Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis.

Defining the contributions and interactions of paternal and maternal genomes during embryo development is critical to understand the fundamental processes involved in hybrid vigor, hybrid sterility, and reproductive isolation. To determine the parental contributions and their regulation during Arabidopsis embryogenesis, we combined deep-sequencing-based RNA profiling and genetic analyses. At the 2-4 cell stage there is a strong, genome-wide dominance of maternal transcripts, although transcripts are contributed by both parental genomes. At the globular stage the relative paternal contribution is higher, largely due to a gradual activation of the paternal genome. We identified two antagonistic maternal pathways that control these parental contributions. Paternal alleles are initially downregulated by the chromatin siRNA pathway, linked to DNA and histone methylation, whereas transcriptional activation requires maternal activity of the histone chaperone complex CAF1. Our results define maternal epigenetic pathways controlling the parental contributions in plant embryos, which are distinct from those regulating genomic imprinting.

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.

Chromatin dynamics during cellular differentiation in the female reproductive lineage of flowering plants.

Sexual reproduction in flowering plants offers a number of remarkable aspects to developmental biologists. First, the spore mother cells - precursors of the plant reproductive lineage - are specified late in development, as opposed to precocious germline isolation during embryogenesis in most animals. Second, unlike in most animals where meiosis directly produces gametes, plant meiosis entails the differentiation of a multicellular, haploid gametophyte, within which gametic as well as non-gametic accessory cells are formed. These observations raise the question of the factors inducing and modus operandi of cell fate transitions that originate in floral tissues and gametophytes, respectively. Cell fate transitions in the reproductive lineage imply cellular reprogramming operating at the physiological, cytological and transcriptome level, but also at the chromatin level. A number of observations point to large-scale chromatin reorganization events associated with cellular differentiation of the female spore mother cells and of the female gametes. These include a reorganization of the heterochromatin compartment, the genome-wide alteration of the histone modification landscape, and the remodeling of nucleosome composition. The dynamic expression of DNA methyltransferases and actors of small RNA pathways also suggest additional, global epigenetic alterations that remain to be characterized. Are these events a cause or a consequence of cellular differentiation, and how do they contribute to cell fate transition? Does chromatin dynamics induce competence for immediate cellular functions (meiosis, fertilization), or does it also contribute long-term effects in cellular identity and developmental competence of the reproductive lineage? This review attempts to review these fascinating questions.

Control of female gamete formation by a small RNA pathway in Arabidopsis.

In the ovules of most sexual flowering plants female gametogenesis is initiated from a single surviving gametic cell, the functional megaspore, formed after meiosis of the somatically derived megaspore mother cell (MMC). Because some mutants and certain sexual species exhibit more than one MMC, and many others are able to form gametes without meiosis (by apomixis), it has been suggested that somatic cells in the ovule are competent to respond to a local signal likely to have an important function in determination. Here we show that the Arabidopsis protein ARGONAUTE 9 (AGO9) controls female gamete formation by restricting the specification of gametophyte precursors in a dosage-dependent, non-cell-autonomous manner. Mutations in AGO9 lead to the differentiation of multiple gametic cells that are able to initiate gametogenesis. The AGO9 protein is not expressed in the gamete lineage; instead, it is expressed in cytoplasmic foci of somatic companion cells. Mutations in SUPPRESSOR OF GENE SILENCING 3 and RNA-DEPENDENT RNA POLYMERASE 6 exhibit an identical defect to ago9 mutants, indicating that the movement of small RNA (sRNAs) silencing out of somatic companion cells is necessary for controlling the specification of gametic cells. AGO9 preferentially interacts with 24-nucleotide sRNAs derived from transposable elements (TEs), and its activity is necessary to silence TEs in female gametes and their accessory cells. Our results show that AGO9-dependent sRNA silencing is crucial to specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA-dependent silencing in plant gametes.

Embryo and endosperm inherit distinct chromatin and transcriptional states from the female gametes in Arabidopsis.

