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.
Stomatal movements via the control of gas exchanges determine plant growth in relation to environmental stimuli through a complex signalling network involving reactive oxygen species that lead to ...post‐translational modifications of Cys and Met residues, and alter protein activity and/or conformation. Thiol‐reductases (TRs), which include thioredoxins, glutaredoxins (GRXs) and peroxiredoxins (PRXs), participate in signalling pathways through the control of Cys redox status in client proteins. Their involvement in stomatal functioning remains poorly characterized. By performing a mass spectrometry‐based proteomic analysis, we show that numerous thiol reductases, like PRXs, are highly abundant in guard cells. When investigating various Arabidopsis mutants impaired in the expression of TR genes, no change in stomatal density and index was noticed. In optimal growth conditions, a line deficient in cytosolic NADPH‐thioredoxin reductases displayed higher stomatal conductance and lower leaf temperature evaluated by thermal infrared imaging. In contrast, lines deficient in plastidial 2‐CysPRXs or type‐II GRXs exhibited compared to WT reduced conductance and warmer leaves in optimal conditions, and enhanced stomatal closure in epidermal peels treated with abscisic acid or hydrogen peroxide. Altogether, these data strongly support the contribution of thiol redox switches within the signalling network regulating guard cell movements and stomatal functioning.
The contribution of thiol reductases to the signalling network governing stomatal functioning remains poorly known. Using proteomics, we show that numerous thiol reductases are abundant in a guard cell‐enriched fraction, and we unveil a stomatal phenotype in Arabidopsis mutants for thiol reductases.
Various physiological imbalances lead to reactive oxygen species (ROS) overproduction and/or increases in lipoxygenase (LOX) activities, both events ending in lipid peroxidation of polyunsaturated ...fatty acids (PUFAs). Besides the quantification of such a process, the development of tools is necessary in order to allow the identification of the primary cause of its development and localization. A biochemical method assessing 9 LOX, 13 LOX and ROS-mediated peroxidation of membrane-bound and free PUFAs has been improved. The assay is based on the analysis of hydroxy fatty acids derived from PUFA hydroperoxides by both the straight and chiral phase high-performance liquid chromatography. Besides the upstream products of peroxidation of the 18:2 and 18:3 PUFAs, products coming from the 16:3 were characterized and their steady-state level quantified. Moreover, the observation that the relative amounts of the ROS-mediated peroxidation isomers of 18:3 were constant in leaves allowed us to circumvent the chiral analyses for the discrimination and quantification of 9 LOX, 13 LOX and ROS-mediated processes in routine experiments. The methodology has been successfully applied to decipher lipid peroxidation in Arabidopsis leaves submitted to biotic and abiotic stresses. We provide evidence of the relative timing of enzymatic and non-enzymatic lipid peroxidation processes. The 13 LOX pathway is activated early whatever the nature of the stress, leading to the peroxidation of chloroplast lipids. Under cadmium stress, the 9 LOX pathway added to the 13 LOX one. ROS-mediated peroxidation was mainly driven by light and always appeared as a late process.
Plant mitogen-activated protein kinases (MAPKs) are involved in important processes, including stress signaling and development. In a functional yeast screen, we identified mutations that render ...Arabidopsis thaliana MAPKs constitutively active (CA). Importantly, CA-MAPKs maintain their specificity toward known activators and substrates. As a proof-of-concept, Arabidopsis MAPK4 (MPK4) function in plant immunity was investigated. In agreement with the phenotype of mpk4 mutants, CA-MPK4 plants were compromised in pathogen-induced salicylic acid accumulation and disease resistance. MPK4 activity was found to negatively regulate pathogen-associated molecular pattern-induced reactive oxygen species production but had no impact on callose deposition, indicating that CA-MPK4 allows discriminating between processes regulated by MPK4 activity from processes indirectly affected by mpk4 mutation. Finally, MPK4 activity was also found to compromise effector-triggered immunity conditioned by the Toll Interleukin-1 Receptor—nucleotide binding (NB)—Leu-rich repeat (LRR) receptors RPS4 and RPP4 but not by the coiled coil—NB-LRR receptors RPM1 and RPS2. Overall, these data reveal important insights on how MPK4 regulates plant defenses and establishes that CA-MAPKs offer a powerful tool to analyze the function of plant MAPK pathways.
New checkpoints in stomatal defense Montillet, Jean-Luc; Hirt, Heribert
Trends in plant science,
06/2013, Letnik:
18, Številka:
6
Journal Article
Recenzirano
Recent reports have revealed new guard cell signaling elements that function in stomatal defense in Arabidopsis thaliana (Arabidopsis). We discuss here the role of oxylipins, salicylic acid (SA), and ...abscisic acid (ABA) in stomatal immunity in response to the bacterial pathogen Pseudomonas syringae.
