Superoxide Anion Generation in Rice Blade Protoplasts with the Blast Fungus Proteoglucomannan Elicitor as Determined by CLA-phenyl Luminescence and Its Suppression by Treating the Elicitor withα-D-Mannosidase

Abstract

A chemiluminescence probe specific for the superoxide anion, 2-methyl-6-phenyl-3, 7-dihydroimidazo-[1, 2-a]pyrazine-3-one, was used to directly demonstrate the O·^-__2 generation in rice blade protoplasts with the purified blast-fungus elicitor (proteoglucomannan). A hyperbolic relationship between the relative photon emission and the amount of elicitor applied, implied the presence of a putative receptor for the elicitor. Pretreatment of the elicitor with α-D-mannosidase completely crossed out the stimulative activity to generate O·^-__2. The presence of mannostatins, inhibitors of α-D-mannosidase, also inhibited O·^-__2 generation. Plasma membrane turbulence was observed by the quenching of fluorescence from a membrane turbulence reporter, bis-(3-propyl-5-oxo-isoxazol-4-yl) pentamethine oxonol, when the protoplasts were stimulated with the elicitor. Although treating rice protoplasts with IAA alone did not stimulate O·^-__2 generaion, protoplasts pretreated with IAA markedly activated the O·^-__2 generation capability when stimulated by the elicitor. The O·^-__2 generation activated by stimulating the elicitor was abruptly blocked by the addition of IAA.