Ryuichi Nishihama

Summary: 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. Mp GC1L induction was retained in Mp erf15 and Mp laxr mutants and was unaffected by OPDA or auxin treatment, whereas Mp ERF15 and Mp LAXR were still induced in Mp gc1l Mp gcam1 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.

The plasma membrane proton pump [PM H ⁺ -adenosine triphosphatase (PM H ⁺ -ATPase)] is essential in plants. C-terminal phosphorylation events regulate proton pump activity, such as Thr ⁸⁸¹ phosphorylation in Arabidopsis AHA1. We discovered a sequential protein phosphorylation pathway in which two distinct types of Raf-like protein kinases, C5-Raf and C7-Raf, form a heterocomplex that phosphorylates Thr ⁸⁸¹ to activate PM H ⁺ -ATPases. This regulatory system is highly conserved across lineages from liverworts to angiosperms. In Arabidopsis , a C5-Raf Raf36 regulates plant growth through the phosphorylation of multiple Arabidopsis H ⁺ -ATPases (AHAs). Additionally, another C5-Raf HT1 functions with C7-Rafs CBC1/2 to phosphorylate AHA1 ^T881 , thereby generating a driving force for light-induced stomatal opening. Our findings provide a framework for understanding PM H ⁺ -ATPase activation in various physiological processes, particularly in elucidating the complete mechanistic understanding of light-induced stomatal opening.

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 that shares the same origin but has evolved and diversified along unique trajectories.

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 for M. polymorpha. 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. The genomic resources of MarpolBase are freely available at https://marchantia.info.

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.

MID1-COMPLEMENTING ACTIVITY (MCA) is a land plant-specific, plasma membrane protein and Ca²⁺ signaling component that responds to exogenous mechanical stimuli, such as touch, gravity, and hypotonic-osmotic stress, in various plant species. MCA is essential for cell proliferation and differentiation during growth and development in rice (Oryza sativa) and maize (Zea mays). However, the mechanism by which MCA mediates cell proliferation and differentiation via Ca²⁺ signaling remains unknown. Here, we address this question using the liverwort Marchantia polymorpha. We show that the M. polymorpha MCA ortholog, MpMCA, is highly expressed in actively dividing regions, such as apical notches in the thalli and developing gametangiophores, and that MpMCA is a plasma membrane protein. In vivo Ca²⁺ imaging using a Ca²⁺ sensor (yellow cameleon) revealed that MpMCA is required for maintaining proper [Ca²⁺]_cyt levels in the apical notch region, egg cells, and antheridium cells. Mpmca mutant plants showed severe cell proliferation and differentiation defects in the thalli, gametangiophores, and gametangia, resulting in abnormal development and unsuccessful fertilization. Furthermore, expression of the Arabidopsis MCA1 gene complemented most of the defects in the growth and development of the Mpmca mutant plants. Our findings indicate that MpMCA is an evolutionarily conserved Ca²⁺-signaling component that regulates cell proliferation and development across the life cycle of land plants.

Chloroplasts accumulate on the cell surface under weak light conditions to efficiently capture light but avoid strong light to minimize photodamage. The blue light receptor phototropin regulates the chloroplast movement in various plant species. In Arabidopsis thaliana, phototropin mediates the light-induced chloroplast movement and positioning via specialized actin filaments on the chloroplasts, chloroplast-actin filaments. KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT (KAC) and CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) are pivotal for chloroplast-actin-based chloroplast movement and positioning in land plants. However, the mechanisms by which KAC and CHUP1 regulate chloroplast movement and positioning remain unclear. In this study, we characterized KAC and CHUP1 orthologs in the liverwort Marchantia polymorpha, MpKAC and MpCHUP1, respectively. Their knockout mutants, Mpkac^k° and Mpchup1^k°, impaired the light-induced chloroplast movement. Although Mpchup1^k° showed mild chloroplast aggregation, Mpkac^k° displayed severe chloroplast aggregation, suggesting the greater contribution of MpKAC to the chloroplast anchorage to the plasma membrane. Analysis of the subcellular localization of the functional MpKAC-Citrine indicated that MpKAC-Citrine formed a punctate structure on the plasma membrane. Structure-function analysis of MpKAC revealed that a deletion of the conserved C-terminal domain abrogates the targeting to the plasma membrane and its function. A deletion of the N-terminal motor domain retained the plasma membrane targeting but abrogates the formation of punctate structure and showed severe defect in the light-induced chloroplast movement. Our findings suggest that the formation of the punctate structure on the plasma membrane of MpKAC is essential for chloroplast movement.

As a model plant for bryophytes, Marchantia polymorpha offers insights into the role of RNA silencing in aiding early land plants navigate the challenges posed by high-temperature environments. Genomic analysis revealed unique ARGONAUTE1 ortholog gene (MpAGO1) in M. polymorpha that is regulated by two species-specific microRNAs (miRNAs), miR11707.1 and miR11707.2. Comparative studies of small RNA profiles from M. polymorpha cellular and MpAGO1 immunoprecipitation (MpAGO1-IP) profiles at various temperatures, along with analyses of Arabidopsis AGO1 (AtAGO1), revealed that MpAGO1 has a low-selectivity for a diverse range of small RNA species than AtAGO1. Protein structural comparisons revealed no discernible differences in the MID domains of MpAGO1 and AtAGO1, suggesting the complexity of miRNA species specificity and necessitating further exploration. Small RNA profiling and size exclusion chromatography have pinpointed a subset of M. polymorpha miRNAs, notably miR11707, that remain in free form within the cell at 22°C but are loaded into MpAGO1 at 28°C to engage in RNA silencing. Investigations into the mir11707 gene editing (mir11707^ge) mutants provided evidence of the regulation of miR11707 in MpAGO1. Notably, while MpAGO1 mRNA expression decreases at 28°C, the stability of the MpAGO1 protein and its associated miRNAs is essential for enhancing the RISC activity, revealing the importance of RNA silencing in enabling M. polymorpha to survive thermal stress. This study advances our understanding of RNA silencing in bryophytes and provides groundbreaking insights into the evolutionary resilience of land plants to climatic adversities.