Biology subjects

Nishihama, R.

Publications and source records attributed to Nishihama, R..

At least 19 recordsLinked to original sources

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

Diversification of functional requirements for proteolysis of Auxin Response Factors

Auxin signaling through the Nuclear Auxin Pathway is essential for plant development and is mediated by competing A-class and B-class Auxin Response Factor (ARF) transcription factors. Recently, proteasomal ARF degradation through a degradation signal embedded within the DNA-Binding Domain, was identified as a key component of auxin response. Here, we investigate the structural requirements and biological relevance of ARF degradation in the bryophyte Marchantia polymorpha. We identify a critical residue for proteolysis of the repressive, B-class MpARF2, and find it to be functionally conserved in degradation of the activating, A-class MpARF1. Unlike MpARF2, however, impaired MpARF1 degradation had little effect on auxin response and development, suggesting differential integration in biological function. We find that MpARF2 degradation occurs across all developmental stages of the life cycle, and is required for MpARF2 function during development. Our findings reveal a degradation mechanism targeting A- and B-class ARFs which shares the same origin, but has evolved along unique evolutionary trajectories.

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

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↗

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

Agrobacterium-Mediated Transient Transformation of Marchantia Liverworts

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying GUS genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.

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↗

bioRxiv2019

Design principles of a minimal auxin response system

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1-3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development2-4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. Expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned, or prevented. This fundamental design likely represents the ancestral system, and formed the basis for inflated, complex systems.

plant biology↗

bioRxiv2019

Lateral organ diversification in plants mediated by the ALOG protein family of transcription factors

Land plant shoot structures evolved a diversity of lateral organs as morphological adaptations to the terrestrial environment, in which lateral organs independently evolved in each lineage in the sporophyte or gametophyte generation. The gametophyte meristem of the basally-diverging plant Marchantia polymorpha produces axes with non-photosynthetic scale-like lateral organs instead of leaves. Here we report that an ALOG (Arabidopsis LSH1 and Oryza G1) family protein in Marchantia, MpTAWAWA1 (MpTAW1), regulates meristem maintenance and lateral organ development. A mutation in MpTAW1, preferentially expressed in lateral organs, induces lateral organs with mis-specified identity and increased cell number, and furthermore, causes defects in apical meristem maintenance. Remarkably, MpTAW1 expression rescued the elongated-spikelet phenotype of a rice mutant of MpTAW1 homologue. This suggests that ALOG genes are co-opted to specify lateral organ identities in both gametophyte and sporophyte shoots by repressing lateral organ growth. We propose that the recruitment of ALOG-mediated lateral organ modification was in part responsible for the convergent evolution of independently-evolved lateral organs among highly divergent plant lineages and contributed to the morphological diversification of land plants.

plant biology↗

bioRxiv2019

An evolutionarily conserved mechanism for production of secondary meristems in land plants

A variety of plants in diverse taxa can reproduce asexually via vegetative propagation, in which clonal propagules with new meristem(s) are generated directly from vegetative organs. A basal land plant, Marchantia polymorpha, develops clonal propagules, gemmae, in a specialized receptacle, gemma cup. Here we report an R2R3-MYB transcription factor, designated GEMMA CUP-ASSOCIATED MYB 1 (GCAM1), which is an essential regulator of gemma cup development in M. polymorpha. Although gemma cups are a characteristic gametophyte organ for vegetative reproduction in a taxonomically restricted group of liverwort species, phylogenetic and interspecific complementation analyses supported the orthologous relationship of GCAM1 to regulatory factors for axillary meristem formation, e.g. Arabidopsis RAXs and tomato Blind, in angiosperm sporophytes. The present findings in M. polymorpha suggest an ancient acquisition of a regulatory mechanism for production of secondary meristems, and the use of the mechanism for diverse developmental programs during land plant evolution.

plant biology↗

bioRxiv2018

Loss of CG methylation in Marchantia polymorpha causes disorganization of cell division and reveals unique DNA methylation regulatory mechanisms of non-CG methylation.

DNA methylation is an epigenetic mark that ensures silencing of transposable elements (TEs) and affects gene expression in many organisms. The function of different DNA methylation regulatory pathways has been largely characterized in the model plant Arabidopsis thaliana. However, far less is known about DNA methylation regulation and functions in basal land plants. Here we focus on the liverwort Marchantia polymorpha, an emerging model species that represents a basal lineage of land plants. We identified MpMET, the M. polymorpha orthologue of the METHYLTRANSFERASE 1 (MET1) gene required for maintenance of methylation at CG sites in angiosperms. We generated Mpmet mutants using the CRISPR/Cas9 system, which showed a significant loss of CG methylation and severe morphological changes and developmental defects. The mutants developed many adventitious shoot-like structures, suggesting that MpMET is required for maintaining differentiated cellular identities in the gametophyte. Numerous TEs were up-regulated, even though non-CG methylation was highly increased at TEs in the Mpmet mutants. Closer inspection of CHG methylation revealed features unique to M. polymorpha. Methylation of CCG sites in M. polymorpha does not depend on MET1, unlike in A. thaliana and Physcomitrella patens. Furthermore, unlike A. thaliana, M. polymorpha shows higher methylation level at CAG sites than at other CHG contexts and CAG/CTG sites are mostly methylated asymmetrically. Interestingly, CAG and CTG methylation reached comparable levels and symmetry upon loss of CG methylation. Our results highlight the diversity of non-CG methylation regulatory mechanisms in plants.

plant biology↗