Mary J. Beilby

Action Potential in Charophytes

The plant action potential (AP) has been studied for more than half a century. The experimental system was provided mainly by the large charophyte cells, which allowed insertion of early large electrodes, manipulation of cell compartments, and inside and outside media. These early experiments were inspired by the Hodgkin and Huxley (HH) work on the squid axon and its voltage clamp techniques. Later, the patch clamping technique provided information about the ion transporters underlying the excitation transient. The initial models were also influenced by the HH picture of the animal AP. At the turn of the century, the paradigm of the charophyte AP shifted to include several chemical reactions, second messenger-activated channel, and calcium ion liberation from internal stores. Many aspects of this new model await further clarification. The role of the AP in plant movements, wound signaling, and turgor regulation is now well documented. Involvement in invasion by pathogens, chilling injury, light, and gravity sensing are under investigation.

Mechanosensory ion channels in charophyte cells: the response to touch and salinity stress.

Abstract. Mechanosensitive (MS) ion channels are activated by mechanical stress and then transduce this information into electrical signals. These channels are involved in the growth, development and response to environmental stress in higher plants. Detailed analyses of the electrophysiology in higher plants are difficult because such plants are composed of complex tissues. The large cells of the charophytes facilitate electrophysiological measurements and allow us to study MS ion channels at the level of single cells. We draw parallels between the process of touch-perception in freshwater Chara, and the turgor-regulating response to osmotic shock in salt-tolerant Lamprothamnium. In terms of electrophysiology, these responses can be considered in three stages: (1) stimulus perception, (2) signal transmission and (3) induction of response. In Chara the first stage is due to the receptor potential (RPD), a transient depolarization with a critical threshold that triggers action potentials, which are responsible for stages (2) and (3). Receptor potentials are generated by MS ion channels. Action potentials involve a transient influx of Ca2+ to the cytoplasm, effluxes of K+ and Cl– and a temporary decrease of turgor pressure. Reducing cell turgor increases sensitivity to mechanical stimulation. In Lamprothamnium, a hypotonic shock produces an extended depolarization that resembles an extended RPD and is responsive to osmotic rather than ionic changes. Like the action potential, a critical threshold depolarization triggers Ca2+ influx, opening of Ca2+-sensitive Cl– channels and K+ channels; effluxes that last over an hour and result in turgor regulation. These processes show us, in primal form and at the level of single cells, how mechanoperception occurs in higher plants. Recent progress in research into the role of MS ion channels in the freshwater and salt-tolerant Characeae is reviewed and the relevance of these findings to plants in general is considered.

Exogenous melatonin affects photosynthesis in characeae Chara australis.

Melatonin was found in the fresh water characeae Chara australis. The concentrations (~4 μg/g of tissue) were similar in photosynthesizing cells, independent of their position on the plant and rhizoids (roots) without chloroplasts. Exogenous melatonin, added at 10 μM to the artificial pond water, increased quantum yield of photochemistry of photosystem II by 34%. The increased efficiency appears to be due to the amount of open reaction centers of photosystem II, rather than increased efficiency of each reaction center. More open reaction centers reflect better functionality of all photosynthetic transport chain constituents. We suggest that melatonin protection against reactive oxygen species covers not only chlorophyll, but also photosynthetic proteins in general.

The effect of an extracellular mucilage on the response to osmotic shock in the charophyte alga Lamprothamnium papulosum.

