PECT1, a rate-limiting enzyme in phosphatidylethanolamine biosynthesis, is involved in the regulation of stomatal movement in Arabidopsis.

An Arabidopsis mutant displaying impaired stomatal responses to CO2 , cdi4, was isolated by a leaf thermal imaging screening. The mutated gene PECT1 encodes CTP:phosphorylethanolamine cytidylyltransferase. The cdi4 exhibited a decrease in phosphatidylethanolamine levels and a defect in light-induced stomatal opening as well as low-CO2 -induced stomatal opening. We created RNAi lines in which PECT1 was specifically repressed in guard cells. These lines are impaired in their stomatal responses to low-CO2 concentrations or light. Fungal toxin fusicoccin (FC) promotes stomatal opening by activating plasma membrane H+ -ATPases in guard cells via phosphorylation. Arabidopsis H+ -ATPase1 (AHA1) has been reported to be highly expressed in guard cells, and its activation by FC induces stomatal opening. The cdi4 and PECT1 RNAi lines displayed a reduced stomatal opening response to FC. However, similar to in the wild-type, cdi4 maintained normal levels of phosphorylation and activation of the stomatal H+ -ATPases after FC treatment. Furthermore, the cdi4 displayed normal localization of GFP-AHA1 fusion protein and normal levels of AHA1 transcripts. Based on these results, we discuss how PECT1 could regulate CO2 - and light-induced stomatal movements in guard cells in a manner that is independent and downstream of the activation of H+ -ATPases. [Correction added on 15 May 2023, after first online publication: The third sentence is revised in this version.].

Two independent cis-acting elements are required for the guard cell-specific expression of SCAP1, which is essential for late stomatal development.

Regulating the stomatal aperture to adapt to environmental changes is critical for plants as stomatal guard cells are responsible for gas exchange between plants and the atmosphere. We previously showed that a plant-specific DNA-binding with one finger (Dof)-type transcription factor, SCAP1, functions as a key regulator in the final stages of guard cell differentiation. In the present study, we performed deletion and gain-of-function analyses with the 5' flanking region of SCAP1 to identify the regulatory region controlling the guard cell-specific expression of SCAP1. The results revealed that two cis-acting elements, 5'-CACGAGA-3' and 5'-CACATGTTTCCC-3', are crucial for the guard cell-specific expression of SCAP1. Consistently, when an 80-bp promoter region including these two cis-elements was fused to a gene promoter that is not active in guard cells, it functioned as a promoter that directed gene expression in guard cells. Furthermore, the promoter region of HT1 encoding the central regulator of stomatal CO2 signaling was also found to contain a 5'-CACGAGA-3' sequence, which was confirmed to function as a cis-element necessary for guard cell-specific expression of HT1. These findings suggest the existence of a novel transcriptional regulatory mechanism that synchronously promotes the expression of multiple genes required for the stomatal maturation and function.

Cuticle permeability is an important parameter for the trade-off strategy between drought tolerance and CO2 uptake in land plants.

To protect against water loss, land plants have developed the cuticle; however, the cuticle strongly restricts CO2 uptake for photosynthesis. Controlling this trade-off relationship is an important strategy for plant survival, but the extent to which the changes in cuticle affects this relationship is not clear. To evaluate this, we measured CO2 assimilation rate and transpiration rate together in the Arabidopsis thaliana mutant excessive transpiration1 (extra1), which exhibited marked evaporative water loss due to an increased cuticle permeability caused by a new allele of ACETYL-COA CARBOXYLASE 1 (ACC1). Under high humidity (85%) conditions, the extra1 mutant exhibited higher CO2 assimilation rate in exchange for decreasing water use efficiency by one-third compared to the slow anion channel-associated 1 (slac1) mutant, whose stomata are continuously open. Our results indicate that the increased cuticle permeability in extra1 affects transpiration rate more than CO2 assimilation rate, but the effect on CO2 assimilation rate is larger than the effect of open stomata in slac1, suggesting that the cuticle permeability is an important parameter for the trade-off relationship between drought tolerance and CO2 uptake in land plants.

The Endoplasmic Reticulum Pathway for Membrane Lipid Synthesis Has a Significant Contribution toward Shoot Removal-Induced Root Chloroplast Development in Arabidopsis.

