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The liverwort Marchantia polymorpha is equipped with a wide range of molecular and genetic tools and resources that have led to its wide use to explore the evo-devo aspects of land plants. Although its diverse transcriptome data are rapidly accumulating, there is no extensive yet user-friendly tool to exploit such a compilation of data and to summarize results with the latest annotations. Here, we have developed a web-based suite of tools, MarpolBase Expression (MBEX, https://marchantia.info/mbex/), where users can visualize gene expression profiles, identify differentially expressed genes, perform co-expression and functional enrichment analyses, and summarize their comprehensive output in various portable formats. Using oil body biogenesis as an example, we demonstrated that the results generated by MBEX were consistent with the published experimental evidence and also revealed a novel transcriptional network in this process. MBEX should facilitate the exploration and discovery of the genetic and functional networks behind various biological processes in M. polymorpha, and promote our understanding of the evolution of land plants.

Background Land plants commonly produce red pigmentation as a response to environmental stressors, both abiotic and biotic. The type of pigment produced varies among different land plant lineages. In the majority of species they are flavonoids, a large branch of the phenylpropanoid pathway. Flavonoids that can confer red colours include 3-hydroxyanthocyanins, 3-deoxyanthocyanins, sphagnorubins and auronidins, which are the predominant red pigments in flowering plants, ferns, mosses and liverworts, respectively. However, some flowering plants have lost the capacity for anthocyanin biosynthesis and produce nitrogen-containing betalain pigments instead. Some terrestrial algal species also produce red pigmentation as an abiotic stress response, and these include both carotenoid and phenolic pigments. Scope In this review, we examine: which environmental triggers induce red pigmentation in non-reproductive tissues; theories on the functions of stress-induced pigmentation; the evolution of the biosynthetic pathways; and structure–function aspects of different pigment types. We also compare data on stress-induced pigmentation in land plants with those for terrestrial algae, and discuss possible explanations for the lack of red pigmentation in the hornwort lineage of land plants. Conclusions The evidence suggests that pigment biosynthetic pathways have evolved numerous times in land plants to provide compounds that have red colour to screen damaging photosynthetically active radiation but that also have secondary functions that provide specific benefits to the particular land plant lineage.

In angiosperms, KANADI transcription factors have roles in the sporophyte generation regulating tissue polarity, organogenesis and shade avoidance responses, but are not required during the gametophyte generation. Whether these roles are conserved in the gametophyte-dominant bryophyte lineages is unknown, which we examined by characterising the sole KANADI ortholog, MpKAN, in the liverwort Marchantia polymorpha. In contrast to angiosperm orthologs, MpKAN functions in the gametophyte generation in Marchantia, where it regulates apical branching and tissue differentiation, but does not influence tissue polarity in either generation. MpKAN can partially rescue the sporophyte polarity defects of kanadi mutants in Arabidopsis, indicating that MpKAN has conserved biochemical activity to its angiosperm counterparts. Mpkan loss-of-function plants display defects in far-red (FR) light responses. Mpkan plants have reduced FR-induced growth tropisms, have a delayed transition to sexual reproduction and fail to correctly form gametangiophores. Our results indicate that MpKAN is a modulator of FR responses, which may reflect a conserved role for KANADI across land plants. Under FR, MpKAN negatively regulates MpDELLA expression, suggesting that MpKAN and MpDELLA act in a pathway regulating FR responses, placing MpKAN in a gene regulatory network exhibiting similarities with those of angiosperms.

Why do some genomes stay small and simple, while others become huge, and why are some genomes more stable? In contrast to angiosperms and gymnosperms, liverworts are characterized by small genomes with low variation in size and conserved chromosome numbers. We quantified genome evolution among five Marchantiophyta (liverworts), measuring gene characteristics, transposable element (TE) landscape, collinearity, and sex chromosome evolution that might explain the small size and limited variability of liverwort genomes. No genome duplications were identified among examined liverworts and levels of duplicated genes are low. Among the liverwort species, Lunularia cruciata stands out with a genome size almost twice that of the other liverwort species investigated here, and most of this increased size is due to bursts of Ty3/Gypsy retrotransposons. Intrachromosomal rearrangements between examined liverworts are abundant but occur at a slower rate compared with angiosperms. Most genes on L. cruciata scaffolds have their orthologs on homologous Marchantia polymorpha chromosomes, indicating a low degree of rearrangements between chromosomes. Still, translocation of a fragment of the female U chromosome to an autosome was predicted from our data, which might explain the uniquely small U chromosome in L. cruciata. Low levels of gene duplication, TE activity, and chromosomal rearrangements might contribute to the apparent slow rate of morphological evolution in liverworts.

