Abstract The liverwort Marchantia polymorpha is equipped with a wide range of molecular and genetic tools and resources that have led to its wide use to explore the evo-devo aspects of land plants. Although its diverse transcriptome data are rapidly accumulating, there is no extensive yet user-friendly tool to exploit such a compilation of data and to summarize results with the latest annotations. Here, we have developed a web-based suite of tools, MarpolBase Expression (MBEX, https://marchantia.info/mbex/), where users can visualize gene expression profiles, identify differentially expressed genes, perform co-expression and functional enrichment analyses, and summarize their comprehensive output in various portable formats. Using oil body biogenesis as an example, we demonstrated that the results generated by MBEX were consistent with the published experimental evidence and also revealed a novel transcriptional network in this process. MBEX should facilitate the exploration and discovery of the genetic and functional networks behind various biological processes in M. polymorpha, and promote our understanding of the evolution of land plants.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Peroxisomes are present in eukaryotic cells and have essential roles in various biological processes. Plant peroxisomes proliferate by de novo biosynthesis or division of pre-existing peroxisomes, degrade, or replace metabolic enzymes, in response to developmental stages, environmental changes, or external stimuli. Defects of peroxisome functions and biogenesis alter a variety of biological processes and cause aberrant plant growth. Traditionally, peroxisomal function-based screening has been employed to isolate Arabidopsis thaliana mutants that are defective in peroxisomal metabolism, such as lipid degradation and photorespiration. These analyses have revealed that the number, subcellular localization, and activity of peroxisomes are closely related to their efficient function, and the molecular mechanisms underlying peroxisome dynamics including organelle biogenesis, protein transport, and organelle interactions must be understood. Various approaches have been adopted to identify factors involved in peroxisome dynamics. With the development of imaging techniques and fluorescent proteins, peroxisome research has been accelerated. Image-based analyses provide intriguing results concerning the movement, morphology, and number of peroxisomes that were hard to obtain by other approaches. This review addresses image-based analysis of peroxisome dynamics in plants, especially A. thaliana and Marchantia polymorpha.

The liverwort Marchantia polymorpha possesses oil bodies in idioblastic oil body cells scattered in its thallus. Oil bodies are subcellular organelles in which specific sesquiterpenes and bisbibenzyls are accumulated. Therefore, a specialized system for the biosynthesis and accumulation of these defense compounds specifically in oil bodies has been implied. A recent study on M. polymorpha genome sequencing revealed 10 genes that shared high similarities with fungal-type terpene synthases (TPSs). Eight of these fungal-type TPS-like genes in M. polymorpha (MpFTPSL1-6, -9 and -10) are located within a 376-kb stretch on chromosome 6 and share similarities of over 94% at the nucleotide level. Therefore, these genes have likely originated from recent gene duplication events. The expression of a subset of MpFTPSLs was induced under non-axenic growth on vermiculite, which increased the amounts of sesquiterpenes and number of oil bodies. The tdTomato fluorescent protein-based in-fusion reporter assay with MpFTPSL2 promoter revealed fluorescent signals specifically in oil body cells of the thallus, indicating that MpFTPSL2 functions in oil body cells. Recombinant MpFTPSL2 expression in Escherichia coli led to sesquiterpene synthesis from farnesyl pyrophosphate. Moreover, suppression of a subset of MpFTPSLs through RNA interference reduced sesquiterpene accumulation in thalli grown on vermiculite. Taken together, these results suggest that at least a subset of MpFTPSLs is involved in sesquiterpene synthesis in oil body cells.

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.

We describe a simple and efficient plastid transformation method for the liverwort, Marchantia polymorpha L. Use of rapidly proliferating cells such as sporelings, which are immature thalli developing from spores, as targets made plastid transformation by particle bombardment efficient. Selection on a sucrose-free medium and linearization of the transformation vector significantly improved the recovery rate of plastid transformants. With the method described here, a few plastid transformants are obtained from a single bombardment of sporelings. Homoplasmic transformants of thalli are obtained immediately after primary selection.

Regulation of the stability and the quality of mitochondrial RNA is essential for the maintenance of mitochondrial and cellular functions in eukaryotes. We have previously reported that the eukaryotic poly(A)-specific ribonuclease (PARN) and the prokaryotic poly(A) polymerase encoded by AHG2 and AGS1, respectively, coordinately regulate the poly(A) status and the stability of mitochondrial mRNA in Arabidopsis. Mitochondrial function of PARN has not been reported in any other eukaryotes. To know how much this PARN-based mitochondrial mRNA regulation is conserved among plants, we studied the AHG2 and AGS1 counterparts of the liverwort, Marchantia polymorpha, a member of basal land plant lineage. We found that M. polymorpha has one ortholog each for AHG2 and AGS1, named MpAHG2 and MpAGS1, respectively. Their Citrine-fused proteins were detected in mitochondria of the liverwort. Molecular genetic analysis showed that MpAHG2 is essential and functionally interacts with MpAGS1 as observed in Arabidopsis. A recombinant MpAHG2 protein had a deadenylase activity in vitro. Overexpression of MpAGS1 and the reduced expression of MpAHG2 caused an accumulation of polyadenylated Mpcox1 mRNA. Furthermore, MpAHG2 suppressed Arabidopsis ahg2-1 mutant phenotype. These results suggest that the PARN-based mitochondrial mRNA regulatory system is conserved in land plants.

Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes, and our knowledge of the evolution of chromatin-related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants circa 400 mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional genome organization in an early diverging land plant lineage. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and other repetitive sequences in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres. Although a large fraction of constitutive heterochromatin was marked by H3K9 methylation as in flowering plants, a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein-coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that repressed B compartments were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that, in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

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 analysis to identify the causal gene, KARAPPO (KAR), which encodes a ROP guanine nucleotide exchange factor (RopGEF) carrying a plant-specific ROP nucleotide exchanger (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 demonstrates 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.

The liverwort <i>Marchantia polymorpha</i> has become one of the model organisms, since it has less genetic redundancy, sexual and asexual modes of reproduction and a range of genomic and molecular genetic resources. Cryopreservation of fertile spermatozoa eliminates time, space and labor for growing and maintaining male plants in reproductive phase, and also provides an optional way to backup lines. Here we report a protocol to cryopreserve spermatozoa of <i>M. polymorpha</i> in liquid nitrogen. A cryoprotective solution containing sucrose, glycerol and egg yolk and controlled cooling and warming processes led to successful recovery of motile <i>M. polymorpha</i> spermatozoa after the cryogenic process. The survival rate and average motility of spermatozoa after cryopreservation were maintained at 71 and 54% of those before cryopreservation, respectively. Cryopreserved spermatozoa were capable of fertilization to form normal spores. The technique presented here confers more versatility to experiments using <i>M. polymorpha</i> and could be applied to preservation of plant spermatozoa in general.

Land plants differentiate germ cells in the haploid gametophyte. In flowering plants, a generative cell is specified as a precursor that subsequently divides into two sperm cells in the developing male gametophyte, pollen. Generative cell specification requires cell-cycle control and microtubule-dependent nuclear relocation (reviewed in [1-3]). However, the generative cell fate determinant and its evolutionary origin are still unknown. In bryophytes, gametophytes produce eggs and sperm in multicellular reproductive organs called archegonia and antheridia, respectively, or collectively called gametangia. Given the monophyletic origin of land plants [4-6], evolutionarily conserved mechanisms may play key roles in these diverse reproductive processes. Here, we showed that a single member of the subfamily VIIIa of basic helix-loop-helix (bHLH) transcription factors in the liverwort Marchantia polymorpha primarily accumulated in the initial cells and controlled their development into gametangia. We then demonstrated that an Arabidopsis thaliana VIIIa bHLH transiently accumulated in the smaller daughter cell after an asymmetric division of the meiosis-derived microspore and was required for generative cell specification redundantly with its paralog. Furthermore, these A. thaliana VIIIa bHLHs were functionally replaceable by the M. polymorpha VIIIa bHLH. These findings suggest the VIIIa bHLH proteins as core regulators for reproductive development, including germ cell differentiation, since an early stage of land plant evolution.

, Copyright © 2018 American Society of Plant Biologists. All rights reserved. Green leaf volatiles (GLVs), including six-carbon (C6) aldehydes, alcohols, and esters, are formed when plant tissues are damaged. GLVs play roles in direct plant defense at wound sites, indirect plant defense via the attraction of herbivore predators, and plant-plant communication. GLV components provoke distinctive responses in their target recipients; therefore, the control of GLV composition is important for plants to appropriately manage stress responses. The reduction of C6-aldehydes into C6-alcohols is a key step in the control of GLV composition and also is important to avoid a toxic buildup of C6-aldehydes. However, the molecular mechanisms behind C6-aldehyde reduction remain poorly understood. In this study, we purified an Arabidopsis (Arabidopsis thaliana) NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980, named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR). CHR T-DNA knockout mutant plants displayed a normal growth phenotype; however, we observed significant suppression of C6-alcohol production following partial mechanical wounding or herbivore infestation. Our data also showed that the parasitic wasp Cotesia vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants compared with GLVs emitted from chr plants, which corresponded with reduced C6-alcohol levels in the mutant. Moreover, chr plants were more susceptible to exogenous high-dose exposure to (Z)-3-hexenal, as indicated by their markedly lowered photosystem II activity. Our study shows that reductases play significant roles in changing GLV composition and, thus, are important in avoiding toxicity from volatile carbonyls and in the attraction of herbivore predators.

Eleocharis vivipara is an amphibious sedge that displays C4 traits under terrestrial environments and C3 traits in submerged environments. This plant is thus potentially advantageous for screening genes indispensable to the development of C4 photosynthesis. In this study, we performed de novo transcriptome analysis of E. vivipara using its terrestrial- and submerged-type plants. By next-generation sequencing (NGS), approximately 90 and 89 million reads were yielded for the terrestrial and submerged types, respectively, and were assembled into 27,249 unigenes. Of these de novo consensus sequences, 94.5% showed similarities to database-registered sequences, and 69.4% were assigned with Gene Ontology terms. Our de novo assembled sequence data should provide a foundation for genetic analysis of the C4 photosynthetic system.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

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.

Most terrestrial plants form green leaf volatiles (GLVs), which are mainly composed of six-carbon (C6) compounds. In our effort to study the distribution of the ability of lipoxygenase (LOX) to form GLVs, we found that a liverwort, Marchantia polymorpha, formed n-hexanal and (Z)-3-hexenal. Some LOXs execute a secondary reaction to form short chain volatiles. One of the LOXs from M. polymorpha (MpLOX7) oxygenized arachidonic and -linolenic acids at almost equivalent efficiency and formed C6-aldehydes during its catalysis; these are likely formed from hydroperoxides of arachidonic and -linolenic acids, with a cleavage of the bond between carbon at the base of the hydroperoxy group and carbon of double bond, which is energetically unfavorable. These lines of evidence suggest that one of the LOXs in liverwort employs an unprecedented reaction to form C6 aldehydes as by-products of its reaction with fatty acid substrates.

The cytoplasm of laticifers, which are plant cells specialized for rubber production and defense against microbes and herbivores, is a latex. Although laticifers share common functions, the protein constituents of latexes are highly variable among plant species and even among organs. In this study, transcriptomic and proteomic analyses of Euphorbia tirucalli's (Euphorbiaceae) latex were conducted to determine the molecular basis of the laticifer's functions in this plant. The hybrid de novo assembly of Illumina mRNA-seq and expressed sequence tags obtained by Sanger's sequencing revealed 26,447 unigenes. A unigene similar to Arabidopsis embryo-specific protein 3 (AT5G62200), which is a PLAT domain-containing protein, and rubber elongation factor showed the highest expression levels. The proteome analysis, studied by liquid chromatography-mass spectrometry with the de novo assembled unigenes as the database, revealed 161 proteins in the latex, 107 of which were not detected in the stem. A gene ontology analysis indicated that the laticifer's proteome was enriched with proteins related to proteolysis, phosphatase, defense against various environmental stresses and lipid metabolisms. D-mannose-binding lectin, ricin (which lacked the N-terminal conserved ribosome-inactivating protein domain), chitinase and peroxidase were highly accumulated, as confirmed by two-dimensional polyacrylamide gel electrophoresis. Thus, the lectins and chitinase may be the major defensive proteins against pests, and the other defense-related proteins and transcripts detected in latex may work in coordination with them. Highly expressing unigenes with unknown functions are candidate novel defense- or rubber production related genes. (C) 2016 Elsevier Masson SAS. All rights reserved.

