Liverwort Conocephalum Conicum Research Articles

Chemical Composition of Salvia fruticosa Mill. Essential Oil and Its Protective Effects on Both Photosynthetic Damage and Oxidative Stress in Conocephalum conicum L. Induced by Environmental Heavy Metal Concentrations.

The genus Salvia L., belonging to the Lamiaceae family, contains more than 900 species distributed in various parts of the world. It is a genus containing aromatic plants used both in the culinary field and above all in the cosmetic area to produce several perfumes. Salvia fruticosa Mill., notoriously known as Greek Salvia, is a plant used since ancient times in traditional medicine, but today cultivated and used in various parts of Europe and Africa. Polar and apolar extracts of this plant confirmed the presence of several metabolites such as abietane and labdane diterpenoids, triterpenoids, steroids, and some flavonoids, causing interesting properties such as sedative, carminative, and antiseptic, while its essential oils (EOs) are mainly characterized by compounds such as 1,8-cineole and camphor. The aim of this work concerns the chemical analysis by GC and GC-MS, and the investigation of the biological properties, of the EO of S. fruticosa plants collected in eastern Sicily. The gas-chromatographic analysis confirmed the presence of 1,8-cineole (17.38%) and camphor (12.81%), but at the same time, also moderate amounts of α-terpineol (6.74%), β-myrcene (9.07%), camphene (8.66%), β-pinene (6.55%), and α-pinene (6.45%). To study the protective effect of EOs from S. fruticosa (both the total mixture and the individual compounds) on possible damage induced by heavy metals, an in vitro system was used in which a model organism, the liverwort Conocephalum conicum, was subjected to the effect of a mix of heavy metals (HM) prepared using values of concentrations actually measured in one of the most polluted watercourses of the Campania region, the Regi Lagni. Finally, the antioxidant response and the photosynthetic damage were examined. The exogenous application of the EO yields a resumption of the oxidative stress induced by HM, as demonstrated by the reduction in the Reactive Oxygen Species (ROS) content and by the increased activity of antioxidant enzyme catalase (CAT) and glutathione-S-transferase (GST). Furthermore, plants treated with HMs and EO showed a higher Fv/Fm (maximal quantum efficiency of PSII in the dark) with respect to HMs-only treated ones. These results clearly indicate the protective capacity of the EO of S. fruticosa against oxidative stress, which is achieved at least in part by modulating the redox state through the antioxidant pathway and on photosynthetic damage.

Tissue localization and the physiological effects induced by an environmentally relevant mix of heavy metals in the liverwort Conocephalum conicum L. Dum

Tissue accumulation, ultrastructural alterations, oxidative stress, and effects on photosynthesis were assessed in the liverwort Conocephalum conicom exposed in vitro to heavy metals (HM) concentrations in three sites of the Savone River, representative of different anthropic impacts. The uptake and accumulation of HM in the thallus of the liverwort was first studied, and the biological effects in relation to the different accumulation sites of HM along thallus, ribs and wings, were then investigated, considering: bioaccumulation (by atomic absorption spectrometry), localization (by X-ray scanning electron microscopy microanalysis), ultrastructural damage of photosynthetic parenchyma (by transmission electron microscopy), oxidative stress (by ROS contents and antioxidant enzymes activities determination), photosynthesis (by chlorophyll fluorescence). The results showed the HM bioaccumulation in C. conicom was dependent by their concentrations in the contaminated water. As for spatial localization, HM preferentially accumulated in the nerve of gametophytes respect to the wings. With respect to tissue localization, HM were mainly found in the hyaline and in the photosynthetic parenchyma. Essential metals (Cu and Zn) were accumulated at higher concentrations with respect to non-essential metals (Pb and Cd). At the ultrastructural level, HM caused alterations of the fine structure of the cells, most evident along the nerve, inducing marked alterations of the chloroplast structure and therefore of the photosynthetic capacity. Based on the results of the presented study, C.conicum can be used as a marker to indicate heavy metal pollution in water natural resources.

