Ethylene mediates brassinosteroid‐induced stomatal closure via Gα protein‐activated hydrogen peroxide and nitric oxide production in Arabidopsis

Brassinosteroids (BRs) are essential for plant growth and development; however, whether and how they promote stomatal closure is not fully clear. In this study, we report that 24‐epibrassinolide (EBR), a bioactive BR, induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering a signal transduction pathway including ethylene synthesis, the activation of Gα protein, and hydrogen peroxide (H₂O₂) and nitric oxide (NO) production. EBR initiated a marked rise in ethylene, H₂O₂ and NO levels, necessary for stomatal closure in the wild type. These effects were abolished in mutant bri1‐301, and EBR failed to close the stomata of gpa1 mutants. Next, we found that both ethylene and Gα mediate the inductive effects of EBR on H₂O₂ and NO production. EBR‐triggered H₂O₂ and NO accumulation were canceled in the etr1 and gpa1 mutants, but were strengthened in the eto1‐1 mutant and the cGα line (constitutively overexpressing the G protein α‐subunit AtGPA1). Exogenously applied H₂O₂ or sodium nitroprusside (SNP) rescued the defects of etr1‐3 and gpa1 or etr1 and gpa1 mutants in EBR‐induced stomatal closure, whereas the stomata of eto1‐1/AtrbohF and cGα/AtrbohF or eto1‐1/nia1‐2 and cGα/nia1‐2 constructs had an analogous response to H₂O₂ or SNP as those of AtrbohF or Nia1‐2 mutants. Moreover, we provided evidence that Gα plays an important role in the responses of guard cells to ethylene. Gα activator CTX largely restored the lesion of the etr1‐3 mutant, but ethylene precursor ACC failed to rescue the defects of gpa1 mutants in EBR‐induced stomatal closure. Lastly, we demonstrated that Gα‐activated H₂O₂ production is required for NO synthesis. EBR failed to induce NO synthesis in mutant AtrbohF, but it led to H₂O₂ production in mutant Nia1‐2. Exogenously applied SNP rescued the defect of AtrbohF in EBR‐induced stomatal closure, but H₂O₂ did not reverse the lesion of EBR‐induced stomatal closure in Nia1‐2. Together, our results strongly suggest a signaling pathway in which EBR induces ethylene synthesis, thereby activating Gα, and then promotes AtrbohF‐dependent H₂O₂ production and subsequent Nia1‐catalyzed NO accumulation, and finally closes stomata.     

Arabidopsis RING E3 ubiquitin ligase JUL1 participates in ABA‐mediated microtubule depolymerization,stomatal closure,and tolerance response to drought stress

Ubiquitination is a critical post‐translational protein modification that has been implicated in diverse cellular processes, including abiotic stress responses, in plants. In the present study, we identified and characterized a T‐DNA insertion mutant in the At5g10650 locus. Compared to wild‐type Arabidopsis plants, at5g10650 progeny were hyposensitive to ABA at the germination stage. At5g10650 possessed a single C‐terminal C3HC4‐type Really Interesting New Gene (RING) motif, which was essential for ABA‐mediated germination and E3 ligase activity in vitro. At5g10650 was closely associated with microtubules and microtubule‐associated proteins in Arabidopsis and tobacco leaf cells. Localization of At5g10650 to the nucleus was frequently observed. Unexpectedly, At5g10650 was identified as JAV1‐ASSOCIATED UBIQUITIN LIGASE1 (JUL1), which was recently reported to participate in the jasmonate signaling pathway. The jul1 knockout plants exhibited impaired ABA‐promoted stomatal closure. In addition, stomatal closure could not be induced by hydrogen peroxide and calcium in jul1 plants. jul1 guard cells accumulated wild‐type levels of H₂O₂ after ABA treatment. These findings indicated that JUL1 acts downstream of H₂O₂ and calcium in the ABA‐mediated stomatal closure pathway. Typical radial arrays of microtubules were maintained in jul1 guard cells after exposure to ABA, H₂O₂, and calcium, which in turn resulted in ABA‐hyposensitive stomatal movements. Finally, jul1 plants were markedly more susceptible to drought stress than wild‐type plants. Overall, our results suggest that the Arabidopsis RING E3 ligase JUL1 plays a critical role in ABA‐mediated microtubule disorganization, stomatal closure, and tolerance to drought stress.     