Abstract. We have used current/voltage (I/V) analysis to investigate the role played by extracellular mucilage in the cellular response to osmotic shock in Lamprothamnium papulosum. Cells lacking extracellular mucilage originated in a brackish environment (1/3 seawater). These were compared, first with cells coated with thick (∼50 μm) extracellular mucilage, collected from a marine lake, and second, with equivalent mucilaginous marine cells, treated with heparinase enzyme to disrupt the mucilage layer. Histochemical stains Toluidine Blue and Alcian Blue at low pH identified the major component of the extracellular mucilage as sulfated polysaccharides. Treating mucilage with heparinase removed the capacity for staining with cationic dyes at low pH, although the mucilage was not removed, and remained as a substantial unstirred layer. Cells lacking mucilage responded to hypotonic shock with depolarization (by ∼95 mV), cessation of cyclosis, due to transient opening of Ca2+ channels, and opening of Ca2+-activated Cl− channels and K+ channels. Cell conductance transiently increased tenfold, but after 60 min was restored to the conductance prior to hypotonic shock. Mucilaginous cells depolarized by a small amount (∼18 mV), but Ca2+ channels failed to open in large enough numbers for cyclosis to cease. Likewise most Ca2+-activated Cl− channels failed to open and conductance increased only ∼1.2-fold above the prehypotonic level. After 60 min conductance was less than the conductance prior to hypotonic shock. Heparinased mucilaginous cells recovered several aspects of the hypotonic response in cells lacking mucilage. These cells depolarized (by ∼103 mV); cyclosis ceased, indicating that Ca2+ channels had opened, and conductance increased to ∼4 times the value prior to hypotonic shock, indicating that Ca2+-activated Cl− channels opened. However, after 60 min, these cells had neither restored membrane potential (and remained at positive values), nor decreased their conductance. It was not possible to determine whether K+ channels had opened. The heparinased cells recovered the normal hypotonic response of mucilaginous cells when heparinase was washed out. Apical seawater cells, which lacked mucilage, were unaffected by heparinase treatment. The results demonstrate that the presence of extracellular sulfated polysaccharide mucilage impacts upon the electrophysiology of the response to osmotic shock in Lamprothamnium cells. The role of such sulfated mucilages in marine algae and animal cells is compared and discussed.

Modeling the Current-Voltage Characteristics of Charophyte Membranes. II.* The Effect of Salinity on Membranes of Lamprothamnium papulosum

Lamprothamnium is a salt-tolerant charophyte that inhabits a broad range of saline environments. The electrical characteristics of Lamprothamnium cell membranes were modeled in environments of different salinity: full seawater (SW), 0.5 SW, 0.4 SW, and 0.2 SW. The cells were voltage-clamped to obtain the I/V (current-voltage) and G/V (conductance-voltage) profiles of the cell membranes. Cells growing at the different salinities exhibited one of three types of I/V profiles (states): pump-, background- and K+-states. This study concentrates on the pump- and background-states. Curved (pump-dominated) I/V characteristics were found in cells with resting membrane PDs (potential differences) of −219 ± 12 mV (in 0.2 SW: 6 cells, 16 profiles), −161 ± 12 mV (in 0.4 SW: 6 cells, 7 profiles), −151 ± 12 mV (in 0.5 SW: 6 cells, 12 profiles) and −137 ± 12 mV (in full SW: 8 cells, 13 profiles). The linear I/V characteristics of the background-state were found in cells with resting PDs of −107 ± 12 mV (in 0.4 SW: 7 cells, 12 profiles), −108 ± 12 mV (in 0.5 SW: 7 cells, 10 profiles) and −104 ± 12 mV (in full SW: 3 cells, 5 profiles). The resting conductance (G) of the cells progressively increased with salinity, from 0.5 S·m−2 (in 0.2 SW) to 22.0 S·m−2 (in full SW). The pump peak conductance only rose from 2 S·m−2 (0.2 SW) to 5 S·m−2 (full SW), accounting for the increasingly depolarized resting PD observed in cells in more saline media.Upon exposure to hypertonic medium, both the pump and an inward K+ rectifier were stimulated. The modeling of the I/V profiles identified the inward K+ rectifier as an early electrical response to hypertonic challenge.