Heavy metal stress induces adaptative responses in the liverwort Conocephalum conicum L. (Dum.): An integrated biologic and metabolomic study

Bioaccumulation, ultrastructural and DNA damage, oxidative stress, and metabolomic analyses were carried out in vitro on the liverwort Conocephalum conicum L. in response to the heavy metal concentrations measured in two sites along the Sarno river (C1 and C2) representative of two different environmental conditions. Bioaccumulation and bioconcentration data confirm C. conicum is a good bioindicator. Our TEM observations showed typically preserved ultrastructure of the chloroplasts in C1 samples, nearly identical to the chloroplasts of the control sample (CTRL; freshly collected and not exposed), whereas in C2 samples they appeared misshaped, swollen and with poorly developed thylakoids in a rather clear stroma. DNA damage, antioxidant enzymatic activity, ROS production and localization showed no difference between CTRL and C1 samples, instead significant differences between C1 and C2 samples. In addition, in this study, we used for the first time the untargeted MS-based molecular networking to examine the role of heavy metal stress in the differential production of the secondary metabolites (SM) in C. conicum. Metabolome analysis evidenced no changes between CTRL and C1 samples, instead a dramatic change in SM production under metal stress conditions, in particular a remodeling of the lipid metabolism through the production of members of betaine and oxidated lipid classes, as well as production of flavonoids was observed. All the occurred variations respond consistently with the heavy metal concentration trend so we can propose them as pollution biomarkers.

Immunomodulatory Constituents of Conocephalum conicum (Snake Liverwort) and the Relationship of Isolepidozenes to Germacranes and Humulanes.

Structural elucidation of three new sesquiterpenoids, namely, (1Z,4E)-lepidoza-1(10),4-dien-14-ol (1), rel-(1(10)Z,4S,5E,7R)-germacra-1(10),6 diene-11,14-diol (2), and rel-(1(10)Z,4S,5E,7R)-humula-1(10),5-diene-7,14-diol (3), isolated from the liverwort Conocephalum conicum, was accomplished by a combination of extensive NMR experiments, 1H NMR simulation, and other means. Additionally, the change of the identity of bicyclogermacren-14-al, previously reported as a C. conicum constituent, to isolepidozen-14-al is proposed. Compounds 2 and 3 appear to be related to 1 via hydration involving a shared intermediate, a substituted cyclopropylmethyl cation, formed by a highly regio- and stereoselective protonation of 1, followed by a stereospecific fission of the three-membered ring. In other words, an isolepidozene derivative might be a branchpoint to humulanes and germacranes; this transformation could be of, up to now, unknown, biosynthetic and/or synthetic relevance. Multivariate statistical analysis of the compositional data of C. conicum extract constituents was used to probe the hypothesized biochemical relations. The immunomodulatory effect of 1-3 and conocephalenol (4) was evaluated in an in vitro model on both nonstimulated and mitogen-stimulated rat splenocytes. The compounds displayed varying degrees of cytotoxicity to nonstimulated splenocytes, whereas 2 and 3 were found to exert immunosuppressive effects on concanavalin A-stimulated splenocytes while not being cytotoxic at the same concentrations.

Biological Responses to Cadmium Stress in Liverwort Conocephalum conicum (Marchantiales).

Oxidative damage (production and localization of reactive oxygen species) and related response mechanisms (activity of antioxidant enzymes), and induction of Heat Shock Protein 70 expression, have been studied in the toxi-tolerant liverwort Conocephalum conicum (Marchantiales) in response to cadmium stress using two concentrations (36 and 360 µM CdCl2). Cadmium dose-dependent production of reactive oxygen species (ROS) and related activity of antioxidant enzymes was observed. The expression level of heat shock protein (Hsp)70, instead, was higher at 36 µM CdCl2 in comparison with the value obtained after exposure to 360 µM CdCl2, suggesting a possible inhibition of the expression of this stress gene at higher cadmium exposure doses. Biological responses were related to cadmium bioaccumulation. Since C. conicum was able to respond to cadmium stress by modifying biological parameters, we discuss the data considering the possibility of using these biological changes as biomarkers of cadmium pollution.

