Enid A. C. MacRobbie

Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells

myo-Inositol hexakisphosphate (InsP6) is the most abundant inositol phosphate in cells, yet it remains the most enigmatic of this class of signaling molecule. InsP6 plays a role in the processes by which the drought stress hormone abscisic acid (ABA) induces stomatal closure, conserving water and ensuring plant survival. Previous work has shown that InsP6 levels in guard cells are elevated in response to ABA, and InsP6 inactivates the plasma membrane inward K+ conductance (IK,in) in a cytosolic calcium-dependent manner. The use of laser-scanning confocal microscopy in dye-loaded patch-clamped guard cell protoplasts shows that release of InsP6 from a caged precursor mobilizes calcium. Measurement of calcium (barium) currents ICa in patch-clamped protoplasts in whole cell mode shows that InsP6 has no effect on the calcium-permeable channels in the plasma membrane activated by ABA. The InsP6-mediated inhibition of IK,in can also be observed in the absence of external calcium. Thus the InsP6-induced increase in cytoplasmic calcium does not result from calcium influx but must arise from InsP6-triggered release of calcium from endomembrane stores. Measurements of vacuolar currents in patch-clamped isolated vacuoles in whole-vacuole mode showed that InsP6 activates both the fast and slow conductances of the guard cell vacuole. These data define InsP6 as an endomembrane-acting calcium-release signal in guard cells; the vacuole may contribute to InsP6-triggered Ca2+ release, but other endomembranes may also be involved.

Membrane transport in stomatal guard cells: The importance of voltage control

SummaryPotassium uptake and export in the resting conditions and in response to the phytohormone abscisic acid (ABA) were examined under voltage clamp in guard cells of Vicia faba L. In 0.1 mm external K+ (with 5 mm Ca2+-HEPES, pH 7.4) two distinct transport states could be identified based on the distribution of the free-running membrane voltage(VM) data in conjunction with the respective I-V and G-V relations. One state was dominated by passive diffusion (mean VM= −143± 4 mV), the other (mean VM= −237± 10 mV) exhibited an appreciable background of primary H+ transport activity. In the presence of pump activity the free-running membrane voltage was negative of the respective K+equilibrium potential (EK+), in 3 and 10 mm external K+. In these cases VMwas also negative of the activation voltage for the inward rectifying K+ current, thus creating a strong bias for passive K+ uptake through inward-rectifying K+ channels. In contrast, when pump activity was absent VMwas situated positive of EK+and cells revealed a bias for K+ efflux. Occasionally spontaneous voltage transitions were observed during which cells switched between the two states. Rapid depolarizations were induced in cells with significant pump activity upon adding 10 μm ABA to the medium. These depolarizations activated current through outward-rectifying K+ channels which was further amplified in ABA by a rise in the ensemble channel conductance. Current-voltage characteristics recorded before and during ABA treatments revealed concerted modulations in current passage through at least four distinct transport processes, results directly comparable to one previous study (Blatt, M.R., 1990, Planta 180:445) carried out with guard cells lacking detectable primary pump activity. Comparative analyses of guard cells in each case are consistent with depolarizations resulting from the activation of an inward-going, as yet unidentified current, rather than an ABA-induced fall in H+-ATPase output. Also observed in a number of cells was an inward-directed current which activated in ABA over a narrow range of voltages positive of -150 mV; this and additional features of the current suggest that it may reflect the ABA-dependent activation of an anion channel previously characterized in Vicia guard cell protoplasts, but rule out its function as the primary mechanism for initial depolarization. The analyses also yield indirect evidence for a rise in cytoplasmic Ca2+ activity in ABA, as well as for a K+ current distinct from the dominant inward and outward-rectifying K+ channels, but neither support nor discount a role for Ca2+ influx in depolarizing the membrane. A striking similarity was found for the modulation of inward currents either in response to ABA or after spontaneous depolarizations. This renders the possibility of an agonist (i.e., ABA) activated Ca2+ current across the plasma membrane as trigger for the voltage transitions unlikely.

FACTS AND MECHANISMS: A COMPARATIVE SURVEY

1. This review aims to survey the process of translocation of solutes in the phloem, including the experimental observations of the process, hypothetical mechanisms with their consequences, and the compatibility of these mechanisms with the experimental information.

THE NATURE OF THE COUPLING BETWEEN LIGHT ENERGY AND ACTIVE ION TRANSPORT IN NITELLA TRANSLUCENS.

