The molecular identity of the characean OH- transporter: a candidate related to the SLC4 family of animal pH regulators.

Characeae are closely related to the ancient algal ancestors of all land plants. The long characean cells display a pH banding pattern to facilitate inorganic carbon import in the acid zones for photosynthetic efficiency. The excess OH-, generated in the cytoplasm after CO2 is taken into the chloroplasts, is disposed of in the alkaline band. To identify the transporter responsible, we searched the Chara australis transcriptome for homologues of mouse Slc4a11, which functions as OH-/H+ transporter. We found a single Slc4-like sequence CL5060.2 (named CaSLOT). When CaSLOT was expressed in Xenopus oocytes, an increase in membrane conductance and hyperpolarization of resting potential difference (PD) was observed with external pH increase to 9.5. These features recall the behavior of Slc4a11 in oocytes and are consistent with the action of a pH-dependent OH-/H+ conductance. The large scatter in the data might reflect intrinsic variability of CaSLOT transporter activation, inefficient expression in the oocyte due to evolutionary distance between ancient algae and frogs, or absence of putative activating factor present in Chara cytoplasm. CaSLOT homologues were found in chlorophyte and charophyte algae, but surprisingly not in related charophytes Zygnematophyceae or Coleochaetophyceae.

Protein assemblages and tight curves in the plasma membranes of photosynthetic eukaryotes.

Protein assemblages in the plasma membrane of photosynthetic organisms include the polar occurrence of PIN proteins permitting polar auxin transport in embryophytes and Charales, and the H+ ATPase in acid zones of Charales cells. Production of small radius of curvature membrane areas in transfer cells and charasomes is incompletely understood.

Surface pH changes suggest a role for H+/OH- channels in salinity response of Chara australis.

To understand salt stress, the full impact of salinity on plant cell physiology has to be resolved. Electrical measurements suggest that salinity inhibits the proton pump and opens putative H+/OH- channels all over the cell surface of salt sensitive Chara australis (Beilby and Al Khazaaly 2009; Al Khazaaly and Beilby 2012). The channels open transiently at first, causing a characteristic noise in membrane potential difference (PD), and after longer exposure remain open with a typical current-voltage (I/V) profile, both abolished by the addition of 1 mM ZnCl2, the main known blocker of animal H+ channels. The cells were imaged with confocal microscopy, using fluorescein isothiocyanate (FITC) coupled to dextran 70 to illuminate the pH changes outside the cell wall in artificial fresh water (AFW) and in saline medium. In the early saline exposure, we observed alkaline patches (bright fluorescent spots) appearing transiently in random spatial distribution. After longer exposure, some of the spots became fixed in space. Saline also abolished or diminished the pH banding pattern observed in the untreated control cells. ZnCl2 suppressed the alkaline spot formation in saline and the pH banding pattern in AFW. The osmotic component of the saline stress did not produce transient bright spots or affect banding. The displacement of H+ from the cell wall charges, the H+/OH- channel conductance/density, and self-organization are discussed. No homologies to animal H+ channels were found. Salinity activation of the H+/OH- channels might contribute to saline response in roots of land plants and leaves of aquatic angiosperms.

Multi-Scale Characean Experimental System: From Electrophysiology of Membrane Transporters to Cell-to-Cell Connectivity, Cytoplasmic Streaming and Auxin Metabolism.

The morphology of characean algae could be mistaken for a higher plant: stem-like axes with leaf-like branchlets anchored in the soil by root-like rhizoids. However, all of these structures are made up of giant multinucleate cells separated by multicellular nodal complexes. The excised internodal cells survive long enough for the nodes to give rise to new thallus. The size of the internodes and their thick cytoplasmic layer minimize impalement injury and allow specific micro-electrode placement. The cell structure can be manipulated by centrifugation, perfusion of cell contents or creation of cytoplasmic droplets, allowing access to both vacuolar and cytoplasmic compartments and both sides of the cell membranes. Thousands of electrical measurements on intact or altered cells and cytoplasmic droplets laid down basis to modern plant electrophysiology. Furthermore, the giant internodal cells and whole thalli facilitate research into many other plant properties. As nutrients have to be transported from rhizoids to growing parts of the thallus and hormonal signals need to pass from cell to cell, Characeae possess very fast cytoplasmic streaming. The mechanism was resolved in the characean model. Plasmodesmata between the internodal cells and nodal complexes facilitate transport of ions, nutrients and photosynthates across the nodes. The internal structure was found to be similar to those of higher plants. Recent experiments suggest a strong circadian influence on metabolic pathways producing indole-3-acetic acid (IAA) and serotonin/melatonin. The review will discuss the impact of the characean models arising from fragments of cells, single cells, cell-to-cell transport or whole thalli on understanding of plant evolution and physiology.

Circadian changes in endogenous concentrations of indole-3-acetic acid, melatonin, serotonin, abscisic acid and jasmonic acid in Characeae (Chara australis Brown).

