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.     

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.     

Carbon monoxide-induced stomatal closure in Vicia faba is dependent on nitric oxide synthesis

Recently, in animals, carbon monoxide (CO), like nitric oxide (NO), was implicated as another important physiological messenger or bioactive molecule. Previous researches indicate that heme oxygenase (HO)-1 (EC 1.14.99.3) catalyzes the oxidative conversion of heme to CO and biliverdin IXa (BV) with the concomitant release of iron. However, little is known about the physiological roles of CO in plant, especially in stomatal movement of guard cells. In the present paper, the regulatory role of CO during stomatal movement in Vicia faba was surveyed. Results indicated that, like sodium nitroprusside (SNP), CO donor hematin induced stomatal closure in dose- and time-dependent manners. These responses were also proved by the addition of gaseous CO aqueous solution with different concentrations, showing for the first time that CO and NO exhibit similar regulation role in the stomatal movement. Moreover, our data showed that 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)/N^G-nitro- l -arginine-methyl ester ( l -NAME) not only reversed stomatal closure by CO, but also suppressed the NO fluorescence induced by CO, implying that CO-induced stomatal closure probably involves NO/nitric oxide synthase (NOS) signal system. Additionally, the CO/NO scavenger hemoglobin (Hb) and CO-specific synthetic inhibitor zinc protoporphyrin IX (ZnPPIX), NO scavenger cPTIO and NOS inhibitor l -NAME reversed the darkness-induced stomatal closure and NO fluorescence. These results show that, maybe like NO, the levels of CO in guard cells of V.   faba is higher in dark than that in light, HO-1 and NOS are the enzyme systems responsible for generating endogenous CO and NO in darkness, respectively, and that CO being from HO-1 mediates darkness-induced NO synthesis in guard cells' stomatal closure of V.   faba .     

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.     

Cytosolic alkalinization is a common and early messenger preceding the production of ROS and NO during stomatal closure by variable signals,including abscisic acid,methyl jasmonate and chitosan

Stomata are unique that they sense and respond to several internal and external stimuli, by modulating signaling components in guard cells. The levels of reactive oxygen species (ROS), nitric oxide (NO) and cytosolic calcium (Ca²⁺) increase significantly during stomatal closure by not only plant hormones [such as abscisic acid (ABA) or methyl jasmonate (MJ)] but also elicitors (such as chitosan). We observed that cytosolic alkalinization preceded the production of ROS as well as NO during ABA induced stomatal closure. We therefore propose that besides ROS and NO, the cytosolic pH is an important secondary messenger during stomatal closure by ABA or MJ. We also noticed that there is either a cross talk or feedback regulation by cytosolic Ca²⁺ and ROS (mostly H₂O₂). Further experiments on the interactions between cytosolic pH, ROS, NO and Ca²⁺ would yield interesting results.Key words: abscisic acid, methyl jasmonate, chitosan, cytosolic pH, reactive oxygen species, H₂O₂, nitric oxide, cytosolic calcium     

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.     

Chitosan-Induced Stomatal Closure Accompanied by Peroxidase-Mediated Reactive Oxygen Species Production in Arabidopsis

Chitosan induced stomatal closure in wild type-plants and NADPH oxidase knock-out mutants (atrbohD atrbohF), and reactive oxygen species (ROS) production in wild-type guard cells. Closure and production were completely abolished by catalase and a peroxidase inhibitor. These results indicate that chitosan induces ROS production mediated by peroxidase, resulting in stomatal closure.     

Chitosan-induced stomatal closure accompanied by peroxidase-mediated reactive oxygen species production in Arabidopsis

Chitosan induced stomatal closure in wild type-plants and NADPH oxidase knock-out mutants (atrbohD atrbohF), and reactive oxygen species (ROS) production in wild-type guard cells. Closure and production were completely abolished by catalase and a peroxidase inhibitor. These results indicate that chitosan induces ROS production mediated by peroxidase, resulting in stomatal closure.     

