Membrane voltage initiates Ca2+ waves and potentiates Ca2+ increases with abscisic acid in stomatal guard cells

In higher plants changes and oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca2+]i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca2+]i to voltage and its interaction with ABA. We recorded [Ca2+]i by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near −50 mV and −200 mV were associated with oscillations in [Ca2+]i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca2+]i to increase, often in excess of 1 μM, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca2+]i that spread centripetally from the peripheral cytoplasm within 5–10 s and relaxed over 40–60 s thereafter. The [Ca2+]i increases showed a voltage threshold near −120 mV and were sensitive to external Ca2+ concentration. Substituting Mn2+ for Ca2+ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca2+]i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca2+]i. These results demonstrate a capacity for voltage to evoke [Ca2+]i increases, they point to a dual interaction with ABA in triggering and propagating [Ca2+]i increases, and they implicate a role for voltage in “conditioning” [Ca2+]i signals that regulate ion channels for stomatal function.

Ozone inhibits guard cell K+ channels implicated in stomatal opening

Ozone (O3) deleteriously affects organisms ranging from humans to crop plants, yet little is understood regarding the underlying mechanisms. In plants, O3 decreases CO2 assimilation, but whether this could result from direct O3 action on guard cells remained unknown. Potassium flux causes osmotically driven changes in guard cell volume that regulate apertures of associated microscopic pores through which CO2 is supplied to the photosynthetic mesophyll tissue. We show in Vicia faba that O3 inhibits (i) guard cell K+ channels that mediate K+ uptake that drives stomatal opening; (ii) stomatal opening in isolated epidermes; and (iii) stomatal opening in leaves, such that CO2 assimilation is reduced without direct effects of O3 on photosynthetic capacity. Direct O3 effects on guard cells may have ecological and agronomic implications for plant productivity and for response to other environmental stressors including drought.

Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells

(RS)-2-cis, 4-trans-abscisic acid (ABA), a naturally occurring plant stress hormone, elicited rapid agonist-specific changes in myo-inositol hexakisphosphate (InsP6) measured in intact guard cells of Solanum tuberosum (n = 5); these changes were not reproduced by (RS)-2-trans, 4-trans-abscisic acid, an inactive stereoisomer of ABA (n = 4). The electrophysiological effects of InsP6 were assessed on both S. tuberosum (n = 14) and Vicia faba (n = 6) guard cell protoplasts. In both species, submicromolar concentrations of InsP6, delivered through the patch electrode, mimicked the inhibitory effects of ABA and internal calcium (Cai2+) on the inward rectifying K+ current, IK,in, in a dose-dependent manner. Steady state block of IK,in by InsP6 was reached much more quickly in Vicia (3 min at ≈1 μM) than Solanum (20–30 min). The effects of InsP6 on IK,in were specific to the myo-inositol isomer and were not elicited by other conformers of InsP6 (e.g., scyllo- or neo-). Chelation of Ca2+ by inclusion of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or EGTA in the patch pipette together with InsP6 prevented the inhibition of IK,in, suggesting that the effect is Ca2+ dependent. InsP6 was ≈100-fold more potent than Ins(1,4,5)P3 in modulating IK,in. Thus ABA increases InsP6 in guard cells, and InsP6 is a potent Ca2+-dependent inhibitor of IK,in. Taken together, these results suggest that InsP6 may play a major role in the physiological response of guard cells to ABA.

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K+ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K+ currents, an inactivating outward-rectifying K+ current was characterized (plant A-type current: IAP). IAP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. IAP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to −100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward IAP was mainly carried by K+ ions. In contrast to the sustained outward-rectifying K+ currents, cytosolic alkaline pH was found to inhibit IAP and extracellular K+ was required for IAP activity. Furthermore, increasing cytosolic free Ca2+ in the physiological range strongly inhibited IAP activity with a half inhibitory concentration of ≈ 94 nM. We present a detailed characterization of an inactivating K+ current in a higher plant cell. Regulation of IAP by diverse factors including membrane potential, cytosolic Ca2+ and pH, and extracellular K+ and Ca2+ implies that the inactivating IAP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.