Whether deposited maternal products are important during early seed development in flowering plants remains controversial. Here, we show that RNA interference-mediated downregulation of transcription is deleterious to endosperm development but does not block zygotic divisions. Furthermore, we show that RNA POLYMERASE II is less active in the embryo than in the endosperm. This dimorphic pattern is established late during female gametogenesis and is inherited by the two products of fertilization. This juxtaposition of distinct transcriptional activities correlates with differential patterns of histone H3 lysine 9 dimethylation, LIKE HETEROCHROMATIN PROTEIN1 localization, and Histone H2B turnover in the egg cell versus the central cell. Thus, distinct epigenetic and transcriptional patterns in the embryo and endosperm are already established in their gametic progenitors. We further demonstrate that the non-CG DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and DEMETER-LIKE DNA glycosylases are required for the correct distribution of H3K9 dimethylation in the egg and central cells, respectively, and that plants defective for CMT3 activity show abnormal embryo development. Our results provide evidence that cell-specific mechanisms lead to the differentiation of epigenetically distinct female gametes in Arabidopsis thaliana. They also suggest that the establishment of a quiescent state in the zygote may play a role in the reprogramming of the young plant embryo.

3D cellular morphometrics of ovule primordium development in Zea mays reveal differential division and growth dynamics specifying megaspore mother cell singleness.

Introduction: Differentiation of spore mother cells marks the somatic-to-reproductive transition in higher plants. Spore mother cells are critical for fitness because they differentiate into gametes, leading to fertilization and seed formation. The female spore mother cell is called the megaspore mother cell (MMC) and is specified in the ovule primordium. The number of MMCs varies by species and genetic background, but in most cases, only a single mature MMC enters meiosis to form the embryo sac. Multiple candidate MMC precursor cells have been identified in both rice and Arabidopsis, so variability in MMC number is likely due to conserved early morphogenetic events. In Arabidopsis, the restriction of a single MMC per ovule, or MMC singleness, is determined by ovule geometry. To look for potential conservation of MMC ontogeny and specification mechanisms, we undertook a morphogenetic description of ovule primordium growth at cellular resolution in the model crop maize. Methods: We generated a collection of 48 three-dimensional (3D) ovule primordium images for five developmental stages, annotated for 11 cell types. Quantitative analysis of ovule and cell morphological descriptors allowed the reconstruction of a plausible developmental trajectory of the MMC and its neighbors. Results: The MMC is specified within a niche of enlarged, homogenous L2 cells, forming a pool of candidate archesporial (MMC progenitor) cells. A prevalent periclinal division of the uppermost central archesporial cell formed the apical MMC and the underlying cell, a presumptive stack cell. The MMC stopped dividing and expanded, acquiring an anisotropic, trapezoidal shape. By contrast, periclinal divisions continued in L2 neighbor cells, resulting in a single central MMC. Discussion: We propose a model where anisotropic ovule growth in maize drives L2 divisions and MMC elongation, coupling ovule geometry with MMC fate.

High-frequency synthetic apomixis in hybrid rice.

Introducing asexual reproduction through seeds - apomixis - into crop species could revolutionize agriculture by allowing F1 hybrids with enhanced yield and stability to be clonally propagated. Engineering synthetic apomixis has proven feasible in inbred rice through the inactivation of three genes (MiMe), which results in the conversion of meiosis into mitosis in a line ectopically expressing the BABYBOOM1 (BBM1) parthenogenetic trigger in egg cells. However, only 10-30% of the seeds are clonal. Here, we show that synthetic apomixis can be achieved in an F1 hybrid of rice by inducing MiMe mutations and egg cell expression of BBM1 in a single step. We generate hybrid plants that produce more than 95% of clonal seeds across multiple generations. Clonal apomictic plants maintain the phenotype of the F1 hybrid along successive generations. Our results demonstrate that there is no barrier to almost fully penetrant synthetic apomixis in an important crop species, rendering it compatible with use in agriculture.

SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria.

Root endosymbioses vitally contribute to plant nutrition and fitness worldwide. Nitrogen-fixing root nodulation, confined to four plant orders, encompasses two distinct types of associations, the interaction of legumes (Fabales) with rhizobia bacteria and actinorhizal symbioses, where the bacterial symbionts are actinomycetes of the genus Frankia. Although several genetic components of the host-symbiont interaction have been identified in legumes, the genetic basis of actinorhiza formation is unknown. Here, we show that the receptor-like kinase gene SymRK, which is required for nodulation in legumes, is also necessary for actinorhiza formation in the tree Casuarina glauca. This indicates that both types of nodulation symbiosis share genetic components. Like several other legume genes involved in the interaction with rhizobia, SymRK is also required for the interaction with arbuscular mycorrhiza (AM) fungi. We show that SymRK is involved in AM formation in C. glauca as well and can restore both nodulation and AM symbioses in a Lotus japonicus symrk mutant. Taken together, our results demonstrate that SymRK functions as a vital component of the genetic basis for both plant-fungal and plant-bacterial endosymbioses and is conserved between legumes and actinorhiza-forming Fagales.