Giant-celled Characeae (Chara australis Brown), grown for 4 months on 12/12 hr day/night cycle and summer/autumn temperatures, exhibited distinct concentration maxima in auxin (indole-3-acetic acid; IAA), melatonin and serotonin about 4 hr after subjective daybreak. These concentration peaks persisted after 3 day pretreatment in continuous darkness: confirming a circadian rhythm, rather than a response to "light on." The plants pretreated for 3 d in continuous light exhibited several large IAA concentration maxima throughout the 24 hr. The melatonin and serotonin concentrations decreased and were less synchronized with IAA. Chara plants grown on 9/15 hr day/night cycle for 4 months and winter/spring temperatures contained much smaller concentrations of IAA, melatonin and serotonin. The IAA concentration maxima were observed in subjective dark phase. Serotonin concentration peaks were weakly correlated with those of IAA. Melatonin concentration was low and mostly independent of circadian cycle. The "dark" IAA concentration peaks persisted in plants treated for 3 d in the dark. The plants pretreated for 3 d in the light again developed more IAA concentration peaks. In this case the concentration maxima in melatonin and serotonin became more synchronous with those in IAA. The abscisic acid (ABA) and jasmonic acid (JA) concentrations were also measured in plants on winter regime. The ABA concentration did not exhibit circadian pattern, while JA concentration peaks were out of phase with those of IAA. The data are discussed in terms of crosstalk between metabolic pathways.

Salt tolerance at single cell level in giant-celled Characeae.

Characean plants provide an excellent experimental system for electrophysiology and physiology due to: (i) very large cell size, (ii) position on phylogenetic tree near the origin of land plants and (iii) continuous spectrum from very salt sensitive to very salt tolerant species. A range of experimental techniques is described, some unique to characean plants. Application of these methods provided electrical characteristics of membrane transporters, which dominate the membrane conductance under different outside conditions. With this considerable background knowledge the electrophysiology of salt sensitive and salt tolerant genera can be compared under salt and/or osmotic stress. Both salt tolerant and salt sensitive Characeae show a rise in membrane conductance and simultaneous increase in Na(+) influx upon exposure to saline medium. Salt tolerant Chara longifolia and Lamprothamnium sp. exhibit proton pump stimulation upon both turgor decrease and salinity increase, allowing the membrane PD to remain negative. The turgor is regulated through the inward K(+) rectifier and 2H(+)/Cl(-) symporter. Lamprothamnium plants can survive in hypersaline media up to twice seawater strength and withstand large sudden changes in salinity. Salt sensitive C. australis succumbs to 50-100 mM NaCl in few days. Cells exhibit no pump stimulation upon turgor decrease and at best transient pump stimulation upon salinity increase. Turgor is not regulated. The membrane PD exhibits characteristic noise upon exposure to salinity. Depolarization of membrane PD to excitation threshold sets off trains of action potentials, leading to further loses of K(+) and Cl(-). In final stages of salt damage the H(+)/OH(-) channels are thought to become the dominant transporter, dissipating the proton gradient and bringing the cell PD close to 0. The differences in transporter electrophysiology and their synergy under osmotic and/or saline stress in salt sensitive and salt tolerant characean cells are discussed in detail.

Salinity-induced noise in membrane potential of Characeae Chara australis: effect of exogenous melatonin.

Salt sensitive Characeae Chara australis responds to 50 mM NaCl by a prompt appearance of noise in the trans-membrane potential difference (PD). The noise diminishes with time in saline and PD depolarization, leading to altered current-voltage characteristics that could be modeled with H(+)/OH(-) channels. Beilby and Al Khazaaly (JMB 230:21-34, 2009) suggested that the noise might arise from cooperative transient opening of H(+)/OH(-) channels. Presoaking cells in 10 µM melatonin over 24 h abolished the noise in some cells, postponed its appearance in others or changed its characteristics. As melatonin is a very effective antioxidant, we postulated opening of H(+)/OH(-) channels by reactive oxygen species (ROS). Measurement of ROS using dihydrodichlorofluorescein diacetate confirmed substantial reduction in ROS production in melatonin-treated cells in saline and sorbitol media. However, ROS concentration decreased as a function of time in saline medium. Possible schemes for activation of H(+)/OH(-) channels under salinity stress are considered.

A study of the native cell wall structures of the marine alga Ventricaria ventricosa (Siphonocladales, Chlorophyceae) using atomic force microscopy.

A substantial proportion of the architecture of the plant cell wall remains unknown with a few cell wall models being proposed. Moreover, even less is known about the green algal cell wall. Techniques that allow direct visualization of the cell wall in as near to its native state are of importance in unravelling the spatial arrangement of cell wall structures and hence in the development of cell wall models. Atomic force microscopy (AFM) was used to image the native cell wall of living cells of Ventricaria ventricosa (V. ventricosa) at high resolution under physiological conditions. The cell wall polymers were identified mainly qualitatively via their structural appearance. The cellulose microfibrils (CMFs) were easily recognizable and the imaging results indicate that the V. ventricosa cell wall has a cross-fibrillar structure throughout. We found the native wall to be abundant in matrix polysaccharides existing in different curing states. The soft phase matrix polysaccharides susceptible by the AFM scanning tip existed as a glutinous fibrillar meshwork, possibly incorporating both the pectic- and hemicellulosic-type substances. The hard phase matrix producing clearer images, revealed coiled fibrillar structures associated with CMFs, sometimes being resolved as globular structures by the AFM tip. The coiling fibrillar structures were also seen in the images of isolated cell wall fragments. The mucilaginous component of the wall was discernible from the gelatinous cell wall matrix as it formed microstructural domains over the surface. AFM has been successful in imaging the native cell wall and revealing novel findings such as the 'coiling fibrillar structures' and cell wall components which have previously not been seen, that is, the gelatinous matrix phase.

