Inhibition of light-induced stomatal opening and of guard-cell-protoplast swelling in Vicia faba L. by tentoxin,an inhibitor of photophosphorylation

The fungal phytotoxin tentoxin and its natural derivative dihydrotentoxin impair light-induced stomatal opening in epidermal strips of broad bean (Vicia faba L.) incubated in a potassium-rich medium. Swelling of guard-cell protoplasts (GCPs) of the same species is inhibited in the presence of both substances. Swollen GCPs shrink after tentoxin or dihydrotentoxin treatment and these effects cannot be fully compensated by the phytoeffector fusicoccin. A comparison with the potassium carrier valinomycin shows that tentoxin acts in a different manner, because it is effective in the light only, whereas valinomycin causes shrinkage of GCPs also in the dark. Determination of adenine nucleotides in GCPs indicates a reduced ATP content and an enhanced ADP level after addition of tentoxin. At the same time, tentoxintreated GCPs contain more NADPH and less NAD⁺ than the control (NADP⁺ and NADH content does not differ). The results presented are consistent with the hypothesis that tentoxin closes stomata as a consequence of its inhibitory action on photophosphorylation.Abbreviations FC fusicoccin - GCP guard-cell protoplast - KIDA potassium iminodiacetate     

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

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

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.     

Regulation of Dark-Induced Stomatal Closure in <Emphasis Type="Italic">Arabidopsis</Emphasis> Dynamin-Like Protein 1E (<Emphasis Type="Italic">adl1e</Emphasis>) Mutant Leaves

Arabidopsis thaliana dynamin-like protein 1E (ADL1E) is known to regulate mitochondrial elongation. The adl1e mutant has no morphological phenotype, and the growth and photosynthetic activity of the mutant are similar to those of the wild type. Leaf O₂ uptake, which is supported by mitochondrial activity in the dark, is increased 1.7-fold by mutation in adl1e gene. The ATP content in the dark of guard and mesophyll cell protoplasts (GCPs and MCPs, respectively) was 2.5- to 4-fold higher in GCPs of the mutant and the wild type, and increased upon the addition of glucose in both genotypes. Oligomycin, an inhibitor of mitochondrial ATPase, suppressed ATP synthesis in both GCPs and MCPS isolated from adl1e plants, indicating that mutant had higher mitochondrial activity. The stomatal apertures of mutant and wild-type plants were then analyzed in vitro. In the light, the stomata of both genotypes showed similar patterns of opening. However, in the dark response, the stomata of the adl1e mutant closed faster than did those of the wild type. Oligomycin severely inhibited dark-induced stomatal closure in both cell types. The results suggest that stomatal closure in the dark is governed by cytosolic ATP concentration, which is stimulated by mitochondrial activity.     

Inhibition of Blue Light-Dependent H+ Pumping by Abscisic Acid in Vicia Guard-Cell Protoplasts

Blue-light (BL)-dependent H+ pumping in guard-cell protoplasts (GCPs) from Vicia faba was inhibited by 65% in the presence of abscisic acid (ABA). The inhibition increased with the time after application of ABA and was concentration dependent with a saturating concentration of 1 [mu]M at pH 6.2. The inhibition was nearly independent of the pH of the medium in the range 5.4 to 7.2 when ABA was applied at 10 [mu]M, whereas it was dependent on pH when the ABA concentration was decreased. The protonated form of ABA was saturating at 40 nM in inhibiting BL-dependent H+ pumping under various experimental conditions, whereas the dissociated form at 500 nM had no inhibitory effect on the pumping, suggesting that the protonated form of ABA is the form active in inhibiting the pumping. Fusicoccin (10 [mu]M), an activator of plasma membrane H+-ATPase, induced H+ pumping from GCPs, and the rate of H+ pumping was decreased to 70% by ABA. In contrast, ABA did not inhibit H+ pumping in isolated microsome vesicles from GCPs. These results suggest that the inhibition of BL-dependent H+ pumping by ABA in GCPs may be due to indirect inactivation of plasma membrane H+-ATPase and/or inhibition of the BL-signaling pathway. The pump inhibition by ABA causes membrane depolarization and can be an initial step to induce stomatal closure and reduces the transpirational water loss under drought stress in the daytime.     

