Peter Ache

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.

GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K(+)-selective, K(+)-sensing ion channel.

Here we report on the molecular identification, guard cell expression and functional characterization of AtGORK, an Arabidopsis thaliana goutward rectifying K+ channel. GORK represents a new member of the plant Shaker K+ channel superfamily. When heterologously expressed in Xenopus oocytes the gene product of GORK mediated depolarization‐activated K+ currents. In agreement with the delayed outward rectifier in intact guard cells and protoplasts thereof, GORK is activated in a voltage‐ and potassium‐dependent manner. Furthermore, the single channel conductance and regulation of GORK in response to pH changes resembles the biophysical properties of the guard cell delayed outward rectifier. Thus GORK very likely represents the molecular entity for depolarization‐induced potassium release from guard cells.

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 Stomatal Response to Reduced Relative Humidity Requires Guard Cell-Autonomous ABA Synthesis

Stomata are pores on the leaf surface, bounded by two guard cells, which control the uptake of CO(2) for photosynthesis and the concomitant loss of water vapor. In 1898, Francis Darwin showed that stomata close in response to reduced atmospheric relative humidity (rh); however, our understanding of the signaling pathway responsible for coupling changes in rh to alterations in stomatal aperture is fragmentary. The results presented here highlight the primacy of abscisic acid (ABA) in the stomatal response to drying air. We show that guard cells possess the entire ABA biosynthesis pathway and that it appears upregulated by positive feedback by ABA. When wild-type Arabidopsis and the ABA-deficient mutant aba3-1 were exposed to reductions in rh, the aba3-1 mutant wilted, whereas the wild-type did not. However, when aba3-1 plants, in which ABA synthesis had been specifically rescued in guard cells, were challenged with dry air, they did not wilt. These data indicate that guard cell-autonomous ABA synthesis is required for and is sufficient for stomatal closure in response to low rh. Guard cell-autonomous ABA synthesis allows the plant to tailor leaf gas exchange exquisitely to suit the prevailing environmental conditions.

KAT1 is not essential for stomatal opening

It is generally accepted that K+ uptake into guard cells via inward-rectifying K+ channels is required for stomatal opening. To test whether the guard cell K+ channel KAT1 is essential for stomatal opening, a knockout mutant, KAT1∷En-1, was isolated from an En-1 mutagenized Arabidopsis thaliana population. Stomatal action and K+ uptake, however, were not impaired in KAT1-deficient plants. Reverse transcription–PCR experiments with isolated guard cell protoplasts showed that in addition to KAT1, the K+ channels AKT1, AKT2/3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inward-rectifying K+ currents across the plasma membrane of both wild-type and KAT1∷En-1 plants. This study demonstrates that multiple K+ channel transcripts exist in guard cells and that KAT1 is not essential for stomatal action.

AtKC1, a silent Arabidopsis potassium channel α-subunit modulates root hair K+ influx

Ion channels in roots allow the plant to gain access to nutrients. The composition of the individual ion channels and the functional contribution of different α-subunits is largely unknown. Focusing on K+-selective ion channels, we have characterized AtKC1, a new α-subunit from the Arabidopsis shaker-like ion channel family. Promoter-β-glucuronidase (GUS) studies identified AtKC1 expression predominantly in root hairs and root endodermis. Specific antibodies recognized AtKC1 at the plasma membrane. To analyze further the abundance and the functional contribution of the different K+ channels α-subunits in root cells, we performed real-time reverse transcription–PCR and patch-clamp experiments on isolated root hair protoplasts. Studying all shaker-like ion channel α-subunits, we only found the K+ inward rectifier AtKC1 and AKT1 and the K+ outward rectifier GORK to be expressed in this cell type. Akt1 knockout plants essentially lacked inward rectifying K+ currents. In contrast, inward rectifying K+ currents were present in AtKC1 knockout plants, but fundamentally altered with respect to gating and cation sensitivity. This indicates that the AtKC1 α-subunit represents an integral component of functional root hair K+ uptake channels.

