Comparative effects of salicylate, 2,4-dinitrophenol and abscisic acid on stomatal resistance of detached tobacco leaves

The effects of salicylate on stomatal resistance (a measure of stomatal opening) were compared with those produced by 2,4-dinitrophenol and abscisic acid. Salicylate and dinitrophenol had the same minimum effective concentration and comparable kinetics, and induced stomatal closure persisting for a long time in the absence of further supply. However, a K and Ca solution prevented the salicylate-induced, but not dinitrophenol-induced, stomatal closure. The effects of salicylate and abscisic acid had very different characteristics. Cytokinins had no relevant effects on the stomatal closure induced by the three compounds. A close correlation was found between stomatal closure and K⁺ leakage from the treated leaves, suggesting that damage to the cell membrane may be involved. Research work supported by CNR, Italy. Special grant I.P.R.A.-Subproject 1. Paper N. 666.     

一氧化氮是脱落酸诱导杨树叶片气孔关闭的信号分子

研究了外源NO和ABA对杨树气孔运动调节作用．结果表明,外源NO和ABA都能诱导杨树离体叶片气孔关闭,且具有剂量效应,NO可加强ABA诱导气孔关闭的作用．NO清除剂(c—PTIO)可大大减弱NO和ABA对气孔关闭的诱导作用．证实了NO参与ABA调控气孔开闭运动过程,不同浓度NO供体SNP和ABA处理杨树离体叶片,SOD活性变化不明显,POD活性受到显著抑制．杨树叶片粗酶液的体外实验表明,不同浓度SNP对POD活性的抑制呈明显的浓度及时间效应;而ABA对POD活性则几乎没有影响．本研究证明,NO调节ABA诱导的树木气孔关闭作用,是ABA诱导树木气孔关闭的一种重要信号分子．     

Hydrogen peroxide production is an early event during bicarbonate induced stomatal closure in abaxial epidermis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

The presence of 2 mM bicarbonate in the incubation medium induced stomatal closure in abaxial epidermis of Arabidopsis. Exposure to 2 mM bicarbonate elevated the levels of H₂O₂ in guard cells within 5 min, as indicated by the fluorescent probe, dichlorofluorescein diacetate (H₂DCF-DA). Bicarbonate-induced stomatal closure as well as H₂O₂ production were restricted by exogenous catalase or diphenylene iodonium (DPI, an inhibitor of NAD(P)H oxidase). The reduced sensitivity of stomata to bicarbonate and H₂O₂ production in homozygous atrbohD/F double mutant of Arabidopsis confirmed that NADP(H) oxidase is involved during bicarbonate induced ROS production in guard cells. The production of H₂O₂ was quicker and greater with ABA than that with bicarbonate. Such pattern of H₂O₂ production may be one of the reasons for ABA being more effective than bicarbonate, in promoting stomatal closure. Our results demonstrate that H₂O₂ is an essential secondary messenger during bicarbonate induced stomatal closure in Arabidopsis.     

Ethylene mediates UV-B-induced stomatal closure via peroxidase-dependent hydrogen peroxide synthesis in Vicia faba L

Ultraviolet B (UV-B) radiation is an important environmental signal for plant growth and development, but its signal transduction mechanism is unclear. UV-B is known to induce stomatal closure via hydrogen peroxide (H(2)O(2)), and to affect ethylene biosynthesis. As ethylene is also known to induce stomatal closure via H(2)O(2) generation, the possibility of UV-B-induced stomatal closure via ethylene-mediated H(2)O(2) generation was investigated in Vicia faba by epidermal strip bioassay, laser-scanning confocal microscopy, and assays of ethylene production. It was found that H(2)O(2) production in guard cells and subsequent stomatal closure induced by UV-B radiation were inhibited by interfering with ethylene biosynthesis as well as ethylene signalling, suggesting that ethylene is epistatic to UV-B radiation in stomatal movement. Ethylene production preceded H(2)O(2) production upon UV-B radiation, while exogenous ethylene induced H(2)O(2) production in guard cells and subsequent stomatal closure, further supporting the conclusion. Inhibitors for peroxidase but not for NADPH oxidase abolished H(2)O(2) production upon UV-B radiation in guard cells, suggesting that peroxidase is the source of UV-B-induced H(2)O(2) production. Taken together, our results strongly support the idea that ethylene mediates UV-B-induced stomatal closure via peroxidase-dependent H(2)O(2) generation.     