Requirements for activation of the signal-transduction network that leads to regulatory phosphorylation of leaf guard-cell phosphoenolpyruvate carboxylase during fusicoccin-stimulated stomatal opening

Leaves regulate gas exchange through control of stomata in the epidermis. Stomatal aperture increases when the flanking guard cells accumulate K+ or other osmolytes. K+ accumulation is stoichiometric with H+ extrusion, which is compensated for by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31)-mediated malate synthesis. Plant PEPCs are regulated allosterically and by phosphorylation. Aspects of the signal-transduction network that control the PEPC phosphorylation state in guard cells are reported here. Guard cells were preloaded with [32P]orthophosphate (32Pi); then stomata were incubated with fusicoccin (FC), which activates the guard-cell plasma membrane H+-ATPase. [32P]PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. In -FC controls, stomatal size, guard-cell malate, and [32P]PEPC were low; maximum values for these parameters were observed in the presence of FC after a 90-min incubation and persisted for an additional 90 min. This high steady-state phosphorylation status resulted from continuous phosphorylation and dephosphorylation, even after the malate-accumulation phase. PEPC phosphorylation was diminished by approximately 80% when K+ uptake was associated with Cl- uptake and was essentially abolished when stomatal opening was sucrose--rather than K+--dependent. Finally, alkalinization by NH4+ in the presence of K+ did not cause PEPC phosphorylation (as it does in C4 plants). As discussed, a role for cytoplasmic protons cannot be completely excluded by this result. In summary, activation of the plasma membrane H+-ATPase was essential, but not sufficient, to cause phosphorylation of guard-cell PEPC. Network components downstream of the H+-ATPase influence the phosphorylation state of this PEPC isoform.     

Crosstalk between blue-light- and aba-signaling pathways in stomatal guard cells

We recently established an immunohistochemical method for the detection of blue light (BL)-induced and phototropin-mediated phosphorylation of plasma-membrane H⁺-ATPase in stomatal guard cells of Arabidopsis thaliana. This technique makes it possible to detect the phosphorylation/activation status of guard-cell H⁺-ATPase in the epidermis of a single rosette leaf, without the need to prepare guard-cell protoplasts (GCPs) from a large number of plants. Moreover, it can detect guard-cell responses under more natural and stress-free conditions compared to using GCPs. Taking advantage of these properties, we examined the effect of abscisic acid (ABA) on BL-induced phosphorylation of guard-cell H⁺-ATPase by using ABA-insensitive mutants. This revealed inhibition of BL-induced phosphorylation of guard-cell H⁺-ATPase via the early ABA-signaling components PYR/PYL/RCAR-PP2Cs-SnRK2s, which are known to be early ABA-signaling components for a wide range of ABA responses in plants.      

Cytosolic Concentration of Ca2+ Regulates the Plasma Membrane H+-ATPase in Guard Cells of Fava Bean

Opening of the stomata is driven by the light-activated plasma membrane proton pumping ATPase, although the activation and inactivation mechanism of the enzyme is not known. In this study, we show that the H+-ATPase in guard cells is reversibly inhibited by Ca2+ at physiological concentrations. Isolated microsomal membranes of guard cell protoplasts from fava bean exhibited vanadate-sensitive, ATP-dependent proton pumping. The activity was inhibited almost completely by 1 [mu]M Ca2+ with a half-inhibitory concentration at 0.3 [mu]M and was restored immediately by the addition of 1,2-bis(2-aminophenoxy)ethane N,N,N[prime],N[prime]-tetraacetic acid, a calcium chelating reagent. Similar reversible inhibition by Ca2+ was shown by the generation of electrical potential in the membranes. Activity of ATP hydrolysis was inhibited similarly by Ca2+ in the same membrane preparations. The addition of 1,2-bis(2-aminophenoxy)ethane N,N,N[prime],N[prime]-tetraacetic acid and EGTA, Ca2+ chelators, to epidermal peels of fava bean induced stomatal opening in the dark, and the opening was suppressed by vanadate. This suggests that the lowered cytosolic Ca2+ activated the proton pump in vivo and that the activated pump elicited stomatal opening. Inhibition of H+-ATPase by Ca2+ may depolarize the membrane potential and could be a key step in the process of stomatal closing through activation of the anion channels. Furthermore, similar inhibition of the proton pumping and ATP hydrolysis by Ca2+ was found in isolated plasma membranes of mesophyll cells of fava bean. These results suggest that Ca2+ regulates the activity of plasma membrane H+-ATPases in higher plant cells, thereby modulating stomatal movement and other cellular processes in plants.     

