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.