Proceedings of the 7th Series of Seminars on Advances in Apomixis Research.

These proceedings contain the abstracts for the presentations given at the 7th biennial Seminars on Advances in Apomixis Research, held virtually on 2-3 and 9 December 2020. The first day hosted the kick-off meeting of the EU-funded Mechanisms of Apomictic Development (MAD) project, while the remaining days were dedicated to oral presentations and in-depth exchanges on the latest progress in the field of apomixis and plant reproductive biology research.

Organ geometry channels reproductive cell fate in the Arabidopsis ovule primordium.

In multicellular organisms, sexual reproduction requires the separation of the germline from the soma. In flowering plants, the female germline precursor differentiates as a single spore mother cell (SMC) as the ovule primordium forms. Here, we explored how organ growth contributes to SMC differentiation. We generated 92 annotated 3D images at cellular resolution in Arabidopsis. We identified the spatio-temporal pattern of cell division that acts in a domain-specific manner as the primordium forms. Tissue growth models uncovered plausible morphogenetic principles involving a spatially confined growth signal, differential mechanical properties, and cell growth anisotropy. Our analysis revealed that SMC characteristics first arise in more than one cell but SMC fate becomes progressively restricted to a single cell during organ growth. Altered primordium geometry coincided with a delay in the fate restriction process in katanin mutants. Altogether, our study suggests that tissue geometry channels reproductive cell fate in the Arabidopsis ovule primordium.

What is quantitative plant biology?

Quantitative plant biology is an interdisciplinary field that builds on a long history of biomathematics and biophysics. Today, thanks to high spatiotemporal resolution tools and computational modelling, it sets a new standard in plant science. Acquired data, whether molecular, geometric or mechanical, are quantified, statistically assessed and integrated at multiple scales and across fields. They feed testable predictions that, in turn, guide further experimental tests. Quantitative features such as variability, noise, robustness, delays or feedback loops are included to account for the inner dynamics of plants and their interactions with the environment. Here, we present the main features of this ongoing revolution, through new questions around signalling networks, tissue topology, shape plasticity, biomechanics, bioenergetics, ecology and engineering. In the end, quantitative plant biology allows us to question and better understand our interactions with plants. In turn, this field opens the door to transdisciplinary projects with the society, notably through citizen science.

H3.1 Eviction Marks Female Germline Precursors in Arabidopsis.

In flowering plants, germline precursors are differentiated from somatic cells. The female germline precursor of Arabidopsis thaliana is located in the internal (nucellar) tissue of the ovule, and is known as the Megaspore Mother Cell (MMC). MMC differentiation in Arabidopsis occurs when a cell in the subepidermal layer of the nucellar apex enters the meiotic program. Increasing evidence has demonstrated that MMC specification is a plastic process where the number and developmental outcome of MMCs are variable. During its differentiation, the MMC displays specific chromatin hallmarks that distinguish it from other cells within the primordium. To date, these signatures have been only analyzed at developmental stages where the MMC is morphologically conspicuous, and their role in reproductive fate acquisition remains to be elucidated. Here, we show that the histone 3 variant H3.1 HISTONE THREE RELATED 13 (HTR13) can be evicted in multiple subepidermal cells of the nucellus, but that H3.1 eviction persists only in the MMC. This pattern is established very early in ovule development and is reminiscent of the specific eviction of H3.1 that marks cell cycle exit in other somatic cell types, such as the root quiescent center (QC) of Arabidopsis. Our findings suggest that cell cycle progression in the subepidermal region of the ovule apex is modified very early in development and is associated with plasticity of reproductive fate acquisition.

Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata.

In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the beta-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP(+) hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.

Localization of Chromatin Marks in Arabidopsis Early Embryos.

During early embryo development, profound changes in chromatin structure and regulation take place. It is difficult to study these changes in plant embryos however, largely because of their relative inaccessibility, which impedes the application of current epigenomic and biochemistry protocols. To circumvent this issue and to analyze the epigenetic status of the embryo at both the cellular and subcellular level, we describe here a simple method to immunolocalize chromatin marks in whole mount early Arabidopsis embryos, either within maternal tissues or isolated from seeds. We show that this protocol can be combined with fluorescent protein markers, allowing for the simultaneous detection of several chromatin components and/or cell fate markers. This new protocol will facilitate deciphering the epigenetic circuits controlling early embryogenesis in plants.

Genomic imprinting in plants: the epigenetic version of an Oedipus complex.

