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Summary: Guard cells control the opening of stomatal pores in the leaf surface, with the use of a network of protein kinases and phosphatases. Loss of function of the CBL‐interacting protein kinase 23 (CIPK23) was previously shown to decrease the stomatal conductance, but the molecular mechanisms underlying this response still need to be clarified.CIPK23 was specifically expressed in Arabidopsis guard cells, using an estrogen‐inducible system. Stomatal movements were linked to changes in ion channel activity, determined with double‐barreled intracellular electrodes in guard cells and with the two‐electrode voltage clamp technique in Xenopus oocytes.Expression of the phosphomimetic variant CIPK23T190D enhanced stomatal opening, while the natural CIPK23 and a kinase‐inactive CIPK23K60N variant did not affect stomatal movements. Overexpression of CIPK23T190D repressed the activity of S‐type anion channels, while their steady‐state activity was unchanged by CIPK23 and CIPK23K60N.We suggest that CIPK23 enhances the stomatal conductance at favorable growth conditions, via the regulation of several ion transport proteins in guard cells. The inhibition of SLAC1‐type anion channels is an important facet of this response. [ABSTRACT FROM AUTHOR]

Summary: Green plants are equipped with photoreceptors that are capable of sensing radiation in the ultraviolet‐to‐blue and the red‐to‐far‐red parts of the light spectrum. However, plant cells are not particularly sensitive to green light (GL), and light which lies within this part of the spectrum does not efficiently trigger the opening of stomatal pores. Here, we discuss the current knowledge of stomatal responses to light, which are either provoked via photosynthetically active radiation or by specific blue light (BL) signaling pathways. The limited impact of GL on stomatal movements provides a unique option to use this light quality to control optogenetic tools. Recently, several of these tools have been optimized for use in plant biological research, either to control gene expression, or to provoke ion fluxes. Initial studies with the BL‐activated potassium channel BLINK1 showed that this tool can speed up stomatal movements. Moreover, the GL‐sensitive anion channel GtACR1 can induce stomatal closure, even at conditions that provoke stomatal opening in wild‐type plants. Given that crop plants in controlled‐environment agriculture and horticulture are often cultivated with artificial light sources (i.e. a combination of blue and red light from light‐emitting diodes), GL signals can be used as a remote‐control signal that controls stomatal transpiration and water consumption. [ABSTRACT FROM AUTHOR]

Summary: Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone‐pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown.We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R‐GECO1.We show that within 1 min, prior to stomatal closure, O3 triggered a drop in whole‐plant CO2 uptake. Within this early phase, O3 pulses (200–1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long‐term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O3 induced cell death, systemic Ca2+ signals and an irreversible drop in stomatal conductance and photosynthetic capacity.We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic calcium ion (Ca2+) level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals. [ABSTRACT FROM AUTHOR]

Summary: Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca2+ signalling has been intensively studied, the role of the vacuolar membrane remains elusive.A newly developed vacuolar voltage clamp technique was used in combination with live‐cell imaging, to study the role of the vacuolar membrane in Ca2+ and pH homeostasis of bulging root hair cells of Arabidopsis.Depolarisation of the vacuolar membrane caused a rapid increase in the Ca2+ concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses.The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H+/Ca2+ exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage‐dependent vacuolar Ca2+ homeostat could contribute to calcium signalling when coupled to a recently discovered K+ channel‐dependent module for electrical excitability of the vacuolar membrane. [ABSTRACT FROM AUTHOR]

Keywords: 1-naphthaleneacetic acid (1-NAA); auxin; root hairs; triiodobenzoic acid (TIBA); transport EN 1-naphthaleneacetic acid (1-NAA) auxin root hairs triiodobenzoic acid (TIBA) transport 286 292 7 06/17/20 20200715 NES 200715 Introduction Auxin represents a key plant hormone that functions to shape plant development and growth (Leyser, [19]). Nuclear auxin perception by F-box proteins of the I TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX i ( I TIR1/AFB i ) family governs the expression of auxin responsive genes through the degradation of I Aux/IAA i class transcriptional repressors (Leyser, [19]). Error bars show ± SD, n = 4. gl The auxin efflux inhibitor TIBA reduces the driving force of auxin uptake Given that IAA and NAA transport are dependent on the proton motive force (pmf), we next tested the effects of auxin transport inhibitors NPA and TIBA on auxin-induced fast root hair plasma membrane depolarization. Fast auxin signalling depends on a nonnuclear fraction of SCF SP TIR1/AFB sp auxin receptors as well as the activity of the IAA uptake carrier AUX1 (Yang I et al. i , [39]; Dindas I et al. i , [6]). [Extracted from the article]

