Roles for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense

Reactive oxygen species (ROS) are both signal molecules and direct participants in plant defense against pathogens. Many fungi synthesize mannitol, a potent quencher of ROS, and there is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS-mediated plant defenses. Here we show induction of mannitol production and secretion in the phytopathogenic fungus Alternaria alternata in the presence of host-plant extracts. Conversely, we show that the catabolic enzyme mannitol dehydrogenase is induced in a non-mannitol-producing plant in response to both fungal infection and specific inducers of plant defense responses. This provides a mechanism whereby the plant can counteract fungal suppression of ROS-mediated defenses by catabolizing mannitol of fungal origin.

Comparison of Binding Properties and Early Biological Effects of Elicitins in Tobacco Cells1

Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca2+ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca2+ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca2+ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.

The Respiratory Burst and Electrolyte Leakage Induced by Sulfhydryl Blockers in Egeria densa Leaves Are Associated with H2O2 Production and Are Dependent on Ca2+ Influx1

In leaves of Egeria densa Planchon, N-ethylmaleimide (NEM) and other sulfhydryl-binding reagents induce a temporary increase in nonmitochondrial respiration (ΔQO2) that is inhibited by diphenylene iodonium and quinacrine, two known inhibitors of the plasma membrane NADPH oxidase, and are associated with a relevant increase in electrolyte leakage (M. Bellando, S. Sacco, F. Albergoni, P. Rocco, M.T. Marré [1997] Bot Acta 110: 388–394). In this paper we report data indicating further analogies between the oxidative burst induced by sulfhydryl blockers in E. densa and that induced by pathogen-derived elicitors in animal and plant cells: (a) NEM- and Ag+-induced ΔQO2 was associated with H2O2 production and both effects depended on the presence of external Ca2+; (b) Ca2+ influx was markedly increased by treatment with NEM; (c) the Ca2+ channel blocker LaCl3 inhibited ΔQO2, electrolyte release, and membrane depolarization induced by the sulfhydryl reagents; and (d) LaCl3 also inhibited electrolyte leakage induced by the direct infiltration of the leaves with H2O2. These results suggest a model in which the interaction of sulfhydryl blockers with sulfhydryl groups of cell components would primarily induce an increase in the Ca2+ cytosolic concentration, followed by membrane depolarization and activation of a plasma membrane NADPH oxidase. This latter effect, producing active oxygen species, might further influence plasma membrane permeability, leading to the massive release of electrolytes from the tissue.

Potentiation of the Oxidative Burst and Isoflavonoid Phytoalexin Accumulation by Serine Protease Inhibitors1

Treatment of soybean (Glycine max L. cv Williams 82) cell-suspension cultures with Pseudomonas syringae pv glycinea (Psg) harboring an avirulence gene (avrA) or with yeast elicitor resulted in an oxidative burst characterized by the accumulation of H2O2. This burst, and the resultant induction of glutathione S-transferase transcripts, occurred more rapidly and was more prolonged if cells were simultaneously treated with serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) or diisopropylfluorophosphate. PMSF and diisopropylfluorophosphate potentiate a large oxidative burst in cells exposed to Psg harboring the avrC avirulence gene, which is not recognized by the soybean cultivar used in this study. The potentiated burst was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor K252a. PMSF treatment of elicited cells or cells exposed to Psg:avrA caused a large increase in the accumulation of the isoflavonoid phytoalexin glyceollin; however, this was not associated with increased levels of transcripts encoding key phytoalexin biosynthetic enzymes. Glyceollin accumulation was inhibited by diphenylene iodonium; however, the oxidative burst in cells treated with Psg:avrC and PMSF was not followed by phytoalexin accumulation. We conclude that active oxygen species from the oxidative burst are necessary but not sufficient for inducing isoflavonoid phytoalexin accumulation in soybean cells.

Oxidative Burst and Hypoosmotic Stress in Tobacco Cell Suspensions

Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca2+ channels. Inhibition of the oxidative response impaired Cl− efflux, K+ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.