Abstract 1. 1. The aim of the work was to decide whether the link between light and the light-driven active ion fluxes in Nitella translucens reflects the consumption of ATP produced by photophosphorylation in the process of ion transport, or is the consequence of a direct coupling between ion transport and photosynthetic electron-transfer reactions. 2. 2. The active uptake of Cl − appears to require the participation of the second light reaction of photosynthesis—that involving the photooxidation of water, for which Cl − is an essential cofactor. Thus the Cl − uptake is not supported by far-red light in which only cyclic photophosphorylation is possible, and is sensitive to low concentrations of dichlorophenyldimethylurea. 3. 3. By contrast the active uptake of K + can be supported by cyclic photophosphorylation alone, in far-red light or in the presence of low concentrations of dichlorophenyldimethylurea. 4. 4. Low concentrations of imidazole, which uncouples photophosphorylation in isolated chloroplasts, inhibit K + uptake while leaving the Cl − flux unaffected. 5. 5. It is suggested that K + uptake is supported by light energy through the utilization of ATP produced in photophosphorylation, but that the Cl − uptake is directly linked to the light-driven electron-transfer reactions associated with the evolution of O 2 in photosynthesis, and does not depend on photophosphorylation. 6. 6. This work provides further evidence for the occurrence of cyclic photophosphorylation in intact cells.

Ion content and aperture in “isolated” guard cells ofCommelina communis L.

Summary“Isolated” guard cells ofCommelina communis L., in epidermal strips in which all cells other than guard cells have been killed by treatment at low pH, will open to a degree dependent on the K (Rb)/Cl(Br) concentration in the bathing medium. Estimates of the changes with aperture of the ion concentrations in the guard cells were made by measurement of86Rb uptake from RbCl, of82Br uptake from K82Br, and of potassium activity with a potassium-sensitive microelectrode. The osmotic effects of such changes were compared with the previous estimates of the osmotic changes required to change the aperture. The results suggest that a substantial fraction of the osmotic pressure of “isolated” guard cells is contributed by solutes other than KCl (or other potassium salts), and that, even in stomata opened by incubation on KCl solutions, a substantial fraction of the increase in osmotic pressure associated with opening is contributed by solutes other than KCl.

Evidence for a role for protein tyrosine phosphatase in the control of ion release from the guard cell vacuole in stomatal closure

Protein tyrosine phosphatases (PTPases) exist in plants, but their role in plant signaling processes is unknown. One of the most important signaling networks in plants concerns the regulation of stomatal aperture, by which closure of stomatal pores restricts water loss in dry conditions, a process essential for plant survival. Closure is achieved by reduction in guard cell volume as a consequence of net efflux of potassium salt at both plasmalemma and tonoplast. To test whether protein tyrosine phosphorylation has any role in guard cell signaling processes, the effects on stomatal aperture and on guard cell K(Rb) fluxes of a number of specific inhibitors of PTPases have been investigated. Stomatal closure induced by abscisic acid, high external Ca2+, hydrogen peroxide, and dark were all prevented by one such inhibitor, phenylarsine oxide, which added to closed stomata promoted reopening. Flux measurements with 86Rb+ identified the efflux across the tonoplast as the sensitive process, implying that protein tyrosine dephosphorylation must occur at or downstream of the Ca2+ signal responsible for triggering ion efflux from the vacuole. There was no inhibition of efflux at the plasmalemma. A second inhibitor of PTPases, 3,4 dephosphatin, gave very similar effects, inhibiting closure induced by abscisic acid, high external Ca2+, and dark, and promoting reopening if added to closed stomata. Again, the efflux of K(Rb) at the tonoplast was the sensitive process. These results provide clear evidence for the involvement of PTPases in a major signaling network in plants.

Potassium content and aperture in “intact” stomatal and epidermal cells ofCommelina communis L

SummaryMeasurements of potassium activity with a potassium-sensitive microelectrode have been made in the cells of the stomatal complex, and in epidermal cells, ofCommelina communis L., as a function of stomatal aperture. The estimated osmotic effects of the changing accumulation of potassium salts in the guard cell have been compared with the previous estimates of the osmotic changes required to open/close the pore. The results suggest that a significant fraction of the osmotic pressure of the guard cells, particularly when closed, is contributed by solutes other than potassium salts. The degree of potassium accumulation may determine the aperture of wide-open stomata, but the potassium changes in the early stages of opening are much too small to account for the osmotic changes required. The difference in potassium contents of “intact” and “isolated” guard cells is close to that required to overcome the previously estimated effect of subsidiary cell turgor on the water relations of the guard cell. In some tissue (but not in all) much more K is lost from epidermal cells than appears in other cells of the complex as the stomata open, and extracellular storage would be required.

