Biology subjects

Kyozuka, J.

Publications and source records attributed to Kyozuka, J..

13 recordsLinked to original sources

bioRxiv2026

Generation of KARRIKIN INSENSITIVE2 loss-of-function mutants in Ceratopteris richardii using a CRISPR/Cas9 system based on ribozyme-gRNA-ribozyme (RGR) technology

Plant hormones regulate almost every aspect of plant growth and development. KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling, which is thought to transduce signals derived from an unidentified ligand known as the KAI2 ligand (KL), regulates numerous traits, including seed germination in angiosperms and vegetative reproduction in bryophytes. The origin of KAI2 is believed to be ancient, and the evolution of its signaling pathways remains of significant interest. Ferns represent critical lineages for elucidating the evolution of land plant traits and growth mechanisms that enabled adaptation to terrestrial environments. Therefore, functional studies of key components of this pathway in ferns are essential for understanding the evolutionary trajectory of KAI2-dependent signaling during vascular plant diversification. However, experimental platforms for the CRISPR/Cas9 system, a powerful tool for investigating gene function, remain undeveloped in ferns. Here, we report an efficient CRISPR/Cas9 system based on ribozyme-gRNA-ribozyme (RGR) technology in the model fern, Ceratopteris richardii (C. richardii). We generated loss-of-function mutants of the KAI2 ortholog in C. richardii (CrKAI2), as well as the signaling components CrMAX2 and CrSMXL. We demonstrate that exogenous application of an artificial KL agonist increases the expression of KAI2-dependent signaling responsive genes in wild type plants; this response is abolished in Crkai2 mutants. These findings indicate that KAI2-dependent signaling is conserved in C. richardii. Furthermore, this study proposes an efficient CRISPR/Cas9 method that will facilitate genetic studies in ferns.

plant biology↗

bioRxiv2025

MAX2-dependent signaling regulates the transition from 2D to 3D growth by suppressing cytokinin accumulation in Physcomitrium patens

Plants undergo distinct developmental phases throughout their life cycle. Proper regulation of the timing of phase transition in response to internal and external cues is crucial to optimize proliferation and reproduction. In Physcomitrium patens (P. patens), a moss, the transition from growth of filamentous tissue growth called protonema to gametophore bud formation represents a shift from two-dimensional (2D) to three-demensional (3D) growrh, ultimately leading to reproductive development. MORE AXILLARY GROWTH2 (MAX2), an F-box protein, targets repressor proteins in the KARRIKIN INSENSITIVE2 (KAI2)-dependent signaling pathway for degradation via the 26S proteasome-dependent pathway. KAI2 perceives an unidentified ligand known as KAI2 ligand (KL) as well as karrikins. The four SMAX1-LIKE (SMXL) family proteins work as suppressors in the MAX2-dependent signaling in P. patens. Gametophore bud formation was accelerated when MAX2-dependent signaling was disrupted, while it was delayed in Ppsmxls, loss of function mutants of the PpSMXLs. These results indicate that MAX2-dependent signaling suppresses gametophore bud formation in P. patens, which has not been previously described. We demonstrate that cytokinin activity and contents are increased in Ppmax2 mutants, and cytokinin application rescued the delay in bud formation in Ppsmxl mutants, suggesting that MAX2-dependent signaling suppresses cytokinin accumulation. The four SMXL genes redundantly act to promote gametophore bud formation; PpSMXLC and PpSMXLD, which contain the conserved degron sequence, act as the repressor of MAX2-dependent signaling, while PpSMXLA and PpSMXLB function independent of proteolysis-dependent signaling. Based on these findings, we propose that modulating cytokinin activity to control developmental phase transition may be a conserved role of MAX2-dependent signaling in bryophytes. Significance StatementPlant hormones, both individually and in combination, regulate almost all aspects of plant growth and development. Gametophore bud formation in Physcomitrium patens, a moss, marks the transition from two-dimensional (2D) filamentous growth to three-dimensional (3D) shoot growth. We demonstrate that gametophore bud formation is controlled by the sequential action of two plant hormones, KL, an unidentified hormone that is transduced through MAX2-dependent signaling, and cytokinin. Four SMXL genes, repressors in MAX2-dependent signaling, redundantly promote bud formation: two function within the signaling pathway, while the other two operate independently of it. Our findings uncover novel mechanisms that directly link MAX2-dependent signaling to developmental phase transition by regulating stem cell activity, achieved through the regulation of cytokinin accumulation in Physcomitrium patens.

