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Calcium (Ca) deficiency causes necrotic symptoms of foliar edges known as tipburn; however, the underlying cellular mechanisms have been poorly understood due to the lack of an ideal plant model and research platform. Using a phenotyping system that quantitates growth and tipburn traits in the model bryophyte Marchantia polymorpha, we evaluated metabolic compounds and the Gβ-null mutant (gpb1) that modulate the occurrence and expansion of the tipburn. Transcriptomic comparisons between wild-type and gpb1 plants revealed the phenylalanine/phenylpropanoid biosynthesis pathway and reactive oxygen species (ROS) important for Ca deficiency responses. gpb1 plants reduced ROS production possibly through transcriptomic regulations of class III peroxidases and induced the expression of phenylpropanoid pathway enzymes without changing downstream lignin contents. Supplementation of intermediate metabolites and chemical inhibitors further confirmed the cell-protective mechanisms of the phenylpropanoid and ROS pathways. Marchantia polymorpha, Arabidopsis thaliana, and Lactuca sativa showed comparable transcriptomic changes where genes related to phenylpropanoid and ROS pathways were enriched in response to Ca deficiency. In conclusion, our study demonstrated unresolved signaling and metabolic pathways of Ca deficiency response. The phenotyping platform can speed up the discovery of chemical and genetic pathways, which could be widely conserved between M. polymorpha and angiosperms.

Sterols are the essential components of the eukaryotic cell membranes. However, studies on sterol biosynthesis in bryophytes are limited. This study analyzed the sterol profiles in the bryophyte model plant Marchantia polymorpha L. The thalli contained typical phytosterols such as campesterol, sitosterol, and stigmasterol. BLASTX analysis of the M. polymorpha genome against the Arabidopsis thaliana sterol biosynthetic genes confirmed the presence of all of the enzymes responsible for sterol biosynthesis in M. polymorpha. In this study, we focused on characterizing two genes, MpDWF5A and MpDWF5B, which showed high homology with A. thaliana DWF5, encoding Δ5,Δ7-sterol Δ7-reductase. Functional analysis using a yeast expression system revealed that MpDWF5A converted 7-dehydrocholesterol to cholesterol, indicating that MpDWF5A is a Δ5,Δ7-sterol Δ7-reductase. Mpdwf5a-knockout lines (Mpdwf5a-ko) were constructed using CRISPR/Cas9 mediated genome editing. GC-MS analysis of Mpdwf5a-ko revealed that phytosterols such as campesterol, sitosterol, and stigmasterol disappeared, and instead, the corresponding Δ7-type sterols accumulated. The thalli of Mpdwf5a-ko grew smaller than those of the wild type, and excessive formation of apical meristem in the thalli was observed. In addition, the gemma cups of the Mpdwf5a-ko were incomplete, and only a limited number of gemma formations were observed. Treatment with 1 µM of castesterone or 6-deoxocastasterone, a bioactive brassinosteroid, partly restored some of these abnormal phenotypes, but far from complete recovery. These results indicate that MpDWF5A is essential for the normal growth and development of M. polymorpha and suggest that the dwarfism caused by the MpDWF5A defect is due to the deficiency of typical phytosterols and, in part, a brassinosteroid-like compound derived from phytosterols.

Plants produce sugars by photosynthesis and use them for growth and development. Sugars are transported from source-to-sink organs via the phloem in the vasculature. It is well known that vascular development is precisely controlled by plant hormones and peptide hormones. However, the role of sugars in the regulation of vascular development is poorly understood. In this study, we examined the effects of sugars on vascular cell differentiation using a vascular cell induction system named ‘Vascular Cell Induction Culture System Using Arabidopsis Leaves’ (VISUAL). We found that sucrose has the strongest inhibitory effect on xylem differentiation, among several types of sugars. Transcriptome analysis revealed that sucrose suppresses xylem and phloem differentiation in cambial cells. Physiological and genetic analyses suggested that sucrose might function through the BRI1-EMS-SUPPRESSOR1 transcription factor, which is the central regulator of vascular cell differentiation. Conditional overexpression of cytosolic invertase led to a decrease in the number of cambium layers due to an imbalance between cell division and differentiation. Taken together, our results suggest that sucrose potentially acts as a signal that integrates environmental conditions with the developmental program.

