On the configuration and occurrence of 2,6-cyclocuparan-3-ols: Resolving a lasting discrepancy.

2,6-Cyclocuparan-3-ols are chemical markers and major volatiles of several liverwort species. Conflicting reports on the structures of these cyclocuparanols can be found in the literature-different research groups assigned the same spectral data to different structures, yet these inconsistencies were never addressed, let alone satisfactorily explained. Following the isolation of all four diastereoisomeric cyclocuparanols from Marchantia polymorpha, their relative and absolute configurations were extensively studied by chemical and spectroscopic methods and definite stereostructures were proposed.

Ligand diversity contributes to the full activation of the jasmonate pathway in Marchantia polymorpha.

In plants, jasmonate signaling regulates a wide range of processes from growth and development to defense responses and thermotolerance. Jasmonates, such as jasmonic acid (JA), (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile), 12-oxo-10,15(Z)-phytodienoic acid (OPDA), and dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), are derived from C18 (18 Carbon atoms) and C16 polyunsaturated fatty acids (PUFAs), which are found ubiquitously in the plant kingdom. Bryophytes are also rich in C20 and C22 long-chain polyunsaturated fatty acids (LCPUFAs), which are found only at low levels in some vascular plants but are abundant in organisms of other kingdoms, including animals. The existence of bioactive jasmonates derived from LCPUFAs is currently unknown. Here, we describe the identification of an OPDA-like molecule derived from a C20 fatty acid (FA) in the liverwort Marchantia polymorpha (Mp), which we term (5Z,8Z)-10-(4-oxo-5-((Z)-pent-2-en-1-yl)cyclopent-2-en-1-yl)deca-5,8-dienoic acid (C20-OPDA). This molecule accumulates upon wounding and, when applied exogenously, can activate known Coronatine Insensitive 1 (COI1) -dependent and -independent jasmonate responses. Furthermore, we identify a dn-OPDA-like molecule (Δ4-dn-OPDA) deriving from C20-OPDA and demonstrate it to be a ligand of the jasmonate coreceptor (MpCOI1-Mp Jasmonate-Zinc finger inflorescence meristem domain [MpJAZ]) in Marchantia. By analyzing mutants impaired in the production of LCPUFAs, we elucidate the major biosynthetic pathway of C20-OPDA and Δ4-dn-OPDA. Moreover, using a double mutant compromised in the production of both Δ4-dn-OPDA and dn-OPDA, we demonstrate the additive nature of these molecules in the activation of jasmonate responses. Taken together, our data identify a ligand of MpCOI1 and demonstrate LCPUFAs as a source of bioactive jasmonates that are essential to the immune response of M. polymorpha.

Mass spectrometry-based quantification and spatial localization of small organic acid exudates in plant roots under phosphorus deficiency and aluminum toxicity.

Low-molecular-weight organic acid (OA) extrusion by plant roots is critical for plant nutrition, tolerance to cations toxicity, and plant-microbe interactions. Therefore, methodologies for the rapid and precise quantification of OAs are necessary to be incorporated in the analysis of roots and their exudates. The spatial location of root exudates is also important to understand the molecular mechanisms directing OA production and release into the rhizosphere. Here, we report the development of two complementary methodologies for OA determination, which were employed to evaluate the effect of inorganic ortho-phosphate (Pi) deficiency and aluminum toxicity on OA excretion by Arabidopsis roots. OA exudation by roots is considered a core response to different types of abiotic stress and for the interaction of roots with soil microbes, and for decades has been a target trait to produce plant varieties with increased capacities of Pi uptake and Al tolerance. Using targeted ultra-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UPLC-HRMS/MS), we achieved the quantification of six OAs in root exudates at sub-micromolar detection limits with an analysis time of less than 5 min per sample. We also employed targeted (MS/MS) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to detect the spatial location of citric and malic acid with high specificity in roots and exudates. Using these methods, we studied OA exudation in response to Al toxicity and Pi deficiency in Arabidopsis seedlings overexpressing genes involved in OA excretion. Finally, we show the transferability of the MALDI-MSI method by analyzing OA excretion in Marchantia polymorpha gemmalings subjected to Pi deficiency.

Phytochemical Profile and Anticancer Potential of Endophytic Microorganisms from Liverwort Species, Marchantia polymorpha L.