Plant cells are surrounded by a cell wall, a rigid structure that is not only important for cell and organ shape, but crucial for intercellular communication and interactions with the environment. In the flowering plant Arabidopsis thaliana, the 17 members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase family are involved in a multitude of physiological and developmental processes, making it difficult to assess their primary or ancestral function. To reduce genetic complexity, we characterized the single CrRLK1L gene of Marchantia polymorpha, MpFERONIA (MpFER). Plants with reduced MpFER levels show defects in vegetative development, i.e., rhizoid formation and cell expansion, and have reduced male fertility. In contrast, Mpfer null mutants and overexpression lines severely affect cell integrity and morphogenesis of the gametophyte. Thus, the CrRLK1L gene family originated from a single gene with an ancestral function in cell expansion and the maintenance of cellular integrity. During land plant evolution, this ancestral gene diversified to fulfil a multitude of specialized physiological and developmental and roles in the formation of both gametophytic and sporophytic structures essential to the life cycle of flowering plants.Summary statementThe CrRLK1L family arose in land plants and the Marchantia polymorpha genome contains a single copy, MpFER, which is broadly expressed and regulates cell expansion and cell wall integrity.

In seed plants, 1-aminocyclopropane-1-carboxylic acid (ACC) is the precursor of the plant hormone ethylene but also has ethylene-independent signaling roles. Nonseed plants produce ACC but do not efficiently convert it to ethylene. In Arabidopsis thaliana, ACC is transported by amino acid transporters, LYSINE HISTIDINE TRANSPORTER 1 (LHT1) and LHT2. In nonseed plants, LHT homologs have been uncharacterized. Here, we isolated an ACC-insensitive mutant (Mpain) that is defective in ACC uptake in the liverwort Marchantia polymorpha. Mpain contained a frameshift mutation (1 bp deletion) in the MpLHT1 coding sequence, and was complemented by expression of a wild-type MpLHT1 transgene. Additionally, ACC insensitivity was re-created in CRISPR/Cas9-Mplht1 knockout mutants. We found that MpLHT1 can also transport l-hydroxyproline and l-histidine. We examined the physiological functions of MpLHT1 in vegetative growth and reproduction based on mutant phenotypes. Mpain and Mplht1 plants were smaller and developed fewer gemmae cups compared to wild-type plants. Mplht1 mutants also had reduced fertility, and archegoniophores displayed early senescence. These findings reveal that MpLHT1 serves as an ACC and amino acid transporter in M. polymorpha and has diverse physiological functions. We propose that MpLHT1 contributes to homeostasis of ACC and other amino acids in M. polymorpha growth and reproduction.

Fruits can be divided into dry and fleshy types. Dry fruits mature through senescence and fleshy fruits through ripening. Previous studies have indicated that partially common molecular networks could govern fruit maturation in these different fruit types. However, the nature of such networks remains obscure. CLASS-II KNOX genes were shown to regulate the senescence of the Arabidopsis (Arabidopsis thaliana) dry fruits, the siliques, but their roles in fleshy-fruit development are unknown. Here, we investigated the roles of the tomato (Solanum lycopersicum) CLASS-II KNOX (TKN-II) genes in fleshy fruit ripening using knockout alleles of individual genes and an artificial microRNA line (35S:amiR-TKN-II) simultaneously targeting all genes. 35S:amiR-TKN-II plants, as well as a subset of tkn-II single and double mutants, have smaller fruits. Strikingly, the 35S:amiR-TKN-II and tknII3 tknII7/+ fruits showed early ripening of the locular domain while their pericarp ripening was stalled. Further examination of the ripening marker-gene RIPENING INHIBITOR (RIN) expression and 35S:amiR-TKN-II rin-1 mutant fruits suggested that TKN-II genes arrest RIN activity at the locular domain and promote it in the pericarp. These findings imply that CLASS-II KNOX genes redundantly coordinate maturation in both dry and fleshy fruits. In tomato, these genes also control spatial patterns of fruit ripening, utilizing differential regulation of RIN activity at different fruit domains.

