Here we report on the molecular identification, guard cell expression and functional characterization of AtGORK, an Arabidopsis thaliana guard cell outward 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. ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

It is generally accepted that K ؉ uptake into guard cells via inwardrectifying 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, AKT2͞3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inwardrectifying K ؉ currents across the plasma membrane of both wildtype 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.

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 ␣-subunits is largely unknown. Focusing on K ؉ -selective ion channels, we have characterized AtKC1, a new ␣-subunit from the Arabidopsis shaker-like ion channel family. Promoter-␤-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 ␣-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 ␣-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 ␣-subunit represents an integral component of functional root hair K ؉ uptake channels.

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 K K-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 GORKmediated potassium transport is essential for osmoregulation and repolarization of the membrane potential in response to elicitors. ß

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 ϩ -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.