K+/Na+ homeostasis is important for land plants, particularly under salt stress. In this study, the structure and ion transport properties of the high-affinity K+ transporter (HKT) of the liverwort Marchantia polymorpha were investigated. Only one HKT gene, MpHKT1, was identified in the genome of M. polymorpha. Phylogenetic analysis of HKT proteins revealed that non-seed plants possess HKTs grouped into a clade independent of the other two clades including HKTs of angiosperms. A distinct long hydrophilic domain was found in the C-terminus of MpHKT1. Complementary DNA (cDNA) of truncated MpHKT1 (t-MpHKT1) encoding the MpHKT_Δ596-812 protein was used to examine the functions of the C-terminal domain. Both MpHKT1 transporters fused with enhanced green fluorescent protein at the N-terminus were localized to the plasma membrane when expressed in rice protoplasts. Two-electrode voltage clamp experiments using Xenopus laevis oocytes indicated that MpHKT1 mediated the transport of monovalent alkali cations with higher selectivity for Na+ and K+, but truncation of the C-terminal domain significantly reduced the transport activity with a decrease in the Na+ permeability. Overexpression of MpHKT1 or t-MpHKT1 in M. polymorpha conferred accumulation of higher Na+ levels and showed higher Na+ uptake rates, compared to those of wild-type plants; however, phenotypes with t-MpHKT1 were consistently weaker than those with MpHKT1. Together, these findings suggest that the hydrophilic C-terminal domain plays a unique role in the regulation of transport activity and ion selectivity of MpHKT1.

Bryophytes occupy a basal position in the monophyletic evolution of land plants and have a life cycle in which the gametophyte generation dominates over the sporophyte generation, offering a significant advantage in conducting genetics. Owing to its low genetic redundancy and the availability of an array of versatile molecular tools, including efficient genome editing, the liverwort Marchantia polymorpha has become a model organism of choice that provides clues to the mechanisms underlying eco-evo-devo biology in plants. Recent analyses of developmental mutants have revealed that key genes in developmental processes are functionally well conserved in plants, despite their morphological differences, and that lineage-specific evolution occurred by neo/subfunctionalization of common ancestral genes. We suggest that M. polymorpha is an excellent platform to uncover the conserved and diversified mechanisms underlying land plant development.

The core G protein signaling module, which consists of Gα and extra-large Gα (XLG) subunits coupled with the Gβγ dimer, is a master regulator of various stress responses. In this study, we compared the basal and salt stress-induced transcriptomic, metabolomic and phenotypic profiles in Gα, Gβ, and XLG-null mutants of two plant species, Arabidopsis thaliana and Marchantia polymorpha, and showed that G protein mediates the shift of transcriptional and metabolic homeostasis to stress readiness status. We demonstrated that such stress readiness serves as an intrinsic protection mechanism against further stressors through enhancing the phenylpropanoid pathway and abscisic acid responses. Furthermore, WRKY transcription factors were identified as key intermediates of G protein-mediated homeostatic shifts. Statistical and mathematical model comparisons between A. thaliana and M. polymorpha revealed evolutionary conservation of transcriptional and metabolic networks over land plant evolution, whereas divergence has occurred in the function of plant-specific atypical XLG subunit. Taken together, our results indicate that the shifts in transcriptional and metabolic homeostasis at least partially act as the mechanisms of G protein-coupled stress responses that are conserved between two distantly related plants.

SummaryDormancy 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.Based on previous transcriptome analysis, we identified the basic helix-loop-helix transcription factor MpHYPNOS (MpHYP) as a key regulator of gemma dormancy.Knock-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.These 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.

The basal land plant Marchantia polymorpha efficiently propagates in favourable environments through clonal progeny called gemmae. Gemmae develop in cup-shaped receptacles known as gemma cups, which are formed on the gametophyte body. Anatomical studies have described the developmental processes involved over a century ago; however, little is known about the underlying molecular mechanisms. Recent studies have started to unravel the mechanism underlying genetic and hormonal regulation of gemma cup and gemma development, showing that it shares some regulatory mechanisms with several sporophytic organs in angiosperms. Further study of these specialized organs will contribute to our understanding of the core regulatory modules underlying organ development in land plants and how these became so diversified morphologically over the course of evolution.

