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.     

The ultraviolet action spectrum for stomatal opening in broad bean

The ultraviolet action spectrum for stomatal opening was measured using epidermal peels from leaves of broad bean (Vicia faba). The spectrum was calculated from hyperbolic fluence response curves using 11 wavelengths ranging from 275 to 459 nm. The action spectrum exhibits a major peak at approximately 280 nm and a minor peak at approximately 360 nm. The response at 280 nm is about three times greater than the response at 459 nm. Under the conditions utilized (i.e. the absence of saturating red light), stomatal opening saturated at extremely low fluence rates: <0.2 μmol m⁻² s⁻¹ at 280 nm, and approximately 1.0 μmol m⁻² s⁻¹ at 459 nm. The threshold for blue-light-induced stomatal opening was approximately 0.02 μmol m⁻² s⁻¹. In light-mixing experiments, the addition of 280 nm light to saturating 650 nm (red) light caused additional stomatal opening, which is indicative of separate photoreceptors. In contrast, adding 280 nm of light to saturating 459 nm (blue) light did not increase stomatal opening, suggesting that they both excite the same receptor. The results with white light were similar to those with blue light. We infer that ultraviolet light acts via the blue light photoreceptor rather than through photosynthesis. The additional absorbance peak at 360 nm suggests that the chromophore is either a flavin or a cis-carotenoid, both of which exhibit peaks in this region. It is proposed that the chromophore can be excited either directly by blue light or by energy transferred from the protein portion of the protein-pigment complex after it absorbs 280 nm light.     

Bioenergetic processes in guard cells related to stomatal function

The energy required for ion uptake in guard cells is provided by two important bioenergetic processes, namely respiration and photosynthesis. The blue light-sensitive plasma membrane redox system is considered as the third bioenergetic phenomenon, since it uses blue light to create a proton gradient across the membrane. The unique features of respiration and photosynthesis in guard cells and their role in stomatal function are emphasized. Evidence for and against the blue light-sensitive components on plasma membrane (ATPase/distinct redox chain) and the photoreceptors (flavins, carotenoids, pterins) in guard cells are presented. The information on ion channels and their response to various kinds of secondary messengers including G-proteins, phosphoinositides, diacylglycerol, calcium, cAMP and protein kinases are reviewed. A model is presented indicating the possible mechanism of perception and transduction by guard cells of external signals and their interaction with different bioenergetic components.     

Lacking chloroplasts in guard cells of crumpled leaf attenuates stomatal opening: both guard cell chloroplasts and mesophyll contribute to guard cell ATP levels

Controversies regarding the function of guard cell chloroplasts and the contribution of mesophyll in stomatal movements have persisted for several decades. Here, by comparing the stomatal opening of guard cells with (crl‐ch) or without chloroplasts (crl‐no ch) in one epidermis of crl (crumpled leaf) mutant in Arabidopsis, we showed that stomatal apertures of crl‐no ch were approximately 65–70% those of crl‐ch and approximately 50–60% those of wild type. The weakened stomatal opening in crl‐no ch could be partially restored by imposing lower extracellular pH. Correspondingly, the external pH changes and K⁺ accumulations following fusicoccin (FC) treatment were greatly reduced in the guard cells of crl‐no ch compared with crl‐ch and wild type. Determination of the relative ATP levels in individual cells showed that crl‐no ch guard cells contained considerably lower levels of ATP than did crl‐ch and wild type after 2 h of white light illumination. In addition, guard cell ATP levels were lower in the epidermis than in leaves, which is consistent with the observed weaker stomatal opening response to white light in the epidermis than in leaves. These results provide evidence that both guard cell chloroplasts and mesophyll contribute to the ATP source for H⁺ extrusion by guard cells.     

