Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

Field Guide to Plant Model Systems.

For the past several decades, advances in plant development, physiology, cell biology, and genetics have relied heavily on the model (or reference) plant Arabidopsis thaliana. Arabidopsis resembles other plants, including crop plants, in many but by no means all respects. Study of Arabidopsis alone provides little information on the evolutionary history of plants, evolutionary differences between species, plants that survive in different environments, or plants that access nutrients and photosynthesize differently. Empowered by the availability of large-scale sequencing and new technologies for investigating gene function, many new plant models are being proposed and studied.

Reflections on the ABC model of flower development.

The formulation of the ABC model by a handful of pioneer plant developmental geneticists was a seminal event in the quest to answer a seemingly simple question: how are flowers formed? Fast forward 30 years and this elegant model has generated a vibrant and diverse community, capturing the imagination of developmental and evolutionary biologists, structuralists, biochemists and molecular biologists alike. Together they have managed to solve many floral mysteries, uncovering the regulatory processes that generate the characteristic spatio-temporal expression patterns of floral homeotic genes, elucidating some of the mechanisms allowing ABC genes to specify distinct organ identities, revealing how evolution tinkers with the ABC to generate morphological diversity, and even shining a light on the origins of the floral gene regulatory network itself. Here we retrace the history of the ABC model, from its genesis to its current form, highlighting specific milestones along the way before drawing attention to some of the unsolved riddles still hidden in the floral alphabet.

The landscape of transcription factor promoter activity during vegetative development in Marchantia.

Transcription factors (TFs) are essential for the regulation of gene expression and cell fate determination. Characterizing the transcriptional activity of TF genes in space and time is a critical step toward understanding complex biological systems. The vegetative gametophyte meristems of bryophytes share some characteristics with the shoot apical meristems of flowering plants. However, the identity and expression profiles of TFs associated with gametophyte organization are largely unknown. With only ∼450 putative TF genes, Marchantia (Marchantia polymorpha) is an outstanding model system for plant systems biology. We have generated a near-complete collection of promoter elements derived from Marchantia TF genes. We experimentally tested reporter fusions for all the TF promoters in the collection and systematically analyzed expression patterns in Marchantia gemmae. This allowed us to build a map of expression domains in early vegetative development and identify a set of TF-derived promoters that are active in the stem-cell zone. The cell markers provide additional tools and insight into the dynamic regulation of the gametophytic meristem and its evolution. In addition, we provide an online database of expression patterns for all promoters in the collection. We expect that these promoter elements will be useful for cell-type-specific expression, synthetic biology applications, and functional genomics.

The single Marchantia polymorpha FERONIA homolog reveals an ancestral role in regulating cellular expansion and integrity.

Plant cells are surrounded by a cell wall, a rigid structure that is not only important for cell and organ shape, but is also 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, cell integrity and morphogenesis of the gametophyte are severely affected in Mpfer null mutants and MpFER overexpression lines. Thus, we conclude that 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 fulfill a multitude of specialized physiological and developmental roles in the formation of both gametophytic and sporophytic structures essential to the life cycle of flowering plants.

The renaissance and enlightenment of Marchantia as a model system.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades, there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Evolution in the Cycles of Life.

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.

DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis.

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.

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.

PIN-FORMED is required for shoot phototropism/gravitropism and facilitates meristem formation in Marchantia polymorpha.

PIN-FORMED auxin efflux transporters, a subclass of which is plasma membrane-localised, mediate a variety of land-plant developmental processes via their polar localisation and subsequent directional auxin transport. We provide the first characterisation of PIN proteins in liverworts using Marchantia polymorpha as a model system. Marchantia polymorpha possesses a single PIN-FORMED gene, whose protein product is predicted to be plasma membrane-localised, MpPIN1. To characterise MpPIN1, we created loss-of-function alleles and produced complementation lines in both M. polymorpha and Arabidopsis. In M. polymorpha, gene expression and protein localisation were tracked using an MpPIN1 transgene encoding a translationally fused fluorescent protein. Overexpression of MpPIN1 can partially complement loss of an orthologous gene, PIN-FORMED1, in Arabidopsis. In M. polymorpha, MpPIN1 influences development in numerous ways throughout its life cycle. Most notably, MpPIN1 is required to establish gemmaling dorsiventral polarity and for orthotropic growth of gametangiophore stalks, where MpPIN1 is basally polarised. PIN activity is largely conserved within land plants, with PIN-mediated auxin flow providing a flexible mechanism to organise growth. Specifically, PIN is fundamentally linked to orthotropism and to the establishment of de novo meristems, the latter potentially involving the formation of both auxin biosynthesis maxima and auxin-signalling minima.

