Petra Dietrich

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.

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.

Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells.

Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch‐clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage‐dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two‐electrode voltage‐clamp and patch‐clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage‐dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range ‐160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)‐selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage‐dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells.

Blue light activates calcium-permeable channels in Arabidopsis mesophyll cells via the phototropin signaling pathway

Light is a central regulator of plant growth and development. Among the processes triggered by blue and UV-A light, phototropism, stomatal movement, and chloroplast orientation rely on the activation of blue-light receptors known as phototropins. So far, these photoreceptors constitute a class of light receptor kinases unique to the plant kingdom. In Arabidopsis thaliana, the two members phot1 and phot2 have been shown to display partially overlapping functions. Up to now little is known about the signaling cascade, which links these phototropins to the physiological responses downstream of blue-light perception. Here, we show that on illumination with blue light, but not red light, voltage-dependent and calcium-permeable channels activate in the plasma membrane of mesophyll cells. Blue-light stimulation in the presence of the photosynthetic electron transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicates that blue-light receptors rather than photosynthesis control channel activity. Sensitivity toward the protein kinase inhibitor K252a further pointed to the possible involvement of light receptor kinases. In support of this hypothesis, in the photoreceptor mutant phot1-5, blue-light induction of calcium currents was dramatically reduced and was eliminated in the double mutant phot1-5 phot2-1. By contrast, in cry1-304 cry2-1, an Arabidopsis mutant lacking another class of plant blue-light receptors, the channel remained sensitive to blue light. We thus conclude that blue light triggers calcium fluxes via the phototropin-activated calcium-permeable channel.

Ca2+-Dependent and -Independent Abscisic Acid Activation of Plasma Membrane Anion Channels in Guard Cells of Nicotiana tabacum

Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca2+ concentration. In Vicia faba, however, guard cell anion channels activate in a Ca2+-independent manner. Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca2+ concentration. ABA triggered transient rises in cytoplasmic Ca2+ in the majority of the guard cells (14 out of 19). In seven out of 14 guard cells, the change in cytoplasmic free Ca2+ closely matched the activation of anion channels, while the Ca2+ rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca2+ level. Even though ABA could activate anion channels in N. tabacum guard cells independent of a rise in the cytoplasmic Ca2+ concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca2+ in an ATP-dependent manner. Guard cells thus seem to have evolved both Ca2+-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca2+-independent pathway.

Physiology and biophysics of plant ligand-gated ion channels.

Small molecules and metabolites often act as intra- or extracellular messengers in signal transduction pathways. Ligand-gated ion channels provide a mean to transduce those biochemical signals at the membrane into electrical events and ion fluxes. In plants, cyclic nucleotides and glutamate represent intra- and extracellular signalling ligands, respectively. While the former have been shown to regulate voltage-dependent ion channels and are supposed to activate cyclic nucleotide gated (CNG) channels, the latter are perceived by ionotropic glutamate receptors (GLRs). This review summarises our current knowledge about CNG channels and glutamate receptors in plants and their proposed roles in plant development and adaptation to biotic and abiotic stresses.

Cation sensitivity and kinetics of guard-cell potassium channels differ among species

Abstract.inward rectifying guard cell K+channel, GCKC1in, from three major crop plants Solanum tuberosum L., Nicotiana tabacum L., and Vicia faba L. Selecting guard cells for our analyses we aimed to test whether K+ channels of the same cell type differ among species. The channels shared basic features including voltage-dependence, selectivity and single-channel conductance. They activated at hyperpolarization (V1/2 ≈ −164 mV) with single channels of 7 pS underlying the whole-cell current. The channel density in S. tuberosum was higher than in V. faba and N. tabacum while the activation and deactivation kinetics were faster in the latter two species. Among different monovalent cations the K+ channels discriminated strongly against Na+, Li+, and Cs+. The sensitivity to Cs+ was similar for the three species. Extracellular Ca2+ blocked the V.␣faba K+ channel at concentrations ≥1 mM but only affected its functional homologs in S. tuberosum and N.␣tabacum at higher concentrations and more-negative membrane potentials. Like the differences in Ca2+-sensitivity, protoplasts from the three species differed remarkably in their response towards extracellular pH changes. Whereas protons neither altered the open probability nor the kinetic parameters of the V. faba GCKC1in, in S. tuberosum and N. tabacum this cation affected the voltage-dependent properties strongly. An increase in proton concentration from pH 8.5 to 4.5 shifted the potential of half-maximal open probability to less-negative values with a maximum effect around pH 6.2. The pH modulation of the K+ channels could be described assuming a two-state model where the open and closed channel can be protonated. The observed differences in cation-sensitivity and voltage-dependent kinetics between K+ channels reflect the diversification of guard-cell channels that may contribute to species-specific variations in the control of stomatal aperture.