The origin of a land flora fundamentally shifted the course of evolution of life on earth, facilitating terrestrialization of other eukaryotic lineages and altering the planet's geology, from changing atmospheric and hydrological cycles to transforming continental erosion processes. Despite algal lineages inhabiting the terrestrial environment for a considerable preceding period, they failed to evolve complex multicellularity necessary to conquer the land. About 470 million years ago, one lineage of charophycean alga evolved complex multicellularity via developmental innovations in both haploid and diploid generations and became land plants (embryophytes), which rapidly diversified to dominate most terrestrial habitats. Genome sequences have provided unprecedented insights into the genetic and genomic bases for embryophyte origins, with some embryophyte-specific genes being associated with the evolution of key developmental or physiological attributes, such as meristems, rhizoids and the ability to form mycorrhizal associations. However, based on the fossil record, the evolution of the defining feature of embryophytes, the embryo, and consequently the sporangium that provided a reproductive advantage, may have been most critical in their rise to dominance. The long timeframe and singularity of a land flora were perhaps due to the stepwise assembly of a large constellation of genetic innovations required to conquer the terrestrial environment.

The liverwort Marchantia polymorpha has been known to man for millennia due to its inclusion Greek herbals. Perhaps due to its familiarity and association with growth in, often, man-made disturbed habitats, it was readily used to address fundamental biological questions of the day, including elucidation of land plant life cycles in the late 18th century, the formulation of cell theory early in the 19th century and the discovery of the alternation of generations in land plants in the mid-19th century. Subsequently, Marchantia was used as model in botany classes. With the arrival of the molecular era, its organellar genomes, the chloroplast and mitochondrial, were some of the first to be sequenced from any plant. In the past two decades, molecular genetic tools have been applied such that genes may be manipulated seemingly at will. Here, are past, present, and some views to the future of Marchantia as a model.

Life on land exposes plants to varied abiotic and biotic environmental stresses. These environmental drivers contributed to a large expansion of metabolic capabilities during land plant evolution and species diversification. In this review we summarize knowledge on how the specialized metabolite pathways of bryophytes may contribute to stress tolerance capabilities. Bryophytes are the non-tracheophyte land plant group (comprising the hornworts, liverworts, and mosses) and rapidly diversified following the colonization of land. Mosses and liverworts have as wide a distribution as flowering plants with regard to available environments, able to grow in polar regions through to hot desert landscapes. Yet in contrast to flowering plants, for which the biosynthetic pathways, transcriptional regulation, and compound function of stress tolerance-related metabolite pathways have been extensively characterized, it is only recently that similar data have become available for bryophytes. The bryophyte data are compared with those available for angiosperms, including examining how the differing plant forms of bryophytes and angiosperms may influence specialized metabolite diversity and function. The involvement of stress-induced specialized metabolites in senescence and nutrient response pathways is also discussed.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1’s primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1., Author summary Up to half of the genes predicted from genome projects lack a known biological and biochemical function. Many of these genes are likely to play essential roles but it is difficult to reveal their function because minor changes in the genetic sequence can result in lethality and genetic redundancy can obscure analysis. Genome projects predict that flowering plants have at least two DEM genes that encode a protein of unknown cellular and biochemical function. In this paper, we use multiple combinations of dem mutants in Arabidopsis to show that DEM genes are essential for cell division and gamete viability. Interestingly, gamete viability depends not only on the number of functional copies of DEM genes in the gametes, but also on the number of functional copies of DEM genes in the parent plant that produces the gametes. We also show that DEM proteins interact with RAN, a highly conserved protein that controls cell division in all eukaryotic organisms.

Plants commonly referred to as "bryophytes" belong to three major lineages of non-vascular plants: the liverworts, the hornworts and the mosses. They are unique among land plants in having a dominant haploid generation and a short-lived diploid sporophytic generation. The dynamics of selection acting on a haploid genome differs from those acting on a diploid genome: new mutations are directly exposed to selection. The general aim of this paper is to investigate the diversification rate of bryophytes - measured as silent site substitution rate representing neutral evolution (mutation rate) and the nonsynonymous to synonymous substitution rate ratio (dN/dS) representing selective evolution - and compare it with earlier studies on vascular plants. Results show that the silent site substitution rate is lower for liverworts as compared to angiosperms, but not as low as for gymnosperms. The selection pressure, measured as dN/dS, is not remarkably lower for bryophytes as compared to other diploid dominant plants as would be expected by the masking hypothesis, indicating that other factors are more important than ploidy.

