Fern and lycophyte guard cells do not respond to endogenous abscisic acid

Stomatal guard cells regulate plant photosynthesis and transpiration. Central to the control of seed plant stomatal movement is the phytohormone abscisic acid (ABA); however, differences in the sensitivity of guard cells to this ubiquitous chemical have been reported across land plant lineages. Using a phylogenetic approach to investigate guard cell control, we examined the diversity of stomatal responses to endogenous ABA and leaf water potential during water stress. We show that although all species respond similarly to leaf water deficit in terms of enhanced levels of ABA and closed stomata, the function of fern and lycophyte stomata diverged strongly from seed plant species upon rehydration. When instantaneously rehydrated from a water-stressed state, fern and lycophyte stomata rapidly reopened to predrought levels despite the high levels of endogenous ABA in the leaf. In seed plants under the same conditions, high levels of ABA in the leaf prevented rapid reopening of stomata. We conclude that endogenous ABA synthesized by ferns and lycophytes plays little role in the regulation of transpiration, with stomata passively responsive to leaf water potential. These results support a gradualistic model of stomatal control evolution, offering opportunities for molecular and guard cell biochemical studies to gain further insights into stomatal control.     

Expression of an mRNA with sequence similarity to pea dehydrin (Psdhn 1) in guard cells of Vicia faba in response to exogenous abscisic acid

The function and location of guard cells uniquely subject them to stress. First, stomatal movements require large fluctuations in the concentration of potassium salts. Second, guard cell inner walls are the first surfaces exposed to evaporation and apoplastic solutes may accumulate there as a result. We have therefore investigated whether guard cells exhibit atypical expression of dehydrin genes because dehydrins accumulate in vegetative tissues in response to water stress. We have also assayed for osmotin mRNA, which is up-regulated in leaves in response to various stresses. mRNA probes for several representative genes were used with RNA extracts from control and water-stressed Vicia faba leaflets. Correlatively, these probes were used with RNA extracts from "isolated' guard cells that had been incubated with combinations of abscisic acid, mannitol and Ca²⁺. (Isolated guard cells are epidermal strips sonicated to destroy cells other than guard cells.) Hybridization with the probe prepared for a dehydrin from Pisum sativum (Psdhn 1) was detected in leaf extracts only if the leaf had been stressed. Similarly, after 1- and 6-h incubations with abscisic acid, isolated guard cells contained an mRNA that hybridized with the probe for Psdhn 1. Appearance of this abscisic acid-dependent mRNA required neither mannitol nor exogenous Ca²⁺. Regardless of the conditions or tissue, no hybridization was detected with the probe against osmotin, but our interpretation of this result is qualified. The simplest conclusion is that atypical expression of dehydrin is not the mechanism by which guard cells cope with their peculiar function and location.     

Evidence for an Extracellular Reception Site for Abscisic Acid in Commelina Guard Cells

The phytohormone abscisic acid (ABA) triggers stomatal closing as a physiological response to drought stress. Several basic questions limit an understanding of the mechanism of ABA reception in guard cells. Whether primary ABA receptors are located on the extracellular side of the plasma membrane, within the intracellular space of guard cells, or both remains unknown. Furthermore, it is not clear whether ABA must be transported into guard cells to exert control over stomatal movements. In the present study, a combination of microinjection into guard cells and physiological assays of stomatal movements have been performed to determine primary sites of ABA reception in guard cells. Microinjection of ABA into guard cells of Commelina communis L. resulted in injected cytosolic concentrations of 50 to 200 [mu]M ABA and in additional experiments in lower concentrations of approximately 1 [mu]M ABA. Stomata with ABA-loaded guard cells (n > 180) showed opening similar to stomata with uninjected guard cells. The viability of guard cells following ABA injection was demonstrated by neutral red staining as well as monitoring of stomatal opening. Extracellular application of 10 [mu]M ABA inhibited stomatal opening by 98% at pH 6.15 and by 57% at pH 8.0. The pH dependence of extracellular ABA action may suggest a contribution of an intracellular ABA receptor to stomatal regulation. The findings presented here show that intracellular ABA alone does not suffice to inhibit stomatal opening under the imposed conditions. Furthermore, these data provide evidence that a reception site for ABA-mediated inhibition of stomatal opening is on the extracellular side of the plasma membrane of guard cells.     