Species-dependent changes in stomatal sensitivity to abscisic acid mediated by external pH

The direct effects of pH changes and/or abscisic acid (ABA) on stomatal aperture were examined in epidermal strips of Commelina communis L. and Arabidopsis thaliana. Stomata were initially opened at pH 7 or pH 5. The stomatal closure induced by changes in external pH and/or ABA (10 microM or 10 nM) was monitored using video microscopy and quantified in terms of changes in stomatal area using image analysis software. Measurements of aperture area enabled stomatal responses and, in particular, small changes in stomatal area to be quantified reliably. Both plant species exhibited a biphasic closure response to ABA: an initial phase of rapid stomatal closure, followed by a second, more prolonged, phase during which stomata closure proceeded at a slower rate. Changes in stomatal sensitivity to ABA were also observed. Comparison of these effects between C. communis and A. thaliana demonstrate that this differential sensitivity of stomata to ABA is species-dependent, as well as being dependent on the pH of the extracellular environment.     

Glucose‐ and mannose‐induced stomatal closure is mediated by ROS production,Ca2+ and water channel in Vicia faba

Sugars act as vital signaling molecules that regulate plant growth, development and stress responses. However, the effects of sugars on stomatal movement have been unclear. In our study, we explored the effects of monosaccharides such as glucose and mannose on stomatal aperture. Here, we demonstrate that glucose and mannose trigger stomatal closure in a dose‐ and time‐dependent manner in epidermal peels of broad bean (Vicia faba). Pharmacological studies revealed that glucose‐ and mannose‐induced stomatal closure was almost completely inhibited by two reactive oxygen species (ROS) scavengers, catalase (CAT) and reduced glutathione (GSH), was significantly abolished by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), whereas they were hardly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM). Furthermore, glucose‐ and mannose‐induced stomatal closure was strongly inhibited by a Ca²⁺ channel blocker, LaCl₃, a Ca²⁺ chelator, ethyleneglycol‐bis(beta‐aminoethylether)‐N,N'‐tetraacetic acid (EGTA) and two water channel blockers, HgCl₂ and dimethyl sulfoxide (DMSO); whereas the inhibitory effects of the water channel blockers were essentially abolished by the reversing agent β‐mercaptoethanol (β‐ME). These results suggest that ROS production mainly via NADPH oxidases, Ca²⁺ and water channels are involved in glucose‐ and mannose‐induced stomatal closure.     

Nitric oxide,actin reorganization and vacuoles change are involved in PEG 6000‐induced stomatal closure in Vicia faba

Water deficit and the resulting osmotic stress affect stomatal movement. There are two types of signals, hydraulic and chemical signals, involving in the regulation of stomatal behavior responses to osmotic stress. Compared with the chemical signals, little has been known about the hydraulic signals and the corresponding signal transduction network and regulatory mechanisms. Here, using an epidermal‐strip bioassay and laser‐scanning confocal microscopy, we provide evidence that nitric oxide (NO) generation in Vicia faba guard cells can be induced by hydraulic signals. We used polyethylene glycol (PEG) 600 to simulate hypertonic conditions. This hydraulic signal led to stomatal closure and rapid promotion of NO production in guard cells. The effects were decreased by NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5, 5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (c‐PTIO) and NO synthase (Enzyme Commission 1.14.13.39) inhibitor N^G‐nitro‐ l ‐Arg‐methyl ester (l ‐NAME). These results indicate that PEG 6000 induces stomatal closure by promoting NO production. Cytochalasin B (CB) inhibited stomatal closure induced by PEG 6000 but did not prevent the increase of endogenous NO levels, indicating that microfilaments polymerization participate in stomatal closure induced by PEG 6000, and may act downstream of NO signaling. In addition, big vacuoles split into many small vacuoles were observed in response to PEG 6000 and sodium nitroprusside (SNP) treatment, and CB inhibited these changes of vacuoles, the stomatal closure was also been inhibited. Collectively, these results suggest that the stomatal closure induced by PEG 6000 may be intimately associated with NO levels, reorganization of actin filaments and the changes of vacuoles, showing a crude outline of guard‐cells signaling process in response to hydraulic signals.     

Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis

Methylglyoxal (MG) is an oxygenated short aldehyde and a glycolytic intermediate that accumulates in plants under environmental stresses. Being a reactive α-oxoaldehyde, MG may act as a signaling molecule in plants during stresses. We investigated whether MG induces stomatal closure, reactive oxygen species (ROS) production, and cytosolic free calcium concentration ([Ca2?](cyt)) to clarify roles of MG in Arabidopsis guard cells. MG induced production of ROS and [Ca2?](cyt) oscillations, leading to stomatal closure. The MG-induced stomatal closure and ROS production were completely inhibited by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), but were not affected by an NAD(P)H oxidase mutation, atrbohD atrbohF. Furthermore, the MG-elicited [Ca2?](cyt) oscillations were significantly suppressed by SHAM but not by the atrbohD atrbohF mutation. Neither endogenous abscisic acid nor endogenous methyl jasmonate was involved in MG-induced stomatal closure. These results suggest that intrinsic metabolite MG can induce stomatal closure in Arabidopsis accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca2?](cyt) oscillations.     

Nitric oxide is a signaling intermediate during bicarbonate-induced stomatal closure in Pisum sativum

The role of nitric oxide (NO) during bicarbonate-induced stomatal closure was studied in the abaxial epidermis of Pisum sativum . A few experiments were done with 10 μ M ABA, for comparison. The presence of 2 m M sodium bicarbonate or 10 μ M ABA induced an increase of NO in guard cells. Elevation of NO by sodium nitroprusside induced stomatal closure and enhanced further the closure by bicarbonate. The bicarbonate induced increase in NO of guard cells, or stomatal closure was prevented partially by 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide, an NO scavenger and N -nitro- l -Arg-methyl ester, an inhibitor of NO synthase (NOS). These results suggested that guard cells generated NO on exposure to bicarbonate and that NOS was involved at least partially in such NO production. Time course experiments revealed that on exposure to bicarbonate or ABA, the rise in guard cell NO production peaked within 10 min. Experiments using pharmacological compounds like wortmannin/LY294002 (phosphatidylinositol 3 kinase inhibitors), 1 H -(1,2,4)-oxadiazole-[4,3 a ]quinoxalin-1-one (guanylyl cyclase inhibitor), nicotinamide (cyclic adenosine diphosphate ribose antagonist), guanosine 5'-O-(2-thiodiphosphate) (G-protein antagonist) suggested a role of phosphatidylinositol 3-phosphate or G-proteins during bicarbonate-induced stomatal closure.     

Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.     

Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure

The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca(2+) ([Ca(2+)](ext))-induced [Ca(2+)](cyt) transients and stomatal closure in Arabidopsis. CAS (Ca(2+) sensing receptor) is a plant-specific putative Ca(2+)-binding protein that was originally proposed to be a plasma membrane-localized external Ca(2+) sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N-terminus acidic Ca(2+)-binding region is likely exposed to the stromal side of the membrane. The phenotype of T-DNA insertion CAS knockout mutants and cDNA mutant-complemented plants revealed that CAS is essential for stomatal closure induced by external Ca(2+). In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca(2+). Furthermore, using the transgenic aequorin system, we showed that [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane-localized CAS is essential for [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients and stomatal closure.     

Polyamines increase nitric oxide and reactive oxygen species in guard cells of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> during stomatal closure

A comprehensive study which was undertaken on the effect of three polyamines (PAs) on stomatal closure was examined in relation to nitric oxide (NO) and reactive oxygen species (ROS) levels in guard cells of Arabidopsis thaliana. Three PAs—putrescine (Put), spermidine (Spd), and spermine (Spm)—induced stomatal closure, while increasing the levels of NO as well as ROS in guard cells. The roles of NO and ROS were confirmed by the reversal of closure by cPTIO (NO scavenger) and catalase (ROS scavenger). The presence of L-NAME (NOS-like enzyme inhibitor) reversed PA-induced stomatal closure, suggesting that NOS-like enzyme played a significant role in NO production during stomatal closure. The reversal of stomatal closure by diphenylene iodonium (DPI, NADPH oxidase inhibitor) or 2-bromoethylamine (BEA, copper amine oxidase inhibitor) or 1,12 diaminododecane (DADD, polyamine oxidase inhibitor) was partial. In contrast, the presence of DPI along with BEA/DADD reversed completely the closure by PAs. We conclude that both NO and ROS are essential signaling components during Put-, Spd-, and Spm-induced stomatal closure. The PA-induced ROS production is mediated by both NADPH oxidase and amine oxidase. The rise in ROS appears to be upstream of NO. Ours is the first detailed study on the role of NO and its dependence on ROS during stomatal closure by three major PAs.     