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 development1,3,4. 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. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems. The minimal regulatory logic and the evolutionary history of the auxin signalling pathway are explored by studying the much simpler auxin network in Marchantia with a combination of genetic, domain shuffling and biochemical strategies.

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 are considered an important strategy to support the 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 real-time radioisotope imaging and the macro- and micro-autoradiography. We analysed 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

Bioactive specialized (secondary) metabolites are indispensable for plant development or adjustment to their surrounding environment. In many plants, these specialized metabolites are accumulated in specifically differentiated cells. Catharanthus roseus is a well-known medicinal plant known for producing many kinds of monoterpenoid indole alkaloids (MIAs). C. roseus has two types of specifically differentiated cells accumulating MIAs, so-called idioblast cells and laticifer cells. In this study, we compared each of the cells as they changed during seedling growth, and found that the fluorescent metabolites accumulated in these cells were differentially regulated. Analysis of fluorescent compounds revealed that the fluorescence observed in these cells was emitted from the compound serpentine. Further, we found that the serpentine content of leaves increased as leaves grew. Our findings suggest that idioblast cells and laticifer cells have different biological roles in MIA biosynthesis and its regulation.

Cytokinins are known to regulate various physiological events in plants. Cytokinin signaling is mediated by the phosphorelay system, one of the most ancient mechanisms controlling hormonal pathways in plants. The liverwort Marchantia polymorpha possesses all components necessary for cytokinin signaling; however, whether they respond to cytokinins and how the signaling is fine-tuned remain largely unknown. Here, we report cytokinin function in Marchantia development and organ formation. Our measurement of cytokinin species revealed that cis-zeatin is the most abundant cytokinin in Marchantia. We reduced the endogenous cytokinin level by overexpressing the gene for cytokinin oxidase, MpCKX, which inactivates cytokinins, and generated overexpression and knockout lines for type-A (MpRRA) and type-B (MpRRB) response regulators to manipulate the signaling. The overexpression lines of MpCKX and MpRRA, and the knockout lines of MpRRB, shared phenotypes such as inhibition of gemma cup formation, enhanced rhizoid formation and hyponastic thallus growth. Conversely, the knockout lines of MpRRA produced more gemma cups and exhibited epinastic thallus growth. MpRRA expression was elevated by cytokinin treatment and reduced by knocking out MpRRB, suggesting that MpRRA is upregulated by the MpRRB-mediated cytokinin signaling, which is antagonized by MpRRA. Our findings indicate that when plants moved onto land they already deployed the negative feedback loop of cytokinin signaling, which has an indispensable role in organogenesis.

The physiological importance of MpUVR8 in UV-B resistance and translocation in a UV-B-dependent manner from the cytosol into the nucleus is characterized in Marchantia polymorpha. UV RESISTANCE LOCUS 8 (UVR8) is an ultraviolet-B (UV-B) light receptor functioning for UV-B sensing and tolerance in Arabidopsis thaliana and other species. It is unclear whether UVR8 physiologically functions in UV-B-induced defense responses in Marchantia polymorpha, which belongs to the earliest diverging group of embryophyte lineages. Here, we demonstrate that UVR8 has a physiological function in UV-B tolerance and that there is a UVR8-dependent pathway involved. In addition, a UVR8-independent pathway is revealed. We examine the tissue-specific expression pattern of M. polymorpha UVR8 (MpUVR8), showing that it is highly expressed in the apical notch in thalli and gametangiophores, as well as in antheridial and archegonial heads. Furthermore, Mpuvr8KO plant transformants, in which the MpUVR8 locus was disrupted, were produced and analyzed to understand the physiological and molecular function of MpUVR8. Analysis using these plants indicates the important roles of MpUVR8 and MpUVR8-regulated genes, and of MpUVR8-independent pathways in UV-B tolerance. Subcellular localization of Citrine-fused MpUVR8 in M. polymorpha cells was also investigated. It was found to translocate from the cytosol into the nucleus in response to UV-B irradiation. Our findings indicate strong conservation of the physiological function of UVR8 and the molecular mechanisms for UVR8-dependent signal transduction through regulation of gene expression in embryophytes.

