Immunochemical Identification of the Main Cell Wall Polysaccharides of the Early Land Plant Marchantia polymorpha.

Plant primary cell walls are composite structures surrounding the protoplast and containing pectins, hemicelluloses, and cellulose polysaccharides, as well as proteins. Their composition changed during the evolution of the green lineage from algae to terrestrial plants, i.e., from an aquatic to a terrestrial environment. The constraints of life in terrestrial environments have generated new requirements for the organisms, necessitating adaptations, such as cell wall modifications. We have studied the cell wall polysaccharide composition of thalli of Marchantia polymorpha, a bryophyte belonging to one of the first land plant genera. Using a collection of specific antibodies raised against different cell wall polysaccharide epitopes, we were able to identify in polysaccharide-enriched fractions: pectins, including low-methylesterified homogalacturonans; rhamnogalacturonan I with arabinan side-chains; and hemicelluloses, such as xyloglucans with XXLG and XXXG modules, mannans, including galactomannans, and xylans. We could also show the even distribution of XXLG xyloglucans and galactomannans in the cell walls of thalli by immunocytochemistry. These results are discussed with regard to the cell wall proteome composition and in the context of the evolution of the green lineage. The cell wall polysaccharides of M. polymorpha illustrate the transition from the charophyte ancestors of terrestrial plants containing xyloglucans, xylans and mannans as hemicelluloses, and embryophytes which do not exhibit mannans as major primary cell wall polysaccharides.

G protein signaling and metabolic pathways as evolutionarily conserved mechanisms to combat calcium deficiency.

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

Marchantia polymorpha L. Flavonoids Protect Liver From CCl4-Induced Injury by Antioxidant and Gene-Regulatory Effects.

Marchantia polymorpha L. (MPL), a common type of liverwort, has been used as herbal medicine to improve liver function in China for many years. Although modern studies revealed that MPL contains various polyphenols, terpenoids, and bis[bibenzyls], its biological effects on liver function have never been systemically studied in any animal model. In this study, flavonoids were extracted from MPL and the components in the MPL flavonoids as well as the antioxidant capacity of MPL flavonoids were analysed. A rat model of liver injury was induced by intraperitoneal injection of 10% carbon tetrachloride (CCl4). MPL flavonoids were administered daily at a dose of 50, 100, and 200 mg/kg body weight to the rats for 2 weeks prior to injection of CC14. Treatment with MPL flavonoids, especially at a dose of 200 mg/kg, attenuated CCl4-induced increases in alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, nitric oxide, malondialdehyde, tumour necrosis factor-α, interleukin-1ß, and interleukin-6, as well as reductions in superoxide dismutase and glutathione peroxidase. Microarray analyses showed that co-treatment with MPL flavonoids and CCl4 up-regulated many antioxidant and anti-apoptotic genes, but down-regulated several pro-inflammatory genes, compared to treatment with CCl4 alone. PCR and western blot assays further identified that MPL flavonoids increased GPX1, TMX1, TXN, and XIAP expression, but decreased IL-1 and IL1RAP expression and inhibited Jak/stat3 signalling. In conclusion, MPL flavonoids exerted hepatoprotective effects via antioxidant and gene regulatory mechanisms. (Altern Ther Health Med.

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.

Oil Body Formation in Marchantia polymorpha Is Controlled by MpC1HDZ and Serves as a Defense against Arthropod Herbivores.

The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a class I homeodomain leucine-zipper (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stress, including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather, Mpc1hdz mutant plants were more susceptible to herbivory, and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time-lapse imaging, we observed that MpC1HDZ expression maxima precede oil body formation, indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory, but not for abiotic stress tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages.

Cryopreservation of Marchantia polymorpha spermatozoa.

The liverwort Marchantia polymorpha 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 M. polymorpha in liquid nitrogen. A cryoprotective solution containing sucrose, glycerol and egg yolk and controlled cooling and warming processes led to successful recovery of motile M. polymorpha 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 M. polymorpha and could be applied to preservation of plant spermatozoa in general.

