Sönke Scherzer

Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair

In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities

In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as –independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca2+-independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein–protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca2+-sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca2+-insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.

Stomatal Closure by Fast Abscisic Acid Signaling Is Mediated by the Guard Cell Anion Channel SLAH3 and the Receptor RCAR1

Plant survival during periods of drought may involve SLAH3, a nitrate-conducting anion channel activated by abscisic acid. Conducting Closure Stomata are pores in the plant epidermis that allow the movement of CO2 into the plant concomitant with the loss of water. The opening and closing of these pores is mediated by the surrounding guard cells, which respond to drought, nutrient availability, and the plant stress hormone abscisic acid (ABA). Geiger et al. identified the anion channel SLAH3 as a player in the guard cell pathway downstream of ABA and defined its mode of regulation through an ABA receptor–phosphatase RCAR1-ABI complex and a calcium-dependent kinase, CPK21. Unlike previously characterized anion channels that are regulated by ABA and contribute to stomatal closure, activation of SLAH3 was promoted by nitrate and was 20 times as permeable to nitrate ions as to chloride ions. Thus, SLAH3 may integrate nitrate signaling and metabolism with signals initiated by drought conditions to control respiration and water loss. S-type anion channels are direct targets of abscisic acid (ABA) signaling and contribute to chloride and nitrate release from guard cells, which in turn initiates stomatal closure. SLAC1 was the first component of the guard cell S-type anion channel identified. However, we found that guard cells of Arabidopsis SLAC1 mutants exhibited nitrate conductance. SLAH3 (SLAC1 homolog 3) was also present in guard cells, and coexpression of SLAH3 with the calcium ion (Ca2+)–dependent kinase CPK21 in Xenopus oocytes mediated nitrate-induced anion currents. Nitrate, calcium, and phosphorylation regulated SLAH3 activity. CPK21-dependent SLAH3 phosphorylation and activation were blocked by ABI1, a PP2C-type protein phosphatase that is inhibited by ABA and inhibits the ABA signaling pathway in guard cells. We reconstituted the ABA-stimulated phosphorylation of the SLAH3 amino-terminal domain by CPK21 in vitro by including the ABA receptor–phosphatase complex RCAR1-ABI1 in the reactions. We propose that ABA perception by the complex consisting of ABA receptors of the RCAR/PYR/PYL family and ABI1 releases CPK21 from inhibition by ABI1, and then CPK21 is further activated by an increase in the cytosolic Ca2+ concentration, leading to its phosphorylation of SLAH3. Thus, the identification of SLAH3 as the nitrate-, calcium-, and ABA-sensitive guard cell anion channel provides insights into the relationship among stomatal response to drought, signaling by nitrate, and nitrate metabolism.

The Venus Flytrap Dionaea muscipula Counts Prey-Induced Action Potentials to Induce Sodium Uptake

Summary Carnivorous plants, such as the Venus flytrap (Dionaea muscipula), depend on an animal diet when grown in nutrient-poor soils. When an insect visits the trap and tilts the mechanosensors on the inner surface, action potentials (APs) are fired. After a moving object elicits two APs, the trap snaps shut, encaging the victim. Panicking preys repeatedly touch the trigger hairs over the subsequent hours, leading to a hermetically closed trap, which via the gland-based endocrine system is flooded by a prey-decomposing acidic enzyme cocktail. Here, we asked the question as to how many times trigger hairs have to be stimulated (e.g., now many APs are required) for the flytrap to recognize an encaged object as potential food, thus making it worthwhile activating the glands. By applying a series of trigger-hair stimulations, we found that the touch hormone jasmonic acid (JA) signaling pathway is activated after the second stimulus, while more than three APs are required to trigger an expression of genes encoding prey-degrading hydrolases, and that this expression is proportional to the number of mechanical stimulations. A decomposing animal contains a sodium load, and we have found that these sodium ions enter the capture organ via glands. We identified a flytrap sodium channel DmHKT1 as responsible for this sodium acquisition, with the number of transcripts expressed being dependent on the number of mechano-electric stimulations. Hence, the number of APs a victim triggers while trying to break out of the trap identifies the moving prey as a struggling Na+-rich animal and nutrition for the plant. Video Abstract

Guard cell SLAC1-type anion channels mediate flagellin-induced stomatal closure

Summary 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.

Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps

Significance The Venus flytrap Dionaea muscipula has been in the focus of scientists since Darwin’s time. Carnivorous plants, with their specialized lifestyle, including insect capture, as well as digestion and absorption of prey, developed unique tools to gain scarce nutrients. In this study, we identified the molecular nature of the uptake machinery for prey-derived potassium and the posttranslational regulation. For the first time, to our knowledge, we functionally characterize DmHAK5 here—a KUP/HAK/KT family member as activated by a CBL-CIPK kinase complex. Detailed electrophysiological characterization identified DmHAK5 as a proton-driven, high-affinity potassium transporter with a weak selectivity. Working hand-in-hand with the low-affinity, high-capacity K+-channel DmKT1 activated by the same kinase, the transporters allow the Venus flytrap to take up prey-derived potassium. The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K+ uptake system in the Venus flytrap. In search of K+ transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K+-transporter genes into Xenopus oocytes, however, both putative K+ transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K+ transporter 1 (AKT1), we coexpressed the putative K+ transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K+ uptake. DmKT1 was found to be a K+-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around −120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K+, reducing its concentration from millimolar levels down to trace levels.