The Effect of Osmotic Stress on the Solutes in Guard Cells of Vicia faba L.

We investigated the changes in the levels of solutes in guardcells under osmotic stress. Epidermal strips peeled from Viciafaba L. leaflets were sonicated and incubated in 0.4 M mannitolsolution (osmotic stress) in either light or dark. Stomata wereclosed by osmotic stress. Under osmotic stress, malate accumulatedlight-dependently and sucrose accumulated light-independentlyin the guard cells. The level of K⁺ in guard cells increasedslightly under osmotic stress in the light, although withoutstatistical significance. The levels of all these solutes werereduced by 10 µM ABA treatment. These results suggestthat osmotic stress affects carbon metabolism in guard cells;this metabolic change is different from that caused by ABA alone.Respiratory activity of guard cells decreased under osmoticstress. Therefore, the accumulation of malate and sucrose maybe caused by reduced respiration under osmotic stress. Accumulationof solutes in guard cells by osmotic stress may result in increasedosmotic pressure of guard cells and may play a role in protectionof guard cells from osmotic stress. (Received December 17, 1998; Accepted May 28, 1999)     

ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells

Increased synthesis and redistribution of the phytohormone abscisic acid (ABA) in response to water deficit stress initiates an intricate network of signalling pathways in guard cells leading to stomatal closure. Despite the large number of ABA signalling intermediates that are known in guard cells, new discoveries are still being made. Recently, the reactive oxygen species hydrogen peroxide (H2O2) and the reactive nitrogen species nitric oxide (NO) have been identified as key molecules regulating ABA-induced stomatal closure in various species. As with many other physiological responses in which H2O2 and NO are involved, stomatal closure in response to ABA also appears to require the tandem synthesis and action of both these signalling molecules. Recent pharmacological and genetic data have identified NADPH oxidase as a source of H2O2, whilst nitrate reductase has been identified as a source of NO in Arabidopsis guard cells. Some signalling components positioned downstream of H2O2 and NO are calcium, protein kinases and cyclic GMP. However, the exact interaction between the various signalling components in response to H2O2 and NO in guard cells remains to be established.     

Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba

One of the most important functions of the plant hormone abscisic acid (ABA) is to induce stomatal closure by reducing the turgor of guard cells under water deficit. Under environmental stresses, hydrogen peroxide (H(2)O(2)), an active oxygen species, is widely generated in many biological systems. Here, using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that H(2)O(2) may function as an intermediate in ABA signaling in Vicia faba guard cells. H(2)O(2) inhibited induced closure of stomata, and this effect was reversed by ascorbic acid at concentrations lower than 10(-5) M. Further, ABA-induced stomatal closure also was abolished partly by addition of exogenous catalase (CAT) and diphenylene iodonium (DPI), which are an H(2)O(2) scavenger and an NADPH oxidase inhibitor, respectively. Time course experiments of single-cell assays based on the fluorescent probe dichlorofluorescein showed that the generation of H(2)O(2) was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than within the other regions of guard cells. The ABA-induced change in fluorescence intensity in guard cells was abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H(2)O(2) production, which preceded stomatal closure. These effects were abolished by CAT or DPI micro-injection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H(2)O(2) production involved, and H(2)O(2) may be an intermediate in ABA signaling.     

Abscisic acid-induced actin reorganization in guard cells of dayflower is mediated by cytosolic calcium levels and by protein kinase and protein phosphatase activities

