Transcription of the Antisense Long Non-Coding RNA, SUPPRESSOR OF FEMINIZATION, Represses Expression of the Female-Promoting Gene FEMALE GAMETOPHYTE MYB in the Liverwort Marchantia polymorpha.

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.

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.

P-MIRU, a Polarized Multispectral Imaging System, Reveals Reflection Information on the Biological Surface.

Reflection light forms the core of our visual perception of the world. We can obtain vast information by examining reflection light from biological surfaces, including pigment composition and distribution, tissue structure and surface microstructure. However, because of the limitations in our visual system, the complete information in reflection light, which we term 'reflectome', cannot be fully exploited. For example, we may miss reflection light information outside our visible wavelengths. In addition, unlike insects, we have virtually no sensitivity to light polarization. We can detect non-chromatic information lurking in reflection light only with appropriate devices. Although previous studies have designed and developed systems for specialized uses supporting our visual systems, we still do not have a versatile, rapid, convenient and affordable system for analyzing broad aspects of reflection from biological surfaces. To overcome this situation, we developed P-MIRU, a novel multispectral and polarization imaging system for reflecting light from biological surfaces. The hardware and software of P-MIRU are open source and customizable and thus can be applied for virtually any research on biological surfaces. Furthermore, P-MIRU is a user-friendly system for biologists with no specialized programming or engineering knowledge. P-MIRU successfully visualized multispectral reflection in visible/non-visible wavelengths and simultaneously detected various surface phenotypes of spectral polarization. The P-MIRU system extends our visual ability and unveils information on biological surfaces.

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.

Identification of the sex-determining factor in the liverwort Marchantia polymorpha reveals unique evolution of sex chromosomes in a haploid system.

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.