Fig. 5. Nod factor-induced axillary root hair formation. Lateral root bases of methylene blue-stained roots, observed 24 h (A–C) or 5 days (D–L) after inoculation. To test whether ethylene and/or ROS (H₂O₂ and superoxide radicals) are involved in Nod factor-induced root hair formation, several agents that interfere with ethylene synthesis or perception, or ROS production were used. 1-Aminocyclopropane-1-carboxylate synthase, involved in the first step of ethylene synthesis, is inhibited by l-a -(2-aminoethoxyvinyl)-glycine or (aminooxy)acetic acid; the second enzyme, 1-aminocyclopropane-1-carboxylate oxidase, is inhibited by a -aminoisobutyric acid or Co²⁺; Ag⁺ ions and 2,5-norbornadiene interfere with perception of ethylene by its receptors (1–3). Ascorbic acid was applied as a H₂O₂ scavenger (4), and diphenyleneiodonium chloride and diethyldithio carbamic acid were used to inhibit oxidative burst. Diphenyleneiodonium chloride blocks the activity of the NADPH oxidase complex (5) that generates superoxide radicals, which are converted to H₂O₂ by a superoxide dismutase. Diphenyleneiodonium chloride also inhibits apoplastic peroxidases that may generate H₂O₂ and NO synthase (6) whereas diethyldithio carbamic acid blocks superoxide dismutases (7). (A) Control, mock-inoculated. (B) Inoculated with ORS571 and (C) with ORS571-V44, unable to produce Nod factors. (D) Treatment with 10^–9 M Nod factors. (E) Nod factor-induced root hairs showing branches and (F) corkscrews. (G) Ethylene treatment. (H) Inhibition of Nod factor-induced root hair formation by pretreatment with l-a -(2-aminoethoxyvinyl)-glycine. (I) Treatment with H₂O₂. (J) Inhibition of Nod factor-induced root hair formation by diethyldithio carbamic acid. (K) Ethylene treatment of roots pretreated with diphenyleneiodonium chloride. (L) H₂O₂ application on roots pretreated with l-a -(2-aminoethoxyvinyl)-glycine. [Bars = 1 mm (A–D and G–L), and 40 m m (E and F).]

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