Biology subjects

Kohchi, T.

Publications and source records attributed to Kohchi, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Functional diversification of ERF transcription factors in land plants

The ERF family is one of the largest families of plant-specific transcription factors, controlling diverse developmental processes and stress responses. Extensive gene duplication and redundancy in flowering plants have generated large ERF repertoires, complicating comparisons of ERF function across land-plant lineages. How ERF transcription factor repertoires and their functions have evolved across land plants remains poorly understood. Here, we reconstruct the phylogenetic relationships and expansion of ERF transcription factors across land plants and exploit the compact ERF repertoire of the model liverwort Marchantia polymorpha to investigate their functional diversification. Systematic gain-of-function analysis reveals that different clades of ERFs can drive distinct cell fate and reprogramming outcomes in Marchantia, in addition to stress-related functions. Interestingly, we found that MpERF2, the sole Group I ERF in Marchantia, phylogenetically related to the WIND clade, promotes rhizoid cell differentiation. This contrast with the role of the WIND clade in wound-induced regeneration in Arabidopsis. Heterologous expression of AtWIND1 in Marchantia was also capable of inducing rhizoid formation, supporting the hypothesis that this reprogramming capacity is conserved at the protein level. On the other hand, loss of MpERF2 reduced rhizoid coverage but did not significantly impair regeneration. These findings provide an evolutionary framework for understanding the functional evolution of ERF transcription factors and how conserved transcriptional regulators are co-opted into novel developmental programs during the radiation of land plants.

plant biology↗

bioRxiv2026

A paralog of a clonal propagation regulator promotes cell-cycle re-entry during thallus regeneration in Marchantia polymorpha

Plants possess a remarkable capacity for regeneration, which involves the redeployment of developmental programs and diverse regulatory mechanisms. However, how related regulators with overlapping functions are differentially deployed during regeneration remains poorly understood. The model liverwort Marchantia polymorpha provides a powerful experimental system for studying regeneration because it readily regenerates apical meristems from basal thallus fragments after removal of the original meristem, even without exogenous plant hormones. Here, we identify the R2R3-MYB transcription factor GEMMA CUP-ASSOCIATED MYB1-LIKE (MpGC1L), the closest paralog of the clonal propagation regulator MpGCAM1, as a positive regulator of regeneration. MpGC1L was rapidly induced at the cut site following meristem removal. Ectopic overexpression of MpGC1L caused the proliferation of undifferentiated cells, whereas Mpgc1l mutants showed delayed regeneration and reduced S-phase entry. Loss of MpGCAM1 alone had little effect on regeneration but markedly enhanced the Mpgc1l phenotype, indicating partially redundant functions. Transcriptome analysis of the double mutant revealed reduced induction of genes associated with ribosome biogenesis and the cell cycle. We next examined the relationship between MpGC1L and the known jasmonate- and auxin- related regeneration regulators, MpERF15 and MpLAXR. MpGC1L induction was retained in Mperf15 and Mplaxr mutants and was unaffected by OPDA or auxin treatment, whereas MpERF15 and MpLAXR were still induced in Mpgc1l Mpgcam1 double mutants. Thus, these regulators are not arranged in a simple linear transcriptional pathway. Our findings reveal that the paralogous MYB transcription factors MpGC1L and MpGCAM1 promote cell proliferation in distinct developmental contexts, thereby linking clonal propagation and wound-induced regeneration.

plant biology↗

bioRxiv2025

A SABATH family enzyme regulates development via the gibberellin-related pathway in the liverwort Marchantia polymorpha

The SABATH family enzymes are a group of plant-specific methyltransferases that catalyze the methylation of many small molecules, including several plant hormones. While this family emerged anciently before the evolution of land plants from streptophyte algae, little is known about their biological function in plant lineages other than angiosperms. Here, we identified 12 SABATH family genes from the liverwort Marchantia polymorpha and found that MpSABATH2 is essential for its development. Mpsabath2 mutants were severely inhibited in thallus growth and gemma cup formation, while spontaneously forming sexual branches under non-inductive conditions. These phenotypes resembled the developmental responses to far-red light, which was also supported by transcriptome analysis. Further genetic analysis connected this phenomenon with gibberellin (GA)-related metabolism. Blocking GA biosynthesis partially rescued Mpsabath2 phenotypes, which were restored by treatment with the GA precursor, ent-kaurenoic acid. Given that bryophyte and angiosperm SABATH proteins fall into distinct phylogenetic clades, our findings suggest that SABATH family enzymes independently acquired roles in developmental regulation through convergent or parallel evolution in land plants.