超氧阴离子通过提高[Ca2+]cyt调节保卫细胞K+通道和气孔运动

氧化信号参与了许多生理过程的调控。用膜片钳和激光共聚焦显微镜,采用可以产生O2^ 的甲基紫精处理蚕豆(Vicia faba L)保卫细胞,测定了O2^ 对气孔运动调节过程中胞质Ca^2 离子浓度和细胞质膜K^ 通道活性的变化,结果表明甲基紫精可以促进气孔的关闭,乙二醇四乙酸酯(Ethylene glycol bis(2-aminoethyl)tetra-acetic acid,EGTA)、抗坏血酸(Ascorbic acid,AsA)和过氧化物酶(Catalase,CAT)可以消除小于10^-5mol／L甲基紫精对气孔运动的影响;10^-2和10^-5mol/L的甲基紫精可使保卫细胞胞质Ca^2 浓度有不同程度提高,并伴随有钙震荡。蚕豆气孔保卫细胞质膜内向K^ 通道可被咆外甲基紫精抑制,而这种抑制和[Ca^2 ]cyt有关。推测甲基紫精产生的O2^-对蚕豆气孔运动的调节,主要是通过O2^ 诱导的胞内游离Ca^2 浓度的升高,从而抑制了通过保卫细胞质膜K^ 内向电流。     

Nitric oxide production occurs after cytosolic alkalinization during stomatal closure induced by abscisic acid

Abscisic acid (ABA) raised the cytosolic pH and nitric oxide (NO) levels in guard cells while inducing stomatal closure in epidermis of Pisum sativum. Butyrate (a weak acid) reduced the cytosolic pH/NO production and prevented stomatal closure by ABA. Methylamine (a weak base) enhanced the cytosolic alkalinization and aggravated stomatal closure by ABA. The rise in guard cell pH because of ABA became noticeable after 6 min and peaked at 12 min, while NO production started at 9 min and peaked at 18 min. These results suggested that NO production was downstream of the rise in cytosolic pH. The ABA-induced increase in NO of guard cells and stomatal closure was prevented by 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (cPTIO, a NO scavenger) and partially by N-nitro-L-Arg-methyl ester (L-NAME, an inhibitor of NO synthase). In contrast, cPTIO or L-NAME had only a marginal effect on the pH rise induced by ABA. Ethylene glycol tetraacetic acid (EGTA, a calcium chelator) prevented ABA-induced stomatal closure while restricting cytosolic pH rise and NO production. We suggest that during ABA-induced stomatal closure, a rise in cytosolic pH is necessary for NO production. Calcium may act upstream of cytosolic alkalinization and NO production, besides its known function as a downstream component.     

Short communication. Abscisic acid is not necessarily required for the induction of patchy stomatal closure

Patchy stomatal closure was observed in leaves of transgenic plants of Nicotiana plumbaginifolia producing antibodies that block the action of abscisic acid. Stomatal patchiness was induced by leaf detachment and subsequent water loss. Stomatal closure was followed by an irreversible reduction of maximal chlorophyll fluorescence. The degree of deviation from the A/ci-curve is correlated with steady-state diffusion conductance before leaf detachment. It is concluded that a heterogeneous sensitivity of stomata to abscisic acid is not directly involved in the induction of patchy stomatal closure.Keywords: Abscisic acid, chlorophyll fluorescence imaging, patchy stomatal closure, Nicotiana plumbaginifolia.      

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA‐induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²⁺]_cyt) oscillations and inward‐rectifying potassium (K⁺_in) channel activity in Arabidopsis. SA‐induced stomatal closure was inhibited by pre‐treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA‐induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA‐induced stomatal closures. 3,3′‐Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂^– production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) suppressed the SA‐induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA‐induced NO production. SA failed to induce [Ca²⁺]_cyt oscillations in guard cells whereas K⁺_in channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM‐sensitive peroxidase, intracellular ROS accumulation and K⁺_in channel inactivation.     

