Optogenetic manipulation of stomatal kinetics improves carbon assimilation, water use, and growth.

Stomata serve dual and often conflicting roles, facilitating carbon dioxide influx into the plant leaf for photosynthesis and restricting water efflux via transpiration. Strategies for reducing transpiration without incurring a cost for photosynthesis must circumvent this inherent coupling of carbon dioxide and water vapor diffusion. We expressed the synthetic, light-gated K+ channel BLINK1 in guard cells surrounding stomatal pores in Arabidopsis to enhance the solute fluxes that drive stomatal aperture. BLINK1 introduced a K+ conductance and accelerated both stomatal opening under light exposure and closing after irradiation. Integrated over the growth period, BLINK1 drove a 2.2-fold increase in biomass in fluctuating light without cost in water use by the plant. Thus, we demonstrate the potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation.

Modelling water use efficiency in a dynamic environment: An example using Arabidopsis thaliana.

Intrinsic water use efficiency (Wi), the ratio of net CO2 assimilation (A) over stomatal conductance to water vapour (gs), is a complex trait used to assess plant performance. Improving Wi could lead in theory to higher productivity or reduced water usage by the plant, but the physiological traits for improvement and their combined effects on Wi have not been clearly identified. Under fluctuating light intensity, the temporal response of gs is an order of magnitude slower than A, which results in rapid variations in Wi. Compared to traditional approaches, our new model scales stoma behaviour at the leaf level to predict gs and A during a diurnal period, reproducing natural fluctuations of light intensity, in order to dissect Wi into traits of interest. The results confirmed the importance of stomatal density and photosynthetic capacity on Wi but also revealed the importance of incomplete stomatal closure under dark conditions as well as stomatal sensitivity to light intensity. The observed continuous decrease of A and gs over the diurnal period was successfully described by negative feedback of the accumulation of photosynthetic products. Investigation into the impact of leaf anatomy on temporal responses of A, gs and Wi revealed that a high density of stomata produces the most rapid response of gs but may result in lower Wi.

Mitochondrial sequestration of BCECF after ester loading in the giant alga Chara australis.

Ratiometric fluorescent dyes are often used to monitor free ion concentrations in vivo, especially in cells that are recalcitrant to transformation with genetically encoded fluorescent markers. Although intracellular dye distributions are often found to be cytosolic, dye localisation has often not been examined in detail. We began exploring the use of BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to monitor pH in the giant alga Chara australis and discovered that younger leaf cells could be loaded using the acetoxymethyl ester of BCECF. However, we were puzzled to find in microphotometric measurements that the fluorescence ratio appeared insensitive to manipulations affecting cytosolic pH. Confocal imaging of C. australis cells loaded with BCECF showed an accumulation of the dye in two locations: (1) on the outside of the chloroplasts in irregularly shaped stationary bodies; (2) within 1-1.5 mum structures that moved rapidly with the pericellular cytoplasmic streaming. Together with the streaming cytoplasm, these organelles were rendered stationary with 50 muM cytochalasin D. Rhodamine 123, a mitochondrionspecific dye, highlighted organelles outside of the chloroplasts, similar to those shown by BCECF in location 1. We conclude that in the cytoplasmic compartment, BCECF was sequestered within cytoplasmic mitochondria in immature and fast-growing cells and within the cortical mitochondrial system in older and slowly growing cells. Thus, BCECF-AM is unsuitable for reporting changes in cytosolic pH in C. australis but might be employed in future to study pH changes in the mitochondria.

Protein-binding partners of the tobacco syntaxin NtSyr1.

