State Of The Art Of Sex Determination In Bryophytes.

With the advent of genomic and other omics technologies the last decades have witnessed a series of steady and important breakthroughs in the understanding of the genetic determinants of the different reproductive systems of vascular plants and especially on how sexual reproduction shaped their evolution. In contrast, the molecular mechanisms of these fundamental aspects of the biology of bryophytes, a group of non-vascular embryophyte plants sister to all tracheophytes, are still largely obscure. The recent characterization of the sex chromosomes and genetic switches determining sex in bryophytes as well as emerging approaches for molecular sexing of gametophytes hold great promise for elucidation of the evolutionary history as well as the conservation of this species-rich but understudied group of land plants.

Analysis of the Qualitative and Quantitative Content of the Phenolic Compounds of Selected Moss Species under NaCl Stress.

The response to salt stress analysed by quantitative and qualitative analyses in three selected moss species was studied. Non-halophytic funaroid Physcomitrium patens and two halophytic mosses, funaroid Entosthodon hungaricus and pottioid Hennediella heimii were exposed to salt stress under controlled in vitro conditions. The results clearly showed various phenolics to be present and included to some extent as a non-enzymatic component of oxidative, i.e., salt stress. The common pattern of responses characteristic of phenolic compounds was not present in these moss species, but in all three species the role of phenolics to stress tolerance was documented. The phenolic p-coumaric acid detected in all three species is assumed to be a common phenolic included in the antioxidative response and salt-stress tolerance. Although the stress response in each species also included other phenolics, the mechanisms were different, and also dependent on the stress intensity and duration.

Extracts Characterization and In Vitro Evaluation of Potential Immunomodulatory Activities of the Moss Hypnum cupressiforme Hedw.

Recently, there has been an increasing interest in the chemistry and biological potential of mosses, since a large number of biologically active compounds have been found within these species. This study aimed at examining the chemical composition and immunomodulatory potential (antioxidant, antidiabetic, anti-neuroinflammatory/antineurodegenerative, and antitumor activities) of moss Hypnum cupressiforme Hedw. extracts. Corresponding extracts have been obtained applying Soxhlet extractor. The chemical characterization was performed using spectrophotometric assays and liquid chromatography-mass spectrometry (LC-MS). The extracts were analyzed for antioxidant activity and for inhibitory activities on α-glucosidase, α-amylase, acetylcholinesterase, and tyrosinase. Additionally, extracts were tested against four cell lines-MRC-5, BV2, HCT-116, and MDA-MB-231-for antitumor and anti-inflammatory activities. Chemical analysis of extracts revealed the presence of flavonoids, phenolic acids, and triterpenoids. Major compounds identified by LC-MS in H. cupressiforme were kaempferol and five phenolic acids: p-hydroxybenzoic, protocatechuic, p-coumaric, gallic, and caffeic acid. According to biochemical assays the investigated extracts exhibited significant immunomodulatory potential. Significant antiproliferative potential against MDA-MB-231 cells has been observed together with the promising anti-neuroinflammatory application. The obtained data suggest that moss H. cupressiforme is a valuable natural source of biologically active compounds with potential application in the pharmaceutical industry.

Is the Binding Pattern of Zinc(II) Equal in Different Bryophyte Species?

Bryophytes are usually taken as good bioindicators. However, they represent a large group of terrestrial plants and they express an enormous range of peculiarities within the plant kingdom. With the aim to search for a common pattern of zinc binding, we established axenical in vitro cultures of a dozen bryophyte species that include hornworts, thallose, and leafy liverworts, as well as acrocarp and pleurocarp mosses. The species were grown free of contaminants for many years prior to the application of different treatments, i.e. offering Zn(II) from solid and liquid media and in combination with different anions. The localization and binding of zinc was detected by confocal microscopy using the zinc-specific dye FluoZin™-3. In one of the species, Hypnum cupressiforme (which is widely used for atmospheric heavy metal deposition studies in biomonitoring), semi-quantitative analyses of zinc were performed by energy dispersive X-ray microspectrometry (EDX) in a scanning electron microscope. The results suggest no common pattern of Zn(II) binding in different bryophyte species. Instead, the binding pattern seems to be species specific. Zinc is located in certain areas or cellular compartments, as clearly shown by the EDX measurements in H. cupressiforme.