In contrast to animals, in which the germ cell lineage is established during embryogenesis, plant germ cells are generated in reproductive organs via reprogramming of somatic cells. The factors that control germ cell differentiation and reprogramming in plants are poorly understood. Members of the RKD subfamily of plant-specific RWP-RK transcription factors have been implicated in egg cell formation in Arabidopsis based on their expression patterns and ability to cause an egg-like transcriptome upon ectopic expression [1]; however, genetic evidence of their involvement is lacking, due to possible genetic redundancy, haploid lethality, and the technical difficulty of analyzing egg cell differentiation in angiosperms. Here we analyzed the factors that govern germ cell formation in the liverwort Marchantia polymorpha. This recently revived model bryophyte has several characteristics that make it ideal for studies of germ cell formation, such as low levels of genetic redundancy, readily accessible germ cells, and the ability to propagate asexually via gemma formation [2, 3]. Our analyses revealed that MpRKD, a single RWP-RK factor closely related to angiosperm RKDs, is preferentially expressed in developing eggs and sperm precursors in M. polymorpha. Targeted disruption of MpRKD had no effect on the gross morphology of the vegetative and reproductive organs but led to striking defects in egg and sperm cell differentiation, demonstrating that MpRKD is an essential regulator of germ cell differentiation. Together with previous findings [1, 4-6], our results suggest that RKD factors are evolutionarily conserved regulators of germ cell differentiation in land plants.

バラ科シモツケ属のキイシモツケ（Spiraea nipponica Maxim. var. ogawae（ Nakai） Yamanaka）は、蛇紋岩地帯にて自生する和歌山県固有の植物である。県北部に位置する龍門山の山頂付近の群落は県の天然記念物に指定されている。キイシモツケは、かつては新種とされSpiraea ogawai Nakai と命名され登録されていた。しかし近年は形態学的比較に基づいて、高知県に自生するトサシモツケ（Spiraea nipponica Maxim. var. tosaensis（ Yatabe） Makino）と共に、近畿以北に自生するイワシモツケ（Spiraea nipponica Maxim.）の変種とされ、学名も上記のように改められている。本研究では、キイシモツケがトサシモツケおよびイワシモツケなどの近縁種とは顕著に異なるのかどうかを初めて分子系統分類学の立場から検討した。同時に、キイシモツケの蛇紋岩土壌との関わりについて若干の検討を行った。まず、3 種類それぞれに特徴的な葉の形態は、種子から同一の環境条件下で生育させても再現され、生来の形質であることがわかった。次に、龍門山の土壌の交換性金属イオンの元素組成を調べ、蛇紋岩土壌特有の組成をもつことが確かめられた。キイシモツケの植物体について、金属イオンの元素分析を行った結果、蛇紋岩土壌の元素の中で最も植物に対する毒性が強いとされるニッケル（Ni）が植物体内に取り込まれていることを認めた。さらにキイシモツケの種子は発芽時に3 者の中で最も強いNi 耐性を示し、若い苗では10 mmol L - 1 のNi 2 + に対しても耐性を示した。葉緑体DNA のtRNA のtrnL – trnF 領域および核ゲノムDNA の5S RNA 遺伝子とその両端のスペーサー配列を含むITS 領域の塩基配列を解析した。その結果、葉緑体のtRNA 領域（約1000 塩基対）は完全に一致し、ITS 領域（約640 塩基対）では3 種は互いに数塩基（1％以下）異なるのみであり、分子系統学的比較においてもキイシモツケはイワシモツケおよびトサシモツケと極めて近縁であることが証明された。したがって、キイシモツケは日本の近畿以北に広く分布するイワシモツケを起源とし、和歌山の限られた蛇紋岩地帯に適応して、地理的に隔絶して自生するにいたったエコタイプであると推測された。A plant called Kiishimotsuke（ in Japanese） is indigenous to Wakayama prefecture in Japan. The plant vegetates only in the three narrow mountain areas consisting of serpentine soils. The largest colony of this plant in the area of Mt. Ryumon higher than 600 m above sea level had been designated as a prefectural natural treasure since May 1971. The plant was first discovered by Y. Ogawa, a botanist in Iwade city, and registered as a new species with a name of Spiraea ogawai Nakai in 1928. However, the validity of this registration was questioned later, because there were two kinds of plants, called Iwashimotsuke and Tosashimotsuke, which were morphologically similar to Kiishimotuke except for the shape of leaves. Iwashimotsuke is vegetating in northern area than Kinki district, and Tosashimotsuke indigenously in Kochi prefecture. Thus at present Iwashimotsuke is named as Spiraea nipponica Maxim, Tosashimotsuke as S. nipponica Maxim var. tosaensis（ Yatabe） Makino, and Kiishimotsuke as S. nipponica Maxim var. ogawae （Nakai） Yamanaka. In view of recent progress of molecular phylogenetics, the conventional method was employed to examine whether Kiishimotsuke is a species intimately related to the other two plants or not. The nucleotide sequences were determined for the regions of trn L – trn F of chloroplast DNA and ITS （Internal Transcribed Spacer） of 5S RNA coding region of nuclear DNA. The results showed that the differences in the sequences were less than 1％ among the three, demonstrating their close evolutionary relationships. Thus it is reasonable to conclude that Kiishimotsuke is a variety or ecotype of S. nipponica based not only on morphological comparison but also on molecular phylogenetic analysis. Unexpectedly significant difference in the sequence of the ITS for Iwashimotsuke（ Spiraea nipponica Maxim） was found between the one published by Potter et al.（ DQ897622） and ours（ LC133174 and LC13375）. The difference remains to be elucidated. Since Kiishimotsuke is endemic to serpentine soils in Wakayama, the interaction of the plant with serpentine soils was also studied. When these three plants were raised from seeds on non-serpentine cultivation soils, all of them grew normally and the differences in the shape of the leaves were retained, indicating the shapes of leaves were not affected by the kind of soil but determined inherently. Nikkel ion is known to be one of the most toxic metal ions in serpentine soils. When the effects of increasing concentrations of Ni 2 + on the sprouting of seeds were tested, Kiishimotsuke was most tolerant among others. In the aboveground parts of Kiishimotsuke plants grown on serpentine soils of Mt. Ryumon, accumulation of Ni 2 + was confirmed and its possible significance of the plant survival was discussed.

Abscisic acid (ABA) is a phytohormone widely distributed among members of the land plant lineage (Embryophyta), regulating dormancy, stomata closure and tolerance to environmental stresses. In angiosperms (Magnoliophyta), ABA-induced gene expression is mediated by promoter elements such as the G-box-like ACGT-core motifs recognized by bZIP transcription factors. In contrast, the mode of regulation by ABA of gene expression in liverworts (Marchantiophyta), representing one of the earliest diverging land plant groups, has not been elucidated. In this study, we used promoters of the liverwort Marchantia polymorpha dehydrin and the wheat Em genes fused to the -glucuronidase (GUS) reporter gene to investigate ABA-induced gene expression in liverworts. Transient assays of cultured cells of Marchantia indicated that ACGT-core motifs proximal to the transcription initiation site play a role in the ABA-induced gene expression. The RY sequence recognized by B3 transcriptional regulators was also shown to be responsible for the ABA-induced gene expression. In transgenic Marchantia plants, ABA treatment elicited an increase in GUS expression in young gemmalings, which was abolished by simultaneous disruption of the ACGT-core and RY elements. ABA-induced GUS expression was less obvious in mature thalli than in young gemmalings, associated with reductions in sensitivity to exogenous ABA during gametophyte growth. In contrast, lunularic acid, which had been suggested to function as an ABA-like substance, had no effect on GUS expression. The results demonstrate the presence of ABA-specific response mechanisms mediated by conserved cis-regulatory elements in liverworts, implying that the mechanisms had been acquired in the common ancestors of embryophytes.

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.

While Marchantia polymorpha has been utilized as a model system to investigate fundamental biological questions for over almost two centuries, there is renewed interest in M. polymorpha as a model genetic organism in the genomics era. Here we outline community guidelines for M. polymorpha gene and transgene nomenclature, and we anticipate that these guidelines will promote consistency and reduce both redundancy and confusion in the scientific literature.

The liverwort Marchantia polymorpha L. is one of the key model plants in evo-devo studies, and an increasing number of transgenic and mutant lines have been established. For reliable long-term preservation of M. polymorpha plants, spores have been used, but crossing is indispensable to obtain them. Gemmae, however, are vegetative clones and readily available in large numbers without crossing, thereby enabling the clonal preservation and rapid propagation of transgenic or mutant lines. Here, we report a simple cryopreservation protocol for in vitro grown M. polymorpha gemmae using aluminum cryoplates. Gemmae were pre-cultured on sucrose-containing medium, embedded in calcium alginate gel on the surface of a cryoplate, moderately dehydrated and stored in liquid nitrogen. After rapid thawing, the stored gemmae showed a 100% survival rate. Our protocol does not require plant growth regulators such as ABA, and takes only 1 h to complete except for 1 d of pre-culture. Furthermore, gemmae treated as described above but then air-dried for 2 h can be stored at -80A degrees C for at least 1 year without a significant decrease in survival rate, which is convenient for most laboratories that have a -80A degrees C freezer but not a liquid nitrogen container for long-term storage. These preservation techniques for M. polymorpha should increase their availability in the research community.

The membrane trafficking pathway has been diversified in a specific way for each eukaryotic lineage, probably to fulfill specific functions in the organisms. In green plants, comparative genomics has supported the possibility that terrestrialization and/or multicellularization could be associated with the elaboration and diversification of membrane trafficking pathways, which have been accomplished by an expansion of the numbers of genes required for machinery components of membrane trafficking, including soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, information regarding membrane trafficking pathways in basal land plant lineages remains limited. In the present study, we conducted extensive analyses of SNARE molecules, which mediate membrane fusion between target membranes and transport vesicles or donor organelles, in the liverwort, Marchantia polymorpha. The M. polymorpha genome contained at least 34 genes for 36 SNARE proteins, comprising fundamental sets of SNARE proteins that are shared among land plant lineages with low degrees of redundancy. We examined the subcellular distribution of a major portion of these SNARE proteins by expressing Citrine-tagged SNARE proteins in M. polymorpha, and the results showed that some of the SNARE proteins were targeted to different compartments from their orthologous products in Arabidopsis thaliana. For example, MpSYP12B was localized to the surface of the oil body, which is a unique organelle in liverworts. Furthermore, we identified three VAMP72 members with distinctive structural characteristics, whose N-terminal extensions contain consensus sequences for N-myristoylation. These results suggest that M. polymorpha has acquired unique membrane trafficking pathways associated with newly acquired machinery components during evolution.

Liverworts occupy a basal position in the evolution of land plants, and are a key group to address a wide variety of questions in plant biology. Marchantia polymorpha is a common, easily cultivated, dioecious liverwort species, and is emerging as an experimental model organism. The haploid gametophytic generation dominates the diploid sporophytic generation in its life cycle. Genetically homogeneous lines in the gametophyte generation can be established easily and propagated through asexual reproduction, which aids genetic and biochemical experiments. Owing to its dioecy, male and female sexual organs are formed in separate individuals, which enables crossing in a fully controlled manner. Reproductive growth can be induced at the desired times under laboratory conditions, which helps genetic analysis. The developmental process from a single-celled spore to a multicellular body can be observed directly in detail. As a model organism, molecular techniques for M. polymorpha are well developed; for example, simple and efficient protocols of Agrobacterium-mediated transformation have been established. Based on them, various strategies for molecular genetics, such as introduction of reporter constructs, overexpression, gene silencing and targeted gene modification, are available. Herein, we describe the technologies and resources for reverse and forward genetics in M. polymorpha, which offer an excellent experimental platform to study the evolution and diversity of regulatory systems in land plants.

