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Ishizaki, K.

Publications and source records attributed to Ishizaki, K..

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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↗

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↗

RHO GTPase of Plants contributes to robust establishment of cellular asymmetry during development from a single cell

A fundamental question in developmental biology is how highly complex, yet reproducible multicellular body plans form from a single cell. The multicellular haploid body of the land plant Marchantia polymorpha develops from a single isolated cell - the spore - that divides asymmetrically. This produces a small terminally differentiating basal cell, and a large proliferative apical cell that gives rise to the multicellular sporeling body. The genetic basis for the establishment of asymmetry, first within the spore, and later within the sporeling body has remained virtually unknown. Here, we show that the plant specific RHO-type GTPase - RHO OF PLANTS (ROP) - polarises to the spore basal pole to ensure spore division is highly and reproducibly asymmetric. We demonstrate this highly asymmetric division is necessary to specify the terminal differentiation of the basal cell. Furthermore, we show that ROP-mediated polarised outgrowth is required to establish asymmetry within the sporeling body derived from the apical cell. Our discovery highlights how ROP function confers developmental robustness and contributes to the establishment of asymmetry during plant development from a single isolated cell.

plant biology↗

Initiation of asexual reproduction by the AP2/ERF gene GEMMIFER in Marchantia polymorpha

Plants can propagate their own clones through asexual reproduction. Genetic and hormonal factors regulating asexual reproduction have begun to be elucidated in the liverwort Marchantia polymorpha, which produce asexual propagules called gemmae within the gemma cups. Here, we report an AP2/ERF family gene, GEMMIFER (MpGMFR), as a key regulator of asexual reproduction in M. polymorpha. Suppression of MpGMFR function using genome editing and amiRNA results in the loss of gemma and gemma cup formation. In contrast, activation of MpGMFR function using a dexamethasone inducible system promotes gemma and/or gemma cup formation depending on the induction conditions. Notably, transient strong activation of MpGMFR induced gemma initial cells at the meristem, which develop into mature gemmae capable of reproducing as new individuals after detachment. These results show that MpGMFR is necessary and sufficient for the initiation of asexual reproduction at the meristem. MpGMFR expression was detected from the meristem through to the early stages of gemma development including the gemma cup floor cells. MpGMFR expression precedes the expression of GEMMA CUP-ASSOCIATED MYB1 (MpGCAM1) in early gemma development, supporting the notion that MpGMFR initiates asexual reproduction. We anticipate this finding to be a foundation to study the evolution of extra meristem formation in plant bodies that may have made a significant contribution to the prosperity of plants on land.

plant biology↗

Evolutionary conserved RLF, a plant cytochrome b5-like heme-binding protein, is essential for organ development in Marchantia polymorpha

In Arabidopsis thaliana, REDUCED LATERAL ROOT FORMATION (RLF), a cytochrome b5-like heme-binding domain (Cytb5-HBD) protein, is necessary for proper lateral root formation. Whereas the other Cytb5-HBD proteins in A. thaliana regulate different metabolic reactions, RLF is unique as it specifically regulates organ development. However, it remains unknown whether heme binding to RLF is necessary for its function, and whether RLF orthologs in different plant species also regulate organ development. We demonstrate that RLF binds to heme in vitro and that the two histidine residues, which are conserved among Cytb5-HBD, are crucial for both heme binding and its biological function in A. thaliana. In addition, MpRLF, a RLF ortholog in Marchantia polymorpha, rescues the lateral root formation phenotype of the A. thaliana rlf mutant. Mprlfge, the loss-of-function mutation in the MpRLF, resulted in delayed thallus growth and inhibited both gemma cup and antheridiophore formation. Transcriptome analysis using Mprlfge revealed that MpRLF affects several metabolic pathways. Our findings indicate that MpRLF is essential for vegetative and reproductive development in M. polymorpha, suggesting that the RLF-dependent redox reaction systems are evolutionarily conserved as crucial mechanisms for organ development across diverse plant species.

plant biology↗

Regulation of ROP GTPase cycling between active/inactive states is essential for vegetative organogenesis in Marchantia polymorpha

