AKT2/3 Subunits Render Guard Cell K+ Channels Ca2+ Sensitive

Inward-rectifying K+ channels serve as a major pathway for Ca2+-sensitive K+ influx into guard cells. Arabidopsis thaliana guard cell inward-rectifying K+ channels are assembled from multiple K+ channel subunits. Following the recent isolation and characterization of an akt2/3-1 knockout mutant, we examined whether the AKT2/3 subunit carries the Ca2+ sensitivity of the guard cell inward rectifier. Quantification of RT-PCR products showed that despite the absence of AKT2 transcripts in guard cells of the knockout plant, expression levels of the other K+ channel subunits (KAT1, KAT2, AKT1, and AtKC1) remained largely unaffected. Patch-clamp experiments with guard cell protoplasts from wild type and akt2/3-1 mutant, however, revealed pronounced differences in Ca2+ sensitivity of the K+ inward rectifier. Wild-type channels were blocked by extracellular Ca2+ in a concentration- and voltage-dependent manner. Akt2/3-1 mutants lacked the voltage-dependent Ca2+ block, characteristic for the K+ inward rectifier. To confirm the akt2/3-1 phenotype, two independent knockout mutants, akt2-1 and akt2::En-1 were tested, demonstrating that the loss of AKT2/3 indeed affects the Ca2+ dependence of guard cell inward rectifier. In contrast to AKT2 knockout plants, AKT1, AtKC1, and KAT1 loss-of-function mutants retained Ca2+ block of the guard cell inward rectifier. When expressed in HEK293 cells, AKT2 channel displayed a pronounced susceptibility toward extracellular Ca2+, while the dominant guard cell K+ channel KAT2 was Ca2+ insensitive. Thus, we conclude that the AKT2/3 subunit constitutes the Ca2+ sensitivity of the guard cell K+ uptake channel

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

Cold Transiently Activates Calcium-Permeable Channels in Arabidopsis Mesophyll Cells

Living organisms are capable of discriminating thermal stimuli from noxious cold to noxious heat. For more than 30 years, it has been known that plant cells respond to cold with a large and transient depolarization. Recently, using transgenic Arabidopsis (Arabidopsis thaliana) expressing the calcium-sensitive protein aequorin, an increase in cytosolic calcium following cold treatment was observed. Applying the patch-clamp technique to Arabidopsis mesophyll protoplasts, we could identify a transient plasma membrane conductance induced by rapid cooling. This cold-induced transient conductance was characterized as an outward rectifying 33 pS nonselective cation channel. The permeability ratio between calcium and cesium was 0.7, pointing to a permeation pore >3.34 Å (ø of cesium). Our experiments thus provide direct evidence for the predicted but not yet measured cold-activated calcium-permeable channel in plants

Diurnal and Light-Regulated Expression of AtSTP1 in Guard Cells of Arabidopsis

Guard cell chloroplasts are unable to perform significant photosynthetic CO(2) fixation via Rubisco. Therefore, guard cells depend on carbon supply from adjacent cells even during the light period. Due to their reversible turgor changes, this import cannot be mediated by plasmodesmata. Nevertheless, guard cells of several plants were shown to use extracellular sugars or to accumulate sucrose as an osmoticum that drives water influx to increase stomatal aperture. This paper describes the first localization of a guard cell-specific Arabidopsis sugar transporter involved in carbon acquisition of these symplastically isolated cells. Expression of the AtSTP1 H(+)-monosacharide symporter gene in guard cells was demonstrated by in situ hybridization and by immunolocalization with an AtSTP1-specific antiserum. Additional RNase protection analyses revealed a strong increase of AtSTP1 expression in the dark and a transient, diurnally regulated increase during the photoperiod around midday. This transient increase in AtSTP1 expression correlates in time with the described guard cell-specific accumulation of sucrose. Our data suggest a function of AtSTP1 in monosaccharide import into guard cells during the night and a possible role in osmoregulation during the day