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.

Exploring In Vitro Immunomodulatory Properties of Moss Atrichum undulatum Extracts.

Bryophytes are rich sources of diverse secondary metabolites with a wide range of biological activities, including anti-inflammatory, antitumor and antimicrobial effects. The aim of this study was to investigate the chemical composition of extracts from two different genotypes (Serbian and Hungarian) of the axenic moss Atrichum undulatum and evaluate the immunomodulatory potential of the prepared extracts in vitro. Both genotypes of moss samples were cultivated in vitro and subsequently extracted in a Soxhlet apparatus with methanol or ethyl acetate. The highest concentration of total phenolic compounds was found in the methanolic extract of the Serbian genotype (54.25 mg GAE/g extract), while the ethyl acetate extract of the Hungarian genotype showed the highest concentration of phenolic acids (163.20 mg CAE/extract), flavonoids (35.57 mg QE/extract), and flavonols (2.25 mg QE/extract). The extracts showed anti-neuroinflammatory properties by reducing the production of reactive oxygen species, nitric oxide, and tumor necrosis factor alpha by lipopolysaccharide-stimulated microglial cells. Moreover, they mitigated the cytotoxic effects of the pro-inflammatory mediators produced by activated microglia on neurons. The data obtained suggest that extracts from A. undulatum moss have promising anti-neuroinflammatory and neuroprotective properties, making them interesting candidates for further research to combat neuroinflammation.

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.

The genetic characterization of grapevines prospected in old Serbian vineyards reveals multiple relationships between traditional varieties of the Balkans.

Serbia preserves a high number of local grape varieties, which have been cultivated across the country for centuries. Now, these ancient varieties are in the spotlight, and there is a global trend towards their recovery and characterization because they can revitalize regional, national and international grape and wine sectors. In addition, their genetic study can be useful to find new pedigree relationships to reveal how local varietal assortment evolved over time. Here, the genetic characterization of 138 grapevines from old Serbian vineyards revealed 59 different genetic profiles, 49 of which were identified as grapevine varieties whose origin in the country could be linked to some major Serbian historical periods. Most of the genetic profiles found in this work arranged in a complex pedigree network that integrates numerous grapevine varieties from diverse Balkan countries, agreeing with an intense exchange of plant material among Balkan regions for centuries. This analysis identified some varieties as important founders of Balkan genetic resources, like 'Alba Imputotato', 'Braghina Rosie', 'Coarna Alba', and 'Vulpea'. After deepening into their genealogy, these major direct founders might have ultimately derived from 'Visparola', an ancient variety of likely Balkan origin with a major founding role in some European regions. Our results also indicated the genetic singularity of the grapevine resources from the Balkans when compared to those from other relevant winemaking regions, supporting the interest of their detailed study to evaluate their oenological potential and for the eventual identification of useful traits to counteract current viticulture challenges.

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.

The AAA-type ATPase AtSKD1 contributes to vacuolar maintenance of Arabidopsis thaliana.

The vacuole is the most prominent organelle of plant cells. Despite its importance for many physiological and developmental aspects of plant life, little is known about its biogenesis and maintenance. Here we show that Arabidopsis plants expressing a dominant-negative version of the AAA (ATPase associated with various cellular activities) ATPase AtSKD1 (SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) under the control of the trichome-specific GLABRA2 (GL2) promoter exhibit normal vacuolar development in early stages of trichome development. Shortly after its formation, however, the large central vacuole is fragmented and finally disappears completely. Secretion assays with amylase fused to the vacuolar sorting signal of Sporamin show that dominant-negative AtSKD1 inhibits vacuolar trafficking of the reporter that is instead secreted. In addition, trichomes expressing dominant-negative AtSKD1 frequently contain multiple nuclei. Our results suggest that AtSKD1 contributes to vacuolar protein trafficking and thereby to the maintenance of the large central vacuole of plant cells, and might play a role in cell-cycle regulation.

The Arabidopsis ESCRT protein-protein interaction network.

In yeast, endosomal sorting of monoubiquitylated transmembrane proteins is performed by a subset of the 19 "class E vacuolar protein sorting" proteins. The core machinery consists of 11 proteins that are organised in three complexes termed ESCRT I-III (endosomal sorting complex required for transport I-III) and is conserved in eukaryotic cells. While the pathway is well understood in yeast and animals, the plant ESCRT system is largely unexplored. At least one sequence homolog for each ESCRT component can be found in the Arabidopsis genome. Generally, sequence conservation between yeast/animals and the Arabidopsis proteins is low. To understand details about participating proteins and complex organization we have performed a systematic pairwise yeast two hybrid analysis of all Arabidopsis proteins showing homology to the ESCRT core machinery. Positive interactions were validated using bimolecular fluorescence complementation. In our experiments, most putative ESCRT components exhibited interactions with other ESCRT components that could be shown to occur on endosomes suggesting that despite their low homology to their yeast and animal counterparts they represent functional components of the plant ESCRT pathway.

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.

In vitro culture and secondary metabolite isolation in bryophytes.