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.

Marchantin A, a macrocyclic bisbibenzyl ether, isolated from the liverwort Marchantia polymorpha, inhibits protozoal growth in vitro.

In vitro anti-plasmodial activity-guided fractionation of a diethyl ether extract of the liverwort species Marchantia polymorpha, collected in Iceland, led to isolation of the bisbibenzyl ether, marchantin A. The structure of marchantin A (1) was confirmed by NMR and HREIMS. Marchantin A inhibited proliferation of the Plasmodium falciparum strains, NF54 (IC(50)=3.41µM) and K1 (IC(50)=2.02µM) and showed activity against other protozoan species Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani with IC(50) values 2.09, 14.90 and 1.59µM, respectively. Marchantin A was tested against three recombinant enzymes (PfFabI, PfFabG and PfFabZ) of the PfFAS-II pathway of P. falciparum for malaria prophylactic potential and showed moderate inhibitory activity against PfFabZ (IC(50)=18.18µM). In addition the cytotoxic effect of marchantin A was evaluated. This is the first report describing the inhibitory effects of the liverwort metabolite marchantin A against these parasites in vitro.

Formation of tetrahydrocurcumin by reduction of curcumin with cultured plant cells of Marchantia polymorpha.

Cultured plant cells of Marchantia polymorpha, Nicotiana tabacum, Phytolacca americana, Catharanthus roseus, and Gossypium hirsutum were examined for their ability to reduce curcumin. Only M. polymorpha cells converted curcumin into tetrahydrocurcumin in 90% yield in one day. Time-course experiment revealed a two-step formation of tetrahydrocurcumin via dihydrocurcumin.

Synergistic cytotoxic effect of the microtubule inhibitor marchantin A from Marchantia polymorpha and the Aurora kinase inhibitor MLN8237 on breast cancer cells in vitro.

Macrocyclic bisbibenzyls are a class of characteristic compounds, exclusively produced by liverworts. They are attracting increasing attention due to their wide range of biological activities, including antibacterial, antifungal, and antioxidative properties as well as cytotoxicity. Marchantin A is a cyclic bisbibenzyl that has previously been isolated from Marchantia polymorpha and other liverwort species and has been shown to exert cytotoxic effects. In the present study we found that the Icelandic M. polymorpha species produces marchantin A and through an in vitro cell growth inhibition assay, marchantin A was shown to induce a reduction in cell viability of breast cancer cell lines A256 (IC50 = 5.5 µM), MCF7 (IC50 = 11.5 µM), and T47D (IC50 = 15.3 µM). The effect was considerably increased in all cell lines in a synergistic manner when the Aurora-A kinase inhibitor MLN8237 was added simultaneously. Fluorescence microscopy confirmed the antimicrotubular effect of marchantin A, and cell cycle analysis indicated enhanced cell division failure when combining this mitotic-spindle inhibitor with the checkpoint modulator.

Syntheses of marchantins C, O and P as promising highly bioactive compounds.

Recently, remarkable microtubule inhibitor and anti-tumor activities of the bis(bibenzyl) marchantin C--isolated from liverworts like Marchantia polymorpha since 1983--were found. In this paper we describe the efficient total synthesis of this subtype of bis(bibenzylic) compounds with two biarylether connections. Two selectively methylated derivatives known as natural compounds marchantin O and P were synthesized for the first time by modification of the arene subunits and can now be considered as promising highly bioactive compounds.

Evolutionarily conserved regulatory mechanisms of abscisic acid signaling in land plants: characterization of ABSCISIC ACID INSENSITIVE1-like type 2C protein phosphatase in the liverwort Marchantia polymorpha.

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 amino-terminal 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.

A new method for antimicrobial susceptibility testing of in vitro-cultured bacteria by means of resonance light scattering technique.