The role of vacuolar ion channels in salt stress tolerance in the liverwort Conocephalum conicum

Liverworts are pioneer plants that colonized lands. They had to cope with frequent sea water flooding causing salt stress. The role of vacuoles and in particular slow-activating (SV) channels in the salt stress tolerance was addressed in the present study. A patch-clamp method was used to study sodium fluxes through the tonoplast of the liverwort Conocephalum conicum. The whole-vacuole measurements carried out in a symmetrical Na+ concentration allowed recording of slowly activated outward currents typical for SV channels. In a Na+ gradient promoting an efflux of Na+ from the vacuole, the outward rectifying properties of SV channels were reduced and inward Na+ currents with different inactivation rates were recorded. Single channel analysis proved that a decrease in cytoplasmic Na+ concentration evoked an increase in the open probability of the channels and shifted the activation voltages towards negative values. The number of SV channels recorded at negative voltages was dependent on the vacuolar calcium and decreased at the high concentration of this ion in the vacuole. In some of the tested patches, the channels exhibited a flickering type of activity and two different conductance levels. The role of SV channels in Na+ accumulation during salt stress and its removal after periods of flooding is discussed in the present paper.

Evolutionary Roots of Plant Microbiomes and Biogeochemical Impacts of Nonvascular Autotroph-Microbiome Systems over Deep Time

Since rooted vascular plants rose to dominance, associated microbiota have powerfully influenced global biogeochemistry by mobilizing N and P and otherwise aiding plant health, thereby fostering sequestration of CO2 into coal and soil organics. Less well understood are evolutionary history and biogeochemical roles of microbiomes of ecologically significant nonvascular plants and related algae, whose lineages penetrate more deeply into time. Because analyses of diverse host-microbiome systems indicate that evolutionary history commonly influences microbiome composition and function, we mapped onto a geological time frame biogeochemical features inferred in our previous metagenomic studies of a phylogenetic spectrum of multicellular, freshwater-terrestrial, nonvascular autotroph-microbiome systems: Nostoc commune, representing microbialite-forming cyanobacteria; the Nostoc lichen Peltigera ponojensis; chlorophyte Cladophora glomerata; streptophyte alga Coleochaete pulvinata; moss Sphagnum fimbriatum; and liverwort Conocephalum conicum. Widespread and often abundant today, these species represent ancient lineages of minimal ages ranging from 1450 to 380 Ma, information we used to estimate present global annual magnitudes of organic/inorganic C sequestration, N fixation, and methane oxidation and to calculate biogeochemical impacts during periods from lineage origin to the rise of vascular plants and from lineage origins to present. Functional comparisons indicated that (1) functionally diverse bacterial associates have likely enhanced global C- and N-cycle roles of cyanobacteria for ∼1.5 billion years; (2) by 700–800 Ma, chlorophyte microbiomes retaining ancient functional features (e.g., vitamin B12 biosynthesis and N fixation) had added diverse methane-oxidizing bacteria and eukaryotes, including early metazoans; (3) by ∼500 Ma, streptophyte algal microbiomes possessing ancient functions had incorporated early-diverging fungi; (4) by 450 Ma, microbiomes of early plants included diverse prokaryotes, protists, fungi, and early terrestrial animals; and (5) by 385 Ma, plant microbiomes included P-mobilizing fungi. Autotroph-microbiome systems appear to have accreted functions of global significance over deep time, a new concept that illuminates the early evolution of terrestrial life and that aids modeling past, present, and future biogeochemical impacts.