Blue-light- and phosphorylation-dependent binding of a 14-3-3 protein to phototropins in stomatal guard cells of broad bean

Phototropins are blue-light (BL) receptor serine (Ser)/threonine kinases, and contain two light, oxygen, and voltage (LOV) domains, and are members of the PAS domain superfamily. They mediate phototropism, chloroplast movement, leaf expansion, and stomatal opening of higher plants in response to BL. In stomatal guard cells, genetic analysis has revealed that phototropins mediate activation of the plasma membrane H+-ATPase by phosphorylation and drive stomatal opening. However, biochemical evidence for the involvement of phototropins in the BL response of stomata is lacking. Using guard cell protoplasts, we showed that broad bean (Vicia faba) phototropins (Vfphots) were phosphorylated by BL, and that this phosphorylation of Vfphots reached to the maximum level earlier than that of the H+-ATPase. Phosphorylation of both Vfphots and H+-ATPase showed similar sensitivity to BL and were similarly suppressed by protein kinase and flavoprotein inhibitors. We found that a 14-3-3 protein was bound to Vfphots upon phosphorylation, and this binding occurred earlier than the H+-ATPase phosphorylation. Vfphots (Vfphot1a and Vfphot1b) were expressed in Escherichia coli, and phosphorylation sites were determined to be Ser-358 for Vfphot1a and Ser-344 for Vfphot1b, which are localized between LOV1 and LOV2. We conclude that Vfphots act as BL receptors in guard cells and that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 protein binding are likely to be key steps of BL response in stomata. The binding of a 14-3-3 protein to Vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that this event was common to phototropin-mediated responses.     

Nitric oxide inhibits blue light-specific stomatal opening via abscisic acid signaling pathways in Vicia guard cells

Recent evidence suggests that nitric oxide (NO) acts as an intermediate of ABA signal transduction for stomatal closure. However, NO's effect on stomatal opening is poorly understood even though both opening and closing activities determine stomatal aperture. Here we show that NO inhibits stomatal opening specific to blue light, thereby stimulating stomatal closure. NO inhibited blue light-specific stomatal opening but not red light-induced opening. NO inhibited both blue light-induced H(+) pumping and H(+)-ATPase phosphorylation. The NO scavenger 2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) restored all these inhibitory effects. ABA and hydrogen peroxide (H(2)O(2)) inhibited all of these blue light-specific responses in a manner similar to NO. c-PTIO partially restored the ABA-induced inhibition of all of these opening responses but did not restore inhibition of the responses by H(2)O(2). ABA, H(2)O(2) and NO had slight inhibitory effects on the phosphorylation of phototropins, which are blue light receptors in guard cells. NO inhibited neither fusicoccin-induced H(+) pumping in guard cells nor H(+) transport by H(+)-ATPase in the isolated membranes. From these results, we conclude that both NO and H(2)O(2) inhibit blue light-induced activation of H(+)-ATPase by inhibiting the component(s) between phototropins and H(+)-ATPase in guard cells and stimulate stomatal closure by ABA.     

Accumulation of malate in guard cells of Vicia faba during stomatal opening

Summary The level of malate in the epidermis from illuminated leaves of Vicia faba was greater than in that from dark-treated leaves. A difference in the malate level was still detected after the epidermis had been treated by rolling so that only the guard cells remained alive. The results suggest that malate may accumulate in guard cells on illumination. In subsequent experiments, stomatal apertures were measured, and potassium as well as malate was analysed in extracts of epidermis. In illuminated leaves, the potassium content of rolled epidermis increased from about 90 to about 335 picoequivalents mm^-2 of epidermis whele malate increased from about zero to about 71 pmoles mm^-2 and the stomata opened; in dark-treated leaves, the potassium content of rolled epidermis decreased slightly, the malate level remained about zero, and the stomata showed very slight further closure. The measured increase in potassium is likely to represent an increase in potassium concentration in the guard cells of about 0.4 Eq l^-1 with stomatal opening; the increase in malate could correspond to 0.23 Eq l^-1 (with respect to potassium) in the guard cells. Thus, malate accumulating in guard cells could balance about half of the potassium taken up by guard cells when stomata open in the light.     

Rubisco activity in guard cells compared with the solute requirement for stomatal opening

We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/oxygenase (Rubisco) were determined by the [¹⁴C]carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to ¹⁴CO₂ for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19 femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.     

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin‐related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin‐D‐induced depolymerization or phalloidin‐induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild‐type (WT) Arabidopsis plants. Light‐induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.     

