Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91 phox and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91 phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca 2þ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern-induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac-Rboh interaction was supported by further studies using in vitro pulldown assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca 2þ concentration may regulate Rac-Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac-Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca 2þ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.

In the previous paper [Kawano et al. (2000a) Plant Cell Physiol. 41: 1251], we demonstrated that addition of phenylethylamine (PEA) and benzylamine can induce an immediate and transient burst of active oxygen species (AOS) in tobacco suspension culture. Detected AOS include H2O2, superoxide anion and hydroxyl radicals. Use of several inhibitors suggested the presence of monoamine oxidase-like H2O2-generating activity in the cellular soluble fraction. It was also suggested that peroxidase(s) or copper amine oxidase(s) are involved in the extracellular superoxide production as a consequence of H2O2 production. Since more than 85% of the PEA-dependent AOS generating activity was localized in the extracellular space (extracellular fluid + cell wall), extracellularly secreted enzymes, probably peroxidases, may largely contribute to the oxidative burst induced by PEA. The PEA-induced AOS generation was also observed in the horseradish peroxidase (HRP) reaction mixture, supporting the hypothesis that peroxidases catalyze the oxidation of PEA leading to AOS generation. In addition to AOS production, we observed that PEA induced an increase in monodehydroascorbate radicals (MDA) in the cell suspension culture and in HRP reaction mixture using electron spin resonance spectroscopy and the newly invented MDA reductase-coupled method. Here we report that MDA production is an indicator of peroxidase-mediated generation of PEA radical species in tobacco suspension culture.

Aromatic monoamines may contribute to both chemical and physical protection of plants. Addition of phenylethylamine (PEA) and benzylamine to tobacco suspension culture (cell line BY-2) induced a very rapid and transient generation of two active oxygen species (AOS), H2O2 and superoxide anion, both detected with chemiluminescence. Electron spin resonance spectroscopy revealed that hydroxy radicals are also produced. With laser-scanning confocal microscopy, fluorescence spectroscopy and microplate fluorescence reading, intracellular H2O2 production was detected using dichlorofluorescin diacetate as a fluorescent probe. Following AOS production, cytosolic Ca2+ concentration ([Ca2+]c) of the tobacco cells, monitored with luminescence of transgenic aequorin, increased and attained to a peak level 12 s after PEA addition. The PEA-induced increase in [Ca2+]c was inhibited by a Ca2+ chelator, Ca2+ antagonists and AOS scavengers, suggesting that PEA-induced AOS triggered a Ca2+ influx across the plasma membrane.

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;We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR. The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein. The predicted 24-kDa protein was named DtNDK1. It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides. The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity. Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus. The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity. Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.

Due to the high prevalence of vascular obstructive diseases, discovering potent, safe, and affordable fibrinolytic agents is of importance. There is particular interest concerning the use of functional foods that have a fibrinolytic activity, such as natto, a Japanese fermented soy-based product made with Bacillus subtilis (natto) strain BEST195. We recently isolated another bacterial strain from natto commercialized in Indonesia, B. subtilis G8, which has proven to exert fibrinolytic activity. Herein, a further characterization of B. subtilis G8 was assessed through a comparison with commercialized nattokinase, the major fibrinolytic enzyme of B. subtilis , by utilizing various in vitro fibrinolytic assays, namely whole blood clot lysis, euglobulin clot lysis, the fibrin plate method, and zymography. Both nattokinase and B. subtilis G8 were able to dissolve both whole blood and euglobulin clots. Furthermore, both nattokinase and B. subtilis G8 were able to lyse blood clots, presumably due to their ability to directly lyse fibrin. Finally, a crude extract of B. subtilis G8 displayed six zymogram bands of approximately 42.0, 35.5, 30.8, 26.7, 20.0, and 13.7 kDa, with the strongest activity observed at 20.0 kDa. This indicates that B. subtilis G8 contained several fibrinolytic enzymes, which might have comprised nattokinase and other fibrinolytic enzymes. In summary, we demonstrated that a crude extract of B. subtilis G8 has potent fibrinolytic activity and that the activity was mediated by various fibrinolytic enzymes.