In guard cells of open stomata under daylight, long actin filaments are arranged at the cortex, radiating out from the stomatal pore. Abscisic acid (ABA), a signal for stomatal closure, induces rapid depolymerization of cortical actin filaments and the slower formation of a new type of actin that is randomly oriented throughout the cell. This change in actin organization has been suggested to be important in signaling pathways involved in stomatal closing movement, since actin antagonists interfere with normal stomatal closing responses to ABA. Here we present evidence that the actin changes induced by ABA in guard cells of dayflower (Commelina communis) are mediated by cytosolic calcium levels and by protein phosphatase and protein kinase activities. Treatment of guard cells with CaCl2 induced changes in actin organization similar to those induced by ABA. Removal of extracellular calcium with EGTA inhibited ABA-induced actin changes. These results suggest that Ca2+ acts as a signal mediator in actin reorganization during guard cell response to ABA. A protein kinase inhibitor, staurosporine, inhibited actin reorganization in guard cells treated with ABA or CaCl2, and also increased the population of cells with long radial cortical actin filaments in untreated control cells. A protein phosphatase inhibitor, calyculin A, induced fragmentation of actin filaments in ABA- or CaCl2-treated cells and in control cells, and inhibited the formation of randomly oriented long actin filaments induced by ABA or CaCl2. These results suggest that protein kinase(s) and phosphatase(s) participate in actin remodeling in guard cells during ABA-induced stomatal closure.     

The guard-cell apoplast as a site of abscisic acid accumulation in Vicia faba L.

Abscisic acid (ABA) integrates the water status of a plant and causes stomatal closure. Physiological mechanisms remain poorly understood, however, because guard cells flanking stomata are small and contain only attomol quantities of ABA. Here, pooled extracts of dissected guard cells of Vicia faba L. were immunoassayed for ABA at sub‐fmol sensitivity. A pulse of water stress was imposed by submerging the roots in a solution of PEG. The water potentials of root and leaf declined during 20 min of water stress but recovered after stress relief. During stress, the ABA concentration in the root apoplast increased, but that in the leaf apoplast remained low. The ABA concentration in the guard‐cell apoplast increased during stress, providing evidence for intra‐leaf ABA redistribution and leaf apoplastic heterogeneity. Subsequently, the ABA concentration of the leaf apoplast increased, consistent with ABA import via the xylem. Throughout, the ABA contents of the guard‐cell apoplast, but not the guard‐cell symplast, were convincingly correlated with stomatal aperture size, identifying an external locus for ABA perception under these conditions. Apparently, ABA accumulates in the guard‐cell apoplast by evaporation from the guard‐cell wall, so the ABA signal in the xylem is amplified maximally at high transpiration rates. Thus, stomata will display apparently higher sensitivity to leaf apoplastic ABA if stomata are widely open in a relatively dry atmosphere.     

Cytoplasmic alkalization precedes reactive oxygen species production during methyl jasmonate- and abscisic acid-induced stomatal closure

Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis wild type, ABA-insensitive (ost1-2), and MJ-insensitive mutants (jar1-1) in order to examine a crosstalk between ABA and MJ signal transduction. Some of the experiments were performed on epidermal strips of Pisum sativum. Stomata of jar1-1 mutant plants are insensitive to MJ but are able to close in response to ABA. However, their sensitivity to ABA is less than that of wild-type plants. Reciprocally, the stomata of ost1-2 are insensitive to ABA but are able to close in response to MJ to a lesser extent compared to wild-type plants. Both MJ and ABA promote H(2)O(2) production in wild-type guard cells, while exogenous application of diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, results in the suppression of ABA- and MJ-induced stomatal closure. ABA elevates H(2)O(2) production in wild-type and jar1-1 guard cells but not in ost1-2, whereas MJ induces H(2)O(2) production in both wild-type and ost1-2 guard cells, but not in jar1-1. MJ-induced stomatal closing is suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Furthermore, MJ induces alkalization in guard cell cytosol, and MJ-induced stomatal closing is inhibited by butyrate. Analyses of the kinetics of cytosolic pH changes and reactive oxygen species (ROS) production show that the alkalization of cytoplasm precedes ROS production during the stomatal response to both ABA and MJ. Our results further indicate that JAR1, as OST1, functions upstream of ROS produced by NAD(P)H oxidases and that the cytoplasmic alkalization precedes ROS production during MJ or ABA signal transduction in guard cells.     

