Rapid Propagation of Ca2+ Waves and Electrical Signals in the Liverwort Marchantia polymorpha.

In response to both biotic and abiotic stresses, vascular plants transmit long-distance Ca2+ and electrical signals from localized stress sites to distant tissues through their vasculature. Various models have been proposed for the mechanisms underlying the long-distance signaling, primarily centered around the presence of vascular bundles. We here demonstrate that the non-vascular liverwort Marchantia polymorpha possesses a mechanism for propagating Ca2+ waves and electrical signals in response to wounding. The propagation velocity of these signals was approximately 1-2 mm s-1, equivalent to that observed in vascular plants. Both Ca2+ waves and electrical signals were inhibited by La3+ as well as tetraethylammonium chloride, suggesting the crucial importance of both Ca2+ channel(s) and K+ channel(s) in wound-induced membrane depolarization as well as the subsequent long-distance signal propagation. Simultaneous recordings of Ca2+ and electrical signals indicated a tight coupling between the dynamics of these two signaling modalities. Furthermore, molecular genetic studies revealed that a GLUTAMATE RECEPTOR-LIKE (GLR) channel plays a central role in the propagation of both Ca2+ waves and electrical signals. Conversely, none of the three two-pore channels were implicated in either signal propagation. These findings shed light on the evolutionary conservation of rapid long-distance Ca2+ wave and electrical signal propagation involving GLRs in land plants, even in the absence of vascular tissue.

Immobilisation of Cellobiose Dehydrogenase and Laccase on Chitosan Particles as a Multi-Enzymatic System for the Synthesis of Lactobionic Acid.

Lactobionic acid (LBA) is a bioactive compound that has become increasingly popular in medicine in recent years due to its unique properties. This chemical can be formed via the enzymatic oxidation of lactose using fungal oxidoreductive enzymes. This study aimed to intensify the synthesis of LBA using immobilised enzymes (cellobiose dehydrogenase from Phanerochaete chrysosporium (PchCDH) and laccase from Cerrena unicolor (CuLAC)) on chitosan microspheres. We used three different crosslinking agents: genipin, glutaraldehyde, and polyethyleneimine to activate the chitosan. The FTIR and CellDrop techniques were used to characterise the activated microspheres. Quantitative (HPLC) and qualitative (TLC) methods were used to determine the obtained LBA. The results show that the type of activator used influences the efficiency of the binding of the enzyme to the matrix. Furthermore, the amount of LBA formed depends on the type of system used. The use of a system in which one of the enzymes is immobilised on a PEI-activated carrier (PchCDH) and the other is free (CuLAC) proved to be the most optimal, as it yielded almost 100% conversion of lactose to lactobionic acid. Summarising the data obtained the following: lactobionic acid immobilised on chitosan microspheres has great potential for medical applications.

Long-Distance Electrical and Calcium Signals Evoked by Hydrogen Peroxide in Physcomitrella.

Electrical and calcium signals in plants are some of the basic carriers of information that are transmitted over a long distance. Together with reactive oxygen species (ROS) waves, electrical and calcium signals can participate in cell-to-cell signaling, conveying information about different stimuli, e.g. abiotic stress, pathogen infection or mechanical injury. There is no information on the ability of ROS to evoke systemic electrical or calcium signals in the model moss Physcomitrella nor on the relationships between these responses. Here, we show that the external application of hydrogen peroxide (H2O2) evokes electrical signals in the form of long-distance changes in the membrane potential, which transmit through the plant instantly after stimulation. The responses were calcium-dependent since their generation was inhibited by lanthanum, a calcium channel inhibitor (2 mM), and EDTA, a calcium chelator (0.5 mM). The electrical signals were partially dependent on glutamate receptor (GLR) ion channels since knocking-out the GLR genes only slightly reduced the amplitude of the responses. The basal part of the gametophyte, which is rich in protonema cells, was the most sensitive to H2O2. The measurements carried out on the protonema expressing fluorescent calcium biosensor GCaMP3 proved that calcium signals propagated slowly (>5 µm/s) and showed a decrement. We also demonstrate upregulation of a stress-related gene that appears in a distant section of the moss 8 min after the H2O2 treatment. The results help understand the importance of both types of signals in the transmission of information about the appearance of ROS in the plant cell apoplast.

Multi-Enzymatic Synthesis of Lactobionic Acid Using Wood-Degrading Enzymes Produced by White Rot Fungi.