Effects of the Salt Stress Duration and Intensity on Developmental and Physiological Features of the Moss Polytrichum formosum.

The two accessions of the polytrichaceous moss species Polytrichum formosum, namely German and Serbian genotypes, were subjected to salt stress, aiming to study the species' developmental and physiological features. Various concentrations of sodium chloride were applied to an axenic in vitro culture of the two moss genotypes, and the growth parameters as well as physiological feature changes were followed. As inferred by the morpho-developmental parameters and survival index, the Serbian genotype showed higher resistance to salt stress as compared to the German one. However, both moss genotypes survived the highest applied concentration (500 mM). As expected, short exposures to salt were rather easily overcome. No clear patterns in sugar content and changes were observed during the stress, but they are surely included in salt stress response and tolerance in P. formosum. Longer stress increased total chlorophyll content in both genotypes. In short-term applied salt stress, the Serbian genotype had a higher total chlorophyll concentration to control unstressed plants, while the German genotype decreased the total amount of chlorophyll. Similarly, carotenoids were shown to be significantly higher in the Serbian genotype, both in unstressed and treated plants, compared to the German one. The contents of tocopherols were higher in the Serbian genotype in controlled unstressed and subsequently short- and long-stressed plantlets compared to the German accession. In general, we can assume that P. formosum is unexpectedly tolerant to salt stress and that there are differences within various accessions of overall European populations, as referred by two randomly selected genotypes, which is most probably a consequence of different genetic structure.

An Insight into the Biology of the Rare and Peculiar Moss Pterygoneurum sibiricum (Pottiaceae): A Conservation Physiology Approach.

The biological features of the recently described peculiar and rare pottioid moss species Pterygoneurum sibiricum have been studied. A conservation physiology approach through in vitro axenic establishment and laboratory-controlled tests was applied to learn more about its development, physiology, and ecology. Additionally, ex situ collection for this species was established, and a micropropagation methodology was developed. The results obtained clearly document its reaction to salt stress in contrast to its sibling bryo-halophyte species P. kozlovii. The reaction to exogenously applied plant growth regulators, auxin and cytokinin, can be used in the different moss propagation phases of this species or for target structure production and development. Inference to the poorly known ecology of this species should also help in recent species records, and thus improve knowledge about its distribution and conservation.

Effects of Cesium on Physiological Traits of the Catherine's Moss Atrichum undulatum Hedw.

Mosses are proven bioindicators of living environments. It is known that mosses accumulate pollutants from precipitates and, to some lesser extent, from the substrate. In this study, the effects of cesium (Cs) on the physiological traits of acrocarp polytrichaceous Catherine's moss (Atrichum undulatum Hedw.) were studied under controlled, in vitro conditions. Cesium can be found in the environment in a stable form (133Cs) and as a radioactive isotope (134Cs and 137Cs). Belonging to the same group of elements, Cs and potassium (K) share various similarities, due to which Cs can interfere with this essential element and thus possibly alter the plant's metabolism. Results have shown that Cs affects the measured physiological characteristics of A. undulatum, although the changes to antioxidative enzyme activities were not drastic following Cs treatments. Therefore, the activities of antioxidative enzymes at lower pH values are more the consequence of pH effects on enzymatic conformation than simply the harmful effects of Cs. Moreover, Cs did not affect the survival of plants grown on the solid substrate nor plants grown in conditions of light and heavy rain simulation using Cs with variable pH, indicating that Cs is not harmful in this form for the studied species A. undulatum.