Chloroplast lipids are synthesized via two distinct pathways: the plastidic pathway and endoplasmic reticulum (ER) pathway. We previously reported that the contribution of the two pathways toward chloroplast development is different between mesophyll cells and guard cells in Arabidopsis leaf tissues and that the ER pathway plays a major role in guard cell chloroplast development. However, little is known about the contribution of the two pathways toward chloroplast development in other tissue cells, and in this study, we focused on root cells. Chloroplast development is normally repressed in roots but can be induced when the roots are detached from the shoots (root greening). We found that, similar to guard cells, root cells exhibit a higher proportion of glycolipid from the ER pathway. Root greening was repressed in the gles1 mutant, which has a defect in ER-to-plastid lipid transportation via the ER pathway, while normal root greening was observed in the ats1 mutant, whose plastidic pathway is blocked. Lipid analysis revealed that the gles1 mutation caused drastic decrease in the ER-derived glycolipids in roots. Furthermore, the gles1 detached roots showed smaller chloroplasts containing less starch than WT. These results suggest that the ER pathway has a significant contribution toward chloroplast development in the root cells.

Increased Cuticle Permeability Caused by a New Allele of ACETYL-COA CARBOXYLASE1 Enhances CO2 Uptake.

Carbon dioxide (CO2) is an essential substrate for photosynthesis in plants. CO2 is absorbed mainly through the stomata in land plants because all other aerial surfaces are covered by a waxy layer called the cuticle. The cuticle is an important barrier that protects against extreme water loss; however, this anaerobic layer limits CO2 uptake. Simply, in the process of adapting to a terrestrial environment, plants have acquired drought tolerance in exchange for reduced CO2 uptake efficiency. To evaluate the extent to which increased cuticle permeability enhances CO2 uptake efficiency, we investigated the CO2 assimilation rate, carbon content, and dry weight of the Arabidopsis (Arabidopsis thaliana) mutant excessive transpiration1 (extra1), whose cuticle is remarkably permeable to water vapor. We isolated the mutant as a new allele of ACETYL-COA CARBOXYLASE1, encoding a critical enzyme for fatty acid synthesis, thereby affecting cuticle wax synthesis. Under saturated water vapor conditions, the extra1 mutant demonstrated a higher CO2 assimilation rate, carbon content, and greater dry weight than did the wild-type plant. On the other hand, the stomatal mutant slow-type anion channel-associated1, whose stomata are continuously open, also exhibited a higher CO2 assimilation rate than the wild-type plant; however, the increase was only half of the amount exhibited by extra1 These results indicate that the efficiency of CO2 uptake via a permeable cuticle is greater than the efficiency via stomata and confirm that land plants suffer a greater loss of CO2 uptake efficiency by developing a cuticle barrier.

Involvement of S-type anion channels in disease resistance against an oomycete pathogen in Arabidopsis seedlings.

Pharmacological indications suggest that anion channel-mediated plasma membrane (PM) anion efflux is crucial in early defense signaling to induce immune responses and programmed cell death in plants. Arabidopsis SLAC1, an S-type anion channel required for stomatal closure, is involved in cryptogein-induced PM Cl- efflux to positively modulate the activation of other ion fluxes, production of reactive oxygen species and a wide range of defense responses including hypersensitive cell death in tobacco BY-2 cells. We here analyzed disease resistance against several pathogens in multiple mutants of the SLAC/SLAH channels of Arabidopsis. Resistance against a biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 was significantly enhanced in the SLAC1-overexpressing plants than in the wild-type, while that against a bacteria Pseudomonas syringae was not affected significantly. Possible regulatory roles of S-type anion channels in plant immunity and disease resistance against bacterial and oomycete pathogens is discussed.

Eukaryotic lipid metabolic pathway is essential for functional chloroplasts and CO2 and light responses in Arabidopsis guard cells.