ARP2/3 complex‐mediated actin dynamics is required for hydrogen peroxide‐induced stomatal closure in Arabidopsis

Multiple cellular events like dynamic actin reorganization and hydrogen peroxide (H₂O₂) production were demonstrated to be involved in abscisic acid (ABA)‐induced stomatal closure. However, the relationship between them as well as the underlying mechanisms remains poorly understood. Here, we showed that H₂O₂ generation is indispensable for ABA induction of actin reorganization in guard cells of Arabidopsis that requires the presence of ARP2/3 complex. H₂O₂‐induced stomatal closure was delayed in the mutants of arpc4 and arpc5, and the rate of actin reorganization was slowed down in arpc4 and arpc5 in response to H₂O₂, suggesting that ARP2/3‐mediated actin nucleation is required for H₂O₂‐induced actin cytoskeleton remodelling. Furthermore, the expression of H₂O₂ biosynthetic related gene AtrbohD and the accumulation of H₂O₂ was delayed in response to ABA in arpc4 and arpc5, demonstrating that misregulated actin dynamics affects H₂O₂ production upon ABA treatment. These results support a possible causal relation between the production of H₂O₂ and actin dynamics in ABA‐mediated guard cell signalling: ABA triggers H₂O₂ generation that causes the reorganization of the actin cytoskeleton partially mediated by ARP2/3 complex, and ARP2/3 complex‐mediated actin dynamics may feedback regulate H₂O₂ production.     

Brassinosteroids accelerate recovery of photosynthetic apparatus from cold stress by balancing the electron partitioning,carboxylation and redox homeostasis in cucumber

The aim of this study was to examine the role of brassinosteroids (BRs) in protecting the photosynthetic apparatus from cold‐induced damage in cucumber (Cucumis sativus) plants. Recovery at both high light (HL) and low light (LL) after a cooling at 10/7°C induced irreversible inhibition of CO₂ assimilation, photoinhibition at photosystem I (PSI) and inhibition of enzyme activities of Calvin cycle and ascorbate (AsA)‐reduced glutathione (GSH) cycle, followed by accumulation of H₂O₂ and malondialdehyde. However, cold‐induced photoinhibition at PSII was fully recovered at LL but not at HL. Meanwhile, recovery at HL increased electron flux to O₂‐dependent alternative pathway [Ja(O₂‐dependent)]. Foliar application of 24‐epibrassinolide (EBR) accelerated recovery from photoinhibition of PSII but not of PSI. EBR also significantly increased CO₂ assimilation, activity of Calvin cycle enzymes and electron flux to carbon reduction [Je(PCR)], with a concomitant decrease in Ja(O₂‐dependent); meanwhile EBR increased the activity of enzymes in AsA‐GSH cycle and cellular redox states. However, the positive effect of EBR on plant recovery was observed only at HL, but not LL. These results indicate that BR accelerates the recovery of photosynthetic apparatus at HL by activation of enzymes in Calvin cycle and increasing the antioxidant capacity, which in turn mitigate the photooxidative stress and the inhibition of plant growth during the recovery.     

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.     

Heterotrimeric G protein mediates ethylene‐induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis

Heterotrimeric G proteins function as key players in hydrogen peroxide (H₂O₂) production in plant cells, but whether G proteins mediate ethylene‐induced H₂O₂ production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H₂O₂ in guard cell ethylene signalling. In wild‐type leaves, ethylene‐triggered H₂O₂ synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene‐induced H₂O₂ production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H₂O₂ production in response to ethylene. Ethylene‐triggered H₂O₂ generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H₂O₂ rescued the defect of RAN1 and EIN4 mutants or etr1‐3 in ethylene‐induced H₂O₂ production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1‐1 and etr1‐9 in ethylene‐induced H₂O₂ production. Stomata of CTR1 mutants showed constitutive H₂O₂ production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H₂O₂, but do generate H₂O₂ following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene‐induced stomatal closure via H₂O₂ production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H₂O₂ signalling in guard cells.     

PP2A and microtubules function in 5-aminolevulinic acid-mediated H2O2 signaling in Arabidopsis guard cells

5-aminolevulinic acid (ALA), a plant growth regulator with great application potential in agriculture and horticulture, induces stomatal opening and inhibits stomatal closure by decreasing guard cell H₂O₂. However, the mechanisms behind ALA-decreased H₂O₂ in guard cells are not fully understood. Here, using type 2A protein phosphatase (PP2A) inhibitors, microtubule-stabilizing/disrupting drugs and green fluorescent protein-tagged α-tubulin 6 transgenic Arabidopsis (GFP-TUA6), we find that PP2A and cortical microtubules (MTs) are involved in ALA-regulated stomatal movement. Then, we analyze stomatal responses of Arabidopsis overexpressing C2 catalytic subunit of PP2A (PP2A-C2) and pp2a-c2 mutant to ALA and abscisic acid (ABA) under both light and dark conditions, and show that PP2A-C2 participates in ALA-induced stomatal movement. Furthermore, using pharmacological methods and confocal studies, we reveal that PP2A and MTs function upstream and downstream, respectively, of H₂O₂ in guard cell signaling. Finally, we demonstrate the role of H₂O₂-mediated microtubule arrangement in ALA inhibiting ABA-induced stomatal closure. Our findings indicate that MTs regulated by PP2A-mediated H₂O₂ decreasing play an important role in ALA guard cell signaling, revealing new insights into stomatal movement regulation.     

