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.     

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.     

Phylogenetic relationships among taxa of the liverwort Conocephalum conicum (Conocephalaceae) revealed by psbA sequence

The 5' flanking and coding regions of the psbA gene were sequenced to clarify relationships among several taxa discovered recently in thalloid liverwort Conocephalum conicum. Twenty six samples included in this study represent five "cryptic species" detected in worldwide collection, five groups discovered from Japan and finally three "chemo-types" with different dominant volatile component. Pairwise differences in nucleotide sequences of coding region between samples of Conocephalum conicum varied greatly depending on the combination, ranging from 0.001 to 0.018 per site. In total, seven amino acid substitutions were found among all samples. NJ and parsimony trees among the taxa were constructed for the first time. The phylogenetic relationships among taxa were basically consistent with those inferred from isozyme and morphological studies. Conocephalum conicum -species FS and T taxon found in Japan have identical sequence of psbA gene, suggesting that they are conspecific. Similarly, YFS and KYT taxa cluster together with FS cryptic species, and "chemo-types" I and II with "cryptic species" J. A chemotype III was very different from any other taxa of C. conicum. These results suggest that morphological species, Conocephalum conicum is highly differentiated at the molecular level and some of the taxa may in fact represents different species as previous studies suggested.     

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.     

Purification and characterization of glutathione reductase from Scots pine needles

Changes in the excitability of the liverwort Conocephalum conicum L. caused by inhibitors of ionic channels and phosphorylation uncouplers, were examined. Action potentials were triggered by electrical and light stimuli. Tetraethylammonium chloride, an inhibitor of K⁺ channels, completely blocked the ability to generate action potentials. Excitability also disappeared under the influence of MnCl₂ and LaCl₃, inhibitors of Ca²⁺ channels. The participation of Ca²⁺ and K⁺ in the electrogenesis of action potentials in C. conicum is discussed. Treatment with phosphorylation uncouplers induced a gradual disappearance of the metabolic component of the resting potential. It was accompanied by some series of excitations, numbering from several to over a dozen impulses characterized by decreasing amplitudes, after which the organism became totally unexcitable. Dicyclohexylcarbodiimide an inhibitor of H⁺-ATPase, also caused depolarization of the transmembrane potential and disappearance of excitability. The results indicated the participation of a metabolic component in the generation of action potentials in C. conicum .     

Cytoplasmic Ca2+, K+, Cl-, and NO3- Activities in the Liverwort Conocephalum conicum L. at Rest and during Action Potentials

Intracellular Ca2+, K+, Cl-, and NO3- activities were measured with ion-selective microelectrodes in the liverwort Conocephalum conicum L. at rest, during dark/light changes, and in the course of action potentials triggered by light or electrical stimuli. The average free cytosolic Ca2+ concentration was 231 [plus or minus] 65 nM. We did not observe any light-dependent changes of the free cytosolic Ca2+ concentration as long as no action potential was triggered. During action potentials, on average a 2-fold increase of the free cytoplasmic Ca2+ concentration was recorded. Intracellular K+ activity was 76 [plus or minus] 10 mM. It did not depend on K+ concentration changes in the bath solution between 0.1 and 10 mM. The average equilibrium potential for K+ in the standard medium containing 1 mM K+ was -110 mV, which differed significantly from the resting potential of -151 [plus or minus] 2 mV. During action potentials, either a slight decrease or no changes in intracellular K+ activity were recorded. The average Cl- activity was 7.4 [plus or minus] 0.2 mM in the cytoplasm and 43.5 [plus or minus] 7 mM in the vacuole. The activities of NO3- were 0.63 [plus or minus] 0.05 mM in the cytoplasm and 3.0 [plus or minus] 0.3 mM in the vacuole. For both anions the vacuolar activity was 5 to 6 times higher than the cytoplasmic activity. After the light was switched off both the Cl- and the NO3- activity showed either no change or a slight increase. Illumination caused a gradual return to previous values or no change. During action potentials a slight decrease of intracellular Cl- activity was recorded. It was concluded that in Conocephalum, as in characean cells, chloride channels are involved in the depolarization phase of the action potentials. We discuss a model for the ion fluxes during an action potential in Conocephalum.     