Summary: During drought, abscisic acid (ABA) induces closure of stomata via a signaling pathway that involves the calcium (Ca2+)‐independent protein kinase OST1, as well as Ca2+‐dependent protein kinases. However, the interconnection between OST1 and Ca2+ signaling in ABA‐induced stomatal closure has not been fully resolved.ABA‐induced Ca2+ signals were monitored in intact Arabidopsis leaves, which express the ratiometric Ca2+ reporter R‐GECO1‐mTurquoise and the Ca2+‐dependent activation of S‐type anion channels was recorded with intracellular double‐barreled microelectrodes.ABA triggered Ca2+ signals that occurred during the initiation period, as well as in the acceleration phase of stomatal closure. However, a subset of stomata closed in the absence of Ca2+ signals. On average, stomata closed faster if Ca2+ signals were elicited during the ABA response. Loss of OST1 prevented ABA‐induced stomatal closure and repressed Ca2+ signals, whereas elevation of the cytosolic Ca2+ concentration caused a rapid activation of SLAC1 and SLAH3 anion channels.Our data show that the majority of Ca2+ signals are evoked during the acceleration phase of stomatal closure, which is initiated by OST1. These Ca2+ signals are likely to activate Ca2+‐dependent protein kinases, which enhance the activity of S‐type anion channels and boost stomatal closure. [ABSTRACT FROM AUTHOR]

ContentsSummary84I.Introduction84II.Stomatal form and biomechanics85III.Stomatal function86IV.Evolution of guard cell ion channels87V.Conclusions88Acknowledgements88Author contributions88References88 Summary: Stomatal pores with apertures that can be adjusted by changes in guard cell turgor have facilitated plant success in dry environments. We explore their evolutionary origins, considering recent findings from bryophytes. Unlike vascular plant stomata, which close to prevent water loss, bryophyte stomata become locked open to promote spore desiccation. We find that the families of ion channels, known to control stomatal movements in angiosperms, are ancient and represented across extant land plants. However, although angiosperm guard cells express specific ion channel genes, none appear specifically expressed in stomata‐bearing moss tissues. Given the evolutionary shift in stomatal function from promotion to prevention of water loss, we postulate that ion channels adopted guard cell‐specific functions after the divergence of bryophytes. [ABSTRACT FROM AUTHOR]

Summary: The membrane‐bound proton‐pumping pyrophosphatase (V‐PPase), together with the V‐type H+‐ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V‐PPases were shown to have improved salinity tolerance, but the relative impact of increasing PPi hydrolysis and proton‐pumping functions has yet to be dissected. For a better understanding of the molecular processes underlying V‐PPase‐dependent salt tolerance, we transiently overexpressed the pyrophosphate‐driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch‐clamp, impalement electrodes and pH imaging. NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt‐untreated conditions, V‐PPase‐overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP‐hyperactive cells from cell death. Furthermore, a salt‐induced rise in V‐PPase but not of V‐ATPase pump currents was detected in nontransformed plants. The results indicate that under normal growth conditions, plants need to regulate the V‐PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V‐PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton‐coupled Na+ sequestration. [ABSTRACT FROM AUTHOR]