Eight-carbon (C8) volatiles, such as 1-octen-3-ol, octan-3-one, and octan-3-ol, are ubiquitously found among fungi and bryophytes. In this study, it was found that the thalli of the common liverwort Marchantia polymorpha, a model plant species, emitted high amounts of C8 volatiles mainly consisting of (R)-1-octen-3-ol and octan-3-one upon mechanical wounding. The induction of emission took place within 40 min. In intact thalli, 1-octen-3-yl acetate was the predominant C8 volatile while tissue disruption resulted in conversion of the acetate to 1-octen-3-ol. This conversion was carried out by an esterase showing stereospecificity to (R)-1-octen-3-yl acetate. From the transgenic line of M. polymorpha (des6(KO)) lacking arachidonic acid and eicosapentaenoic acid, formation of C8 volatiles was only minimally observed, which indicated that arachidonic and/or eicosapentaenoic acids were essential to form C8 volatiles in M. polymorpha. When des6(KO) thalli were exposed to the vapor of 1-octen-3-ol, they absorbed the alcohol and converted it into 1-octen-3-yl acetate and octan-3-one. Therefore, this implied that 1-octen-3-ol was the primary C8 product formed from arachidonic acid, and further metabolism involving acetylation and oxidoreduction occurred to diversify the C8 products. Octan-3-one was only minimally formed from completely disrupted thalli, while it was formed as the most abundant product in partially disrupted thalli. Therefore, it is assumed that the remaining intact tissues were involved in the conversion of 1-octen-3-ol to octan-3-one in the partially disrupted thalli. The conversion was partly promoted by addition of NAD(P)H into the completely disrupted tissues, suggesting an NAD(P)H-dependent oxidoreductase was involved in the conversion. (C) 2014 Elsevier Ltd. All rights reserved.

Blue-light-induced chloroplast photorelocation movement is observed in most land plants. Chloroplasts move toward weak-light-irradiated areas to efficiently absorb light (the accumulation response) and escape from strong-light-irradiated areas to avoid photodamage (the avoidance response). The plant-specific kinase phototropin (phot) is the blue-light receptor for chloroplast movements. Although the molecular mechanisms for chloroplast photorelocation movement have been analyzed, the overall aspects of signal transduction common to land plants are still unknown. Here, we show that the liverwort Marchantia polymorpha exhibits the accumulation and avoidance responses exclusively induced by blue light as well as specific chloroplast positioning in the dark. Moreover, in silico and Southern-blot analyses revealed that the M. polymorpha genome encodes a single PHOT gene, MpPHOT, and its knockout line displayed none of the chloroplast photorelocation movements, indicating that the sole MpPHOT gene mediates all types of movement. Mpphot was localized on the plasma membrane and exhibited blue-light-dependent autophosphorylation both in vitro and in vivo. Heterologous expression of MpPHOT rescued the defects in chloroplast movement of phot mutants in the fern Adiantum capillus-veneris and the seed plant Arabidopsis (Arabidopsis thaliana). These results indicate that Mpphot possesses evolutionarily conserved regulatory activities for chloroplast photorelocation movement. M. polymorpha offers a simple and versatile platform for analyzing the fundamental processes of phototropin-mediated chloroplast photorelocation movement common to land plants.

The liverwort Marchantia polymorpha L. is one of extant species of the first land plants that appeared about 450 Myr ago. The genomes of plastid, mitochondria and Y chromosome in M. polymorpha were the first to be published among all plant species, making significant contributions to plant biology. M. polymorpha has now become a fascinating model organism for plant biology, because of its crucial position in the evolution of land plants and molecular genetic tools that have been recently developed. M. polymorpha is dioecious, and its complete haploid set of chromosomes (approximately 280 Mb) consists of eight autosomes and a single sex chromosome: an X chromosome for a female (n=8+X) and a Y chromosome for a male (n=8+Y). For sexual reproduction, male plants of M. polymorpha produce biflagellated spermatozoa, which swim in the water toward female plants to fertilize eggs. How spermatozoa detect, approach, and unite with the eggs at the molecular level is still a major issue in biology. A number of organisms, mostly animals and some algae, have been intensively studied to address these questions. Only a few of them, however, receive the advantage of molecular genetic approaches, such as genomic resources, genetics, transformation and gene targeting. M. polymorpha is one such organism with a variety of molecular genetic tools. The potential of M. polymorpha as a model organism for reproduction research will be discussed.

Plant disease resistance gene (R gene)-like sequences were screened from the Physcomitrella patens genome. We found 603 kinase-like, 475 Nucleotide Binding Site (NBS)-like and 8594 Leucine Rich Repeat (LRR)-like sequences by homology searching using the respective domains of PpC24 (Accession No. BAD38895), which is a candidate kinase-NBS-LRR (kinase-NL) type R-like gene, as a reference. The positions of these domains in the genome were compared and 17 kinase-NLs were predicted. We also found four TIR-NBS-LRR (TIR-NL) sequences with homology to Arabidopsis TIR-NL (NM_001125847), but three out of the four TIR-NLs had tetratricopeptide repeats or a zinc finger domain in their predicted C-terminus. We also searched for kinase-LRR (KLR) type sequences by homology with rice OsXa21 and Arabidopsis thaliana FLS2. As a result, 16 KLRs with similarity to OsXa21 were found. In phylogenetic analysis of these 16 KLRs, PpKLR36, PpKLR39, PpKLR40, and PpKLR43 formed a cluster with OsXa21. These four PpKLRs had deduced transmembrane domain sequences and expression of all four was confirmed. We also found 14 homologs of rice OsXB3, which is known to interact with OsXa21 and is involved in signal transduction. Protein-protein interaction was observed between the four PpKLRs and at least two of the XB3 homologs in Y2H analysis.

Photoperiodic control of the phase transition from vegetative to reproductive growth is critical for land plants. The GIGANTEA (GI) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) protein complex controls this process in angiosperms. However, little is known about how plants evolved this regulatory system. Here, we report that orthologues of GI and FKF1 are present in a basal plant, the liverwort Marchantia polymorpha, and describe the molecular interaction between their products. Knockout of either the GI or FKF1 orthologue completely abolishes the long-day-dependent growth-phase transition in M. polymorpha. Overexpression of either gene promotes growth-phase transition, even under short-day conditions. Introduction of the GI orthologue partially rescues the late-flowering phenotype of the Arabidopsis thaliana gi mutant. Our findings suggest that plants had already acquired the GI-FKF1 system to regulate growth-phase transition when they colonized land, and that this system was co-opted from gametophyte to sporophyte generation during evolution.

The phytohormone auxin plays a pivotal role in various developmental aspects in land plants. However, little is known of the auxin response and distribution in non-vascular plants. In this study, we made transgenic plants of the liverwort Marchantia polymorpha which express the uidA (GUS) reporter gene under control of the soybean auxin-inducible promoter, ProGH3, and used it to indirectly monitor auxin-mediated transcriptional activation in planta. Transgenic plants carrying ProGH3:GUS showed GUS activity in an auxin-dependent manner. Histochemical GUS staining was observed at the bottom of gemma cups in the process of vegetative propagation. Significant GUS activity was also detected around the gametophyte-sporophyte junction as well as the developing sporophyte after fertilization. These results suggest that the activity of auxin is crucial in both gametophyte and sporophyte development in M. polymorpha, and that the mechanism for auxin-mediated transcriptional activation had already been established when plants emerged on the terrestrial environment.

A laticifer is a cell involved in plant defense against biotic stresses such as herbivores and microorganisms; however, its gene expression is poorly understood. We compared protein accumulation and transcriptomes among laticifers of lignified and unlignified organs of mulberry (Morus alba), which has a non-articulated, branched type of laticifer. LA-a (equivalent to MLX56) and its homolog LA-b (insecticidal chitinase-like proteins containing two chitin-binding domains) were major proteins in laticifers of unlignified organs, and another protein (LA-c) was a major protein in laticifers of lignified organs. Purification, cDNA cloning, and bioassay of LA-c revealed that LA-c was an acidic class I chitinase having antifungal but not insecticidal activity. Comparative mRNA-Seq analysis using a GS-FLX revealed transcripts of other possible defense-related proteins. Jacalin-like lectin, galacturonase-inhibitor, and pathogenesis-related proteins were also abundant; however, the relative amounts differed among laticifers of lignified and unlignified organs. The results suggest a discontinuous laticifer network in planta and adaptation to different potential enemies among these organs.

CtBP/BARS is a unique protein family in having quite diversified cellular functions, intercellular localizations, and developmental roles. ANGUSTIFOLIA (AN) is the sole homolog of CtBP/BARS from Arabidopsis thaliana, although it has plant AN-specific motifs and a long C-terminus. Previous studies suggested that AN would function in the nucleus as a transcriptional co-repressor, as CtBPs function in animals; however, precise verification has been lacking. In this paper, we isolated a homologous gene (MAN) of AN from liverwort, Marchantia polymorpha. Transformation of the Arabidopsis an-1 mutant with 35S-driven MAN completely complemented the an-1 phenotype, although it lacks the putative nuclear localization signal (NLS) that exists in AN proteins isolated from other plant species. We constructed several plasmids for expressing modified ANs with amino acid substitutions in known motifs. The results clearly indicated that modified AN with mutations in the putative NLS-like domain could complement the an-1 phenotype. Therefore, we re-examined localization of AN using several techniques. Our results demonstrated that AN localizes on punctuate structures around the Golgi, partially overlapping with a trans-Golgi network resident, which highlighted an unexpected link between leaf development and membrane trafficking. We should reconsider the roles and evolutionary traits of AN based on these findings.

The use of fast-growing oleaginous algal strains is indispensable to achieve low-cost production system for microalgal biodiesel. We therefore previously isolated a new algal species within new genus " Pseudochoricystisas" (invalid name, class Trebouxiophyceae), "Pseudochoricystis eHipsoidea" (MBIC11204), which possesses oil vesicles stainable with a selective fluorescent dye for intracellular lipid droplets, Nile Red (Sekiguchi et al., 12th Annu. Meet. Jpn. Microbiol. Cult. Coll. 2005). In the present study we attempted to investigate growth and lipid accumulation of this new isolate. The maximum growth rate of P. ellipsoidea was 3.46 g dry weight 1-1 day-1. Nile Red fluorescence reached maximum intensity within 5-10 days after transferring P. ellipsoidea cells to nitrogen starvation conditions in the light, but not in the dark. Total lipid content made up 32% of normal-grown (+N) and 26% of nitrogen-starved (-N, 8 days) dry weight algal cells, and the hydrocarbon fraction was more than 10 times higher in -N cells. Fatty acid composition changes and an increase in triglycerides to 82% of total lipid were also observed with nitrogen starvation. These results suggest that P. ellipsoidea is a fast-growing oleaginous algal strain in which hydrocarbons and triglycerides can be produced photoautotrophically up to 30% of the dried biomass.

Abscisic acid (ABA) is postulated to be a ubiquitous hormone that plays a central role in seed development and responses to environmental stresses of vascular plants. However, in liverworts (Marchantiophyta), which represent the oldest extant lineage of land plants, the role of ABA has been least emphasized; thus, very little information is available on the molecular mechanisms underlying ABA responses. In this study, we isolated and characterized MpABI1, an ortholog of ABSCISIC ACID INSENSITIVE1 (ABI1), from the liverwort Marchantia polymorpha. The MpABI1 cDNA encoded a 568-amino acid protein consisting of the carboxy-terminal protein phosphatase 2C (PP2C) domain and a novel amino-terminal regulatory domain. The MpABI1 transcript was detected in the gametophyte, and its expression level was increased by exogenous ABA treatment in the gemma, whose growth was strongly inhibited by ABA. Experiments using green fluorescent protein fusion constructs indicated that MpABI1 was mainly localized in the nucleus and that its nuclear localization was directed by the aminoterminal domain. Transient overexpression of MpABI1 in M. polymorpha and Physcomitrella patens cells resulted in suppression of ABA-induced expression of the wheat Em promoter fused to the beta-glucuronidase gene. Transgenic P. patens expressing MpABI1 and its mutant construct, MpABI1-d2, lacking the amino-terminal domain, had reduced freezing and osmotic stress tolerance, and associated with reduced accumulation of ABA-induced late embryogenesis abundant-like boiling-soluble proteins. Furthermore, ABA-induced morphological changes leading to brood cells were not prominent in these transgenic plants. These results suggest that MpABI1 is a negative regulator of ABA signaling, providing unequivocal molecular evidence of PP2C-mediated ABA response mechanisms functioning in liverworts.

Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a LifeactVenus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used LifeactVenus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages.

Euphorbia tirucalli L., which is also known as a petroleum plant, produces a large amount of phytosterols and triterpenes. During their biosynthesis, squalene synthase converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. An E. tirucalli cDNA clone of a putative squalene synthase gene (EtSS) was isolated by RT-PCR followed by 5'- and 3'-RACE. The restriction fragment polymorphisms revealed by Southern blot analysis suggest that EtSS is a single copy gene. The glycine at the 287th residue from the N-terminal end of domain C has replaced alanine, which is conserved among all the other SS sequences deposited in the Genbank database. The N-terminal 380 residues of the hydrophilic sequence was expressed as a peptide-tagged protein in E. coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. E. tirucalli transgenic callus lines, in which EtSS was overexpressed, accumulated increased amounts of phytosterols as compared with that of wild type callus. RT-PCR analysis of wild type E. tirucalli plants revealed that the EtSS transcript accumulated in almost equal amounts in the stems and the leaves with a stalk, while a lower amount was detected in the roots. In situ hybridization analysis revealed that prominent antisense-probe signal was detected in the cambia within bundle sheathes. These results indicate that EtSS functions prominently in cambia, which are located adjacent to conductive tubes, and that this gene plays important roles in phytosterol accumulation in petroleum plants.

The complete nucleotide sequence of chloroplast DNA (121;025 base pairs, bp) from a liverwort, Marchantia polymorpha, has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. We also determined the complete nucleotide sequence of the liverwort mitochondrial DNA and deduced 96 possible genes in the sequence of 186,608 bp. The complete chloroplast geuome encodes twenty introns (19 group II and I group I) in 18 different, genes. One of the chloroplast group II introns separates a ribosomal protein gene in a trans-position. The mitochondrial genome contains thirty-two introns (25 group II and 7 group I) in the coding regions of 17 genes. From the evolutionary point of view, we describe the origin of organellar introns and give evidence for vertical and horizontal intron transfers and their intragenomic propagation. Furthermore, we describe the gene organization of the Y chromosome in the dioecious liverwort M. polymorpha, the first detailed view of a Y chromosome in a haploid organism. On the 10 megabase (Mb) Y chromosome, 64 genes are identified, 14 of which are detected only in the male genome. These 14 genes are expressed in reproductive organs but not in vegetative thalli, suggesting their participation in male reproductive functions. These findings indicate that the Y and X chromosomes share the same ancestral autosome and support the prediction that in a haploid organism essential genes on sex chromosomes are more likely to persist than in a diploid organism.

The liverwort, Marchantia polymorpha L., belongs to a group of basal land plants and is an emerging model for plant biology. We established a procedure to prepare sporangia of M. polymorpha under laboratory conditions by promoting its transition to reproductive development by far-red light irradiation. Here we report an improved direct transformation system of M. polymorpha using immature thalli developing from spores. Hygromycin-resistant transformants were obtained on selective media by transformation with a plasmid carrying the hygromycin-phosphotransferase gene (hpt) conferring hygromycin resistance in 4 weeks. The aminoglycoside-3 ''-adenyltransferase gene (aadA) conferring spectinomycin resistance was also successfully used as an additional selectable marker for nuclear transformation of M. polymorpha. The availability of the aadA gene in addition to the hpt gene should make M. polymorpha a versatile host for genetic manipulation. DNA gel-blot analyses indicated that transformed thalli carried a variable number of copies of the transgene integrated into the genome. Although the previous system using thalli grown from gemmae required a two-step selection in liquid and solid media for 8 weeks, the system reported here using thalli developing from spores allows generation of transformants in half the time by direct selection on solid media, facilitating genetic analyses in this model plant.

Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the beta-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.

The liverwort Marchantia polymorpha L. synthesizes arachidonic (ARA) and eicosapentaenoic acids (EPA) from linoleic and alpha-linolenic acids respectively by a series of reactions catalyzed by Delta 6-desaturase, Delta 6-elongase, and Delta 5-desaturase. Overexpression of the M. polymorpha genes encoding these enzymes in transgenic M. polymorpha plants resulted in 3- and 2-fold accumulation of ARA and EPA respectively, as compared to those in the wild type. When these three genes were introduced and co-expressed in tobacco plants, in which long-chain polyunsaturated fatty acids (LCPU-FAs) are not native cellular components, ARA and EPA represented up to 15.5% and 4.9% respectively of the total fatty acid in the leaves. Similarly in soybean, C20-LCPUFAs represented up to 19.5% of the total fatty acids in the seeds. These results suggest that M. polymorpha can provide genes crucial to the production of C20-LCPUFAs in transgenic plants.

Y chromosomes are different from other chromosomes because of a lack of recombination. Until now, complete sequence information of Y chromosomes has been available only for some primates, although considerable information is available for other organisms, e.g., several species of Drosophila. Here, we report the gene organization of the Y chromosome in the dioecious liverwort Marchantia polymorpha and provide a detailed view of a Y chromosome in a haploid organism. On the 10-Mb Y chromosome, 64 genes are identified, 14 of which are detected only in the male genome and are expressed in reproductive organs but not in vegetative thalli, suggesting their participation in male reproductive functions. Another 40 genes on the Y chromosome are expressed in thalli and male sexual organs. At least six of these genes have diverged X-linked counterparts that are in turn expressed in thalli and sexual organs in female plants, suggesting that these X-and Y-linked genes have essential cellular functions. These findings indicate that the Y and X chromosomes share the same ancestral autosome and support the prediction that in a haploid organism essential genes on sex chromosomes are more likely to persist than in a diploid organism.

We have established a simple and efficient plastid transformation system for liverwort, Marchantia polymorpha L., suspension-culture cells, which are homogenous, chloroplast-rich and rapidly growing. Plasmid pCS31 was constructed to integrate an aadA expression cassette for spectinomycin-resistance into the trnI-trnA intergenic region of the liverwort plastid DNA by homologous recombination. Liverwort suspension-culture cells were bombarded with pCS31-coated gold projectiles and selected on a medium containing spectinomycin. Plastid transformants were reproducibly isolated from the obtained spectinomycin-resistant calli. Selection on a sucrose-free medium greatly improved the efficiency of selection of plastid transformants. Homoplasmic plastid transformant lines were established by successive subculturing for 14 weeks or longer on the spectinomycin-containing medium. The plastid transformation system of liverwort suspension-culture cells should facilitate the investigation of the fundamental genetic systems of plastid DNA, such as replication.

Pinolenic acid (PA; 18:3 Delta(5,9,12)) and coniferonic acid (CA; 18:4 Delta(5,9,12,15)) are Delta(5)-unsaturated bis-methylene-interrupted fatty acids (Delta(5)-UBIFAs) commonly found in pine seed oil. They are assumed to be synthesized from linoleic acid (LA; 18:2 Delta(9,12)) and alpha-linolenic acid (ALA; 18:3 Delta(9,12,15)), respectively, by Delta(5)-desaturation. A unicellular green microalga Chlamydomonas reinhardtii also accumulates PA and CA in a betain lipid. The expressed sequence tag (EST) resource of C. reinhardtii led to the isolation of a cDNA clone that encoded a putative fatty acid desaturase named as CrDES containing a cytochrome b5 domain at the N-terminus. When the coding sequence was expressed heterologously in the methylotrophic yeast Pichia pastoris, PA and CA were newly detected and comparable amounts of LA and ALA were reduced, demonstrating that CrDES has Delta(5)-desaturase activity for both LA and ALA. CrDES expressed in the yeast showed Delta(5)-desaturase activity on 18:1 Delta(9) but not 18: 1 Delta(11). Unexpectedly, CrDES also showed Delta(5)-desaturase activity on 20:2 Delta(11,14) and 20:3 Delta(11,14,17) to produce 20:3 Delta(7,11,14) and 20:4 Delta(7,11,14,17), respectively. Since both the Delta(5) bond in C18 and the Delta(7) bond in C20 fatty acids are 'omega 13' double bonds, these results indicate that CrDES has omega 13 desaturase activity for omega 9 unsaturated C18/C20 fatty acids, in contrast to the previously reported front-end desaturases. In order to evaluate the activity of CrDES in higher plants, transgenic tobacco plants expressing CrDES were created. PA and CA accumulated in the leaves of transgenic plants. The highest combined yield of PA and CA was 44.7% of total fatty acids, suggesting that PA and CA can be produced in higher plants on a large scale.

Bryophyte Marchantia polymorpha L. produces C22 very-long-chain polyunsaturated fatty acid (VLCPUFA). Thus far, no enzyme that mediates elongation of C20 VLCPUFAs has been identified in land plants. Here, we report the isolation and characterization of the gene MpELO2, which encodes an ELO-like fatty acid elongase in M. polymorpha. Heterologous expression in yeast demonstrated that MpELO2 encodes Delta 5-elongase, which mediates elongation of arachidonic (20:4) and eicosapentaenoic acids (20:5). Phylogenetic and gene structural analysis indicated that the MpELO2 gene is closely related to bryophyte Delta 6-elongase genes for C18 fatty acid elongation and diverged from them by local gene duplication. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Euphorbia tirucalli L., known as the petroleum plant, produces a large amount of triterpenes, such as beta-amyrin. Degenerate RTPCR based on the sequences conserved among known beta-amyrin synthases led to cloning of a putative triterpene synthase cDNA, EtAS, from leaves of E tirucalli. The deduced amino acid sequence of the EtAS cDNA showed the highest identity of 82% to the Panax ginseng P-amyrin synthase. Heterologous expression of the EtAS ORF in the methylotrophic yeast, Pichia pastoris, resulted in production of beta-amyrin, revealing that the EtAS cDNA codes for a P-amyrin synthase. This is the first report of a gene involved in the triterpene synthetic pathway from Euphorbiaceae plants. (c) 2005 Elsevier Ltd. All rights reserved.

Photosynthetic acclimation to CO2-limiting stress is associated with control of genetic and physiological responses through a signal transduction pathway, followed by integrated monitoring of the environmental changes. Although several CO2-responsive genes have been previously isolated, genome-wide analysis has not been applied to the isolation of CO2-responsive genes that may function as part of a carbon-concentrating mechanism (CCM) in photosynthetic eukaryotes. By comparing expression profiles of cells grown under CO2-rich conditions with those of cells grown under CO2-limiting conditions using a cDNA membrane array containing 10,368 expressed sequence tags, 51 low-CO2 inducible genes and 32 genes repressed by low CO2 whose mRNA levels were changed more than 2.5-fold in Chlamydomonas reinhardtii Dangeard were detected. The fact that the induction of almost all low-CO2 inducible genes was impaired in the ccm1 mutant suggests that CCM1 is a master regulator of CCM through putative low-CO2 signal transduction pathways. Among low-CO2 inducible genes, two novel genes, LciA and LciB, were identified, which may be involved in inorganic carbon transport. Possible functions of low-CO2 inducible and/or CCM1-regulated genes are discussed in relation to the CCM.

