Light-triggered action potentials in the liverwort Conocephalum conicum

The response to light of a liverwort, Conocephalum conicum L., measured as a change in the resting potential, consists of two stages. The first stage is a slight depolarization dependent on light intensity. This plays the role of a generator potential (GP) which induces the second stage - an action potential of the all-or-none character. Action potentials induced by light and by electrical stimuli have the same properties, i.e. identical time course, propagation velocity, and refractory periods. A summation occurs of sub threshold light stimuli and of light and electrical stimuli. The presence of 5⋅10-⁻⁶ M DCMU cancelled the light response and blocked - by inhibition of the electron transport chain - the mechanism leading to GP generation. However, this effect did not produce any change in the response to electrical stimuli.     

Airborne sperm of Conocephalum conicum (Conocephalaceae)

We have obtained the first momentary photographs of sperms just as they are discharged from the antheridium of a liverwort, Conocephalum conicum, and have succeeded in monitoring the airborne sperms of bryophytes under field conditions. Airborne sperm of liverworts seems to be an effective strategy for raising the efficiency of fertilization between male and female plants separated in a drought environment. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The influence of apigenin on membrane and action potential in the liverwort <Emphasis Type="Italic">Conocephalum conicum</Emphasis>

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.     

Characteristics of the light-induced generator potentials in the liverwort Conocephalum conicum

Light-induced generator potentials were examined in the liverwort Conocephalum conicum L. by applying sub-threshold light stimuli (400–750 nm). The fluenceresponse dependence was determined for various wavelengths. At low values of quantum flux density (less than 1 μmol m⁻² s⁻¹) the plant reacts to the light pulses with hyperpolarization which, with a rise in the pulse intensity, is masked by the depolarization of the transmembrane potential. The action spectrum of depolarization shares many features with the absorption specturm of photosynthetic pigments. Application of inhibitors and electron donors showed a predominant role of photosystem II in the formation of the generator potential. No significant Emerson enhancement was observed after simultaneous illumination with light beams of 650 and 700 nm.     

Measurements of intra-and extracellular pH in the liverwort Conocephalum conicum during action potentials

During action potentials triggered by electricity and light, measurements of intra- and extracellular pH in the liverwort Conocephalum conicum L. were carried out by the use of antimony-filled H⁺-sensitive microelectrodes. Intracellular pH increased transiently by about 0.05 unit in the course of an action potential, while extracellular pH, measured at the surface of the thallus, remained unchanged. Switching the light off caused a transient increase in intracellular pH by less than 0.1 unit. Turning the light on produced a fast pH decrease by about 0.15 unit followed by a slow increase. When the light was intensive enough, the action potential thus triggered caused a slight increase in intracellular pH superimposed on the phase of a slow pH increment.
The magnitude and time course of the intracellular pH changes seem insufficient for a role as messenger between action potential and the up to 100% increase in the rate of respiration that has been registered in Conocephalum conicum as a consequence of excitation.     

Extra- and intracellular measurements of action potentials in the liverwort Conocephalum conicum

Action potentials induced by electrical stimuli were recorded from the thalli of tfie liverwort, Conocephalum conicum L. An analysis of an equivalent circuit of Conocephalum conicum thallus in situ has shown that the multiphasic time course of extracellular recordings is caused by a branching of extracellular conduction layers and by an overlapping of action currents. As compared to the velocity of excitation propagation without short-circuiting, more than 10 times higher rates of action potential conduction have been recorded when the plants are immersed in a low resistaace solution. Furthermore, simultaneous extra- and intracellular recordings from thalli in situ are presented.     

The effect of excitation on the rate of respiration in the liverwort Conocephalum conicum

Simultaneous measurements were taken of the electrical activity and the rate of respiration of thalli of Conocephalum conicum L. stimulated electrically and mechanically (by cutting). The measurements of the rate of respiration employed a modified Warburg apparatus for O₂ consumption and an infra-red gas analyzer with computer recording and data processing for CO₂ evolution. The action potential, produced by either a cut (a damaging stimulus) or an electrical stimulus (a non-damaging stimulus), caused a transient rise in the rate of respiration. The course of changes in the rate of respiration depends on the character of the excitation and the area of the thallus covered by it. If stimulation does not produce excitation, the increase in the rate of respiration does not take place, regardless of the magnitude and type of the stimulus applied.     

Electrical activity of the liverwort Conocephalum conicum: The all-or-nothing law, strength-duration relation, refractory periods and intracellular potentials

The characteristics of the potential changes caused by excitation of the thallus of Conocephalum conicum L. were investigated and compared with the basic criteria of an action potential. Electrical stimulation (square DC pulses) was applied, and extraand intracellular potentials were recorded. The all-or-nothing law, strength-duration relation, refractory periods, and general characteristics of the changes of transmembrane potential difference were examined. The electrical phenomena occurring during the excitation of Conocephalum fulfil the basic criteria of an action potential. The results contribute additional evidence for the hypothesis that action potentials occur both in plants and in animals.     

