Dominant negative guard cell K+ channel mutants reduce inward-rectifying K+ currents and light-induced stomatal opening in arabidopsis

Inward-rectifying potassium (K+(in)) channels in guard cells have been suggested to provide a pathway for K+ uptake into guard cells during stomatal opening. To test the proposed role of guard cell K+(in) channels in light-induced stomatal opening, transgenic Arabidopsis plants were generated that expressed dominant negative point mutations in the K+(in) channel subunit KAT1. Patch-clamp analyses with transgenic guard cells from independent lines showed that K+(in) current magnitudes were reduced by approximately 75% compared with vector-transformed controls at -180 mV, which resulted in reduction in light-induced stomatal opening by 38% to 45% compared with vector-transformed controls. Analyses of intracellular K+ content using both sodium hexanitrocobaltate (III) and elemental x-ray microanalyses showed that light-induced K+ uptake was also significantly reduced in guard cells of K+(in) channel depressor lines. These findings support the model that K+(in) channels contribute to K+ uptake during light-induced stomatal opening. Furthermore, transpirational water loss from leaves was reduced in the K+(in) channel depressor lines. Comparisons of guard cell K+(in) current magnitudes among four different transgenic lines with different K+(in) current magnitudes show the range of activities of K+(in) channels required for guard cell K+ uptake during light-induced stomatal opening.     

The stomata of the fern Adiantum capillus-veneris do not respond to CO2 in the dark and open by photosynthesis in guard cells

The stomata of the fern Adiantum capillus-veneris lack a blue light-specific opening response but open in response to red light. We investigated this light response of Adiantum stomata and found that the light wavelength dependence of stomatal opening matched that of photosynthesis. The simultaneous application of red (2 micromol m(-2) s(-1)) and far-red (50 micromol m(-2) s(-1)) light synergistically induced stomatal opening, but application of only one of these wavelengths was ineffective. Adiantum stomata did not respond to CO2 in the dark; the stomata neither opened under a low intercellular CO2 concentration nor closed under high intercellular CO2 concentration. Stomata in Arabidopsis (Arabidopsis thaliana), which were used as a control, showed clear sensitivity to CO2. In Adiantum, stomatal conductance showed much higher light sensitivity when the light was applied to the lower leaf surface, where stomata exist, than when it was applied to the upper surface. This suggests that guard cells likely sensed the light required for stomatal opening. In the epidermal fragments, red light induced both stomatal opening and K+ accumulation in guard cells, and both of these responses were inhibited by a photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The stomatal opening was completely inhibited by CsCl, a K+ channel blocker. In intact fern leaves, red light-induced stomatal opening was also suppressed by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that Adiantum stomata lack sensitivity to CO2 in the dark and that stomatal opening is driven by photosynthetic electron transport in guard cell chloroplasts, probably via K+ uptake.     

Expression of a Cs(+)-resistant guard cell K+ channel confers Cs(+)-resistant, light-induced stomatal opening in transgenic arabidopsis.

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+in 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+in channel, KAT1, 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+(in) currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KAT1 functions as a plasma membrane K+ channel in vivo and that K+in channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+in channels in guard cells indicate that components in addition to KAT1 may contribute to the formation of K+in channels in vivo.     

Actin Filaments Modulate Both Stomatal Opening and Inward K+-Channel Activities in Guard Cells of Vicia faba L

Actin antagonists have previously been shown to alter responses of Commelina communis stomata to physiological stimuli, implicating actin filaments in the control of guard cell volume changes (M. Kim, P.K. Hepler, S.-O. Eun, K.S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Since K+ channels in the guard cell play an important role in stomatal movements, we examined the possible regulation of K+-channel activities by the state of actin polymerization. Agents affecting actin polymerization altered light-induced stomatal opening and inward K+-channel activities measured by patch clamping in Vicia faba. Cytochalasin D, which induces depolymerization of actin filaments, promoted light-induced stomatal opening and potentiated the inward K+ current in guard cell protoplasts. Phalloidin, a stabilizer of filamentous actin, inhibited both light-induced stomatal opening and inward K+ current. Inward K+-channel activities in outside-out membrane patches showed responses to these agents that support results at the whole-cell current level, suggesting that cytochalasin D facilitates and phalloidin inhibits K+ influx in intact guard cells, thus resulting in enhancement and inhibition of stomatal opening, respectively. To our knowledge, this is the first report that provides evidence that actin filaments may regulate an important physiological process by modulating the activities of ion channels in plant cells.     

