Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

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.

Aromatic monoamine-induced immediate oxidative burst leading to an increase in cytosolic Ca2+ concentration 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.

Phenylethylamine-induced generation of reactive oxygen species and ascorbate free radicals in tobacco suspension culture: mechanism for oxidative burst mediating Ca2+ influx.

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.

Biopotency of fetal bovine serum, and insulin and insulin-like growth factors I and II in enhancing whole-body protein synthesis of chicken embryos cultured in vitro.

Whole-body protein synthesis in chicken embryos was measured to examine the biopotency of fetal bovine serum, bovine insulin, recombinant human insulin, and recombinant human insulin-like growth factors (IGF) I and II. In all experiments chicken embryos at 7 days of incubation age were used and cultured in a synthetic serum-free medium in the presence or absence of the testing substances with a rotatory whole-embryo culture apparatus for up to 30 min. Whole-body protein synthesis was measured from the time course of specific radioactivities of free and protein-bound L-[4-3H]phenylalanine. In addition, the effect of bovine insulin on phenylalanine extraction rate from the culture medium was investigated. Supplementing the medium with fetal bovine serum and human IGF-I significantly enhanced whole-body protein synthesis. In contrast, human IGF-II, bovine insulin, or human insulin did not affect whole-body protein synthesis, although bovine insulin increased phenylalanine extraction rate from the culture medium in a proportional fashion as the dose increased. Therefore, from these results it was concluded that IGF-I, but not IGF-II, might be one of the anabolic factors in fetal bovine serum for stimulating whole-body protein synthesis, and that insulin may play a role in stimulating amino acid transport, but it may not be directly involved in protein synthesis of chicken embryos.

Protein synthesis measured in the whole body of chick embryos cultured in vitro.

1. Whole body protein synthesis was measured in chick embryos cultured in vitro. On day 7 of incubation chick embryos were cultured for 60 min in synthetic serum-free medium containing 4-[3H]phenylalanine. Specific radioactivities in free and protein-bound phenylalanine in the whole embryo were measured, starting 2 min after commencement of the culture process. 2. The values for fractional synthesis rate (FSR) estimated in vitro at 20, 30, 45 and 60 min during the embryo culture agreed well, ranging from 35 to 40%/d, suggesting that the method would serve as a useful model for studying the effect of growth promoters in chick embryos. 3. Bovine insulin in the synthetic medium did not affect FSR of protein in chick embryos cultured in vitro.