plant biology↗

bioRxiv2025

MarpolBase: Genome database for Marchantia polymorpha featuring high quality reference genome sequences

The liverwort Marchantia polymorpha is a key model organism for understanding land plant evolution, development, and gene regulation. To support the growing demand for high-quality genomic resources, we present MarpolBase, a comprehensive and integrated genome database that hosts newly assembled, high-accuracy reference genomes for both the male Tak-1 and female Tak-2 accessions, designated as ver. 7.1 reference genomes. These new assemblies, generated using PacBio HiFi long-read sequencing, represent nearly telomere-to-telomere chromosome-level genomes, with improvements in assembly continuity, annotation accuracy, and structural resolution--especially for repeat-rich regions and sex chromosomes. MarpolBase offers not only access to genome sequences and gene annotations but also provides a unified platform for data exploration, comparative analysis, and community-driven gene nomenclature. It includes keyword-searchable gene pages with structural and functional annotations, expression data integration, genome browser visualization, and online analytical and utility tools. By unifying genome assembly, annotation, nomenclature, and analysis tools in a single platform, MarpolBase serves as a central resource for functional genomics and evolutionary studies in M. polymorpha, and a model for future plant genome databases.

genomics↗

bioRxiv2024

Evolutionary acquisition of a primitive light-dependent nuclear relocation in Marchantia polymorpha

The terrestrialization of plants was accompanied by exposure to several environmental stresses. Adaptation to these stresses required numerous changes at the cellular and molecular level. One such adaptation in the leaves of Arabidopsis thaliana is the movement of cell nuclei to avoid UV damage. In the dark, the nuclei locate to the bottom walls of leaf cells to distance genetic material from external stresses, but in response to intense blue light (an indication of the presence of UV), they move to the side walls to escape UV-induced DNA damage1. The movement is driven by the photoreceptor phototropin and the actin cytoskeleton2. However, how this protective mechanism evolved in land plants remains unclear. Here, we show that in the liverwort Marchantia polymorpha, nuclei show a similar, but less stable movement in response to intense blue light. In the dark, M. polymorpha positioned nuclei on the upper walls of epidermal cells in young thalli, but in response to intense blue light, the nuclei immediately moved to the side walls, similar to A. thaliana. However, the movement was transient and the nuclei returned to the upper walls through both the actin and microtubule cytoskeletons. Unlike A. thaliana, M. polymorpha responded to prolonged (> 1 day) exposure to low light by moving nuclei from the upper to the side walls through both the actin and microtubule cytoskeletons and two photoreceptors (phototropin and phytochrome). However, no light-dependent nuclear relocation was observed in charophyte algae, suggesting that light-dependent nuclear relocation was initially established in the common ancestor of land plants as a result of terrestrialization and then diverged during land plant evolution.

plant biology↗

bioRxiv2023

Evolutionarily Conserved CKI1-Mediated Two-Component Signaling is Required for Female Germline Specification in Marchantia polymorpha

In land plants, gametes derive from a small number of dedicated haploid cells. 1 In angiosperms, one central cell and one egg cell are differentiated in the embryo sac as female gametes for double fertilization, while in non-flowering plants, only one egg cell is generated in the female sexual organ, called the archegonium. 2,3 The central cell specification of Arabidopsis thaliana is controlled by the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1), which is a two-component signaling (TCS) activator sharing downstream regulatory components with the cytokinin signaling pathway. 4-7 Our phylogenetic analysis suggested that CKI1 orthologs broadly exist in land plants. However, the role of CKI1 in the archegonium-bearing non-flowering plants remains unclear. Here we found that the sole CKI1 ortholog in the liverwort Marchantia polymorpha, MpCKI1, which functions through conserved downstream TCS components, regulates the female germline specification for egg cell development in the archegonium. In M. polymorpha, the archegonium develops three-dimensionally from a single cell, accumulating MpBONOBO (MpBNB), a master regulator for germline initiation and differentiation. 8 We visualized female germline specification by capturing the distribution pattern of MpBNB in the discrete stages of early archegonium development, and found that the MpBNB accumulation is restricted to female germline cells. MpCKI1 is required for the proper MpBNB accumulation in the female germline, and is critical for the asymmetric cell divisions that specify the female germline cells. These results suggest that CKI1-mediated TCS originated during early land plant evolution and participates in female germ cell specification in deeply-diverged plant lineages.