Inhibitory effects of methylglyoxal on light-induced stomatal opening and inward K+ channel activity in Arabidopsis

Methylglyoxal (MG) is a reactive aldehyde derived by glycolysis. In Arabidopsis, MG inhibited light-induced stomatal opening in a dose-dependent manner. It significantly inhibited both inward-rectifying potassium (K(in)) channels in guard-cell protoplasts and an Arabidopsis K(in) channel, KAT1, heterologously expressed in Xenopus oocytes. Thus it appears that MG inhibition of stomatal opening involves MG inhibition of K(+) influx into guard cells.     

Expression and manipulation of phosphoenolpyruvate carboxykinase 1 identifies a role for malate metabolism in stomatal closure

Malate, along with potassium and chloride ions, is an important solute for maintaining turgor pressure during stomatal opening. Although malate is exported from guard cells during stomatal closure, there is controversy as to whether malate is also metabolised. We provide evidence that phosphoenolpyruvate carboxykinase (PEPCK), an enzyme involved in malate metabolism and gluconeogenesis, is necessary for full stomatal closure in the dark. Analysis of the Arabidopsis PCK1 gene promoter indicated that this PEPCK isoform is specifically expressed in guard cells and trichomes of the leaf. Spatially distinct promoter elements were found to be required for post-germinative, vascular expression and guard cell/trichome expression of PCK1. We show that pck1 mutant plants have reduced drought tolerance, and show increased stomatal conductance and wider stomatal apertures compared with the wild type. During light-dark transients the PEPCK mutant plants show both increased overall stomatal conductance and less responsiveness of the stomata to darkness than the wild type, indicating that stomata get 'jammed' in the open position. These results show that malate metabolism is important during dark-induced stomatal closure and that PEPCK is involved in this process.     

Reversal by green light of blue light-stimulated stomatal opening in intact, attached leaves of Arabidopsis operates only in the potassium-dependent, morning phase of movement

Green light reversal of blue light-stimulated stomatal opening was discovered in isolated stomata. The present study shows that the response also occurs in stomata from intact leaves. Arabidopsis thaliana plants were grown in a growth chamber under blue, red and green light. Removal of the green light opened the stomata and restoration of green light closed them to baseline values under experimental conditions that rule out a mesophyll-mediated effect. Assessment of the response to green light over a daily time course showed that the stomatal sensitivity to green light was observed only in the morning, which coincided with the use of potassium as a guard cell osmoticum. Sensitivity to green light was absent during the afternoon phase of stomatal movement, which was previously shown to be dominated by sucrose osmoregulation in Vicia faba. Hence, the shift away from potassium-based osmoregulation in guard cells is further postulated to entail a shift from blue light to photosynthesis as the primary component of the stomatal response to light. Stomata from intact leaves of the zeaxanthin-less, npq1 mutant of Arabidopsis failed to respond to the removal or restoration of green light in the growth chamber, or to short, high fluence pulses of blue or green light. These data confirm previous studies showing that npq1 stomata are devoid of a specific blue light response. In contrast, stomata from intact leaves of phot1 phot2 double mutant plants had a reduced but readily detectable response to the removal of green light and to blue and green pulses.     