Mechano‐perception in Chara cells: the influence of salinity and calcium on touch‐activated receptor potentials, action potentials and ion transport

This paper investigates the impact of increased salinity on touch-induced receptor and action potentials of Chara internodal cells. We resolved underlying changes in ion transport by current/voltage analysis. In a saline medium with a low Ca(2+) ion concentration [(Ca(2+))(ext)], the cell background conductance significantly increased and proton pump currents declined to negligible levels, depolarizing the membrane potential difference (PD) to the excitation threshold [action potential (AP)(threshold)]. The onset of spontaneous repetitive action potentials further depolarized the PD, activating K(+) outward rectifying (KOR) channels. K(+) efflux was then sustained and irrevocable, and cells were desensitized to touch. However, when [Ca(2+)](ext) was high, the background conductance increased to a lesser extent and proton pump currents were stimulated, establishing a PD narrowly negative to AP(threshold). Cells did not spontaneously fire, but became hypersensitive to touch. Even slight touch stimulus induced an action potential and further repetitive firing. The duration of each excitation was extended when [Ca(2+)](ext) was low. Cell viability was prolonged in the absence of touch stimulus. Chara cells eventually depolarize and die in the saline media, but touch-stimulated and spontaneous excitation accelerates the process in a Ca(2+)-dependent manner. Our results have broad implications for understanding the interactions between mechano-perception and salinity stress in plants.

The Physiology of Characean Cells

The aim of this chapter is to give physiologists a thorough grounding in the morphology, taxonomy and ecology of the characean plant. The morphology of characean plants is depicted and explained, with specific examples of the morphological characteristics of different species or species groups that are used in physiological studies. The details of characean cellular structure in growing plants and in the reproductive organs are reviewed. The history of taxonomy and nomenclature is outlined, along with the most recent approaches to systematics (and what name to use for characean plants in physiological studies), and finally the patterns of characean plant distribution and requirements for growth in natural situations are explained and related to the culture and growth of characean plants for physiological studies. 1.1 General Morphology The characean thallus (or plant body) is similar in appearance and size to the plant body of other submerged plants such as Ceratophyllum or Myriophyllum. Characean plants consist of long photosynthetic stem-like structures (axes) anchored in the soil, with whorls of leaf-like organs (branchlets) along the stem (Fig. 1.1a). Close examination reveals that the structure of characean plants is very different to that of flowering plants. Instead of roots they have colourless rhizoids, instead of leaves they have whorls of branchlets of limited growth, instead of stems they have an axis of giant cells joined end on end and instead of flowers and fruit they have relatively simple reproductive structures (the oogonium and antheridium, Fig. 1.1b) that produce gametes. The product of fertilisation of the gametes is an oospore (Fig. 1.1c) rather than a seed. The thallus of characean plants is essentially filamentous. The axes (stems) are made up of long, multinucleate, single cells interrupted by multicellular nodes (Fig. 1.2). There is no development of tissues such as parenchyma, although the axial nodes approach such an arrangement (Sect. 4.3). Several organs of limited M.J. Beilby and M.T. Casanova, The Physiology of Characean Cells, DOI 10.1007/978-3-642-40288-3_1, # Springer-Verlag Berlin Heidelberg 2014 1 growth (branchlets, stipulodes and cortical filaments) arise in whorls at the nodes (Fig. 1.2). Branchlets are the “leaf-like” organs that occur in spreading whorls, and below these there are often whorls of smaller cells called stipulodes. In many species of Chara, the stipulodes occur in two whorls, the upper whorl pointing upwards and the lower whorl pointing downwards (Fig. 1.2a, b). In the genera Lamprothamnium and Tolypella, and some species of Chara, the axial node can also be the site of gametangial development (Fig. 1.2c). Branchlet arrangement (Fig. 1.3) and morphology (Fig. 1.4) varies among the genera but is characterised by elongate multinucleate cells interrupted by multicellular branchlet nodes. Other cellular structures can be produced at the branchlet nodes, namely bract cells (Figs. 1.3a, b, and 1.4a, b), secondary and tertiary (et seq.) branchlet segments or rays (Figs. 1.3c, d, and 1.4c, d), cortical filaments (Fig. 1.4a) and gametangial initials (Fig. 1.4). Some species of Chara have elongate bract cells Apex Branch Reproductive organs

