A charged existence: A century of transmembrane ion transport in plants

Abstract

If the past century marked the birth of membrane transport as a focus for research in plants, the past 50 years has seen the field mature from arcane interest to a central pillar of plant physiology. Ion transport across plant membranes accounts for roughly 30% of the metabolic energy consumed by a plant cell, and it underpins virtually every aspect of plant biology, from mineral nutrition, cell expansion, and development to auxin polarity, fertilization, plant pathogen defense, and senescence. The means to quantify ion flux through individual transporters, even single channel proteins, became widely available as voltage clamp methods expanded from giant algal cells to the fungus Neurospora crassa in the 1970s and the cells of angiosperms in the 1980s. Here, I touch briefly on some key aspects of the development of modern electrophysiology with a focus on the guard cells of stomata, now without dispute the premier plant cell model for ion transport and its regulation. Guard cells have proven to be a crucible for many technical and conceptual developments that have since emerged into the mainstream of plant science. Their study continues to provide fundamental insights and carries much importance for the global challenges that face us today.

Figure 1.

Alternative models for high-affinity K⁺ transport and data critical for selecting between them. A) The H⁺/K⁺-ATPase “slippage” model effectively postulated two interchangeable modes of operation for the pump: one that hydrolyses ATP to transport H⁺ out of the cell (above) and contributes to membrane voltage, and the second that additionally transports H⁺ out of the cell in exchange for K⁺ uptake (below). The second mode was proposed to engage on K⁺ starvation and to operate concurrently with the first mode in order to explain membrane hyperpolarization. B) The chemiosmotic model proposes that the H⁺-ATPase (above) and a separate H⁺–K⁺ symporter (below) are coupled across a common membrane. Disabling the symporter by removing K⁺ outside would cause the H⁺-ATPase to hyperpolarise the membrane toward its equilibrium voltage, offset only by a small background conductance (not shown). In the presence of K⁺ outside, the membrane would depolarize, with the current through the H⁺-ATPase balanced by the symporter so that two H⁺ ions would pass through the H⁺-ATPase for every pair of charges, one H⁺ and one K⁺, returning through the symporter. C) Current–voltage (IV) curves recorded from K⁺-starved Neurospora before (❍), upon adding 5 µM K⁺ (♦), and after removing K⁺ from the bath (●). The current introduced at the free-running voltage on adding K⁺ (I_K) is indicated (red arrow). Insets: Simplified circuit for recordings from spherical cells of Neurospora and the free-running voltage before, during, and after adding 5 µM K⁺ (free-running voltages and time scale indicated by trace). The simplified circuit comprises an amplifier (triangle) that reports the membrane voltage (V_m) from electrode impaled in the spherical cell (circle) referenced to the earthed bath solution. D) Net chemical flux (⁴²K⁺, ●) and current at the free-running voltage (❍) determined from concurrent radiotracer and voltage clamp recordings of Neurospora such as shown in (C). Current here converted to units of pmol cm⁻² s⁻¹ for direct comparison with chemical flux. The results show that two charges move across the membrane with each K⁺ ion. Data in (C) and (D) are redrawn from Rodriguez-Navarro et al. (1986).

Figure 2.