Enzymes produced by white rot fungi are involved in the synthesis of secondary metabolites with valuable biotechnological properties. One of these metabolites is lactobionic acid (LBA). The aim of this study was to characterize a novel enzyme system consisting of a cellobiose dehydrogenase from Phlebia lindtneri (PlCDH), a laccase from Cerrena unicolor (CuLAC), a redox mediator (ABTS or DCPIP), and lactose as a substrate. We used quantitative (HPLC) and qualitative methods (TLC, FTIR) to characterise the obtained LBA. The free radical scavenging effect of the synthesised LBA was assessed with the DPPH method. Bactericidal properties were tested against Gram-negative and Gram-positive bacteria. We obtained LBA in all the systems tested; however, the study showed that the temperature of 50 °C with the addition of ABTS was the most advantageous condition for the synthesis of lactobionic acid. A mixture with 13 mM LBA synthesised at 50 °C with DCPIP showed the best antioxidant properties (40% higher compared with the commercial reagent). Furthermore, LBA had an inhibitory effect on all the bacteria tested, but the effect was better against Gram-negative bacteria with growth inhibition no lower than 70%. Summarizing the obtained data, lactobionic acid derived in a multienzymatic system is a compound with great biotechnological potential.

Impact of Mammalian Two-Pore Channel Inhibitors on Long-Distance Electrical Signals in the Characean Macroalga Nitellopsis obtusa and the Early Terrestrial Liverwort Marchantia polymorpha.

Inhibitors of human two-pore channels (TPC1 and TPC2), i.e., verapamil, tetrandrine, and NED-19, are promising medicines used in treatment of serious diseases. In the present study, the impact of these substances on action potentials (APs) and vacuolar channel activity was examined in the aquatic characean algae Nitellopsis obtusa and in the terrestrial liverwort Marchantia polymorpha. In both plant species, verapamil (20-300 µM) caused reduction of AP amplitudes, indicating impaired Ca2+ transport. In N. obtusa, it depolarized the AP excitation threshold and resting potential and prolonged AP duration. In isolated vacuoles of M. polymorpha, verapamil caused a reduction of the open probability of slow vacuolar SV/TPC channels but had almost no effect on K+ channels in the tonoplast of N. obtusa. In both species, tetrandrine (20-100 µM) evoked a pleiotropic effect: reduction of resting potential and AP amplitudes and prolongation of AP repolarization phases, especially in M. polymorpha, but it did not alter vacuolar SV/TPC activity. NED-19 (75 µM) caused both specific and unspecific effects on N. obtusa APs. In M. polymorpha, NED-19 increased the duration of repolarization. However, no inhibition of SV/TPC channels was observed in Marchantia vacuoles, but an increase in open probability and channel flickering. The results indicate an effect on Ca2+ -permeable channels governing plant excitation.

Glutamate-Induced Electrical and Calcium Signals in the Moss Physcomitrella patens.

The mode of transmission of signals between plant cells is an important aspect of plant physiology. The main role in the generation of long-distance signals is played by changes in the membrane potential and cytoplasm calcium concentration, but the relationship between these responses evoked by the same stimuli in the same plant remains unknown. As one of the first plants that colonized land, the moss Physcomitrella patens is a suitable model organism for studying the evolution of signaling pathways in plants. Here, by the application of glutamate as a stimulus, we demonstrated that electrical but not calcium signals can be true carriers of information in long-distance signaling in Physcomitrella. The generation of electrical signals in a form of propagating transient depolarization seems to be dependent on the opening of calcium channels since the responses were reduced or totally blocked by calcium channel inhibitors. While the microelectrode measurements demonstrated the transmission of electric signals between leaf cells and juvenile cells (protonema), the fluorescence imaging of cytoplasmic calcium changes indicated that calcium response occurs only locally-at the site of glutamate application, and only in protonema cells. This study indicates different involvement of glutamate-induced electrical and calcium signals in cell-to-cell communication in these evolutionarily old terrestrial plants.

Cold- and menthol-evoked membrane potential changes in the moss Physcomitrella patens: influence of ion channel inhibitors and phytohormones.

Microelectrode measurements carried out on leaf cells from Physcomitrella patens revealed that a sudden temperature drop and application of menthol evoked two types of different-shaped membrane potential changes. Cold stimulation evoked spike-type responses. Menthol depolarized the cell membrane with different rates. When it reached above 1 mV s-1 , the full response was recorded. Characteristic for the full responses was also a few-minute plateau of the membrane potential recorded after depolarization. The influence of inhibitors of calcium channels (5 mM Gd3+ ), potassium channels (5 mM Ba2+ ), chloride channels (200 µM Zn2+ , 50 µM niflumic acid) and proton pumps (10 µM DES), an activator of calcium release from intracellular stores (Sr2+ ), calcium chelation (by 400 µM EGTA) and phytohormones (50 µM auxin, 50 µM abscisic acid (ABA), 500 µM salicylic acid) on cold- and menthol-evoked responses was tested. Both responses are different in respect to the ion mechanism: cold-evoked depolarizations were influenced by Ba2+ and DES; in turn, menthol-evoked potential changes were most effectively blocked by Zn2+ . Moreover, the effectiveness of menthol in generation of full responses was reduced after administration of auxin or ABA, i.e. phytohormones known for their participation in responses to cold and regulation of proton pumps. The effects of DES indicated that one of the main conditions for generation of menthol-evoked responses is inhibition of the proton pump activity. Our results indicate that perception of cold and menthol by plants proceeds in different ways due to the differences in ionic mechanism and hormone dependence of cold- and menthol-evoked responses.