plant biology↗

bioRxiv2023

An ancient role for the CYP73 gene family in t-cinnamic acid 4-hydroxylation, phenylpropanoid biosynthesis and embryophyte development

The phenylpropanoid pathway is a plant metabolism intimately linked to the transition to terrestrial life. It produces phenolic compounds that play essential roles in stress mitigation and ecological interactions. The pathway also provides the building blocks for hydrophobic polymers that form apoplastic diffusion barriers and make up a significant fraction of the land plant biomass. Despite its significance in embryophytes (i.e., land plants), the origin and evolutionary history of the phenylpropanoid pathway remain poorly understood. In particular, little is known about the organization and function of the pathway in bryophytes, the non-vascular embryophytes. In this study, we conducted a multidisciplinary analysis of the CYP73 gene family that encodes t-cinnamic acid 4-hydroxylase (C4H), the first plant-specific enzyme in the pathway. Our results indicate that C4H activity originated with the emergence of the CYP73 gene family in an ancestor of land plants and was supported by an arginine residue that stabilizes its substrate in the active site. C4H deficiency in the moss Physcomitrium patens, the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis resulted in a shortage of phenylpropanoids and abnormal plant development. The latter could be rescued in the moss by the exogenous supply of p-coumaric acid, the product of C4H. Our findings establish the emergence of the CYP73 gene family as a foundational event for the development of the canonical plant phenylpropanoid pathway and underscores the deep-rooted conservation of the C4H enzyme function in embryophyte biology.

plant biology↗

bioRxiv2023

Thermospermine is an evolutionarily ancestral phytohormone required for organ development and stress responses in the basal land plant Marchantia polymorpha

Thermospermine, a structural isomer of spermine, suppresses auxin-inducible xylem differentiation, whereas spermine is implicated in stress responses in angiosperms. Thermospermine synthase ACAULIS5 (ACL5) is well conserved from algae to land plants, but its physiological function remains elusive in non-vascular plants. Here we focused on MpACL5, a gene in the liverwort Marchantia polymorpha, which rescued the dwarf phenotype of the acl5 mutant of Arabidopsis. In the Mpacl5 mutants generated by genome editing, growth of the vegetative organ, thallus, and the sexual reproductive organ, gametangiophore, was severely retarded. The mutant gametangiophore exhibited remarkable morphological defects such as short stalks, fasciation, and indeterminate growth; it was formed as a fusion of two gametangiophores and a new gametangiophore was often initiated from the old one. Furthermore, Mpacl5 was shown to be hypersensitive to heat and salt stresses. Given the absence of spermine in liverworts including M. polymorpha, these results reveal that thermospermine has a dual primordial function in organ development and stress responses in the basal land plant lineage, the latter of which may have eventually been assigned to spermine during the land plant evolution.

plant biology↗

bioRxiv2023

Harnessing deep learning to analyze cryptic morphological variability of Marchantia polymorpha

Characterizing phenotypes is a fundamental aspect of biological sciences, although it can be challenging due to various factors. For instance, the liverwort (Marchantia polymorpha), a model system for plant biology, exhibits morphological variability, making it difficult to identify and quantify distinct phenotypic features using objective measures. To address this issue, we utilized a deep learning-based image classifier that can handle plant images directly without manual extraction of phenotypic features, and analyzed bright-field images of M. polymorpha. This dioicous plant species exhibits morphological differences between male and female wild accessions at an early stage of gemmaling growth, although it remains elusive whether the differences are attributable to sexual dimorphism or autosomal genetic variation. To dissect the genomic factors, we established a male and female set of recombinant inbred lines (RILs) from a set of male and female wild accessions. We then trained deep-learning models to classify the sexes of the RILs and the wild accessions. Our results showed that the trained classifiers accurately classified male and female gemmalings of wild accessions in the first week of growth, confirming the intuition of plant researchers in a reproducible and objective manner. In contrast, the RILs were less distinguishable, indicating that the differences between the parental wild accessions arose from autosomal variations instead of sexual dimorphism. Furthermore, we validated our trained models by an "explainable AI" technique that highlights image regions relevant to the classification. Our findings demonstrate that the classifier-based approach provides a powerful tool for analyzing plant species that lack standardized phenotyping metrics.