Arabidopsis mutants of AtABCG22, an ABC transporter gene, increase water transpiration and drought susceptibility

In plants, water vapour is released into the atmosphere through stomata in a process called transpiration. Abscisic acid (ABA) is a key phytohormone that facilitates stomatal closure through its action on guard cells. Recently, ATP-binding cassette (ABC) transporter genes, AtABCG25 and AtABCG40, were shown to be involved in ABA transport and responses. However, the functions of many other AtABCG family genes are still unknown. Here, we identified another ABCG gene (AtABCG22) that is required for stomatal regulation in Arabidopsis. The atabcg22 mutant plants had lower leaf temperatures and increased water loss, implying elevated transpiration through an influence on stomatal regulation. We also found that atabcg22 plants were more suspectible to drought stress than wild-type plants. AtABCG22 was expressed in aerial organs, mainly guard cells, in which the gene expression pattern was consistent with the mutant phenotypes. Using double mutants, we investigated the genetic relationships between the mutations. The atabcg22 mutation further increased the water loss of srk2e/ost1 mutants, which were defective in ABA signalling in guard cells. Also, the atabcg22 mutation enhanced the phenotype of nced3 mutants, which were defective in ABA biosynthesis. Accordingly, the additive roles of AtABCG22 functions in ABA signalling and ABA biosynthesis are discussed.     

Abscisic Acid Accumulation by in Situ and Isolated Guard Cells of Pisum sativum L. and Vicia faba L. in Relation to Water Stress

Isolated guard cells, prepared by sonication of epidermal peels, were used to investigate the endogenous level of abscisic acid (ABA) in the guard cells of turgid and stressed leaves of Vicia faba L. and the argenteum (arg) mutant of Pisum sativum L. The guard cells of V. faba and arg were found to contain 18 and 8 times more ABA, respectively, when isolated from stressed leaves than from turgid leaves. Isolated guard cells of V. faba were also directly stressed with the osmoticum Aquacide III. These guard cells were capable of producing stress-induced ABA to at least 3 times their ABA level when non-stressed.     

ABA-regulated promoter activity in stomatal guard cells

CDeT6-19 is an ABA-regulated gene which has been isolated from Craterostigma plantagineum . The CDeT6-19 gene promoter has been fused to the β- glucuronidase reporter gene ( GUS ) and used to stably transform Arabidopsis thaliana and Nicotiana tabacum . This construct has been shown to be expressed in stomatal guard cells and often in the adjacent epidermal cells of both species in response to both exogenous ABA and drought stress. These results indicate that the stomatal guard cell is competent to relay an ABA signal to the nucleus. In contrast GUS expression directed by the promoter from a predominantly seed-specific, ABA-regulated gene, Em , or the promoter from the ABA-regulated CDeT27-45 gene is not detectable in the epidermal or guard cells of tobacco or Arabidopsis in response to ABA. The fact that not all ABA-regulated gene promoters are active in stomatal guard cells suggests that effective transduction of the signal is dependent upon particular regions within the gene promoter or that guard cells lack all or part of the specific transduction apparatus required to couple the ABA signal to these promoters. This suggests that there are multiple ABA stimulus response coupling pathways. The identification of a regulatory sequence from an ABA-induced gene which is expressed in stomatal guard cells creates the possibility of examining the role of Ca²⁺ and other second messengers in ABA-induced gene expression.     

EFFECTS OF WATER STRESS,ABSCISIC ACID,AND GIBBERELLIC ACID ON FLOWER PRODUCTION AND DIFFERENTIATION IN THE CLEISTOGAMOUS SPECIES COLLOMIA GRANDIFLORA DOUGL. EX LINDL. (POLEMONIACEAE)

The effects of water stress, abscisic acid (ABA), and gibberellic acid (GA₃) on flower production and differentiation by Collomia grandiflora were investigated. An untreated plant typically produced both small, closed cleistogamous (CL) and large, open chasmogamous (CH) flowers. The larger corolla of CH flowers was due to a greater cell number and size. When plants were water-stressed or sprayed with ABA, both the percentage of CH flowers and the total number of flowers were reduced significantly. The corolla dimensions and epidermal cell numbers and sizes of CL flowers produced by water-stressed and ABA-sprayed plants did not differ from those of CL flowers produced by control plants. Application of GA₃ to both well-watered and water-stressed plants significantly increased the percentage of CH flowers formed compared to well-watered controls. In the absence of GA₃, water-stressed plants produced almost entirely CL flowers. GA₃-sprayed plants produced CH flowers whose corolla dimensions were intermediate between those of CL and CH flowers formed by control plants. Epidermal cells of these intermediate corollas were reduced only in number and not in size when compared to control CH flowers. Endogenous levels of ABA and gibberellins may control the type of flower produced by C. grandiflora and may mediate some of the observable effects of water stress on flowering.     