The Conservation Physiology of Bryophytes.

An introduction to the conservation physiology of bryophytes is given. The insights into the problems, solutions and examples of the physiological approach to conservation within bryophyte representatives are discussed. The significance of experimental treatments of bryophytes is highlighted. The documentation of bryophyte functional traits and eco-physiological mechanisms in the conservation background for protection purposes is highlighted by the selected examples. The introduction of bryophytes into a new scientific field is resumed and some insights from specific case studies are presented.

The Influence of Seasonality on Secondary Metabolite Profiles and Neuroprotective Activities of Moss Hypnum cupressiforme Extracts: In Vitro and In Silico Study.

Numerous representatives of mosses, including Hypnum cupressiforme, have been used to alleviate different inflammation-related conditions. However, the mode of action underlying this anti-inflammatory potential has been poorly understood. Moreover, the influence of seasonality on the chemical composition and biological activity of mosses is generally overlooked. This study aimed to investigate the influence of seasonal changes (spring, summer, and autumn) on secondary metabolite composition and biological activities of ethyl acetate H. cupressiforme extracts. Antioxidant activity was measured using ß-carotene bleaching assay, while MTT, NBT, ELISA, and Griess assays were carried out to explore the anti-neuroinflammatory and neuroprotective potential of extracts. Inhibitory activities on acetylcholinesterase and tyrosinase were assessed experimentally and by docking analysis. The highest content of secondary metabolites and antioxidant activity were observed in moss during the summer. Extracts inhibited the secretion of ROS, NO, TNF-α, and IL-6, alleviating the inflammatory potential of H2O2 and LPS in microglial and neuronal cells. Strong inhibitory effects on acetylcholinesterase and tyrosinase were observed in vitro. Docking analyses revealed high-affinity interactions of secondary metabolites present in H. cupressiforme with important enzyme residues. Altogether, these results reveal the neuroprotective potential and the significance of seasonal fluctuations on secondary metabolite content and biological activities in moss H. cupressiforme.

Chemical characterization and in vitro immunomodulatory effects of different extracts of moss Hedwigia ciliata (Hedw.) P. Beauv. from the Vrsacke Planine Mts., Serbia.

Bioactive compounds from natural sources are of great importance because of their potential pharmacological activity and tremendous structural diversity. In this study, the chemical composition of different moss extracts of Hedwigia ciliata P. Beauv. have been examined, as well as their antioxidant, antineurodegenerative/anti-neuroinflammatory, antidiabetic, and antiproliferative potential. The extracts were prepared by Soxhlet extractor using solvents of different polarity. Chemical characterization of the extracts revealed the presence of phenolics and flavonoid compounds, together with triterpenoids as secondary metabolites of high biological activity. Significant antioxidant properties of all the extracts were exhibited using the ß-carotene assay. The highest activities were found for water:ethanol extract (with the highest inhibition rate of 96%), but also significant inhibition was measured for ethanol and ethyl acetate extracts (80% and 70%, respectively). Confirmation of biocompatibility of investigated moss extracts has been performed using normal human fibroblast cell line, MRC-5. The H. ciliata extracts exhibited significant antiproliferative activity (~ 50%) against the MDA-MB-231 (human breast adenocarcinoma cell line), which has not previously been reported elsewhere. The Griess assay confirmed the potential anti-neuroinflammatory activity of the extracts, as significant effects in reducing NO production by LPS-stimulated BV2 (normal murine microglia cell line) was observed. This data is in line with noted antineurodegenerative potential measured by the inhibition of acetylcholinesterase (with the highest inhibition rate of 60% for ethyl acetate extract) and tyrosinase (with the highest inhibition rate of 70% for ethanol extract). Additionally, the H. ciliata extracts exhibited significant antidiabetic effect mediated by α-glucosidase inhibition (with the highest inhibition rate of 80% for ethyl acetate extract). The obtained data suggest the presence of immunomodulatory effects of the moss extracts in vitro, which allows the design of new experiments aimed at detecting and characterizing bioactive compounds of the extracts and additionally elucidate detailed mechanisms of their effects.