plant biology↗

bioRxiv2023

Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha

The phytohormone gibberellins (GAs) are key regulators of growth, development and environmental responses in angiosperms. From an evolutionary perspective, all major steps of GA biosynthesis are conserved among vascular plants, while GA biosynthetic 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 altered thallus growth allometry, delayed the initiation of gametogenesis, and affected the morphology of gamete-bearing structures (gametangiophores). By chemical treatments and LC-MS/MS analyses, we confirmed these phenotypes were caused by 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 the canonical GA receptors in bryophytes, we hypothesize that GA-related compounds are commonly synthesized in land plants but co-opted independently to regulate responses to light quality change in different lineages during the past 450 million years of evolution.

plant biology↗

bioRxiv2023

Overexpression of NIMA-related kinase suppresses cell proliferation and tip growth in a liverwort Marchantia polymorpha

NIMA-related kinases (NEKs) regulate a series of mitotic events in fungi and animals, whereas plant NEKs regulate growth direction of cells and organs. The liverwort Marchantia polymorpha has a single functional MpNEK1 gene, whose knockout leads to twisted growth of rhizoids. MpNEK1 is also expressed in the meristem of vegetative flat organ, thallus, while its function remains unknown. Here, we generated transgenic lines for the inducible expression of MpNEK1 using an estrogen receptor mediated system. Estradiol treatment efficiently induced the accumulation of MpNEK1 mRNA and MpNEK1-Citrine fusion protein throughout plant body. Overexpression of MpNEK1 severely suppressed growth of rhizoids and thalli, eventually causing the lethality of juvenile plants. The effect of estradiol was reversible until 3 days, whereas 7-days treatment resulted in irreversible suppression of growth. This severe effect was observed even at the nanomolar level of estradiol. EdU staining and microtubule imaging clearly indicated the suppression of cell proliferation by estradiol-induced MpNEK1. Unexpectedly, the overexpression of kinase-deficient MpNEK1 also suppressed thallus growth and rhizoid formation, despite their slightly mild effect than the full length MpNEK1, indicating phosphorylation-independent mechanism of growth suppression. In conclusion, overexpression of MpNEK1 suppresses cell division and elongation, leading to growth cessation and lethality. Our results imply that the expression of MpNEK1 is tightly regulated and plant NEKs might control cell division as in fungi and animals.

plant biology↗

bioRxiv2023

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. We here 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 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. In 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 suggests a tightly coupled progression of germline segregation and sexual dimorphism development in M. polymorpha.

plant biology↗

bioRxiv2022

Genetic and phosphoproteomic basis of LysM-mediated immune signaling in Marchantia polymorpha highlights conserved elements and new aspect of pattern-triggered immunity in land plants

Pattern-recognition receptor (PRR)-triggered immunity (PTI) wards off a wide range of pathogenic microbes, playing a pivotal role in plant immunity. The model liverwort Marchantia polymorpha is emerging as a popular model for investigating the evolution of plant-microbe interactions. M. polymorpha triggers defense-related gene expression upon sensing components of bacterial and fungal extracts, suggesting the existence of PTI in this plant model. However, the molecular components of the putative PTI in M. polymorpha have not yet been described. We show that, in M. polymorpha, which has four LysM receptor homologs, lysin motif (LysM) receptor-like kinase (LYK) MpLYK1 and LYK-related (LYR) MpLYR are required for sensing chitin and peptidoglycan fragments, triggering a series of characteristic immune responses. Comprehensive phosphoproteomic analysis of M. polymorpha in response to chitin treatment identified regulatory proteins that potentially shape LysM-mediated PTI. The identified proteins included homologs of well-described PTI components in angiosperms as well as proteins whose roles in PTI are not yet determined, including the blue-light receptor phototropin MpPHOT. We revealed that MpPHOT is required for a negative feedback of defense-related gene expression during PTI. Taken together, this study outlines the basic framework of LysM-mediated PTI in M. polymorpha and demonstrates the utility of M. polymorpha as a plant model for discovering novel or fundamental molecular mechanisms underlying PRR-triggered immune signaling in plants.