Syntaxins and other SNARE (soluble NSF-attachment protein receptor) complex proteins play a key role in the cellular processes of vesicle trafficking, vesicle fusion and secretion. Intriguingly, the SNARE NtSyr1 (=NtSyp121) from Nicotiana tabacum also appears to have a role in signalling evoked by the plant stress hormone abscisic acid. However, partner proteins contributing to its function(s) remain unknown. We used an affinity chromatography approach to identify proteins from tobacco leaf microsomes that directly interact with the hydrophilic (cytosolic) domains of NtSyr1 and report several interacting proteins with sensitivities to the endopeptidase activity of Clostridium botulinum neurotoxins, including one protein that was recognised by alphaAtSNAP33 antiserum, raised against the Arabidopsis SNAP25 homologue. Treatment of microsomal membrane fractions indicated a protein near 55 kDa was sensitive to proteolysis by BotN/A and BotN/E, yielding degradation products of approximately 34 and 23 kDa. Expressed and purified AtSNAP33 also bound directly to the cytosolic domain of NtSyr1 and was sensitive to proteolysis by these toxins, suggesting that NtSyr1, a tobacco homologue of AtSNAP33, and coordinate SNAREs are likely to associate as partners for function in vivo.

Extracellular Ba2+ and voltage interact to gate Ca2+ channels at the plasma membrane of stomatal guard cells.

Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] ([Ca2+](i)) that regulate K+ and Cl- channels for stomatal closure in higher-plant leaves. Under voltage clamp, the initial rate of increase in [Ca2+](i) in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating. To test this idea, we recorded single-channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages. We found that the open probability (P(o)) increased strongly with external Ba2+ concentration, consistent with a 4-fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state. Dwell time analyses indicated the presence of a single open state and at least three closed states of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state. Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage. We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate.

Overexpression of auxin-binding protein enhances the sensitivity of guard cells to auxin.

To explore the role of auxin-binding protein (ABP1) in planta, a number of transgenic tobacco (Nicotiana tabacum) lines were generated. The wild-type KDEL endoplasmic reticulum targeting signal was mutated to HDEL, another common retention sequence in plants, and to KEQL or KDELGL to compromise its activity. The auxin-binding kinetics of these forms of ABP1 were found to be similar to those of ABP1 purified from maize (Zea mays). To test for a physiological response mediated by auxin, intact guard cells of the transgenic plants were impaled with double-barreled microelectrodes, and auxin-dependent changes in K(+) currents were recorded under voltage clamp. Exogenous auxin affected inwardly and outwardly rectifying K(+) currents in a dose-dependent manner. Auxin sensitivity was markedly enhanced in all plants overexpressing ABP1, irrespective of the form present. Immunogold electron microscopy was used to investigate the localization of ABP1 in the transgenic plants. All forms were detected in the endoplasmic reticulum and the KEQL and KDELGL forms passed further across the Golgi stacks than KDEL and HDEL forms. However, neither electron microscopy nor silver-enhanced immunogold epipolarization microscopy revealed differences in cell surface ABP1 abundance for any of the plants, including control plants, which indicated that overexpression of ABP1 alone was sufficient to confer increased sensitivity to added auxin. Jones et al. ([1998] Science 282: 1114-1117) found increased cell expansion in transgenic plants overexpressing wild-type ABP1. Single cell recordings extend this observation, with the demonstration that the auxin sensitivity of guard cell K(+) currents is mediated, at least in part, by ABP1.

Localization and control of expression of Nt-Syr1, a tobacco SNARE protein.

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion and secretion. Previously, we isolated the syntaxin-related protein Nt-Syr1 from Nicotiana in a screen for ABA-related signalling elements, and demonstrated its role in determining the ABA sensitivity of stomatal guard cells. Because the location and expression of SNAREs are often important clues to their functioning, we have examined the distribution and stimulus-dependent expression of Nt-Syr1 between tissues, as well as its location within the cell, using antisera raised against purified recombinant peptides corresponding to overlapping cytosolic domains of Nt-Syr1. The Nt-Syr1 epitope was strongly represented in roots and to lesser extents in stems, leaves and flowers of well-watered plants. Biochemical analysis and examination of immunogold labelling under the electron microscope indicated Nt-Syr1 to be located primarily at the plasma membrane. Expression of the protein in leaves and to a lesser extent in flowers and stems was transiently enhanced by ABA, but not by auxin, kinetin or gibberellic acid. Expression in leaves was promoted by salt stress and wounding, but not by cold. By contrast, Nt-Syr1 levels in the root were unaffected by ABA. In the leaves, enhanced expression of Nt-Syr1 by salt stress was not observed in aba1 mutant Nicotiana, which is deficient in ABA synthesis, and in plants carrying the Arabidopsis abi1 transgene that suppresses a number of ABA-evoked responses in these plants. However, an enhanced expression in response to wounding was observed, even in the mutant backgrounds. We conclude that Nt-Syr1 expression at the plasma membrane is important for its function and is subject to control by parallel, stress-related signalling pathways, both dependent on and independent of ABA. Nt-Syr1 may be associated with additional functions, especially in the roots, that are unrelated to ABA or stress responses in the plant.

