ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H(2)O(2) have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H(2)O(2) in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H(2)O(2) production. Stomatal closure induced by H(2)O(2) is inhibited by the removal of NO with NO scavenger, and both ABA and H(2)O(2) stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H(2)O(2) synthesis nor does NO treatment induce H(2)O(2) production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H(2)O(2) and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H(2)O(2) synthesis. In the NR double mutant nia1, nia2 both ABA and H(2)O(2) fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H(2)O(2) are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H(2)O(2) production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H(2)O(2) and NO-induced stomatal closure.     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.     

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.     

Guard-cell signalling for hydrogen peroxide and abscisic acid

Guard cells can integrate and process multiple complex signals from the environment and respond by opening and closing stomata in order to adapt to the environmental signal. Over the past several years, considerable research progress has been made in our understanding of the role of reactive oxygen species (ROS) as essential signal molecules that mediate abscisic acid (ABA)-induced stomatal closure. In this review, we discuss hydrogen peroxide (H2O2) generation and signalling, H2O2-induced gene expression, crosstalk and the specificity between ABA and H2O2 signalling, and the cellular mechanism for ROS sensing in guard cells. This review focuses especially on the points of connection between ABA and H2O2 signalling in guard cells. The fundamental progress in understanding the role of ABA and ROS in guard cells will continue to provide a rational basis for biotechnological improvements in the development of drought-tolerant crop plants with improved water-use efficiency.     

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.     

NO可能作为H2O2的下游信号介导ABA诱导的蚕豆气孔关闭

ABA、H2O2和硝普钠(SNP)均能诱导蚕豆气孔关闭.NO的清除剂c-PTIO可以减轻由ABA或H2O2所诱导的蚕豆气孔关闭的程度,而过氧化氢酶(CAT)则不能减轻NO诱导的气孔关闭程度.激光共聚焦显微检测结果显示,10μmo1/L的ABA处理后,胞内H2O2的产生速率明显高于NO的产生速率;CAT几乎可完全抑制ABA所诱导的DAF的荧光增加;外源H2O2能显著诱导胞内DAF的荧光增加;c-PTIO对ABA诱导的DCF荧光略有促进作用,但外源SNP不能诱导胞内DCF荧光增加.这些结果表明,在ABA诱导气孔关闭过程中,H2O2可能在NO的上游起作用并受NO的负反馈调节.     

Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide

MAP kinases have been linked to guard cell signalling. Arabidopsis thaliana MAP Kinase 3 (MPK3) is known to be activated by abscisic acid (ABA) and hydrogen peroxide (H(2)O(2)), which also control stomatal movements. We therefore studied the possible role of MPK3 in guard cell signalling through guard cell-specific antisense inhibition of MPK3 expression. Such transgenic plants contained reduced levels of MPK3 mRNA in the guard cells and displayed partial insensitivity to ABA in inhibition of stomatal opening, but responded normally to this hormone in stomatal closure. However, ABA-induced stomatal closure was reduced compared with controls when cytoplasmic alkalinization was prevented with sodium butyrate. MPK3 antisense plants were less sensitive to exogenous H(2)O(2), both in inhibition of stomatal opening and in promotion of stomatal closure, thus MPK3 is required for the signalling of this compound. ABA-induced H(2)O(2) synthesis was normal in these plants, indicating that MPK3 probably acts in signalling downstream of H(2)O(2). These results provide clear evidence for the important role of MPK3 in the perception of ABA and H(2)O(2) in guard cells.     

Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells

Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are important signalling agents in both animals and plants. In plants, G proteins modulate numerous responses, including abscisic acid (ABA) and pathogen-associated molecular pattern (PAMP) regulation of guard cell ion channels and stomatal apertures. Previous analyses of mutants deficient in the sole canonical Arabidopsis Gα subunit, GPA1, have shown that Gα-deficient guard cells are impaired in ABA inhibition of K(+) influx channels, and in pH-independent activation of anion efflux channels. ABA-induced Ca(2+) uptake through ROS-activated Ca(2+)-permeable channels in the plasma membrane is another key component of ABA signal transduction in guard cells, but the question of whether these channels are also dependent on Gα for their ABA response has not been evaluated previously. We used two independent Arabidopsis T-DNA null mutant lines, gpa1-3 and gpa1-4, to investigate this issue. We observed that gpa1 mutants are disrupted both in ABA-induced Ca(2+)-channel activation, and in production of reactive oxygen species (ROS) in response to ABA. However, in response to exogenous H(2)O(2) application, I(Ca) channels are activated normally in gpa1 guard cells. In addition, H(2)O(2) inhibition of stomatal opening and promotion of stomatal closure are not disrupted in gpa1 mutant guard cells. These data indicate that absence of GPA1 interrupts ABA signalling between ABA reception and ROS production, with a consequent impairment in Ca(2+)-channel activation.     

