Dominant Negative Guard Cell K+ Channel Mutants Reduce Inward-Rectifying K+ Currents and Light-Induced Stomatal Opening in Arabidopsis (original) (raw)
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THE PLANT CELL ONLINE, 1997
Inward-rectifying K+ (K+in) channels in the guard cell plasma membrane have been suggested to function as a major pathway for K+ influx into guard cells during stomatal opening. When K+, channels were blocked with external Cs+ in wild-type Arabidopsis guard cells, light-induced stomatal opening was reduced. Transgenic Arabidopsis plants were generated that expressed a mutant of the guard cell K+, channel, KATl, which shows enhanced resistance to the Cs+ block. Stomata in these transgenic lines opened in the presence of external Cs+. Patch-clamp experiments with transgenic guard cells showed that inward K+i, currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KATl functions as a plasma membrane K+ channel in vivo and that K+, channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+, channels in guard cells indicate that components in addition to KATl may contribute to the formation of K+in channels in vivo.
Proceedings of the National Academy of Sciences, 2008
At least four genes encoding plasma membrane inward K ؉ channels (Kin channels) are expressed in Arabidopsis guard cells. A double mutant plant was engineered by disruption of a major K in channel gene and expression of a dominant negative channel construct. Using the patch-clamp technique revealed that this mutant was totally deprived of guard cell Kin channel (GCKin) activity, providing a model to investigate the roles of this activity in the plant. GCKin activity was found to be an essential effector of stomatal opening triggered by membrane hyperpolarization and thereby of blue light-induced stomatal opening at dawn. It improved stomatal reactivity to external or internal signals (light, CO2 availability, and evaporative demand). It protected stomatal function against detrimental effects of Na ؉ when plants were grown in the presence of physiological concentrations of this cation, probably by enabling guard cells to selectively and rapidly take up K ؉ instead of Na ؉ during stomatal opening, thereby preventing deleterious effects of Na ؉ on stomatal closure. It was also shown to be a key component of the mechanisms that underlie the circadian rhythm of stomatal opening, which is known to gate stomatal responses to extracellular and intracellular signals. Finally, in a meteorological scenario with higher light intensity during the first hours of the photophase, GCKin activity was found to allow a strong increase (35%) in plant biomass production. Thus, a large diversity of approaches indicates that GCKin activity plays pleiotropic roles that crucially contribute to plant adaptation to fluctuating and stressing natural environments.
The Plant Journal, 2004
To gain insights into the performance of poplar guard cells, we have measured stomatal conductance and aperture, guard cell K content and K -channel activity of the guard cell plasma membrane in intact poplar leaves. In contrast to Arabidopsis, broad bean and tobacco grown under same conditions, poplar stomata operated just in the dynamic range ± any change in conductance altered the rate of photosynthesis. In response to light, CO 2 and abscisic acid (ABA), the stomatal opening velocity was two to ®ve times faster than that measured for Arabidopsis thaliana, Nicotiana tabacum and Vicia faba. When stomata opened, the K content of guard cells increased almost twofold, indicating that the very fast stomatal opening in this species is mediated via potassium uptake. Following impalement of single guard cells embedded in their natural environment of intact leaves with triple-barrelled microelectrodes, time-dependent inward and outward-rectifying K -channel-mediated currents of large amplitude were recorded. To analyse the molecular nature of genes encoding guard cell K -uptake channels, we cloned K -transporter Populus tremula (KPT)1 and functionally expressed this potassium channel in a K -uptake-de®cient Escherichia coli mutant. In addition to guard cells, this K -transporter gene was expressed in buds, where the KPT1 gene activity strongly correlated with bud break. Thus, KPT1 represents one of only few poplar genes associated with bud¯ush.
Proceedings of the National Academy of Sciences, 2003
Microscopic pores present in the epidermis of plant aerial organs, called stomata, allow gas exchanges between the inner photosynthetic tissue and the atmosphere. Regulation of stomatal aperture, preventing excess transpirational vapor loss, relies on turgor changes of two highly differentiated epidermal cells surrounding the pore, the guard cells. Increased guard cell turgor due to increased solute accumulation results in stomatal opening, whereas decreased guard cell turgor due to decreased solute accumulation results in stomatal closing. Here we provide direct evidence, based on reverse genetics approaches, that the Arabidopsis GORK Shaker gene encodes the major voltage-gated outwardly rectifying K ؉ channel of the guard cell membrane. Expression of GORK dominant negative mutant polypeptides in transgenic Arabidopsis was found to strongly reduce outwardly rectifying K ؉ channel activity in the guard cell membrane, and disruption of the GORK gene (T-DNA insertion knockout mutant) fully suppressed this activity. Bioassays on epidermal peels revealed that disruption of GORK activity resulted in impaired stomatal closure in response to darkness or the stress hormone azobenzenearsonate. Transpiration measurements on excised rosettes and intact plants (grown in hydroponic conditions or submitted to water stress) revealed that absence of GORK activity resulted in increased water consumption. The whole set of data indicates that GORK is likely to play a crucial role in adaptation to drought in fluctuating environments.
