Compound stress response in stomatal closure: a mathematical model of ABA and ethylene interaction in guard cells.
ABSTRACT: Stomata are tiny pores in plant leaves that regulate gas and water exchange between the plant and its environment. Abscisic acid and ethylene are two well-known elicitors of stomatal closure when acting independently. However, when stomata are presented with a combination of both signals, they fail to close.Toshed light on this unexplained behaviour, we have collected time course measurements of stomatal aperture and hydrogen peroxide production in Arabidopsis thaliana guard cells treated with abscisic acid, ethylene, and a combination of both. Our experiments show that stomatal closure is linked to sustained high levels of hydrogen peroxide in guard cells. When treated with a combined dose of abscisic acid and ethylene, guard cells exhibit increased antioxidant activity that reduces hydrogen peroxide levels and precludes closure. We construct a simplified model of stomatal closure derived from known biochemical pathways that captures the experimentally observed behaviour.Our experiments and modelling results suggest a distinct role for two antioxidant mechanisms during stomatal closure: a slower, delayed response activated by a single stimulus (abscisic acid 'or' ethylene) and another more rapid 'and' mechanism that is only activated when both stimuli are present. Our model indicates that the presence of this rapid 'and' mechanism in the antioxidant response is key to explain the lack of closure under a combined stimulus.
Project description:Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone--an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation and climate change. The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes. Despite the vital role of guard cells in controlling plant water loss, ozone sensitivity and CO2 supply, the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli. However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1. We show that SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO2, abscisic acid, ozone, light/dark transitions, humidity change, calcium ions, hydrogen peroxide and nitric oxide. Mutations in SLAC1 impair slow (S-type) anion channel currents that are activated by cytosolic Ca2+ and abscisic acid, but do not affect rapid (R-type) anion channel currents or Ca2+ channel function. A low homology of SLAC1 to bacterial and fungal organic acid transport proteins, and the permeability of S-type anion channels to malate suggest a vital role for SLAC1 in the function of S-type anion channels.
Project description:Stomatal closure is affected by various stimuli such as light, atmospheric carbon dioxide concentration, humidity and phytohormones. Our research focuses on phytohormones, specifically: abscisic acid (ABA), ethylene (ET) and methyl jasmonate (MeJA) that are responsible for the regulation of several plant processes, especially in guard cell signalling. While several studies show that these three phytohormones cause stomatal closure in plants, only two studies are notable for establishing a mathematical model of guard cell signalling involving phytohormones. Those two studies employed Boolean modelling and mechanistic ordinary differential equations modelling. In this study, we propose a new mathematical model of guard cell transduction network for stomatal closure using continuous logical modelling framework. Results showed how the different components of the network function. Furthermore, the model verified the role of antioxidants in the closure mechanism, and the diminished closure level of stomata with combined ABA-ET stimulus. The analysis was extended to ABA-ET-MeJA crosstalk.
Project description: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.
Project description:Stomatal movements are crucial for the control of plant water status and protection against pathogens. Assays on epidermal peels revealed that, similar to abscisic acid (ABA), pathogen-associated molecular pattern (PAMP) flg22 requires the AtPIP2;1 aquaporin to induce stomatal closure. Flg22 also induced an increase in osmotic water permeability (Pf) of guard cell protoplasts through activation of AtPIP2;1. The use of HyPer, a genetic probe for intracellular hydrogen peroxide (H2O2), revealed that both ABA and flg22 triggered an accumulation of H2O2 in wild-type but not pip2;1 guard cells. Pretreatment of guard cells with flg22 or ABA facilitated the influx of exogenous H2O2 Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) and open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6) were both necessary to flg22-induced Pf and both phosphorylated AtPIP2;1 on Ser121 in vitro. Accumulation of H2O2 and stomatal closure as induced by flg22 was restored in pip2;1 guard cells by a phosphomimetic form (Ser121Asp) but not by a phosphodeficient form (Ser121Ala) of AtPIP2;1. We propose a mechanism whereby phosphorylation of AtPIP2;1 Ser121 by BAK1 and/or OST1 is triggered in response to flg22 to activate its water and H2O2 transport activities. This work establishes a signaling role of plasma membrane aquaporins in guard cells and potentially in other cellular context involving H2O2 signaling.
Project description:Reactive oxygen species (ROS), including hydrogen peroxide (H(2)O(2)), are among the important second messengers in abscisic acid (ABA) signaling in guard cells. In this study, to investigate specific roles of H(2)O(2) in ABA signaling in guard cells, we examined the effects of mutations in the guard cell-expressed catalase (CAT) genes, CAT1 and CAT3, and of the CAT inhibitor 3-aminotriazole (AT) on stomatal movement. The cat3 and cat1 cat3 mutations significantly reduced CAT activities, leading to higher basal level of H(2)O(2) in guard cells, when assessed by 2',7'-dichlorodihydrofluorescein, whereas they did not affect stomatal aperture size under non-stressed condition. In addition, AT-treatment at concentrations that abolish CAT activities, showed trivial affect on stomatal aperture size, while basal H(2)O(2) level increased extensively. In contrast, cat mutations and AT-treatment potentiated ABA-induced stomatal closure. Inducible ROS production triggered by ABA was observed in these mutants and wild type as well as in AT-treated guard cells. These results suggest that ABA-inducible cytosolic H(2)O(2) elevation functions in ABA-induced stomatal closure, while constitutive increase of H(2)O(2) do not cause stomatal closure.