Microbiomes of Streptophyte Algae and Bryophytes Suggest That a Functional Suite of Microbiota Fostered Plant Colonization of Land

Premise of research. The origin of land plants catalyzed key changes in Earth’s atmosphere and biota. Microbial associations likely nurtured earliest plants and influenced their biogeochemical roles. Because angiosperm and animal microbiomes—bacteria, archaea, microbial eukaryotes, and genes that promote host survival—are known to display lineage effects, we hypothesized that microbiomes of early-diverging modern bryophytes and phylogenetically closely related green algae might likewise reveal commonalities reflecting ancestral traits.Methodology. New metagenomic sequence data were obtained for the late-diverging streptophyte algae Chaetosphaeridium globosum and Coleochaete pulvinata and the liverwort Conocephalum conicum, representing early-diverging land plants. New 16S rDNA amplicon sequences were acquired for the charalean Nitella tenuissima. Sequence data were used to infer bacterial genera and fungi for comparisons among streptophyte microbiota and with our published microbiome data for the outgroup...

Phytosociological Analysis Of Communities With Adiantum Capillusveneris In The Foothills Of The Julian Alps (Western Slovenia)

Abstract We conducted a phytosociological study of the communities hosting the rare and endangered fern Adiantum capillus-veneris in the foothills of the Julian Alps, in Karst and in Istria. Based on a comparison with similar communities elsewhere in the southern Alps (northern Italy) we classified most of the recorded stands into the syntaxa Eucladio-Adiantetum eucladietosum and -cratoneuretosum commutati. Releves from the southern Julian Alps, located in comparatively slightly colder and moister local climate and the dolomite bedrock are classified into the new subassociation -hymenostylietosum recurvirostri subass. nova. Stands with the abundant occurrence of the liverwort Conocephalum conicum, are classified in to the new subassociation -conocephaletosum conici subass. nova. Stands in conglomerate rock shelters along the Soča at Solkan are classified into the new association Phyteumato columnae-Adiantetum ass. nova, a community of transitional character between the classes Adiantetea capilli-veneris and Asplenietea trichomanis.

Menthol-induced action potentials in Conocephalum conicum as a result of unspecific interactions between menthol and the lipid phase of the plasma membrane.

Our previous study has shown that the liverwort Conocephalum conicum generates action potentials (APs) in response to both temperature drop and menthol, which are also activators of the TRPM8 (transient receptor potential melastatin 8) receptor in animals. Not only similarities but also differences between electrical reactions to menthol and cooling observed in the liverwort aroused our interest in the action of menthol at the molecular level. Patch-clamp investigations have shown that menthol causes a reduction of current flowing through slow vacuolar (SV) channels to 29 ± 10% of the initial value (n = 9); simultaneously, it does not influence magnitudes of currents passing through a single SV channel. This may point to an unspecific interaction between menthol and the lipid phase of the membrane. An influence of menthol on lipid organization in membranes was investigated in two-component monomolecular layers formed with menthol and dipalmitoylphosphatidylcholine (DPPC) at the argon-water interface. Analyses of the mean molecular area parameters vs the molar fraction of the menthol component have shown over-additivity (approximately 20 Å(2) ) in the region of high molar fractions of menthol. Infrared absorption spectroscopy studies have shown that menthol, most probably, induces breaking of a hydrogen bond network formed by ester carbonyl groups and water bridges in the lipid membrane and binds to the polar head group region of DPPC. We conclude that the disruption in the lipid phase of the membrane influences ion channels and/or pumps and subsequently causes generation of APs in excitable plants such as C. conicum.

Insecticidal Activity of Fatty Acid-Rich Turkish Bryophyte Extracts Against Sitophilus granarius (Coleoptera: Curculionidae)