Rho/Rac of plant (ROP) GTPases are a plant-specific subfamily of Rho small GTP-binding proteins that function as molecular switches by being converted to the active state by guanine nucleotide exchange factors (GEFs) and to the inactive state by GTPase-activating proteins (GAPs). The bryophyte Marchantia polymorpha contains single-copy genes encoding ROP (MpROP), two types of GEFs (ROPGEF and SPIKE (SPK)), and two types of GAPs (ROPGAP and ROP enhancer (REN)). MpROP regulates the development of various organs, including the air chambers, rhizoids, and clonal propagule gemmae. While the sole PRONE-type ROPGEF, KARAPPO (MpKAR), plays an essential role in gemma initiation, little is known about the in-planta functions of other ROP regulatory factors in M. polymorpha. In this study, we focused on the functions of two types of GAPs: MpROPGAP and MpREN. Loss-of-function Mprenge single mutants showed pleiotropic defects in thallus growth, air chamber formation, rhizoid tip growth, and gemma development, whereas MpROPGAP mutants showed no detectable abnormalities. Despite the distinctive domain structures of MpROPGAP and MpREN, MpropgapgeMprenge double mutants showed more severe phenotypes than the Mprenge single mutants, suggesting redundant functions of MpROPGAP and MpREN in gametophyte organogenesis. Interestingly, overexpression of MpROPGAP, MpREN, and dominant-negative MpROP (MpROPDN) resulted in similar air chamber defects, as well as loss-of-function of MpREN and MpROPGAP and overexpression of constitutively active MpROP (MpROPCA), suggesting importance of activation/inactivation cycling (or balancing) of MpROP. Furthermore, we proved the contributions of the sole DOCK family GEF, MpSPK, to MpROP-regulated air chamber formation. In summary, our results demonstrate a significant role of the two GAPs in the development of various organs and that the two GEFs are responsible for organogenesis through the control of the MpROP active/inactive cycle in the vegetative growth of M. polymorpha.

plant biology↗

Control of cell fate specification and patterning by an ancestral microRNA

The formation of an organized body requires the establishment and maintenance of cells with structural and functional distinctive characteristics. A central question in developmental biology is how changes in the regulation of genes drive cell specification and patterning1. microRNAs (miRNAs) are small non-coding RNAs that regulate development through mRNA cleavage and/or translational repression2. In plants, miRNAs regulate key aspects including growth, development, stem cell maintenance, vegetative phase change, leaf morphogenesis, floral organ formation and flowering time3. Biogenesis of plant miRNAs depends on the activity of DICER-LIKE 1 (DCL1), an RNase type III endonuclease that processes double stranded RNA to give rise to mature miRNAs 4. The genomes of todays flora contain at least one bona fide copy of DCL1 5,6. Using Marchantia polymorpha -a model bryophyte that allows comparative approaches to infer characteristics of the ancestral land plant-, we demonstrate that MpDCL1a is required for the biogenesis of miRNAs and uncovered a central role for miR166/Homeodomain Zipper Class III-regulated auxin synthesis in the specification of cell identity, patterning, meristem function, laminar expansion and the development of the body in the last common ancestor of extant land plants.

plant biology↗

The bHLH transcription factor MpHYPNOS is a critical regulator of gemma dormancy in the liverwort Marchantia polymorpha.

O_LIDormancy is a key process employed by land plants to adapt to harsh terrestrial environments. The liverwort Marchantia polymorpha produces dormant propagules called gemmae for asexual reproduction. The plant hormone abscisic acid (ABA) plays important roles in regulating dormancy in both the seeds of flowering plants and the gemmae of M. polymorpha. C_LIO_LIBased on previous transcriptome analysis, we identified the basic helix-loop-helix transcription factor MpHYPNOS (MpHYP) as a key regulator of gemma dormancy. C_LIO_LIKnock-out mutants of MpHYP showed much higher germination rates of gemmae in gemma cups than ABA-related mutants, while the growth and development of these mutants resembled that of the wild type. Transient induction of MpHYP caused irreversible growth arrest of gemmae and thalli. Transcriptome and RT-qPCR analyses revealed that MpHYP represses the expression of cell cycle-related genes and induces ABA biosynthesis and ABA-responsive genes. Indeed, ABA levels increased in MpHYP overexpression lines and decreased in Mphyp knock-out lines. However, the growth arrest caused by MpHYP overexpression was not suppressed by a mutation in an ABA receptor gene. C_LIO_LIThese findings suggest that MpHYP regulates gemma dormancy and thallus growth mainly through the ABA-independent pathway, providing clues about ABA-dependent and independent regulation of dormancy in land plants. C_LI

plant biology↗

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↗

Transcriptional and morpho-physiological responses of Marchantia polymorpha upon phosphate starvation