The orientation of the transition dipole moments of a polyene antibiotic Amphotericin B under UV-VIS studies.

Amphotericin B (AmB) belongs to naturally occurring fluorescent antibiotics, commonly used in the treatment of life-threatening fungal infections. Open question regarding mechanism of action of this molecule calls for its orientation and organization studies in biomembranes. Here, we present studies on linear dichroism and fluorescence polarization of AmB embedded in isotropic and oriented poly(vinyl) alcohol films to characterize their transition dipole moments to low energy excited electronic transitions S1 (2 Ag(-)) and S2 (1 Bu(+)). The dichroic ratio and fluorescence anisotropy data were analyzed for stretched PVA films doped AmB. The results show that the transition moment for absorption makes an angle φ = 27° ± 2° with the molecular axis of AmB defined by the film stretching direction. The angles between the absorption and emission transition moments have been found for both the low excited electronic states, S2 (ß = 4° ± 5°) and S1 (ß = 6° ± 5°). The fluorescence anisotropy analysis from the S2 state reveals additional component assigned to antiparallel AmB dimeric structure.

Self-association of amphotericin B: spontaneous formation of molecular structures responsible for the toxic side effects of the antibiotic.

Amphotericin B (AmB) is a lifesaving antibiotic used to treat deep-seated mycotic infections. Both the pharmaceutical activity and highly toxic side effects of the drug rely on its interaction with biomembranes, which is governed by the molecular organization of AmB. In the present work, we present a detailed analysis of self-assembly of AmB molecules in different environments, interesting from the physiological standpoint, based on molecular spectroscopy techniques: electronic absorption, circular dichroism, steady state and time-resolved fluorescence and molecular dynamic calculations. The results show that, in the water medium, AmB self-associates to dimeric structures, referred to as "parallel" and "antiparallel". AmB dimers can further assemble into tetramers which can play a role of transmembrane ion channels, affecting electrophysiological homeostasis of a living cell. Understanding structural determinants of self-assembly of AmB opens a way to engineering preparations of the drug which retain pharmaceutical effectiveness under reduced toxicity.

Molecular organization of polyene antibiotic amphotericin B studied by means of fluorescence technique.

Amphotericin B (AmB) is a polyene antibiotic used to treat deep-seated mycoses. Both the pharmaceutical and toxic activities of AmB depend on the molecular organization of the drug. The fluorescence of AmB has proven to be a powerful technique of studying the drug's association state. In particular, fluorescence lifetime appeared to be sensitive to the formation of AmB dimers and aggregated structures. This paper addresses the application of the fluorescence technique in the study of the molecular organization of AmB, and perspectives on future application of this approach are addressed briefly.

Toward understanding of toxic side effects of a polyene antibiotic amphotericin B: fluorescence spectroscopy reveals widespread formation of the specific supramolecular structures of the drug.

Amphotericin B (AmB) is a lifesaving polyene antibiotic used widely to treat deep-seated mycoses. Both the pharmaceutical effectiveness as well as toxic side effects depend on molecular organization of the drug. In the present study, we analyzed steady-state fluorescence, fluorescence anisotropy spectra, fluorescence lifetimes, and fluorescence anisotropy decays of AmB in the systems believed to ensure monomeric organization of the drug and in model lipid membranes. The results of the analyses show that in all of the systems studied, the drug appears in, at least, two spectral forms, interpreted as monomeric and aggregated. Spectroscopic and fluorescence lifetime characteristics of both forms are provided. Interpretation of the fluorescence anisotropy spectra of AmB incorporated into liposomes formed with dipalmitoylphosphatidylcholine let us conclude that monomers of the drug are more tightly bound to the lipid membranes as compared to the aggregates and that AmB aggregates destabilize the membrane structure. Structural model analysis, compared to the analysis of spectral shifts, leads to the conclusion that basic constituents of AmB aggregated structure is a tetramer composed of two hydrogen-bond-stabilized dimers, each dimer formed by molecules twisted by ca. 170°. The tetramer itself can span lipid bilayers and can act as a transmembrane ion channel. Specific aggregate formation of AmB has been concluded as a universal and ubiquitous form of molecular organization of the drug. This process is discussed in terms of toxic side effects of AmB.