Conocephalum salebrosum (Marchantiopsida, Conocephalaceae) – a new Holarctic liverwort species

Detailed analysis of 270 specimens of Conocephalum conicum (L.) Dumort., mainly from the POZW Herbarium revealed some diagnostic differences between two cryptic species originally detected on the basis of isozyme studies. Several diagnostic characters were found in the structure of the archegoniophore, sporophyte and sterile thallus. The most reliable diagnostic features are size and structure of stomatal apparatus of the archegoniophore ‘heads’, type of junction of the air chamber walls with epidermal cells, and the thallus surface details. On the basis of morphological and anatomical diagnostic characters, two formerly cryptic species are recognized following the rules of formal taxonomy. The lectotype of C. conicum preserved in FI was found to possess characteristics of cryptic species C. conicum- species L. Accordingly, the species previously named as C. conicum–species L must bear the name Conocephalum conicum (L.) Dumort. No published name was related to C. conicum – species S, which is therefore described as a new species: Conocephalum salebrosum Szweykowski, Buczkowska &; Odrzykoski. Both species are fully described and their diagnostic characters are illustrated. Distribution maps for both species are given.     

Fatty acid ethyl esters in the liverwort Conocephalum conicum

A series of fatty acid ethyl esters ranging from C₁₄ to C₂₄ was isolated from a hexane extract of the liverwort Conocephalum conicum, these esters accounted for 77% of the extract. The ethyl esters consisting of even-numbered fatty acids were predominant and ethyl palmitate was the major constituent.     

Incorporation of [1-(13)C]1-deoxy-D-xylulose into isoprenoids of the liverwort Conocephalum conicum

The incorporation of (13)C labeled 1-deoxy-D-xylulose into the monoterpene bornyl acetate, the sesquiterpene cubebanol, and the diterpene phytol has been studied in axenic cultures of the liverwort Conocephalum conicum. Quantitative (13)C NMR spectroscopic analysis of the labeling patterns of the sesquiterpene indicated a possible degradation of 1-deoxy-D-xylulose to acetate and subsequent incorporation via the mevalonic acid pathway. In bornyl acetate, the labeling occurred only in the acetate moiety whereas the isoprene units remained unlabelled. The isoprene units of the diterpene phytol showed incorporation of intact deoxy-D-xylulose. These results indicate the involvement of both IPP biosynthetic pathways and two independently operating compartments/cell types with MEP pathway machinery. One MEP compartment is presumably the plastid where phytol is formed; the second, involved in the build-up of the isoprene part of bornyl acetate, might be located in the oil cells. The acetylation of borneol to bornyl acetate in turn occurs in a cellular compartment that is not involved in the build-up of the isoprene units of borneol.     

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.     

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.     

Allozyme variability within and divergence among populations of the liverwortConocephalum conicum (Marchantiales: Hepaticae) in Japan

Genetic variation within, and divergence among, populations of the liverwortConocephalum conicum were estimated from the study of 17 populations and 23 putative gene loci. Two additional multilocus genotypes (“T” and “FS”) were detected in Japan, along with the previously reported “J” type. These three multilocus genotypes differed both morphologically and ecologically. All eight populations from western Japan included only the J-type and exhibited low genetic variation within populations: Nei's (1973) average gene diversity (Ĥ)=0.080±0.029. In contrast, co-occurrence of several multilocus genotypes in each population from the Kanto District resulted in much higher levels of genetic variation (Ĥ=0.218±0.037). If the three genotypes are distinguished,Ĥ values are 0.113±0.030 for T-type, 0.107±0.033 for FS-type, and 0.083±0.018 for J-type. UsingC. japonicum, which showed low genetic variation (0.014±0.010) as an outgroup, each genotype formed a monophyletic clade, and the J- and FS-types were more closely related to each other than to the T-type. Populations of western Japan and the Kanto District also differed in the degree of gene diversity among populations, but the reasons for these differences are obscure.     

The influence of glutamic and aminoacetic acids on the excitability of the liverwort Conocephalum conicum

Intracellular microelectrode measurements revealed that a resting potential (RP), an action potential (AP) and a calcium component of AP (named voltage transient, VT) can be influenced by glutamic acid (Glu) and aminoacetic acid (glycine, Gly) in the liverwort Conocephalum conicum. In the continuous presence of 5mM Glu or 5mM Gly, the RP hyperpolarized constantly and the plants became desensitized to the excitatory amino acids (Glu or Gly). Under such circumstances, the amplitudes of APs evoked by stimuli other than Glu or Gly grew, as did their calcium components (VTs). The sudden application of 1-15 mM Glu or Gly to a thallus not yet desensitized resulted in an excitation, i.e. a single AP or AP series. Aspartate (Asp) could not substitute for Glu in any way. Simultaneous action of both amino acids acted synergically to trigger APs. The same phenomenon was observed when glycine solution was enriched with N-methyl-D-aspartic acid (NMDA). Gly-induced APs were totally hindered by 1mM D-amino-5-phosphonopentanoic acid (AP5)--an inhibitor of ionotropic glutamate receptors of the NMDA kind. Glu-induced APs could be totally suppressed by 1mM AP5 as well as by 1mM 6,7-dinitroquinoxaline-2,3-dione (DNQX)--an inhibitor of AMPA/KA receptors. DNQX also completely blocked the calcium component of Glu-evoked APs. After DNQX treatment, the only response to Glu was a membrane potential hyperpolarization (like the Glu response in a desensitized plant). It was concluded that the Glu-induced depolarization and hyperpolarization are separate phenomena. The stimulatory effects of both Glu and Gly on liverwort excitability may be the consequences of an activation of a variety of ionotropic Glu receptor subtypes.     