The composition of fatty acids and insecticidal effects was performed for the Turkish mosses Dicranum scoparium, Hypnum cupressiforme, Polytrichastrum formosum, Homalothecium lutescens and the Turkish liverwort Conocephalum conicum. All structures were determined by means of gas chromatography and gas chromatography-mass spectrometry techniques. The determination of fatty acids was done using a simple and mild method that utilized different solvent extractions ranging from nonpolar to polar solvents (hexane, dichloromethane, chloroform, ethyl acetate and methanol, respectively), and the samples were powdered with and without liquid nitrogen. The correlations between the saturated and unsaturated fatty acid contents depending on the solvent polarity and their crushing process by liquid nitrogen were observed. The insecticidal activity of the bryophytes was analyzed by using the methanol, hexane and esterified methanol extracts. The hexane extracts of Polytrichastrum formosum showed the highest insecticidal activity (70.33%) against Sitophilus granarius. Contact toxicity activities of lauric, myristic and palmitic acids besides single dose studies of the solvent extracts were carried out. The highest mortality rate (53.34%) was obtained from the myristic acid among the tested pure fatty acids. The activities of palmitic and lauric acids were 17.75% and 4.32%, respectively.

Characteristics of quercetin interactions with liposomal and vacuolar membranes

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is claimed to exert many beneficial health effects. With application of 1H NMR (nuclear magnetic resonance) and FTIR (Fourier-transform infrared) techniques, quercetin interaction with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was analyzed. Patch-clamp technique was employed to study quercetin effects at single channel level of vacuolar membranes in the liverwort Conocephalum conicum. Light and electron microscopy were applied to study quercetin effects on human negroid cervix carcinoma cells (HeLa). Enzymatic measurements along with DPPH (1,1-diphenyl-2-picrylhydrazyl) bioassay were performed to investigate the influence of quercetin on antioxidant enzymes and reactive oxygen species (ROS) production. The inclusion of quercetin to the membrane exerted pronounced ordering effect on the motional freedom of lipids in the head group region as manifested by broadening of the 1H NMR spectral line representing the choline groups. FTIR analysis revealed quercetin incorporation into DPPC liposomes via hydrogen bonding between its own hydroxyl groups and lipid polar head groups in the C–O–P–O–C segment. Both, FTIR and NMR techniques indicated also quercetin spectral effects in the region corresponding to alkyl chains. Patch-clamp experiments showed that quercetin stabilizes tonoplast and promotes a close state of SV channels. Microscopic observations of HeLa cells revealed characteristic changes in ultrastructure and morphology of the examined cells in comparison to control cells. Pretreatment of HeLa cells with quercetin alleviated H2O2-induced cell injury by improving redox balance as indicated by the increase in glutathione content and SOD (superoxide dismutase) levels as well as by the decrease in ROS level. \\In conclusion, the incorporation, distribution and the changes of biophysical properties of the membranes are very important for the effectiveness of phenolic compounds as antioxidant and anticancer factors.

Identification of cryptic species within liverwort Conocephalum conicum based on the volatile components

Cryptic speciation refers to cases where species are clearly genetically differentiated, but show little or no morphological differences. This study investigated whether volatile components can be used to distinguish the cryptic species within the Conocephalum conicum complex. Thirteen samples were included in this study representing four cryptic species (A, F, J, L) and the recently described Conocephalum salebrosum (formerly cryptic species S) detected in a worldwide collection. Results received from GC-MS analyses showed some differences between samples, indicating the existence of chemical polymorphism. It is evident from both statistical analyses (CA and PCA), that cryptic species L appears to be the most differentiated species, with the volatile components not significantly shared with the rest of the types. The most characteristic compound of this type is the sesquiterpene alcohol, conocephalenol. Distinction between other examined cryptic species were also indicated. However species F and J, as well as species A and C. salebrosum, share a great similarity in their volatile composition and are grouped together in both CA and PCA. Despite their similarities, we were able to find chemical markers which can describe each species. Cubebol is characteristic for C. salebrosum, while cryptic species A produces (E)-methylcinnamate. The presence of a large amount of the monoterpene hydrocarbon, sabinene, is characteristic for cryptic species J. In one of the analyzed specimens belonging to the cryptic species F, a large amount of cyclocolorenone was detected. Chemical differentiation was correlated with the geographical distribution of the analyzed samples.