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.     

Chitosan signaling in guard cells requires endogenous salicylic acid

An elicitor chitosan (CHT) induces stomatal closure but the mechanism remains to be clarified. A phytohormone salicylic acid (SA) is crucial for elicitor-induced defense signaling in plants. Here we investigated whether endogenous SA is required for CHT signaling in guard cells. In the SA-deficient nahG mutant, treatment of CHT did not induce either apoplastic reactive oxygen species (ROS) production or stomatal closure but co-treatment of CHT and SA induced both apoplastic ROS production and stomatal closure, indicating the involvement of endogenous SA in CHT-induced apoplastic ROS production and CHT-induced stomatal closure. Furthermore, CHT induced transient cytosolic free calcium concentration increments in the nahG mutant in the presence of exogenous SA but not in the absence of exogenous SA. These results provide evidence that endogenous SA is a crucial element in CHT-induced stomatal closure.     

NO和H2O2在光／暗调控蚕豆气孔运动中的作用及其相互关系

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

NO和H2O2在光/暗调控蚕豆气孔运动中的作用及其相互关系

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

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.     

Abscisic acid (ABA) inhibits light-induced stomatal opening through calcium- and nitric oxide-mediated signaling pathways

Nitric oxide (NO) is an important signaling component of ABA-induced stomatal closure. However, only fragmentary data are available about NO effect on the inhibition of stomatal opening. Here, we present results supporting that, in Vicia faba guard cells, there is a critical Ca2+-dependent NO increase required for the ABA-mediated inhibition of stomatal opening. Light-induced stomatal opening was inhibited by exogenous NO in V. faba epidermal strips. Furthermore, ABA-mediated inhibition of stomatal opening was blocked by the specific NO scavenger cPTIO, supporting the involvement of endogenous NO in this process. Since the raise in Ca2+ concentration is a pre-requisite in ABA-mediated inhibition of stomatal opening, it was interesting to establish how does Ca2+, NO and ABA interact in the inhibition of light-induced stomatal opening. The permeable Ca2+ specific buffer BAPTA-AM blocked both ABA- and Ca2+- but not NO-mediated inhibition of stomatal opening. The NO synthase (NOS) specific inhibitor L-NAME prevented Ca2+-mediated inhibition of stomatal opening, indicating that a NOS-like activity was required for Ca2+ signaling. Furthermore, experiments using the NO specific fluorescent probe DAF-2DA indicated that Ca2+ induces an increase of endogenous NO. These results indicate that, in addition to the roles in ABA-triggered stomatal closure, both NO and Ca2+ are active components of signaling events acting in ABA inhibition of light-induced stomatal opening. Results also support that Ca2+ induces the NO production through the activation of a NOS-like activity.     

NO可能作为Ca^2＋的下游信号介导乙烯诱导的蚕豆气孔关闭

以蚕豆（Vicia faba L．）为材料,利用药理学实验,结合激光共聚焦显微技术和分光光度法．探讨Ca^2＋和一氧化氮（nitric oxide,NO）在乙烯（ethylene,Eth）调控气孔运动信号转导中的作用。结果表明．光下乙烯利（0．004％,0．04％,0．4％）可诱导蚕豆叶片气孔关闭,且具有时间和剂量效应：NO清除剂cPTIO、硝酸还原酶（nitrate reductase,NR）抑制剂NaN3及胞外Ca^2＋螯合剂EGTA可部分逆转乙烯诱导的气孔关闭;乙烯能够明显增加气孔保卫细胞NO水平;提高蚕豆叶片NO含量和NR活性．并且NO的含量变化与NR活性的变化趋势基本一致;NR抑制剂NaN3可抑制乙烯诱导的气孔保卫细胞和叶片NO含量的增加;清除胞外Ca^2＋可减弱乙烯对NO含量和NR活性的诱导效应。说明Ca^2＋和NO均参与乙烯诱导的蚕豆气孔关闭,且NO（主要由NR途径合成）可能位于Ca^2＋下游参与调控这一信号转导过程。     