Bryophytes, the second largest group of land plants, are extremely rich in terpenoids, phenols, glycosides, and fatty acids. Although bryophytes could be used in medicine, their chemistry is not very well known and the problem remains to obtain enough quantity of same species for analysis. In vitro cultivation of bryophytes is the most appropriate way for large biomass production and isolate of numerous useful compounds showing some interesting biologic activities. This technique is also useful in developmental, cellular, molecular, biochemical, and eco-physiologic studies.

Antimicrobial activity of Bryum argenteum.

The antimicrobial activity of Bryum argenteum ethanol extracts was evaluated by microdilution method against four bacterial (Escherichia coli, Bacillus subtilis, Micrococcus luteus and Staphilococcus aureus) and four fungal species (Aspergillus niger, Penicillium ochrochloron, Candida albicans and Trichophyton mentagrophyes). All the investigated ethanol extracts have been proved to be active against all bacteria and fungi tested.

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.

In vitro micropropagation of rare and endangered moss Entosthodon hungaricus (Funariaceae) / Micropropagação in vitro do musgo raro Entosthodon hungaricus, ameaçado de extinção (Funariaceae)

The moss Entosthodon hungaricus (Boros) Loeske is an European endemic species typical of dry and saline soils extending from the Iberian Peninsula to Aral-Caspian steppes, similarly to some other xerothermic bryophytes. However, the distribution range is fragmented and localities are quite scattered and the species is considered as rare and vulnerable because of its ephemeral characteristics and specialized ecology. With the aim to develop an active protection plan for this species, the ex situ conservation requirements of E. hungaricus were developed. The axenic culture in in vitro conditions were established, and the optimal growth parameters were adjusted to achieve fully developed gametophytes ready to be reintroduced to its native range and other potentially native areas, where this species was once reported but has not been collected in recent times, suggesting its local extinction (i.e. some areas in Vojvodina, N. Serbia). Starting materials were derived from recent herbarium specimens and from fresh materials collected from Hungarian populations. Several means for sterilization of stating material and growing nutritive media were assayed in different regimes of light and temperature. Here we describe the conditions to achieve full plant development and for its micropropagation. Such materials are adequate for ex situ conservation purposes and for experimental introductions in native and potentially native areas. The first axenical culture of E. hungaricus was successfully established, and the first in vitro micropropagation of this rare and endangered species was achieved. Our study contributes to the conservation biology as well as for the potential use of this moss species in biotechnological research.

O musgo Entosthodon hungaricus (Boros) Loeske é uma espécie endêmica Européia típica de solos secos e salinos que se estendem da Península Ibérica até as estepes Aral-Cáspias, similar a outras briófitas de clima seco. Entretanto, a distribuição é bastante dispersa e fragmentada e a espécie é considerada muito rara e vulnerável devido às suas características efêmeras e ecologia especializada. Com o intuito de desenvolver um plano de proteção a essa espécie, foram elaborados os requisitos de preservação ex situ das E. hungaricus. As condições para a cultura axênica in vitro foram estabelecidas e os parâmetros ideais de crescimento foram atingidos para conseguir gametófitos completamente desenvolvidos, prontos para serem reintroduzidos em suas áreas nativas e em outras áreas potencialmente nativas, onde essa espécie já foi relatada. Porém, não houve coleta da mesma nos últimos anos, o que sugere uma extinção local (por exemplo, algumas áreas em Voivodina, Norte da Sérvia). Os materiais iniciais foram derivados de espécies recentes de herbários e de materiais frescos coletados de populações Húngaras. Várias formas de assepsia do material inicial e dos meios de crescimento nutritivo foram ensaiadas em diferentes regimes de luz e temperatura. No trabalho descrevemos as condições para obter desenvolvimento completo da planta e sua micropropagação. Os materiais são adequados para os fins de conservação ex situ e para as introduções experimentais em áreas nativas e/ou potencialmente nativas. O estudo contribui para a conservação biológica bem como para o potencial uso dessa espécie de musgo em pesquisas biotecnológicas.

The Arabidopsis elch mutant reveals functions of an ESCRT component in cytokinesis.

Recently, an alternative route to the proteasomal protein-degradation pathway was discovered that specifically targets transmembrane proteins marked with a single ubiquitin to the endosomal multivesicular body (MVB) and, subsequently, to the vacuole (yeast) or lysosome (animals), where they are degraded by proteases. Vps23p/TSG101 is a key component of the ESCRT I-III machinery in yeast and animals that recognizes mono-ubiquitylated proteins and sorts them into the MVB. Here, we report that the Arabidopsis ELCH (ELC) gene encodes a Vps23p/TSG101 homolog, and that homologs of all known ESCRT I-III components are present in the Arabidopsis genome. As with its animal and yeast counterparts, ELC binds ubiquitin and localizes to endosomes. Gel-filtration experiments indicate that ELC is a component of a high-molecular-weight complex. Yeast two-hybrid and immunoprecipitation assays showed that ELC interacts with Arabidopsis homologs of the ESCRT I complex. The elc mutant shows multiple nuclei in various cell types, indicating a role in cytokinesis. Double-mutant analysis with kaktus shows that increased ploidy levels do not influence the cytokinesis effect of elc mutants, suggesting that ELC is only important during the first endoreduplication cycle. Double mutants with tubulin folding cofactor a mutants show a synergistic phenotype, suggesting that ELC regulates cytokinesis through the microtubule cytoskeleton.