Bryophytes occupy a basal position in the monophyletic evolution of land plants and have a life cycle in which the gametophyte generation dominates over the sporophyte generation, offering a significant advantage in conducting genetics. Owing to its low genetic redundancy and the availability of an array of versatile molecular tools, including efficient genome editing, the liverwort

KARRIKIN INSENSITIVE2 (KAI2) was first identified as a receptor of karrikins, smoke-derived germination stimulants. KAI2 is also considered a receptor of an unidentified endogenous molecule called the KAI2 ligand. Upon KAI2 activation, signals are transmitted through the degradation of D53/SMXL proteins via MAX2-dependent ubiquitination. Although components in the KAI2-dependent signaling pathway, namely MpKAI2A and MpKAI2B, MpMAX2, and MpSMXL, exist in the genome of the liverwort Marchantia polymorpha, their functions remain unknown. Here, we show that early thallus growth is retarded and gemma dormancy in the dark is suppressed in Mpkai2a and Mpmax2 loss-of-function mutants. These defects are counteracted in Mpkai2a Mpsmxl and Mpmax2 Mpsmxl double mutants indicating that MpKAI2A, MpMAX2, and MpSMXL act in the same genetic pathway. Introduction of MpSMXLd53, in which a domain required for degradation is mutated, into wild-type plants mimicks Mpkai2a and Mpmax2 plants. In addition, the detection of citrine fluorescence in Nicotiana benthamiana cells transiently expressing a SMXL-Citrine fusion protein requires treatment with MG132, a proteasome inhibitor. These findings imply that MpSMXL is subjected to degradation, and that the degradation of MpSMXL is crucial for MpKAI2A-dependent signaling in M. polymorpha. Therefore, we claim that the basic mechanisms in the KAI2-dependent signaling pathway are conserved in M. polymorpha.

Plant hormones are master regulators of developmental and genetic mechanisms to deal with diverse environmental cues. Upon phosphate (Pi) limitation, vascular plants modify phytohormone metabolism to coordinate diverse mechanisms to overcome such stress. However, the transcriptional program underlying the hormonal signaling in response to Pi scarcity in early branches of land plant phylogeny remains unclear. Therefore, we explored the transcriptional dynamics of key genes involved in auxin, cytokinin, ethylene, jasmonate, gibberellin, and abscisic acid metabolism in the early divergent land plant Marchantia polymorpha upon Pi starvation. Our RNAseq approach revealed major changes in genes associated with auxin and ethylene biosynthesis. Genes involved in cytokinin synthesis are repressed. Interestingly, genes involved in auxin and ethylene signaling, such as MpARF1 and MpARF2, are upregulated. In contrast, MpARRb is down-regulated. Moreover, genes involved in the synthesis of jasmonates were highly upregulated, but those related to signaling did not change in expression. Our data suggest that auxin and ethylene act as positive regulators of rhizoid development under Pi-limited conditions, whereas cytokinin may act as a negative regulator. The transcriptional behavior of some hormone-related genes in Marchantia is similar to those described in controlling root hair development in arabidopsis, maize, and rice, upon Pi scarcity.

KARRIKIN INSENSITIVE2 (KAI2) was first identified inArabidopsis thalianaas 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 liverwortMarchantia polymorpha, namely MpKAI2Aand MpKAI2B, MpMAX2encoding 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, MpMAX2and MpSMXLact 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 Mpkai2aand Mpmax2mutants. In addition, Citrine fluorescence was detected in tobacco cells transiently transformed with the35S:MpSMXL-Citrinegene construct and treated with MG132, a proteasome inhibitor. On the other hand, introduction of35S:MpSMXLd53-Citrineconferred Citrine fluorescence without MG132 treatment. These findings imply that MpSMXL is subjected to degradation, and that degradation of MpSMXL is crucial for KL signaling inM. 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 inM. polymorpha.

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.