K+ transport properties of K+ channels in the plasma membrane of Vicia faba guard cells

Electrical properties of the plasma membrane of guard cell protoplasts isolated from stomates of Vicia faba leaves were studied by application of the whole-cell configuration of the patch-clamp technique. The two types of K+ currents that have recently been identified in guard cells may allow efflux of K+ during stomatal closing, and uptake of K+ during stomatal opening (Schroeder et al., 1987). A detailed characterization of ion transport properties of the inward-rectifying (IK+,in) and the outward-rectifying (IK+,out) K+ conductance is presented here. The permeability ratios of IK+,in and IK+,out currents for K+ over monovalent alkali metal ions were determined. The resulting permeability sequences (PK+ greater than PRb+ greater than PNa+ greater than PLi+ much greater than PCs+) corresponded closely to the ion specificity of guard cell movements in V. faba. Neither K+ currents exhibited significant inactivation when K+ channels were activated for prolonged periods (greater than 10 min). The absence of inactivation may permit long durations of K+ fluxes, which occur during guard cell movements. Activation potentials of inward K+ currents were not shifted when external K+ concentrations were changed. This differs strongly from the behavior of inward-rectifying K+ channels in animal tissue. Blue light and fusicoccin induce hyperpolarization by stimulation of an electrogenic pump. From slow-whole-cell recordings it was concluded that electrogenic pumps require cytoplasmic substrates for full activation and that the magnitude of the pump current is sufficient to drive K+ uptake through IK+,in channels. First, direct evidence was gained for the hypothesis that IK+,in channels are a molecular pathway for K+ accumulation by the finding that IK+,in was blocked by Al3+ ions, which are known to inhibit stomatal opening but not closing. The results presented in this study strongly support a prominent role for IK+,in and IK+,out channels in K+ transport across the plasma membrane of guard cells.     

Potassium Loss from Stomatal Guard Cells at Low Water Potentials

The potassium content of guard cells and the resistance to viscousflow of air through the leaf were determined in sunflower (Helianthusannuus) subjected to low leaf water potentials under illuminatedconditions. In intact plants desiccated slowly by withholdingwater from the soil, large losses in guard cell K occurred asleaf water potentials decreased. Leaf viscous resistance increased,indicating stomatal closure. Similar results were obtained whendetached leaf segments were desiccated rapidly. Upon rehydrationof leaves, no stomatal opening was observed initially, despiteleaf water potentials at predesiccated levels. After severalhours, however, re-entry of K occurred and stomata became fullyopen. Turgid leaf segments floated on an ABA solution showedlosses of guard cell K and closure of stomata as rapidly andcompletely as those brought about by desiccation. It is concludedthat stomatal closure at low water potentials under illuminatedconditions is not controlled solely by water loss from the tissuebut involves the loss of osmoticum from the guard cells as well.This in turn decreases the turgor difference between the guardcells and the surrounding cells, and closing occurs.     

Guard cell inward K+ channel activity in arabidopsis involves expression of the twin channel subunits KAT1 and KAT2

Stomatal opening, which controls gas exchanges between plants and the atmosphere, results from an increase in turgor of the two guard cells that surround the pore of the stoma. KAT1 was the only inward K(+) channel shown to be expressed in Arabidopsis guard cells, where it was proposed to mediate a K(+) influx that enables stomatal opening. We report that another Arabidopsis K(+) channel, KAT2, is expressed in guard cells. More than KAT1, KAT2 displays functional features resembling those of native inward K(+) channels in guard cells. Coexpression in Xenopus oocytes and two-hybrid experiments indicated that KAT1 and KAT2 can form heteromultimeric channels. The data indicate that KAT2 plays a crucial role in the stomatal opening machinery.     

Effect of Phenolic Compounds on ABA-induced Changes in K+ Concentration of Guard Cells and in Epidermal Diffusive Resistance

The study of the structure-activity relationship of phenoliccompounds in reversing the ABA-effect on stomata led us to investigatethe changes in K⁺ concentrations in guard cells and in the epidermaldiffusive resistance of leaves, after treatment with ABA andphenolics. The amount of potassium localized in guard cells usually correspondsto stomatal aperture in different treatments. Umbelliferone,however, permits stomatal opening without retention of potassiumin the guard cells, which is an exception. The effect of phenolicsin retaining K⁺ in epidermal peels is matched by recorded epidermaldiffusive resistance changes in the leaves.Although flavonoidsand some other phenolics behave differently showing recoveryin epidermal peels with K⁺ in guard cells, epidermal diffusiveresistance is not recovered. Key words: Epidermal diffusive resistance, K⁺, ABA, phenolics, stomata     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

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 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.     