Cellular signaling and volume control in stomatal movements in plants.

Stomatal guard cells are unique as a plant cell model and, because of the depth of present knowledge on ion transport and its regulation, offer a first look at signal integration in higher plants. A large body of data indicates that Ca(2+) and H(+) act independently, integrating with protein kinases and phosphatases, to control the gating of the K(+) and Cl(-) channels that mediate solute flux for stomatal movements. Oscillations in the cytosolic-free concentration of Ca(2+) contribute to a signaling cassette, integrated within these events through an unusual coupling with membrane voltage for solute homeostasis. Similar cassettes are anticipated to include control pathways linked to cytosolic pH. Additional developments during the last two years point to events in membrane traffic that play equally important roles in stomatal control. Research in these areas is now adding entirely new dimensions to our understanding of guard cell signaling.

Ca(2+) signalling and control of guard-cell volume in stomatal movements.

Stomatal guard cells are unique as a plant cell model and, because of the depth of knowledge now to hand on ion transport and its regulation, serve as an excellent model for the analysis of stimulus-response coupling in higher plants. Parallel controls - mediated by Ca(2+), H(+) protein kinases and phosphatases - regulate the gating of the K(+) and Cl(-) channels that facilitate solute flux for stomatal movements. A growing body of evidence now indicates that oscillations in the cytosolic free concentration of Ca(2+) contribute to a 'signalling cassette', which is integrated within these events through an unusual coupling with membrane voltage. Additional developments during the past two years point to events in membrane traffic that play complementary roles in stomatal control. Research in these areas, especially, is now adding entirely new dimensions to our understanding of guard cell signalling.

Ca2+ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid.

In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by means of Ca(2+) entry from outside and release from intracellular stores. The mechanism(s) for Ca(2+) flux across the plasma membrane is poorly understood. Because [Ca(2+)](i) increases are voltage-sensitive, we suspected a Ca(2+) channel at the guard cell plasma membrane that activates on hyperpolarization and is regulated by ABA. We recorded single-channel currents across the Vicia guard cell plasma membrane using Ba(2+) as a charge-carrying ion. Both cell-attached and excised-patch measurements uncovered single-channel events with a maximum conductance of 12.8 +/- 0.4 pS and a high selectivity for Ba(2+) (and Ca(2+)) over K(+) and Cl(-). Unlike other Ca(2+) channels characterized to date, these channels rectified strongly toward negative voltages with an open probability (P(o)) that increased with [Ba(2+)] outside and decreased roughly 10-fold when [Ca(2+)](i) was raised from 200 nM to 2 microM. Adding 20 microM ABA increased P(o), initially by 63- to 260-fold; in both cell-attached and excised patches, it shifted the voltage sensitivity for channel activation, and evoked damped oscillations in P(o) with periods near 50 s. A similar, but delayed response was observed in 0.1 microM ABA. These results identify a Ca(2+)-selective channel that can account for Ca(2+) influx and increases in [Ca(2+)](i) triggered by voltage and ABA, and they imply a close physical coupling at the plasma membrane between ABA perception and Ca(2+) channel control.

Functional conservation between yeast and plant endosomal Na(+)/H(+) antiporters.

Vacuolar compartmentation of Na(+) is an essential mechanism for salinity tolerance since it lowers cytosolic Na(+) levels while contributing to osmotic adjustment for cell turgor and expansion. The AtNHX1 protein of Arabidopsis thaliana substituted functionally for ScNHX1, the endosomal Na(+)/H(+) antiporter of yeast. Ion tolerance conferred by AtNHX1 and ScNHX1 correlated with ion uptake into an intracellular pool that was energetically dependent on the vacuolar (H(+))ATPase. AtNHX1 localized to vacuolar membrane fractions of yeast. Hence, both transporters share an evolutionarily conserved function in Na(+) compartmentation. AtNHX1 mRNA levels were upregulated by ABA and NaCl treatment in leaf but not in root tissue.