Summary: Recent studies have revealed that some responses of fern stomata to environmental signals differ from those of their relatives in seed plants. However, it is unknown whether the biophysical properties of guard cells differ fundamentally between species of both clades. Intracellular micro‐electrodes and the fluorescent Ca2+ reporter FURA2 were used to study voltage‐dependent cation channels and Ca2+ signals in guard cells of the ferns Polypodium vulgare and Asplenium scolopendrium. Voltage clamp experiments with fern guard cells revealed similar properties of voltage‐dependent K+ channels as found in seed plants. However, fluorescent dyes moved within the fern stomata, from one guard cell to the other, which does not occur in most seed plants. Despite the presence of plasmodesmata, which interconnect fern guard cells, Ca2+ signals could be elicited in each of the cells individually. Based on the common properties of voltage‐dependent channels in ferns and seed plants, it is likely that these key transport proteins are conserved in vascular plants. However, the symplastic connections between fern guard cells in mature stomata indicate that the biophysical mechanisms that control stomatal movements differ between ferns and seed plants. [ABSTRACT FROM AUTHOR]

This article is a commentary on Franks & Britton‐Harper, 211: 819–827. [ABSTRACT FROM AUTHOR]

During infection plants recognize microbe-associated molecular patterns ( MAMPs), and this leads to stomatal closure. This study analyzes the molecular mechanisms underlying this MAMP response and its interrelation with ABA signaling., Stomata in intact Arabidopsis thaliana plants were stimulated with the bacterial MAMP flg22, or the stress hormone ABA, by using the noninvasive nanoinfusion technique. Intracellular double-barreled microelectrodes were applied to measure the activity of plasma membrane ion channels., Flg22 induced rapid stomatal closure and stimulated the SLAC1 and SLAH3 anion channels in guard cells. Loss of both channels resulted in cells that lacked flg22-induced anion channel activity and stomata that did not close in response to flg22 or ABA. Rapid flg22-dependent stomatal closure was impaired in plants that were flagellin receptor ( FLS2)-deficient, as well as in the ost1-2 ( Open Stomata 1) mutant, which lacks a key ABA-signaling protein kinase. By contrast, stomata of the ABA protein phosphatase mutant abi1-1 ( ABscisic acid Insensitive 1) remained flg22-responsive., These data suggest that the initial steps in flg22 and ABA signaling are different, but that the pathways merge at the level of OST1 and lead to activation of SLAC1 and SLAH3 anion channels. [ABSTRACT FROM AUTHOR]

Contents [ABSTRACT FROM AUTHOR]

Information about several papers discussed at the 29th New Phytologist Symposium: Stomata in Manchester, England in July 2012 on the global impact of stomatic pores and guard cells is presented. Topics include stomatal development and evolution, climate models and improved crop production. Its logo is also provided which shows the role of such cells in regulating worldwide issues such as climate change and breeding of sustainable crop.

Summary Stomatal closure is known to be associated with early defence responses of plant cells triggered by microbe-associated molecular patterns (MAMPs). However, the molecular mechanisms underlying these guard-cell responses have not yet been elucidated. We therefore studied pathogen-induced changes in ion channel activity in Hordeum vulgare guard cells. Barley mildew ( Blumeria graminis) hyphae growing on leaves inhibited light-induced stomatal opening, starting at 9 h after inoculation, when appressoria had developed. Alternatively, stomatal closure was induced by nano-infusion of chitosan via open stomata into the sub-stomatal cavity. Experiments using intracellular double-barreled micro-electrodes revealed that mildew stimulated S-type (slow) anion channels in guard cells. These channels enable the efflux of anions from guard cells and also promote K+ extrusion by altering the plasma membrane potential. Stimulation of S-type anion channels was also provoked by nano-infusion of chitosan. These data suggest that MAMPs of mildew hyphae penetrating the cuticle provoke activation of S-type anion channels in guard cells. In response, guard cells extrude K+ salts, resulting in stomatal closure. Plasma membrane anion channels probably represent general targets of MAMP signaling in plants, as these elicitors depolarize the plasma membrane of various cell types. [ABSTRACT FROM AUTHOR]

Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae ( Eremosphaera and Chlamydomonas), with those of a multicellular alga ( Chara), liverworts ( Conocephalum), mosses ( Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K+ channels during membrane potential changes. In most other species, the Ca2+-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels. [ABSTRACT FROM AUTHOR]