The charge and ionic circuits typical of plant membranes. A) A minimal charge circuit for a plant membrane, incorporating a H⁺-ATPase and K⁺ channel, illustrates the codependence of the two arms of the current cycle, outward via the H⁺-ATPase and inward via the channel (central schematic and green arrows), and their impacts on membrane voltage. In the steady state, charge movement (current) through the two arms must always be equal in amplitude and opposite in direction across the membrane (vertical black and red arrows). Thus, from the central current–voltage (IV) plot (1), membrane hyperpolarization is possible (2) by increasing the H⁺-ATPase activity (black curve), and/or (3) by decreasing the activity of the current return pathway through the K⁺ channel (red curve). Conversely, membrane depolarization is possible (4) by increasing the activity of the current return pathway through the K⁺ channel and/or (5) by decreasing the H⁺-ATPase activity. The initial and final free-running voltages in each case is marked by a red circle and the shift in voltage marked by horizontal blue arrows in each plot. Previous H⁺-ATPase and K⁺ channel activities from the central plot are shown as dotted lines in each case. B) Simplified charge and chemical (H⁺) circuits of the plasma membrane illustrated with arrows weighted to show the overall balance of chemical (H⁺) and charge flux. A similar pair of circuits occurs at the tonoplast. Physical laws require that the net charge flux (green arrows) across the membrane in the steady state must sum to zero. [Note: The chemical (H⁺, red arrows) circuit does not need to balance in the same way.] In other words, at the free-running membrane voltage charge passing out of the cell, here shown as charge movement through the H⁺-ATPase (I_P), must be the same as the sum of charges passing back into the cell through the other transporters (active charge-carrying transport, I_HCl, I_Ki, I_HK). As a consequence, a change in charge flux through any one transport pathway will affect the balance of charge movement through all of the other transporters that move charge. Note that one-to-one exchange transport (antiport) of a positively charged solute with H⁺, here shown as a Na⁺/H⁺ antiporter (Quintero et al. 2000; Pardo et al. 2006; Bassil and Blumwald 2014), does not result in net charge movement across the membrane and therefore does not contribute to charge balance. C) The simplified charge circuit of the plasma membrane in (B) illustrated as current–voltage (IV) curves for each of the transporters to show the capacity for current through each transporter as a function of voltage. Total membrane current (I_tot) comprises the sum of the transporter currents, here the H⁺-ATPase (I_P), the H⁺–K⁺ (I_HK), and H⁺–Cl⁻ (I_HCl) symports, and the inward- and outward-rectifying K⁺ channels (I_Ki and I_Ko). At the free-running voltage, the point at which I_tot crosses the voltage axis, the vector sum of these currents is zero (right). Note that the outward-rectifying K⁺ channels contribute to I_tot but, because of their gating properties, only at voltages well positive of the free-running voltage.

Figure 3.

Current spread under voltage clamp in different cell geometries. Cell geometry is a critical factor in voltage clamp studies that make use of microelectrodes for clamp-current injection. Shown in each illustration is the current spread over the cell membrane as red arrows, with the arrow weight indicating the current density. In every case, the membrane is the primary resistance to ground (the external bath). The voltage clamp circuit in each illustration comprises two amplifiers (triangles). A voltage follower amplifier reports the membrane voltage from the right-most electrode; a comparator amplifier, labeled with C, compares this voltage with a command voltage input (not shown) and then returns a current to the cell through the other electrode(s) to correct for any difference between the recorded and command voltages. Also shown is a voltage step (ΔV) reported at the follower amplifier output, with the command step indicated in red and offset for visibility. A) Voltage clamp of a spherical or near-spherical cell ensures a uniform current distribution over the cell membrane. B) Voltage clamp of a near-cylindrical cell, here shown as a root hair cell, leads to current dissipation along the length of the cell as if it were an infinite cable. Thus, current passing across the membrane is reduced with distance away from the point of injection as current passes across the more proximal surfaces. The decline in the voltage step (ΔV) as the membrane current falls off with distance x is shown below. The follower amplifier recording voltage near the root hair tip sees only a fraction of this voltage change. C) Voltage clamp of a 3D matrix (x, y, z frame, below), comprising cells interconnected by plasmodesmata, leads to an ill-defined spread of current over the surfaces of the connected cells. Individual mesophyll cells may present a near-spherical shape, but their interconnections will draw down current locally. Most estimates (Spanswick 1972; Goldsmith and Goldsmith 1978) suggest that 10% to 20% of the current injected into a cell passes to the next through these connections. Thus, the challenge becomes one of defining the cellular interconnections and their spatial distributions.

Figure 4.