K+ channel profile and electrical properties of Arabidopsis root hairs

Ion channels and solute transporters in the plasma membrane of root hairs are proposed to control nutrient uptake, osmoregulation and polar growth. Here we analyzed the molecular components of potassium transport in Arabidopsis root hairs by combining K+‐selective electrodes, reverse transcription‐PCR, and patch‐clamp measurements. The two inward rectifiers AKT1 and ATKC1 as well as the outward rectifier GORK dominated the root hair K+ channel pool. Root hairs of AKT1 and ATKC1 loss‐of‐function plants completely lack the K+ uptake channel or exhibited altered properties, respectively. Upon oligochitin‐elicitor treatment of root hairs, transient changes in K+ fluxes and membrane polarization were recorded in wild‐type plants, while akt1‐1 root hairs showed a reduced amplitude and pronounced delay in the potassium re‐uptake process. This indicates that AKT1 and ATKC1 represent essential α‐subunits of the inward rectifier. Green fluorescent protein (GFP) fluorescence following ballistic bombardment with GORK promoter‐GFP constructs as well as analysis of promoter‐GUS lines identified this K+ outward rectifier as a novel ion channel expressed in root hairs. Based on the expression profile and the electrical properties of the root hair plasma membrane we conclude that AKT1‐, ATKC‐ and GORK‐mediated potassium transport is essential for osmoregulation and repolarization of the membrane potential in response to elicitors.

The Arabidopsis Plastidic Glucose 6-Phosphate/Phosphate Translocator GPT1 Is Essential for Pollen Maturation and Embryo Sac Development

Plastids of nongreen tissues can import carbon in the form of glucose 6-phosphate via the glucose 6-phosphate/phosphate translocator (GPT). The Arabidopsis thaliana genome contains two homologous GPT genes, AtGPT1 and AtGPT2. Both proteins show glucose 6-phosphate translocator activity after reconstitution in liposomes, and each of them can rescue the low-starch leaf phenotype of the pgi1 mutant (which lacks plastid phosphoglucoisomerase), indicating that the two proteins are also functional in planta. AtGPT1 transcripts are ubiquitously expressed during plant development, with highest expression in stamens, whereas AtGPT2 expression is restricted to a few tissues, including senescing leaves. Disruption of GPT2 has no obvious effect on growth and development under greenhouse conditions, whereas the mutations gpt1-1 and gpt1-2 are lethal. In both gpt1 lines, distorted segregation ratios, reduced efficiency of transmission in males and females, and inability to complete pollen and ovule development were observed, indicating profound defects in gametogenesis. Embryo sac development is arrested in the gpt1 mutants at a stage before the fusion of the polar nuclei. Mutant pollen development is associated with reduced formation of lipid bodies and small vesicles and the disappearance of dispersed vacuoles, which results in disintegration of the pollen structure. Taken together, our results indicate that GPT1-mediated import of glucose 6-phosphate into nongreen plastids is crucial for gametophyte development. We suggest that loss of GPT1 function results in disruption of the oxidative pentose phosphate cycle, which in turn affects fatty acid biosynthesis.

Identification of Arabidopsis thaliana phloem RNAs provides a search criterion for phloem‐based transcripts hidden in complex datasets of microarray experiments

SUMMARY Phloem-mobile signals play a major role in plant nutrition, development and communication. In the latter context, phloem-mobile RNAs have been associated with signalling between plant tissues. In this study, we focused on the identification of transcripts in the shoot phloem of the model plant Arabidopsis thaliana. To isolate transcripts expressed in phloem parenchyma cells and in companion cell-sieve element complexes, we used laser microdissection coupled to laser pressure catapulting (LMPC). Mobile transcripts in sieve elements were isolated from leaf phloem exudates. After optimization of sampling and fixation, RNA of high quality was isolated from both sources. The modifications to the RNA amplification procedure described here were well suited to production of RNA of sufficient yield and quality for microarray experiments. Microarrays hybridized with LMPC-derived phloem tissue or phloem sap RNA allowed differentiation between phloem-expressed and mobile transcript species. Using this set of phloem transcripts and comparing them with microarrays derived from databases of light, hormone and nutrient treatment experiments, we identified phloem-derived RNAs as mobile, potential long-distance signals. Our dataset thus provides a search criterion for phloem-based signals hidden in the complex datasets of microarray experiments. The availability of these comprehensive phloem transcript profiles will facilitate reverse-genetic studies and forward-genetic screens for phloem and long-distance RNA signalling mutants.