Role of H2O2 dynamics in brassinosteroid‐induced stomatal closure and opening in Solanum lycopersicum

Brassinosteroids (BRs) are essential for plant growth and development; however, their roles in the regulation of stomatal opening or closure remain obscure. Here, the mechanism underlying BR‐induced stomatal movements is studied. The effects of 24‐epibrassinolide (EBR) on the stomatal apertures of tomato (Solanum lycopersicum) were measured by light microscopy using epidermal strips of wild type (WT), the abscisic acid (ABA)‐deficient notabilis (not) mutant, and plants silenced for SlBRI1, SlRBOH1 and SlGSH1. EBR induced stomatal opening within an appropriate range of concentrations, whereas high concentrations of EBR induced stomatal closure. EBR‐induced stomatal movements were closely related to dynamic changes in H₂O₂ and redox status in guard cells. The stomata of SlRBOH1‐silenced plants showed a significant loss of sensitivity to EBR. However, ABA deficiency abolished EBR‐induced stomatal closure but did not affect EBR‐induced stomatal opening. Silencing of SlGSH1, the critical gene involved in glutathione biosynthesis, disrupted glutathione redox homeostasis and abolished EBR‐induced stomatal opening. The results suggest that transient H₂O₂ production is essential for poising the cellular redox status of glutathione, which plays an important role in BR‐induced stomatal opening. However, a prolonged increase in H₂O₂ facilitated ABA signalling and stomatal closure.     

Stomatal closure induced by phytosphingosine-1-phosphate and sphingosine-1-phosphate depends on nitric oxide and pH of guard cells in <Emphasis Type="Italic">Pisum sativum</Emphasis>

Main conclusion

Phyto-S1P and S1P induced stomatal closure in epidermis of pea ( Pisum sativum ) by raising the levels of NO and pH in guard cells. Phosphosphingolipids, such as phytosphingosine-1-phosphate (phyto-S1P) and sphingosine-1-phosphate (S1P), are important signaling components during drought stress. The biosynthesis of phyto-S1P or S1P is mediated by sphingosine kinases (SPHKs). Although phyto-S1P and S1P are known to be signaling components in higher plants, their ability to induce stomatal closure has been ambiguous. We evaluated in detail the effects of phyto-S1P, S1P and SPHK inhibitors on signaling events leading to stomatal closure in the epidermis of Pisum sativum. Phyto-S1P or S1P induced stomatal closure, along with a marked rise in nitric oxide (NO) and cytoplasmic pH of guard cells, as in case of ABA. Two SPHK inhibitors, DL-threo dihydrosphingosine and N’,N’-dimethylsphingosine, restricted ABA-induced stomatal closure and prevented the increase of NO or pH by ABA. Modulators of NO or pH impaired both stomatal closure and increase in NO or pH by phyto-S1P/S1P. The stomatal closure by phyto-S1P/S1P was mediated by phospholipase D and phosphatidic acid (PA). When present, PA elevated the levels of pH, but not NO of guard cells. Our results demonstrate that stomatal closure induced by phyto-S1P and S1P depends on rise in pH as well as NO of guard cells. A scheme of signaling events initiated by phyto-S1P/S1P, and converging to cause stomatal closure, is proposed.

     

2R,3R-butanediol, a bacterial volatile produced by Pseudomonas chlororaphis O6, is involved in induction of systemic tolerance to drought in Arabidopsis thaliana

Root colonization of plants with certain rhizobacteria, such as Pseudomonas chlororaphis O6, induces tolerance to biotic and abiotic stresses. Tolerance to drought was correlated with reduced water loss in P. chlororaphis O6-colonized plants and with stomatal closure, indicated by size of stomatal aperture and percentage of closed stomata. Stomatal closure and drought resistance were mediated by production of 2R,3R-butanediol, a volatile metabolite of P. chlororaphis O6. Root colonization with bacteria deficient in 2R,3R-butanediol production showed no induction of drought tolerance. Studies with Arabidopsis mutant lines indicated that induced drought tolerance required the salicylic acid (SA)-, ethylene-, and jasmonic acid-signaling pathways. Both induced drought tolerance and stomatal closure were dependent on Aba-1 and OST-1 kinase. Increases in free SA after drought stress of P. chlororaphis O6-colonized plants and after 2R,3R-butanediol treatment suggested a primary role for SA signaling in induced drought tolerance. We conclude that the bacterial volatile 2R,3R-butanediol was a major determinant in inducing resistance to drought in Arabidopsis through an SA-dependent mechanism.