Metabolomics of red‐light‐induced stomatal opening in Arabidopsis thaliana: Coupling with abscisic acid and jasmonic acid metabolism

Environmental stimuli‐triggered stomatal movement is a key physiological process that regulates CO₂ uptake and water loss in plants. Stomata are defined by pairs of guard cells that perceive and transduce external signals, leading to cellular volume changes and consequent stomatal aperture change. Within the visible light spectrum, red light induces stomatal opening in intact leaves. However, there has been debate regarding the extent to which red‐light‐induced stomatal opening arises from direct guard cell sensing of red light versus indirect responses as a result of red light influences on mesophyll photosynthesis. Here we identify conditions that result in red‐light‐stimulated stomatal opening in isolated epidermal peels and enlargement of protoplasts, firmly establishing a direct guard cell response to red light. We then employ metabolomics workflows utilizing gas chromatography mass spectrometry and liquid chromatography mass spectrometry for metabolite profiling and identification of Arabidopsis guard cell metabolic signatures in response to red light in the absence of the mesophyll. We quantified 223 metabolites in Arabidopsis guard cells, with 104 found to be red light responsive. These red‐light‐modulated metabolites participate in the tricarboxylic acid cycle, carbon balance, phytohormone biosynthesis and redox homeostasis. We next analyzed selected Arabidopsis mutants, and discovered that stomatal opening response to red light is correlated with a decrease in guard cell abscisic acid content and an increase in jasmonic acid content. The red‐light‐modulated guard cell metabolome reported here provides fundamental information concerning autonomous red light signaling pathways in guard cells.     

ACTION OF FUSICOCCIN ON THE POTASSIUM BALANCE OF GUARD CELLS OF PHASEOLUS VULGARIS

Fusicoccin induces stomatal opening in both the light and dark. The stomatal aperture and K content of guard cells was measured to determine whether the action of fusicoccin in inducing stomatal opening is directly related to the uptake of K by the guard cells. Both detached and attached epidermis was treated with fusicoccin and the K content was determined by staining with cobalt sodium nitrite or by electron probe microanalysis. The K content of guard cells in detached epidermal strips floated on 10 μm fusicoccin in 10 mm KCl and aqueous CH₃OH (0.02%, v/v) increased in the light and dark as the stomata opened. After exposure to fusicoccin for 6 hr in the light, however, the stomata were closed and no K could be detected in the guard cells. The K content of guard cells of attached epidermis painted with fusicoccin also increased as the stomata opened, but the concentration of K in the subsidiary cells was not significantly altered by fusicoccin-stimulated opening. Moreover, painting with fusicoccin did not significantly change the Ca and P content of the guard or subsidiary cells. Stomata of epidermal strips, opened to their maximum width by fusicoccin, showed only a small and temporary closure when transferred to a solution of 10 μm abscisic acid. The use of metabolic inhibitors suggested that energy for the uptake of the K may be provided by both photophosphorylation and oxidative phosphorylation.     

Characterisation of abscisic acid inhibition of stomatal opening in isolated epidermal strips

Abscisic acid (ABA) inhibited the light-induced opening of stomata in isolated epidermal strips of Commelina benghalensis. It did not alter stomatal closure in the dark. The ABA-induced inhibition in light was released under conditions conducive for cyclic photophosphorylation and remarkably reversed by ATP in the presence of pyruvate. Cyclic photophosphorylation rates of isolated guard cell chloroplasts were significantly reduced by ABA. It is proposed that the direct effect of ABA on stomatal opening was mediated in two ways: (1) by inhibition of cyclic photophosphorylation activities of guard cell chloroplasts and (2) by blocking organic acid formation in guard cells.     

