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Sexual differentiation is a fundamental process in the life cycles of land plants, ensuring successful sexual reproduction and thereby contributing to species diversity and survival. In the dioicous liverwort Marchantia polymorpha , this process is governed by an autosomal sex-differentiation locus comprising FEMALE GAMETOPHYTE MYB (FGMYB), a female-promoting gene, and SUPPRESSOR OF FEMINIZATION (SUF), an antisense strand-encoded long non-coding RNA (lncRNA). SUF is specifically transcribed in male plants and suppresses the expression of FGMYB , leading to male differentiation. However, the molecular mechanisms underlying this process remain elusive. Here, we show that SUF acts through its transcription to suppress FGMYB expression. Transgene complementation analysis using CRISPR/Cas9D10A-based large-deletion mutants identified a genomic region sufficient for the sex differentiation switch function in the FGMYB-SUF locus. Inserting a transcriptional terminator sequence into the SUF- transcribed region resulted in the loss of SUF function and allowed expression of FGMYB in genetically male plants, leading to conversion of the sex phenotype from male to female. Partial deletions of SUF had no obvious impact on its function. Replacement of the FGMYB sequence with that of an unrelated gene did not affect the ability of SUF transcription to suppress sense-strand expression. Taken together, our findings suggest that the process of SUF transcription, rather than the resulting transcripts, is required for controlling sex differentiation in M. polymorpha. [ABSTRACT FROM AUTHOR]

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 Mp FGMYB. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Regeneration in land plants is accompanied by the establishment of new stem cells, which often involves reactivation of the cell division potential in differentiated cells. The phytohormone auxin plays pivotal roles in this process. In bryophytes, regeneration is enhanced by the removal of the apex and repressed by exogenously applied auxin, which has long been proposed as a form of apical dominance. However, the molecular basis behind these observations remains unexplored. Here, we demonstrate that in the liverwort Marchantia polymorpha , the level of endogenous auxin is transiently decreased in the cut surface of decapitated explants, and identify by transcriptome analysis a key transcription factor gene, LOW- AUXIN RESPONSIVE (Mp LAXR), which is induced upon auxin reduction. Loss of Mp LAXR function resulted in delayed cell cycle reactivation, and transient expression of Mp LAXR was sufficient to overcome the inhibition of regeneration by exogenously applied auxin. Furthermore, ectopic expression of Mp LAXR caused cell proliferation in normally quiescent tissues. Together, these data indicate that decapitation causes a reduction of auxin level at the cut surface, where, in response, Mp LAXR is up-regulated to trigger cellular reprogramming. Mp LAXR is an ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION 1/DORNR Ö SCHEN , which has dual functions as a shoot regeneration factor and a regulator of axillary meristem initiation, the latter of which requires a low auxin level. Thus, our findings provide insights into stem cell regulation as well as apical dominance establishment in land plants. [ABSTRACT FROM AUTHOR]

Key message: The liverwort Marchantia polymorpha regulates gametangia and gametangiophore development by using evolutionarily conserved regulatory modules that are shared with angiosperm mechanisms regulating flowering and germ cell differentiation. Bryophytes, the earliest diverged lineage of land plants comprised of liverworts, mosses, and hornworts, produce gametes in gametangia, reproductive organs evolutionarily conserved but lost in extant angiosperms. Initiation of gametangium development is dependent on environmental factors such as light, although the underlying mechanisms remain elusive. Recent studies showed that the liverwort Marchantia polymorpha regulates development of gametangia and stalked receptacles called gametangiophores by using conserved regulatory modules which, in angiosperms, are involved in light signaling, microRNA-mediated flowering regulation, and germ cell differentiation. These findings suggest that these modules were acquired by a common ancestor of land plants before divergence of bryophytes, and were later recruited to flowering mechanism in angiosperms. [ABSTRACT FROM AUTHOR]