Photosystem I Is an Early Target of Photoinhibition in Barley Illuminated at Chilling Temperatures1

Light-induced damage to photosystem I (PSI) was studied during low-light illumination of barley (Hordeum vulgare L.) at chilling temperatures. A 4-h illumination period induced a significant inactivation of PSI electron transport activity. Flash-induced P700 absorption decay measurements revealed progressive damage to (a) the iron-sulfur clusters FA and FB, (b) the iron-sulfur clusters FA, FB, and FX, and (c) the phylloquinone A1 and the chlorophyll A0 or P700 of the PSI electron acceptor chain. Light-induced PSI damage was also evidenced by partial degradation of the PSI-A and PSI-B proteins and was correlated with the appearance of smaller proteins. Aggravated photodamage was observed upon illumination of barley leaves infiltrated with KCN, which inhibits Cu,Zn-superoxide dismutase and ascorbate peroxidase. This indicates that the photodamage of PSI in barley observed during low-light illumination at chilling temperatures arises because the defense against active oxygen species by active oxygen-scavenging enzymes is insufficient at these specific conditions. The data obtained demonstrate that photoinhibition of PSI at chilling temperatures is an important phenomenon in a cold-tolerant plant species.

Ozone-induced responses in Arabidopsis thaliana: the role of salicylic acid in the accumulation of defense-related transcripts and induced resistance.

Exposure of Arabidopsis thaliana to ozone results in the expression of a number of defense-related genes that are also induced during a hypersensitive response. A potential common link between the activation of defense gene expression during a hypersensitive response and by ozone treatment is the production of active oxygen species and the accumulation of hydrogen peroxide. Here we report that salicylic acid accumulation, which can be induced by hydrogen peroxide and is required for the expression of both a hypersensitive response and systemic acquired resistance, is also required for the induction of some, but not all, ozone-induced mRNAs examined. In addition, we show that ozone exposure triggers induced resistance of A. thaliana to infection with virulent phytopathogenic Pseudomonas syringae strains. Infection of transgenic plants expressing salicylate hydroxylase, which prevents the accumulation of salicylic acid, or npr1 mutant plants, which are defective in the expression of systemic acquired resistance at a step downstream of salicylic acid, demonstrated that the signaling pathway activated during ozone-induced resistance overlaps with the systemic acquired resistance activation pathway and is salicylic acid dependent. Interestingly, plants expressing salicylate hydroxylase exhibited increased sensitivity to ozone exposure. These results demonstrate that ozone activates at least two distinct signaling pathways, including a salicylic acid dependent pathway previously shown to be associated with the activation of pathogen defense reactions, and that this latter pathway also induces a protective response to ozone.

Elicitins from Phytophthora and basic resistance in tobacco.

Elicitins are a family of small proteins secreted by species of Phytophthora. They are thought to be major determinants of the resistance response of tobacco against these oomycetes, since purified elicitins, alone and at low concentrations, can induce vigorous defense responses in tobacco (i.e., hypersensitive cell death and resistance against subsequent pathogen attack), and in vitro elicitin production by Phytophthora isolates is strongly negatively correlated with their pathogenicity on tobacco plants. A number of elicitins have been purified and their amino acid sequences have been determined and found to be conserved. A three-dimensional structure for elicitin is emerging from nuclear magnetic resonance studies. Two structural classes, alpha and beta, are distinguished by their biological effects when applied to decapitated stems or petioles; the beta class causes more necrosis on leaves and provides better subsequent protection against pathogen attack. However, both these classes of elicitins will similarly cause necrosis when each is, instead, directly infiltrated into tobacco leaf panels. Effects of elicitins on tobacco cells include rapid electrolyte leakage, changes in protein phosphorylation and amounts of active oxygen species, and later production of ethylene and capsidiol. The sites of initial interaction with tobacco cells are unknown, but the interaction appears to induce general defense-related responses.

Three metal ions at the active site of the Tetrahymena group I ribozyme

Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3′-oxygen of the oligonucleotide substrate and the 3′- and 2′-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA.