Biochemical evidence for the requirement of 14-3-3 protein binding in activation of the guard-cell plasma membrane H+-ATPase by blue light

Blue light (BL) activates the plasma membrane H(+)-ATPase via phosphorylation of the C-terminus with concomitant binding of 14-3-3 protein to the terminus in stomatal guard cells. However, the binding site and role of 14-3-3 protein in this physiological response have not been elucidated. We investigated the above using synthetic phosphopeptides designed from the C-terminus of Vicia H(+)-ATPase (isoform 1; VHA1). The presence of KGLDIDTIQQHYphospho-T(950)V peptide (P-950) prevented binding of 14-3-3 protein to the phosphorylated H(+)-ATPase. Dephosphorylated P-950 and other phosphopeptides, including typical phosphorylation sites in the C-terminus, had no effect on the binding. Incubation of BL-activated plasma membrane H(+)-ATPase with P-950 dissociated the 14-3-3 protein from the H(+)-ATPase without affecting phosphorylation levels and decreased the H(+)-ATPase activity. By contrast, incubation of P-950 with the activated H(+)-ATPase from fusicoccin-treated guard-cell protoplasts neither dissociated the 14-3-3 protein nor decreased the H(+)-ATPase activity. These results indicate that BL induces phosphorylation on threonine residue (Thr(950)) in the C-terminus of H(+)-ATPase, and that the binding of 14-3-3 to this site is required for the activation of H(+)-ATPase in stomatal guard cells.     

Inhibition of blue light-dependent H+ pumping by abscisic acid through hydrogen peroxide-induced dephosphorylation of the plasma membrane H+-ATPase in guard cell protoplasts

Blue light (BL)-dependent H+ pumping by guard cells, which drives stomatal opening, is inhibited by abscisic acid (ABA). We investigated this response with respect to the activity of plasma membrane H+-ATPase using Vicia guard cell protoplasts. ATP hydrolysis by the plasma membrane H+-ATPase, phosphorylation of the H+-ATPase, and the binding of 14-3-3 protein to the H+-ATPase stimulated by BL were inhibited by ABA at 10 microm. All of these responses were similarly inhibited by hydrogen peroxide (H2O2) at 1 mm. The ABA-induced inhibitions of BL-dependent H+ pumping and phosphorylation of the H+-ATPase were partially restored by ascorbate, an intracellular H2O2 scavenger. A single-cell analysis of the cytosolic H2O2 using 2',7'-dichlorofluorescin revealed that H2O2 was generated by ABA in guard cell protoplasts. We also indicated that H+ pumping induced by fusicoccin and the binding of 14-3-3 protein to the H+-ATPase were inhibited slightly (approximately 20%) by both ABA and H2O2. By contrast, H2O2 at 1 mm did not affect H+ pumping by the H+-ATPase in microsomal membranes. From these results, we concluded that inhibition of BL-dependent H+ pumping by ABA was due to a decrease in the phosphorylation levels of H+-ATPase and that H2O2 might be involved in this response. Moreover, there are at least two inhibition sites by ABA in the BL signaling pathway of guard cells.     