Cytosolic acidification precedes nitric oxide removal during inhibition of ABA-induced stomatal closure by fusicoccin

The role of nitric oxide (NO) and the relationship between NO and cytosolic pH during inhibition of ABA effect by fusicoccin (FC) in guard cells of Vicia faba were analyzed. ABA induced NO generation and stomatal closure, but FC inhibited the effects of ABA. Treatment with 2-(4-carboxyphenyl)-4,4,5,5-tetra-methylimidazoline-1-oxyl-3-oxide (cPTIO) and N^G-nitro-L-Arg-methyl ester (L-NAME) mimicked the effects of FC. These data suggest that inhibition of ABA effect by FC is possibly related to the decreasing in the NO level. Furthermore, like cPTIO, FC not only suppressed stomatal closure and NO level in guard cells treated with NO donor sodium nitroprusside (SNP), but also reopened stomata, which had been closed by ABA, and reduced the level of NO in guard cells that had been produced by ABA, indicating that FC caused NO removal. Butyric acid simulated the effects of FC on the stomatal aperture and increased NO levels in guard cells treated with SNP and had been closed by ABA, and both FC and butyric acid surely reduced cytosolic pH, which demonstrates that cytosolic acidification mediates FC-induced NO removal. Taken together, our results show that FC induces NO removal and reduces NO level via cytosolic acidification in guard cells, thus inhibiting ABA effect.     

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.     

星型孢菌素(STS)对蚕豆气孔运动的调控效应

用激光扫描共聚焦显微技术,初步研究广谱性蛋白激酶抑制剂星型孢菌素(STS)对蚕豆气孔运动的调控效应.结果表明:(1)光下STS对气孔开度无影响但暗中显著促进气孔开放,表明蛋白激酶参与光/暗对气孔运动的调控,光下蛋白激酶活性低而暗中高;(2)与H2O2清除剂抗坏血酸(ASA)和NO清除剂羧基-2-苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)一样,STS既降低暗处理和光下外源H2O2、硝普钠(SNP)处理保卫细胞H2O2、NO水平,也促进气孔开放,表明暗中蛋白激酶通过抑制H2O2、NO清除机制提高保卫细胞内源H2O2、NO水平并促进气孔关闭.     

Calcium is involved in nitric oxide- and auxin-induced lateral root formation in rice

In the present study, the role of nitric oxide (NO) in the regulation of lateral root (LR) formation in rice was examined. Application of sodium nitroprusside (SNP; a NO donor) and indole-3-butyric acid (IBA; a naturally occurring auxin) to rice seedlings induced LR formation. The effect is specific for NO because the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (cPTIO) blocked the action of SNP and IBA. Endogenous NO was detected by the specific fluorescence probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. SNP- and IBA-induced NO fluorescence was specifically suppressed by cPTIO. Nitrate reductase (NR) inhibitor sodium tungstate completely inhibited IBA-induced LR formation and NO fluorescence. However, nitric oxide synthase inhibitor N ^G-nitro-l-arginine methyl ester hydrochloride slightly reduced IBA-induced LR formation and NO generation. It appears that NO generation that occurs in response to IBA might primarily involve NR activity. Moreover, NO production caused by SNP and IBA was localized in root area corresponding to LR emergence. The effects of Ca²⁺ chelators, Ca²⁺-channel inhibitors, and calmodulin antagonists on LR formation induced by SNP and IBA were also examined. All these inhibitors were effective in reducing the action of SNP and IBA. However, Ca²⁺ chelators and Ca²⁺-channel inhibitors had no effect on SNP- and IBA-induced NO generation. It is concluded that cytosolic levels of Ca²⁺ may regulate SNP and IBA action through calmodulin-dependent mechanism.