Mutations in the yeast two pore K+ channel YKC1 identify functional differences between the pore domains.

The K+ channel of Saccharomyces cerevisiae encoded by the YKC1 gene includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarisation and is regulated by the extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Our previous work showed that substitutions of equivalent residues L293 and A428 within the pore-loops had qualitatively similar effects on both the [K+]o-sensitivity of channel gating and its voltage-dependence. Here, we report that mutations of equivalent residues N275 and N410, N-terminal from the K+ channel signature sequences of the two pores, have very different actions on channel gating and, in this case, are without effect on its voltage-sensitivity. The mutation N410D slowed current activation in a [K+]o-dependent manner and it accelerated deactivation, but without significant effect on the apparent affinity for K+. The N275D mutant, by contrast, had little effect on the [K+]o-sensitivity for activation and it greatly altered the. [K+]o-dependence of current deactivation. Neither mutant affected the voltage-dependence of the steady-state current nor the ability for other alkali cations to substitute for K+ in regulating gating. The double mutant N410D-N275D showed characteristics of N410D in the [K+]o-sensitivity of current activation and of N275D in the [K+]o-sensitivity of deactivation, suggesting that little interaction occurs between pore domains with mutations at these sites. The results indicate that the two pore domains are not functionally equivalent and they suggest that the regulation of gating by external K+ is mediated by K+ binding at two physically distinct sites with different actions.

K+ channels of Cf-9 transgenic tobacco guard cells as targets for Cladosporium fulvum Avr9 elicitor-dependent signal transduction.

The Cf-9 gene encodes an extracytosolic leucine-rich repeat (LRR) protein that is membrane anchored near its C-terminus. The protein confers resistance in tomato to races of the fungus Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In Nicotiana tabacum the Cf-9 transgene confers sensitivity to the Avr9 elicitor, and leads on elicitation to a subset of defence responses qualitatively similar to those normally seen in the tomato host. One of the earliest responses, both in the native and transgenic hosts, results in K+ salt loss from the infected tissues. However, the mechanism(s) underlying this solute flux and its control is poorly understood. We have explored the actions of Avr9 on Cf-9 transgenic Nicotiana using guard cells as a model. Much detail of guard cell ion channels and their regulation is already known. Measurements were carried out on intact guard cells in epidermal peels, and the currents carried by inward- (IK,in) and outward-rectifying (IK,out) K+ channels were characterized under voltage clamp. Exposures to Avr9-containing extracts resulted in a 2.5- to 3-fold stimulation of IK,out and almost complete suppression of IK,in within 3-5 min. The K+ channel responses were irreversible. They were specific for the Avr9 elicitor, were not observed in guard cells of Nicotiana lacking the Cf-9 transgene and, from kinetic analyses, could be ascribed to changes in channel gating. Both K+ channel responses were found to be saturable functions of Avr9 concentration and were completely blocked in the presence of 0.5 microM staurosporine and 100 microM H7, both broad-range protein kinase antagonists. These results demonstrate the ability of the Cf-9 transgene to couple Avr9 elicitation specifically to a concerted action on two discrete K+ channels and they indicate a role for protein phosphorylation in Avr9/Cf-9 signal transduction leading to transport control.

A tobacco syntaxin with a role in hormonal control of guard cell ion channels.

The plant hormone abscisic acid (ABA) regulates potassium and chloride ion channels at the plasma membrane of guard cells, leading to stomatal closure that reduces transpirational water loss from the leaf. The tobacco Nt-SYR1 gene encodes a syntaxin that is associated with the plasma membrane. Syntaxins and related SNARE proteins aid intracellular vesicle trafficking, fusion, and secretion. Disrupting Nt-Syr1 function by cleavage with Clostridium botulinum type C toxin or competition with a soluble fragment of Nt-Syr1 prevents potassium and chloride ion channel response to ABA in guard cells and implicates Nt-Syr1 in an ABA-signaling cascade.