Land plant shoot structures evolved a diversity of lateral organs as morphological adaptations to the terrestrial environment, with lateral organs arising independently in different lineages. Vascular plants and bryophytes (basally diverging land plants) develop lateral organs from meristems of sporophytes and gametophytes, respectively. Understanding the mechanisms of lateral organ development among divergent plant lineages is crucial for understanding the evolutionary process of morphological diversification of land plants. However, our current knowledge of lateral organ differentiation mechanisms comes almost entirely from studies of seed plants, and thus, it remains unclear how these lateral structures evolved and whether common regulatory mechanisms control the development of analogous lateral organs. Here, we performed a mutant screen in the liverwort Marchantia polymorpha, a bryophyte, which produces gametophyte axes with nonphotosynthetic scalelike lateral organs. We found that an Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1 (ALOG) family protein, named M. polymorpha LATERAL ORGAN SUPRESSOR 1 (MpLOS1), regulates meristem maintenance and lateral organ development in Marchantia. A mutation in MpLOS1, preferentially expressed in lateral organs, induces lateral organs with misspecified identity and increased cell number and, furthermore, causes defects in apical meristem maintenance. Remarkably, MpLOS1 expression rescued the elongated spikelet phenotype of a MpLOS1 homolog in rice. This suggests that ALOG genes regulate the development of lateral organs in both gametophyte and sporophyte shoots by repressing cell divisions. We propose that the recruitment of ALOG-mediated growth repression was in part responsible for the convergent evolution of independently evolved lateral organs among highly divergent plant lineages, contributing to the morphological diversification of land plants., Ancestral land plants lacked leaves; instead, these evolved independently in each lineage and were key innovations that allowed the radiation of plants on land during the lower Palaeozoic. This study of the liverwort Marchantia polymorpha reveals that each time they evolved they used the same molecular mechanism to control leaf development.

Sugar metabolism pathways such as photosynthesis produce dicarbonyls, e.g. methylglyoxal (MG), which can cause cellular damage. The glyoxalase (GLX) system comprises two enzymes GLX1 and GLX2, and detoxifies MG; however, this system is poorly understood in the chloroplast, compared with the cytosol. In the present study, we determined GLX1 and GLX2 activities in spinach chloroplasts, which constituted 40% and 10%, respectively, of the total leaf glyoxalase activity. In Arabidopsis thaliana, five GFP-fusion GLXs were present in the chloroplasts. Under high CO2 concentrations, where increased photosynthesis promotes the MG production, GLX1 and GLX2 activities in A. thaliana increased and the expression of AtGLX1-2 and AtGLX2-5 was enhanced. On the basis of these findings and the phylogeny of GLX in oxygenic phototrophs, we propose that the GLX system scavenges MG produced in chloroplasts during photosynthesis.

Seasonal recycling of nutrients is an important strategy for deciduous perennials. Deciduous perennials maintain and expand their nutrient pools by the autumn nutrient remobilization and the subsequent winter storage throughout their long life. Phosphorus (P), one of the most important elements in living organisms, is remobilized from senescing leaves during autumn in deciduous trees. However, it remains unknown how phosphate is stored over winter. Here we show that in poplar trees (Populus alba L.), organic phosphates are accumulated in twigs from late summer to winter, and that IP6 (myo-inositol-1,2,3,4,5,6-hexakis phosphate: phytic acid) is the primary storage form. IP6 was found in high concentrations in twigs during winter and quickly decreased in early spring. In parenchyma cells of winter twigs, P was associated with electron-dense structures, similar to globoids found in seeds of higher plants. Various other deciduous trees were also found to accumulate IP6 in twigs during winter. We conclude that IP6 is the primary storage form of P in poplar trees during winter, and that it may be a common strategy for seasonal P storage in deciduous woody plants.

In land plants, photosystem I (PSI) photoinhibition limits carbon fixation and causes growth defects. In addition, recovery from PSI photoinhibition takes much longer than PSII photoinhibition when the PSI core-complex is degraded by oxidative damage. Accordingly, PSI photoinhibition should be avoided in land plants, and land plants should have evolved mechanisms to prevent PSI photoinhibition. However, such protection mechanisms have not yet been identified, and it remains unclear whether all land plants suffer from PSI photoinhibition in the same way. In the present study, we focused on the susceptibility of PSI to photoinhibition and investigated whether mechanisms of preventing PSI photoinhibition varied among land plant species. To assess the susceptibility of PSI to photoinhibition, we used repetitive short-pulse (rSP) illumination, which specifically induces PSI photoinhibition. Subsequently, we found that land plants possess a wide variety of tolerance mechanisms against PSI photoinhibition. In particular, gymnosperms, ferns and mosses/liverworts exhibited higher tolerance to rSP illumination-induced PSI photoinhibition than angiosperms, and detailed analyses indicated that the tolerance of these groups could be partly attributed to flavodiiron proteins, which protected PSI from photoinhibition by oxidizing the PSI reaction center chlorophyll (P700) as an electron acceptor. Furthermore, we demonstrate, for the first time, that gymnosperms, ferns and mosses/liverworts possess a protection mechanism against photoinhibition of PSI that differs from that of angiosperms.

