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.

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.

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.

Characterization of callase (ß-1,3-D-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum.

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.

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. The nature of the primary step of its action (alteration in membrane composition, transient influx of Ca2+ etc.,) has not been elicited yet. Our electrophysiological experiments conducted on the liverwort Conocephalum conicum showed that its cells respond not only to sudden cooling 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. TRP receptors are detected, among others, in green and brown algae. Possible existence of TRPM8-like channel-receptor in Conocephalum conicum is discussed here.

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.

Can membrane-bound carotenoid pigment zeaxanthin carry out a transmembrane proton transfer?

Polar carotenoid pigment zeaxanthin (beta,beta-carotene-3,3'-diol) incorporated into planar lipid membranes formed with diphytanoyl phosphatidylcholine increases the specific electric resistance of the membrane from ca. 4 to 13 x 10(7) Omega cm2 (at 5 mol% zeaxanthin with respect to lipid). Such an observation is consistent with the well known effect of polar carotenoids in decreasing fluidity and structural stabilization of lipid bilayers. Zeaxanthin incorporated into the lipid membrane at 1 mol% has very small effect on the overall membrane resistance but facilitates equilibration of the transmembrane proton gradient, as demonstrated with the application of the H+-sensitive antimony electrodes. Relatively low changes in the electrical potential suggest that the equilibration process may be associated with a symport/antiport activity or with a transmembrane transfer of the molecules of acid. UV-Vis linear dichroism analysis of multibilayer formed with the same lipid-carotenoid system shows that the transition dipole moment of the pigment molecules forms a mean angle of 21 degrees with respect to the axis normal to the plane of the membrane. This means that zeaxanthin spans the membrane and tends to have its two hydroxyl groups anchored in the opposite polar zones of the membrane. Detailed FTIR analysis of beta-carotene and zeaxanthin indicates that the polyene chain of carotenoids is able to form weak hydrogen bonds with water molecules. Possible molecular mechanisms responsible for proton transport by polyenes are discussed, including direct involvement of the polyene chain in proton transfer and indirect effect of the pigment on physical properties of the membrane.