Mutations in the pore regions of the yeast K+ channel YKC1 affect gating by extracellular K+.

The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Analysis of the wild-type current now shows that: (i) [K+]o suppresses a very slowly relaxing component, accelerating activation; (ii) [K+]o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating. We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the [K+]o sensitivity. Substitution of these residues gave channels with reduced sensitivity to [K+]o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance. In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low [K+]o. The results indicate that these residues are involved in [K+]o sensing. Their situation close to the permeation pathway points to an interaction between gating and permeation.

Membrane voltage initiates Ca2+ waves and potentiates Ca2+ increases with abscisic acid in stomatal guard cells.

In higher plants changes and oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca2+]i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca2+]i to voltage and its interaction with ABA. We recorded [Ca2+]i by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near -50 mV and -200 mV were associated with oscillations in [Ca2+]i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca2+]i to increase, often in excess of 1 microM, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca2+]i that spread centripetally from the peripheral cytoplasm within 5-10 s and relaxed over 40-60 s thereafter. The [Ca2+]i increases showed a voltage threshold near -120 mV and were sensitive to external Ca2+ concentration. Substituting Mn2+ for Ca2+ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca2+]i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca2+]i. These results demonstrate a capacity for voltage to evoke [Ca2+]i increases, they point to a dual interaction with ABA in triggering and propagating [Ca2+]i increases, and they implicate a role for voltage in "conditioning" [Ca2+]i signals that regulate ion channels for stomatal function.

The role of ABI1 in abscisic acid signal transduction: from gene to cell.

The semi-dominant abi1-1 mutation of Arabidopsis interferes with multiple aspects of abscisic acid signal transduction resulting in reduced seed dormancy and sensitivity of root growth in ABA. Furthermore, the mutant transpires excessively as a result of abnormal stomatal regulation leading to a wilty phenotype. The ABI1 gene has been cloned. The carboxyl-terminal domain of the predicted ABI1 protein is related to the 2C class of serine-threonine phosphatases while no overt homology was found in the extended amino terminus. A combination of in vitro assays and yeast mutant complementation studies confirmed that ABI1 is a functional protein phosphatase 2C. The abi1-1 mutation converts the amino acid glycine180 to aspartic acid, and in the above test systems, causes a partial loss of the phosphatase activity. In transgenic Nicotiana benthamiana guard cells, the abi1-1 gene causes a reduction in the background current of the outward-rectifying potassium channels, and also in the abscisic acid-sensitivity of both the outward- and the inward-rectifying potassium channels in the plasma membrane. However, normal sensitivity of both potassium channels to, and stomatal closure in, abscisic acid was recovered in the presence of H7 and staurosporine, both broad-range protein kinase antagonists. These results suggest the aberrant potassium channel behavior as a major consequence of abi1-1 action and implicate ABI1 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell potassium channels to abscisic acid-evoked signal cascades.

High-affinity NO(3-)-H+ cotransport in the fungus Neurospora: induction and control by pH and membrane voltage.

High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO3- challenge and to quantify transport activity. The NO3(-)-associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO3(-)-free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 microM NO3-. In the latter, induction showed a latency of 40-80 min and rose in scalar fashion with full transport activity measurable approx. 100 min after first exposure to NO3-; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO3- additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 microM NO3-; and it was suppressed when NH4+ was present during the first, inductive exposure to NO3-. Voltage clamp measurements carried out immediately before and following NO3- additions showed that the NO3(-)-evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages (-400 to +100 mV). Measurements of NO3- uptake using NO3(-)-selective macroelectrodes indicated a charge stoichiometry for NO3- transport of 1(+):1(NO3-) with common K(m) and Jmax values around 25 microM and 75 pmol NO3- cm-2sec-1, respectively, and combined measurements of pHo and [NO3-]o showed a net uptake of approx. 1 H+ with each NO3- anion. Analysis of the NO3- current demonstrated a pronounced voltage sensitivity within the normal physiological range between -300 and -100 mV as well as interactions between the kinetic parameters of membrane voltage, pHo and [NO3-]o. Increasing the bathing pH from 5.5 to 8.0 reduced the current and the associated membrane depolarizations 2- to 4-fold. At a constant pHo of 6.1, driving the membrane voltage from -350 to -150 mV resulted in an approx. 3-fold reduction in the maximum current and a 5-fold rise in the apparent affinity for NO3-. By contrast, the same depolarization effected an approx. 20% fall in the K(m) for transport as a function in [H+]o. These, and additional results are consistent with a charge-coupling stoichiometry of 2(H+) per NO3- anion transported across the membrane, and implicate a carrier cycle in which NO3- binding is kinetically adjacent to the rate-limiting step of membrane charge transit. The data concur with previous studies demonstrating a pronounced voltage-dependence to high-affinity NO3- transport system in Arabidopsis, and underline the importance of voltage as a kinetic factor controlling NO3- transport; finally, they distinguish metabolite repression of NO3- transport induction from its sensitivity to metabolic blockade and competition with the uptake of other substrates that draw on membrane voltage as a kinetic substrate.