Regulation of the stability and the quality of mitochondrial RNA is essential for the maintenance of mitochondrial and cellular functions in eukaryotes. We have previously reported that the eukaryotic poly(A)-specific ribonuclease (PARN) and the prokaryotic poly(A) polymerase encoded by AHG2 and AGS1 , respectively, coordinately regulate the poly(A) status and the stability of mitochondrial mRNA in Arabidopsis. Mitochondrial function of PARN has not been reported in any other eukaryotes. To know how much this PARN-based mitochondrial mRNA regulation is conserved among plants, we studied the AHG2 and AGS1 counterparts of the liverwort, Marchantia polymorpha , a member of basal land plant lineage. We found that M. polymorpha has one ortholog each for AHG2 and AGS1 , named Mp AHG2 and Mp AGS1 , respectively. Their Citrine-fused proteins were detected in mitochondria of the liverwort. Molecular genetic analysis showed that Mp AHG2 is essential and functionally interacts with Mp AGS1 as observed in Arabidopsis. A recombinant MpAHG2 protein had a deadenylase activity in vitro. Overexpression of Mp AGS1 and the reduced expression of Mp AHG2 caused an accumulation of polyadenylated Mp cox1 mRNA. Furthermore, Mp AHG2 suppressed Arabidopsis ahg2-1 mutant phenotype. These results suggest that the PARN-based mitochondrial mRNA regulatory system is conserved in land plants. [ABSTRACT FROM AUTHOR]

Presents a functional analysis of a beta-ketoacyl-CoA synthase (KCS) gene, MpFAE2, by gene silencing in the liverwort Marchantia polymorpha L. Isolation of a KCS gene homologue, MpFAE2, from M. polymorpha; Characterization of the putative MpFAE2 product; Transgenic M. polymorpha plants for gene silencing and overexpression of the MpFAE2 gene.

Gibberellins (GAs) are key phytohormones that regulate growth, development, and environmental responses in angiosperms. From an evolutionary perspective, all major steps of GA biosynthesis are conserved among vascular plants, while GA biosynthesis intermediates such as ent-kaurenoic acid (KA) are also produced by bryophytes. Here, we show that in the liverwort Marchantia polymorpha, KA and GA12 are synthesized by evolutionarily conserved enzymes, which are required for developmental responses to far-red light (FR). Under FR-enriched conditions, mutants of various biosynthesis enzymes consistently exhibited altered thallus growth allometry, delayed initiation of gametogenesis, and abnormal morphology of gamete-bearing structures (gametangiophores). By chemical treatments and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses, we confirmed that these phenotypes were caused by the deficiency of some GA-related compounds derived from KA, but not bioactive GAs from vascular plants. Transcriptome analysis showed that FR enrichment induced the up-regulation of genes related to stress responses and secondary metabolism in M. polymorpha, which was largely dependent on the biosynthesis of GA-related compounds. Due to the lack of canonical GA receptors in bryophytes, we hypothesize that GA-related compounds are commonly synthesized in land plants but were co-opted independently to regulate responses to light quality change in different plant lineages during the past 450 million years of evolution. Bryophytes do not generate canonical gibberellins, but M. polymorpha needs to produce gibberellin precursors to control anisotropic growth and accelerate gametogenesis under far-red enriched light. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Feminizer (BPCU) linked to the liverwort female sex chromosome was identified • BPCU regulates the autosomal sex-determining locus, FGMYB /S UF • BPCU additionally shares a function for reproductive induction with its gametolog • The liverwort sex chromosomes are the oldest among any other known systems Iwasaki et al. identify a female sex determinant, Feminizer , on the female sex chromosome of a haploid plant, Marchantia polymorpha. Unexpectedly, Feminizer also plays a role in reproductive induction, which is shared by its male gametolog. The liverwort sex chromosomes diverged around 430 mya, older than any known sex chromosome systems. [ABSTRACT FROM AUTHOR]