Sex determination is a central process for sexual reproduction and is often regulated by a sex determinant encoded on a sex chromosome. Rules that govern the evolution of sex chromosomes via specialization and degeneration following the evolution of a sex determinant have been well studied in diploid organisms. However, distinct predictions apply to sex chromosomes in organisms where sex is determined in the haploid phase of the life cycle: both sex chromosomes, female U and male V, are expected to maintain their gene functions, even though both are non-recombining. This is in contrast to the X-Y (or Z-W) asymmetry and Y (W) chromosome degeneration in XY (ZW) systems of diploids. Here, we provide evidence that sex chromosomes diverged early during the evolution of haploid liverworts and identify the sex determinant on the Marchantia polymorpha U chromosome. This gene, Feminizer, encodes a member of the plant-specific BASIC PENTACYSTEINE transcription factor family. It triggers female differentiation via regulation of the autosomal sex-determining locus of FEMALE GAMETOPHYTE MYB and SUPPRESSOR OF FEMINIZATION. Phylogenetic analyses of Feminizer and other sex chromosome genes indicate dimorphic sex chromosomes had already been established 430 mya in the ancestral liverwort. Feminizer also plays a role in reproductive induction that is shared with its gametolog on the V chromosome, suggesting an ancestral function, distinct from sex determination, was retained by the gametologs. This implies ancestral functions can be preserved after the acquisition of a sex determination mechanism during the evolution of a dominant haploid sex chromosome system., 半数体生物の性染色体上の性決定遺伝子を解明 --コケがもつ現生生物最古の起源の性染色体--. 京都大学プレスリリース. 2021-11-08.

Plant hormones are master regulators of developmental and genetic mechanisms to deal with diverse environmental cues. Upon phosphate (Pi) limitation, vascular plants modify phytohormone metabolism to coordinate diverse mechanisms to overcome such stress. However, the transcriptional program underlying the hormonal signaling in response to Pi scarcity in early branches of land plant phylogeny remains unclear. Therefore, we explored the transcriptional dynamics of key genes involved in auxin, cytokinin, ethylene, jasmonate, gibberellin, and abscisic acid metabolism in the early divergent land plant Marchantia polymorpha upon Pi starvation. Our RNAseq approach revealed major changes in genes associated with auxin and ethylene biosynthesis. Genes involved in cytokinin synthesis are repressed. Interestingly, genes involved in auxin and ethylene signaling, such as MpARF1 and MpARF2, are upregulated. In contrast, MpARRb is down-regulated. Moreover, genes involved in the synthesis of jasmonates were highly upregulated, but those related to signaling did not change in expression. Our data suggest that auxin and ethylene act as positive regulators of rhizoid development under Pi-limited conditions, whereas cytokinin may act as a negative regulator. The transcriptional behavior of some hormone-related genes in Marchantia is similar to those described in controlling root hair development in arabidopsis, maize, and rice, upon Pi scarcity.

Phosphate (Pi) is a pivotal nutrient that constraints plant development and productivity in natural ecosystems. Land colonization by plants, more than 470 million years ago, evolved adaptive mechanisms to conquer Pi-scarce environments. However, little is known about the molecular basis underlying such adaptations at early branches of plant phylogeny. To shed light on how early divergent plants respond to Pi limitation, we analyzed the morpho-physiological and transcriptional dynamics of Marchantia polymorpha upon Pi starvation. Our phylogenomic analysis highlights some gene networks present since the Chlorophytes and others established in the Streptophytes (e.g., PHR1–SPX1 and STOP1–ALMT1, respectively). At the morpho-physiological level, the response is characterized by the induction of phosphatase activity, media acidification, accumulation of auronidins, reduction of internal Pi concentration, and developmental modifications of rhizoids. The transcriptional response involves the induction of MpPHR1, Pi transporters, lipid turnover enzymes, and MpMYB14, which is an essential transcription factor for auronidins biosynthesis. MpSTOP2 up-regulation correlates with expression changes in genes related to organic acid biosynthesis and transport, suggesting a preference for citrate exudation. An analysis of MpPHR1 binding sequences (P1BS) shows an enrichment of this cis regulatory element in differentially expressed genes. Our study unravels the strategies, at diverse levels of organization, exerted by M. polymorpha to cope with low Pi availability.

