Superoxide production as a stress response of wounded root cells: ESR spin-trap and acceptor methods

The Superoxide generation in the plant root cells in response to wound stress has been studied by the electron spin resonance (ESR) spin-trap and epinephrine-adrenochrome acceptor methods. Tiron readily oxidized by O₂^⋅− to a rather stable free radical semiquinone was used as a spin trap. Wound stress was shown to activate the root cells inducing an increase in Superoxide production. The largest amount of Superoxide was registered in the early stage after excision of the roots from the seedlings (over 1–2 h). Further incubation of the roots for 5 and 6 h resulted in the lowering of the Superoxide level. Electron donors NADH and NADPH, nonpenetrating via plasma membrane, caused the amplification of Superoxide production in root cells, whereas oxidized nucleotide NAD did not affect the O₂^⋅− synthesis. Treatment of the roots with a water-soluble analog of naphthoquinone, vitamin K₃, led to the total disappearance of the ESR signal from Tiron semiquinone and suppression of epinephrine-adrenochrome conversion. An excessive amount of calcium ions in the root cells induced a powerful increase in the Superoxide release and disturbed the adaptation. The data obtained give us a further indication that the redox system of plasma membrane, comprising a flavoprotein, is likely involved in the production of Superoxide occurring in the response to wound stress in root cells.