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe-associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN-SENSING 2 (FLS2) and EF-Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode-impaled mesophyll cells and root hairs of Col-0 Arabidopsis plants revealed rapid, dose-dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion-selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species-independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2-associated receptor-like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1-dependent, calcium-associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway. [ABSTRACT FROM AUTHOR]

Rapid stomatal closure is driven by the activation of S-type anion channels in the plasma membrane of guard cells. This response has been linked to Ca2+ signalling, but the impact of transient Ca2+ signals on S-type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca2+ level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S-type anion channels were monitored using intracellular triple-barrelled micro-electrodes. In cells kept at a holding potential of −100 mV, voltage steps to −180 mV triggered elevation of the cytosolic free Ca2+ concentration. The increase in the cytosolic Ca2+ level was accompanied by activation of S-type anion channels. Guard cell anion channels were activated by Ca2+ with a half maximum concentration of 515 nm (SE = 235) and a mean saturation value of −349 pA (SE = 107) at −100 mV. Ca2+ signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to −100 mV, a transient increase in the cytosolic Ca2+ level was observed, activating S-type channels without measurable delay. These data show that cytosolic Ca2+ elevation can activate S-type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca2+ transport proteins, resulting in an overshoot of the cytosolic Ca2+ level after returning the membrane potential to −100 mV. [ABSTRACT FROM AUTHOR]

Plant cells maintain high Ca2+ concentration gradients between the cytosol and the extracellular matrix, as well as intracellular compartments. During evolution, the regulatory mechanisms, maintaining low cytosolic free Ca2+ concentrations, most likely provided the backbone for the development of Ca2+-dependent signalling pathways. In this review, the current understanding of molecular mechanisms involved in Ca2+ homeostasis of plants cells is evaluated. The question is addressed to which extent the mechanisms, controlling the cytosolic Ca2+ concentration, are linked to Ca2+-based signalling. A large number of environmental stimuli can evoke Ca2+ signals, but the Ca2+-induced responses are likely to differ depending on the stimulus applied. Two mechanisms are put forward to explain signal specificity of Ca2+-dependent responses. A signal may evoke a specific Ca2+ signature that is recognized by downstream signalling components. Alternatively, Ca2+ signals are accompanied by Ca2+-independent signalling events that determine the specificity of the response. The existence of such parallel-acting pathways explains why guard cell responses to abscisic acid (ABA) can occur in the absence, as well as in the presence, of Ca2+ signals. Future research may shed new light on the relation between parallel acting Ca2+-dependent and -independent events, and may provide insights in their evolutionary origin. [ABSTRACT FROM AUTHOR]

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of NtMPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of NtMPK4-silenced plants do not close in elevated atmospheric CO2, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO2 activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in NtMPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca2+ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca2+ than in those with steady Ca2+ levels. Both the darkness-induced anion channel activation and Ca2+ signals were repressed in NtMPK4-silenced guard cells. These data show that CO2 and darkness can activate anion channels in a Ca2+-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca2+ concentration. NtMPK4 plays an essential role in CO2- and darkness-induced activation of guard-cell anion channels, through Ca2+-independent as well as Ca2+-dependent signalling pathways. [ABSTRACT FROM AUTHOR]

Potassium is an inevitable component of plant life, and potassium channels play a pivotal role in plant growth and development. The role of potassium and of K+ channels in plant cell division and cell-cycle progression, however, has not been determined so far. K+ channel blocker studies with synchronized tobacco BY-2 cells revealed that K+ uptake is required for proper cell-cycle progression during the transition from G1 to S phase. Electrophysiological studies (patch-clamp and voltage-clamp techniques) showed a cell-cycle dependency of K+ channel activities and reduced driving force for K+ uptake in dividing cells. Among the four Shaker-like K+ channel genes expressed in BY-2 cells, NKT1 represents an inwardly rectifying K+ channel that mediates K+ uptake. NKT1 is transcriptionally induced during G1 phase, while transcripts of the outward-rectifier NTORK1 dominate S phase. Elongating BY-2 cells appeared hyperpolarized (−101 ± 11 mV), and had elevated osmotic pressure and approximately twice the turgor pressure when compared with depolarized (−64 ± 8 mV) dividing cells. This indicates that cells have to gain a threshold K+ level to re-enter the cell cycle. Based on these findings, turgor regulation through modulation of K+ channel density in plant cell division and cell-cycle progression is discussed. [ABSTRACT FROM AUTHOR]