The stomatal response to CO2 is linked to changes in guard cell zeaxanthin*

The mechanisms mediating CO₂ sensing and light–CO₂ interactions in guard cells are unknown. In growth chamber-grown Vicia faba leaves kept under constant light (500 μ mol m^–2 s^–1) and temperature, guard cell zeaxanthin content tracked ambient [CO₂] and stomatal apertures. Increases in [CO₂] from 400 to 1200 cm³ m^–3 decreased zeaxanthin content from 180 to 80 mmol mol^–1 Chl and decreased stomatal apertures by 7·0 μ m. Changes in zeaxanthin and aperture were reversed when [CO₂] was lowered. Guard cell zeaxanthin content was linearly correlated with stomatal apertures. In the dark, the CO₂-induced changes in stomatal aperture were much smaller, and guard cell zeaxanthin content did not change with chamber [CO₂]. Guard cell zeaxanthin also tracked [CO₂] and stomatal aperture in illuminated stomata from epidermal peels. Dithiothreitol (DTT), an inhibitor of zeaxanthin formation, eliminated CO₂-induced zeaxanthin changes in guard cells from illuminated epidermal peels and reduced the stomatal CO₂ response to the level observed in the dark. These data suggest that CO₂-dependent changes in the zeaxanthin content of guard cells could modulate CO₂-dependent changes of stomatal apertures in the light while a zeaxanthin-independent CO₂ sensing mechanism would modulate the CO₂ response in the dark.     

PP2A and microtubules function in 5-aminolevulinic acid-mediated H2O2 signaling in Arabidopsis guard cells

5-aminolevulinic acid (ALA), a plant growth regulator with great application potential in agriculture and horticulture, induces stomatal opening and inhibits stomatal closure by decreasing guard cell H₂O₂. However, the mechanisms behind ALA-decreased H₂O₂ in guard cells are not fully understood. Here, using type 2A protein phosphatase (PP2A) inhibitors, microtubule-stabilizing/disrupting drugs and green fluorescent protein-tagged α-tubulin 6 transgenic Arabidopsis (GFP-TUA6), we find that PP2A and cortical microtubules (MTs) are involved in ALA-regulated stomatal movement. Then, we analyze stomatal responses of Arabidopsis overexpressing C2 catalytic subunit of PP2A (PP2A-C2) and pp2a-c2 mutant to ALA and abscisic acid (ABA) under both light and dark conditions, and show that PP2A-C2 participates in ALA-induced stomatal movement. Furthermore, using pharmacological methods and confocal studies, we reveal that PP2A and MTs function upstream and downstream, respectively, of H₂O₂ in guard cell signaling. Finally, we demonstrate the role of H₂O₂-mediated microtubule arrangement in ALA inhibiting ABA-induced stomatal closure. Our findings indicate that MTs regulated by PP2A-mediated H₂O₂ decreasing play an important role in ALA guard cell signaling, revealing new insights into stomatal movement regulation.     

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.     

Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney.

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r     

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 function of guard cells does not require an intact array of cortical microtubules

The development of stomatal guard cells is known to require cortical microtubules; however, it is not known if microtubules are also required by mature guard cells for stomatal function. To study the role of microtubules in guard cell function, epidermal peels of Vicia faba were subjected to conditions known to open or close stomata in the presence or absence of microtubule inhibitors. To verify the action of the inhibitors, microtubules in appropriately treated epidermal peels were localized by cryofixation followed by freeze substitution and embedding in butyl-methyl methacrylate. Mature guard cells had a radial array of microtubules, focused toward the thick cell wall of the pore, and the appearance of this array was the same for stomata remaining closed in darkness or induced to open by light. Treatment of epidermal peels with 1 mM colchicine for 1 h depolymerized nearly all cortical microtubules. Measurements of stomatal aperture showed that neither 1 mM colchicine nor 20 M taxol affected any of the responses tested: remaining closed in the dark, opening in response to light or fusicoccin, and closing in response to calcium and darkness. We conclude that intact microtubule arrays are not invariably required for guard cell function.     