Stomatal guard cells develop unique chloroplasts in land plant species. However, the developmental mechanisms and function of chloroplasts in guard cells remain unclear. In seed plants, chloroplast membrane lipids are synthesized via two pathways: the prokaryotic and eukaryotic pathways. Here we report the central contribution of endoplasmic reticulum (ER)-derived chloroplast lipids, which are synthesized through the eukaryotic lipid metabolic pathway, in the development of functional guard cell chloroplasts. We gained insight into this pathway by isolating and examining an Arabidopsis mutant, gles1 (green less stomata 1), which had achlorophyllous stomatal guard cells and impaired stomatal responses to CO2 and light. The GLES1 gene encodes a small glycine-rich protein, which is a putative regulatory component of the trigalactosyldiacylglycerol (TGD) protein complex that mediates ER-to-chloroplast lipid transport via the eukaryotic pathway. Lipidomic analysis revealed that in the wild type, the prokaryotic pathway is dysfunctional, specifically in guard cells, whereas in gles1 guard cells, the eukaryotic pathway is also abrogated. CO2-induced stomatal closing and activation of guard cell S-type anion channels that drive stomatal closure were disrupted in gles1 guard cells. In conclusion, the eukaryotic lipid pathway plays an essential role in the development of a sensing/signaling machinery for CO2 and light in guard cell chloroplasts.

Contribution of the S-type Anion Channel SLAC1 to Stomatal Control and Its Dependence on Developmental Stage in Rice.

Rice production depends on water availability and carbon fixation by photosynthesis. Therefore, optimal control of stomata, which regulate leaf transpiration and CO2 absorption, is important for high productivity. SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) is an S-type anion channel protein that controls stomatal closure in response to elevated CO2. Rice slac1 mutants showed significantly increased stomatal conductance (gs) and enhanced CO2 assimilation. To discern the contribution of stomatal regulation to rice growth, we compared gs in the wild type (WT) and two mutants, slac1 and the dominant-positive mutant SLAC1-F461A, which expresses a point mutation causing an amino acid substitution (F461A) in SLAC1, at different growth stages. Because the side group of F461 is estimated to function as the channel gate, stomata in the SLAC1-F461A mutant are expected to close constitutively. All three lines had maximum gs during the tillering stage, when the gs values were 50% higher in slac1 and 70% lower in SLAC1-F461A, compared with the WT. At the tillering stage, the gs values were highest in the first leaves at the top of the stem and lower in the second and third leaves in all three lines. Both slac1 and SLAC1-F461A retained the ability to change gs in response to the day-night cycle, and showed differences in tillering rate and plant height compared with the WT, and lower grain yield. These observations show that SLAC1 plays a crucial role in regulating stomata in rice at the tillering stage.

Differential Effects of Phosphatidylinositol 4-Kinase (PI4K) and 3-Kinase (PI3K) Inhibitors on Stomatal Responses to Environmental Signals.

Specific cellular components including products of phosphatidylinositol (PI) metabolism play an important role as signaling molecules in stomatal responses to environmental signals. In this study, pharmacological inhibitors of a set of cellular components, including PI4-kinase (PI4K) and PI3K, were used to investigate stomatal closure in response to CO2, darkness, and abscisic acid (ABA). Treatment with PAO, a specific inhibitor of PI4K, specifically inhibited the stomatal response to CO2 compared with that to darkness and ABA. In contrast, treatment with LY294002, a PI3K-specific inhibitor, specifically inhibited the stomatal response to darkness compared with that to CO2 and ABA. The specific inhibitory effects of PAO and LY294002 were also observed as changes in the spatial density of dot-like structures labeled by green fluorescent protein-tagged PATROL1, a protein that controls stomatal aperture possibly via regulation of H+-ATPase amount in guard cell plasma membranes. Our results suggest an important role for PI4K and PI3K in the CO2 and darkness signal transduction pathways, respectively, that mediate PATROL1 dynamics.

Dominant and recessive mutations in the Raf-like kinase HT1 gene completely disrupt stomatal responses to CO2 in Arabidopsis.