Ca²⁺-dependent gating of Ca²⁺ channels needed to support Ca²⁺-induced Ca²⁺ release. A) The cycle of Ca²⁺-induced Ca²⁺ release and its recovery progresses clockwise through four steps in guard cells (see Blatt 2000). Outer insets at each step illustrate the component current-voltage curves for the plasma membrane H⁺-ATPase (I_P, black), the two K⁺ channels (I_Ki, blue; I_Ko, red), and the Cl⁻ channel (I_Cl, green). The free-running membrane voltage in each is indicated by the position of the opposing arrows along the voltage axis (color-coded to the dominant current in each case) and below each cartoon frame (V, dark green arrows). (1) Ca²⁺ influx across the plasma membrane, driven by the H⁺-ATPase and membrane hyperpolarisation, triggers endomembrane Ca²⁺-permeable channels to activate. (2) Endomembrane Ca²⁺ release floods the cytosol to raise [Ca²⁺]_i to micromolar concentrations, suppressing inward-rectifying K⁺ channel and H⁺-ATPase activities and promoting Cl⁻ channel activity, thereby depolarizing the plasma membrane. (3) [Ca²⁺]_i elevation and membrane depolarization promote Ca²⁺-ATPases to resequester the cation and remove it to the apoplast. (4) The lowered [Ca²⁺]_i reduces Cl⁻ channel activity, promotes inward-rectifying K⁺ channel activity and allows the H⁺-ATPase to re-activate, thereby repolarising the plasma membrane. The outward-rectifying K⁺ channel activity is insensitive to [Ca²⁺]_i and parallels that of the Cl⁻ channels, driven principally by the changes in membrane voltage [see also Chen et al (2012) and Blatt (2000)]. B) Ca²⁺-dependent gating characteristics of the 13 pS, plasma membrane Ca²⁺ channel (Ca_in) is consistent with Ca²⁺-dependent inhibition expected of a channel that overlaps with, and triggers, Ca²⁺-induced Ca²⁺ release [data redrawn from Hamilton et al (2000, 2001)]. The Ca²⁺-dependent gating characteristics of the dominant Ca²⁺ release pathway (CICR) requires both Ca²⁺-dependent activation at submicromolar [Ca²⁺]_i, overlapping with the activity of the triggering Ca²⁺ channels, and Ca²⁺-dependent inactivation at supramicromolar [Ca²⁺]_i [curve redrawn from Bezprozvanny et al (1991)]. These characteristics are not a feature of TPC1, which shows only Ca²⁺-dependent activation at [Ca²⁺]_i beyond the effective range for CICR [curve redrawn from data of Schulz-Lessdorf et al (1995) and Pei et al (1999)].

Figure 5.

OnGuard correctly predicts slowed stomatal opening and closing in the ost2 H⁺-ATPase mutant in response to steps in relative humidity. OnGuard modeling outputs (left) and experimental data (right) connect slowed stomatal reopening in the Arabidopsis ost2 mutant with the greatly reduced activity of the inward-rectifying K⁺ channels when compared with wild-type Arabidopsis (wt). Modeling and experimental data consolidated and redrawn from Figs 3, 5, and 6 of Wang et al (2017). A) Stomatal conductances (g_s) predicted (left) and measured (right) as means ± SE of 4 independent experiments. Relative humidity (%RH) steps indicated (above). Corresponding halftimes (t_1/2) for closing and opening (below) were calculated by nonlinear least-squares fitting of model outputs and experimental data to a single exponential function. B) Current–voltage (IV) curves predicted (left) and recorded (right) under voltage clamp as means ± SE of 5 independent experiments. Steady-state currents shown are for the outward- (above) and inward-rectifying (below) K⁺ channels. Insets (right): Representative current relaxations with voltage steps recorded under voltage clamp. Additional data and analysis (Wang et al. 2017) ascribes the enhanced outward-rectifying K⁺ current to a 0.3 unit rise in cytosolic pH and the reduced inward-rectifying K⁺ current to this pH rise and an increase in [Ca²⁺]_i to near 500 nM at rest. Slowed reopening in the ost2 mutant was predicted as a direct consequence of reducing the inward K⁺ current by roughly 90%; the slowed closure was the predicted consequence of hyperpolarizing the membrane and raising pH, thereby suppressing activation of the R-type channels that engage the [Ca²⁺]_i and voltage oscillations needed to promote the outward-rectifying K⁺ channels and accelerate stomatal closure.