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•The biochemical activity of chloroplast ceQORH protein was unknown.•ceQORH reduces the double bond of stress-related oxidized lipids named γ-ketols.•ceQORH is a γ-ketol reductase ...identified in the chloroplast.
Under oxidative stress conditions the lipid constituents of cells can undergo oxidation whose frequent consequence is the production of highly reactive α,β-unsaturated carbonyls. These molecules are toxic because they can add to biomolecules (such as proteins and nucleic acids) and several enzyme activities cooperate to eliminate these reactive electrophile species. CeQORH (chloroplast envelope Quinone Oxidoreductase Homolog, At4g13010) is associated with the inner membrane of the chloroplast envelope and imported into the organelle by an alternative import pathway. In the present study, we show that the recombinant ceQORH exhibits the activity of a NADPH-dependent α,β-unsaturated oxoene reductase reducing the double bond of medium-chain (C⩾9) to long-chain (18 carbon atoms) reactive electrophile species deriving from poly-unsaturated fatty acid peroxides. The best substrates of ceQORH are 13-lipoxygenase-derived γ-ketols. γ-Ketols are spontaneously produced in the chloroplast from the unstable allene oxide formed in the biochemical pathway leading to 12-oxo-phytodienoic acid, a precursor of the defense hormone jasmonate. In chloroplasts, ceQORH could detoxify 13-lipoxygenase-derived γ-ketols at their production sites in the membranes. This finding opens new routes toward the understanding of γ-ketols role and detoxification.
Cadmium is suspected to exert its toxic action on cells through oxidative damage. However, the transition metal is unable to directly generate reactive oxygen species (ROS) via redox reactions with ...molecular oxygen in a biological environment. Here, we show that bright yellow-2 (BY-2) tobacco cells exposed to millimolar concentrations of CdCl₂ developed cell death within 2-3 h. The death process was preceded by two successive waves of ROS differing in their nature and subcellular localization. Firstly, these consisted in the transient NADPH oxidase-dependent accumulation of H₂O₂ followed by the accumulation of O₂⁻. in mitochondria. A third wave of ROS consisting in fatty acid hydroperoxide accumulation was concomitant with cell death. Accumulation of H₂O₂ was preceded by an increase in cytosolic free calcium concentration originating from internal pools that was essential to activate the NADPH oxidase. The cell line gp3, impaired in NADPH oxidase activity, and that was unable to accumulate H₂O₂ in response to Cd²⁺, was nevertheless poisoned by the metal. Therefore, this first wave of ROS was not sufficient to trigger all the cadmium-dependent deleterious effects. However, we show that the accumulation of O₂⁻. of mitochondrial origin and membrane peroxidation are key players in Cd²⁺-induced cell death.
We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species ...(ROS)-mediated lipid peroxidation from 9- and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H₂O₂-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H₂O₂ plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H₂O₂ and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H₂O₂ and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.
Cytosolic/nuclear molecular chaperones of the heat shock protein families HSP90 and HSC70 are conserved and essential proteins in eukaryotes. These proteins have essentially been implicated in the ...innate immunity and abiotic stress tolerance in higher plants. Here, we demonstrate that both chaperones are recruited in Arabidopsis (Arabidopsis thaliana) for stomatal closure induced by several environmental signals. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are compromised in the dark-, CO₂-, flagellin 22 peptide-, and abscisic acid (ABA)-induced stomatal closure. HSC70-1 and IISP90 proteins are needed to establish basal expression levels of several ABA-responsive genes, suggesting that these chaperones might also be involved in ABA signaling events. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are hypersensitive to ABA in seed germination assays, suggesting that several chaperone complexes with distinct substrates might tune tissue-specific responses to ABA and the other biotic and abiotic stimuli studied. This study demonstrates that the HSC70/HSP90 machinery is important for stomatal closure and serves essential functions in plants to integrate signals from their biotic and abiotic environments.
Hypersensitive response (HR) is a programmed cell death that is commonly associated with disease resistance in plants. Among the different HR-related early induced genes, the AtMYB30 gene is ...specifically, rapidly, and transiently expressed during incompatible interactions between Arabidopsis and bacterial pathogens. Its expression was also shown to be deregulated in Arabidopsis mutants affected in the control of cell death initiation. Here, we demonstrate that overexpression in Arabidopsis and tobacco of AtMYB30 (i) accelerates and intensifies the appearance of the HR in response to different avirulent bacterial pathogens, (ii) causes HR-like responses to virulent strains, and (iii) increases resistance against different bacterial pathogens, and a virulent biotrophic fungal pathogen, Cercospora nicotianae. In antisense AtMYB30 Arabidopsis lines, HR cell death is strongly decreased or suppressed in response to avirulent bacterial strains, resistance against different bacterial pathogens decreased, and the expression of HR- and defense-related genes was altered. Taken together, these results strongly suggest that AtMYB30 is a positive regulator of hypersensitive cell death.
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