The liverwort Marchantia polymorpha contains high proportions of arachidonic and eicosapentaenoic acids. In general, these C20 polyunsaturated fatty acids (PUFA) are synthesized from linoleic and alpha-linolenic acids, respectively, by a series of reactions catalyzed by Delta(6)-desaturase, an ELO-like enzyme involved in Delta(6) elongation and Delta(5)-desaturase. Here we report the isolation and characterization of the cDNAs, MpDES6, MpELO1 and MpDES5, coding for the respective enzymes from M. polymorpha. Coexpression of the MpDES6, MpELO1 and MpDES5 cDNAs resulted in the accumulation of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris. Interestingly, D6 desaturation by the expression of the MpDES6 cDNA appears to occur both in glycerolipids and the acyl-CoA pool, although other lower-plant Delta(6)-desaturases are known to have a strong preference for glycerolipids.

We report generation of 9,301 expressed sequence tags (ESTs) derived from callus cells of Euphorbia tirucalli in search of candidate genes involved in the triterpenoid and sterol biosyntheses. After assembling 4,342 redundant ESTs into 1,252 clusters, a total of 6,211 non-redundant sequences were obtained. Database search revealed that 4,449 out of the 6,211 sequences shared significant similarities to known nucleotide or amino acid sequences, while the remaining 1,762 showed no significant matches and appear to represent novel genes in E. tirucalli. The annotations assigned to the hit database entries suggest that 48 of the unique sequences are involved in triterpenoid and sterol biosyntheses. Although functions of genes tagged by the 48 sequences are yet to be determined, the EST resource described here should contribute to identification of genes participating in the triterpenoid and sterol biosyntheses in E. tirucalli.

Euphorbia tirucalli is a potential source of commercially important chemicals such as sterols. Here we report the first successful plant regeneration from internode explants of E. tirucalli. Adventitious buds were efficiently induced on LS medium supplemented with 0.02 mg l-1 thidiazuron. On average of four experiments, 17.3 adventitious buds were induced from 12 explants on this medium. The adventitious buds grew into shoots during subsequent cultures on a hormone-free LS medium. For rooting treatment, we cultured these shoots on the LS medium containing 0.02 mg l-1 naphthalenacetic acid, followed by on the half-strength LS medium without vitamins, and were successful to obtain whole plantlets.

In lower land plants, genes controlling the transition from vegetative growth to sexual reproduction have not yet been identified. In the dioecious liverwort Marchantia polymorpha, the transition to sexual reproduction accompanied by the formation of sexual organs on the gametophytic thallus is initiated under long-day conditions. By particle bombardment-mediated mutagenesis, we generated a mutant of M. polymorpha that constitutively forms sexual organs. This mutant is fully fertile, showing that the mutation does not affect formation of male or female sexual organs per se. Genetic analysis reveals that this phenotype is caused by mutation of a single autosomal locus, suggesting that this mutation defines or controls a gene regulating the transition to sexual reproduction in M. polymorpha.

Fatty acid chain elongation is a crucial step in the biosynthesis of long chain fatty acids. An essential reaction in the elongation process is condensation of malonyl-CoA with acyl-CoA, which is catalyzed by beta-ketoacyl-CoA synthase (KCS) in plants. We have isolated and characterized the MpFAE3 gene, one of the KCS gene family in the liverwort Marchantia polymorpha. Transgenic M. polymorpha plants overexpressing MpFAE3 accumulate fatty acids 18:0, 20:0, and 22:0. In these plants, the amount of 16:0 is reduced to 50% of wild type. In a heterologous assay, transgenic methylotrophic yeast expressing the MpFAE3 gene accumulates fatty acid 18:0 and generates several longer fatty acids which are not detectable in the control, accompanied by a decrease of 16:0. These observations indicate that the MpFAE3 protein is preferentially involved in the elongation of 16:0 to 18:0 and also in the subsequent steps of 18:0 to 20:0 and 20:0 to 22:0 in M. polymorpha.

We have isolated a beta-ketoacyl CoA synthase (KCS) gene, MpFAE2, from a liverwort, Marchantia polymorpha, and identified its substrate specificity using the technique of dsRNA-mediated gene silencing and overexpression. KCS catalyzes an essential reaction in the fatty acid elongation process, i.e., condensation of malonyl-CoA with acyl-CoA. By introducing a construct with a hairpin structure containing a partial MpFAE2 gene, the level of the MpFAE2 gene expression was suppressed constitutively. The transgenic plants showed a specific accumulation of fatty acid 18:0. In contrast, in transgenic M. polymorpha plants overexpressing the MpFAE2 gene, fatty acid 22:0 is accumulated. These results indicate that the MpFAE2 gene product catalyzes the elongation steps of 18:0 to 20:0 and possibly also of 20:0 to 22:0.

In the haploid dioecious liverwort, Marchantia polymorpha, the X chromosome, but not the Y, carries a cluster of ribosomal RNA genes (rDNAs). Here we show that sequences of 5S, 17S, 5.8S and 26S rDNAs are highly conserved(> 99% identity) between the X chromosomal and autosomal rDNA repeat units, but the intergenic spacer sequences differ considerably. The most prominent difference is the presence of a 615-bp DNA fragment in the intergenic spacer, X615, which has accumulated predominantly in the rDNA cluster of the X chromosome. These observations suggest that the rDNA repeat unit on the X chromosome evolved independently of that on autosomes, incorporating sex chromosome-specific sequences.

Sex of the liverwort Marchantia polymorpha is determined by the sex chromosomes Y and X, in male and female plant, respectively. Approximately half of the Y chromosome is made up of unique repeat sequences. Here, we report that part of the Y chromosome, represented by a 90-kb insert of a genomic clone pMM2D3, contains five putative genes in addition to the ORF162 gene, which is present also within the Y chromosome-specific repeat region. One of the five putative genes shows similarity to a male gamete-specific protein of lily and is expressed predominantly in male sex organs, suggesting that this gene has a male reproductive function. Furthermore, Southern blot analysis revealed that these five putative genes are amplified on the Y chromosome, but they also probably have homologs on the X chromosome and/or autosomes. These observations suggest that the Y chromosome evolved by co-amplifying protein-coding genes with unique repeat sequences.

The liverwort Marchantia polymorpha has X and Y chromosomes in the respective female and male haploids. Here we report the Successful exploitation of representational difference analyses to isolate DNA markers for the sex chromosomes. Two female-specific and six male-specific DNA fragments were genetically confirmed to originate from the X and Y chromosomes, respectively.

The haploid liverwort Marchantia polymorpha has heteromorphic sex chromosomes, an X chromosome in the female and a Y chromosome in the male. We here report on the repetitive structure of the liverwort Y chromosome through the analysis of male-specific P1-derived artificial chromosome (PAC) clones, pMM4G7 and pMM23-130F12. Several chromosome-specific sequence elements of approximate to 70 to 400 nt are combined into larger arrangements, which in turn are assembled into extensive Y chromosome-specific stretches. These repeat sequences contribute 2-3 Mb to the Y chromosome based on the observations of three different approaches: fluorescence in situ hybridization, dot blot hybridization, and the frequency of clones containing the repeat sequences in the genomic library. A novel Y chromosome-specific gene family was found embedded among these repeat sequences. This gene family encodes a putative protein with a RING finger motif and is expressed specifically in male sexual organs. To our knowledge, there have been no other reports for an active Y chromosome-specific gene in plants. The chromosome-specific repeat sequences possibly contribute to determining the identity of the Y chromosome in M. polymorpha as well as to maintaining genes required for male functions, as in mammals such as human.

Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male-specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.

A total of 935 expressed sequence tags (ESTs) from male immature sexual organ were determined, of which 600 ESTs were assembled into 110 non-redundant groups, resulting in 445 unique EST sequences. Of these, 244 sequences shared significant similarities to known nucleotide or amino acid sequences in other organisms. The remaining 201 unique sequences showed no significant matches and thus are likely to be novel transcripts. ESTs from male and female immature sexual. organs of a liverwort, Marchantia polymorpha, were compared to characterize gene expression patterns during sex differentiation. Ninety-nine male ESTs turned out to be common genes found also in the female library. Interestingly, one of the ESTs found only in male shows a significant similarity to the transformer-2 gene involved in sex determination in Drosophila. In female, several unique lectin ESTs were found that are not present in the male library.

Thalli of the haploid liverwort Marchantia polymorpha were successfully used for direct particle bombardment with plasmid pMT, which carries a hygromycin phosphotransferase gene (hpt) controlled by the CaMV 35S promoter and the NOS polyadenylation region. Hygromycin-resistant cell masses arose from the thallus surface and developed directly into hygromycin-resistant thalli. Southern blot analyses indicated that these thalli carried at least 1-4 copies of the hpt gene, which were stably transmitted to their asexual thallus progenies via gemma propagation for three generations. This transformation and direct plant regeneration protocol is expected to be a valuable tool for the molecular analysis of this lower land plant.

The 5S ribosomal RNA genes (5S rDNA) are located independently from the 45S rDNA repeats containing 18S, 5.8S and 26S ribosomal RNA genes in higher eukaryotes. Southern blot and fluorescence in situ hybridization analyses demonstrated that the 5S rDNAs are encoded in the 45S rDNA repeat unit of a liverwort, Marchantia polymorpha, in contrast to higher plants. Sequencing analyses revealed that a single-repeat unit of the M. polymorpha nuclear rDNA, which is 16 103 bp in length, contained a 5S rDNA downstream of 18S, 5.8S and 26S rDNA. To our knowledge, this is the first report on co-localization of the 5S and 45S rDNAs in the rDNA repeat of land plants. Furthermore, we detected a 5S rDNA in the rDNA repeat of a moss, Funaria hygrometrica, by a homology search in a database. These findings suggest that there has been structural re-organization of the rDNAs after divergence of the bryophytes from the other plant species in the course of evolution.

The nonchromosomal stripe (NCS) mutants of maize are a set of mitochondrial deletion mutants. In aerobic organisms, deletions of essential mitochondrial genes are lethal. Therefore, most plants carrying deletions of part of essential mitochondrial genes survive only heteroplasmically; that is, the plants contain mixtures of the deleted and normal mitochondrial genomes. The severity of the mutant phenotype depends on which mitochondrial gene is altered, which also influences the extent of homoplasmic tissues on the plants. NCS2 plants carry a partial deletion of the nad4 gene that codes for a subunit of complex I. Unlike animals, plants have additional enzymes that can partially compensate for the loss of complex I function. Kernels that are homoplasmic for the nad4 deletion mutation usually abort, although we report that homoplasmic mutant cultures can be initiated and maintained from immature embryos of such kernels. We also report the first example of a rare homoplasmic mutant NCS2 plant. The plant is very short, pale green, with a reduced number of narrow leaves. It is apparently male and female sterile, The possibility that the kernel that gave rise to this plant had a heteroplasmic endosperm is discussed.

A total of 970 expressed sequence tag (EST) clones were generated from immature female sexual organ of a liverwort, Marchantia polymorpha. The 376 ESTs resulted in 123 redundant groups, thus the total number of unique sequences in the EST set was 717. Database search by BLAST algorithm showed that 302 of the unique sequences shared significant similarities to known nucleotide or amino acid sequences. Six unique sequences showed significant similarities to genes that are involved in flower development and sexual reproduction, such as cynarase, fimbriata-associated protein and S-receptor kinase genes. The remaining unique 415 sequences have no significant similarity with any database-registered genes or proteins. The redundant 123 ESTs implied the presence of gene families and abundant transcripts of unknown identity. Analyses of the coding sequences of 61 unique sequences, which contained no ambiguous bases in the predicted coding regions, highly homologous to known sequences at the amino acid level with a similarity score greater than 400, and with stop codons at similar positions as their possible orthologues, indicated the presence of biased codon usage and higher GC content within the coding sequences (50.4%) than that within 3′ flanking sequences (41.9%).