Yeast chorismate mutase in the R state: Simulations of the active site

The isomerization of chorismate to prephenate by chorismate mutase in the biosynthetic pathway that forms Tyr and Phe involves C5—O (ether) bond cleavage and C1—C9 bond formation in a Claisen rearrangement. Development of negative charge on the ether oxygen, stabilized by Lys-168 and Glu-246, is inferred from the structure of a complex with a transition state analogue (TSA) and from the pH-rate profile of the enzyme and the E246Q mutant. These studies imply a protonated Glu-246 well above pH 7. Here, several 500-ps molecular dynamics simulations test the stability of enzyme–TSA complexes by using a solvated system with stochastic boundary conditions. The simulated systems are (i) protonated Glu-246 (stable), (ii) deprotonated Glu-246 (unstable), (iii) deprotonated Glu-246 plus one H2O between Glu-246 and the ether oxygen (unstable), (iv) the E246Q mutant (stable), and (v) addition of OH− between protonated Glu-246 and the ether oxygen. In (v), a local conformational change of Lys-168 displaced the OH− into the solvent region, suggesting a possible rate-determining step that precedes the catalytic step. In a 500-ps simulation of the enzyme complexed with the reactant chorismate or the product prephenate, no water molecule remained near the oxygen of the ligand. Calculations using the linearized Poisson–Boltzmann equation show that the effective pKa of Glu-246 is shifted from 5.8 to 8.1 as the negative charge on the ether oxygen of the TSA is changed from −0.56 electron to −0.9 electron. Altogether, these results support retention of a proton on Glu-246 to high pH and the absence of a water molecule in the catalytic steps.

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b595 was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the α-band of heme b595. The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b595 within a few ps, pointing to a direct interaction between hemes b595 and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b595 initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b595 provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b595 and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.

Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate. Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C--O bond. To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected. The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential. Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement. The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.

Location of the active site of allosteric chorismate mutase from Saccharomyces cerevisiae, and comments on the catalytic and regulatory mechanisms.

The active site of the allosteric chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5) from yeast Saccharomyces cerevisiae (YCM) was located by comparison with the mutase domain (ECM) of chorismate mutase/prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] (the P protein) from Escherichia coli. Active site domains of these two enzymes show very similar four-helix bundles, each of 94 residues which superimpose with a rms deviation of 1.06 A. Of the seven active site residues, four are conserved: the two arginines, which bind to the inhibitor's two carboxylates; the lysine, which binds to the ether oxygen; and the glutamate, which binds to the inhibitor's hydroxyl group in ECM and presumably in YCM. The other three residues in YCM (ECM) are Thr-242 (Ser-84), Asn-194 (Asp-48), and Glu-246 (Gln-88). This Glu-246, modeled close to the ether oxygen of chorismate in YCM, may function as a polarizing or ionizable group, which provides another facet to the catalytic mechanism.

A heme-protein-based oxygen-sensing mechanism controls the expression and suppression of multiple proteins in anoxia-tolerant turtle hepatocytes.

The O2 sensitivity of protein expression was assessed in hepatocytes from the western painted turtle. Anoxic cells consistently expressed proteins of 83.0, 70.4, 42.5, 35.3, and 16.1 kDa and suppressed proteins of 63.7, 48.2, 36.9, 29.5, and 17.7 kDa. Except for the 70.4-kDa protein, this pattern was absent during aerobic incubation with 2 mM NaCN, suggesting a specific requirement for O2. Aerobic incubation with Co2+ or Ni2+ increased expression of the 42.5-, 35.3-, and 16.1-kDa protein bands which was diminished with the heme synthesis inhibitor 4,6-dioxoheptanoic acid. Proteins suppressed in anoxia were also suppressed during aerobic incubation with Co2+ or Ni2+ but this was not relieved by 4,6-dioxoheptanoic acid. The anoxia- and Co2+/Ni2+-induced expression of the 42.5-, 35.3-, and 16.1-kDa protein bands was antagonized by 10% CO; however, with the exception of the 17.7-kDa protein, this was not found for any of the O2- or Co2+/Ni2+-suppressed proteins. Anoxia-induced proteins were compared with proteins expressed during heat shock. Heat shock proteins appeared at 90.2, 74.8, 63.4, 25, and 15.5 kDa and were of distinct molecular masses compared with the anoxia-induced proteins. These results suggest that O2-sensing mechanisms are active in the control of protein expression and suppression during anoxia and that, in the case of the 42.5-, 35.3-, 17.7-, and 16.1-kDa proteins, a conformational change in a ferro-heme protein is involved in transducing the O2 signal.