Three-Dimensional Morphological Analysis Revealed the Cell Patterning Bases for the Sexual Dimorphism Development in the Liverwort Marchantia polymorpha.

In land plants, sexual dimorphism can develop in both diploid sporophytes and haploid gametophytes. While developmental processes of sexual dimorphism have been extensively studied in the sporophytic reproductive organs of model flowering plants such as stamens and carpels of Arabidopsis thaliana, those occurring in gametophyte generation are less well characterized due to the lack of amenable model systems. In this study, we performed three-dimensional morphological analyses of gametophytic sexual branch differentiation in the liverwort Marchantia polymorpha, using high-depth confocal imaging and a computational cell segmentation technique. Our analysis revealed that the specification of germline precursors initiates in a very early stage of sexual branch development, where incipient branch primordia are barely recognizable in the apical notch region. Moreover, spatial distribution patterns of germline precursors differ between males and females from the initial stage of primordium development in a manner dependent on the master sexual differentiation regulator MpFGMYB. At later stages, distribution patterns of germline precursors predict the sex-specific gametangia arrangement and receptacle morphologies seen in mature sexual branches. Taken together, our data suggest a tightly coupled progression of germline segregation and sexual dimorphism development in M. polymorpha.

A cis-acting bidirectional transcription switch controls sexual dimorphism in the liverwort.

Plant life cycles alternate between haploid gametophytes and diploid sporophytes. While regulatory factors determining male and female sexual morphologies have been identified for sporophytic reproductive organs, such as stamens and pistils of angiosperms, those regulating sex-specific traits in the haploid gametophytes that produce male and female gametes and hence are central to plant sexual reproduction are poorly understood. Here, we identified a MYB-type transcription factor, MpFGMYB, as a key regulator of female sexual differentiation in the haploid-dominant dioicous liverwort, Marchantia polymorpha MpFGMYB is specifically expressed in females and its loss resulted in female-to-male sex conversion. Strikingly, MpFGMYB expression is suppressed in males by a cis-acting antisense gene SUF at the same locus, and loss-of-function suf mutations resulted in male-to-female sex conversion. Thus, the bidirectional transcription module at the MpFGMYB/SUF locus acts as a toggle between female and male sexual differentiation in M. polymorpha gametophytes. Arabidopsis thaliana MpFGMYB orthologs are known to be expressed in embryo sacs and promote their development. Thus, phylogenetically related MYB transcription factors regulate female gametophyte development across land plants.

The ancestral chromatin landscape of land plants.

Recent studies have shown that correlations between chromatin modifications and transcription vary among eukaryotes. This is the case for marked differences between the chromatin of the moss Physcomitrium patens and the liverwort Marchantia polymorpha. Mosses and liverworts diverged from hornworts, altogether forming the lineage of bryophytes that shared a common ancestor with land plants. We aimed to describe chromatin in hornworts to establish synapomorphies across bryophytes and approach a definition of the ancestral chromatin organization of land plants. We used genomic methods to define the 3D organization of chromatin and map the chromatin landscape of the model hornwort Anthoceros agrestis. We report that nearly half of the hornwort transposons were associated with facultative heterochromatin and euchromatin and formed the center of topologically associated domains delimited by protein coding genes. Transposons were scattered across autosomes, which contrasted with the dense compartments of constitutive heterochromatin surrounding the centromeres in flowering plants. Most of the features observed in hornworts are also present in liverworts or in mosses but are distinct from flowering plants. Hence, the ancestral genome of bryophytes was likely a patchwork of units of euchromatin interspersed within facultative and constitutive heterochromatin. We propose this genome organization was ancestral to land plants.

Compensation: a key to clarifying the organ-level regulation of lateral organ size in plants.