Over-reduction of the photosynthetic electron transport (PET) chain should be avoided, because the accumulation of reducing electron carriers produces reactive oxygen species (ROS) within photosystem I (PSI) in thylakoid membranes and causes oxidative damage to chloroplasts. To prevent production of ROS in thylakoid membranes the H+ gradient (ΔpH) needs to be built up across the thylakoid membranes to suppress the over-reduction state of the PET chain. In this study, we aimed to identify the critical component that stimulates ΔpH formation under illumination in higher plants. To do this, we screened ethyl methane sulfonate (EMS)-treated Arabidopsis thaliana, in which the formation of ΔpH is impaired and the PET chain caused over-reduction under illumination. Subsequently, we isolated an allelic mutant that carries a missense mutation in the γ-subunit of chloroplastic CF0 CF1 -ATP synthase, named hope2. We found that hope2 suppressed the formation of ΔpH during photosynthesis because of the high H+ efflux activity from the lumenal to stromal side of the thylakoid membranes via CF0 CF1 -ATP synthase. Furthermore, PSI was in a more reduced state in hope2 than in wild-type (WT) plants, and hope2 was more vulnerable to PSI photoinhibition than WT under illumination. These results suggested that chloroplastic CF0 CF1 -ATP synthase adjusts the redox state of the PET chain, especially for PSI, by modulating H+ efflux activity across the thylakoid membranes. Our findings suggest the importance of the buildup of ΔpH depending on CF0 CF1 -ATP synthase to adjust the redox state of the reaction center chlorophyll P700 in PSI and to suppress the production of ROS in PSI during photosynthesis.

The processes involved in sexual reproduction have been diversified during plant evolution. Whereas charales, bryophytes, pteridophytes, and some gymnosperms utilize motile sperm as male gametes, in other gymnosperms and angiosperms the immotile sperm cells are delivered to the egg cells through elongated pollen tubes. During formation of the motile sperms, cells undergo a dynamic morphological transformation including drastic changes in shape and the generation of locomotor architecture. The molecular mechanism involved in this process remains mostly unknown. Membrane trafficking fulfills the exchange of various proteins and lipids among single membrane-bound organelles in eukaryotic cells, contributing to various biological functions. RAB GTPases and SNARE proteins are evolutionarily conserved key machineries of membrane trafficking mechanisms, which regulate tethering and fusion of the transport vesicles to target membranes. Our observation of fluorescently tagged plasma membrane-resident SNARE proteins demonstrated that these proteins relocalize to spherical structures during the late stages in spermiogenesis. Similar changes in subcellular localization were also observed for other fluorescently tagged SNARE proteins and a RAB GTPase, which acts on other organelles including the Golgi apparatus and endosomes. Notably, a vacuolar SNARE, MpVAMP71, was localized on the membrane of the spherical structures. Electron microscopic analysis revealed that there are many degradation-related structures such as multi-vesicular bodies, autophagosomes, and autophagic bodies containing organelles. Our results indicate that the cell-autonomous degradation pathway plays a crucial role in the removal of membrane components and the cytoplasm during spermiogenesis of Marchantia polymorpha. This process differs substantially from mammalian spermatogenesis in which phagocytic removal of excess cytoplasm involves neighboring cells.