Phosphate (Pi) is a pivotal nutrient that constraints plant development and productivity in natural ecosystems. Land colonization by plants, more than 470 million years ago, evolved adaptive mechanisms to conquer Pi-scarce environments. However, little is known about the molecular basis underlying such adaptations at early branches of plant phylogeny. To shed light on how early divergent plants respond to Pi limitation, we analyzed the morpho-physiological and transcriptional dynamics of Marchantia polymorpha upon Pi starvation. Our phylogenomic analysis highlights some gene networks present since the Chlorophytes and others established in the Streptophytes (eg. PHR1-SPX1 and STOP1-ALMT1, respectively). At the morpho-physiological level, the response is characterized by the induction of phosphatase activity, media acidification, accumulation of auronidins, reduction of internal Pi concentration and developmental modifications of rhizoids. The transcriptional response involves the induction of MpPHR1, Pi transporters, lipid turnover enzymes and MpMYB14, an essential transcription factor for auronidins biosynthesis. MpSTOP2 up-regulation correlates with expression changes in genes related to organic acid biosynthesis and transport, suggesting preference for citrate exudation. Analysis of MpPHR1 binding sequences (P1BS) shows enrichment of this cis regulatory element in differentially expressed genes. Our study unravels the strategies, at diverse levels of organization, exerted by M. polymorpha to cope with low Pi availability. Significance StatementThis study unravels the transcriptional and morphophysiological mechanisms executed by the non-vascular, and rootless, plant Marchantia polymorpha upon phosphate starvation conditions. The findings in this study shed light on the mechanisms that early land plants may have developed for the conquest of substrates poor in available phosphate, some of which are still conserved by current-day plants. Moreover, our results open several working hypotheses and novel perspectives for the study of Pi-starvation responses along plant evolution.

plant biology↗

Visualization of seasonal shifts of phosphorus re-translocation and overwintering recycling in a shortened annual cycle system of the deciduous poplar tree (Populus alba L.).

Phosphorus (P) is an essential macronutrient for plant growth. In deciduous trees, P is remobilized from senescing leaves and stored in perennial tissues during winter for further growth. Annual internal recycling and accumulation of P is considered an important strategy to support vigorous growth of trees. However, the pathways of seasonal re-translocation of P and the molecular mechanisms of this transport have not been clarified. Here we show the seasonal P re-translocation route visualized using the real-time radioisotope imaging and the macro- and micro-autoradiography. We analyzed the seasonal re-translocation P in poplar (Populus alba. L) cultivated under "a shortened annual cycle system", which mimicked seasonal phenology in a laboratory. From growing to senescing season, sink tissues of 32P and/or 33P shifted from young leaves and the apex to the lower stem and roots. The radioisotope P re-translocated from a leaf was stored in phloem and xylem parenchyma cells and redistributed to new shoots after dormancy. Seasonal expression profile of phosphate transporters (PHT1, PHT5 and PHO1 family) was obtained in the same system. Our results reveal the seasonal P re-translocation routes at the organ and tissue levels and provide a foothold for elucidating its molecular mechanisms.

plant biology↗

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↗

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↗

The RopGEF KARAPPO is Essential for the Initiation of Vegetative Reproduction in Marchantia

Many plants can reproduce vegetatively, producing clonal progeny from vegetative cells; however, little is known about the molecular mechanisms underlying this process. Liverwort (Marchantia polymorpha), a basal land plant, propagates asexually via gemmae, which are clonal plantlets formed in gemma cups on the dorsal side of the vegetative thallus [1]. The initial stage of gemma development involves elongation and asymmetric divisions of a specific type of epidermal cell, called a gemma initial, which forms on the floor of the gemma cup [2, 3]. To investigate the regulatory mechanism underlying gemma development, we focused on two allelic mutants in which no gemma initial formed; these mutants were named karappo, meaning \"empty\". We used whole-genome sequencing of both mutants, and molecular genetic analyses to identify the causal gene, KARAPPO (KAR), which encodes a Rop guanine nucleotide exchange factor (RopGEF) carrying a PRONE catalytic domain. In vitro GEF assays showed that the full-length KAR protein and the PRONE domain have significant GEF activity toward MpRop, the only Rop GTPase in M. polymorpha. Moreover, genetic complementation experiments showed a significant role for the N- and C-terminal variable regions in gemma development. Our investigation demonstrated an essential role for KAR/RopGEF in the initiation of plantlet development from a differentiated cell, which may involve cell polarity formation and subsequent asymmetric cell division via activation of Rop signaling, implying a similar developmental mechanism in vegetative reproduction of various land plants.

plant biology↗