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.Key words: action potential, cold, liverwort, menthol, thermoreceptors, voltage transient     

Degradation and release from the thylakoid membrane of Photosystem II subunits after UV-B irradation of the liverwort Conocephalum conicum

The effect of ultraviolet-B (UV-B) radiation on the amount of various Photosystem (PS) II subunits has been studied in the thalloid liverwort Conocephalum conicum. UV-B irradiation led to a drastic decrease of the reaction center proteins D1 and D2 and the outer light harvesting antenna (LHC II). A minor reduction was found in the levels of the CP 43 polypeptide of the inner antenna and the 33, 23 and 16 kDa extrinsic polypeptides of PS II. During UV-B irradiation, the extrinsic polypeptides accumulated in the soluble protein fraction, but D1 and D2 were not dedectable. Streptomycin, but not cycloheximide inhibited the repair process of PS II, indicating that only protein synthesis in the chloroplast is necessary for recovery. This indicates that the extrinsic proteins of PS II dissociate from the membrane during UV-B treatment and reassociate with PS II in the course of the repair process. We conclude that the reaction center core is a target of UV-B radiation in C. concicum. The extrinsic proteins of PS II are not directly affected by UV-B, but their release is the consequence of UV-B-induced degradation of the D1 and D2 proteins.     

Genetic differentiation without concordant morphological divergence in the thallose liverwortConocephalum conicum

Allozyme variation was examined at 33 enzyme loci for 96 populations from throughout the geographical range ofConocephalum conicum (L.)Dum. (Marchantiales, Hepaticae). Variation was partitioned into five discrete groups, suggesting that this morphologically defined species is genetically heterogeneous. The degree of differentiation among these groups, as measured by genetic distance, is as large as is commonly reported between different vascular plant species, and much larger than that between conspecific populations. In Europe, two of these genetically distinct groups (S and L) occur sympatrically, but apparently do not interbreed. Geographical ranges of the other three groups (A, C, and J) are probably allopatric with the exception that the range of S, the most genetically divergent group, overlaps with group A in N. America. It is suggested that these five natural assemblages constitute different sibling species.     

Effects of quercetin and enhanced UV-B radiation on the soybean (Glycine max) leaves

The possible ameliorative effects of quercetin on soybean [Glycine max (L.) Merr.] leaves exposed to UV-B radiation were conducted in greenhouse. The symmetrical leaves supplied with quercetin solution (0.2%, 1%) were exposed to UV-B radiation (0, 3.5, 6.5 kJ m⁻² d⁻¹). 0.2% quercetin ameliorated leaf photosynthesis, improved leaf water content (LWC), and decreased lipid oxidation. The unfavorable effect on photosynthetic parameter was displayed in 1% quercetin treatment. The effect of quercetin on phenylalanine ammonia lyase (PAL) activity varied with the quercetin concentration, UV-B radiation intensity and leaf development. In the later development polyphenol oxidase (PPO) activity was increased significantly by quercetin treatments. We suggested that quercetin with suitable concentration could serve as UV-B protective agent partly due to its antioxidant capacity.     

Effects of membrane potential on calcium fluxes of Pelvetia eggs

⁴⁵Ca²⁺ fluxes across the plasma membrane of zygotes of the fucoid alga, Pelvetia fastagiata (J. Ag.) De Toni, were studied in artificial sea waters of various potassium concentrations. Except for two cases, hyperpolarization of the cell membrane (with low [K⁺]) increases, and depolarization (with high [K⁺]) decreases the influx of Ca²⁺ over the range of [K⁺] studied (1–100 mM). The fractional increases of influx during hyperpolarization are close to the fractional increases in membrane potential but the decreases during depolarization are much smaller than those in membrane potential. In two anomalous cases, the influxes of ⁴⁵Ca²⁺ at a potassium concentration of 30 mM were about 20% higher than the control value instead of being 10% lower.The effluxes of ⁴⁵Ca²⁺ are increased by both hyperpolarization and by depolarization. On balance (and excepting the two anomalous cases) the net result of hyperpolarization should be to increase and that of depolarization to decrease intracellular [Ca²⁺].