plant biology↗

bioRxiv2024

Fine-Tuning TAWAWA1-Mediated Panicle Architecture by Genome Editing of a Downstream Conserved Noncoding Sequence in Rice

Genome editing is a promising tool to enhance plant breeding, particularly for the generation of practical quantitative traits. We focused on the rice yield-related gene TAWAWA1 (TAW1) and produced various degrees of increased panicle branching phenotypes by modifying its downstream conserved noncoding sequence (CNS). Differential modification of two highly conserved regions containing putative repressive elements in this CNS caused transcriptional upregulation of TAW1, resulting in diverse phenotypes.

plant biology↗

bioRxiv2024

KAI2-dependent signaling controls vegetative reproduction in Marchantia polymorpha through activation of LOG-mediated cytokinin synthesis

Marchantia polymorpha reproduces vegetatively (asexually) by producing propagules known as gemmae within gemma cups and sexually through spores. We previously reported that KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling promotes gemma cup and gemma formation. KAI2A perceives unidentified endogenous ligand(s), tentatively referred to as KAI2 ligands (KL). Perception of KL by KAI2 triggers MAX2-dependent proteolysis of MpSMXL. In this study, we identified genes working downstream of KAI2-dependent signaling in M. polymorpha. We found that KAI2-dependent signaling positively controls the expression of MpLONLEY GUY (MpLOG), encoding a cytokinin biosynthesis enzyme. Disruption of the MpLOG function decreased endogenous cytokinin levels and caused defects similar to KAI2-dependent signaling mutants. Moreover, supplying exogenous cytokinins rescued the defects of Mplog and KAI2-dependent signaling mutants, implying that cytokinins work downstream of KAI2-dependent signaling. Activation of MpLOG by KAI2-dependent signaling occurs in a highly cell-type-specific manner, leading to cell-specific induction of GEMMA CUP-ASSOCIATED MYB1 (GCAM1), the master regulator of vegetative reproduction of M. polymorpha. We propose a genetic cascade, starting from KAI2-dependent signaling, that promotes vegetative reproduction through the induction of MpLOG and GCAM1. The interaction between KAI2-dependent signaling and cytokinin in M. polymorpha provides a novel insight into the function and evolution of KAI2-dependent signaling.

plant biology↗

bioRxiv2024

snRNA-seq analysis of the moss Physcomitrium patens reveals a conserved cytokinin-ESR module promoting pluripotent stem cell identity

The shoot apical meristem (SAM), which contains pluripotent stem cells, serves as the source of the entire shoot system in land plants. To uncover the mechanisms underlying SAM development and its orign, we employed single-nucleus RNA sequencing technology in Physcomitrium patens, which contains a single stem cell known as the gametophore apical cell. We identified distinct cell clusters representing major cell types of the P. patens gametophyte, including the gametophore apical cells. We revealed dynamic gene expression changes during cell fate progression in the gametophore apical cell and found upregulation of cytokinin biosynthesis genes in this cell. We also identified ENHANCER OF SHOOT REGENERATION 1 (ESR1) orthologs as key regulators of gametophore apical cells downstream of cytokinin. Given that ESRs promote SAM formation under cytokinin in angiosperms, we propose that the cytokinin-ESR module represents a conserved mechanism promoting stem cell identity that evolved in the common ancestor of land plants.