The evolution of land plants from a charophycean algal ancestor was accompanied by an increased diversity of regulatory networks, including signaling pathways mediating cellular communication within plants and between plants and the environment. Canonical land plant hormone signaling pathways were originally identified in angiosperms, and comparative studies in basal taxa show that they have been assembled from both ancient and newly evolved components, both before and during land plant evolution. In this review we present our current understanding, and highlight several uncertainties, of the evolution of hormone signaling pathways, focusing on the biosynthetic pathways generating putative ligands and the downstream perception and signaling pathways often leading to transcriptional responses.

The plant hormone ethylene has many roles in growth and development1. In seed plants, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is converted into ethylene by ACC oxidase (ACO), and treatment with ACC induces ethylene responses2. However, non-seed plants lack ACO homologues3–8, which led us to examine the relationship between ACC and ethylene in the liverwort Marchantia polymorpha. Here, we demonstrate that ACC and ethylene can induce divergent growth responses in Marchantia. Ethylene increases plant and gemma size, induces more gemma cups and promotes gemmae dormancy. As predicted, Mpctr1-knockout mutants display constitutive ethylene responses, whereas Mpein3-knockout mutants exhibit ethylene insensitivity. Compared with the wild type, Mpctr1 gemmae have more and larger epidermal cells, whereas Mpein3 gemmae have fewer and smaller epidermal cells, suggesting that ethylene promotes cell division and growth in developing gemmae. By contrast, ACC treatment inhibits gemma growth and development by suppressing cell division, even in the Mpein3-knockout alleles. Knockout mutants of one or both ACC SYNTHASE (ACS) gene homologues produce negligible levels of ACC, have more and larger gemma cups, and have more-expanded thallus branches. Mpacs2 and Mpacs1 Mpacs2 gemmae also display a high frequency of abnormal apical notches (meristems) that are not observed in ethylene mutants. These findings reveal that ethylene and ACC have distinct functions, and suggest that ACC is a signalling molecule in Marchantia. ACC may be an evolutionarily conserved signal that predates its efficient conversion to ethylene in higher plants. Marchantia polymorpha lacks the enzyme that converts ACC into ethylene in higher plants. Genetic characterization of ethylene mutants and treatments with exogenous molecules suggest that, in this species, ACC and ethylene have independent functions.

Eukaryotic life cycles alternate between haploid and diploid phases and in phylogenetically diverse unicellular eukaryotes, expression of paralogous homeodomain genes in the two gametes directs the haploid-to-diploid transition. In the unicellular Chlorophyte alga Chlamydomonas KNOX and BELL TALE-homeodomain genes mediate the transition. Here we demonstrate that in the liverwort Marchantia polymorpha paternal (sperm) expression three of the five phylogenetically diverse BELL genes, MpBELL234, and maternal (egg) expression of MpKNOX1 mediate the haploid-to-diploid transition. Loss-of-function alleles of either result in zygotic or early embryonic arrest. In land plants both the haploid gametophyte and diploid sporophyte are complex multicellular organisms. Expression of MpKNOX1 and two other paralogs, MpBELL1 and MpKNOX2, during sporophyte development is consistent with a later role in patterning the sporophyte. These results indicate that the ancestral mechanism to activate diploid gene expression was retained in early diverging land plants and subsequently co-opted during evolution of the diploid sporophyte body.