We have characterized two promoters of the cytochrome oxidase subunit 2 (cox2) gene in Zea perennis mitochondria present in maize lines. Initiation at a site 907 bases upstream of the start codon results in the major similar to 1900 nt cox2 transcript. A sequence just upstream of this site conforms to the consensus described for maize mitochondrial promoters and its transcription is correctly initiated in a maize mitochondrial in vitro transcription extract. A second transcription initiation site (-347) is used only when the dominant allele of a nuclear gene, Mct, is present and its use results in an additional, shorter major transcript, Sequences flanking the Mct-dependent transcription initiation site, which we have termed the conditional promoter of cox2 (cpc), do not fit the maize mitochondrial promoter consensus and do not function in the maize in vitro transcription extract. The cpc region does not hybridize with mitochondrial, chloroplast or nuclear DNAs from most maize or teosinte lines, However, the cpc sequence is found in the same position upstream of the cox2 gene in Zea diploperennis mtDNA and it has striking similarity to the previously reported 'ORF of unknown origin' fused to the ATPase subunit 6 gene in maize CMS-C mitochondria, cpc appears to represent a new type of mitochondrial promoter, Further analysis of both conditional and constitutive promoters should help us to better understand the control of transcription in plant mitochondria.

The genes encoding subunits 1, 3, 4L, and 6 of NADH dehydrogenase (nad1, nad3, nad4L, nad6) in the mitochondrial genome of a liverwort, Marchantia polymorpha, were characterized by comparing homologies of the amino-acid sequences of the subunits with those of other organisms. The nad3 and nad4L genes are split by single and double group 11 introns, respectively. The 5'-half portion of the nad6 gene was repeated at an identity of 89% to form a reading frame consisting of 100 amino-acid residues. The Northern hybridization analysis showed that all four genes are transcribed in the liverwort mitochondria.

Three genes for the subunits of the NADH dehydrogenase (nad5, nad4, and nad2) are tandemly clustered on the liverwort mitochondrial genome. Their gene products showed high levels of amino acid sequence identity with the corresponding subunits from higher plant mitochondria (82.8-84.4%), and significant levels of identity with those from liverwort chloroplast (32.0-33.5%), Podospora anserina mitochondria (21.4-45.9%), and human mitochondria (18.4-27.9%). In addition, these three subunits from liverwort mitochondria have conserved amino acid residues in their central regions. The gene nad5 is interrupted by a 672 bp group I intron, while genes nad4 and nad2 are interrupted by group II introns of 899 bp and 1418 bp, respectively. Northern blot analysis using exon-intron specific probes indicated that these three genes are transcribed as a single precursor mRNA of 9.6 kb in length and are processed into mature mRNA molecules in liverwort mitochondria. Several regions of this nad gene cluster are repeated in the liverwort mitochondrial genome.

The complete nucleotide sequence of the mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, contains thirty-two introns. Twenty-five of these introns possess the characteristic secondary structures and consensus sequences of group II introns. The remaining seven are group I introns, six of which happen to interrupt the gene coding for subunit 1 of cytochrome oxidase (cox1). Interestingly, the insertion sites of one group II and four group I introns in the cox1 gene coincide with those of the respective fungal mitochondrial introns. Moreover, comparison of the four group I introns with their fungal counterparts shows that group I introns inserted at identical genomic sites in different organisms are indeed related to one another, in terms of the peptide sequences generated from the complete or fragmental ORFs encoded by these introns. At the same time, the liverwort introns turned out to be more divergent from their fungal cognates than the latter are from one another. We therefore conclude that vertical transmission from a common ancestor organism is the simplest explanation for the presence of cognate introns in liverwort and fungal mitochondrial genomes.

Twenty-nine genes for 27 species of tRNAs were deduced from the complete nucleotide sequence of the mitochondrial genome from a liverwort, Marchantia Polymorpha. One to three species of tRNA genes corresponded to each of 20 amino acids including three species for leucine and arginine, two species for serine and glycine, and one for the rest of the amino acids. Interestingly, all tRNA genes were located in the semicircle of the liverwort mitochondrial genome except for the trnY and trnR genes. The region containing these tRNA genes was originally duplicated, and two trnR genes have diverged from each other. On the other hand, trnY and trnfM are present as two identical copies. The G:U and U:N wobbling between the first nucleotide of the anticodon and the third nucleotide of the codon permit the 27 tRNA identified species to translate almost all codons. However, at least two additional tRNA genes, trnI-GAU for AUY codon and trnT-UGU for ACR codon, are required to read all codons used in the liverwort mitochondrial genome. All of the identified tRNA genes are 'native' in liverwort mitochondria, not 'chloroplast-like' tRNAs as are found in the mitochondria of higher plants. This result implies that the tRNA gene transfer from chloroplast to mitochondrial genome in higher plants has occurred after the divergence from bryophytes.

We detected 16 genes for ribosomal proteins in the complete sequence of the mitochondrial DNA from a liverwort, Marchantia polymorpha. The genes formed two major clusters, rps12-rps7 and rps10-rp12-rps19-rps3-rpl16-rp15-rps14-rps8-rp16-rps13-rps11-rps1, very similar in organization to Escherichia coli ribosomal protein operons (str and S10-spc-alpha operons, respectively). In contrast, rps2 and rps4 genes were located separately in the liverwort mitochondrial genome (the latter was part of the alpha-operon in E. coli). Furthermore, several ribosomal proteins encoded by the liverwort mitochondrial genome differed substantially in size from their counterparts in E. coli and liverwort chloroplast.

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwort Marchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins ( 16 ribosomal proteins, 3 subunits of cytochrome coxidase, apocytochrome b protein, 3 subunits of H*-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request.

A gene, frxC, which is unique to the chloroplast genome of the liverwort Marchantia polymorpha, has sequence similarity to nifH, the product of which is an iron protein of a nitrogenase. Although frxC is expressed to produce a protein in liverwort chloroplasts, its function is not known. Using a probe of liverwort chloroplast DNA, a 10.1-kb region containing a gene cluster consisting of open reading frames (ORF278-frxC-ORF469-ORF248) was isolated from the cyanobacterium Synechocystis PCC6803. In this region, frxC and ORF469 showed sequence similarities to liverwort chloroplast frxC (83%) and immediately downstream ORF465 (74%), respectively. Synechocystis frxC showed 31% amino acid sequence identity with nifH1 from Clostridium pasteurianum. Additionally, Synechocystis ORF469 showed a sequence similarity (19% identity) to C. pasteurianum nifK product, which is the beta-subunit of a molybdenum-iron protein of a nitrogenase complex. Conservation of the gene arrangement between liverwort and Synechocystis suggests that the liverwort chloroplast frxC-ORF465 cluster may have evolved from an ancestor common to Synechocystis, and that these two genes may have been transferred to the nuclear genome in tobacco and rice during evolution.

The mitochondrial DNA (mtDNA) from the cultured cells of a cytoplasmic male-sterile line (A-58CMS) of rice (Oryza sativa) was cloned and its physical map was constructed. There was structural alteration on the mitochondrial genome during the cell culture. Detailed restriction analysis of cosmid clones having mtDNA fragments suggested either that the master genome has a 100-kb duplication (the genome size becomes 450 kb) or that a master circle is not present in the genome (the net structural complexity becomes 350 kb). The physical map of plant mitochondrial genomes thus far reported is illustrated in a single circle, namely a master circle. However, no circular DNA molecule corresponding to a master circle has yet been proved. In the present report, representation of plant mitochondrial genomes and a possibility for mitochondrial genome without a master circle are discussed.

「ミトコンドリアDNA」「オルガネラ」の２項目について解説

本研究ではゼニゴケをモデルとして、植物における精子走化性の理解を目指している。これまでの研究でゼニゴケの精子走化性においてCa2+の関与が強く示唆されている。そこで、Ca2+チャネルのホモログMpVICSPER1 (voltage-gated ion channel of sperm 1)、Ca2+トランスポーターのホモログMpPMCA (Plasma Membrane Ca2+-ATPase) およびCa2+結合ドメインであるEF-Handを有するMpCAPS (Ca2+-dependent activator protein for secretion) 遺伝子に注目して解析を実施している。 MpVICSPER1の機能的性格付けを目的として動物細胞での発現・解析を計画しているが、まだコンストラクト作成の段階にある。 MpPMCAの精子走化性への関与を調べるために、ゲノム編集によって遺伝子を破壊し、その表現型を観察した。機能欠損変異体において葉状体および生殖器、精子の形態に明らかな異常は認められなかった。しかし、変異体精子の遊泳速度は低下し、造卵器への明確な誘引も観察されなかった。しかし、野生株よりやや少ない傾向が観察されたものの、変異体精子により胞子のうが形成された。 同様に、MpCAPSについてもゲノム編集によって機能欠損変異株を作成・観察した。変異体精子は野生株精子と同等の速さで遊泳していたが、最終的な移動距離は顕著に減少していた。しかし、造卵器への誘引を示し、受精によって胞子も形成された。 以上の結果より、MpCAPSおよびMpPMCAは精子の正常な遊泳に必要とされるものの、精子走化性および受精には必須ではないことが示された。

陸上植物は多細胞の配偶体世代nと胞子体世代2nを繰り返すという特徴がある（世代交代）。これまでに被子植物を材料として有性生殖の誘導や雌雄性分化の仕組みが調べられてきたが、これらは主に胞子体世代の器官分化を扱った研究であり、配偶体世代の有性生殖誘導の制御機構の解析は材料的な制約のために遅れていた。そこで、陸上植物進化の基部に位置する苔類のモデルであるゼニゴケを用いて、環境依存的な生殖細胞系列決定、可塑的な性分化、遺伝的頑健性を与える性決定機構に関する研究を進めた。 ゼニゴケは実験室環境では遠赤色光補光により、有性生殖を誘導する。遠赤色光を必要としない機能獲得型変異体より、bHLH転写因子MpBONOBOを同定した。この因子が生殖細胞系列を決定するのに必要十分な因子であることを明らかにした。また、シロイヌナズナの相同遺伝子は、花粉における雄原細胞分化に必須な因子であることもわかった。つまり、陸上植物に共通した生殖細胞決定の機構を明らかにし、成果を論文として公表した。 生殖細胞系列決定の下流には雌雄分化がある。雌性分化を制御する転写因子に注目し、逆遺伝学的に遺伝子の機能解析を進めた。雌特異的に発現するFGMYB遺伝子を破壊すると、遺伝的な雌株が雄器托と造精器を分化した。この遺伝子座には、アンチセンス長鎖非翻訳RNAであるSUFがコードされ、雄特異的に発現していた。この遺伝子の破壊雄株は、雄株であるにも関わらず、FGMYBが発現し雌器托と造卵器を分化した。本年度はFGMYBとSUFからなる性分化制御モジュールの機能解析のため、遺伝子のプロモーターや転写物の蓄積に関して実験を進めた。 ゼニゴケは性染色体により遺伝的な性が決定する。ゲノム解析により明らかにされた性染色体上の遺伝子について、遺伝子の構造および発現特異性に関する解析を進め、ゲノム編集株の作出などの実験に着手した。

Genome editing vectors for large deletion in Marchantia polymorpha were constructed. Using these vectors, the transformants harboring the 4.5 kbp deletion were obtained at the rate of 25% of the T1 transformants. The vectors were deposited and the research paper about the efficient genome editing in M. polymorpha was open at preprint server. In addition, we implemented the software that can automatically design gRNAs for all the gene, including sex chromosomes, in M. polymorpha. The presentation about the software was done at the international meeting.

Here we identified a master transcription factor MpBONOBO (MpBNB) that controls sexual organ development in the liverwort Marchantia polymorpha. MpBNB is expressed in the initial cells that are destined to develop into archegonia and antheridia, suggesting its major role in archegonial/antheridial development. Phylogenetic analysis suggested that MpBNB is a member of an evolutionarily conserved transcription factor family. Arabidopsis BNB1 and BNB2 are redundantly required for specification of generative cell in developing pollen, and are functionally replaceable with MpBNB. These findings suggest evolutionarily conserved role of BNB family proteins in the regulation of germ cell differentiation from gametophytes in land plants.