Guard cells respond to light through two independent signalling pathways. The first pathway is initiated by photosynthetically active radiation and has been associated with changes in the intercellular CO2 concentration, leading to inhibition of plasma membrane anion channels. The second response is blue-light-specific and so far has been restricted to the activation of plasma membrane H+-ATPases. In a search for interactions of both signalling pathways, guard cells of Vicia faba and Arabidopsis thaliana were studied in intact plants. Vicia faba guard cells recorded in CO2-free air responded to blue light with a transient outward plasma membrane current that had an average peak value of 17 pA. In line with previous reports, changes in the current–voltage relation of the plasma membrane indicate that this outward current is based on the activation of H+-ATPases. However, when V. faba guard cells were blue-light-stimulated in air with 700 μl l−1 CO2, the outward current increased to 56 pA. The increase in current was linked to inhibition of S-type anion channels. Blue light also inhibited plasma membrane anion channels in A. thaliana guard cells, but not in the phot1 phot2 double mutant. These results show that blue light inhibits plasma membrane anion channels through a pathway involving phototropins, in addition to the stimulation of guard cell plasma membrane H+-ATPases. [ABSTRACT FROM AUTHOR]

Stomatal openings can be stimulated by light through two signalling pathways. The first pathway is blue light specific and involves phototropins, while the second pathway mediates a response to photosynthetically active radiation (PAR). This second pathway was studied with the use of albino Vicia faba plants and variegated leaves of Chlorophytum comosum. Treatment of V. faba with norflurazon (Nf) inhibits the synthesis of carotenoids and leads to albino leaves with guard cells that lack functional green chloroplasts. Guard cells in albino leaf patches of C. comosum, however, do contain photosynthetically active chloroplasts. Stomata in albino leaf patches of both plants did not respond to red light, although blue light could still induce stomatal opening. This shows that the response to PAR is not functioning in albino leaf patches, even though guard cells of C. comosum harbour chloroplasts. Stomata of Nf-treated plants still responded to CO2 and abscisic acid (ABA). The size of Nf-treated guard cells was increased, but impalement studies with double-barrelled microelectrodes revealed no changes in ion-transport properties at the plasma membrane of guard cells. Blue light could hyperpolarize albino guard cells by triggering outward currents with peak values of 37 pA in albino plants and 51 pA in green control cells. Because of the inhibition of carotenoid biosynthesis, Nf-treated V. faba plants contained only 4% of the ABA content found in green control plants. The ABA dose dependence of anion channel activation in guard cells was shifted in these plants, causing a reduced response to 10 µm ABA. These data show that despite the dramatic changes in physiology caused by Nf, the gross responsiveness of guard cells to blue light, CO2 and ABA remains unaltered. Stomata in albino leaf patches, however, do not respond to PAR, but require photosynthetically active mesophyll cells for this response. [ABSTRACT FROM AUTHOR]

Summary 1 I. Introduction 1 II. The hydrodynamic valve 2 III. Regulation of ion transport in guard cells 14 IV. Interaction between guard cell signaling pathways 18 V. Outlook 20 Acknowledgements 21 References 21 Stomata can be regarded as hydraulically driven valves in the leaf surface, which open to allow CO2 uptake and close to prevent excessive loss of water. Movement of these ‘Watergates’ is regulated by environmental conditions, such as light, CO2 and humidity. Guard cells can sense environmental conditions and function as motor cells within the stomatal complex. Stomatal movement results from the transport of K+ salts across the guard cell membranes. In this review, we discuss the biophysical principles and mechanisms of stomatal movement and relate these to ion transport at the plasma membrane and vacuolar membrane. Studies with isolated guard cells, combined with recordings on single guard cells in intact plants, revealed that light stimulates stomatal opening via blue light-specific and photosynthetic-active radiation-dependent pathways. In addition, guard cells sense changes in air humidity and the water status of distant tissues via the stress hormone abscisic acid (ABA). Guard cells thus provide an excellent system to study cross-talk, as multiple signaling pathways induce both short- and long-term responses in these sensory cells. New Phytologist (2005) doi: 10.1111/j.1469-8137.2005.01460.x© New Phytologist (2005) [ABSTRACT FROM AUTHOR]