plant biology↗

bioRxiv2022

A RAF-like kinase mediates a deeply conserved, ultra-rapid auxin response

The plant signaling molecule auxin triggers both fast and slow cellular responses across the plant kingdom, including both land plants and algae. A nuclear response pathway mediates auxin-dependent gene expression, and controls a range of growth and developmental processes in land plants. It is unknown what mechanisms underlie both the physiological responses occurring within seconds, and the responses in algae, that lack the nuclear auxin response pathway. We discovered an ultra-fast proteome-wide phosphorylation response to auxin across 5 land plant and algal species, converging on a core group of shared target proteins. We find conserved rapid physiological responses to auxin in the same species and identified a RAF-like protein kinase as a central mediator of auxin-triggered phosphorylation across species. Genetic analysis allowed to connect this kinase to both auxin-triggered protein phosphorylation and a rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.

plant biology↗

bioRxiv2022

Nuclear auxin signaling is essential for organogenesis but not for cell survival in the liverwort Marchantia polymorpha

Auxin plays pleiotropic roles in plant development via gene regulation upon perception by the receptors TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFBs). This nuclear auxin signaling (NAS) originated in the common ancestor of land plants. Although complete loss of TIR1/AFBs causes embryonic lethality in Arabidopsis thaliana, it is unclear whether the requirement for TIR1-mediated auxin perception in cell viability can be generalized. The model liverwort Marchanita polymorpha has a minimal NAS system with only a single TIR1/AFB, MpTIR1. Here we show by genetic, biochemical, and transcriptomic analyses that MpTIR1 functions as an evolutionarily conserved auxin receptor. Null mutants and conditionally knocked-out mutants of MpTIR1 were viable but incapable of forming any organs and grew as cell masses. Principal component analysis performed using transcriptomes at various developmental stages indicated that MpTIR1 is involved in the developmental transition from spores to organized thalli, during which apical notches containing stem cells are established. In Mptir1 cells, stem-cell- and differentiation-related genes were up- and down-regulated, respectively. Our findings suggest that, in M. polymorpha, NAS is dispensable for cell division but essential for three-dimensional body plans by establishing pluripotent stem cells for organogenesis, a derived trait of land plants.

plant biology↗

bioRxiv2022

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.

plant biology↗

bioRxiv2022

A transcription factor module regulating stomatal formation is co-opted for the development of setae in the astomatous liverwort Marchantia polymorpha

The evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, a TF module formed by subfamilies Ia and IIIb basic helix-loop-helix TFs (Ia-IIIb bHLH) regulates stomatal formation; however, how this module evolved during land plant diversification remains unclear. Here, we show that, in the astomatous liverwort Marchantia polymorpha, a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the seta, which is found in mosses and liverworts. The sole Ia bHLH gene, MpSETA, and a IIIb bHLH gene, MpICE2, regulate the cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana, suggesting that a common regulatory mechanism underlies the setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating cell fate determination and/or cell division of distinct types of cells during land plant evolution.

plant biology↗

bioRxiv2021

Coordination between growth and stress responses by DELLA in the liverwort Marchantia polymorpha

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 loss of DELLA function is associated with increased plant size and sensitivity towards stress1. Given that DELLA protein stability is dependent on gibberellin (GA) levels2, and GA metabolism is influenced by the environment3, this pathway is proposed to relay environmental information to the transcriptional programs that regulate growth and stress responses in angiosperms4,5. However, DELLA genes are also found in bryophytes, whereas canonical GA receptors appeared only in vascular plants6-10. 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 the tolerance towards oxidative stress. 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 DELLA from the common ancestor of land plants, and the importance of this function is justified by its conservation over the past 450 M years.

plant biology↗