Damaging UVB radiation is a major abiotic stress facing land plants. In angiosperms the UV RESISTANCE LOCUS8 (UVR8) photoreceptor coordinates UVB responses, including inducing biosynthesis of protective flavonoids. We characterised the UVB responses of Marchantia polymorpha (marchantia), the model species for the liverwort group of basal plants. Physiological, chemical and transcriptomic analyses were conducted on wild-type marchantia exposed to three different UVB regimes. CRISPR/Cas9 was used to obtain plant lines with mutations for components of the UVB signal pathway or the flavonoid biosynthetic pathway, and transgenics overexpressing the marchantia UVR8 sequence were generated. The mutant and transgenic lines were analysed for changes in flavonoid content, their response to UVB exposure, and transcript abundance of a set of 48 genes that included components of the UVB response pathway characterised for angiosperms. The marchantia UVB response included many components in common with Arabidopsis, including production of UVB-absorbing flavonoids, the central activator role of ELONGATED HYPOCOTYL5 (HY5), and negative feedback regulation by REPRESSOR OF UV-B PHOTOMORPHOGENESIS1 (RUP1). Notable differences included the greater importance of CHALCONE ISOMERASE-LIKE (CHIL). Mutants disrupted in the response pathway (hy5) or flavonoid production (chalcone isomerase, chil) were more easily damaged by UVB. Mutants (rup1) or transgenics (35S:MpMYB14) with increased flavonoid content had increased UVB tolerance. The results suggest that UVR8-mediated flavonoid induction is a UVB tolerance character conserved across land plants and may have been an early adaptation to life on land.

Microbiomes influence plant establishment, development, nutrient acquisition, pathogen defense, and health. Plant microbiomes are shaped by interactions between the microbes and a selection process of host plants that distinguishes between pathogens, commensals, symbionts and transient bacteria. In this work, we explore the microbiomes through massive sequencing of the 16S rRNA genes of microbiomes two Marchantia species of liverworts. We compared microbiomes from M. polymorpha and M. paleacea plants collected in the wild relative to their soils substrates and from plants grown in vitro that were established from gemmae obtained from the same populations of wild plants. Our experimental setup allowed identification of microbes found in both native and in vitro Marchantia species. The main OTUs (97% identity) in Marchantia microbiomes were assigned to the following genera: Methylobacterium, Rhizobium, Paenibacillus, Lysobacter, Pirellula, Steroidobacter, and Bryobacter. The assigned genera correspond to bacteria capable of plant-growth promotion, complex exudate degradation, nitrogen fixation, methylotrophs, and disease-suppressive bacteria, all hosted in the relatively simple anatomy of the plant. Based on their long evolutionary history Marchantia is a promising model to study not only long-term relationships between plants and their microbes but also the transgenerational contribution of microbiomes to plant development and their response to environmental changes.

Contents Summary 409 I. Introduction 409 II. RNA silencing machinery in Marchantia polymorpha 410 III. miRNA prediction by integrating omics approach 410 IV. miRNAs and their targets in Marchantia polymorpha 410 V. Mpo-miR390-mediated MpTAS3 tasiRNA biogenesis and potential tasiARF target MpARF2 414 VI. Artificial miRNA and CRISPR-CAS9 edited MIR gene in Marchantia polymorpha 414 VII. Conclusions 415 Acknowledgements 415 References 415 SUMMARY: The liverwort Marchantia polymorpha occupies an important phylogenetic position for comparative studies of land plant gene regulation. Multiple gene regulatory pathways mediated by small RNAs, including microRNAs (miRNAs), trans-acting short-interfering RNAs, and heterochromatic siRNAs often associated with RNA-dependent DNA methylation, have been characterized in flowering plants. Genes for essential components for all of these small RNA-mediated gene silencing pathways are found in M. polymorpha as well as the moss Phsycomitrella patens, indicating that these pathways existed in the ancestral land plant. However, only seven miRNAs are conserved across land plants, with both ancestral and novel targets identified in M. polymorpha. There is little or no evidence that any of these conserved miRNAs are present in algae. As with other plants investigated, most miRNAs in M. polypmorpha exhibit lineage-specific evolution. Application of artificial miRNA and CRISPR-Cas9 technologies in genetic studies of M. polymorpha provide avenues to further investigate miRNA biology.

Bryophytes are close extant relatives of the ancestral land plant. As such, they have retained many innovations that had enabled the adaptation of early land plants to the terrestrial environment. ...

Eukaryotic life cycles alternate between haploid and diploid phases and in phylogenetically diverse unicellular eukaryotes, expression of paralogous homeodomain genes in gametes primes the haploid-to-diploid transition. In the unicellular chlorophyte alga Chlamydomonas, KNOX and BELL TALE-homeodomain genes mediate this transition. We demonstrate that in the liverwort Marchantia polymorpha, paternal (sperm) expression of three of five phylogenetically diverse BELL genes, MpBELL234, and maternal (egg) expression of both MpKNOX1 and MpBELL34 mediate the haploid-to-diploid transition. Loss-of-function alleles of MpKNOX1 result in zygotic arrest, whereas a loss of either maternal or paternal MpBELL234 results in variable zygotic and early embryonic arrest. Expression of MpKNOX1 and MpBELL34 during diploid sporophyte development is consistent with a later role for these genes in patterning the sporophyte. These results indicate that the ancestral mechanism to activate diploid gene expression was retained in early diverging land plants and subsequently co-opted during evolution of the diploid sporophyte body.