Plant survival depends on the optimal use of resources under variable environmental conditions. Among the mechanisms that mediate the balance between growth, differentiation, and stress responses, the regulation of transcriptional activity by DELLA proteins stands out. In angiosperms, DELLA accumulation promotes defense against biotic and abiotic stress and represses cell division and expansion, while the loss of DELLA function is associated with increased plant size and sensitivity toward stress. 1 Given that DELLA protein stability is dependent on gibberellin (GA) levels 2 and GA metabolism is influenced by the environment, 3 this pathway is proposed to relay environmental information to the transcriptional programs that regulate growth and stress responses in angiosperms. 4,5 However, DELLA genes are also found in bryophytes, whereas canonical GA receptors have been identified only in vascular plants. 6–10 Thus, it is not clear whether these regulatory functions of DELLA predated or emerged with typical GA signaling. Here, we show that, as in vascular plants, the only DELLA in the liverwort Marchantia polymorpha also participates in the regulation of growth and key developmental processes and promotes oxidative stress tolerance. Moreover, part of these effects is likely caused by the conserved physical interaction with the MpPIF transcription factor. Therefore, we suggest that the role in the coordination of growth and stress responses was already encoded in the DELLA protein of the common ancestor of land plants, and the importance of this function is underscored by its conservation over the past 450 million years. [Display omitted] • MpDELLA accumulation impairs thallus growth and cell division • MpDELLA interacts with MpPIF to modulate developmental traits • Mp DELLA expression increases under oxidative stress and promotes stress tolerance • Coordination between growth and stress responses by DELLA predates GA signaling DELLA proteins coordinate growth and stress responses as part of gibberellin signaling in angiosperms. Hernández-García, Sun, et al. show that this role is also conserved in Marchantia polymorpha despite the lack of a bona fide gibberellin perception module in bryophytes. [ABSTRACT FROM AUTHOR]

In angiosperms, the phase transition from vegetative to reproductive growth involves the de-repression of the squamosa promoter-binding-protein-like (SPL) class of transcription factors, which is negatively regulated by the specific microRNAs (miRNAs/miRs) miR156/529 [ 1 ]. Non-vascular land plants also undergo growth-phase transition to the reproductive state, but knowledge regarding the controlling mechanisms is limited. Here, we investigate the reproductive transition in the liverwort Marchantia polymorpha , focusing on the roles of miR529c [ 2–4 ] and MpSPL2. First, we established mir529c -null mutants using CRISPR/Cas9. Even in the absence of far-red light-supplemented long-day condition, which is usually needed to induce reproductive development [ 5, 6 ], the mutant thalli developed sexual reproductive organs (gametangia) and produced gametes. Transgenic plants expressing a miR529-resistant Mp SPL2 transgene also showed a similar phenotype of reproductive transition in the absence of inductive far-red light signals. In these mutants and transgenic plants, the Mp SPL2 mRNA abundance was elevated. Mp spl2 ko mutant plants showed successful gamete development and fertilization, which suggests that Mp SPL2 is involved in, but not essential for, sexual reproduction in M. polymorpha. Furthermore, analysis of Mp spl2 ko mutant and its complemented lines suggests that Mp SPL2 may have a role in promotion of reproductive transition. These findings support the notion that the transition to reproductive development in liverworts is controlled by a system similar to that in angiosperms, and the miR156/529- SPL module has common significance in the control of the vegetative-to-reproductive transition during development in many land plants, including liverworts. • Loss of miR529c causes gamete production in the absence of environmental signals • Repression of Mp SPL2 expression by miR529c prevents the reproductive transition • Mp SPL2 is involved in, but not essential for, reproductive development • The miR156/529- SPL module controls the reproductive transition in land plants Tsuzuki et al. report that the loss of miR529c in the liverwort Marchantia polymorpha causes gametangium formation and gamete production without the far-red light signaling needed for the reproductive transition, because of de-repressed expression of Mp SPL2. The miR156/529- SPL module generally controls the reproductive transition in land plants. [ABSTRACT FROM AUTHOR]