Channel-mediated high-affinity K+ uptake into guard cells from Arabidopsis
Proceedings of the National Academy of Sciences, 1999
Potassium uptake by higher plants is the result of high-or low-affinity transport accomplished by different sets of transporters. Although K ؉ channels were thought to mediate low-affinity uptake only, the molecular mechanism of the high-affinity, proton-dependent K ؉ uptake system is still scant. Taking advantage of the high-current resolution of the patch-clamp technique when applied to the small Arabidopsis thaliana guard cells densely packed with voltage-dependent K ؉ channels, we could directly record channels working in the concentration range of high-affinity K ؉ uptake systems. Here we show that the K ؉ channel KAT1 expressed in Arabidopsis guard cells and yeast is capable of mediating potassium uptake from media containing as little as 10 M of external K ؉ . Upon reduction of the external K ؉ content to the micromolar level the voltage dependence of the channel remained unaffected, indicating that this channel type represents a voltage sensor rather than a K ؉ -sensing valve. This behavior results in K ؉ release through K ؉ uptake channels whenever the Nernst potential is negative to the activation threshold of the channel. In contrast to the H ؉coupled K ؉ symport shown to account for high-affinity K ؉ uptake in roots, pH-dependent K ؉ uptake into guard cells is a result of a shift in the voltage dependence of the K ؉ channel. We conclude that plant K ؉ channels activated by acid pH may play an essential role in K ؉ uptake even from dilute solutions.
The Plant Journal, 2006
Plant outward-rectifying K þ channels mediate K þ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K þ concentration, although the ions carrying the current are derived from inside the cell. This K þ dependence confers a sensitivity to the extracellular K þ concentration ([K þ ]) that ensures that the channels mediate K þ efflux only, regardless of the [K þ ] prevailing outside. We investigated the mechanism of K þ -dependent gating of the K þ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K þ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K þ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K þ -sensory process in plants.
A dual role for the OsK5.2 ion channel in stomatal movements and K+ loading into xylem sap
Plant physiology, 2017
The roles of potassium channels from the Shaker family in stomatal movements have been investigated by reverse genetics analyses in Arabidopsis, but corresponding information is lacking outside this model species. Rice and other cereals possess stomata that are more complex than those of Arabidopsis. We examined the role of the outward Shaker K+ channel gene OsK5.2. Expression of the OsK5.2 gene (GUS reporter strategy) was observed in the whole stomatal complex (guard cells and subsidiary cells), in the root vasculature and root cortex. In stomata, loss of OsK5.2 functional expression resulted in lack of time-dependent outward potassium currents in guard cells, higher rates of water loss through transpiration, and severe slowdown of stomatal closure. In line with the expression of OsK5.2 in the plant vasculature, mutant plants displayed a reduced K+ translocation from the root system towards the leaves via the xylem. The comparison between rice and Arabidopsis show that despite the ...
Expression of an Arabidopsis potassium channel gene in guard cells
PLANT PHYSIOLOGY, 1995
The Arabidopsis thaliana KATl cDNA encodes a voltage-gated inward-rectifying K+ channel. A KATl genomic DNA clone was isolated and sequenced, and a 5' promoter and coding sequences containing eight introns were identified. Reporter gene analysis of transgenic plants containing the KATl promoter fused to bacterial P-glucuronidase showed robust P-glucuronidase activity primarily in guard cells.
1995
The swelling of plant motor cells is regulated by various signals with almost unknown mediators. One of the obligatory steps in the signaling cascade is the activation of K+-influx channels -K+ channels activated by hyperpolarization (KH channels). We thus explored the regulation of these channels in our model system, motor cell protoplasts from Samanea saman, using patch-clamp in the "whole cell" configuration. (a) The most novel finding was that the activity of KH channels in situ varied with the time of the day, in positive correlation with cell swelling: in Extensor cells KH channels were active in the earlier part of the day, while in Flexor cells only during the later part of the day; (b) High internal pH promoted the activity of these channels in Extensor cells, opposite to the behavior of the equivalent channels in guard cells, but in conformity with the predicted behavior of the putative KH channel, cloned from S. saman recently; (c) HIgh external K+ concentration...
Molecular basis of plant-specific acid activation of K+ uptake channels
Proceedings of the National Academy of Sciences, 1997
During stomatal opening potassium uptake into guard cells and K ؉ channel activation is tightly coupled to proton extrusion. The pH sensor of the K ؉ uptake channel in these motor cells has, however, not yet been identified. Electrophysiological investigations on the voltage-gated, inward rectifying K ؉ channel in guard cell protoplasts from Solanum tuberosum (KST1), and the kst1 gene product expressed in Xenopus oocytes revealed that pH dependence is an intrinsic property of the channel protein. Whereas extracellular acidification resulted in a shift of the voltage-dependence toward less negative voltages, the single-channel conductance was pH-insensitive. Mutational analysis allowed us to relate this acid activation to both extracellular histidines in KST1. One histidine is located within the linker between the transmembrane helices S3 and S4 (H160), and the other within the putative pore-forming region P between S5 and S6 (H271). When both histidines were substituted by alanines the double mutant completely lost its pH sensitivity. Among the single mutants, replacement of the pore histidine, which is highly conserved in plant K ؉ channels, increased or even inverted the pH sensitivity of KST1. From our molecular and biophysical analyses we conclude that both extracellular sites are part of the pH sensor in plant K ؉ uptake channels.