Liverwort endophytes could be a source of new biologically active substances, especially when these spore-forming plants are known to produce compounds that are not found in other living organisms. Despite the significant development of plant endophytes research, there are only a few studies describing liverwort endophytic microorganisms and their metabolites. In the presented study, the analysis of the volatile compounds obtained from thallose liverwort species, Marchantia polymorpha L., and its endophytes was carried out. For this purpose, non-polar extracts of plant material and symbiotic microorganisms were obtained. The extracts were analyzed using gas chromatography coupled to mass spectrometry. Compounds with the structure of diketopiperazine in the endophyte extract were identified. Liverwort volatile extract was a rich source of cuparane-, chamigrane-, acorane-, and thujopsane-type sesquiterpenoids. The cytotoxicity of ethyl acetate extracts from endophytic microorganisms was evaluated on a panel of cancer (FaDu, HeLa, and SCC-25) cell lines and normal (VERO), and revealed significant anticancer potential towards hypopharyngeal squamous cell carcinoma and cervical adenocarcinoma.

Differential localization of cell wall polymers across generations in the placenta of Marchantia polymorpha.

To further knowledge on cell wall composition in early land plants, we localized cell wall constituents in placental cells of the liverwort Marchantia polymorpha L. using monoclonal antibodies (MAbs) in the transmission electron microscope and histochemical staining. The placenta of M. polymorpha is similar to the majority of bryophytes in that both generations contain transfer cells with extensive wall ingrowths. Although the four major cell wall polymers, i.e., cellulose, pectins, hemicelluloses, and arabinogalactan proteins, are present, there are variations in the richness and specificity across generations. An abundance of homogalacturonan pectins in all placental cell walls is consistent with maintaining cell wall permeability and an acidic apoplastic pH necessary for solute transport. Although similar in ultrastructure, transfer cell walls on the sporophyte side in M. polymorpha are enriched with xyloglucans and diverse AGPs not detected on the gametophyte side of the placenta. Gametophyte wall ingrowths are more uniform in polymer composition. Lastly, extensins and callose are not components of transfer cell walls of M. polymorpha, which deviates from studies on transfer cells in other plants. The difference in polymer localizations in transfer cell walls between generations is consistent with directional movement from gametophyte to sporophyte in this liverwort.

DIX Domain Polymerization Drives Assembly of Plant Cell Polarity Complexes.

Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.

Molecular Diversity of Alkenal Double Bond Reductases in the Liverwort Marchantia paleacea.

Alkenal double bond reductases (DBRs), capable of catalyzing the NADPH-dependent reduction of the α,ß-unsaturated double bond, play key roles in the detoxication of alkenal carbonyls. Here, the isolation and characterization of two DBRs encoded by the liverwort species Marchantia paleacea are described. The two DBRs share a relatively low similarity, and phylogenetic analysis indicated that MpMDBRL is more closely related to microbial DBRs than to other plant DBRs, while MpDBR shares common ancestry with typical plant DBRs. Both DBR proteins exhibited hydrogenation ability towards hydroxycinnamyl aldehydes; however, their temperature optimums were strikingly different. MpMDBRL demonstrated slightly weaker catalytic efficiency compared to MpDBR, and the structural models of their active binding sites to the substrate may provide a parsimonious explanation. Furthermore, both DBRs significantly responded to phytohormone treatment. In conclusion, M. paleacea produces two distinct types of functional DBRs, both of which participate in the protection against environmental stress in liverwort. The presence of a microbial type of DBR in a plant is herein reported for the first time.

Droplet-based microfluidic analysis and screening of single plant cells.

Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.

n-Hexanal and (Z)-3-hexenal are generated from arachidonic acid and linolenic acid by a lipoxygenase in Marchantia polymorpha L.

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 α-linolenic acids at almost equivalent efficiency and formed C6-aldehydes during its catalysis; these are likely formed from hydroperoxides of arachidonic and α-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.

Occurrence of brassinosteroids in non-flowering land plants, liverwort, moss, lycophyte and fern.