The diffusion efficiency of oxygen in the atmosphere, like that of CO2, is approximately 104 times greater than that in aqueous environments. Consequently, terrestrial photosynthetic organisms need mechanisms to protect against potential oxidative damage. The liverwort Marchantia polymorpha, a basal land plant, has habitats where it is exposed to both water and the atmosphere. Furthermore, like cyanobacteria, M. polymorpha has genes encoding flavodiiron proteins (FLV). In cyanobacteria, FLVs mediate oxygen-dependent alternative electron flow (AEF) to suppress the production of reactive oxygen species. Here, we investigated whether FLVs are required for the protection of photosynthesis in M. polymorpha A mutant deficient in the FLV1 isozyme (ΔMpFlv1) sustained photooxidative damage to photosystem I (PSI) following repetitive short-saturation pulses of light. Compared with the wild type (Takaragaike-1), ΔMpFlv1 showed the same photosynthetic oxygen evolution rate but a lower electron transport rate during the induction phase of photosynthesis. Additionally, the reaction center chlorophyll in PSI, P700, was highly reduced in ΔMpFlv1 but not in Takaragaike-1. These results indicate that the gene product of MpFlv1 drives AEF to oxidize PSI, as in cyanobacteria. Furthermore, FLV-mediated AEF supports the production of a proton motive force to possibly induce the nonphotochemical quenching of chlorophyll fluorescence and suppress electron transport in the cytochrome b6/f complex. After submerging the thalli, a decrease in photosystem II operating efficiency was observed, particularly in ΔMpFlv1, which implies that species living in these sorts of habitats require FLV-mediated AEF.

Cell division patterning is important to determine body shape in plants. Nuclear auxin signaling mediated by AUXIN RESPONSE FACTOR (ARF) transcription factors affects plant growth and development through regulation of cell division, elongation and differentiation. The evolutionary origin of the ARF-mediated pathway dates back to at least the common ancestor of bryophytes and other land plants. The liverwort Marchantia polymorpha has three phylogenetically distinct ARFs: MpARF1, the sole ‘activator’ ARF; and MpARF2 and MpARF3, two ‘repressor’ ARFs. Genetic screens for auxin-resistant mutants revealed that loss of MpARF1 function conferred auxin insensitivity. Mparf1 mutants showed reduced auxin-inducible gene expression and various developmental defects, including thallus twisting and gemma malformation. We further investigated the role of MpARF1 in gemma development, which is traceable at the cellular level. In wild-type plants, a gemma initial first undergoes several transverse divisions to generate a single-celled stalk and a gemma proper, followed by rather synchronous longitudinal divisions in the latter. Mparf1 mutants often contained multicelled stalks and showed defects in the execution and timing of the longitudinal divisions. While wild-type gemmae finally generate two meristem notches, Mparf1 gemmae displayed various numbers of ectopic meristems. These results suggest that MpARF1 regulates formative cell divisions and axis formation through auxin responses. The mechanism for activator ARF regulation of pattern formation may be shared in land plants and therefore important for the general acquisition of three-dimensional body plans.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity

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.

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.

Catharanthus roseus is a medicinal plant well known for producing bioactive compounds such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). Although the leaves of this plant are the main source of these antitumour drugs, much remains unknown on how TIAs are biosynthesised from a central precursor, strictosidine, to various TIAs in planta. Here, we have succeeded in showing, for the first time in leaf tissue of C. roseus, cell-specific TIAs localisation and accumulation with 10 μm spatial resolution Imaging mass spectrometry (Imaging MS) and live single-cell mass spectrometry (single-cell MS). These metabolomic studies revealed that most TIA precursors (iridoids) are localised in the epidermal cells, but major TIAs including serpentine and vindoline are localised instead in idioblast cells. Interestingly, the central TIA intermediate strictosidine also accumulates in both epidermal and idioblast cells of C. roseus. Moreover, we also found that vindoline accumulation increases in laticifer cells as the leaf expands. These discoveries highlight the complexity of intercellular localisation in plant specialised metabolism.

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, on the gametophyte thallus from the basal epidermis of 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. Targeted disruption of GCAM1 conferred a complete loss of gemma cup formation and gemmae generation. Ectopic overexpression of GCAM1 resulted in formation of cell clumps, suggesting a function of GCAM1 in suppression of cell differentiation. 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 new meristems, and the use of the mechanism for diverse developmental programs during land plant evolution.