CLE9 peptide‐induced stomatal closure is mediated by abscisic acid,hydrogen peroxide,and nitric oxide in Arabidopsis thaliana

CLE peptides have been implicated in various developmental processes of plants and mediate their responses to environmental stimuli. However, the biological relevance of most CLE genes remains to be functionally characterized. Here, we report that CLE9, which is expressed in stomata, acts as an essential regulator in the induction of stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 effectively led to stomatal closure and enhanced drought tolerance, whereas CLE9 loss‐of‐function mutants were sensitivity to drought stress. CLE9‐induced stomatal closure was impaired in abscisic acid (ABA)‐deficient mutants, indicating that ABA is required for CLE9‐medaited guard cell signalling. We further deciphered that two guard cell ABA‐signalling components, OST1 and SLAC1, were responsible for CLE9‐induced stomatal closure. MPK3 and MPK6 were activated by the CLE9 peptide, and CLE9 peptides failed to close stomata in mpk3 and mpk6 mutants. In addition, CLE9 peptides stimulated the induction of hydrogen peroxide (H₂O₂) and nitric oxide (NO) synthesis associated with stomatal closure, which was abolished in the NADPH oxidase‐deficient mutants or nitric reductase mutants, respectively. Collectively, our results reveal a novel ABA‐dependent function of CLE9 in the regulation of stomatal apertures, thereby suggesting a potential role of CLE9 in the stress acclimatization of plants.     

Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid‐mediated systemic virus resistance in Nicotiana benthamiana

Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H₂O₂ and NO. Scavenging of H₂O₂ or NO in upper leaves blocked BR‐induced systemic virus resistance. BR‐induced systemic H₂O₂ accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite‐dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR‐triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR‐activated H₂O₂ is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H₂O₂ generation blocked BR‐induced systemic NO production, but BR‐induced H₂O₂ production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR‐induced systemic virus defense in NbRBOHB‐silenced plants, but H₂O₂ did not reverse the effect of NbNR silencing on BR‐induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR‐mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR‐induced H₂O₂ and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR‐mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)‐dependent H₂O₂ production and subsequent systemic NR‐dependent NO generation.     

PeCHYR1, a ubiquitin E3 ligase from Populus euphratica,enhances drought tolerance via ABA‐induced stomatal closure by ROS production in Populus

Drought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba × Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H₂O₂ production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild‐type) controls. Moreover, up‐regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA‐induced stomatal closure caused by hydrogen peroxide (H₂O₂) production in transgenic poplar plants.     

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.     

Copper amine oxidase and phospholipase D act independently in abscisic acid (ABA)-induced stomatal closure in Vicia faba and Arabidopsis

Recent evidence has demonstrated that both copper amine oxidase (CuAO; EC 1.4.3.6) and phospholipase D (PLD; EC 3.1.4.4) are involved in abscisic acid (ABA)-induced stomatal closure. In this study, we investigated the interaction between CuAO and PLD in the ABA response. Pretreatment with either CuAO or PLD inhibitors alone or that with both additively led to impairment of ABA-induced H₂O₂ production and stomatal closure in Vicia faba. ABA-stimulated PLD activation could not be inhibited by the CuAO inhibitor, and CuAO activity was not affected by the PLD inhibitor. These data suggest that CuAO and PLD act independently in the ABA response. To further examine PLD and CuAO activities in ABA responses, we used the Arabidopsis mutants cuaoζ and pldα1. Ablation of guard cell-expressed CuAOζ or PLDα1 gene retarded ABA-induced H₂O₂ generation and stomatal closure. As a product of PLD, phosphatidic acid (PA) substantially enhanced H₂O₂ production and stomatal closure in wide type, pldα1, and cuaoζ. Moreover, putrescine (Put), a substrate of CuAO as well as an activator of PLD, induced H₂O₂ production and stomatal closure in WT but not in both mutants. These results suggest that CuAO and PLD act independently in ABA-induced stomatal closure.     