During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate increase in plasma membrane conductance (Δ G < 5 nS), ABA predominantly activated voltage-independent (slow (S)-type) anion channels. During strong responses (Δ G > 5 nS), however, ABA activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels. We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl− from guard cells and a rapid but confined reduction in stomatal aperture. [ABSTRACT FROM AUTHOR]

Guard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K[SUP+]-transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO[SUB2]. Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO[SUB2]-free air, depolarized after a switch to air with 700 μI I[SUP-1] CO[SUB2], in a reversible manner. As a result, K[SUP+]-fluxes across the plasma membrane changed direction, to favour K[SUP+] extrusion and stomatal closure in the presence of CO[SUB2]. Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO[SUB2] could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO[SUB2] concentration by about 250 μI I[SUP-1], as measured with a miniature CO[SUB2] sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO[SUB2] concentration. [ABSTRACT FROM AUTHOR]

Summary Here, we discuss why guard cells in intact plants respond to environmental signals in a different way than guard cells in epidermal strips, or protoplasts thereof. In intact leaves stomatal opening is counteracted by epidermal cells that press against the guard cells. Changes in the turgor of epidermal cells therefore can alter the stomatal aperture. In addition, stomatal opening may be modulated by the solute composition of the guard cell wall. Changes in apoplastic K+ , Cl- and Ca2+ occur after light–dark transitions, but not in such a way that it would support stomatal opening. Organic anions may play a role, since they enhance the open probability of anion channels in the plasma membrane. Furthermore, studies with auxin-resistant and abscisic acid-insensitive mutants show that light-induced stomatal opening is modulated by these hormones. Using the newly developed method in which guard cells in the intact plant are impaled with double-barreled electrodes, the role of these apoplastic factors now can be studied on single guard cells that are still in their natural environment. [ABSTRACT FROM AUTHOR]

Summary Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit three physiological states. A minority of cells were classified as far-depolarized cells. These cells exhibited positive membrane potentials and were dominated by the activity of voltage-dependent anion channels. All other cells displayed both outward and inward rectifying K+-channel activity. These cells were either depolarized or hyperpolarized, with average membrane potentials of -41 mV (SD 16) and -112 mV (SD 19), respectively. Depolarized guard cells extrude K+ through outward rectifying channels, while K+ is taken up via inward rectifying channels in hyperpolarized cells. Upon a light/dark transition, guard cells that were hyperpolarized in the light switched to the depolarized state. The depolarization was accompanied by a 35 pA decrease in pump current and an increase in the conductance of inward rectifying channels. Both an increase in pump current and a decrease in the conductance of the inward rectifier were triggered by blue light, while red light was ineffective. From these studies we conclude that light modulates plasma membrane transport through large membrane potential changes, reversing the K+-efflux via outward rectifying channels to a K+-influx via inward rectifying channels. [ABSTRACT FROM AUTHOR]

Abscisic acid‐insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi‐1 and abi‐2, display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA. Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis. The largest light‐induced opening of A. thaliana stomates was found at an external KCl concentration of 50 mM. Stomatal apertures were increased by lowering external Ca2+ to 0.05 mM. The apertures of stomates incubated with 10 μM ABA were not altered by changes in Ca2+ from 0.05 to 1.0 mM. Stomates of all abi mutants showed a light‐stimulated stomatal opening. The opening of wild type and abi‐3 stomates was inhibited by ABA, while stomates of abi‐1 and abi‐2 did not respond to ABA. The insensitivity of abi‐1 and abi‐2 stomates to ABA may thus explain the observed disturbed water relations. [ABSTRACT FROM AUTHOR]