Deficient glutathione in guard cells facilitates abscisic acid-induced stomatal closure but does not affect light-induced stomatal opening

We investigated the role of glutathione (GSH) in stomatal movements using a GSH deficient mutant, chlorinal-1 (ch1-1). Guard cells of ch1-1 mutants accumulated less GSH than wild types did. Light induced stomatal opening in ch1-1 and wild-type plants. Abscisic acid (ABA) induced stomatal closure in ch1-1 mutants more than wild types without enhanced reactive oxygen species (ROS) production. Therefore, GSH functioned downstream of ROS production in the ABA signaling cascade.     

The Arabidopsis small G protein ROP2 is activated by light in guard cells and inhibits light-induced stomatal opening

ROP small G proteins function as molecular switches in diverse signaling processes. Here, we investigated signals that activate ROP2 in guard cells. In guard cells of Vicia faba expressing Arabidopsis thaliana constitutively active (CA) ROP2 fused to red fluorescent protein (RFP-CA-ROP2), fluorescence localized exclusively at the plasma membrane, whereas a dominant negative version of RFP-ROP2 (DN-ROP2) localized in the cytoplasm. In guard cells expressing green fluorescent protein-ROP2, the relative fluorescence intensity at the plasma membrane increased upon illumination, suggesting that light activates ROP2. Unlike previously reported light-activated factors, light-activated ROP2 inhibits rather than accelerates light-induced stomatal opening; stomata bordered by guard cells transformed with CA-rop2 opened less than controls upon light irradiation. When introduced into guard cells together with CA-ROP2, At RhoGDI1, which encodes a guanine nucleotide dissociation inhibitor, inhibited plasma membrane localization of CA-ROP2 and abolished the inhibitory effect of CA-ROP2 on light-induced stomatal opening, supporting the negative effect of active ROP2 on stomatal opening. Mutant rop2 Arabidopsis guard cells showed phenotypes similar to those of transformed V. faba guard cells; CA-rop2 stomata opened more slowly and to a lesser extent, and DN-rop2 stomata opened faster than wild-type stomata in response to light. Moreover, in rop2 knockout plants, stomata opened faster and to a greater extent than wild-type stomata in response to light. Thus, ROP2 is a light-activated negative factor that attenuates the extent of light-induced changes in stomatal aperture. The inhibition of light-induced stomatal opening by light-activated ROP2 suggests the existence of feedback regulatory mechanisms through which stomatal apertures may be finely controlled.     

Reactive oxygen species and nitric oxide are involved in ABA inhibition of stomatal opening

Although nitric oxide (NO) and reactive oxygen species (ROS) are essential signalling molecules required for mediation of abscisic acid (ABA)-induced stomatal closure, it is not known whether these molecules also mediate the ABA inhibition of stomatal opening. In this study, we investigated the role of NO and ROS in the ABA inhibition of stomatal opening in Vicia faba. ABA induced both NO and ROS synthesis, and the NO scavenger reduced the ABA inhibition of stomatal opening. Exogenous NO and hydrogen peroxide (H2O2) also inhibited stomatal opening, indicating that NO and ROS are involved in the inhibition signalling process. An inhibitor of nitric oxide synthase (NOS) reversed the ABA inhibition of stomatal opening. Either the NO scavenger or the NOS inhibitor also reversed the process in the H2O2 inhibition of stomatal opening. We found that in the ABA inhibition of stomatal opening, NO is downstream of ROS in the signalling process, and NO is synthesized by a NOS-like enzyme.     

Propofol-induced vascular permeability change is related to the nitric oxide signaling pathway and occludin phosphorylation

The present study was undertaken to elucidate the mechanism of intra-arterial propofol-induced vascular permeability change resulting in tissue edema. The mechanism of propofol-induced hyperpermeability was examined in a rat femoral artery injection model. Vascular permeability was determined by measuring the Evans blue content of the dorsal skin of the infused limb at 15, 30, 45 and 60 min after propofol injection. The total content of the tight junction proteins occludin, ZO-1 and claudin-5 under experimental conditions was also determined by western blotting. Intra-arterial injection with propofol resulted in a marked dose-dependent increase in vascular permeability of the rat hindpaw. Pretreatment with 10 mg/kg of N-nitro-l-arginine methyl ester (l-NAME) but not aminoguanidine significantly inhibited the change in vascular permeability after challenge with propofol. Pretreatment with l-arginine and nitroprusside increased the propofol-induced permeability change. Intra-arterial injection of propofol significantly increased occludin phosphorylation after 15 min, which was consistent with the time profile of the vascular permeability change. l-NAME partially reversed the change in occludin phosphorylation, whereas aminoguanidine had no effect compared with that in the controls. Our observations indicate that nitric oxide (NO) is an important mediator in the induction of vascular permeability induced by propofol. Occludin phosphorylation is a determining factor in the vascular permeability change induced by propofol. NO synthase (NOS) inhibitors might be useful in the treatment of accidental intra-arterial injection of propofol, in the reduction of any adverse effects.     