The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a class I homeodomain leucine-zipper (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stress, including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather, Mpc1hdz mutant plants were more susceptible to herbivory, and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time-lapse imaging, we observed that MpC1HDZ expression maxima precede oil body formation, indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory, but not for abiotic stress tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages. Fil: Romani, Facundo Alihuen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Banic, Elizabeta. Max Planck Institute For Plant Breeding Research; Alemania Fil: Florent, Stevie N.. Monash University. Faculty Of Science. School Of Biological Sciences; Australia Fil: Kanazawa, Takehiko. National Institute For Basic Biology; Japón Fil: Goodger, Jason Q. D.. School Of Sciences; Australia Fil: Mentink, Remco A.. Max Planck Institute For Plant Breeding Research; Alemania Fil: Dierschke, Tom. Monash University. Faculty Of Science. School Of Biological Sciences; Australia Fil: Zachgo, Sabine. Onasbruck University; Alemania Fil: Ueda, Takashi. National Institute For Basic Biology; Japón Fil: Bowman, John L.. Monash University. Faculty Of Science. School Of Biological Sciences; Australia Fil: Tsiantis, Miltos. Max Planck Institute For Plant Breeding Research; Alemania Fil: Moreno, Javier Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina

The flavonoid pathway is one of the best characterized specialized metabolite pathways of plants. In angiosperms, the flavonoids have varied roles in assisting with tolerance to abiotic stress and are also key for signaling to pollinators and seed dispersal agents. The pathway is thought to be specific to land plants and to have arisen during the period of land colonization around 550–470 million years ago. In this review we consider current knowledge of the flavonoid pathway in the bryophytes, consisting of the liverworts, hornworts, and mosses. The pathway is less characterized for bryophytes than angiosperms, and the first genetic and molecular studies on bryophytes are finding both commonalities and significant differences in flavonoid biosynthesis and pathway regulation between angiosperms and bryophytes. This includes biosynthetic pathway branches specific to each plant group and the apparent complete absence of flavonoids from the hornworts.

Complex multicellular plant bodies evolved in both generations of land plants. A new study demonstrates that CLAVATA3-like peptides function via conserved receptors in Physcomitrella patens as key molecules for morphological innovation of 3D growth in land plants.

International audience; Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes, and our knowledge of the evolution of chromatin-related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants circa 400 mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional genome organization in an early diverging land plant lineage. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and other repetitive sequences in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres. Although a large fraction of constitutive heterochromatin was marked by H3K9 methylation as in flowering plants, a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein-coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that repressed B compartments were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that, in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

This paper reports results from the second phase of a two-phase FHWA-sponsored project to test and demonstrate empirically the transferability of activity-based model (ABM) systems between regions. With data obtained from the 2008–2009 National Household Travel Survey, researchers estimated ABMs simultaneously for 13 metropolitan regions in seven U.S. states. Statistical tests were used to test transferability, including tests of regional differences in the model coefficients, likelihood ratio tests of model equivalence, and transferability indexes, which measure the degree of model differences. In addition, differences in prediction sensitivity between locally estimated and transferred models were tested. The project overall found evidence in favor of transferability. It also found that parameters associated with land use, logsum accessibilities, and travel time and cost caused the biggest problems with transferability. Finally, the study found that transferring within a state or between regions with similar urban density improves transferability. This paper presents the data, models, and testing methods used in the project and includes details of all tests and results related to the improved transferability associated with model transfers from regions within the same state or with similar urban density. The conclusion of the study was that agencies considering transfer of an ABM from another region would do well to find a region within the same state or with similar urban density that has a model that is well supported by a large household travel survey data set.