Evidence for the existence of a sulfonylurea-receptor-like protein in plants: Modulation of stomatal movements and guard cell potassium channels by sulfonylureas and potassium channel openers

Limitation of water loss and control of gas exchange is accomplished in plant leaves via stomatal guard cells. Stomata open in response to light when an increase in guard cell turgor is triggered by ions and water influx across the plasma membrane. Recent evidence demonstrating the existence of ATP-binding cassette proteins in plants led us to analyze the effect of compounds known for their ability to modulate ATP-sensitive potassium channels (K-ATP) in animal cells. By using epidermal strip bioassays and whole-cell patch-clamp experiments with Vicia faba guard cell protoplasts, we describe a pharmacological profile that is specific for the outward K+ channel and very similar to the one described for ATP-sensitive potassium channels in mammalian cells. Tolbutamide and glibenclamide induced stomatal opening in bioassays and in patch-clamp experiments, a specific inhibition of the outward K+ channel by these compounds was observed. Conversely, application of potassium channel openers such as cromakalim or RP49356 triggered stomatal closure. An apparent competition between sulfonylureas and potassium channel openers occurred in bioassays, and outward potassium currents, previously inhibited by glibenclamide, were partially recovered after application of cromakalim. By using an expressed sequence tag clone from an Arabidopsis thaliana homologue of the sulfonylurea receptor, a 7-kb transcript was detected by Northern blot analysis in guard cells and other tissues. Beside the molecular evidence recently obtained for the expression of ATP-binding cassette protein transcripts in plants, these results give pharmacological support to the presence of a sulfonylurea-receptor-like protein in the guard-cell plasma membrane tightly involved in the outward potassium channel regulation during stomatal movements.

DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry

Sets of RNA ladders can be synthesized by transcription of a bacteriophage-encoded RNA polymerase using 3′-deoxynucleotides as chain terminators. These ladders can be used for sequencing of DNA. Using a nicked form of phage SP6 RNA polymerase in this study substantially enhanced yields of transcriptional sequencing ladders. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of chain-terminated RNA ladders allowed DNA sequence determination of up to 56 nt. It is also demonstrated that A→G and C→T variations in heterozygous and homozygous samples can be unambiguously identified by the mass spectrometric analysis. As a step towards single-tube sequencing reactions, α-thiotriphosphate nucleotide analogs were used to overcome problems caused by chain terminator-independent, premature termination and by the small mass difference between natural pyrimidine nucleotides.

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells1

Inactivation of inward-rectifying K+ channels (IK,in) by a rise in cytosolic free [Ca2+] ([Ca2+]i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca2+]i action on IK,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca2+]i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording IK,in under a voltage clamp and [Ca2+]i by Fura-2 fluorescence ratiophotometry. IK,in inactivation correlated positively with [Ca2+]i and indicated a Ki of 329 ± 31 nm with cooperative binding of four Ca2+ ions per channel. IK,in was promoted by the Ca2+ channel antagonists Gd3+ and calcicludine, both of which suppressed the [Ca2+]i rise, but the [Ca2+]i rise was unaffected by the K+ channel blocker Cs+. We also found that ryanodine, an antagonist of intracellular Ca2+ channels that mediate Ca2+-induced Ca2+ release, blocked the [Ca2+]i rise, and Mn2+ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca2+]i control of IK,in and to roles for discrete Ca2+ flux pathways in feedback control of the K+ channels by membrane voltage.

Differential Responses of Abaxial and Adaxial Guard Cells of Broad Bean to Abscisic Acid and Calcium1

Regulation by abscisic acid (ABA) and Ca2+ of broad bean (Vicia faba) abaxial and adaxial guard cell movements and inward K+ currents were compared. One millimolar Ca2+ in the bathing medium inhibited abaxial stomatal opening by 60% but only inhibited adaxial stomatal opening by 15%. The addition of 1 μm ABA in the bathing medium resulted in 80% inhibition of abaxial but only 45% inhibition of adaxial stomatal opening. Similarly, ABA and Ca2+ each stimulated greater abaxial stomatal closure than adaxial stomatal closure. Whole-cell patch-clamp results showed that the inward K+ currents of abaxial guard cells were inhibited by 60% (−180 mV) in the presence of 1.5 μm Ca2+ in the cytoplasm, whereas the inward K+ currents of adaxial guard cells were not affected at all by the same treatment. Although 1 μm ABA in the cytoplasm inhibited the inward K+ currents to a similar extent for both abaxial and adaxial guard cells, the former were more sensitive to ABA applied externally. These results suggest that the abaxial stomata are more sensitive to Ca2+ and ABA than adaxial stomata in regard to stomatal opening and closing processes and that the regulation of the inward K+ currents by ABA may not proceed via a Ca2+-signaling pathway in adaxial guard cells. Therefore, there may be different pathways for ABA- and Ca2+-mediated signal transduction in abaxial and adaxial guard cells.

Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel1

Increasing evidence suggests that changes in cytosolic Ca2+ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca2+ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca2+-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca2+ dependent. CDPK exhibits a Ca2+-induced electrophoretic mobility shift and its Ca2+-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca2+ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca2+-dependent protein phosphatase calcineurin enhances the Ca2+-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K+ channel KAT1 protein in a Ca2+-dependent manner. These results suggest that CDPK may be an important component of Ca2+ signaling in guard cells.

Close correspondence between the action spectra for the blue light responses of the guard cell and coleoptile chloroplasts, and the spectra for blue light-dependent stomatal opening and coleoptile phototropism.

Fluorescence spectroscopy was used to characterize blue light responses from chloroplasts of adaxial guard cells from Pima cotton (Gossypium barbadense) and coleoptile tips from corn (Zea mays). The chloroplast response to blue light was quantified by measurements of the blue light-induced enhancement of a red light-stimulated quenching of chlorophyll a fluorescence. In adaxial (upper) guard cells, low fluence rates of blue light applied under saturating fluence rates of red light enhanced the red light-stimulated fluorescence quenching by up to 50%. In contrast, added blue light did not alter the red light-stimulated quenching from abaxial (lower) guard cells. This response pattern paralleled the blue light sensitivity of stomatal opening in the two leaf surfaces. An action spectrum for the blue light-induced enhancement of the red light-stimulated quenching showed a major peak at 450 nm and two minor peaks at 420 and 470 nm. This spectrum matched closely an action spectrum for blue light-stimulated stomatal opening. Coleoptile chloroplasts also showed an enhancement by blue light of red light-stimulated quenching. The action spectrum of this response, showing a major peak at 450 nm, a minor peak at 470 nm, and a shoulder at 430 nm, closely matched an action spectrum for blue light-stimulated coleoptile phototropism. Both action spectra match the absorption spectrum of zeaxanthin, a chloroplastic carotenoid recently implicated in blue light photoreception of both guard cells and coleoptiles. The remarkable similarity between the action spectra for the blue light responses of guard cells and coleoptile chloroplasts and the spectra for blue light-stimulated stomatal opening and phototropism, coupled to the recently reported evidence on a role of zeaxanthin in blue light photoreception, indicates that the guard cell and coleoptile chloroplasts specialize in sensory transduction.

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels--inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels--at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior--including channel insensitivity to ABA-dependent alkalinization of pHi--as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

Strong regulation of slow anion channels and abscisic acid signaling in guard cells by phosphorylation and dephosphorylation events.

Recent evidence suggests that slow anion channels in guard cells need to be activated to trigger stomatal closing and efficiently inactivated during stomatal opening. The patch-clamp technique was employed here to determine mechanisms that produce strong regulation of slow anion channels in guard cells. MgATP in guard cells, serving as a donor for phosphorylation, leads to strong activation of slow anion channels. Slow anion-channel activity was almost completely abolished by removal of cytosolic ATP or by the kinase inhibitors K-252a and H7. Nonhydrolyzable ATP, GTP, and guanosine 5'-[gamma-thio]triphosphate did not replace the ATP requirement for anion-channel activation. In addition, down-regulation of slow anion channels by ATP removal was inhibited by the phosphatase inhibitor okadaic acid. Stomatal closures in leaves induced by the plant hormone abscisic acid (ABA) and malate were abolished by kinase inhibitors and/or enhanced by okadaic acid. These data suggest that ABA signal transduction may proceed by activation of protein kinases and inhibition of an okadaic acid-sensitive phosphatase. This modulation of ABA-induced stomatal closing correlated to the large dynamic range for up- and down-regulation of slow anion channels by opposing phosphorylation and dephosphorylation events in guard cells. The presented opposing regulation by kinase and phosphatase modulators could provide important mechanisms for signal transduction by ABA and other stimuli during stomatal movements.