A novel hydrogen sulfide donor causes stomatal opening and reduces nitric oxide accumulation

Effects of hydrogen sulfide (H₂S) on plant physiology have been previously studied, but such studies have relied on the use of NaSH as a method for supplying H₂S to tissues. Now new compounds which give a less severe H₂S shock and a more prolonged exposure to H₂S have been developed. Here the effects of one such compound, GYY4137, has been investigated to determine its effects on stomatal closure in Arabidopsis thaliana. It was found that both NaSH and GYY4137 caused stomatal opening in the light and prevented stomatal closure in the dark. Nitric oxide (NO) has been well established as a mediator of stomatal movements and here it was found that both NaSH and GYY4137 reduced the accumulation of NO in guard cells, perhaps suggesting a mode of action for H₂S in this system. GYY4137, and future related compounds, will be important tools to unravel the effects of plant exposure to H₂S and to determine how H₂S may fit into plant cell signalling pathways.     

Inhibition of stomatal opening during uptake of carbohydrates by guard cells in isolated epidermal tissues

The uptake of glucose and other carbohydrates into the guard cells of Commelina communis L. was found to inhibit the opening of the stomata. The concentration of glucose necessary to achieve about 50% inhibition was of the same order of magnitude as the potassium concentration required for opening; the uptake systems for potassium and glucose appear to be competitive and to exhibit the same degree of affinity. It is suggested that the uptake of glucose occurs via a proton cotransport, which, depolarizing the membrane potential, slows down the electrogenic import of potassium ions. The process of stomatal closure, in contrast, appears not to be affected by carbohydrate uptake. In guard cells of Tulipa gesneriana L. and Vicia faba L., which do not possess subsidiary cells, import of glucose or other carbohydrates did not interfere with the regulation of stomatal movements.     