Electrical activity of the liver-wort Conocephalum conicum.: Method of investigation and general characteristics of excitation

The excitation of the thallus of Conocephalum conicum L. produced by a tissue damaging stimulus (incision of thallus edge) was investigated. Changes in potential were measured with extracellular contact electrodes applied at various points of the thallus surface. Non-stimulated plants showed no electrical activity. After stimulation a series of impulses occurred and were propagated all over the thallus. This plant electrical activity (PEA) depends on the magnitude of the stimulus. PEA is characterized by changes of frequency, time of duration and changes in the values of the amplitude. A similar phenomenon is well known in animal organisms as frequency coding. The general character of PEA does not depend on the kind of damaging stimulus (burn, puncture). PEA may also be induced by an electrical stimulus which does not damage the tissue.     

蛇苔细胞色素C（Cyt C）的序列分析及功能预测

通过核酸序列比对,在蛇苔cDNA文库中获得细胞色素C（Cyt C）基因序列,并对其编码的蛋白质产物从同源性、氨基酸组成、理化性质、亚细胞位点、结构和功能等进行生物信息学分析和预测。结果表明,该cDNA序列具有完整的开放阅读框（ORF,104—442bp）,推测编码蛋白为112个氨基酸,与向日葵、荞麦、玉米等Cyt C存在着较高的保守性（相似性分别为84％、85％和84％）,同属于细胞色素C超家族;蛇苔CytC蛋白的分子量为11998．7Da,不含信号肽,成熟的CytC起始于Ala2,其活性位点为Cys23、Cys26、His27和Met89;对蛋白质的翻译后修饰预测表明Ala2存在乙酰化修饰,Lys81和Lys95分别进行三甲基赖氨酸修饰。这些特点与其他植物CytC保持一致,表明该基因为蛇苔cyt c基因,在进化上相对保守。     

蛇苔过氧化物酶基因的序列分析与功能预测

过氧化物酶在生物界分布极广,在细胞代谢的氧化还原过程中起重要的作用.为深入研究蛇苔过氧化物酶的理化性质及功能,对其同源性,亚细胞定位,结构和功能等进行了生物信息学预测和分析.结果表明;该序列有1 050 bp长的开放阅读框,编码349个氨基酸,与拟南芥等的POD相似性较高(相似性为71％).预测R68、F71、H72、...     

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.     

Low-temperature induced transmembrane potential changes in the liverwort Conocephalum conicum

Intracellular microelectrode measurements revealed that the liverwort Conocephalum conicum generates all-or-none action potentials (APs) in response to a sudden temperature drop. In plants with anion and potassium conductance blocked, dose-dependent voltage transients (VTs) were evoked by cold stimuli. These VTs did not propagate. When the external concentration of Ca(2+) was decreased or calcium channel inhibitors (La(3+), Gd(3+), verapamil, Mg(2+), Mn(2+)) were used, inhibition of VTs was observed. Amplitudes of both APs and VTs grew when Sr(2+) ions, known to release calcium from internal stores, were added to the medium. Neomycin, which suppresses phospholipase C and indirectly affects inositol triphosphate formation, caused substantial inhibition of both APs and VTs. It is concluded that a temperature drop elucidated membrane potential changes due to calcium influx both from external and internal stores.     

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.     

Quercetin and genistein hindering effect of neomycin action in the liverwort <Emphasis Type="Italic">Conocephalum conicum</Emphasis>

Apigenin, quercetin and genistein are members of the family of plant flavonoids suspected to prevent a number of human diseases, for instance cancer development. They display a number of activities, and part of their beneficial effects may be due to their affinity to cellular membranes. In this study, we used Conocephalum conicum, a well-elaborated model of liverworts. Intracellular microelectrode measurements were carried out to examine the effects of flavonoids in combination with neomycin on the resting and action potentials. Neomycin triggered gradual decline of action potential amplitudes through a membrane potential decrease (membrane potential became less negative) and a decrease of the action potential peak value. Additionally, duration of action potential amplitudes measured at half of the amplitude increased in neomycin-treated plants. However, the simultaneous use of quercetin or genistein (but not apigenin) with neomycin hindered neomycin-specific actions. Hence, the membrane resting potential and action potential amplitudes regained neomycin-free values. It may be concluded that application of at least some flavonoids (namely quercetin and genistein) exerts strong influence on electrical membrane responses in C. conicum.     

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.     