Endogenous brassinosteroids (BRs) in non-flowering land plants were analyzed. BRs were found in a liverwort (Marchantia polymorpha), a moss (Physcomitrella patens), lycophytes (Selaginella moellendorffii and S. uncinata) and 13 fern species. A biologically active BR, castasterone (CS), was identified in most of these non-flowering plants but another biologically active BR, brassinolide, was not. It may be distinctive that levels of CS in non-flowering plants were orders of magnitude lower than those in flowering plants. 22-Hydroxycampesterol and its metabolites were identified in most of the non-flowering plants suggesting that the biosynthesis of BRs via 22-hydroxylation of campesterol occurs as in flowering plants. Phylogenetic analyses indicated that M. polymorpha, P. patens and S. moellendorffii have cytochrome P450s in the CYP85 clans which harbors BR biosynthesis enzymes, although the P450 profiles are simpler as compared with Arabidopsis and rice. Furthermore, these basal land plants were found to have multiple P450s in the CYP72 clan which harbors enzymes to catabolize BRs. These findings indicate that green plants were able to synthesize and inactivate BRs from the land-transition stage.

Direct evidence of specific localization of sesquiterpenes and marchantin A in oil body cells of Marchantia polymorpha L.

Liverworts are a rich source of a diverse array of specialized metabolites, such as terpenoids and benzenoids, which are potentially useful for pharmaceutical or agrochemical applications, and also provide clues to elucidate the strategy by which liverworts adapt to the terrestrial environment. Liverworts, belonging to orders Marchantiales and Jungermanniales, possess oil bodies. In Marchantia polymorpha L., oil bodies are confined to scattered idioblastic oil body cells. It has been assumed that the specialized metabolites in M. polymorpha specifically accumulate in the oil bodies in oil body cells; however, no direct evidence was previously available for this specific accumulation. In this study, direct evidence was obtained using micromanipulation techniques coupled with MS analysis that demonstrated the specific accumulation of sesquiterpenoids and marchantin A in the oil body cells of M. polymorpha thalli. It was also observed that the number of oil body cells increased in thalli grown in low-mineral conditions. The amounts of sesquiterpenoids and marchantin A detected in crude extract prepared from the whole thallus were roughly proportional to the number of oil body cells found in a given volume of thallus, suggesting that oil body cell differentiation and sesquiterpenoid and marchantin A biosynthetic pathways are coordinated with each other.

Flavonoids, Antioxidant Potential, and Acetylcholinesterase Inhibition Activity of the Extracts from the Gametophyte and Archegoniophore of Marchantia polymorpha L.

Marchantia polymorpha L. is a representative bryophyte used as a traditional Chinese medicinal herb for scald and pneumonia. The phytochemicals in M. polymorpha L. are terpenoids and flavonoids, among which especially the flavonoids show significant human health benefits. Many researches on the gametophyte of M. polymorpha L. have been reported. However, as the reproductive organ of M. polymorpha L., the bioactivity and flavonoids profile of the archegoniophore have not been reported, so in this work the flavonoid profiles, antioxidant and acetylcholinesterase inhibition activities of the extracts from the archegoniophore and gametophyte of M. polymorpha L. were compared by radical scavenging assay methods (DPPH, ABTS, O(2-)), reducing power assay, acetylcholinesterase inhibition assay and LC-MS analysis. The results showed that the total flavonoids content in the archegoniophore was about 10-time higher than that of the gametophyte. Differences between the archegoniophore and gametophyte of M. polymorpha L. were observed by LC-MS analysis. The archegoniophore extracts showed stronger bio-activities than those of the gametophyte. The archegoniophore extract showed a significant acetylcholinesterase inhibition, while the gametophyte extract hardly inhibited it.

Bis-bibenzyls from the Cameroon Liverwort Marchantia debilis.

Liverworts are rich sources of terpenoids and aromatic compounds among which bis-bibenzyls are well known for their wide spectrum of biological activities. This is the first report of chemical analysis of the African liverwort Marchantia debilis Goebel. From the methanol extract marchantinquinone-l'-methyl ether was newly isolated together with three known bis-bibenzyls, marchantin C, marchantinquinone and perrottetin E. The presence of bis-bibenzyls with a quinone moiety is noted for the first time in the Marchantia genus.

Management of Diabetic Bacterial Foot Infections with Organic Extracts of Liverwort Marchantia debilis from Cameroon.