Phytochromes are red light (R) and far-red light (FR) receptors that play important roles in many aspects of plant growth and development. Phytochromes mainly function in the nucleus and regulate sets of genes by inhibiting negatively acting basic helix-loop-helix transcription factors named PHYTOCHROME INTERACTING FACTORs (PIFs) in Arabidopsis thaliana. Although R/FR photoreversible responses and phytochrome genes are well documented in diverse lineages of plants, the extent to which phytochrome signaling is mediated by gene regulation beyond angiosperms remains largely unclear. Here, we show that the liverwort Marchantia polymorpha, an emerging model basal land plant, has only one phytochrome gene, Mp-PHY, and only one PIF gene, Mp-PIF. These genes mediate typical low fluence responses, which are reversibly elicited by R and FR, and regulate gene expression. Mp-phy is light-stable and translocates into the nucleus upon irradiation with either R or FR, demonstrating that the single phytochrome Mp-phy exhibits combined biochemical and cell-biological characteristics of type I and type II phytochromes. Mp-phy photoreversibly regulates gemma germination and downstream gene expression by interacting with Mp-PIF and targeting it for degradation in an R-dependent manner. Our findings suggest that the molecular mechanisms for light-dependent transcriptional regulation mediated by PIF transcription factors were established early in land plant evolution.

In land plants, there are two types of male gametes: one is a non-motile sperm cell which is delivered to the egg cell by a pollen tube, and the other is a motile sperm cell with flagella. The molecular mechanism underlying the sexual reproduction with the egg and pollen-delivered sperm cell is well understood from studies using model plants such as Arabidopsis and rice. On the other hand, the sexual reproduction with motile sperm has remained poorly characterized, due to the lack of suitable models. Marchantia polymorpha L. is a model basal land plant with sexual reproduction involving an egg cell and bi-flagellated motile sperm. To understand the differentiation process of plant motile sperm, we analyzed the gene expression profile of developing antheridia of M. polymorpha. We performed RNA-sequencing experiments and compared transcript profiles of the male sexual organ (antheridiophore and antheridium contained therein), female sexual organ (archegoniophore) and a vegetative organ (thallus). Transcriptome analysis showed that the antheridium expresses nearly half of the protein-coding genes predicted in the genome, but it also has unique features. The antheridium transcriptome shares some common features with male gamete transcriptomes of angiosperms and animals, and homologs of genes involved in male gamete formation and function in angiosperms and animals were identified. In addition, we showed that some of them had distinct expression patterns in the spermatogenous tissue of developing antheridia. This study provides a transcriptional framework on which to study the molecular mechanism of plant motile sperm development in M. polymorpha as a model.

Bryophytes (liverworts, hornworts and mosses) comprise the three earliest diverging lineages of land plants (embryophytes). Marchantia polymorpha, a complex thalloid Marchantiopsida liverwort that has been developed into a model genetic system, occupies a key phylogenetic position. Therefore, M. polymorpha is useful in studies aiming to elucidate the evolution of gene regulation mechanisms in plants. In this study, we used computational, transcriptomic, small RNA and degradome analyses to characterize microRNA (miRNA)-mediated pathways of gene regulation in M. polymorpha. The data have been integrated into the open access ContigViews-miRNA platform for further reference. In addition to core components of the miRNA pathway, 129 unique miRNA sequences, 11 of which could be classified into seven miRNA families that are conserved in embryophytes (miR166a, miR390, miR529c, miR171-3p, miR408a, miR160 and miR319a), were identified. A combination of computational and degradome analyses allowed us to identify and experimentally validate 249 targets. In some cases, the target genes are orthologous to those of other embryophytes, but in other cases, the conserved miRNAs target either paralogs or members of different gene families. In addition, the newly discovered Mpo-miR11707.1 and Mpo-miR11707.2 are generated from a common precursor and target MpARGONAUTE1 (LW1759). Two other newly discovered miRNAs, Mpo-miR11687.1 and Mpo-miR11681.1, target the MADS-box transcription factors MpMADS1 and MpMADS2, respectively. Interestingly, one of the pentatricopeptide repeat (PPR) gene family members, MpPPR_66 (LW9825), the protein products of which are generally involved in various steps of RNA metabolism, has a long stem-loop transcript that can generate Mpo-miR11692.1 to autoregulate MpPPR_66 (LW9825) mRNA. This study provides a foundation for further investigations of the RNA-mediated silencing mechanism in M. polymorpha as well as of the evolution of this gene silencing pathway in embryophytes.