HT1 (HIGH LEAF TEMPERATURE 1) is the first component associated with changes in stomatal aperture in response to CO2 to be isolated by forward genetic screening. The HT1 gene encodes a protein kinase expressed mainly in guard cells. The loss-of-function ht1-1 and ht1-2 mutants in Arabidopsis thaliana have CO2-hypersensitive stomatal closure with concomitant reductions in their kinase activities in vitro In addition to these mutants, in this study we isolate or obtaine five new ht1 alleles (ht1-3, ht1-4, ht1-5, ht1-6, and ht1-7). Among the mutants, only ht1-3 has a dominant mutant phenotype and has widely opened stomata due to CO2 insensitivity. The ht1-3 mutant has a missense mutation affecting a non-conserved residue (R102K), whereas the other six recessive mutants have mutations in highly conserved residues in the catalytic domains required for kinase activity. We found that the dominant mutation does not affect the expression of HT1 or the ability to phosphorylate casein, a universal kinase substrate, but it does affect autophosphorylation activity in vitro A 3D structural model of HT1 also shows that the R102 residue protrudes from the surface of the kinase, implying a role for the formation of oligomers and/or interaction with its targets. We demonstrate that both the loss-of-function and gain-of-function ht1 mutants have completely disrupted CO2 responses, although they have normal responses to ABA. Furthermore, light-induced stomatal opening is smaller in ht1-3 and much smaller in ht1-2 Taken together, these results indicate that HT1 is a critical regulator for CO2 signaling and is partially involved in the light-induced stomatal opening pathway.

Enhanced Stomatal Conductance by a Spontaneous Arabidopsis Tetraploid, Me-0, Results from Increased Stomatal Size and Greater Stomatal Aperture.

The rate of gas exchange in plants is regulated mainly by stomatal size and density. Generally, higher densities of smaller stomata are advantageous for gas exchange; however, it is unclear what the effect of an extraordinary change in stomatal size might have on a plant's gas-exchange capacity. We investigated the stomatal responses to CO2 concentration changes among 374 Arabidopsis (Arabidopsis thaliana) ecotypes and discovered that Mechtshausen (Me-0), a natural tetraploid ecotype, has significantly larger stomata and can achieve a high stomatal conductance. We surmised that the cause of the increased stomatal conductance is tetraploidization; however, the stomatal conductance of another tetraploid accession, tetraploid Columbia (Col), was not as high as that in Me-0. One difference between these two accessions was the size of their stomatal apertures. Analyses of abscisic acid sensitivity, ion balance, and gene expression profiles suggested that physiological or genetic factors restrict the stomatal opening in tetraploid Col but not in Me-0. Our results show that Me-0 overcomes the handicap of stomatal opening that is typical for tetraploids and achieves higher stomatal conductance compared with the closely related tetraploid Col on account of larger stomatal apertures. This study provides evidence for whether larger stomatal size in tetraploids of higher plants can improve stomatal conductance.

The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis.

The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response.

CO2 Sensing and CO2 Regulation of Stomatal Conductance: Advances and Open Questions.

Guard cells form epidermal stomatal gas-exchange valves in plants and regulate the aperture of stomatal pores in response to changes in the carbon dioxide (CO2) concentration ([CO2]) in leaves. Moreover, the development of stomata is repressed by elevated CO2 in diverse plant species. Evidence suggests that plants can sense [CO2] changes via guard cells and via mesophyll tissues in mediating stomatal movements. We review new discoveries and open questions on mechanisms mediating CO2-regulated stomatal movements and CO2 modulation of stomatal development, which together function in the CO2 regulation of stomatal conductance and gas exchange in plants. Research in this area is timely in light of the necessity of selecting and developing crop cultivars that perform better in a shifting climate.

Natural variation in stomatal responses to environmental changes among Arabidopsis thaliana ecotypes.

Stomata are small pores surrounded by guard cells that regulate gas exchange between plants and the atmosphere. Guard cells integrate multiple environmental signals and control the aperture width to ensure appropriate stomatal function for plant survival. Leaf temperature can be used as an indirect indicator of stomatal conductance to environmental signals. In this study, leaf thermal imaging of 374 Arabidopsis ecotypes was performed to assess their stomatal responses to changes in environmental CO2 concentrations. We identified three ecotypes, Köln (Kl-4), Gabelstein (Ga-0), and Chisdra (Chi-1), that have particularly low responsiveness to changes in CO2 concentrations. We next investigated stomatal responses to other environmental signals in these selected ecotypes, with Col-0 as the reference. The stomatal responses to light were also reduced in the three selected ecotypes when compared with Col-0. In contrast, their stomatal responses to changes in humidity were similar to those of Col-0. Of note, the responses to abscisic acid, a plant hormone involved in the adaptation of plants to reduced water availability, were not entirely consistent with the responses to humidity. This study demonstrates that the stomatal responses to CO2 and light share closely associated signaling mechanisms that are not generally correlated with humidity signaling pathways in these ecotypes. The results might reflect differences between ecotypes in intrinsic response mechanisms to environmental signals.