Leaves are ideal model systems to study the organ size regulation of multicellular plants. Leaf cell number and cell size are determinant factors of leaf size which is controlled through cell proliferation and post-mitotic cell expansion, respectively. To achieve a proper leaf size, cell proliferation and post-mitotic cell expansion should be co-ordinated during leaf morphogenesis. Compensation, which is enhanced post-mitotic cell expansion associated with a decrease in cell number during lateral organ development, is suggestive of such co-ordination. Genetic and kinematic studies revealed at least three classes of modes of compensation, indicating that compensation is a heterogeneous phenomenon. Recent studies have increased our understanding about the molecular basis of compensation by identifying the causal genes of each compensation-exhibiting mutant. Furthermore, analyses using chimeric leaves revealed that a type of compensated cell expansion requires cell-to-cell communication. Information from recent advances in molecular and genetic studies on compensation has been integrated here and its role in organ size regulation is discussed.

The ATM-dependent DNA damage response acts as an upstream trigger for compensation in the fas1 mutation during Arabidopsis leaf development.

During leaf development, a decrease in cell number often triggers an increase in cell size. This phenomenon, called compensation, suggests that some system coordinates cell proliferation and cell expansion, but how this is mediated at the molecular level is still unclear. The fugu2 mutants in Arabidopsis (Arabidopsis thaliana) exhibit typical compensation phenotypes. Here, we report that the FUGU2 gene encodes FASCIATA1 (FAS1), the p150 subunit of Chromatin Assembly Factor1. To uncover how the fas1 mutation induces compensation, we performed microarray analyses and found that many genes involved in the DNA damage response are up-regulated in fas1. Our genetic analysis further showed that activation of the DNA damage response and the accompanying decrease of cell number in fas1 depend on ATAXIA TELANGIECTASIA MUTATED (ATM) but not on ATM AND RAD3 RELATED. Kinematic analysis suggested that the delay in the cell cycle leads to a decrease in cell number in fas1 and that loss of ATM partially restores this phenotype. Consistently, both cell size phenotypes and high ploidy phenotypes of fas1 are also suppressed by atm, supporting that the ATM-dependent DNA damage response leads to these phenotypes. Altogether, these data suggest that the ATM-dependent DNA damage response acts as an upstream trigger in fas1 to delay the cell cycle and promote entry into the endocycle, resulting in compensated cell expansion.

Plant reproduction: Ancient origins of male germline differentiation.

Despite the wide diversity in male sexual development across land plants, new work reveals the conservation of a heterodimer of transcription factors as master regulators of the male germline.

The Polycomb repressive complex 2 deposits H3K27me3 and represses transposable elements in a broad range of eukaryotes.

The mobility of transposable elements (TEs) contributes to evolution of genomes. Their uncontrolled activity causes genomic instability; therefore, expression of TEs is silenced by host genomes. TEs are marked with DNA and H3K9 methylation, which are associated with silencing in flowering plants, animals, and fungi. However, in distantly related groups of eukaryotes, TEs are marked by H3K27me3 deposited by the Polycomb repressive complex 2 (PRC2), an epigenetic mark associated with gene silencing in flowering plants and animals. The direct silencing of TEs by PRC2 has so far only been shown in one species of ciliates. To test if PRC2 silences TEs in a broader range of eukaryotes, we generated mutants with reduced PRC2 activity and analyzed the role of PRC2 in extant species along the lineage of Archaeplastida and in the diatom P. tricornutum. In this diatom and the red alga C. merolae, a greater proportion of TEs than genes were repressed by PRC2, whereas a greater proportion of genes than TEs were repressed by PRC2 in bryophytes. In flowering plants, TEs contained potential cis-elements recognized by transcription factors and associated with neighbor genes as transcriptional units repressed by PRC2. Thus, silencing of TEs by PRC2 is observed not only in Archaeplastida but also in diatoms and ciliates, suggesting that PRC2 deposited H3K27me3 to silence TEs in the last common ancestor of eukaryotes. We hypothesize that during the evolution of Archaeplastida, TE fragments marked with H3K27me3 were selected to shape transcriptional regulation, controlling networks of genes regulated by PRC2.

Polycomb-mediated repression of paternal chromosomes maintains haploid dosage in diploid embryos of Marchantia.