Calcium-sensing receptor regulates stomatal closure through hydrogen peroxide and nitric oxide in response to extracellular calcium in Arabidopsis

The Arabidopsis calcium-sensing receptor CAS is a crucial regulator of extracellular calcium-induced stomatal closure. Free cytosolic Ca(2+) (Ca(2+)(i)) increases in response to a high extracellular calcium (Ca(2+)(o)) level through a CAS signalling pathway and finally leads to stomatal closure. Multidisciplinary approaches including histochemical, pharmacological, fluorescent, electrochemical, and molecular biological methods were used to discuss the relationship of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) signalling in the CAS signalling pathway in guard cells in response to Ca(2+)(o). Here it is shown that Ca(2+)(o) could induce H(2)O(2) and NO production from guard cells but only H(2)O(2) from chloroplasts, leading to stomatal closure. In addition, the CASas mutant, the atrbohD/F double mutant, and the Atnoa1 mutant were all insensitive to Ca(2+)(o)-stimulated stomatal closure, as well as H(2)O(2) and NO elevation in the case of CASas. Furthermore, it was found that the antioxidant system might function as a mediator in Ca(2+)(o) and H(2)O(2) signalling in guard cells. The results suggest a hypothetical model whereby Ca(2+)(o) induces H(2)O(2) and NO accumulation in guard cells through the CAS signalling pathway, which further triggers Ca(2+)(i) transients and finally stomatal closure. The possible cross-talk of Ca(2+)(o) and abscisic acid signalling as well as the antioxidant system are discussed.     

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.     

cGMP-dependent ABA-induced stomatal closure in the ABA-insensitive Arabidopsis mutant abi1-1

? The drought hormone abscisic acid (ABA) is widely known to produce reductions in stomatal aperture in guard cells. The second messenger cyclic guanosine 3', 5'-monophosphate (cGMP) is thought to form part of the signalling pathway by which ABA induces stomatal closure. ? We have examined the signalling events during cGMP-dependent ABA-induced stomatal closure in wild-type Arabidopsis plants and plants of the ABA-insensitive Arabidopsis mutant abi1-1. ? We show that cGMP acts downstream of hydrogen peroxide (H(2) O(2) ) and nitric oxide (NO) in the signalling pathway by which ABA induces stomatal closure. H(2) O(2) - and NO-induced increases in the cytosolic free calcium concentration ([Ca(2+) ](cyt) ) were cGMP-dependent, positioning cGMP upstream of [Ca(2+) ](cyt) , and involved the action of the type 2C protein phosphatase ABI1. Increases in cGMP were mediated through the stimulation of guanylyl cyclase by H(2) O(2) and NO. We identify nucleoside diphosphate kinase as a new cGMP target protein in Arabidopsis. ? This study positions cGMP downstream of ABA-induced changes in H(2) O(2) and NO, and upstream of increases in [Ca(2+) ](cyt) in the signalling pathway leading to stomatal closure.     

Cytoplasmic alkalization precedes reactive oxygen species production during methyl jasmonate- and abscisic acid-induced stomatal closure

Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis wild type, ABA-insensitive (ost1-2), and MJ-insensitive mutants (jar1-1) in order to examine a crosstalk between ABA and MJ signal transduction. Some of the experiments were performed on epidermal strips of Pisum sativum. Stomata of jar1-1 mutant plants are insensitive to MJ but are able to close in response to ABA. However, their sensitivity to ABA is less than that of wild-type plants. Reciprocally, the stomata of ost1-2 are insensitive to ABA but are able to close in response to MJ to a lesser extent compared to wild-type plants. Both MJ and ABA promote H(2)O(2) production in wild-type guard cells, while exogenous application of diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, results in the suppression of ABA- and MJ-induced stomatal closure. ABA elevates H(2)O(2) production in wild-type and jar1-1 guard cells but not in ost1-2, whereas MJ induces H(2)O(2) production in both wild-type and ost1-2 guard cells, but not in jar1-1. MJ-induced stomatal closing is suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Furthermore, MJ induces alkalization in guard cell cytosol, and MJ-induced stomatal closing is inhibited by butyrate. Analyses of the kinetics of cytosolic pH changes and reactive oxygen species (ROS) production show that the alkalization of cytoplasm precedes ROS production during the stomatal response to both ABA and MJ. Our results further indicate that JAR1, as OST1, functions upstream of ROS produced by NAD(P)H oxidases and that the cytoplasmic alkalization precedes ROS production during MJ or ABA signal transduction in guard cells.     

Differential requirement for NO during ABA-induced stomatal closure in turgid and wilted leaves

Abscisic acid (ABA)-induced stomatal closure is mediated by a complex, guard cell signalling network involving nitric oxide (NO) as a key intermediate. However, there is a lack of information concerning the role of NO in the ABA-enhanced stomatal closure seen in dehydrated plants. The data herein demonstrate that, while nitrate reductase (NR)1-mediated NO generation is required for the ABA-induced closure of stomata in turgid leaves, it is not required for ABA-enhanced stomatal closure under conditions leading to rapid dehydration. The results also show that NO signalling in the guard cells of turgid leaves requires the ABA-signalling pathway to be both capable of function and active. The alignment of this NO signalling with guard cell Ca²⁺-dependent/independent ABA signalling is discussed. The data also highlight a physiological role for NO signalling in turgid leaves and show that stomatal closure during the light-to-dark transition requires NR1-mediated NO generation and signalling.     

Pyrabactin, an ABA agonist, induced stomatal closure and changes in signalling components of guard cells in abaxial epidermis of Pisum sativum

Pyrabactin, a synthetic agonist of abscisic acid (ABA), inhibits seed germination and hypocotyl growth and stimulates gene expression in a very similar way to ABA, implying the possible modulation of stomatal function by pyrabactin as well. The effect of pyrabactin on stomatal closure and secondary messengers was therefore studied in guard cells of Pisum sativum abaxial epidermis. Pyrabactin caused marked stomatal closure in a pattern similar to ABA. In addition, pyrabactin elevated the levels of reactive oxygen species (ROS), nitric oxide (NO), and cytoplasmic pH levels in guard cells, as indicated by the respective fluorophores. However, apyrabactin, an inactive analogue of ABA, did not affect either stomatal closure or the signalling components of guard cells. The effects of pyrabactin-induced changes were reversed by pharmalogical compounds that modulate ROS, NO or cytoplasmic pH levels, quite similar to ABA effects. Fusicoccin, a fungal toxin, could reverse the stomatal closure caused by pyrabactin, as well as that caused by ABA. Experiments on stomatal closure by varying concentrations of ABA, in the presence of fixed concentration of pyrabactin, and vice versa, revealed that the actions of ABA and pyrabactin were additive. Further kinetic analysis of data revealed that the apparent K(D) of ABA was increased almost 4-fold in the presence of ABA, suggesting that pyrabactin and ABA were competing with each other either at the same site or close to the active site. It is proposed that pyrabactin could be used to examine the ABA-related signal-transduction components in stomatal guard cells as well as in other plant tissues. It is also suggested that pyrabactin can be used as an antitranspirant or as a priming agent for improving the drought tolerance of crop plants.     

Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba

One of the most important functions of the plant hormone abscisic acid (ABA) is to induce stomatal closure by reducing the turgor of guard cells under water deficit. Under environmental stresses, hydrogen peroxide (H(2)O(2)), an active oxygen species, is widely generated in many biological systems. Here, using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that H(2)O(2) may function as an intermediate in ABA signaling in Vicia faba guard cells. H(2)O(2) inhibited induced closure of stomata, and this effect was reversed by ascorbic acid at concentrations lower than 10(-5) M. Further, ABA-induced stomatal closure also was abolished partly by addition of exogenous catalase (CAT) and diphenylene iodonium (DPI), which are an H(2)O(2) scavenger and an NADPH oxidase inhibitor, respectively. Time course experiments of single-cell assays based on the fluorescent probe dichlorofluorescein showed that the generation of H(2)O(2) was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than within the other regions of guard cells. The ABA-induced change in fluorescence intensity in guard cells was abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H(2)O(2) production, which preceded stomatal closure. These effects were abolished by CAT or DPI micro-injection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H(2)O(2) production involved, and H(2)O(2) may be an intermediate in ABA signaling.     