1) Genes for biosynthetic enzymes, especially novel cytochrome P450 and O-methyltransferases, and transcription factors in isoquinoline alkaloid (IQA) biosynthesis were identified based on the draft genome sequence and trascriptome analysis of Eschscholzia californica. 2) Post-transcriptional regulation of WRKY transcription factor in Coptis japonica were characterized for the stable metabolite production. 3) Transcriptomes were characterized in several IQA producing plants to isolate IQA biosynthetic enzyme genes. 4) Common origin of methylenedioxy ring degradation and demethylation enzyme was identified in bacteria. 5) For the microbial production of useful IQAs, total biosynthesis of opiates by stepwise fermentation was established in engineered Escherichia coli, beside the efficient microbial production of stylopine using a Pichia pastoris expression system. 6) 13-Methylberberine, a berberine analogue with stronger anti-adipogenic effects on mouse 3T3-L1 cells was identified.

In order to understand underlying logic of plant developmental process leading to successful sexual reproduction, (1) metabolic basis of regulation of floral transition (in Arabidopsis) and (2) whole process of sexual reproduction from the induction to fertilization and embryogenesis (in Marchantia) were studied. Main achievement includes elucidation of a regulatory pathway of flowering by potassium, elucidation of temporal aspect of florigen (FT protein) transport, identification of specific amino acid residues involved in FT transport, establishment of transcriptional framework for male sexual organ and gamete development, identification of factors involved in germline segregation, identification of factors involved in sperm cell differentiation, identification of factors involved in regulation of induction of sexual development in response to environmental stimuli, and novel findings on fertilization, early stages of embryogenesis and sporogenesis.

DNA-free genome editing allows target-specific manipulation of a given genome. CRISPR RNA and Cas9 protein can be directly introduced into target cells and lead to genome-editing. We have successfully performed DNA-free genome editing mediated by microinjection of CRISPR/Cas9 complex in the liverwort Marchantia polymorpha. NOPPERABO1 (NOP1), of which loss-of-function mutation causes impaired air-chamber formation and thus can be found readily, was selected as a target gene. Custom gRNA and commercially available Cas9 protein were first allowed to form RNA-protein complex and then injected into single-cell sporelings by a laser thermal microinjector. One of the thalli grown from microinjected sporelings formed a sector that showed the nop1 mutant phenotype, and DNA isolated from the sector showed 5-bp deletion in the target coding sequence. This is the first demonstration of DNA-free genome-editing by microinjection in plants.

Vegetative propagation is a form of asexual reproduction in plants, in which new individuals with functional meristems arise without fertilization and develop directly from vegetative tissues, such as leaves, stems, and roots. The liverwort, Marchantia polymorpha produces gemmae on their gametophytes as a means of vegetative propagation. Each gemma is originated from a single epidermal cell at the bottomof the gemma-cup, developed into a mature gemma with two meristems at symmetrical position. In this study, we focused on auxin in the process of vegetative propagation. We demonstrated that machinaries of auxin signaling and biosynthesis in M. polymorpha was more or less the same as that in angiosperms. The auxin biosynthesis and responses was significantly up-regulated in the base of gemma-cup. Molecular genetic studies indicated the critical role of auxin in the both development and dormancy of gemma in M. polymorpha.

Five sequences of a female genomic data were identified as X-linked by comparing with a male genomic data. RNA-seq data obtained from a variety of tissues were mapped onto the X-linked sequences, and genes expressed specifically in female sexual organs were searched. RT-PCR revealed one of the candidate genes was indeed expressed specifically in female sexual organs. The candidate X-linked gene is similar to those involved in regulation of DNA methylation.

トランスクリプトーム解析で得られた雄器床特異的配列のうち，実際に精子でタンパク質として発現しているものを探索した。得られた候補配列の中に，電位依存性イオンチャンネル遺伝子が含まれていた。興味深いことに，この遺伝子のホモログは動物や藻類など鞭毛を有する生物種には広く分布し，被子植物には存在しない。従って，このイオンチャネルの鞭毛機能への関与が示唆される。雄器床特異的に発現する転写産物に，塩基性アミノ酸に富むプロタミン様タンパク質をコードする遺伝子MpPRMが見いだされた。一般に，プロタミンは精子においてヒストンの代わりにDNAに結合している。MpPRM遺伝子に蛍光タンパク質Citrine遺伝子を融合させたコンストラクトPRMCを発現させたところ，精子そのものに強い蛍光が観察された。目視ではPRMC発現精子の運動能に異常は見られず，KClに対する正の走化性も観察された。野生株雌株とPRMC発現株を交配したところ，正常に胞子が形成された。得られた胞子の約半数がハイグロマイシン耐性を示した。以上の結果より，PRMC精子の稔性は正常であると結論づけた。さらに，野生株雌器床にPRMC精子懸濁液をかけ，30分後に造卵器を観察したところ，多数のPRMC精子が造卵器内に認めらた。一部のPRMC精子は卵に到達していた。コケ植物やシダ植物において，これまで造卵器内進入後の精子の挙動をリアルタイムで追跡することは難しかったが，今回作成したPRMC精子によりそれが容易になると期待される。さらに，配偶子が融合した後の精子核の挙動の観察にも利用できる。

The liverwort Marchantia polymorpha is an emerging model of evodevo studies in land plants. We developed molecular tools for forward genetics for M. polymorpha. DNA polymorphism between the standard accession Takaragaike-1 and the reference accession Kitashirakawa was systematically detected and used for genetic mapping with high resolution. We also applied the T-DNA tagging to isolate developmental mutants and to identify causal genes in M. polymorpha.

被子植物に見られる重複受精のしくみは複雑であるが,元は基部陸上植物の生殖機構に近いものから進化したと考えられる。従って,陸上植物に共通する配偶子認識・融合のしくみを理解するために,基部陸上植物ゼニゴケは有用なモデルとなりうると考えた。精子が卵に到達・融合するには,精子細胞膜に存在する膜タンパク質が重要である。そこで,ゼニゴケ精子特異的に発現する膜タンパク質遺伝子を探索するため,まず次世代シーケンサを用いて約60万リードの雄器床由来ESTを取得した。これまでに得た葉状体や受精前後の雌器床などに由来する約250万リードのESTとの比較および整列化により,雄器床由来ESTのみに存在する4,099配列を得た。これらのうち,37配列は他生物種の膜タンパク質と相同性を示し,かつ推定膜貫通ドメインを有していた。これらのうち,少なくとも10配列は実際に雄器床特異的に発現していることをRT-PCRにより確認した。米国Joint Genome Instituteとのゲノムプロジェクトで予備的なゲノム情報が利用可能となり,推定膜貫通ドメインをもつ他の配列についても同様に解析している。さらに,ゼニゴケ精子が卵そのものではなく,造卵器基部に強く誘引されることを見いだし,精子誘引実験系として利用できるようになった。また,Kイオンに対する走化性をもつという古い報告があったが,その再現性も確認した。最近可能となったゼニゴケ遺伝子破壊系と併せて,精子膜タンパク質遺伝子の機能解析を行っている。

Among the genes identified in the Y chromosomal region under investigation, eight appears to have essential functions and thus are expected to have their homologs on the X chromosome. In fact, the Y-chromosomal M547D3.1 gene has its X-chromosomal partner M547D3.1F. Additional 39 X-chromosomal counterparts were obtained from female genomic data provided from JGI.

The novel zinc finger proteins were identified by protein interaction screening with phytochromes. The mutants having T-DNA insertion in the corresponding genes showed late flowering phenotype. The genetic analysis indicated the genes regulated FT expression as downstream factors of phytochrome B. The studies of temporal and spatial expression, phenotypic complementation with the modified genes indicated the proteins functioned in the nucleus in vascular tissue. Our finding gives insight on the link between light environment and development in growth control.

By determining sequences of newly established genomic libraries (BAC and FOSMID) of Chlamydomonas reinhardtii, we have identified new genes on the genomic R-domain regions responsible to the sex determination. We have developed genome viewers which display the detail information on locations of EST, BAC, FOSMID clones as well as the Illumina or SOLid short reads (36-76 nucleotide) on the chromosomes of Chlamydomonas reinhardtii as well as of a moss, Physcomitrella patenswas. Functions of CO_2-responsive proteins and CO_2-signal transduction pathway was. EST and SNPs/SSRs markers were isolated from a liverwort, Marchantia polymorpha. Genes for morphogenesis were phylogenetically analyzed, revealed the evolution of land plants.

さまざまな光照射条件下でゼニゴケの発生過程を観察したところ、1細胞の胞子から多細胞としてメリステムをもつ葉状体への発生や切断した葉状体からの再分化過程が赤色光で促進されることがわかった。顕微鏡を用いて容易に細胞の変化を追跡できることから実験材料として極めて扱いやすい。遠赤色光照射では細胞分裂が抑制され、発芽した胞子が伸長することや、赤色光照射直後に遠赤色光を照射することで赤色光の効果が打ち消されることから、この細胞分裂と分化を制御する光受容体はフィトクロムであることがわかった。ゼニゴケから赤色光-遠赤色光受容体フィトクロム、青色光受容体フォトトロピンとクリプトクロムに対する遺伝子を単離し、それぞれが1分子種である可能性が高いことを示した。単離した遺伝子をもとに、機能欠損型や機能獲得型の形質転換系統を作出し、今後の光応答実験に利用する材料を準備した。植物の発生を制御する主要な植物ホルモンであるオーキシンと葉状体の発生や切断面の再分化過程の関係を調べた。低濃度のオーキシンでは促進、高濃度のオーキシンでは抑制という植物の生長に与える基本的な応答をゼニゴケももつこと、オーキシン、アンチオーキシン、極性輸送阻害剤の添加実験が効果的であるなど、ゼニゴケがオーキシン生理応答の観察材料としても有望であるごとがわかった。オーキシン受容体であるTIR1、信号伝達因子である、AUX/IAA、転写因子であるARFのゼニゴケオルソログ遺伝子をそれぞれが1、1、2分子種単離し、ゼニゴケのオーキシン信号伝達が基本的なものであることを分子系統樹の解析から推定した。

光独立栄養生物であり固着生物を営む植物は、生育する場所の光環境を光受容体を介して正確に感知し、成長を最適化している。フィトクロムを単一分子種としてもつ苔類ゼニゴケ(Marchantia polymorpha)と役割分担が進み複数の分子種をもつシロイヌナズナ(Arabidopsis thaliana)を材料にフィトクロム信号伝達原理と分子進化研究を進めた。信号伝達の研究は、フィトクロム相互作用タンパク質シロイヌナズナVOZについて研究を進めた。VOZは、ジンクフィンガータンパク質であり、フィトクロム制御の特徴のひとつである遺伝子発現変化に関与すると考えられる。相互作用は、in vitro pull downとY2Hアッセーで確認された。更に、in vivoフィトクロムとの相互作用が、Split Luciferaseアッセーでも検出された。Split LUCの系を利用して、相互作用の光質特異性を検討した。VOZはゲノムに2コピー存在し、単一変異では表現型は観察されない。二重変異体では、フィトクロムが関わる花成時期の表現型を示す。花成に重要なフロリゲンをコードするFT遺伝子の発現を調べたところ、その発現が低下すること、特に、概日リズムのピークが消失することがわかった。生体時計の下流ET遺伝子を制御CO遺伝子発現には変化が見られなかった。最近、植物の低温耐性にフィトクロムによる赤色光と遠赤色光の比率の感知が大きく関わることが示されている。そこで、voz変異体において低温応答を調べたところ、耐凍結性が向上していた。これは、VOZが光のみならず、低温のクロストークに関わる信号伝達因子であると期待された。

