Immunostimulant Activity of Marchantia paleacea Bertol. Herb Liverwort Ethanol Extract in BALB/c Mice

Immunostimulants are compounds that can stimulate an immune response by increasing the activity of non-specific and specific components of the immune system (humoral and cellular) against certain infections and diseases. The liverwort plant species Marchantia paleacea Bertol. has long been used as a source of nutrition and empirical medicine. However, scientifically there is still not much research data on immunomodulators in these plants. This study aims to determine the activity of immunomodulators in the ethanol extract of the herb Marchantia paleacea Bertol. in male mice of BALB/c strain. Bioactive compounds from this plant were extracted by maceration method using 96% ethanol. Extract characterization and phytochemical screening were determined according to WHO guidelines and standard procedures from previous studies. The immunomodulatory activity of the extract was tested by carbon clearance method and lymphoid organ index (non-specific responses), primary and secondary antibody titer tests (humoral specific responses), IL-2 cytokine levels and IFN-ɣ from serum secondary antibodies and delayed-type hypersensitivity reaction/DTH (cellular specific response). The results of qualitative phytochemical screening contained flavonoid compounds, saponins, phenolics, tannins and steroids/triterpenoids. The results of the non-specific immune response immunomodulator test showed that the dose of 52 mg/kg bw had the largest phagocytic index of 1.52 which included strong immunostimulation (K > 1.5) and the organ spleen index of 0.55 ± 0.11 which increased significantly compared to the control (p<0.05). The data on the acquisition of specific immune responses in the primary and secondary antibody titer test in the three test extracts resulted in increased titer levels compared to the control and at a dose of 52 mg/kg bw could significantly increase the levels of IL-2 cytokines in the control group (p<0,05). Meanwhile, in the DTH test, doses of 13 and 26 mg/kg bw could significantly increase the thickness of the soles of mice compared to controls (p<0.05).

An Ancestral Function of Strigolactones as Symbiotic Rhizosphere Signals

In flowering plants, carotenoid-derived strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), a previously unidentified SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating that it is ancestral in land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

Lipid exchanges drove the evolution of mutualism during plant terrestrialization

Symbiosis with arbuscular mycorrhizal fungi (AMF) improves plant nutrition in most land plants, and its contribution to the colonization of land by plants has been hypothesized. Here, we identify a conserved transcriptomic response to AMF among land plants, including the activation of lipid metabolism. Using gain of function, we show the transfer of lipids from the liverwort Marchantia paleacea to AMF and its direct regulation by the transcription factor WRINKLED (WRI). Arbuscules, the nutrient-exchange structures, were not formed in loss-of-function wri mutants in M. paleacea, leading to aborted mutualism. Our results show the orthology of the symbiotic transfer of lipids across land plants and demonstrate that mutualism with arbuscular mycorrhizal fungi was present in the most recent ancestor of land plants 450 million years ago.

Transcriptional responses to arbuscular mycorrhizal symbiosis development are conserved in the early divergent Marchantia paleacea

Arbuscular mycorrhizal symbiosis (AMS) arose in land plants more than 400 million years ago, perhaps acting as a major contributor to plant terrestrialization. The ability to engage in AMS is evolutionarily conserved across most clades of extant land plants, including early diverging bryophytes. Despite its broad taxonomic distribution, little is known about the molecular components that underpin AMS in early diverging land plants as the mechanisms regulating the symbiosis were primarily characterized in angiosperms. Several AMS associated genes were recently shown to be conserved in liverworts and hornworts, but evidence of them being associated with symbiosis in bryophytes is scarce. In this study, we characterised the dynamic response of the liverwort Marchantia paleacea to Rhizophagus irregularis colonization by time-resolved transcriptomics across progressive stages of symbiosis development. Gene orthology inference and comparative analysis of the M. paleacea transcriptional profile with a well characterised legume model -Medicago truncatula - revealed a deep conservation of transcriptional responses to AMS across distantly related species. We identified evolutionarily conserved patterns of expression of genes required for pre-symbiotic signalling, intracellular colonization and symbiotic nutrient exchange. Our study demonstrates that the genetic machinery regulating key aspects of symbiosis in plant hosts is largely conserved and coregulated across distantly related land plants. If bryophytes are confirmed to be monophyletic, our analysis provides novel insights on the first molecular pathways associated with symbiosis at the dawn of plant colonization of land.Significance StatementArbuscular mycorrhizal symbiosis (AMS) between plants and soil fungi was proposed as one of the key adaptations enabling land colonization by plants. The symbiosis is widespread across most extant plant clades, including early-diverging bryophytes, suggesting that it evolved before the last common ancestor of land plants. Recent phylogenetic analyses uncovered that genes regulating AMS in angiosperms are present in the genomes of bryophytes. Our work shows that a set of these genes are transcriptionally induced during AMS in liverworts. Based on the conservation of their transcriptional profiles across land plants, we propose that these genes acquired an AMS-associated function before the last common ancestor of land plants.

Bryophyte diversity hotspot: the Marmore Waterfalls Regional Park (Umbria, central Italy)

A study of the bryophyte of Marmore Waterfall Natural Park a very peculiar territory for its geological, biogeographical and bioclimatic traits, was carried out. The reported data were collected in the valley area of ‘Parco Fluviale del Nera’ Regional Park, where the waters are flowing and the humidity of the rocks is oozing. Research led to the identification of 101 taxa of bryophytes (21 liverworts and 80 mosses), among which two liverwort and 10 moss species are new records for the Umbria region, while four are confirmed. The study offers new outcomes on neglected aspects of the flora of central Italy and represents a considerable improvement of the floristic, biogeographical and ecological understanding of its bryophytic component. Additionally, the area turn out to be significant from the conservation point of view both nationally and internationally considering that it harbours high diversity of bryophytes species among which many rare, threatened and interesting species occur. Eighteen species of conservation interest in Italy are recorded during the latest investigation (all in category NT – near threatened). Additionally two species that are considered data deficient in Italy are present in the researched area (Conocephalum salebrosum and Fissidens curvatus). Also, there is a small populations of a species vulnerable (Marchantia paleacea), endangered (Bryum calophyllum) and data deficient (Fissidens curvatus and Barbula bolleana) in European scale.

Isolation of Native Arbuscular Mycorrhizal Fungi within Young Thalli of the Liverwort Marchantia paleacea

Arbuscular mycorrhizal fungi (AMF) are a group of soil microorganisms that establish symbioses with most land plant species. “Root trap culture” generally has been used for isolating a single regenerated spore in order to establish a monospecific, native AMF line. Roots may be co-colonized with multiple AMF species; however, only a small portion of AMF within roots sporulate, and do so only under certain conditions. In this study, we tested whether young thalli (<2 mm) of the liverwort Marchantia paleacea harbour monospecific AMF, and can be used as a vegetative inoculant line. When M. paleacea gemmae were co-cultivated with roots obtained from the field, the young thalli were infected by AMF via rhizoids and formed arbuscules after 18 days post-sowing. Ribosomal DNA sequencing of the AMF-colonized thalli (mycothalli) revealed that they harboured phylogenetically diverse AMF; however, new gemmae sown around transplanted mycothalli showed evidence of colonization from phylogenetically uniform Rhizophagus species. Of note, mycothalli can also be used as an inoculum. These results suggest that the young thalli of M. paleacea can potentially isolate monospecific AMF from field soil in a spore-independent manner.