Cytosine methylation at CG sites is proposed to be maintained during DNA replication through a semiconservative mechanism. In mammals, this maintenance involves replication-coupled and replication-uncoupled steps; however, the underlying mechanisms and their possible conservation across eukaryotes remain unclear. Using live-cell sensors developed in this study to track CG methylation (mCG) dynamics, we uncovered that mCG maintenance extends far beyond DNA replication in the model plant Arabidopsis, with the major CG methyltransferase 1 (MET1) remaining associated with chromatin from S phase till G2 phase. Genetic dissection demonstrated that MET1, like its mammalian homolog DNA methyltransferase 1 (DNMT1), methylates nucleosome-associated DNA. Single-molecule duplex sequencing revealed that MET1/DNMT1-dependent methylation follows the rotational accessibility of nucleosomal DNA and is only partly templated by hemimethylated sites. This nucleosome-constrained de novo activity not only promotes mCG recovery after accidental loss but also shapes short- and long-term DNA methylation variation in plants and mammals.

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.

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.

Fertilization in Arabidopsis thaliana is a highly coordinated process that begins with a pollen tube delivering the two 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 still remains elusive as DNA quantification and DNA replication assays have given conflicting results 1–4 . Here, to reconcile these results, we quantified the DNA replication state by DNA sequencing and performed microscopic analyses of fluorescent markers covering all the phases of the cell cycle. We show that male and female gametes in Arabidopsis are both arrested prior to DNA replication at maturity and initiate their DNA replication only during fertilization.

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.

Double haploid production is the most effective way of creating true-breeding lines in a single generation. In Arabidopsis , haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when outcrossed to wild-type. Here we report that a mutant of the cenH3 assembly factor KNL2 can be used as a haploid inducer. We elucidated that short temperature stress of the knl2 mutant increased the efficiency of haploid induction from 1 to 10%. Moreover, we have 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 GMO approach at any stage. In addition, we have shown that the cenh3-4 mutant, which does not induce haploids under standard growth conditions, functions as a haploid inducer after exposure to short temperature stress.

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.

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.

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.

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.

Plants' ability to respond to environmental stimuli and developmental cues depends upon changes in gene expression. In eukaryotes, genetic information encoded by DNA is packed in a highly regulated and dynamic nucleoprotein complex known as chromatin that is subject to epigenetic modifications. Historically, several biological phenomena relying on epigenetic mechanisms were first characterized in plants. Seminal discoveries such as paramutation and silencing of transposable elements were made in maize (Zea mays). Later rice (Oryza sativa) was selected as a good model for monocotyledons owing to its relatively small genome size and well-annotated sequenced genome. In the past few years an increasing number of epigenetic and epigenomic studies were performed in both maize and rice. In this chapter we will first compare the basic knowledge acquired on epigenetic regulation in rice and maize versus Arabidopsis, then we will describe cereals-specific aspects of epigenetic regulations.

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.

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 ( Os PIP1;1, Os PIP2;4, Os PIP2;5, Os TIP1;1, Os TIP2;2) and three endosomal trafficking proteins ( Os Rab5a, Os GAP1, Os SCAMP1) 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 Os PIPs is concomitant with their high cycling dynamics.

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.

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.

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.

In most eukaryotes, the HISTONE 3 family comprises several variants distinguished by their amino acid sequence, localization, and correlation with transcriptional activity [1-3]. Transgenerational inheritance of epigenetic information carried by histones is still unclear [4, 5]. 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 [6]. 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 [7]. 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.

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.