Endogenous abscisic acid is involved in methyl jasmonate-induced reactive oxygen species and nitric oxide production but not in cytosolic alkalization in Arabidopsis guard cells

We recently demonstrated that endogenous abscisic acid (ABA) is involved in methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we investigated whether endogenous ABA is involved in MeJA-induced reactive oxygen species (ROS) and nitric oxide (NO) production and cytosolic alkalization in guard cells using an ABA-deficient Arabidopsis mutant, aba2-2, and an inhibitor of ABA biosynthesis, fluridon (FLU). The aba2-2 mutation impaired MeJA-induced ROS and NO production. FLU inhibited MeJA-induced ROS production in wild-type guard cells. Pretreatment with 0.1 μM ABA, which does not induce stomatal closure in the wild type, complemented the insensitivity to MeJA of the aba2-2 mutant. However, MeJA induced cytosolic alkalization in both wild-type and aba2-2 guard cells. These results suggest that endogenous ABA is involved in MeJA-induced ROS and NO production but not in MeJA-induced cytosolic alkalization in Arabidopsis guard cells.     

Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis

Ethylene is a plant hormone that regulates many aspects of growth and development. Despite the well-known association between ethylene and stress signalling, its effects on stomatal movements are largely unexplored. Here, genetic and physiological data are provided that position ethylene into the Arabidopsis guard cell signalling network, and demonstrate a functional link between ethylene and hydrogen peroxide (H(2)O(2)). In wild-type leaves, ethylene induces stomatal closure that is dependent on H(2)O(2) production in guard cells, generated by the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase AtrbohF. Ethylene-induced closure is inhibited by the ethylene antagonists 1-MCP and silver. The ethylene receptor mutants etr1-1 and etr1-3 are insensitive to ethylene in terms of stomatal closure and H(2)O(2) production. Stomata of the ethylene signalling ein2-1 and arr2 mutants do not close in response to either ethylene or H(2)O(2) but do generate H(2)O(2) following ethylene challenge. Thus, the data indicate that ethylene and H(2)O(2) signalling in guard cells are mediated by ETR1 via EIN2 and ARR2-dependent pathway(s), and identify AtrbohF as a key mediator of stomatal responses to ethylene.     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H2O2的产生（简报）

The methods of confocal laser scanning microscopy (CLSM) and microinjection were used to study ABA-induced H2O2 in guard cells (Vicia faba), which were labeled with H2O2 specific probe-2, 7-dichlorofluorescin diacetate(H2DCFDA). The results indicated 100 U/mL catalase (CAT) could inhibit partly stomatal closure induced by ABA. 10(-3) mmol/L ABA could significantly induce H2O2 production in chloroplast in guard cells of Vicia faba following microinjection, and 100 U/mL CAT could partly abolish the effects following simultaneous microinjection of ABA and CAT. These suggest that H2O2 is possibly involved in ABA signaling leading to stomatal closure.     

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.     

Phospholipase Dα1 and Phosphatidic Acid Regulate NADPH Oxidase Activity and Production of Reactive Oxygen Species in ABA-Mediated Stomatal Closure in Arabidopsis

We determined the role of Phospholipase Dα1 (PLDα1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The pldα1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in pldα1 cells, whereas PA stimulated NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst oxidase homolog D) and RbohF. The PA binding motifs were identified, and mutation of the Arg residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. Furthermore, ABA-induced production of nitric oxide (NO) was impaired in pldα1 guard cells. Disruption of PA binding to ABI1 protein phosphatase 2C did not affect ABA-induced production of ROS or NO, but the PA–ABI1 interaction was required for stomatal closure induced by ABA, H₂O₂, or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.