Diabetic bacterial foot infections (DBFIs) are limb-threatening complications in patients with diabetes mellitus, accounting for 50% of diabetes related lower limb amputations in developing countries, representing approximately 20 percent of all diabetes-related hospital admissions with significant healthcare-related costs involved. The widespread problem of bacterial resistance to most commonly used antibiotics places a huge economic burden on the healthcare system, with both increased morbidity and mortality among diabetic patients with foot infections. In this study, the antibacterial activity of organic extracts of the fresh liverwort Marchantia debilis from the North West Region of Cameroon is reported. An exit pool system, where patients presenting with DBFIs consented to be involved in the use of phytomedicines, after long term treatment of ulcers with antibiotics and not yielding significant long term benefit, presented themselves at the Phytobiotechnology Research clinic (PRF). Continuous culture of swabs from foot and toe wounds from 30 infected patients on nutrient agar and MacConkey agars in triplicate as well as Gram stain microscopy, revealed the presence of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Bacillus species. Light petroleum and methanol extracts of the whole bryophyte plants at 100% concentration were tested. In vitro inhibition of the tested bacterial isolates from the diabetic foot ulcers by M debilis was observed only with the light petroleum extract. No inhibition by the extracts was observed for the Pseudomonas aeruginosa isolate. The light petroleum extract of M debilis was formulated into a petroleum oil based cream named BryoCream™. This was administered to 20 of the patients with 90% cure rate in a three week time period. The main nonpolar components were determined by GCMS as lepidozene and ß-barbatene, and by NMR as stigmasterol and ß-sitosterol. In conclusion, nonpolar extracts from bryophytes from Cameroon could, potentially, be used to treat diabetic bacterial foot infections.

Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha.

12-Oxo-phytodienoic acid (OPDA) is an intermediate in jasmonic acid (JA) biosynthesis. OPDA exerts JA-dependent and JA-independent biological effects; therefore, it is considered a signaling molecule in flowering plants. OPDA is induced by bacterial infection and wounding and inhibits growth in the moss Physcomitrella patens. The functions of OPDA and allene oxide cyclase (AOC) in the liverwort Marchantia polymorpha were explored, which represents the most basal lineage of extant land plants. The analysis of OPDA showed that it is present in M. polymorpha and is increased by wounding. OPDA has been suggested to be involved in the response to environmental stresses. Moreover, OPDA showed growth inhibitory activity in M. polymorpha. Nonetheless JA in M. polymorpha was not found in this study. AOC synthesizes OPDA from an unstable allene oxide. A database search of the M. polymorpha genome identified only a putative gene encoding allene oxide cyclase (MpAOC). Recombinant MpAOC showed AOC activity similar to that in flowering plants. MpAOC was localized to chloroplasts, as in flowering plants. Expression of MpAOC was induced by wounding and OPDA treatment, and positive feedback regulation of OPDA was demonstrated in M. polymorpha. Overexpression of MpAOC increased the endogenous OPDA level and suppressed growth in M. polymorpha. These results indicate the role of OPDA as a signaling molecule regulating growth and the response to wounding in the liverwort M. polymorpha.

Biochemical and structural characterization of an endoplasmic reticulum-localized late embryogenesis abundant (LEA) protein from the liverwort Marchantia polymorpha.

Late embryogenesis abundant (LEA) proteins, which accumulate to high levels in seeds during late maturation, are associated with desiccation tolerance. A member of the LEA protein family was found in cultured cells of the liverwort Marchantia polymorpha; preculture treatment of these cells with 0.5M sucrose medium led to their acquisition of desiccation tolerance. We characterized this preculture-induced LEA protein, designated as MpLEA1. MpLEA1 is predominantly hydrophilic with a few hydrophobic residues that may represent its putative signal peptide. The protein also contains a putative endoplasmic reticulum (ER) retention sequence, HEEL, at the C-terminus. Microscopic observations indicated that GFP-fused MpLEA1 was mainly localized in the ER. The recombinant protein MpLEA1 is intrinsically disordered in solution. On drying, MpLEA1 shifted predominantly toward α-helices from random coils. Such changes in conformation are a typical feature of the group 3 LEA proteins. Recombinant MpLEA1 prevented the aggregation of α-casein during desiccation-rehydration events, suggesting that MpLEA1 exerts anti-aggregation activity against desiccation-sensitive proteins by functioning as a "molecular shield". Moreover, the anti-aggregation activity of MpLEA1 was ten times greater than that of BSA or insect LEA proteins, which are known to prevent aggregation on drying. Here, we show that an ER-localized LEA protein, MpLEA1, possesses biochemical and structural features specific to group 3 LEA proteins.