Stomatal movement and potassium transport in epidermal strips of Zea mays: The effect of CO2

Summary The correlation between stomatal action and potassium movement in the epidermis of Zea mays was examined in isolated epidermal strips floated on distilled water. Stomatal opening in the isolated epidermis is reversible in response to alternate periods of light or darkness, and is always correlated with a shift in the potassium content of the guard cells. K accumulates in guard cells during stomatal opening, and moves from the guard cells into the subsidiary cells during rapid stomatal closure. When epidermal strips are illuminated in normal air, as against CO₂-free air, the stomata do not open and there is a virtually complete depletion of K from the stomatal apparatus. In darkness CO₂-containing air inhibits stomatal opening and K accumulation in guard cells, but does not lead to a depletion of K from the stomata as observed in the light.     

Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection

Oxalic acid is a virulence factor of several phytopathogenic fungi, including Sclerotinia sclerotiorum (Lib.) de Bary, but the detailed mechanisms by which oxalic acid affects host cells and tissues are not understood. We tested the hypothesis that oxalate induces foliar wilting during fungal infection by manipulating guard cells. Unlike uninfected leaves, stomatal pores of Vicia faba leaves infected with S. sclerotiorum are open at night. This cellular response appears to be dependent on oxalic acid because stomatal pores are partially closed when leaves are infected with an oxalate-deficient mutant of S. sclerotiorum. In contrast to oxalate-deficient S. sclerotiorum, wild-type fungus causes an increase in stomatal conductance and transpiration as well as a decrease in plant biomass. Green fluorescent protein-tagged S. sclerotiorum emerges through open stomata from the uninfected abaxial leaf surface for secondary colonization. Exogenous application of oxalic acid to the detached abaxial epidermis of V. faba leaves induces stomatal opening. Guard cells treated with oxalic acid accumulate potassium and break down starch, both of which are known to contribute to stomatal opening. Oxalate interferes with abscisic acid (ABA)-induced stomatal closure. The Arabidopsis (Arabidopsis thaliana) L. Heynh. mutants abi1, abi3, abi4, and aba2 are more susceptible to oxalate-deficient S. sclerotiorum than wild-type plants, suggesting that Sclerotinia resistance is dependent on ABA. We conclude that oxalate acts via (1) accumulation of osmotically active molecules to induce stomatal opening and (2) inhibition of ABA-induced stomatal closure.     

Microtubules of guard cells are light sensitive

Guard cells of stomata are characterized by ordered bundles of microtubules radiating from the ventral side toward the dorsal side of the cylindrical cell. It was suggested that microtubules play a role in directing the radial arrangement of the cellulose micro-fibrils of guard cells. However, the role of microtubules in daily cycles of opening and closing of stomata is not clear. The organization of microtubules in guard cells of Commelina communis leaves was studied by analysis of three-dimensional immunofluorescent images. It was found that while guard cell microtubules in the epidermis of leaves incubated in the light were organized in parallel, straight and dense bundles, in the dark they were less straight and oriented randomly near the stomatal pore. The effect of blue and red light on the organization of guard cell microtubules resembled the effects of white light and dark respectively. When stomata were induced to open in the dark with fusicoccin, microtubules remained in the dark configuration. Furthermore, when incubated in the light, guard cell microtubules were more resistant to oryzalin. Similarly, microtubules of Arabidopsis guard cells, expressing green fluorescent protein-tubulin alpha 6, were disorganized in the dark, but were organized in parallel arrays in the presence of white light. The dynamics of microtubule rearrangement upon transfer of intact leaves from dark to light was followed in single stomata, showing that an arrangement of microtubules typical for light conditions was obtained after 1 h in the light. Our data suggest that microtubule organization in guard cells is responsive to light signals.     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.