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.

Zinc ions block H⁺/OH⁻ channels in Chara australis.

Chara australis cells exposed to media of pH 10 and above exhibit high conductance, arising from the opening of H⁺/OH⁻ channels in the plasma membrane. This high conductance can be totally inhibited by 1.0 mm ZnCl2 and restored by 0.5 mm 2-mercaptoethanol (ME). Important for carbon fixation, H⁺/OH⁻ channels play a key role in cell pH banding. Banding was also shown to be abolished by 1.0 mm ZnCl2 and restored in some cells by ME. The proton pump is also involved in banding, but was little affected by ZnCl2 over the periods needed for the inhibition of H⁺/OH⁻ channels. Previously, we postulated that H⁺/OH⁻ channels open transiently at the onset of saline stress in salt-sensitive C. australis, causing membrane potential difference (PD) noise; and remain open in latter stages of saline stress, contributing to cell deterioration. ZnCl2 totally inhibited the saline noise and the upwardly concave I/V characteristics associated with the putative H⁺/OH⁻ currents. Again, ME reversed both these effects. We discuss the mode of action of zinc ions and ME with reference to animal voltage-gated H⁺ channels and water channels.

The role of H(+)/OH(-) channels in the salt stress response of Chara australis.

We investigate the electrophysiological salt stress response of the salt-sensitive charophyte Chara australis as a function of time in saline artificial pond water (saline APW) containing 50 mM NaCl and 0.1 mM CaCl(2). The effects are due to an increase in Na(+) concentration rather than an increase in Cl(-) concentration or medium osmolarity. A previous paper (Shepherd et al. Plant Cell Environ 31:1575-1591, 2008) described the rise in the background conductance and inhibition of proton pumping in saline APW in the first 60 min. Here we investigate the shift of membrane potential difference (PD) to levels above -100 mV and the change of shape of the current-voltage (I/V) profiles to upwardly concave. Arguing from thermodynamics, the I/V characteristics can be modeled by channels that conduct H(+) or OH(-). OH(-) was chosen, as H(+) required an unrealistic increase in the number/permeability of the channels at higher pH levels. Prolonged exposure to saline APW stimulated opening of more OH(-) channels. Recovery was still possible even at a PD near -50 mV, with partial return of proton pumping and a decrease in OH(-) current following APW wash. Upon change of pH from 7 to 9, the response was consistent with previously observed I/V characteristics of OH(-) channels. For a pH change to 6, the response was transient before channel closure but could still be modeled. The consequences of opening of H(+) or OH(-) channels while the cell is under salt stress are discussed.

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 characteristics of Ca -activated Cl- channels of the salt-tolerant charophyte Lamprothamnium.

The dependence of the Ca++-activated Cl- channels on potential difference (PD) was extracted from current-voltage (I/V) profiles recorded at the time of hypotonic regulation while the large conductance (G) K+ channels were blocked by tetraethylammonium (TEA). The total clamp current (I) was dominated by the Cl- I, i(Cl), with small contribution from the background I (i(background)). The i(Cl) was fitted by the Goldman-Hodgkin-Katz (GHK) model with enhanced PD dependence simulated by Boltzmann probability distributions. The i(background) was modelled by an empirical equation. The i(Cl) responded to PD changes within tens of milliseconds. The G maxima were located between -20 and -150 mV. The Cl- channel number and channel permeability parameter, N(Cl)P(Cl), decreased as a function of time in a hypotonic medium (from 0.45 x 10(-7) to 0.17 x 10(-7) ms(-1) in 19 min), with the positive half activation PD, V50+, shifting from +35 to -65 mV, and the negative half activation PD, V50-, shifting from -134 to -310 mV. The fitted Cl- concentration [Cl-]cyt at the time of hypotonic regulation indicated rapid equalization of vacuolar and cytoplasmic concentrations. Excellent data obtained under similar experimental conditions in a previous study enabled us to infer [Ca++]cyt influences on the Cl- channel characteristics. Thick sulphated polysaccharide mucilage, found on Lamprothamnium cells acclimated to more saline media, eliminated the activation of the i(Cl) at the time of the hypotonic regulation. This effect was reversed by the application of the enzyme heparinase. The characteristics of the i(Cl) were found to be consistent with a component of the excitation Is at the time of the action potential (AP). The short duration of the excitation transients was contrasted with that of the hypotonic regulation. The mechanisms for Cl- channel activation (and hence the Ca++ channel activation) were considered.