ABSTRACT 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 ([Ca 2+ ] 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 H 2 O 2 and O 2 – 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 [Ca 2+ ] 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.
Abstract Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDα1 and PLDδ, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, pldα1 and pldδ single mutants, and a pldα1 pldδ double mutant. ABA-induced stomatal closure was suppressed in the pldα1 pldδ double mutant but not in the pld single mutants. The pldα1 and pldδ mutations reduced ABA-induced phosphatidic acid production in epidermal tissues. Expression of either PLDα1 or PLDδ complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both pldα1 and pldδ single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDα1 and PLDδ function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the pldα1 pldδ double mutant. Inward-rectifying K+ channel currents of guard cells were inhibited by ABA in the wild type but not in the pldα1 pldδ double mutant. ABA inhibited stomatal opening in the wild type and the pldδ mutant but not in the pldα1 mutant. In wild-type rosette leaves, ABA significantly increased PLDδ transcript levels but did not change PLDα1 transcript levels. Furthermore, the pldα1 and pldδ mutations mitigated ABA inhibition of seed germination. These results suggest that PLDα1 and PLDδ cooperate in ABA signaling in guard cells but that their functions do not completely overlap.
We have demonstrated that reactive carbonyl species (RCS) function as an intermediate downstream of hydrogen peroxide (H2O2) production in abscisic acid (ABA) signaling for stomatal closure in guard cells using transgenic tobacco plants overexpressing alkenal reductase. We investigated the conversion of the RCS production into downstream signaling events in the guard cells. Both ABA and H2O2 induced production of the RCS, such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), in epidermal tissues of wild-type Arabidopsis thaliana plants. Application of the RCS scavengers, carnosine and pyridoxamine, did not affect the ABA-induced H2O2 production but inhibited the ABA- and H2O2-induced stomatal closure. Both acrolein and HNE induced stomatal closure in a plasma membrane NAD(P)H oxidase mutant atrbohD atrbohF as well as in the wild type, but not in a calcium-dependent kinase mutant cpk6. Acrolein activated plasma membrane Ca2+-permeable cation channels, triggered cytosolic free Ca2+ concentration ([Ca2+]cyt) elevation, and induced stomatal closure accompanied by depletion of glutathione in the guard cells. These results suggest that RCS production is a signaling event between the ROS production and [Ca2+]cyt elevation during guard cell ABA signaling.
The expression of plasma membrane K+ channels of NaCl-adapted tobacco suspension cells and effects of extracellular Ca2+ on plasma membrane K+ channels were investigated. Reverse transcription-PCR (RT-PCR) analysis showed that expression of TORK1, which encodes a K+ channel, was much lower in NaCl-adapted cells than in NaCl-unadapted cells. The magnitude of the outward K+ currents of NaCl-adapted as well as NaCl-unadapted cells decreased with increasing extracellular Ca2+ but there is no significant difference in Ca2+ dependency of the K+ current. These analyses suggest that reduction of the number of K+ channels might cause NaCl adaptation of cells through the decrease of outward K+ currents.
Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.
Isothiocyanates, nitriles, and thiocyanates are degradation products of glucosinolates in crucifer plants. In this study, we investigated the stomatal response to allyl isothiocyanate (AITC), 3-butenenitrile (3BN), and ethyl thiocyanate (ESCN) in Arabidopsis. AITC, 3BN, and ESCN induced stomatal closure in the wild type and the atrbohD atrbohF mutant. Stomatal closure was inhibited by catalase and salicylhydroxamic acid (SHAM). The degradation products induced extracellular reactive oxygen species (ROS) production in the rosette leaves, and intracellular ROS accumulation, NO production, and cytosolic free calcium concentration ([Ca(2+)]cyt) oscillations in guard cells, which were inhibited by SHAM. These results suggest that glucosinolate degradation products induce stomatal closure accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca(2+)]cyt oscillation in Arabidopsis.
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