Complex mechanisms regulate gene dosage throughout eukaryotic life cycles. Mechanisms controlling gene dosage have been extensively studied in animals, however it is unknown how generalizable these mechanisms are to diverse eukaryotes. Here, we use the haploid plant Marchantia polymorpha to assess gene dosage control in its short-lived diploid embryo. We show that throughout embryogenesis, paternal chromosomes are repressed resulting in functional haploidy. The paternal genome is targeted for genomic imprinting by the Polycomb mark H3K27me3 starting at fertilization, rendering the maternal genome in control of embryogenesis. Maintaining haploid gene dosage by this new form of imprinting is essential for embryonic development. Our findings illustrate how haploid-dominant species can regulate gene dosage through paternal chromosome inactivation and initiates the exploration of the link between life cycle history and gene dosage in a broader range of organisms.

The reproductive cells of organisms that reproduce sexually ­ the egg and the sperm ­ each contain one copy of the organism's genome. An embryo forms upon fertilization of an egg by a sperm cell. This embryo contains two copies of the genome, one from each parent. Under most circumstances, it does not matter which parent a gene copy came from: both gene copies are expressed. However, in some species genes coming from only one of the parents are switched on. This unusual mode of gene expression is called genomic imprinting. The best-known example of this occurs in female mammals, which repress the genes on the paternal X chromosome. Genomic imprinting also exists in flowering plants. Both mammals and flowering plants evolved tissues that channel nutrients from the mother to the embryo during development; the placenta and the endosperm, respectively. Genomic imprinting had, until now, only been described in these two types of organisms. It was unknown whether imprinting also happens in other organisms, and specifically those in which embryos develop inside the mother but without the help of a placenta or endosperm. Here Montgomery et al. addressed this question by studying the liverwort, Marchantia polymorpha, a moss-like plant. Initial experiments showed that cells in the liverwort embryo mostly expressed the genes coming from the egg, and not the sperm. All the genetic material coming from the sperm had a molecular marker or tag called H3K27me3. This mark, which also appears on the paternal X chromosome in female mammals, switches off the genes it tags. M. polymorpha embryos thus suppress gene expression from all of the genetic material from the father, relying only on maternal genetic material for development. When Montgomery et al. deleted the maternal genes necessary for making the H3K27me3 mark, the paternal genes switched on, and this led to the death of the embryos. The survival of M. polymorpha embryos therefore depended on keeping only one set of genes active. Taken together these experiments indicate that genomic imprinting evolved about 480 million years ago, about 320 million years earlier than previously thought, in organisms for which embryo development depended only on one parent. This means there are likely many more organisms that control gene expression in this way, opening up opportunities for further research. Understanding imprinting in more detail will also shed light on how sexual reproduction evolved.

Deep evolutionary origin of gamete-directed zygote activation by KNOX/BELL transcription factors in green plants.

KNOX and BELL transcription factors regulate distinct steps of diploid development in plants. In the green alga Chlamydomonas reinhardtii, KNOX and BELL proteins are inherited by gametes of the opposite mating types and heterodimerize in zygotes to activate diploid development. By contrast, in land plants such as Physcomitrium patens and Arabidopsis thaliana, KNOX and BELL proteins function in meristem maintenance and organogenesis during the later stages of diploid development. However, whether the contrasting functions of KNOX and BELL were acquired independently in algae and land plants is currently unknown. Here, we show that in the basal land plant species Marchantia polymorpha, gamete-expressed KNOX and BELL are required to initiate zygotic development by promoting nuclear fusion in a manner strikingly similar to that in C. reinhardtii. Our results indicate that zygote activation is the ancestral role of KNOX/BELL transcription factors, which shifted toward meristem maintenance as land plants evolved.

Building new insights in plant gametogenesis from an evolutionary perspective.