A new method for antimicrobial susceptibility testing of in vitro-cultured bacteria on an ordinary fluorescence spectrometer was developed. The viable bacteria reduced 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to produce insoluble particles that displayed intense resonance scattering light (RSL). The assay showed a linear relationship between the number of viable bacteria and the intensity of resonance scattering light. Dead bacteria were unable to reduce MTT. Methicillin-resistant Staphylococcus aureus exposed to flavonoids from Marchantia convoluta showed a flavonoids concentration-dependent inhibition of the ability to reduce MTT. In the assay, less than 12 h were required to attain susceptibility results and fewer bacteria were utilized than in traditional methods. The RLS technique could, in combination with the MTT assay, be a rapid and sensitive measuring method to determine the in vitro activity of new antimicrobials.

Cytotoxicity of Marchantia convoluta leaf extracts to human liver and lung cancer cells.

The cytotoxicity of three extracts (petroleum ether, ethyl acetate and n-butanol) from a plant used in folk medicine, Marchantia convoluta, to human non-small cell lung carcinoma (H1299) and liver carcinoma (HepG2) cell lines was tested. After 72-h incubation of lung and liver cancer cell cultures with varying concentrations of extracts (15 to 200 microg/mL), cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and reported in terms of cell viability. The extracts that showed a significant cytotoxicity were subjected to gas chromatography-mass spectrometry analysis to identify the components. The ethyl acetate, but not the petroleum ether or n-butanol extract, had a significant cytotoxicity against lung and liver carcinoma cells with IC50 values of 100 and 30 microg/mL, respectively. A high concentration of ethyl acetate extract (100 microg/mL) rapidly reduced the number of H1299 cells. At lower concentrations of ethyl acetate extract (15, 30, and 40 microg/mL), the numbers of HepG2 cells started to decrease markedly. Gas chromatography-mass spectrometry analysis of the ethyl acetate extract revealed the presence of several compounds such as phytol (23.42%), 1,2,4-tripropylbenzene (13.09%), 9-cedranone (12.75%), ledene oxide (7.22%), caryophyllene (1.82%), and caryophyllene oxide (1.15%). HPLC analysis result showed that there were no flavonoids in ethyl acetate extract, but flavonoids are abundant in n-butanol extract. Further studies are needed regarding the identification, toxicity, and mechanism of action of active compounds.

Cytotoxicity of Marchantia convoluta leaf extracts to human liver and lung cancer cells

The cytotoxicity of three extracts (petroleum ether, ethyl acetate and n-butanol) from a plant used in folk medicine, Marchantia convoluta, to human non-small cell lung carcinoma (H1299) and liver carcinoma (HepG2) cell lines was tested. After 72-h incubation of lung and liver cancer cell cultures with varying concentrations of extracts (15 to 200 æg/mL), cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and reported in terms of cell viability. The extracts that showed a significant cytotoxicity were subjected to gas chromatography-mass spectrometry analysis to identify the components. The ethyl acetate, but not the petroleum ether or n-butanol extract, had a significant cytotoxicity against lung and liver carcinoma cells with IC50 values of 100 and 30 æg/mL, respectively. A high concentration of ethyl acetate extract (100 æg/mL) rapidly reduced the number of H1299 cells. At lower concentrations of ethyl acetate extract (15, 30, and 40 æg/mL), the numbers of HepG2 cells started to decrease markedly. Gas chromatography-mass spectrometry analysis of the ethyl acetate extract revealed the presence of several compounds such as phytol (23.42 percent), 1,2,4-tripropylbenzene (13.09 percent), 9-cedranone (12.75 percent), ledene oxide (7.22 percent), caryophyllene (1.82 percent), and caryophyllene oxide (1.15 percent). HPLC analysis result showed that there were no flavonoids in ethyl acetate extract, but flavonoids are abundant in n-butanol extract. Further studies are needed regarding the identification, toxicity, and mechanism of action of active compounds.