植物細胞は、組織や環境に応じて、プラズチドの機能発現が高度に制御されている。DNAをもつプラスチドの機能制御には、核とプラスチドの協調的な遺伝子発現制御が重要である。本研究では、ヘム・ビリンの代謝異常がフィトクロム変異表現型以外にプラスチドシグナル異常を示すことに注目し、これらの系統のテトラピロールとプラスチド機能制御について解析した。また、陸上植物の進化の鍵となる苔類ゼニゴケに注目し、葉緑体ゲノム複製や維持に関してオルガネラ研究の新たな展開を探った。hy1やhy2などのビリン生合成変異は、フィトクロム機能欠損となるが、CAB遺伝子の発現を指標にみるとgun表現型として知られる機能が見える。フィトクロム発色団として機能しないフィコエリトロビリンの合成酵素の発現により代謝を撹乱した系統においてCAB発現を解析した。この系統は、胚軸伸長に加えて、gun表現型も保持していた。これはヘムプールの蓄積によるフィードバックによってテトラピロール代謝が制御されるためビリン生合成変異はgun表現型を示すという説明、すなわち、ヘムブランチの効果は間接的であるという従来の説明に矛盾する結果であった。ゼニゴケの実験系では、プラスチドDNA上の複製に関連する領域を特定するために、二次元電気泳動を用いた構造解析を行った。プラスチドDNAには、恒常的に機能する複製開始点は存在しない可能性と、複製フォーク障壁として機能する構造がゲノム存在することがわかった。また、ゼニゴケプラスチド形質転換により、雌雄のプラスチド形質転換系統を準備し、野生型と交配した。導入遺伝子による薬剤耐性を指標としてゼニゴケの母性遺伝が明確に示された。プラスチドDNAを定量したところ、精子形成時期にコピー数が著しく減少し、精子では数コピーのDNAが検出されるに過ぎなかった。これは、精子のプラスチドDNA含量の低さが母性遺伝の要因であることを示している。

On the Y chromosome of the liverwort, Marchantia polymorpha L., 64 genes are identified, 14 of which are detected only in the male genome. These 14 genes are expressed in reproductive organs but not in vegetative thalli, suggesting their participation in male reproductive functions. The aim of this project is to prove this hypothesis.For all but one of the 14 genes, cDNA sequences were determined by RACE, and their promoter regions were predicted by comparing the cDNA sequences and genomic sequences. To facilitate transgenic analyses, we have developed a rapid Agrobacterium-mediated transformation system for M. polymorpha using immature thalli developed from spores (Ishizaki, et. al., submitted). First, the M350E4.4 gene, which encodes F-box protein related to Arabidopsis UFO, was selected as a model case. To examine its expression pattern, a construct which carries the predicted promoter region of M350E4.4 and the β -glucuronidase gene (GUS) was introduced to immature thalli. No GUS activity was detected in transgenic thalli, which is consistent with our previous result of RT-PCR. Expression of 3135054.4 in sexual organ is under investigation. Four types of constructs were tested to examine the function of M350E4.4: (1) ectopic expression by CaMV35S promoter, (2) silencing by expressing dsRNA, (3) overexpression of F-box domain, and (4) overexpression of the coding sequence without F-box domain. Thalli of transgenic plants examined thus far were morphologically indistinguishable from wild-type. Morphology of sexual organ of transgenic plants are being investigated.

Chlamydomonas modulates photosynthetic characteristics to acclimate to CO_2-limiting stress by sensing CO_2 availability and light by inducing a set of genes for carbon-concentrating mechanism. We have analyzed regulation of a gene Cah1 encoding a carbonic anhydrase to elucidate the CO_2-signal transduction pathways. By using Ars-reporter fused with Cah1 promoter, cis-acting elements and DNA-binding proteins involved in CO_2-regulation were elucidated (Kucho et al. Plant Physiol. 2003, Yoshioka et al. Plant Cell, 2004). Genome wide analysis of gene expression of regulatory mutants revealed that CO_2-responsive genes were grouped into several categories (Miura Plant Physiol., 2004). Although functions of a master regulator CCM1 (Fukuzawa et al. PNAS 2001) is not known, several low-CO_2 inducible genes encoding chloroplast envelop proteins and anion transporters were good candidates for inorganic carbon transporters in eukaryotic algae, Chlamydomonas reinhardtii.

植物において、花成などの有性生殖器官の分化誘導は、栄養生長相から生殖生長相への転換点であり、その遺伝的制御機構の解明は発生学上重要な研究対象である。本研究では、下等植物における有性生殖器官の分化誘導の制御に関わる遺伝子を探索するためにゼニゴケ生殖器官誘導変異株の解析を行った。ゼニゴケ変異株hpt2040は、野性型が生殖器官を形成しない培養条件でも生殖器官を形成する。昨年度、2040p-169株(hpt2040株と野生株との交配で得られたF1株の1つ)において、ゲノム中の3箇所の欠失がhpt2040株の表現型と連鎖していることを明らかにした。そこで、これらの欠失領域近傍にあたる野生株のゲノム配列中のタンパク質コード遺伝子を探索した。複数の遺伝子モデル予測プログラムを用いて配列を解析し、エキソンの予測が集中する7個の領域を見いだした。RT-PCRを行ったところ、いずれの領域についても2040p-169では転写産物は検出されなかったが、野生株においては3個の領域で検出した。これら3領域が変異原因遺伝子の候補であると仮定し、二重鎖RNA発現コンストラクトの導入によるRNA干渉実験、およびそれぞれの遺伝子候補を含むゲノミッククローンの導入による相補実験を実施した。いずれのコンストラクトについても形質転換体は得られたが、RNA干渉による変異表現型の再現および変異の相補を示す形質転換体は得られなかった。変異原因遺伝子を同定するには、他の変異アレルを単離することが必要であると考えられる。

YR1を構成する31クローンのドラフト塩基配列を得た。YR2については、コンティグをさらに伸長させ(合計6.29Mb)、57クローンの塩基配列を得た。 YR1中に14個の遺伝子を見いだした。YR2については、Contig-Aで22個、Contig-Bで21個の遺伝子を見いだした。これらのうち少なくとも17個は雄生殖器官および葉状体で発現しており、残り7個は雄生殖器官特異的に発現していた。雄生殖器官特異的に発現していた遺伝子の中には、マウス精子の鞭毛に局在するタンパク質と相同性を示すアミノ酸配列をコードするものも含まれていた。局在するタンパク質と相同性を示すアミノ酸配列をコードするものも含まれていた。 雌雄生殖器官および葉状体由来のcDNAライブラリーを作成し、遺伝学研究所にて配列解読を依頼している。今後の課題として、今回の相同性検索は、BLASTアルゴリズムによる単純なものであったが、今後はWise2を用いてより詳細に遺伝子を探索する。またEST情報を取得した後、遺伝子コード領域を正確かつ網羅的に探索する。同時にアレイ作成に着手し、Y染色体遺伝子の発現解析を行う。ゼニゴケY染色体には、他の植物種の遺伝子とは相同性を示さず、動物の遺伝子のみと相同性を示す遺伝子が存在した。今後、ゼニゴケY染色体遺伝子と動物遺伝子とを比較することで、例えば精子全般に共通して必要とされる因子を同定することが可能になると考えられる。

本研究では、陸上植物における性の決定・分化に必要な遺伝子群の解明を目指し、雌雄異株植物であるゼニゴケのY染色体について全遺伝情報の解読を試みた。 ゼニゴケY染色体のサイズは約10Mbと見積もられ、Y染色体特異的反復配列が蓄積している領域YR1(約4Mb)と、その他の領域(約6Mb)に大別できる。総括班の支援事業を活用し、YR1については、制限酵素切断パターンの異なる24個の代表PACクローンのうち15個および全長約470kbのコンティグについて、合計約2Mbに相当する配列情報を得た。YR2については、全長約6Mbのコンティグ地図から重なりが最小となる56個のPACクローンを選び、41個について合計約?Mbに相当する配列情報を得た。既知の遺伝子とアミノ酸配列レベルで相同性を示し、かつRT-PCRで発現が認められる領域が、YR1では少なくとも6箇所、YR2では少なくとも14箇所存在した。これらの中には、遺伝子発現調節やシグナル伝達に関わると推測されるものがあった。 興味深いことに、これまでゲノミックサザン解析を行ったYR1の遺伝子については、いずれもY染色体に多コピー存在することが明らかになった。一方、これまでゲノミックサザン解析を行ったYR2の遺伝子は、Y染色体のみに存在する単一コピー遺伝子であった。このようなYR1とYR2における遺伝子の存在様式の違いは、両者の機能的な違いを反映している可能性がある。

In the genome of the hpt2040 mutant, which constitutively develops sexual organs, three genomic regions represented by three PAC clones, pMM23-195C11, pMM23-475F7, and pMM24-34B2, in the wild-type genome, have been affected following the introduction of tag DNA. These rearrangement events are accompanied by deletion of at least three regions and recombination at four sites. All of the detected deletions and two of the recombination sites are linked to the hpt2040 phenotype, suggesting that the gene involved in the mutant phenotype is present in the regions. RT-PCR showed that three of putative exons which were computationally predicted in the regions are transcribed in wild type but not in the hpt2040 mutant. A single-locus mutation in the hpt2040 mutant suggests that the affection of the gene is non-redundant and sufficient to regulate the whole process of sexual organ development. Since the hpt2040 mutant was generated by the introduction of tag DNA into the genome, it is likely that the hpt2040 phenotype is caused by a loss-of-function mutation of a gene which represses the transition to sexual reproduction in M. polymorpha. The affected gene of the hpt2040 mutant may encode a novel plant regulator that acts as an "all-or-none" switch for initiating the reproductive growth without affecting the process of sexual organ development. Role of the gene in the transition to sexual reproduction in M. polymorpha should be clarified in detail by molecular cloning and functional analysis.

We constructed PAC genomic libraries for both male and female liverwort, Marchantia polymorpha. A male-specific clone, pMM4G7, was isolated from the male genomic library by differential screening and shown to originate from the Y chromosome by FISH.The entire nucleotide sequence of pMM4G7 was determined to find Y-chromosome-specific repeated sequences and a gene coding for a protein with a RING-finger motif. Another sequenced male-specific PAC clone, pMM2D3, containd six genes which showed significant similarities to genes registered in the public databases. Four of the six genes were present only in the male genome, and RT-PCR showed that these were actually expressed. Five novel markers for the Y chromosome and two for the X chromosome were isolated by representational difference analysis (RDA). Total sizes of contigs constructed thus far are approximately 3Mb and 150kb for the Y and X chromosomes, respectively. Separate cDNA libraries were constructed for male and female sexual organs. Approximately 1000 ESTs were obtained from each of the libraries, revealing gene expression profiles in sexual organs as well as differences in the expression profiles between male and female sexual organs. In order to elucidate functions of novel genes encoded by the sex chromosomes by gene manipulation, we developed a transformation system for M.polymorpha applying particle bombardment. We have constructed a library of over 2000 tagged lines of M.polymorpha and isolated a mutant which was affected in the development of sexual organs.

前年度から単離したY染色体由来PACクローンpMM4G7には、BamHl2.4kbの反復配列が存在する。この反復配列はサザンハイブルダイゼーションおよびPCRの結果より、Y染色体に特異的であることが明らかになった。すなわちBamHl2.4kb断片と相同な配列を持つPACクローンはY染色体由来であると考えられる。そこで、この反復配列をプローブとしてコロニーハイブリダイゼーションを行い、PACクローン429個を得た。これらのうち、雄ゲノムDNAにのみハイブリダイズするPACクローンは70個であった。クローン整列化に必要な分子マーカーを得るため、Y特異的PACクローンからpMM4G7とそれを含むpMM29D7を除いた68個のDNA混合物のショットガンシークエンスを行い、その塩基配列情報からマーカーとして利用できるプライマーセットを53組設計した。これらのプライマーセットを利用し、pMM4G7を起点に総全長1.6Mbpにわたる5つのコンティングを作製した。これらのコンティングに属する雄特異的PACクローンの一部については塩基配列を決定し、遺伝子コード領域を発見した。この中には、転写因子やリングフィンガーモチーフをもつタンパク質がコードされていた。Y染色体DNA中に見いだされた新奇な遺伝子の機能を解析するため、ゼニゴケの形質転換系を開発した。無性芽播種後約2週間のゼニゴケ葉状体に、パーティクルガンを用いてCaMV35Sプロモ-タで発現するハイグロマイシン耐性遺伝子を導入した。約2ヶ月でハイグロマイシン耐性株が得られ、その過程で植物ホルモンの調製による脱分化・再分化操作の必要がないことから、遺伝子操作による機能解析に極めて有用であると言える。

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