Light-gated channelrhodopsin sparks proton-induced calcium release in guard cells.

Although there has been long-standing recognition that stimuli-induced cytosolic pH alterations coincide with changes in calcium ion (Ca2+) levels, the interdependence between protons (H+) and Ca2+ remains poorly understood. We addressed this topic using the light-gated channelrhodopsin HcKCR2 from the pseudofungus Hyphochytrium catenoides, which operates as a H+ conductive, Ca2+ impermeable ion channel on the plasma membrane of plant cells. Light activation of HcKCR2 in Arabidopsis guard cells evokes a transient cytoplasmic acidification that sparks Ca2+ release from the endoplasmic reticulum. A H+-induced cytosolic Ca2+ signal results in membrane depolarization through the activation of Ca2+-dependent SLAC1/SLAH3 anion channels, which enabled us to remotely control stomatal movement. Our study suggests a H+-induced Ca2+ release mechanism in plant cells and establishes HcKCR2 as a tool to dissect the molecular basis of plant intracellular pH and Ca2+ signaling.

Cracking the code of plant herbivore defense.

In 1916, Ricca hypothesized that plant defense mediators are transported by xylem vessels. While it was discovered that electrical waves generated at plant wounds also transmit information over great distances, the molecular nature of the so-called Ricca factor remained unclear. In this issue of Cell, Gao et al. identify thioglucoside glucohydrolases as a Ricca factor in Arabidopsis.

The CIPK23 protein kinase represses SLAC1-type anion channels in Arabidopsis guard cells and stimulates stomatal opening.

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.

The green light gap: a window of opportunity for optogenetic control of stomatal movement.

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.

Rapid depolarization and cytosolic calcium increase go hand-in-hand in mesophyll cells' ozone response.

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.

Optogenetic control of the guard cell membrane potential and stomatal movement by the light-gated anion channel GtACR1.

Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO2 and water. In turn, guard cell anion channels are seen as key players for stomatal closure, but is activation of these channels sufficient to limit plant water loss? To answer this open question, we used an optogenetic approach based on the light-gated anion channelrhodopsin 1 (GtACR1). In tobacco guard cells that express GtACR1, blue- and green-light pulses elicit Cl- and NO3 - currents of -1 to -2 nA. The anion currents depolarize the plasma membrane by 60 to 80 mV, which causes opening of voltage-gated K+ channels and the extrusion of K+ As a result, continuous stimulation with green light leads to loss of guard cell turgor and closure of stomata at conditions that provoke stomatal opening in wild type. GtACR1 optogenetics thus provides unequivocal evidence that opening of anion channels is sufficient to close stomata.

A voltage-dependent Ca2+ homeostat operates in the plant vacuolar membrane.

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.

Publisher Correction: The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity.

Perception of biotic and abiotic stresses often leads to stomatal closure in plants1,2. Rapid influx of calcium ions (Ca2+) across the plasma membrane has an important role in this response, but the identity of the Ca2+ channels involved has remained elusive3,4. Here we report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid-a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses.

CATION-CHLORIDE CO-TRANSPORTER 1 (CCC1) Mediates Plant Resistance against Pseudomonas syringae.

Plasma membrane (PM) depolarization functions as an initial step in plant defense signaling pathways. However, only a few ion channels/transporters have been characterized in the context of plant immunity. Here, we show that the Arabidopsis (Arabidopsis thaliana) Na+:K+:2Cl- (NKCC) cotransporter CCC1 has a dual function in plant immunity. CCC1 functions independently of PM depolarization and negatively regulates pathogen-associated molecular pattern-triggered immunity. However, CCC1 positively regulates plant basal and effector-triggered resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. In line with the compromised immunity to Pst DC3000, ccc1 mutants show reduced expression of genes encoding enzymes involved in the biosynthesis of antimicrobial peptides, camalexin, and 4-OH-ICN, as well as pathogenesis-related proteins. Moreover, genes involved in cell wall and cuticle biosynthesis are constitutively down-regulated in ccc1 mutants, and the cell walls of these mutants exhibit major changes in monosaccharide composition. The role of CCC1 ion transporter activity in the regulation of plant immunity is corroborated by experiments using the specific NKCC inhibitor bumetanide. These results reveal a function for ion transporters in immunity-related cell wall fortification and antimicrobial biosynthesis.

