

Retinoblastoma and its binding partner MSI1 control imprinting in Arabidopsis.

Parental genomic imprinting causes preferential expression of one of the two parental alleles. In mammals, differential sex-dependent deposition of silencing DNA methylation marks during gametogenesis initiates a new cycle of imprinting. Parental genomic imprinting has been detected in plants and relies on DNA methylation by the methyltransferase MET1. However, in contrast to mammals, plant imprints are created by differential removal of silencing marks during gametogenesis. In Arabidopsis, DNA demethylation is mediated by the DNA glycosylase DEMETER (DME) causing activation of imprinted genes at the end of female gametogenesis. On the basis of genetic interactions, we show that in addition to DME, the plant homologs of the human Retinoblastoma (Rb) and its binding partner RbAp48 are required for the activation of the imprinted genes FIS2 and FWA. This Rb-dependent activation is mediated by direct transcriptional repression of MET1 during female gametogenesis. We have thus identified a new mechanism required for imprinting establishment, outlining a new role for the Retinoblastoma pathway, which may be conserved in mammals.

Double fertilization - caught in the act.

In flowering plants, fertilization is unique because it involves two pairs of male and female gametes, a process known as double fertilization. Here, we provide an overview of the field and a detailed review of the outstanding recent advances, including in vivo imaging of double fertilization and the identification of a signaling pathway controlling the release of the male gametes and of a protein involved in gamete membrane fusion. These recent results are stepping stones for further research; our knowledge of double fertilization is expanding as newly discovered molecular pathways are explored and new mutants are characterized. Controlling plant fertilization is essential for seed production, and molecular understanding of double fertilization will provide the tools to improve crops and breeding programs.

Chromatin assembly factor 1 regulates the cell cycle but not cell fate during male gametogenesis in Arabidopsis thaliana.

The interdependence of cell cycle control, chromatin remodeling and cell fate determination remains unclear in flowering plants. Pollen development provides an interesting model, as it comprises only two cell types produced by two sequential cell divisions. The first division separates the vegetative cell from the generative cell. The generative cell divides and produces the two sperm cells, transported to the female gametes by the pollen tube produced by the vegetative cell. We show in Arabidopsis thaliana that loss of activity of the Chromatin assembly factor 1 (CAF1) pathway causes delay and arrest of the cell cycle during pollen development. Prevention of the second pollen mitosis generates a fraction of CAF1-deficient pollen grains comprising a vegetative cell and a single sperm cell, which both express correctly cell fate markers. The single sperm is functional and fertilizes indiscriminately either female gamete. Our results thus suggest that pollen cell fate is independent from cell cycle regulation.

Analysis of CATMA transcriptome data identifies hundreds of novel functional genes and improves gene models in the Arabidopsis genome.

BACKGROUND: Since the finishing of the sequencing of the Arabidopsis thaliana genome, the Arabidopsis community and the annotator centers have been working on the improvement of gene annotation at the structural and functional levels. In this context, we have used the large CATMA resource on the Arabidopsis transcriptome to search for genes missed by different annotation processes. Probes on the CATMA microarrays are specific gene sequence tags (GSTs) based on the CDS models predicted by the Eugene software. Among the 24 576 CATMA v2 GSTs, 677 are in regions considered as intergenic by the TAIR annotation. We analyzed the cognate transcriptome data in the CATMA resource and carried out data-mining to characterize novel genes and improve gene models. RESULTS: The statistical analysis of the results of more than 500 hybridized samples distributed among 12 organs provides an experimental validation for 465 novel genes. The hybridization evidence was confirmed by RT-PCR approaches for 88% of the 465 novel genes. Comparisons with the current annotation show that these novel genes often encode small proteins, with an average size of 137 aa. Our approach has also led to the improvement of pre-existing gene models through both the extension of 16 CDS and the identification of 13 gene models erroneously constituted of two merged CDS. CONCLUSION: This work is a noticeable step forward in the improvement of the Arabidopsis genome annotation. We increased the number of Arabidopsis validated genes by 465 novel transcribed genes to which we associated several functional annotations such as expression profiles, sequence conservation in plants, cognate transcripts and protein motifs.