Metal accumulation in the acrocarp moss Atrichum undulatum under controlled conditions.

Mosses are frequently used to monitor atmospheric metal contamination but few studies on metal adsorption under controlled conditions are available. Here, the accumulation of the heavy metals copper and zinc was studied in the acrocarp moss Atrichum undulatum. An in vitro culture of A. undulatum was established and the same line, size and equally old remets were exposed to six different treatments representing various metal exposure times and washing scenarios as rain simulation. The metal treatments were done in copper and zinc salts (Cu-acetate, CuSO4, ZnSO4 and ZnCl2, respectively). Energy-Dispersive X-ray microanalysis (EDX) was employed to detect bound heavy metals on the moss plantlets. Element distribution in stems and leaves was measured separately. The aqueous solution of metal salts facilitated an adsorption of both elements in the moss tissue as compared to solid medium. Furthermore, A. undulatum can tolerate pollution of zinc and copper in a distinctive extent; our data point towards a higher zinc tolerance whereas copper is rather harmful. However, semi-quantitatively, less zinc was detected within the moss tissue compared to copper. Interestingly, a strong positive correlation between the accumulation of copper/zinc and iron, and a strong negative correlation between copper/zinc and magnesium, respectively, was documented.

Growth supression of plant pathogenic fungi using bryophite extracts / Supressão de crescimento de fungos patogênicos de plantas usando extratos de briófitas

Chemicals are often used in attempts to control diseases caused by plant pathogenic fungi during food production. However, chemicals can have adverse effects not just on food, but they also remain active for a long time within ecosystems, and thus are not environmentally friendly. Therefore,development of bio-treatment and avoiding use of chemicals are urgently needed. With the aim of studying and developing new environmentally-friendly treatments, we tested extracts from selected bryophyte species(Porella platyphylla, Cinclidotus fontinaloides and Anomodon viticulosus) on five plant pathogenic fungi under controlled conditions. The fungi (Botryosphaeria dothidea, Phomopsis viticola, Calosphaeria sp., Colletotrichum acutatum and Monilinia laxa) were selected based on common diseases they cause on fruits and grapevine. They were isolated in cultures and treated with bryophyte extracts. Bryophyte extracts were shown to be effective in suppression of certain plant pathogenic fungi growth and to have a huge potential in development of novel biotechnological treatments and biofungicides. The best results were achieved in inhibition of B. dothidea, P. viticola and Calosphaeria sp.

Fungos fitopatogênicos são controlados com produtos químicos para combater doenças causadas por eles durante a produção de alimentos. Tais produtos são ruins não apenas para a alimentação, mas também podem permanecer por muito tempo nos ecossistemas, portanto, não são ecologicamente corretos. Desta forma, os biotratamentos e a prevenção de usos químicos são urgentemente necessários no futuro próximo. Com o objetivo de estudar e desenvolver nova alternative decontrole de doenças de plantas, testamos os extratos de espécies de briófitas selecionadas (Porella platyphylla, Cinclidotus fontinaloides e Anomodon viticulosus) em cinco fungos fitopatogênicos (Botryosphaeria dothidea, Phomopsis viticola, Calosphaeria sp., Colletotrichum acutatum e Monilinia laxa) em condição controlada. Estes fungos, selecionados com base nas doenças que causam em frutos e videiras, foram isolados em culturas puras e tratados com os respectivos extratos de briófitas. Os extratos de briófitas demonstraram ser eficazes na supressão de certos fungos fitopatogênicos e têm um enorme potencial no desenvolvimento de novos biofungicidas e tratamentos biotecnológicos. Os mais interessantes resultados foram obtidos na inibição de B. dothidea, P. viticola eCalosphaeria sp.