The life cycles of eukaryotes alternate between haploid and diploid phases, which are initiated by meiosis and gamete fusion, respectively. In both ascomycete and basidiomycete fungi and chlorophyte algae, the haploid-to-diploid transition is regulated by a pair of paralogous homeodomain protein encoding genes. That a common genetic program controls the haploid-to-diploid transition in phylogenetically disparate eukaryotic lineages suggests this may be the ancestral function for homeodomain proteins. Multicellularity has evolved independently in many eukaryotic lineages in either one or both phases of the life cycle. Organisms, such as land plants, exhibiting a life cycle whereby multicellular bodies develop in both the haploid and diploid phases are often referred to as possessing an alternation of generations. We review recent progress on understanding the genetic basis for the land plant alternation of generations and highlight the roles that homeodomain-encoding genes may have played in the evolution of complex multicellularity in this lineage.

Land plant bodies develop from meristems, groups of pluripotent stem cells, which may persist throughout the life of a plant or, alternatively, have a transitory existence. Early diverging land plants exhibit indeterminate (persistent) growth in their haploid gametophytic generation, whereas later diverging lineages exhibit indeterminate growth in their diploid sporophytic generation, raising the question of whether genetic machinery directing meristematic functions was co-opted between generations. Class III HD-Zip (C3HDZ) genes are required for the establishment and maintenance of shoot apical meristems in flowering plants. We demonstrate that in the moss Physcomitrella patens, C3HDZ genes are expressed in transitory meristems in both the gametophytic and sporophytic generations, but not in the persistent shoot meristem of the gametyphyte. Loss-of-function of P. patens C3HDZ was engineered using ectopic expression of miR166, an endogenous regulator of C3HDZ gene activity. Loss of C3HDZ gene function impaired the function of gametophytic transitory meristematic activity but did not compromise the functioning of the persistent shoot apical meristem during the gametophyte generation. These results argue against a wholesale co-option of meristematic gene regulatory networks from the gametophyte to the sporophyte during land plant evolution, instead suggesting that persistent meristems with a single apical cell in P. patens and persistent complex meristems in flowering plants are regulated by different genetic programs.

Unlike in animals, the life cycle of land plants alternates between two multicellular generations, the haploid gametophyte and the diploid sporophyte [1]. Gamete differentiation initiates the transition from the gametophyte to the sporophyte generation and, upon maturation, the egg cell establishes a quiescent state that is maintained until fertilization. This quiescence represents a hallmark of the gametophyte-sporophyte transition. The underlying molecular mechanisms are complex and best characterized in the flowering plant Arabidopsis thaliana [2-4]. However, only few genes with egg cell-specific expression or defects have been identified [5-10]. Intriguingly, ectopic expression of members of a clade of RWP-RK domain (RKD)-containing transcription factors, which are absent from animal genomes [11-13], can induce an egg cell-like transcriptome in sporophytic cells of A. thaliana. Yet, to date, loss-of-function experiments have not produced phenotypes affecting the egg cell, likely due to genetic redundancy and/or cross-regulation among the five RKD genes of A. thaliana [10]. To reduce genetic complexity, we explored the genome of Marchantia polymorpha, a liverwort belonging to the basal lineage of extant land plants [14-17]. Based on sequence homology, we identified a single M. polymorpha RKD gene, MpRKD, which is orthologous to all five A. thaliana RKD genes. Analysis of the MpRKD expression pattern and characterization of lines with reduced MpRKD activity indicate that it functions as a regulator of gametophyte development and the gametophyte-sporophyte transition. In particular, MpRKD is required to establish and/or maintain the quiescent state of the egg cell in the absence of fertilization.

Indole-3-acetic acid, that is, auxin, is a molecule found in a broad phylogenetic distribution of organisms, from bacteria to eukaryotes. In the ancestral land plant auxin was co-opted to be the paramount phytohormone mediating tropic responses and acting as a facilitator of developmental decisions throughout the life cycle. The evolutionary origins of land plant auxin biology genes can now be traced with reasonable clarity. Genes encoding the two enzymes of the land plant auxin biosynthetic pathway arose in the ancestral land plant by a combination of horizontal gene transfer from bacteria and possible neofunctionalization following gene duplication. Components of the auxin transcriptional signaling network have their origins in ancestral alga genes, with gene duplication and neofunctionalization of key domains allowing integration of a portion of the preexisting transcriptional network with auxin. Knowledge of the roles of orthologous genes in extant charophycean algae is lacking, but could illuminate the ancestral functions of both auxin and the co-opted transcriptional network.