Metabolic energy for stomatal opening. Roles of photophosphorylation and oxidative phosphorylation

The supply of energy for stomatal opening was investigated with epidermal peels of Commelina communis L. and Vicia faba L., under white, blue and red irradiation or in darkness. Fluencerate response curves of stomatal opening under blue and red light were consistent with the operation of two photosystems, one dependent on photosynthetic active radiation (PAR) and the other on blue light, in the guard cells. The PAR-dependent system was 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-sensitive and KCN-resistant and showed a relatively high threshold irradiance for its activation; its activity was most prominent at moderate to high irradiances. The blue-light-dependent photosystem was KCN-sensitive, was active at low irradiances, and interacted with the PAR-dependent photosystem at high blue irradiances. Stomatal opening in darkness, caused by CO₂-free air, fusicoccin or high KCl concentrations, was KCN-sensitive and DCMU-resistant. These data indicate that stomatal opening in darkness depends on oxidative phosphorylation for the supply of high-energy equivalents driving proton extrusion. Light-dependent stomatal opening appears to require photophosphorylation from guard-cell chloroplasts and the activation of the blue-light photosystem which could rely either on oxidative phosphorylation or a specific, membrane-bound electron-transport carrier.Abbreviations DCMU 3(3,4-dichlorophenyl)-1-1-dimethylurea - FC fusicoccin - KCN potassium cyanide - PAR photosynthetic active radiation - WL white light     

Immunohistochemical detection of blue light-induced phosphorylation of the plasma membrane H+-ATPase in stomatal guard cells

Blue light (BL) receptor phototropins activate the plasma membrane H(+)-ATPase in guard cells through phosphorylation of a penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated C-terminus of H?-ATPase, mediating stomatal opening. To date, detection of the phosphorylation level of the guard cell H?-ATPase has been performed biochemically using guard cell protoplasts (GCPs). However, preparation of GCPs from Arabidopsis for this purpose requires >5,000 rosette leaves and takes >8 h. Here, we show that BL-induced phosphorylation of guard cell H?-ATPase is detected in the epidermis from a single Arabidopsis rosette leaf via an immunohistochemical method using a specific antibody against the phosphorylated penultimate threonine of H?-ATPase. BL-induced phosphorylation of the H?-ATPase was detected immunohistochemically in the wild type, but not in a phot1-5 phot2-1 double mutant. Moreover, we found that physiological concentrations of the phytohormone ABA completely inhibited BL-induced phosphorylation of guard cell H?-ATPase in the epidermis, and that inhibition by ABA in the epidermis is more sensitive than in GCPs. These results indicate that this immunohistochemical method is very useful for detecting the phosphorylation status of guard cell H?-ATPase. Thus, we applied this technique to ABA-insensitive mutants (abi1-1, abi2-1 and ost1-2) and found that ABA had no effect on BL-induced phosphorylation in these mutants. These results indicate that inhibition of BL-induced phosphorylation of guard cell H?-ATPase by ABA is regulated by ABI1, ABI2 and OST1, which are known to be early ABA signaling components for a wide range of ABA responses in plants.     

Blue light activates the plasma membrane H(+)-ATPase by phosphorylation of the C-terminus in stomatal guard cells.

The opening of stomata, which is driven by the accumulation of K(+) salt in guard cells, is induced by blue light (BL). The BL activates the H(+) pump; however, the mechanism by which the perception of BL is transduced into the pump activation remains unknown. We present evidence that the pump is the plasma membrane H(+)-ATPase and that BL activates the H(+)-ATPase via phosphorylation. A pulse of BL (30 s, 100 micromol/m(2)/s) increased ATP hydrolysis by the plasma membrane H(+)-ATPase and H(+) pumping in Vicia guard cell protoplasts with a similar time course. The H(+)-ATPase was phosphorylated reversibly by BL, and the phosphorylation levels paralleled the ATP hydrolytic activity. The phosphorylation occurred exclusively in the C-termini of H(+)-ATPases on both serine and threonine residues in two isoproteins of H(+)-ATPase in guard cells. An endogenous 14-3-3 protein was co-precipitated with H(+)-ATPase, and the recombinant 14-3-3 protein bound to the phosphorylated C-termini of H(+)-ATPases. These findings demonstrate that BL activates the plasma membrane H(+)-ATPase via phosphorylation of the C-terminus by a serine/threonine protein kinase, and that the 14-3-3 protein has a key role in the activation.     