Jasmonate‐mediated stomatal closure under elevated CO2 revealed by time‐resolved metabolomics

Foliar stomatal movements are critical for regulating plant water loss and gas exchange. Elevated carbon dioxide (CO₂) levels are known to induce stomatal closure. However, the current knowledge on CO₂ signal transduction in stomatal guard cells is limited. Here we report metabolomic responses of Brassica napus guard cells to elevated CO₂ using three hyphenated metabolomics platforms: gas chromatography‐mass spectrometry (MS); liquid chromatography (LC)‐multiple reaction monitoring‐MS; and ultra‐high‐performance LC‐quadrupole time‐of‐flight‐MS. A total of 358 metabolites from guard cells were quantified in a time‐course response to elevated CO₂ level. Most metabolites increased under elevated CO₂, showing the most significant differences at 10 min. In addition, reactive oxygen species production increased and stomatal aperture decreased with time. Major alterations in flavonoid, organic acid, sugar, fatty acid, phenylpropanoid and amino acid metabolic pathways indicated changes in both primary and specialized metabolic pathways in guard cells. Most interestingly, the jasmonic acid (JA) biosynthesis pathway was significantly altered in the course of elevated CO₂ treatment. Together with results obtained from JA biosynthesis and signaling mutants as well as CO₂ signaling mutants, we discovered that CO₂‐induced stomatal closure is mediated by JA signaling.     

Stomatal responses to light and leaf-air water vapor pressure difference show similar kinetics in sugarcane and soybean

Stomatal responses to light and humidity (vapor pressure difference, VPD) are important determinants of stomatal conductance. Stomatal movements induced by light are the result of a transduction of the light stimulus into modulated ion fluxes in guard cells and concomitant osmotic adjustments and turgor changes. It is generally assumed that this transduction process is a general stomatal property, with different environmental stimuli integrated into guard cell metabolism through their modulation of ion fluxes. In contrast with this notion, the VPD response, which is unique because both its triggering signal and the turgor changes required for aperture modulations involve water molecules, has been considered to be hydropassive and thus independent of guard cell metabolism. We used a kinetic approach to compare the light and VPD responses in order to test the hypothesis that hydropassive changes in guard cell turgor could be faster than the metabolism-dependent light responses. Changes in stomatal conductance in intact leaves of sugarcane and soybean were measured after application of step changes in VPD and in light. In spite of a 5-fold difference in overall rates between the two species, the response rates following light or VPD steps were similar. Although a coincidental kinetic similarity between two mechanistically different responses cannot be ruled out, the data suggest a common mechanism controlling stomatal movements, with the VPD stimulus inducing metabolic modulations of ion fluxes analogous to other stomatal responses.     

Regulation of stomatal movement by cortical microtubule organization in response to darkness and ABA signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Microtubule dynamics are essential for plant cell development and in producing responses to external stimuli. However, little is known about the regulation of microtubule dynamics or crosstalk between microtubule and stomatal movement. Here we identified microtubule reorganization as a crucial factor determining guard cell responses to dark and abscisic acid (ABA) signaling. As stomata opened, guard cells exhibited radially arranged cortical microtubules, which depolymerized into the cytosol when exposed to darkness and ABA. Suppression of microtubule disassembly by paclitaxel, a microtubule-stabilizing drug, significantly enhanced stomatal aperture under light, and partially blocked ABA- or darkness-induced stomatal closure. However, treatment with only the anti-microtubule drug, oryzalin, did not affect stomatal movement with or without external stimuli. Phosphatidic acid (PA) bound to a clade A type 2C protein phosphatase (PP2C), PP2CA, and deletion of PP2CA partially inhibited PA-induced microtubule depolymerization and stomatal closure. Moreover, microtubule reorganization was altered in the ABA-insensitive mutant pldα1, but not in the ABA-hypersensitive mutant pp2ca. We propose that a faithfully balanced reorganization of microtubules fulfills fundamental functions to enable the fast change of stomata in plant adaptive responses to developmental and environmental cues.     

Actin Filaments in Mature Guard Cells Are Radially Distributed and Involved in Stomatal Movement

Stomatal movements, which regulate gas exchange in plants, involve pronounced changes in the shape and volume of the guard cell. To test whether the changes are regulated by actin filaments, we visualized microfilaments in mature guard cells and examined the effects of actin antagonists on stomatal movements. Immunolocalization on fixed cells and microinjection of fluorescein isothiocyanate-phalloidin into living guard cells of Commelina communis L. showed that cortical microfilaments were radially distributed, fanning out from the stomatal pore site, resembling the known pattern of microtubules. Treatment of epidermal peels with phalloidin prior to stabilizing microfilaments with m-maleimidobenzoyl N-hydroxysuccimimide caused dense packing of radial microfilaments and an accumulation of actin around many organelles. Both stomatal closing induced by abscisic acid and opening under light were inhibited. Treatment of guard cells with cytochalasin D abolished the radial pattern of microfilaments; generated sparse, poorly oriented arrays; and caused partial opening of dark-closed stomata. These results suggest that microfilaments participate in stomatal aperture regulation.     