Electrophysiological approach to examine a putative cold- and menthol-sensitive channel in the liverwort Conocephalum conicum

The mechanism of cold perception by plants is still poorly understood. It was found that temperature drop evokes changes in the activity of ion pumps and channels, which leads to plasma membrane depolarization.1, 2 The nature of the primary step of its action (alteration in membrane composition,3 transient influx of Ca2+ etc.2) has not been elicited yet. Our electrophysiological experiments conducted on the liverwort Conocephalum conicum showed that its cells respond not only to sudden cooling4 but also to menthol, generating depolarization of the plasma membrane and action potentials (APs). Similar results are well documented in mammals; cold or “cooling compounds” including menthol cause activation of thermosenstitive channel TRPM8 permeable to Ca2+ and generation of AP series.5 TRP receptors are detected, among others, in green and brown algae. Possible existence of TRPM8-like channel-receptor in Conocephalum conicum is discussed here.

Allopatric distribution and diversification without niche shift in a bryophyte-feeding basal moth lineage (Lepidoptera: Micropterigidae)

The Lepidoptera represent one of the most successful radiations of plant-feeding insects, which predominantly took place within angiosperms beginning in the Cretaceous period. Angiosperm colonization is thought to underlie the evolutionary success of the Lepidoptera because angiosperms provide an enormous range of niches for ecological speciation to take place. By contrast, the basal lepidopteran lineage, Micropterigidae, remained unassociated with angiosperms since Jurassic times but nevertheless achieved a modest diversity in the Japanese Archipelago. We explored the causes and processes of diversification of the Japanese micropterigid moths by performing molecular phylogenetic analysis and extensive ecological surveying. Phylogenetic analysis recovered a monophyletic group of approximately 25 East Asian endemic species that feed exclusively on the liverwort Conocephalum conicum, suggesting that niche shifts hardly played a role in their diversification. Consistent with the low flying ability of micropterigid moths, the distributions of the Conocephalum specialists are each localized and allopatric, indicating that speciation by geographical isolation has been the major process shaping the diversity of Japanese Micropterigidae. To our knowledge, this is the largest radiation of herbivorous insects that does not accompany any apparent niche differentiation. We suggest that the significance of non-ecological speciation during the diversification of the Lepidoptera is commonly underestimated.

Effect of cold and menthol on membrane potential in plants

In animals, cooling substances such as menthol are perceived as cold sensation because they bind to the same receptor TRPM8 (transient receptor potential melastatin) that activates upon temperature drops. We investigated the effect of menthol on the plant membrane potential to search for analogies between animal and plant perception systems. The study was conducted on the liverwort Conocephalum conicum- a non-vascular plant generating action potentials (APs) in response to different stimuli including cold. (+)Menthol, (-)menthol and (+/-)menthol induced one or more APs, depending on the concentration. In contrast to animal reactions to menthol, threshold concentrations of these isomers were the same (1 mM). The presence of menthol in medium shortened cold-induced APs, whereas low temperature prolonged the repolarization phase of AP evoked by menthol. Cells of C. conicum with anion and potassium channels blocked by anthracene-9-carboxylic acid (A9C) and tetraethylammonium chloride (TEACl) generate short spike-like voltage transients (VTs) in response to cold and light stimulation. Membrane potential changes evoked by menthol in A9C- and TEACl-treated plants differed significantly from VTs - lasted much longer and frequently occurred in series. 5 mM LaCl(3) , 1 mM EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (0 Ca(2+) ) but not 0.2 mM verapamil blocked the putative calcium component of AP induced by menthol. Similar inhibitory effect was observed after the application of proton pump inhibitors: 0.05 mM N,N-dicyclohexylcarbodiimide (DCCD), 0.05 mM diethylstilbestrol (DES) or 0.01 mM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). Our results indicate that cold and menthol act independently, activating different membrane transporters in C. conicum cells.