Proteolytic activation of the plant plasma membrane H(+)-ATPase by removal of a terminal segment

Incubation of oat root plasma membrane vesicles in the presence of ATP with trypsin or chymotrypsin increased the rate of ATP hydrolysis and ATP-dependent proton pumping by the plasma membrane H(+)-ATPase. Proton pumping was stimulated more than 200%, whereas ATP hydrolytic activity was stimulated about 30%. The Km (ATP) for both proton pumping and ATP hydrolysis was lowered from about 0.3 mM to below 0.1 mM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trypsin-treated plasma membranes revealed a decrease in a 100-kDa band and the appearance of a 93-kDa band. Western blot analysis using antibodies against the H(+)-ATPase showed that both of these bands represented the H(+)-ATPase and suggested that a 7-kDa segment was released. Extensive treatment with carboxypeptidase A also activated the H(+)-ATPase indicating that the 7-kDa segment originated from the C terminus.     

Regulation of blue-light-induced proton pumping by Vicia faba L. guard-cell protoplasts: Energetic contributions by chloroplastic and mitochondrial activities

An initial response during signal transduction in guard cells, following absorption of blue light, is the extrusion of protons. Translocation of protons across the guard-cell plasmalemma is an energy-requiring activity. The present study has investigated the energetic contribution from guard-cell chloroplasts and mitochondria to blue-light-induced proton pumping by Vicia faba guard-cell protoplasts. The addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea to the protoplast suspension had a minimal effect on rates of acidification when oxygen concentrations of the medium were maintained close to near-saturating levels. Under the same conditions, oligomycin reduced both the rates of blue-light-induced acidification and total proton efflux. Lowering the oxygen concentration of the suspending medium to approximately 20 M resulted in complete inhibition of blue-light-induced acidification activity. Swelling of protoplasts induced by blue light was also inhibited by low oxygen levels. Levels of ATP from whole-protoplast extracts were reduced by about 64% when exposed to low levels of oxygen. Increasing oxygen levels to near-saturating levels restored both blue-light-induced acidification rates and swelling of the protoplasts within a 60-min recovery period. Levels of ATP also increased during the recovery period. Addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea or oligomycin to the suspending medium prior to increasing the oxygen concentration caused a reduction in acidification rates after the recovery period by 40 and 80%, respectively. Levels of ATP in guard-cell protoplasts were also reduced by both inhibitors after a 60-min recovery period. The results demonstrate that both guard-cell chloroplasts and mitochondria contribute energetically to blue-light-induced proton pumping by guard-cell protoplasts. Furthermore, both energy sources are inhibited by low oxygen concentrations, suggesting coordinated metabolic regulation between photo- and oxidative phosphorylation in guard cells.Abbreviations BL blue light - Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - GCPs guard-cell protoplasts This research was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada and a University Research Grant from The University of Calgary. Dr. L. Gedamu (University of Calgary) is thanked for providing access to the bioluminometer. Technical assistance by C. Chmielewski, C. Turnnir, S. Ham and K. Meyer is gratefully acknowledged.     

The effect of blue light on energy levels in epidermal strips

Red light applied together with blue enhanced stomatal opening in epidermal strips of Commelina communis L. more than red light alone. In red light, stomatal opening was enhanced by exogenously applied ATP and was inhibited by 3-(3,4-dichlorophe-nyl)-l,l-dimethylurea (DCMU), while in the presence of blue light external ATP was almost without effect, and DCMU stimulated stomatal opening. Blue light increased the ATP levels in the epidermal strips. DCMU diminished the amount of ATP in both red light and red + blue light treatments, but did not abolish the stimulatory effect of blue light. Blue light also stimulated the respiration rate of the epidermal strips. Rotenone, which inhibited stomatal opening and respiration rate, abolished the effect of blue light in both processes. These results imply that blue light increases the ATP levels by stimulation of oxidative phosphorylation.     