Salt-dependent regulation of a CNG channel subfamily in Arabidopsis

BackgroundIn Arabidopsis thaliana, the family of cyclic nucleotide-gated channels (CNGCs) is composed of 20 members. Previous studies indicate that plant CNGCs are involved in the control of growth processes and responses to abiotic and biotic stresses. According to their proposed function as cation entry pathways these channels contribute to cellular cation homeostasis, including calcium and sodium, as well as to stress-related signal transduction. Here, we studied the expression patterns and regulation of CNGC19 and CNGC20, which constitute one of the five CNGC subfamilies.ResultsGUS, GFP and luciferase reporter assays were used to study the expression of CNGC19 and CNGC20 genes from Arabidopsis thaliana in response to developmental cues and salt stress. CNGC19 and CNGC20 were differentially expressed in roots and shoots. The CNGC19 gene was predominantly active in roots already at early growth stages. Major expression was observed in the phloem. CNGC20 showed highest promoter activity in mesophyll cells surrounding the veins. Its expression increased during development and was maximal in mature and senescent leaves. Both genes were upregulated in the shoot in response to elevated NaCl but not mannitol concentrations. While in the root, CNGC19 did not respond to changes in the salt concentration, in the shoot it was strongly upregulated in the observed time frame (6-72 hours). Salt-induction of CNGC20 was also observed in the shoot, starting already one hour after stress treatment. It occurred with similar kinetics, irrespective of whether NaCl was applied to roots of intact plants or to the petiole of detached leaves. No differences in K and Na contents of the shoots were measured in homozygous T-DNA insertion lines for CNGC19 and CNGC20, respectively, which developed a growth phenotype in the presence of up to 75 mM NaCl similar to that of the wild type.ConclusionTogether, the results strongly suggest that both channels are involved in the salinity response of different cell types in the shoot. Upon salinity both genes are upregulated within hours. CNGC19 and CNGC20 could assist the plant to cope with toxic effects caused by salt stress, probably by contributing to a re-allocation of sodium within the plant.

Interconversion of fast and slow gating modes of GCAC1, a guard cell anion channel

For guard-cell protoplasts of Vicia faba L. we elucidated whether the slow (S-type) and rapid (R-type) activating anion channels represent different gating modes of GCAC1, the Guard Cell Anion Channel. In the whole-cell configuration of the patch-clamp technique, GCAC1 was activated in a Ca2+- and nucleotide-dependent manner. Cell-free outside-out membrane patches were isolated to determine the relative contribution of different gating modes or channel types to the overall anion current in this cell type. Within 2–15 min after the loss of cytoplasmic control through patch excision, depolarization-activated 38-pS channels characterized by flickering openings in the millisecond range convert into a channel of similar conductance but prolonged open times (hundreds of milliseconds) and lack of pronounced voltage-dependence. The rapid (R-type) and slow (S-type) gating modes exhibited similar ion selectivity but different susceptibility towards block by the stilbene derivative DNDS (4,4′-dinitrostilbene-2,2′-disulfonic acid). On R-type channels DNDS caused a flickering block and a shift in the threshold potential of activation whereas S-type channels remained unaffected. Because of the striking similarities of both channels with respect to single-channel conductance and relative permeability sequence on the one hand, but time-dependent conversion of R- into S-type gating after patch-excision on the other, we conclude that the mode of action of GCAC1 is under the control of cytoplasmic factors.

An IQ Domain Mediates the Interaction with Calmodulin in a Plant Cyclic Nucleotide-Gated Channel

Cyclic nucleotide-gated channels (CNGCs) form non-selective cation entry pathways regulated by calmodulin (CaM), a universal Ca2+ sensor in eukaryotes. Although CaM binding has been shown to be important for Ca2+-dependent feedback regulation of CNGC activity, the CaM-binding properties of these channels have been investigated in a few cases only. We show that CNGC20 from Arabidopsis thaliana binds CaM in a Ca2+-dependent manner and interacts with all AtCaM isoforms but not with the CaM-like proteins CML8 and CML9. CaM interaction with the full-length channel was demonstrated in planta, using bimolecular fluorescence complementation. This interaction occurred at the plasma membrane, in accordance with our localization data of green fluorescent protein (GFP)-fused CNGC20 proteins. The CaM-binding site was mapped to an isoleucine glutamine (IQ) motif, which has not been characterized in plant CNGCs so far. Our results show that compared with the overlapping binding sites for cyclic nucleotides and CaM in CNGCs studied so far, they are sequentially organized in CNGC20. The presence of two alternative CaM-binding modes indicates that ligand regulation of plant CNGCs is more complex than previously expected. Since the IQ domain is conserved among plant CNGCs, this domain adds to the variability of Ca2+-dependent channel control mechanisms underlining the functional diversity within this multigene family.