A new family of K+ transporters from Arabidopsis that are conserved across phyla.

Transport of K+ in higher plants, as in bacteria and fungi, is mediated by two broad classes of transport proteins that operate in the millimolar and micromolar K+ concentration ranges. A search of the Expressed Sequence Tag database using amino acid consensus sequences for the K+ transporters HAK1 from Schwanniomyces and Kup of Escherichia coli yielded two homologous sequences for Arabidopsis. Cloning and sequencing of these genes gave single open reading frames for the putative transporters, AtKT1 and AtKT2, with predicted molecular weights of 79 and 88 kDa. The predicted gene products showed a high degree of homology at the amino acid level (56% identity) and exhibited significant hydrophobic stretches in their N-terminal halves, consistent with 12 membrane-spanning, alpha-helical domains. Database searches using AtKT1 and AtKT2 identified 10 additional sequences in Arabidopsis as well as additional homologous sequences in the plant species Oryza and Allium, the bacterium Lactococcus lactis, and in Homo sapiens. Expression of AtKT2 rescued growth on low millimolar [K+] in Saccharomyces cerevisiae carrying deletions for the genes encoding the K+ transporters TRK1 and TRK2. Rescue was associated with a 2-fold stimulation of Rb+ uptake and was sensitive to competition with external Na+ but not to extracellular pH, indicating that the gene encodes a low-affinity K+ transporter. These and additional results suggest that AtKT1 and AtKT2 belong to a superfamily of cation transporters that have been conserved through evolution.

K(+)-sensitive gating of the K+ outward rectifier in Vicia guard cells.

The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K+ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K+ current was monitored under voltage clamp in 0.1-30 mM K+ and in equivalent concentrations of Rb+, Cs+ and Na+. From a conditioning voltage of -200 mV, clamp steps to voltages between -150 and +50 mV in 0.1 mM K+ activated current through outward-rectifying K+ channels (IK,out) at the plasma membrane in a voltage-dependent fashion. Increasing [K+]o shifted the voltage-sensitivity of IK,out in parallel with the equilibrium potential for K+ across the membrane. A similar effect of [K+]o was evident in the kinetics of IK,out activation and deactivation, as well as the steady-state conductance-(g kappa-) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near -130 mV in 0.1 mM K+, -80 mV in 1.0 mM K+, and -20 mV in 10 mM K+. Similar data were obtained with Rb+ and Cs+, but not with Na+, consistent with the relative efficacy of cation binding under equilibrium conditions (K+ > or = Rb+ > Cs+ > > Na+). Changing Ca2+ or Mg2+ concentrations outside between 0.1 and 10 mM was without effect on the voltage-dependence of g kappa or on IK,out activation kinetics, although 10 mM [Ca2+]o accelerated current deactivation at voltages negative of -75 mV. At any one voltage, increasing [K+]o suppressed g kappa completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K+ was sensitive to voltage, varying from 0.5 to 20 mM with clamp voltages near -100 to 0 mV, respectively. These, and additional data indicate that extracellular K+ acts as a ligand and alters the voltage-dependence of IK,out gating; the results implicate K(+)-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K+ ions outside affects the distribution between closed states of the channel.