Two Transduction Pathways Mediate Rapid Effects of Abscisic Acid in Commelina Guard Cells

Commelina guard cells can be rapidly closed by abscisic acid (ABA), and it is thought that this signal is always transduced through increases in cytosolic calcium. However, when Commelina plants were grown at 10 to 17[deg]C, most guard cells failed to exhibit any ABA-induced increase in cytosolic calcium even though all of these cells closed. At growth temperatures of 25[deg]C or above, ABA-induced closure was always associated with an increase in cytosolic calcium. This suggests that there may be two transduction routes for ABA in guard cells; only one involves increases in cytosolic calcium. Activation of either pathway on its own appears to be sufficient to cause closure. Because the rates of ABA accumulation and transport in plants grown at different temperatures are likely to be different, we synthesized and microinjected caged ABA directly into guard cells. ABA was released internally by UV photolysis and subsequently caused stomatal closure. This result suggests a possible intracellular locale for the hypothesized ABA receptor.     

Ca2+-dependent and -independent abscisic acid activation of plasma membrane anion channels in guard cells of Nicotiana tabacum

Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca(2+) concentration. In Vicia faba, however, guard cell anion channels activate in a Ca(2+)-independent manner. Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca(2+) concentration. ABA triggered transient rises in cytoplasmic Ca(2+) in the majority of the guard cells (14 out of 19). In seven out of 14 guard cells, the change in cytoplasmic free Ca(2+) closely matched the activation of anion channels, while the Ca(2+) rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca(2+) level. Even though ABA could activate anion channels in N. tabacum guard cells independent of a rise in the cytoplasmic Ca(2+) concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca(2+) in an ATP-dependent manner. Guard cells thus seem to have evolved both Ca(2+)-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca(2+)-independent pathway.     

The coronatine-insensitive 1 mutation reveals the hormonal signaling interaction between abscisic acid and methyl jasmonate in Arabidopsis guard cells. Specific impairment of ion channel activation and second messenger production

Methyl jasmonate (MeJA) elicits stomatal closing similar to abscisic acid (ABA), but whether the two compounds use similar or different signaling mechanisms in guard cells remains to be clarified. We investigated the effects of MeJA and ABA on second messenger production and ion channel activation in guard cells of wild-type Arabidopsis (Arabidopsis thaliana) and MeJA-insensitive coronatine-insensitive 1 (coi1) mutants. The coi1 mutation impaired MeJA-induced stomatal closing but not ABA-induced stomatal closing. MeJA as well as ABA induced production of reactive oxygen species (ROS) and nitric oxide (NO) in wild-type guard cells, whereas MeJA did not induce production of ROS and NO in coi1 guard cells. The experiments using an inhibitor and scavengers demonstrated that both ROS and NO are involved in MeJA-induced stomatal closing as well as ABA-induced stomatal closing. Not only ABA but also MeJA activated slow anion channels and Ca(2+) permeable cation channels in the plasma membrane of wild-type guard cell protoplasts. However, in coi1 guard cell protoplasts, MeJA did not elicit either slow anion currents or Ca(2+) permeable cation currents, but ABA activated both types of ion channels. Furthermore, to elucidate signaling interaction between ABA and MeJA in guard cells, we examined MeJA signaling in ABA-insensitive mutant ABA-insensitive 2 (abi2-1), whose ABA signal transduction cascade has some disruption downstream of ROS production and NO production. MeJA also did not induce stomatal closing but stimulated production of ROS and NO in abi2-1. These results suggest that MeJA triggers stomatal closing via a receptor distinct from the ABA receptor and that the coi1 mutation disrupts MeJA signaling upstream of the blanch point of ABA signaling and MeJA signaling in Arabidopsis guard cells.     