plant biology↗

bioRxiv2024

Conservation of symbiotic signalling across 450 million years of plant evolution

HighlightO_LIThe common symbiotic pathway is activated during arbuscular mycorrhizal symbiosis in Marchantia paleacea C_LIO_LIThe three core members of the common symbiotic pathway are essential for symbiosis in Marchantia paleacea C_LIO_LIThe molecular function of the CCaMK/CYCLOPS module is conserved across land plants C_LIO_LISymbiotic signalling has been conserved in plants for 450 million years C_LI The colonization of land by plants 450 million years ago revolutionized life on Earth1. The fossil record2 and genetic evidence in extant species3 suggest that this transition was facilitated by interactions with symbiotic arbuscular mycorrhizal (AM) fungi4. This ancestral symbiosis relied on the biosynthesis of chemicals by the host plant, both as signals5 and as nutrients3. In angiosperms, a signalling pathway involving the receptor-like kinase SYMRK/DMI26,7, the Calcium and Calmodulin-dependent protein kinase CCaMK/DMI38 and the transcription factor CYCLOPS/IPD39,10 has been described as the common symbiosis pathway (CSP), essential for the establishment of the AM symbiosis and the root-nodule symbiosis11. Phylogenetic and comparative phylogenomic analyses indicated an ancient origin of the CSP, present in all extant land plants forming intracellular symbioses12-15. Trans-complementation assays of the angiosperm mutants with orthologs from diverse species further indicated the conservation of the molecular function of the CSP across the embryophytes9,12,14-16. However, this correlative evidence did not allow testing the ancestral biological function of the CSP. In this study we demonstrate that SYMRK, CCaMK and CYCLOPS are essential for the colonization by AM fungi in bryophytes, indicating that plants have maintained a dedicated signalling pathway to support symbiotic interactions for 450 million years.

plant biology↗

bioRxiv2023

Cytokinins specify pluripotent stem cell identity in the moss Physcomitrium patens

The shoot apical meristem (SAM) contains pluripotent stem cells that produce all the aerial parts of the plant. Stem cells undergo asymmetric cell divisions to self-renew and to produce differentiating cells. Our research focused on unraveling the mechanisms governing the specification of these two distinct cell fates following the stem cell division. For this purpose, we used the model organism Physcomitrium patens, which features a singular pluripotent stem cell known as the gametophore apical cell. We show that the activity of cytokinins, critical stem cell regulators, is restricted to the gametophore apical cell due to the specific localization of PpLOG, the enzyme responsible of cytokinin activation. In turn, PpTAW, which promotes differentiating cell identity of the merophyte, is excluded from the gametophore apical cell by the action of cytokinins. We propose a cytokinin-based model for the establishment of asymmetry in the pluripotent stem cell division. One Sentence SummaryCytokinins are confined to the pluripotent stem cells and exclude the differentiation factor TAW1 to establish the SAM from a single stem cell.

plant biology↗

bioRxiv2023

The transcriptional changes underlying the flowering phenology shift in response to climate warming

Climate warming is causing shifts in key life-history events, such as flowering time. To assess the impacts of climate warming on flowering phenology, it is crucial to understand the transcriptional changes of genes underlying the phenological shifts. Here, we comprehensively explore genes that contribute to the flowering phenology shifts under climate warming by monitoring the latitudinal gradient of seasonal expression dynamics of 293 flowering-time genes in a perennial herb, Arabidopsis halleri. Through transplant experiments conducted at northern, southern, and subtropical study sites in Japan, we have demonstrated that the flowering period was shortened with decreasing latitude and ultimately, the plants lost the opportunity to flower in subtropical climates. The key transcriptional changes underlying the shift in flowering phenology and the loss of flowering opportunity were the diminished expression of floral pathway integrator genes and genes in the gibberellin synthesis and aging pathways, all of which are suppressed by increased expression of FLOWERING LOCUS C, a central repressor of flowering. These results suggest that the upper-temperature threshold beyond which plants cannot reproduce in response to warming is governed by a relatively small number of genes that suppress reproduction in the absence of winter cold.

plant biology↗

bioRxiv2022

KARRIKIN INSENSITIVE2 (KAI2)-dependent signaling pathway controls vegetative reproduction in Marchantia polymorpha