The Dionaea muscipula Ammonium Channel DmAMT1 Provides NH4+ Uptake Associated with Venus Flytrap’s Prey Digestion

BACKGROUND Ammonium transporter (AMT/MEP/Rh) superfamily members mediate ammonium uptake and retrieval. This pivotal transport system is conserved among all living organisms. For plants, nitrogen represents a macronutrient available in the soil as ammonium, nitrate, and organic nitrogen compounds. Plants living on extremely nutrient-poor soils have developed a number of adaptation mechanisms, including a carnivorous lifestyle. This study addresses the molecular nature, function, and regulation of prey-derived ammonium uptake in the Venus flytrap, Dionaea muscipula, one of the fastest active carnivores. RESULTS The Dionaea muscipula ammonium transporter DmAMT1 was localized in gland complexes where its expression was upregulated upon secretion. These clusters of cells decorating the inner trap surface are engaged in (1) secretion of an acidic digestive enzyme cocktail and (2) uptake of prey-derived nutrients. Voltage clamp of Xenopus oocytes expressing DmAMT1 and membrane potential recordings with DmAMT1-expressing Dionaea glands were used to monitor and compare electrophysiological properties of DmAMT1 in vitro and in planta. DmAMT1 exhibited the hallmark biophysical properties of a NH4(+)-selective channel. At depolarized membrane potentials (Vm = 0), the Km (3.2 ± 0.3 mM) indicated a low affinity of DmAMT1 for ammonium that increased systematically with negative going voltages. Upon hyperpolarization to, e.g., -200 mV, a Km of 0.14 ± 0.015 mM documents the voltage-dependent shift of DmAMT1 into a NH4(+) transport system of high affinity. CONCLUSIONS We suggest that regulation of glandular DmAMT1 and membrane potential readjustments of the endocrine cells provide for effective adaptation to varying, prey-derived ammonium sources.

A Single-Pore Residue Renders the Arabidopsis Root Anion Channel SLAH2 Highly Nitrate Selective

Here, SLAH2 was characterized as a nitrate-selective root anion channel in Arabidopsis. Comparison of SLAH2 with chloride and nitrate permeable SLAC1 by 3D-model-based mutagenesis approaches elucidated the molecular nature of the selectivity filter within the S-type anion channel family. In contrast to animal cells, plants use nitrate as a major source of nitrogen. Following the uptake of nitrate, this major macronutrient is fed into the vasculature for long-distance transport. The Arabidopsis thaliana shoot expresses the anion channel SLOW ANION CHANNEL1 (SLAC1) and its homolog SLAC1 HOMOLOGOUS3 (SLAH3), which prefer nitrate as substrate but cannot exclude chloride ions. By contrast, we identified SLAH2 as a nitrate-specific channel that is impermeable for chloride. To understand the molecular basis for nitrate selection in the SLAH2 channel, SLAC1 and SLAH2 were modeled to the structure of HiTehA, a distantly related bacterial member. Structure-guided site-directed mutations converted SLAC1 into a SLAH2-like nitrate-specific anion channel and vice versa. Our findings indicate that two pore-occluding phenylalanines constrict the pore. The selectivity filter of SLAC/SLAH anion channels is determined by the polarity of pore-lining residues located on alpha helix 3. Changing the polar character of a single amino acid side chain (Ser-228) to a nonpolar residue turned the nitrate-selective SLAH2 into a chloride/nitrate-permeable anion channel. Thus, the molecular basis of the anion specificity of SLAC/SLAH anion channels seems to be determined by the presence and constellation of polar side chains that act in concert with the two pore-occluding phenylalanines.

Venus flytrap HKT1-type channel provides for prey sodium uptake into carnivorous plant without conflicting with electrical excitability.

The animal diet of the carnivorous Venus flytrap, Dionaea muscipula, contains a sodium load that enters the capture organ via an HKT1-type sodium channel, expressed in special epithelia cells on the inner trap lobe surface. DmHKT1 expression and sodium uptake activity is induced upon prey contact. Here, we analyzed the HKT1 properties required for prey sodium osmolyte management of carnivorous Dionaea. Analyses were based on homology modeling, generation of model-derived point mutants, and their functional testing in Xenopus oocytes. We showed that the wild-type HKT1 and its Na+- and K+-permeable mutants function as ion channels rather than K+ transporters driven by proton or sodium gradients. These structural and biophysical features of a high-capacity, Na+-selective ion channel enable Dionaea glands to manage prey-derived sodium loads without confounding the action potential-based information management of the flytrap.

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.Auxin regulates multiple aspects of plant growth and development. Here Dindas et al. show that in root-hair cells, the AUX1 auxin influx carrier mediates proton-driven auxin import that is perceived by auxin receptors and coupled to Ca2+ waves that may modulate adaptive responses in the root.