Blue light‐induced apoplastic acidification of Arabidopsis thaliana guard cells: Inhibition by ABA is mediated through protein phosphatases

The phytohormone abscisic acid (ABA) inhibits blue light‐induced apoplastic acidification of guard cells. The signal transduction pathway of ABA, mediating this response, was studied using ABA‐insensitive ( abi ) mutants of Arabidopsis thaliana . Apoplastic acidification was monitored with a flat tipped pH‐electrode placed on epidermal strips, in which only guard cells were viable. Blue light‐induced apoplastic acidification was reduced by vanadate and diethylstilbestrol (DES), indicating involvement of plasma membrane‐bound H⁺‐ATPases. In wild type epidermal strips, ABA reduced blue light‐induced acidification to 63%. The inhibition did not result from an increased cytoplasmic free Ca²⁺ concentration in guard cells, since factors that increase the Ca²⁺ concentration stimulated apoplastic acidification. Apoplastic acidification was not inhibited by ABA in abi1 and abi2 mutants. In abi1 epidermal strips ABA had no effect on the acidification rate, while it stimulated apoplastic acidification in abi2 . The ABA response in both mutants could be partially restored with protein kinase and phosphatase inhibitors. The abi1 guard cells became ABA responsive in the presence of okadaic acid, a protein phosphatase inhibitor. In abi2 guard cells the wild type ABA response was partially restored by K‐252a, a protein kinase inhibitor. Apoplastic inhibition is thus mediated through the protein phosphatases encoded by ABI1 and ABI2 . The results with protein kinase and protein phosphatase inhibitors indicate that ABI1 and ABI2 are involved in separate signal transduction pathways.     

Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants.

Abscisic acid (ABA) regulates vital physiological responses, and a number of events in the ABA signaling cascade remain to be identified. To allow quantitative analysis of genetic signaling mutants, patch-clamp experiments were developed and performed with the previously inaccessible Arabidopsis guard cells from the wild type and ABA-insensitive (abi) mutants. Slow anion channels have been proposed to play a rate-limiting role in ABA-induced stomatal closing. We now directly demonstrate that ABA strongly activates slow anion channels in wild-type guard cells. Furthermore, ABA-induced anion channel activation and stomatal closing were suppressed by protein phosphatase inhibitors. In abi1-1 and abi2-1 mutant guard cells, ABA activation of slow anion channels and ABA-induced stomatal closing were abolished. These impairments in ABA signaling were partially rescued by kinase inhibitors in abi1 but not in abi2 guard cells. These data provide cell biological evidence that the abi2 locus disrupts early ABA signaling, that abi1 and abi2 affect ABA signaling at different steps in the cascade, and that protein kinases act as negative regulators of ABA signaling in Arabidopsis. New models for ABA signaling pathways and roles for abi1, abi2, and protein kinases and phosphatases are discussed.     

Guard cells in albino leaf patches do not respond to photosynthetically active radiation, but are sensitive to blue light, CO2 and abscisic acid

Stomatal openings can be stimulated by light through two signalling pathways. The first pathway is blue light specific and involves phototropins, while the second pathway mediates a response to photosynthetically active radiation (PAR). This second pathway was studied with the use of albino Vicia faba plants and variegated leaves of Chlorophytum comosum. Treatment of V. faba with norflurazon (Nf) inhibits the synthesis of carotenoids and leads to albino leaves with guard cells that lack functional green chloroplasts. Guard cells in albino leaf patches of C. comosum, however, do contain photosynthetically active chloroplasts. Stomata in albino leaf patches of both plants did not respond to red light, although blue light could still induce stomatal opening. This shows that the response to PAR is not functioning in albino leaf patches, even though guard cells of C. comosum harbour chloroplasts. Stomata of Nf-treated plants still responded to CO2 and abscisic acid (ABA). The size of Nf-treated guard cells was increased, but impalement studies with double-barrelled microelectrodes revealed no changes in ion-transport properties at the plasma membrane of guard cells. Blue light could hyperpolarize albino guard cells by triggering outward currents with peak values of 37 pA in albino plants and 51 pA in green control cells. Because of the inhibition of carotenoid biosynthesis, Nf-treated V. faba plants contained only 4% of the ABA content found in green control plants. The ABA dose dependence of anion channel activation in guard cells was shifted in these plants, causing a reduced response to 10 microM ABA. These data show that despite the dramatic changes in physiology caused by Nf, the gross responsiveness of guard cells to blue light, CO2 and ABA remains unaltered. Stomata in albino leaf patches, however, do not respond to PAR, but require photosynthetically active mesophyll cells for this response.