Genistein and changes of resting and action potentials in Conocephalum conicum

Genistein (4',5,7-trixydroxyflavone) is a member of the family of plant flavonoids that widely occurs in crop plants. It is involved in a wide spectrum of pharmacological activities, and is suggested to have anti-cancer dietary properties. Cell membranes are one of the targets of anti-cancer drugs. In the present study, we used the liverwort Conocephalum conicum as a model plant in an electrophysiological study. Intracellular microelectrode measurements were carried out to examine the effects of genistein alone and in combination with verapamil on resting and action potentials. The application of isoflavone genistein resulted in a statistically significant elevation in action potential amplitudes. An increase of 13-62% compared with the control was noted. An increase was also found in the membrane resting potentials in genistein-treated plants. Verapamil, the known calcium channel inhibitor, caused a gradual decline of AP amplitudes, whereas preincubation of Conocephalum thalli with genistein prevented inhibition of APs by verapamil. It is concluded that genistein strongly affects the membranes, and the effect of genistein in canceling the activity of verapamil is discussed.

Vacuolar ion channels in the liverwort Conocephalum conicum

As a liverwort Conocephalum conicum belongs to the oldest terrestrial plants1 and is phylogenetically located between green algae and higher plants. Recent patch-clamp recordings on Conocephalum vacuoles demonstrate ion channels very similar to higher plants and clearly different from vacuolar ion channels described in green algae. Here we summarize the features of a vacuolar cation channel and a vacuolar anion channel that both are common in terrestrial plants but are not detected in green algae, and we speculate about the molecular identity of these channels in the liverwort Conocephalum.

Characteristics of Anion Channels in the Tonoplast of the Liverwort Conocephalum conicum

Isolated vacuoles of the liverwort Conocephalum conicum thallus cells were investigated using the patch-clamp technique. At high cytosolic Ca(2+) activities, slowly activating currents were evoked by positive potentials. The currents were conducted by the SV (slow-vacuolar) channel. When isolation of vacuoles was carried out at high Mg(2+) and low Ca(2+) concentration and the same proportion of the cations was kept in the bath, currents were recorded at negative potentials. Once activated, these currents persisted even after replacing Mg(2+) with K(+) in the bath. Sr(2+) and Ba(2+) were also effective activators of the currents. With a Cl(-) gradient, 10 mM in the bath and 100 mM in the lumen, currents were significantly reduced and the current-voltage characteristics shifted towards the reversal potential of Cl(-), indicating Cl(-) selectivity. Currents almost vanished after substituting Cl(-) with gluconate. They were strongly reduced by anion channel inhibitors 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (A9C; 2 mM) and ethacrinic acid (0.5 mM). Single-channel recordings revealed a 32 pS channel activating at negative voltages. It is concluded that the currents at negative potentials are carried by anion channels suitable for conducting anions from the cytosol to the vacuole. The anion channels were weakly calcium dependent, remaining active at physiological calcium concentration. The channels were almost equally permeable to Cl(-), NO3(-) and SO4(2-), and much less permeable to malate(2-). Anion channels did not respond to ATP addition. cAMP (10 microM) had a weak effect on anion channels. Protein kinase A (0.4 U) added to the medium caused no significant effect on anion channels.

The influence of apigenin on membrane and action potential in the liverwort Conocephalum conicum

Apigenin (4′,5,7-trixydroxyflavone) is a member of the family of plant flavonoids considered to prevent a number of human diseases, for instance cancer development. It displays a lot of activities and part of its beneficial effects could come from its affinity to the cellular membranes. In the present study we used the liverwort Conocephalum conicum, a model plant in electrophysiological study. Intracellular microelectrode measurements were carried out to examine the effects of apigenin alone and in combination with verapamil on the resting and action potentials. The application of apigenin caused an increase of action potential amplitudes. An increase even by 110–131% with respect to the control was observed. Little increase was also found in the membrane potentials in apigenin treated plants. Verapamil, the known calcium channel inhibitor, caused gradual decline of AP amplitudes. When apigenin was used simultaneously with verapamil, still high APs were observed. Duration of action potentials amplitudes measuerd at a half of the amplitude decreased in either apigenin or apigenin and verapamil treated plants to 56–62% of the control. It is concluded that apigenin strongly affects the membranes and prevents inhibitory effect of verapamil probably interacting with calcium channel protein.