四川省都江堰龙池地区苔藓植物染色体的研究

报道了四川省都江堰市龙池地区６种苔藓植物配子体的以体研究结果,５种材料均取其生长点或茎尖,观察和计数了细胞有丝分裂中期的染色体数,１．中华扁萼苔（Ｒａｄｕｌｌａｃｈｉｎｅｎｓｉｓ Ｓｔｅｐｈ．ｖａｒ．ｒｏｐｔｕｄｉｓｔｉｐａ）ｎ＝８;２．蛇苔（Ｃｏｎｏｃｅｐｈａｌｕｍ ｃｏｎｉｃｕｍ（Ｌ．）Ｄｕｎ．）＝９;３．长齿匍灯藓（Ｐｌａｇｉｏｍｎｉｕｍ ｄｒｕｍｍｏｎｄｉｉ （Ｂｒｕｃｈ．＆ Ｓｃｈｉｍｐ     

Glomeromycotean associations in liverworts: a molecular, cellular, and taxonomic analysis

Liverworts form endophytic associations with fungi that mirror mycorrhizal associations in tracheophytes. Here we report a worldwide survey of liverwort associations with glomeromycotean fungi (GAs), together with a comparative molecular and cellular analysis in representative species. Liverwort GAs are circumscribed by a basal assemblage embracing the Haplomitriopsida, the Marchantiopsida (except a few mostly derived clades), and part of the Metzgeriidae. Fungal endophytes from Haplomitrium, Conocephalum, Fossombronia, and Pellia were related to Glomus Group A, while the endophyte from Monoclea was related to Acaulospora. An isolate of G. mosseae colonized axenic thalli of Conocephalum, producing an association similar to that in the wild. Fungal colonization in marchantialean liverworts suppressed cell wall autofluorescence and elicited the deposition of a new wall layer that specifically bound the monoclonal antibody CCRC-M1 against fucosylated side groups associated with xyloglucan and rhamnogalacturonan I. The interfacial material covering the intracellular fungus contained the same epitopes present in host cell walls. The taxonomic distribution and cytology of liverwort GAs suggest an ancient origin and multiple more recent losses, but the occurence in widely separated liverwort taxa of fungi related to glomeromycotean lineages that form arbuscular mycorrhizas in tracheophytes, notably the Glomus Group A, is better explained by host shifting from tracheophytes to liverworts.     

Employing ICA and SOM for spike sorting of multielectrode recordings from CNS

For classification of action potential shapes in multineuron recordings, we present a spike sorting system employing independent component analysis (ICA) and an unsupervised artificial neural network (Kohonen's self-organizing map, SOM). We focus on how ICA in the first stage of the spike sorting system can be used to address specific problems arising in recordings using multielectrode arrays in the CNS. Using real data recorded from the pontine nuclei in rats and simulated data, we evaluate the performance of several ICA algorithms to remove cross-talk between electrodes using data from continuous recording (or simulation). When using cut-out data, the standard format of extracellular spike recordings, new problems emerge and robust algorithms are needed. We demonstrate that several ICA algorithms show a good performance on cut-out data from multielectrode array recordings (simulated and real data). In tetrode recordings the same neuron is purposely recorded by several electrodes simultaneously and we show, how independent component analysis can be used in this case to identify redundant information and hence to compress relevant information, improving subsequent clustering of a SOM.     

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.     

Extracellular potentials from single spinal motoneurons

Extracellular action potentials found close to the surface of motoneurons are related to the intracellular spikes. Evidence is cited to support the assumption that the extracellular spikes have the same time course as the membrane current at the site of recording. Simultaneously recorded intracellular and extracellular spikes are compared. Intracellular spikes are transformed, by means of a circuit which is equivalent to the extracellular recording situation, into transients that are like those appearing extracellularly. Evidence is given that the recordings are from the cell bodies of motoneurons. The results show that the membrane at the extracellular recording site does not produce a spike since the time course of the extracellular potentials is determined by the passive properties of the membrane.     

六种叶状体苔藓植物叶绿素含量和光合效率研究

以六种叶状体苔藓为材料,通过测定叶绿素含量及实际光合量子产量,检验叶状体苔藓植物叶绿素含量与实际光合量子产量之间的关系。结果表明,六种叶状体苔藓总叶绿素含量中值6.06 mg·g-1,其中花叶溪苔Pellia endiviifolia最高(9.72 mg·g-1),楔瓣地钱Marchantia emarginata最低(4.46 mg·g-1);叶绿素a/b值变化范围1.61～2.04,林氏黄角苔Phaeoceros carolinianus最高,毛地钱Dumortiera hirsuta最低;紫背苔Plagiochasma rupestre、楔瓣地钱和蛇苔Conocephalum conicum实际光合量子产量明显高于毛地钱、花叶溪苔和林氏黄角苔。叶状体苔藓叶绿素含量与其实际光合量子产量之间并非简单的线性关系,气室结构及生境可能影响其实际光合效率。这一结论进一步丰富了苔藓植物对光照的生态适应理论。