In vegetative reproduction of Marchantia polymorpha, propagules, called gemmae, are formed in gemma cups. Despite its significance for survival, control of gemma and gemma cup formation by environmental cues is not well understood. We show here that the number of gemmae formed in a gemma cup is a genetic trait. Gemma formation starts from the central region of the floor of the gemma cup, proceeds to the periphery, and terminates when the appropriate number of gemmae are initiated. The MpKARRIKIN INSENSITIVE2 (MpKAI2)-dependent signaling pathway promotes gemma cup formation and gemma initiation. The number of gemmae in a cup is controlled by modulating the ON/OFF switch of the KAI2-dependent signaling. Termination of the signaling results in the accumulation of MpSMXL, a suppressor protein. In the Mpsmxl mutants, gemma initiation continues, leading to the formation of a highly increased number of gemmae in a cup. Consistent with its function, the MpKAI2-dependent signaling pathway is active in gemma cups where gemmae initiate, as well as in the notch region of the mature gemma and midrib of the ventral side of the thallus. In this work, we also show that GEMMA CUP-ASSOCIATED MYB1 works downstream of this signaling pathway to promote gemma cup formation and gemma initiation. We also found that the availability of potassium affects gemma cup formation independently from the KAI2-dependent signaling pathway in M. polymorpha. We propose that the ancestral function of the KAI2-dependent signaling pathway may be to optimize vegetative reproduction by adapting to the environment.

plant biology↗

bioRxiv2022

Contrasting and conserved roles of NPR proteins in diverged land plant lineages

The NPR proteins function as salicylic acid (SA) receptors in Arabidopsis thaliana. AtNPR1 plays a central role in SA-induced transcriptional reprogramming whereby positively regulates SA-mediated defense. NPRs are found in the genomes of nearly all land plants. However, we know little about the molecular functions and physiological roles of NPRs in most plant species. Our phylogenetic and alignment analyses show that Brassicaceae NPR1-like proteins have characteristically gained or lost functional residues or motifs identified in AtNPRs, pointing to the possibility of a unique evolutionary trajectory for the Brassicaceae NPR1-like proteins that has resulted in peculiar functions. In line with this observation, we find that the only NPR in Marchantia polymorpha, MpNPR, is not the master regulator of SA-induced transcriptional reprogramming and negatively regulates bacterial resistance in this species. Interspecies complementation analysis indicated that the molecular properties of AtNPR1 and MpNPR are partially conserved, implying the diversification of NPR-associated pathways contributed to distinct roles of NPR in different species. The Mpnpr transcriptome suggested potential roles of MpNPR in heat and far-red light responses. We identify both Mpnpr and Atnpr1-1 display enhanced thermomorphogenesis. NPRs and NPR-associated pathways clearly have evolved distinctively in diverged land plant lineages to cope with different terrestrial environments.

plant biology↗

bioRxiv2021

An ancestral function of strigolactones as symbiotic rhizosphere signals

In flowering plants, carotenoid-derived strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), a previously unidentified SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating that it is ancestral in land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

plant biology↗

bioRxiv2020

Major components in the KARRIKIN INSENSITIVE2-ligand signaling pathway are conserved in the liverwort, Marchantia polymorpha

KARRIKIN INSENSITIVE2 (KAI2) was first identified in Arabidopsis thaliana as a receptor of karrikin, a smoke-derived germination stimulant. KAI2 is also considered a receptor of an unidentified endogenous molecule called the KAI2-ligand (KL). Upon KAI2 activation, signals are transmitted through degradation of D53/SMXL proteins via ubiquitination by a Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. All components in the KL signaling pathway exist in the liverwort Marchantia polymorpha, namely MpKAI2A and MpKAI2B, MpMAX2 encoding the F-box protein, and MpSMXL, indicating that the signaling pathway became functional in the common ancestor of bryophytes and seed plants. Genetic analysis using knock-out mutants of these KL signaling genes, produced using the CRISPR system, indicated that MpKAI2A, MpMAX2 and MpSMXL act in the same genetic pathway and control early gemma growth. Introduction of MpSMXLd53, in which a domain required for degradation is mutated, into wild-type plants caused phenotypes resembling those of the Mpkai2a and Mpmax2 mutants. In addition, Citrine fluorescence was detected in tobacco cells transiently transformed with the 35S:MpSMXL-Citrine gene construct and treated with MG132, a proteasome inhibitor. On the other hand, introduction of 35S:MpSMXLd53-Citrine conferred Citrine fluorescence without MG132 treatment. These findings imply that MpSMXL is subjected to degradation, and that degradation of MpSMXL is crucial for KL signaling in M. polymorpha. We also showed that MpSMXL is negatively regulated by KL signaling. Taken together, this study demonstrates that basic mechanisms in the KL signaling pathway are conserved in M. polymorpha.

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↗