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

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

Extracellular Ca²+ alleviates NaCl-induced stomatal opening through a pathway involving H₂O₂-blocked Na+ influx in Vicia guard cells

To gain further insights into the function of extracellular Ca2+ in alleviating salt stress, Vicia faba guard cell protoplasts (GCPs) were patch-clamped in a whole-cell configuration. The results showed that 100 mM NaCl clearly induced Na+ influx across the plasma membrane in GCPs and promoted stomatal opening. Extracellular Ca2+ at 10 mM efficiently blocked Na+ influx and inhibited stomatal opening, which was partially abolished by La3+ (an inhibitor of plasma membrane Ca2+ channel) or catalase (CAT, a H?O? scavenger), respectively. These results suggest that the plasma membrane Ca2+ channels and H?O? possibly mediate extracellular Ca2+-blocked Na+ influx in GCPs. Furthermore, extracellular Ca2+ activated the plasma membrane Ca2+ channels under NaCl stress, which was partially abolished by CAT. These results, taken together, indicate that hydrogen peroxide (H?O?) likely regulates Na+ uptake by activating plasma membrane Ca2+ channels in GCPs. In accordance with this hypothesis, H?O? could mimic extracellular Ca2+ to activate Ca2+ channels and block Na+ influx in guard cells. A single-cell analysis of cytosolic free Ca2+ ([Ca2+](cyt)) using Fluo 3-AM revealed that extracellular Ca2+ induced the accumulation of cytosolic Ca2+ under NaCl stress, but had few effects on the accumulation of cytosolic Ca2+ under non-NaCl conditions. All of these results, together with our previous studies showing that extracellular Ca2+ induced the generation of H?O? in GCPs during NaCl stress, indicate that extracellular Ca2+ alleviates salt stress, likely by activating the H?O?-dependent plasma membrane Ca2+ channels, and the increase in cytosolic Ca2+ appears to block Na+ influx across the plasma membrane in Vicia guard cells, leading to stomatal closure and reduction of water loss.     

FLOWERING LOCUS T regulates stomatal opening

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange for photosynthesis in response to light, CO(2), and phytohormone abscisic acid. Phototropins (phot1 and phot2) are plant blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H(+)-ATPase. However, the signaling mechanism from phototropins to the H(+)-ATPase has yet to be determined. Here, we show that FLOWERING LOCUS T (FT) is expressed in guard cells and regulates stomatal opening. We isolated an scs (suppressor of closed-stomata phenotype in phot1 phot2) 1-1 mutant of Arabidopsis thaliana and showed that scs1-1 carries a novel null early flowering 3 (elf3) allele in a phot1 phot2 background. scs1-1 (elf3 phot1 phot2 triple mutant) had an open-stomata phenotype with high H(+)-ATPase activity and showed increased levels of FT mRNA in guard cells. Transgenic plants overexpressing FT in guard cells showed open stomata, whereas a loss-of-function FT allele, ft-1, exhibited closed stomata and failed to activate the H(+)-ATPase in response to blue light. Our results define a new cell-autonomous role for FT and demonstrate that the flowering time genes ELF3 and FT are involved in the regulation of H(+)-ATPase by blue light in guard cells.     

Microtubules are essential for guard-cell function in Vicia and Arabidopsis

Radially arranged cortical microtubules are a prominent feature of guard cells. Guard cells expressing GFP-tubulin showed consistent changes in the appearance of microtubules when stomata opened or closed. Guard cells showed fewer microtubule structures as stomata closed, whether induced by transfer to darkness, ABA, hydrogen peroxide, or sodium hydrogen carbonate. Guard cells kept in the dark (closed stomata) showed increases in microtubule structures and stomatal aperture on light treatment. GFP-EB1, marking microtubule growing plus ends, showed no change in number of plus ends or velocity of assembly on stomatal closure. Since the number of growing plus ends and the rate of plus-end growth did not change when microtubule structure numbers declined, microtubule instability and/or rearrangement must be responsible for the apparent loss of microtubules. Guard cells with closed stomata showed more cytosolic GFP-fluorescence than those with open stomata as cortical microtubules became disassembled, although with a large net loss in total fluorescence. Microtubule-targeted drugs blocked guard-cell function in Vicia and Arabidopsis. Oryzalin disrupted guard-cell microtubules and prevented stomatal opening and taxol stabilized guard-cell microtubules and delayed stomatal closure. Gas exchange measurements indicated that the transgenes for fluorescent-labeled proteins did not disrupt normal stomatal function. These dynamic changes in guard-cell microtubules combined with our inhibitor studies provide evidence for an active role of microtubules in guard-cell function.