Regulation of stomatal opening by the guard cell expansin AtEXPA1

Stomatal movement is strictly regulated by various intracellular and extracellular factors in response environmental signals. In our recent study, we found that an Arabidopsis guard cell expressed expansin, AtEXPA1, regulates stomatal opening by altering the structure of the guard cell wall. This addendum proposes a mechanism by which guard cell expansins regulate stomatal movement.Key words: expansin, stomatal movement, AtEXPA1, guard cell, wall looseningStomatal movement is the most popular model system for cellular signaling transduction research. A complicated complex containing many proteins has been proposed to control stomatal responses to outside stimuli. The known regulation factors are primarily located in the nucleus, cytoplasm, plasma membrane and other intracellular organelles.^1,2 Although the cell wall structure of the stomata is different from that of other cells,^3,4 the presence of stomatal movement regulation factors in the cell wall has seldom been reported in reference ⁵. In our previous work, we found that extracellular calmodulin stimulates a cascade of intracellular signaling events to regulate stomatal movement.⁶ The involvement of this signaling pathway is the first evidence that cell wall proteins play an important role in regulation of stomatal opening. Cell wall-modifying factors constitute a major portion of cell wall proteins. However, the role of these factors in the regulation of stomatal movement is not yet known.Expansins are nonenzymatic proteins that participate in cell wall loosening.^7–9 Expansins were first identified as “acid-growth” factors because they have much higher activities at acidic pHs.^10,11 It has been reported that expansins play important roles in plant cell growth, fruit softening, root hair emergence and other developmental processes in which cell wall loosening is involved.^7–9,12,13 Wall loosening is an essential step in guard cell swelling and the role of stomatal expansins was investigated. AtEXPA1 is an Arabidopsis guard-cell-specific expansin.^13,14 Over-expressing AtEXPA1 increases the rate of light-induced stomatal opening,^14,15 while a potential inhibitor of expansin activity, AtEXPA1 antibody, reduces the sensitivity of stomata to stimuli.¹⁴ We showed that the transpiration rate and the photosynthesis rate in plant lines overexpressing AtEXPA1 were nearly two times the rates for wild-type plants (Fig. 1). These in plant data revealed that expansins accelerated stomatal opening under normal physiological conditions. In addition, the increases in the transpiration and photosynthesis rates strongly suggested the possibility of exploiting expansin-regulated stomatal sensitivity to modify plant drought tolerance. Compared with the effect of hydrolytic cell wall enzymes, the destruction of cell wall structures induced by expansins is minimal. In addition, it is very difficult to directly observe the changes in the guard cell wall structure caused by expansins during stomatal movement. Our recent work showed that, in AtEXPA1-overexpressing plants, the volumetric elastic modulus is lower than in wild-type plants,¹⁴ which indicates the wall structure was loosened and that the cell wall was easier to extend. Taken together, our data suggest that expansins participate in the regulation of stomatal movement by modifying the cell walls of guard cells.Open in a separate windowFigure 1Effects of AtEXPA1 overexpression on transpiration rates and photosynthesis rates. The transpiration rate (left) and photosynthesis rate (right) of wild-type and transgenic AtEXPA1 lines were measured at 10:00 AM in the greenhouse after being watered overnight. The illumination intensity was 180 µmol/m²·s. Bars represent the standard error of the mean of at least five plants per line.It is well known that the activation of proton-pumping ATPase (H⁺-ATPase) in the plasma membrane is an early and essential step in stomatal opening.¹⁶ The action of the pump results in an accumulation of H⁺ outside of the cell, increases the inside-negative electrical potential across the plasma membrane and drives potassium uptake through the voltage-gated, inward-rectifying K⁺ channels.^17–19 The main function of the H⁺ pump is well accepted to create an electrochemical gradient across the plasma membrane; however, the other result is the acidification of the guard cell wall, which may also contribute to stomatal opening. A possible mechanism responsible for this effect is as follows. Expansins are in an inactive state when the stomata are in the resting state. Stomatal opening signals induce wall acidification and activate expansins. Then, the expansins move along with cellulose microfibrils and transiently break down hydrogen bonding between hemicellulose and the surface of cellulose microfibrils,^20,21 facilitating the slippage of cell wall polymers under increasing guard cell turgor pressure. The guard cell then swells and the stomata open (Fig. 2).Open in a separate windowFigure 2Model of how guard cell wall expansins regulate stomatal opening. Environmental stimuli, e.g., light, activate guard cell plasma membrane H⁺-ATPases to pump H⁺ into the extracellular wall space. The accumulation H⁺ acidifies the cell wall and induces the activation of expansin. The active expansin disrupts non-covalent bonding between cellulose microfibrils and matrix glucans to enable the slippage of the cell wall. The wall is loosened coincident with guard cell swelling and without substantial breakdown of the structure.Although our results indicate that AtEXPA1 regulates stomatal movement, the biochemical and structural mechanism by which AtEXPA1 loosens the cell wall remains to be discovered. It remains to figure out the existing of other expansins or coordinators involving in this process. In addition, determining the roles of expansins and the guard cell wall in stomatal closing is another main goal of future research.     

Extracellular calcium is involved in stomatal movement through the regulation of water channels in broad bean

Involvement of extracellular Ca²⁺ in stomatal movement through the regulation of water channels was investigated in broad bean (Vicia faba L.). Leaf peels were first incubated to open stomata, and then transferred to buffers in the presence of different CaCl₂ concentrations. Stomatal status was observed under magnification and stomatal aperture (pore width/length) was measured. Stomatal closure was significantly induced and aperture oscillation occurred at lower extracellular concentrations of calcium ([Ca²⁺]_ext), while at higher concentrations, no significant change in stomatal aperture was observed, which was similar to the response recorded with HgCl₂. Lower [Ca²⁺]_ext-induced stomatal closure could be reversed using depolarizing buffer. It is suggested that lower [Ca²⁺]_ext regulates water channels through an indirect way and at higher concentrations, extracellular Ca²⁺ is involved in regulating stomatal aperture by directly influencing water channels to retard aperture change.