Control of Volume and Turgor in Stomatal Guard Cells

Water loss from plants is determined by the aperture of stomatal pores in the leaf epidermis, set by the level of vacuolar accumulation of potassium salt, and hence volume and turgor, of a pair of guard cells. Regulation of ion fluxes across the tonoplast, the key to regulation of stomatal aperture, can only be studied by tracer flux measurements. There are two transport systems in the tonoplast. The first is a Ca2+-activated channel, inhibited by phenylarsine oxide (PAO), responsible for the release of vacuolar K+(Rb+) in response to the “drought” hormone, abscisic acid (ABA). This channel is sensitive to pressure, down-regulated at low turgor and up-regulated at high turgor, providing a system for turgor regulation. ABA induces a transient stimulation of vacuolar ion efflux, during which the flux tracks the ion content (volume, turgor), suggesting ABA reduces the set-point of a control system. The second system, which is PAO-insensitive, is responsible for an ion flux from vacuole to cytoplasm associated with inward water flow following a hypo-osmotic transfer. It is suggested that this involves an aquaporin as sensor, and perhaps also as responder; deformation of the aquaporin may render it ion-permeable, or, alternatively, the deformed aquaporin may signal to an associated ion channel, activating it. Treatment with inhibitors of aquaporins, HgCl2 or silver sulfadiazine, produces a large transient increase in ion release from the vacuole, also PAO-insensitive. It is suggested that this involves the same aquaporin, either rendered directly ion-permeable, or signalling to activate an associated ion channel.

Calcium-dependent and calcium-independent events in the initiation of stomatal closure by abscisic acid

The signal transduction mechanisms by which abscisic acid (ABA) induces net loss of potassium salts from guard cells and closes stomata are not understood. This paper describes the detailed timecourse of the ABA-induced K+(Rb+) efflux transient in guard cells of Commelina communis L. and its dependence on external Ca2+, and compares the effects of short pulses of ABA with that of continuous ABA. The use of short pulses allows the separation of the two phases of the biphasic response. The results show that stimulation of 86Rb+ efflux, in fact, precedes the reported increase in cytoplasmic Ca2+. Furthermore, ABA need not be present on the receptors throughout the full response. Once initiated by a threshold exposure to ABA the response takes over, and the efflux can rise after the removal of ABA. The initial stimulation of efflux requires ABA-occupied receptor sites but is insensitive to external Ca2+. The slower component, likely to reflect the release of vacuolar tracer, and with a timecourse similar to the reported changes in cytoplasmic Ca2+, does not require the continued presence of ABA, but is accelerated by Ca2+. There is also rapid desensitization of the ABA receptors, leaving them unable to respond to reapplication of ABA. The results show that the sequence of events involved in the stomatal closure initiated by ABA is complex, and that only some of the processes initiated are Ca2+-dependent. The initial rapid stimulation of K(Rb) efflux is Ca2+-independent, and is the fastest response to ABA yet reported. This paper clarifies some of the events involved, but the details of the signal transduction remain to be elucidated.

Raising the intracellular level of inositol 1,4,5-trisphosphate changes plasma membrane ion transport in characean algae.

Inositol 1,4,5‐trisphosphate (InsP3) was introduced into the cytoplasm of characean algae in two different ways: (i) by iontophoretic injection into cytoplasm‐enriched fragments from Chara and (ii) by adding InsP3 to the permeabilization medium of locally permeabilized cells of Nitella. In both systems this operation induced a depolarization of the membrane potential, ranging from a few mV to sequences of action potentials. The effect of InsP3 on locally permeabilized Nitella cells was abolished when InsP3 was added together with 30 mM EGTA. When inositol 1,4‐bisphosphate or myo‐inositol were substituted for InsP3 in this system, there was no change in the membrane potential. On the other hand, increasing the free Ca2+ concentration in the permeabilization medium induced, in a similar fashion to InsP3, action potentials. Similarities between InsP3 and Ca2+ action were also observed upon injection into Chara fragments. Both injections increased an inward current. In the first few seconds after injection the current/voltage characteristics of the InsP3‐induced current resembled those of the Ca2(+)‐sensitive current. Subsequently, differences between the InsP3‐ and Ca2(+)‐induced phenomena became apparent in that the InsP3‐induced current continued to increase while the Ca2(+)‐induced current declined, returning to the resting level. Our results suggest that these plant cells contain an InsP3 sensitive system that, under experimental conditions, is able to affect membrane transport via an increase in cytoplasmic free Ca2+.