Genomic imprinting is a mitotically stable epigenetic modification that results in the functional non-equivalency of both parental genomes following fertilization. In flowering plants, studies of parent-of-origin effects have mostly identified genes that are only transcribed from a maternally inherited allele. In Arabidopsis, the Polycomb group protein MEDEA regulates seed development through the expression of the MADS-box gene PHERES1. Activation of the maternal MEDEA allele requires the function of DEMETER, a plant DNA glycosylase that also controls the transcriptional activity of the maternally inherited allele of the late-flowering gene FWA. Current studies of parent-of-origin effects have mostly identified genes that are only transcribed from a maternally inherited allele. Our current understanding of parent-of-origin effects could represent a new form of an Oedipus complex in which flowering plants prefer to rely transcriptionally on their maternal rather than their paternal chromosomes to ensure normal initiation of seed development.

Enhancer detection and gene trapping as tools for functional genomics in plants.

Although more than 25,000 genes of Arabidopsis thaliana have been sequenced and mapped, adequate expression or functional information is available for less than 15% of them. In the case of Oryza sativa (rice), about half of more than 55,000 predicted genes have been assigned to a vague functional category on the basis of their sequence, but fewer than 100 have been ascribed a precise, verified function after the identification of a mutant phenotype caused by the molecular disruption of the corresponding gene. Enhancer detection and gene trapping represent insertional mutagenesis strategies that report random expression of many genes and often generate loss-of-function mutations. Several trapping vectors have been designed in a limited number of species, and large-scale enhancer detection and gene trap screens that aim to generate a wide range of spatially and temporally restricted expression patterns have been initiated in both Arabidopsis and rice. These strategies are proving to be essential to the functional annotation of completely sequenced genomes, enabling the analysis of gene function in the context of the entire plant life cycle and substantially expanding our understanding of plant growth and development.

Parental contributions to the transcriptome of early plant embryos.

In plants and animals, embryo development becomes ultimately controlled by zygotic genes, but the timing of zygotic genome activation (ZGA) varies greatly between organisms. We recently showed that the transcriptome of young Arabidopsis embryos is dominated by maternal transcripts with a progressive ZGA under the maternal control of epigenetic pathways. In contrast, another study reported that both parental genomes contribute equally to the transcriptome of young embryos, suggesting that ZGA occurs immediately after fertilization. How to explain such dramatic differences? We propose that the discrepancies between these two studies likely reflect genuine biological differences between the two experiments, paving the road towards exciting discoveries on ZGA mechanisms in plants.

A role for CHROMOMETHYLASE3 in mediating transposon and euchromatin silencing during egg cell reprogramming in Arabidopsis.

During embryogenesis there is a major switch from dependence upon maternally-deposited products to reliance on products of the zygotic genome. In animals, this so-called maternal-to-zygotic transition occurs following a period of transcriptional quiescence. Recently, we have shown that the early embryo in Arabidopsis is also quiescent, a state inherited from the female gamete and linked to specific patterns of H3K9 dimethylation and TERMINAL FLOWER2 (TFL2) localization. We also demonstrated that CHROMOMETHYLASE 3 (CMT3) is required for H3K9 dimethylation in the egg cell and for normal embryogenesis during the first few divisions of the zygote. Subsequent analysis of CMT3 mutants points to a key role in egg cell reprogramming by controlling silencing in both transposon and euchromatic regions. A speculative model of the CMT3-induced egg cell silencing is presented here, based on these results and current data from the literature suggesting the potential involvement of small RNAs targeted to the egg cell, a process conceptually similar to the division of labor described in the male gametophyte for which we show that H3K9 modifications and TFL2 localization are reminiscent of the female gametophyte.

Cell numbers and leaf development in Arabidopsis: a functional analysis of the STRUWWELPETER gene.

The struwwelpeter (swp) mutant in Arabidopsis shows reduced cell numbers in all aerial organs. In certain cases, this defect is partially compensated by an increase in final cell size. Although the mutation does not affect cell cycle duration in the young primordia, it does influence the window of cell proliferation, as cell number is reduced during the very early stages of primordium initiation and a precocious arrest of cell proliferation occurs. In addition, the mutation also perturbs the shoot apical meristem (SAM), which becomes gradually disorganized. SWP encodes a protein with similarities to subunits of the Mediator complex, required for RNA polymerase II recruitment at target promoters in response to specific activators. To gain further insight into its function, we overexpressed the gene under the control of a constitutive promoter. This interfered again with the moment of cell cycle arrest in the young leaf. Our results suggest that the levels of SWP, besides their role in pattern formation at the meristem, play an important role in defining the duration of cell proliferation.