NO和H2O2在光／暗调控蚕豆气孔运动中的作用及其相互关系

借助表皮条分析和激光扫描共聚焦显微镜技术,对NO和H_2O_2在光/暗调控蚕豆(Vicia faba L.)气孔运动中的作用及其相互关系进行了探索。结果显示,光下外源NO供体硝普钠(SNP)和H_2O_2促进气孔关闭的效应明显大于暗中,暗中NO专一性清除剂2,4-羧基苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)、一氧化氮合酶(NOS)抑制剂N~G-氮-L-精氨酸-甲酯(L-NAME)和H_2O_2清除剂抗坏血酸(Vc)、过氧化氢酶(CAT)对气孔开度的效应明显大于光下,而且光下蚕豆保卫细胞NO和H_2O_2水平比暗中明显降低。上述结果表明,光/暗通过影响保卫细胞NO和H_2O_2的水平调控气孔运动。研究还发现,光下H_2O_2既诱导NO水平增加,也诱导气孔关闭,cPTIO和L-NAME有效地逆转H_2O_2的这些效应;光下SNP既诱导H_2O_2水平增加,也诱导气孔关闭,SNP的上述效应又被Vc和CAT有效逆转。这些结果表明,NO和H_2O_2在生成及效应上均存在明显的相互作用。另外,L-NAME显著逆转暗和光下H_2O_2处理对气孔关闭和NO生成的效应表明,蚕豆保卫细胞中可能存在NOS,暗和光下H_2O_2处理可能通过提高NOS的活性促进NO水平增加,进而诱导气孔关闭。     

In the light of stomatal opening: new insights into 'the Watergate'

Stomata can be regarded as hydraulically driven valves in the leaf surface, which open to allow CO2 uptake and close to prevent excessive loss of water. Movement of these 'Watergates' is regulated by environmental conditions, such as light, CO2 and humidity. Guard cells can sense environmental conditions and function as motor cells within the stomatal complex. Stomatal movement results from the transport of K+ salts across the guard cell membranes. In this review, we discuss the biophysical principles and mechanisms of stomatal movement and relate these to ion transport at the plasma membrane and vacuolar membrane. Studies with isolated guard cells, combined with recordings on single guard cells in intact plants, revealed that light stimulates stomatal opening via blue light-specific and photosynthetic-active radiation-dependent pathways. In addition, guard cells sense changes in air humidity and the water status of distant tissues via the stress hormone abscisic acid (ABA). Guard cells thus provide an excellent system to study cross-talk, as multiple signaling pathways induce both short- and long-term responses in these sensory cells.     

Involvement of Calyculin A- and Okadaic Acid-sensitive Protein Phosphatase in the Blue Light Response of Stomatal Guard Cells

Calyculin A (CA) and okadaic acid (OA), inhibitors of proteinphosphatases, inhibited blue light (BL)-dependent H⁺pumpingin Vicia guard cell protoplasts at half-inhibitory concentrationsof 4.5 nM and 400 nM, respectively. Light-induced stomatal openingin Viciaepidermis was completely suppressed by CA at 100 nMand by OA at 1 µM. These results suggest that CA- andOA-sensitive protein phosphatase is involved in the BL responseof stomatal guard cells. (Received June 27, 1997; Accepted September 2, 1997)     

Inhibition of dark‐induced stomatal closure by fusicoccin involves a removal of hydrogen peroxide in guard cells of Vicia faba