Extant bryophytes are thought to preserve characteristics of ancestral land plants, with a life cycle dominated by the haploid gametophyte. The gametophyte produces gametes in specialized organs that differentiate after an extensive phase of vegetative development. During land plant evolution, these organs became extremely reduced. As a result, in flowers of angiosperms the haploid phase of the life cycle is reduced to few-celled gametophytes, namely the embryo sac (female) and pollen (male). Although many factors contributing to gametogenesis have been identified in flowering plants, the extreme reduction of the gametophytes has prevented a clear molecular dissection of key processes of gametogenesis. Recent studies in the model bryophyte Marchantia polymorpha have identified conserved transcription factors regulating the equivalent steps in the sexual reproduction of land plants. These include FEMALE GAMETOPHYTE MYB for female gametophyte development, BONOBO for gamete progenitor cell specification, DUO POLLEN1 for sperm differentiation and members of the RWP-RK domain family for female gamete formation. These studies demonstrate that M. polymorpha is a powerful model to untangle the core processes of gametogenesis in land plants. We anticipate that a deeper understanding of gametogenesis in bryophytes will circumscribe the origin of plant germ cells and define the differentiation programmes of sperm and eggs.

Plant-inspired pipettes.

The female sex organ of the liverwort (Marchantia polymorpha) has a characteristic parasol-like form highly suitable for collecting water droplets containing sperm for fertilization. Motivated by this observation and using three-dimensional printing techniques, we develop a parasol-like rigid object that can grab, transport and release water droplets of a maximum size of about 1 cm. By combining experiments and scaling theory, we quantify the object's fundamental wetting and fluid dynamical properties. We construct a stability phase diagram and suggest that it is largely insensitive to properties of liquids such as surface tension and viscosity. A simple scaling argument is developed to explain the phase boundary. Our study provides basic design rules of a simple pipette-like device with bubble-free capture and drop of liquids, which can be used in laboratory settings and has applications within soft robotics. Through systematic experimental investigations, we suggest the optimal design criteria of the liverwort-inspired object to achieve maximal pipetting performance. We also provide, based on our scalable model experiments, a biological implication for the mechanistic advantage of this structure in liverwort reproduction.

An Evolutionarily Conserved Plant RKD Factor Controls Germ Cell Differentiation.

In contrast to animals, in which the germ cell lineage is established during embryogenesis, plant germ cells are generated in reproductive organs via reprogramming of somatic cells. The factors that control germ cell differentiation and reprogramming in plants are poorly understood. Members of the RKD subfamily of plant-specific RWP-RK transcription factors have been implicated in egg cell formation in Arabidopsis based on their expression patterns and ability to cause an egg-like transcriptome upon ectopic expression [1]; however, genetic evidence of their involvement is lacking, due to possible genetic redundancy, haploid lethality, and the technical difficulty of analyzing egg cell differentiation in angiosperms. Here we analyzed the factors that govern germ cell formation in the liverwort Marchantia polymorpha. This recently revived model bryophyte has several characteristics that make it ideal for studies of germ cell formation, such as low levels of genetic redundancy, readily accessible germ cells, and the ability to propagate asexually via gemma formation [2, 3]. Our analyses revealed that MpRKD, a single RWP-RK factor closely related to angiosperm RKDs, is preferentially expressed in developing eggs and sperm precursors in M. polymorpha. Targeted disruption of MpRKD had no effect on the gross morphology of the vegetative and reproductive organs but led to striking defects in egg and sperm cell differentiation, demonstrating that MpRKD is an essential regulator of germ cell differentiation. Together with previous findings [1, 4-6], our results suggest that RKD factors are evolutionarily conserved regulators of germ cell differentiation in land plants.

Small RNAs as positional signal for pattern formation.

Pattern formation in plant relies on intimate cell-cell communication exchanging positional information. While ligand-receptor interaction is commonly used by plants and animals as a means to transmit positional information, plant cells can directly exchange regulatory molecules such as transcription factors through a cytoplasmic continuum called the plasmodesmata. Recently endogenous small RNAs (sRNAs) of various biogenetic origins have been shown to function non-cell-autonomously. To date, non-cell-autonomous sRNAs have been shown to regulate leaf polarity, root vascular patterning, meristem formation in embryos, shoot meristem maintenance and female gametogenesis. All these developmental processes are fundamental to the life cycle and architecture of flowering plants, suggesting that sRNA-mediated cell-to-cell signaling has been adopted to achieve novel morphology in the course of plant evolution.