New approaches to the biology of stomatal guard cells.

CO2 acts as an environmental signal that regulates stomatal movements. High CO2 concentrations reduce stomatal aperture, whereas low concentrations trigger stomatal opening. In contrast to our advanced understanding of light and drought stress responses in guard cells, the molecular mechanisms underlying stomatal CO2 sensing and signaling are largely unknown. Leaf temperature provides a convenient indicator of transpiration, and can be used to detect mutants with altered stomatal control. To identify genes that function in CO2 responses in guard cells, CO2-insensitive mutants were isolated through high-throughput leaf thermal imaging. The isolated mutants are categorized into three groups according to their phenotypes: (i) impaired in stomatal opening under low CO2 concentrations; (ii) impaired in stomatal closing under high CO2 concentrations; and (iii) impaired in stomatal development. Characterization of these mutants has begun to yield insights into the mechanisms of stomatal CO2 responses. In this review, we summarize the current status of the field and discuss future prospects.

An S-type anion channel SLAC1 is involved in cryptogein-induced ion fluxes and modulates hypersensitive responses in tobacco BY-2 cells.

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl(-) and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl(-) efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl(-) efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.

A Munc13-like protein in Arabidopsis mediates H+-ATPase translocation that is essential for stomatal responses.

Plants control CO2 uptake and water loss by modulating the aperture of stomata located in the epidermis. Stomatal opening is initiated by the activation of H(+)-ATPases in the guard-cell plasma membrane. In contrast to regulation of H(+)-ATPase activity, little is known about the translocation of the guard cell H(+)-ATPase to the plasma membrane. Here we describe the isolation of an Arabidopsis gene, PATROL1, that controls the translocation of a major H(+)-ATPase, AHA1, to the plasma membrane. PATROL1 encodes a protein with a MUN domain, known to mediate synaptic priming in neuronal exocytosis in animals. Environmental stimuli change the localization of plasma membrane-associated PATROL1 to an intracellular compartment. Plasma membrane localization of AHA1 and stomatal opening require the association of PATROL1 with AHA1. Increased stomatal opening responses in plants overexpressing PATROL1 enhance the CO2 assimilation rate, promoting plant growth.

A Dof transcription factor, SCAP1, is essential for the development of functional stomata in Arabidopsis.

Stomata are highly specialized organs that consist of pairs of guard cells and regulate gas and water vapor exchange in plants [1-3]. Although early stages of guard cell differentiation have been described [4-10] and were interpreted in analogy to processes of cell type differentiation in animals [11], the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, stomatal carpenter 1 (scap1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells, but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as K(+) channel protein, MYB60 transcription factor, and pectin methylesterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation.

Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action.

The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca(2+)-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.

Environmental regulation of stomatal response in the Arabidopsis Cvi-0 ecotype.

The Arabidopsis Cape Verde Islands (Cvi-0) ecotype is known to differ from other ecotypes with respect to environmental stress responses. We analyzed the stomatal behavior of Cvi-0 plants, in response to environmental signals. We investigated the responses of stomatal conductance and aperture to high [CO2] in the Cvi-0 and Col-0 ecotypes. Cvi-0 showed constitutively higher stomatal conductance and more stomatal opening than Col-0. Cvi-0 stomata opened in response to light, but the response was slow. Under low humidity, stomatal opening was increased in Cvi-0 compared to Col-0. We then assessed whether low humidity affects endogenous ABA levels in Cvi-0. In response to low humidity, Cvi-0 had much higher ABA levels than Col-0. However, epidermal peels experiments showed that Cvi-0 stomata were insensitive to ABA. Measurements of organic and inorganic ions in Cvi-0 guard cell protoplasts indicated an over-accumulation of osmoregulatory anions (malate and Cl⁻). This irregular anion homeostasis in the guard cells may explain the constitutive stomatal opening phenotypes of the Cvi-0 ecotype, which lacks high [CO2]-induced and low humidity-induced stomatal closure.