When is a cell not a cell? A theory relating coenocytic structure to the unusual electrophysiology of Ventricaria ventricosa (Valonia ventricosa)

Summary.Ventricaria ventricosa and its relatives have intrigued cell biologists and electrophysiologists for over a hundred years. Historically, electrophysiologists have regarded V. ventricosa as a large single plant cell with unusual characteristics including a small and positive vacuole-to-outside membrane potential difference. However, V. ventricosa has a coenocytic construction, with an alveolate cytoplasm interpenetrated by a complex vacuole containing sulphated polysaccharides. We present a theory relating the coenocytic structure to the unusual electrophysiology of V. ventricosa. The alveolate cytoplasm of V. ventricosa consists of a collective of uninucleate cytoplasmic domains interconnected by fine cytoplasmic strands containing microtubules. The cytoplasm is capable of disassociating into single cytoplasmic domains or aggregations of domains that can regenerate new coenocytes. The cytoplasmic domains are enclosed by outer (apical) and inner (basolateral) faces of a communal membrane with polarised K+-transporting functions, stabilised by microtubules and resembling a tissue such as a polarised epithelium. There is evidence for membrane trafficking through endocytosis and exocytosis and so “plasmalemma” and “tonoplast” do not have fixed identities. Intra- and extracellular polysaccharide mucilage has effects on electrophysiology through reducing the activity of water and through ion exchange. The vacuole-to-outside potential difference, at which the cell membrane conductance is maximal, reverses its sign from positive under hypertonic conditions to negative under hypotonic conditions. The marked mirror symmetry of the characteristics of current as a function of voltage and conductance as a function of voltage is interpreted as a feature of the communal membrane with polarised K+ transport. The complex inhomogeneous structure of the cytoplasm places in doubt previous measurements of cytoplasm-to-outside potential difference.

The proton pump, high pH channels, and excitation: voltage clamp studies of intact and perfused cells ofNitellopsis obtusa

SummaryThe current-voltage (I/V) and conductance-voltage (G/ V) characteristics were recorded for intact and perfused (tonoplast-free) cells ofNitellopsis obtusa. In the pH0 range 5 to 8, the I/V profile was sigmoidal and the G/V profile exhibited a maximum — these characteristics are attributed to the proton pump at the plasmalemma. The pH0 dependence in this range was very similar to that found inChara corallina. At very alkaline pH0 (11.0) the high conductance due to H+/OH− channels was observed in intact cells but not in perfused cells. Young plants ofNitellopsis did not display bands of calcification, but did exhibit pH banding patterns in petri dishes. The pH bands were less than 5mm wide. The excitation transients in intact cells featured two peaks near the excitation threshold, but more peaks could be observed in the p.d. (potential difference) range −90 to −60 mV. The amplitude of the transients was strongly inhibited at pH0 11.0. In the perfused cells the currents lacked complete inhibition at some p.d. levels, but still exhibited one or two peaks. At high pH0 this lack of inactivation was accentuated. The addition of 5 mM TEA to the outside medium abolished the excitation transients in perfused cells.

pH Banding in Charophyte Algae

The internodal cells of Characean algal species have long served as a model for membrane processes in plants, because their large size (up to several centimetres in length), simple geometry (cylinder) and clear separation from other cells in the plant have allowed experimental techniques such as multielectrode electrophysiological techniques and cell perfusion. However, the membranes of these cells are not homogeneous, but show distinct differences in their electrophysiological characteristics and transport capabilities. The most obvious example of this non uniformity is the pH difference seen in the external medium surrounding the cells, the “acid bands”, with a pH similar or slightly acid to the bulk medium, and “alkaline bands”, which can support a pH of 10 or higher. We explore here the transport properties that underlie these differences and their relation to photosynthesis.