CLASS-II KNOX genes coordinate spatial and temporal ripening in tomato.

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.

Green land: Multiple perspectives on green algal evolution and the earliest land plants.

Green plants, broadly defined as green algae and the land plants (together, Viridiplantae), constitute the primary eukaryotic lineage that successfully colonized Earth's emergent landscape. Members of various clades of green plants have independently made the transition from fully aquatic to subaerial habitats many times throughout Earth's history. The transition, from unicells or simple filaments to complex multicellular plant bodies with functionally differentiated tissues and organs, was accompanied by innovations built upon a genetic and phenotypic toolkit that have served aquatic green phototrophs successfully for at least a billion years. These innovations opened an enormous array of new, drier places to live on the planet and resulted in a huge diversity of land plants that have dominated terrestrial ecosystems over the past 500 million years. This review examines the greening of the land from several perspectives, from paleontology to phylogenomics, to water stress responses and the genetic toolkit shared by green algae and plants, to the genomic evolution of the sporophyte generation. We summarize advances on disparate fronts in elucidating this important event in the evolution of the biosphere and the lacunae in our understanding of it. We present the process not as a step-by-step advancement from primitive green cells to an inevitable success of embryophytes, but rather as a process of adaptations and exaptations that allowed multiple clades of green plants, with various combinations of morphological and physiological terrestrialized traits, to become diverse and successful inhabitants of the land habitats of Earth.

MarpolBase Expression: A Web-Based, Comprehensive Platform for Visualization and Analysis of Transcriptomes in the Liverwort Marchantia polymorpha.

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.

KANADI promotes thallus differentiation and FR-induced gametangiophore formation in the liverwort Marchantia.

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.

A transporter of 1-aminocyclopropane-1-carboxylic acid affects thallus growth and fertility in Marchantia polymorpha.

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.

Stress, senescence, and specialized metabolites in bryophytes.

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.

Evolution and function of red pigmentation in 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.

Control of proliferation in the haploid meristem by CLE peptide signaling in Marchantia polymorpha.

The homeostasis of meristems in flowering plants is maintained by cell-to-cell communication via CLE (CLAVATA3/EMBRYO SURROUNDING REGION-related) peptide hormones. In contrast, cell signals that regulate meristem activity remains elusive in bryophytes that maintain apical meristems in the gametophyte (haploid) body and undergo a gametophyte-dominant life cycle. We here show that MpCLE1 confines the proliferative activity of gametophytic meristem and affects the overall size of gametangiophores (reproductive organs) in Marchantia polymorpha, which is in sharp contrast with the meristem-promoting function of its ortholog TDIF/CLE41/CLE44 in Arabidopsis vascular meristems. Expression analysis suggests that MpCLE1 and its receptor gene MpTDR are expressed in distinct patterns across the apical meristem. These data suggest that local CLE peptide signaling may have had a role in regulating cell proliferation in the shoot meristem in the ancestral land plant and acts in both sporophytic and gametophytic meristems of extant plants.

Rates and patterns of molecular evolution in bryophyte genomes, with focus on complex thalloid liverworts, Marchantiopsida.

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 rateof 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, isnot 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.

The KNOXI Transcription Factor SHOOT MERISTEMLESS Regulates Floral Fate in Arabidopsis.

Plants have evolved a unique and conserved developmental program that enables the conversion of leaves into floral organs. Elegant genetic and molecular work has identified key regulators of flower meristem identity. However, further understanding of flower meristem specification has been hampered by redundancy and by pleiotropic effects. The KNOXI transcription factor SHOOT MERISTEMLESS (STM) is a well-characterized regulator of shoot apical meristem maintenance. Arabidopsis thaliana stm loss-of-function mutants arrest shortly after germination; therefore, the knowledge on later roles of STM in later processes, including flower development, is limited. Here, we uncover a role for STM in the specification of flower meristem identity. Silencing STM in the APETALA1 (AP1) expression domain in the ap1-4 mutant background resulted in a leafy-flower phenotype, and an intermediate stm-2 allele enhanced the flower meristem identity phenotype of ap1-4 Transcriptional profiling of STM perturbation suggested that STM activity affects multiple floral fate genes, among them the F-box protein-encoding gene UNUSUAL FLORAL ORGANS (UFO). In agreement with this notion, stm-2 enhanced the ufo-2 floral fate phenotype, and ectopic UFO expression rescued the leafy flowers in genetic backgrounds with compromised AP1 and STM activities. This work suggests a genetic mechanism that underlies the activity of STM in the specification of flower meristem identity.