Anion channel SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure.

In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.

Calcium signals in guard cells enhance the efficiency by which abscisic acid triggers stomatal closure.

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.

Acquiring Control: The Evolution of Stomatal Signalling Pathways.

In vascular plants, stomata balance two opposing functions: they open to facilitate CO2 uptake and close to prevent excessive water loss. Here, we discuss the evolution of three major signalling pathways that are known to control stomatal movements in angiosperms in response to light, CO2, and abscisic acid (ABA). We examine the evolutionary origins of key signalling genes involved in these pathways, and compare their expression patterns between an angiosperm and moss. We propose that variation in stomatal sensitivity to stimuli between plant groups are rooted in differences in: (i) gene presence/absence, (ii) specificity of gene spatial expression pattern, and (iii) protein characteristics and functional interactions.

On the origins of osmotically driven stomatal movements.

Contents Summary 84 I. Introduction 84 II. Stomatal form and biomechanics 85 III. Stomatal function 86 IV. Evolution of guard cell ion channels 87 V. Conclusions 88 Acknowledgements 88 Author contributions 88 References 88 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.

High V-PPase activity is beneficial under high salt loads, but detrimental without salinity.

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.

Mycorrhizal lipochitinoligosaccharides (LCOs) depolarize root hairs of Medicago truncatula.

Arbuscular Mycorrhiza and Root Nodule Symbiosis are symbiotic interactions with a high benefit for plant growth and crop production. Thus, it is of great interest to understand the developmental process of these symbioses in detail. We analysed very early symbiotic responses of Medicago truncatula root hair cells, by stimulation with lipochitinoligosaccharides specific for the induction of nodules (Nod-LCOs), or the interaction with mycorrhiza (Myc-LCOs). Intracellular micro electrodes were used, in combination with Ca2+ sensitive reporter dyes, to study the relations between cytosolic Ca2+ signals and membrane potential changes. We found that sulfated Myc- as well as Nod-LCOs initiate a membrane depolarization, which depends on the chemical composition of these signaling molecules, as well as the genotype of the plants that were studied. A successive application of sulfated Myc-LCOs and Nod-LCOs resulted only in a single transient depolarization, indicating that Myc-LCOs can repress plasma membrane responses to Nod-LCOs. In contrast to current models, the Nod-LCO-induced depolarization precedes changes in the cytosolic Ca2+ level of root hair cells. The Nod-LCO induced membrane depolarization thus is most likely independent of cytosolic Ca2+ signals and nuclear Ca2+ spiking.

Guard cells in fern stomata are connected by plasmodesmata, but control cytosolic Ca2+ levels autonomously.

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.

AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling.

Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca2+ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA- triggered calcium signals, are blocked by treatment with the SCFTIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCFTIR1/AFB receptor and the CNGC14 Ca2+ channel, mediate fast auxin signaling in roots.

A Dual Role for the OsK5.2 Ion Channel in Stomatal Movements and K+ Loading into Xylem Sap.

The roles of potassium channels from the Shaker family in stomatal movements have been investigated by reverse genetics analyses in Arabidopsis (Arabidopsis thaliana), but corresponding information is lacking outside this model species. Rice (Oryza sativa) and other cereals possess stomata that are more complex than those of Arabidopsis. We examined the role of the outward Shaker K+ channel gene OsK5.2. Expression of the OsK5.2 gene (GUS reporter strategy) was observed in the whole stomatal complex (guard cells and subsidiary cells), root vasculature, and root cortex. In stomata, loss of OsK5.2 functional expression resulted in lack of time-dependent outward potassium currents in guard cells, higher rates of water loss through transpiration, and severe slowdown of stomatal closure. In line with the expression of OsK5.2 in the plant vasculature, mutant plants displayed a reduced K+ translocation from the root system toward the leaves via the xylem. The comparison between rice and Arabidopsis show that despite the strong conservation of Shaker family in plants, substantial differences can exist between the physiological roles of seemingly orthologous genes, as xylem loading depends on SKOR and stomatal closure on GORK in Arabidopsis, whereas both functions are executed by the single OsK5.2 Shaker in rice.