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 WITH ALPHA-D-MANNOSIDASE

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-radical-anion 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 alpha-D-mannosidase completely crossed out the stimulative activity to generate O-2-radical-anion. The presence of mannostatins, inhibitors of alpha-D-mannosidase, also inhibited O-2-radical-anion 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-radical-anion generation, protoplasts pretreated with IAA markedly activated the O-2-radical-anion generation capability when stimulated by the elicitor, The O-2-radical-anion generation activated by stimulating the elicitor was abruptly blocked by the addition of IAA.