WD40‐REPEAT 5a functions in drought stress tolerance by regulating nitric oxide accumulation in Arabidopsis

Nitric oxide (NO) generation by NO synthase (NOS) in guard cells plays a vital role in stomatal closure for adaptive plant response to drought stress. However, the mechanism underlying the regulation of NOS activity in plants is unclear. Here, by screening yeast deletion mutants with decreased NO accumulation and NOS‐like activity when subjected to H₂O₂ stress, we identified TUP1 as a novel regulator of NOS‐like activity in yeast. Arabidopsis WD40‐REPEAT 5a (WDR5a), a homolog of yeast TUP1, complemented H₂O₂‐induced NO accumulation of a yeast mutant Δtup1, suggesting the conserved role of WDR5a in regulating NO accumulation and NOS‐like activity. This note was further confirmed by using an Arabidopsis RNAi line wdr5a‐1 and two T‐DNA insertion mutants of WDR5a with reduced WDR5a expression, in which both H₂O₂‐induced NO accumulation and stomatal closure were repressed. This was because H₂O₂‐induced NOS‐like activity was inhibited in the mutants compared with that of the wild type. Furthermore, these wdr5a mutants were more sensitive to drought stress as they had reduced stomatal closure and decreased expression of drought‐related genes. Together, our results revealed that WDR5a functions as a novel factor to modulate NOS‐like activity for changes of NO accumulation and stomatal closure in drought stress tolerance.     

The Arabidopsis heterotrimeric G‐protein β subunit,AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Ca_o responses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cyt oscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double‐mutants, as well as those of the agg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast with ABA‐regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6‐detected [Ca²⁺]_cyt oscillations in response to Ca_o, initiating only a single [Ca²⁺]_cyt spike. Experimentally imposed [Ca²⁺]_cyt oscillations restored stomatal closure in agb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Ca_o induced InsP3 production in Col but not in agb1. In sum, G‐protein signaling via AGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_o apparently require Ca²⁺‐induced Ca²⁺ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.     

UV-B对拟南芥叶片不同来源H2O2的活化和气孔关闭的诱导

在UV-B调控植物许多生理过程中过氧化氢(H2O2)作为第二信使发挥着重要作用,但H2O2来源途径并不清楚。该研究借助气孔开度分析和激光扫描共聚焦显微镜技术,探讨H2O2在介导不同剂量UV-B诱导拟南芥叶片气孔关闭过程中的酶学来源途径。结果发现:0.5W.m-2 UV-B能诱导野生型拟南芥叶片保卫细胞的H2O2产生和气孔关闭,且该效应能被NADPH氧化酶抑制剂二苯基碘(DPI)抑制,而不能被细胞壁过氧化物酶抑制剂水杨基氧肟酸(SHAM)抑制,同时该剂量UV-B也不能诱导NADPH氧化酶功能缺失单突变体AtrbohD和AtrbohF以及双突变体AtrbohD/F保卫细胞的H2O2产生和气孔关闭;相反,0.65 W.m-2 UV-B既能诱导野生型也能诱导NADPH氧化酶突变体保卫细胞的H2O2产生和气孔关闭,且该效应能被SHAM抑制,却不能被DPI抑制。结果表明,不同剂量UV-B通过活化不同生成途径的H2O2来诱导拟南芥叶片气孔关闭,即低剂量UV-B主要诱导NADPH氧化酶AtrbohD和AtrbohF途径来源的H2O2生成,而高剂量UV-B主要活化细胞壁过氧化酶途径来源的H2O2。     

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.     

The mechanism of SO2‐induced stomatal closure differs from O3 and CO2 responses and is mediated by nonapoptotic cell death in guard cells

Plants closing stomata in the presence of harmful gases is believed to be a stress avoidance mechanism. SO₂, one of the major airborne pollutants, has long been reported to induce stomatal closure, yet the mechanism remains unknown. Little is known about the stomatal response to airborne pollutants besides O₃. SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1) and OPEN STOMATA 1 (OST1) were identified as genes mediating O₃‐induced closure. SLAC1 and OST1 are also known to mediate stomatal closure in response to CO₂, together with RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs). The overlaying roles of these genes in response to O₃ and CO₂ suggested that plants share their molecular regulators for airborne stimuli. Here, we investigated and compared stomatal closure event induced by a wide concentration range of SO₂ in Arabidopsis through molecular genetic approaches. O₃‐ and CO₂‐insensitive stomata mutants did not show significant differences from the wild type in stomatal sensitivity, guard cell viability, and chlorophyll content revealing that SO₂‐induced closure is not regulated by the same molecular mechanisms as for O₃ and CO₂. Nonapoptotic cell death is shown as the reason for SO₂‐induced closure, which proposed the closure as a physicochemical process resulted from SO₂ distress, instead of a biological protection mechanism.