Arachidonic acid-dependent carbon-eight volatile synthesis from wounded liverwort (Marchantia polymorpha).

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 40min. 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.

Evolution of oleosin in land plants.

Oleosins form a steric barrier surface on lipid droplets in cytoplasm, preventing them from contacting and coalescing with adjacent droplets. Oleosin genes have been detected in numerous plant species. However, the presence of oleosin genes in the most basally diverging lineage of land plants, liverworts, has not been reported previously. Thus we explored whether liverworts have an oleosin gene. In Marchantia polymorpha L., a thalloid liverwort, one predicted sequence was found that could encode oleosin, possessing the hallmark of oleosin, a proline knot (-PX5SPX3P-) motif. The phylogeny of the oleosin gene family in land plants was reconstructed based on both nucleotide and amino acid sequences of oleosins, from 31 representative species covering almost all the main lineages of land plants. Based on our phylogenetic trees, oleosin genes were classified into three groups: M-oleosins (defined here as a novel group distinct from the two previously known groups), low molecular weight isoform (L-oleosin), and high molecular weight isoform (H-oleosin), according to their amino-acid organization, phylogenetic relationships, expression tissues, and immunological characteristics. In liverworts, mosses, lycophytes, and gymnosperms, only M-oleosins have been described. In angiosperms, however, while this isoform remains and is highly expressed in the gametophyte pollen tube, two other isoforms also occur, L-oleosins and H-oleosins. Phylogenetic analyses suggest that the M-oleosin isoform is the precursor to the ancestor of L-oleosins and H-oleosins. The later two isoforms evolved by successive gene duplications in ancestral angiosperms. At the genomic level, most oleosins possess no introns. If introns are present, in both the L-isoform and the M-isoform a single intron inserts behind the central region, while in the H-isoform, a single intron is located at the 5'-terminus. This study fills a major gap in understanding functional gene evolution of oleosin in land plants, shedding new light on evolutionary transitions of lipid storage strategies.

chlB requirement for chlorophyll biosynthesis under short photoperiod in Marchantia polymorpha L.

Chlorophylls (Chls) play pivotal roles in energy absorption and transduction and also in charge separation in reaction centers in all photosynthetic organisms. In Chl biosynthesis steps, only a step for the enzymatic reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is mediated by both nuclear- and chloroplast-encoded genes in land plants. Many plants encode the genes for light-dependent Pchlide reductase (LPOR) and light-independent Pchlide reductase (DPOR) in the nucleus and chloroplast genome, respectively. During the diversification of land plants, the reduction step of Pchlide to Chlide has become solely dependent on LPOR, and the genes for DPOR have been lost from chloroplast genome. It remains unclear why DPOR persists in some land plants, how they were eliminated from chloroplast genomes during the diversification of land plants, and under what environmental conditions DPOR was required. We demonstrate that DPOR is functional in liverwort (Marchantia polymorpha L.) and plays an important role in Chl biosynthesis. Having established a plastid transformation system in liverwort, we disrupted chlB, which encodes a subunit of DPOR in the M. polymorpha chloroplast genome. Morphological and Chl content analysis of a chlB mutant grown under different photoperiods revealed that DPOR is particularly required for Chl biosynthesis under short-day conditions. Our findings suggest that an environmental condition in the form of photoperiod is an important factor that determines the loss or retention of chloroplast-encoded genes mediating Pchlide reduction to Chlide.

Bioproduction of prostaglandins in a transgenic liverwort, Marchantia polymorpha.

Prostaglandins are biologically active substances used in a wide range of medical treatments. Prostaglandins have been supplied mainly by chemical synthesis; nevertheless, the high cost of prostaglandin production remains a factor. To lower the cost of prostaglandin production, we attempted to produce prostaglandins using a liverwort, Marchantia polymorpha L., which accumulates arachidonic acid, which is known as a substrate of prostaglandins. Here we report the first bioproduction of prostaglandins in plant species by introducing a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla into the liverwort. The transgenic liverworts accumulated prostaglandin F2α, prostaglandin E2 and prostaglandin D2 which were not detected in the wild-type liverwort. Moreover, we succeeded in drastically increasing the bioproduction of prostaglandins using an in vitro reaction system with the extracts of transgenic liverworts.