Project description:Microbes trigger stomatal closure through microbe-associated molecular patterns (MAMPs). The bacterial pathogen Pseudomonas syringae pv. tomato (Pst) synthesizes the polyketide toxin coronatine, which inhibits stomatal closure by MAMPs and by the hormone abscisic acid (ABA). The mechanism by which coronatine, a jasmonic acid-isoleucine analog, achieves this effect is not completely clear. Reactive oxygen species (ROS) are essential second messengers in stomatal immunity, therefore we investigated the possible effect of coronatine on their production. We found that coronatine inhibits NADPH oxidase-dependent ROS production induced by ABA, and by the flagellin-derived peptide flg22. This toxin also inhibited NADPH oxidase-dependent stomatal closure induced by darkness, however, it failed to prevent stomatal closure by exogenously applied H2O2 or by salicylic acid, which induces ROS production through peroxidases. Contrary to what was observed on stomata, coronatine did not affect the oxidative burst induced by flg22 in leaf disks. Additionally, we observed that in NADPH oxidase mutants atrbohd and atrbohd/f, as well as in guard cell ABA responsive but flg22 insensitive mutants mpk3, mpk6, npr1-3, and lecrk-VI.2-1, the inhibition of ABA stomatal responses by both coronatine and the NADPH oxidase inhibitor diphenylene iodonium was markedly reduced. Interestingly, coronatine still impaired ABA-induced ROS synthesis in mpk3, mpk6, npr1-3, and lecrk-VI.2-1, suggesting a possible feedback regulation of ROS on other guard cell ABA signaling elements in these mutants. Altogether our results show that inhibition of NADPH oxidase-dependent ROS synthesis in guard cells plays an important role during endophytic colonization by Pst through stomata.
Project description:Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.
Project description:Plant copper amine oxidases (CuAOs) are involved in wound healing, defense against pathogens, methyl-jasmonate-induced protoxylem differentiation, and abscisic acid (ABA)-induced stomatal closure. In the present study, we investigated the role of the Arabidopsis thaliana CuAOδ (AtCuAOδ; At4g12290) in the ABA-mediated stomatal closure by genetic and pharmacological approaches. Obtained data show that AtCuAOδ is up-regulated by ABA and that two Atcuaoδ T-DNA insertional mutants are less responsive to this hormone, showing reduced ABA-mediated stomatal closure and H2O2 accumulation in guard cells as compared to the wild-type (WT) plants. Furthermore, CuAO inhibitors, as well as the hydrogen peroxide (H2O2) scavenger N,N1-dimethylthiourea, reversed most of the ABA-induced stomatal closure in WT plants. Consistently, AtCuAOδ over-expressing transgenic plants display a constitutively increased stomatal closure and increased H2O2 production compared to WT plants. Our data suggest that AtCuAOδ is involved in the H2O2 production related to ABA-induced stomatal closure.
Project description:The phytohormone abscisic acid (ABA) induces stomatal closure in response to drought stress, leading to reduction of transpirational water loss. A thiol tripeptide glutathione (GSH) is an important regulator of cellular redox homeostasis in plants. Although it has been shown that cellular redox state of guard cells controls ABA-mediated stomatal closure, roles of GSH in guard cell ABA signaling were largely unknown. Recently we demonstrated that GSH functions as a negative regulator of ABA signaling in guard cells. In this study we performed more detailed analyses to reveal how GSH regulates guard cell ABA signaling using the GSH-deficient Arabidopsis mutant cad2-1. The cad2-1 mutant exhibited reduced water loss from rosette leaves. Whole-cell current recording using patch clamp technique revealed that the cad2-1 mutation did not affect ABA regulation of S-type anion channels. We found enhanced activation of Ca(2+) permeable channels by hydrogen peroxide (H2O2) in cad2-1 guard cells. The cad2-1 mutant showed enhanced H2O2-induced stomatal closure and significant increase of ROS accumulation in whole leaves in response to ABA. Our findings provide a new understanding of guard cell ABA signaling and a new strategy to improve plant drought tolerance.
Project description:Abiotic stresses, such as drought and salinity, lead to crop growth damage and a decrease in crop yields. Stomata control CO(2) uptake and optimize water use efficiency, thereby playing crucial roles in abiotic stress tolerance. Hydrogen peroxide (H(2)O(2)) is an important signal molecule that induces stomatal closure. However, the molecular pathway that regulates the H(2)O(2) level in guard cells remains largely unknown. Here, we clone and characterize DST (drought and salt tolerance)-a previously unknown zinc finger transcription factor that negatively regulates stomatal closure by direct modulation of genes related to H(2)O(2) homeostasis-and identify a novel pathway for the signal transduction of DST-mediated H(2)O(2)-induced stomatal closure. Loss of DST function increases stomatal closure and reduces stomatal density, consequently resulting in enhanced drought and salt tolerance in rice. These findings provide an interesting insight into the mechanism of stomata-regulated abiotic stress tolerance, and an important genetic engineering approach for improving abiotic stress tolerance in crops.