Fusicoccin (FC) treatment prevents dark‐induced stomatal closure, the mechanism of which is still obscure. By using pharmacological approaches and laser‐scanning confocal microscopy, the relationship between FC inhibition of dark‐induced stomatal closure and the hydrogen peroxide (H₂O₂) levels in guard cells in broad bean was studied. Like ascorbic acid (ASA), a scavenger of H₂O₂ and diphenylene iodonium (DPI), an inhibitor of H₂O₂‐generating enzyme NADPH oxidase, FC was found to inhibit stomatal closure and reduce H₂O₂ levels in guard cells in darkness, indicating that FC‐caused inhibition of dark‐induced stomatal closure is related to the reduction of H₂O₂ levels in guard cells. Furthermore, like ASA, FC not only suppressed H₂O₂‐induced stomatal closure and H₂O₂ levels in guard cells treated with H₂O₂ in light, but also reopened the stomata which had been closed by darkness and reduced the level of H₂O₂ that had been generated by darkness, showing that FC causes H₂O₂ removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomata treated with H₂O₂ and had been closed by dark, and on H₂O₂ levels in guard cells of stomata treated with H₂O₂ and had been closed by dark, and both FC and butyric acid reduced cytosol pH in guard cells of stomata treated with H₂O₂ and had been closed by dark, which demonstrates that cytosolic acidification mediates FC‐induced H₂O₂ removal. Taken together, our results provide evidence that FC causes cytosolic acidification, consequently induces H₂O₂ removal, and finally prevents dark‐induced stomatal closure.     

NO和H2O2在光/暗调控蚕豆气孔运动中的作用及其相互关系

借助表皮条分析和激光扫描共聚焦显微镜技术,对NO和H2O2在光/暗调控蚕豆(Vicia faba L.)气孔运动中的作用及其相互关系进行了探索.结果显示,光下外源NO供体硝普钠(SNP)和H2O2促进气孔关闭的效应明显大于暗中,暗中NO专一性清除剂2,4-羧基苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)、一氧化氮合酶(NOS)抑制剂NG-氮-L-精氨酸-甲酯(L-NAME)和H2O2清除剂抗坏血酸(Vc)、过氧化氢酶(CAT)对气孔开度的效应明显大于光下,而且光下蚕豆保卫细胞NO和H2O2水平比暗中明显降低.上述结果表明,光/暗通过影响保卫细胞NO和H2O2的水平调控气孔运动.研究还发现,光下H2O2既诱导NO水平增加,也诱导气孔关闭,cPTIO和L-NAME有效地逆转H2O2的这些效应;光下SNP既诱导H2O2水平增加,也诱导气孔关闭,SNP的上述效应又被Vc和CAT有效逆转.这些结果表明,NO和H2O2在生成及效应上均存在明显的相互作用.另外,L-NAME显著逆转暗和光下H2O2处理对气孔关闭和NO生成的效应表明,蚕豆保卫细胞中可能存在NOS,暗和光下H2O2处理可能通过提高NOS的活性促进NO水平增加,进而诱导气孔关闭.     

Stomatal response to humidity in sugarcane and soybean: effect of vapour pressure difference on the kinetics of the blue light response

Abstract. The effect of atmospheric humidity on the kinetics of stomatal responses was quantified in gas exchange experiments using sugarcane ( Saccharum spp. hybrid) and soybean ( Glycine max ). Pulses of blue light were used to elicit pulses of stomatal conductance that were mediated by the specific blue light response of guard cells. Kinetic parameters of the conductance response were more closely related to leaf-air vapour pressure difference (VPD) than to relative humidity or transpiration. Increasing VPD significantly accelerated stomatal opening in both sugarcane and soybean, despite an approximately five-fold faster response in sugarcane. In contrast, the kinetics of stomatal recovery (closure) following the pulse were similar in the two species. Acceleration of opening by high VPD was observed even under conditions where soybean exhibited a feedforward response of decreasing transpiration (E) with increasing evaporative demand (VPD). This result suggests that epidermal, rather than bulk leaf, water status mediates the VPD effect on stomatal kinetics. The data are consistent with the hypothesis that increased cpidermal water loss at high VPD decreases the backpressure exerted by neighbouring cells on guard cells. allowing more rapid stomatal opening per unit of guard cell metabolic response to blue light.