In vitro activation of NADPH-dependent O 2- generating system in a plasma membrane-rich fraction of potato tuber tissues by treatment with an elicitor from Phytophthora infestans or with digitonin

Abstract

NADPH-dependent O 2 - generating activity of a plasma membrane-rich (PM) fraction from potato tuber tissue was measured in vitro using polarographic measurements of superoxide dismutase (SOD) and catalase-sensitive O 2 consumption and/or spectrophotometric measurements of SOD-sensitive cytochrome c (acetylated) reduction. The enhanced NADPH-dependent O 2 - generating activity was found predominantly in the plasma membrane fraction isolated from the potato tuber slices which had been inoculated with an incompatible race of Phytophthora infestans or treated with hypal wall elicitor (HWC-elicitor) from the fungus. No enhanced activity was detected in plasma membrane fractions prepared from tuber tissues inoculated with a compatible race or left untreated. The enhanced activity in the isolated PM fraction was not dependent on Ca 2+. In an in vitro system containing the plasma membrane fraction and a soluble cytosol protein fraction from control tuber tissue slices, CaCl 2, MgCl 2 and ATP, the NADPH-dependent O 2 - generating reaction was slowly or rapidly activated by treatment with HWC-elicitor or digitonin, respectively. In the in vitro system, from which one of either plasma membrane fraction, soluble cytosol protein, Ca 2+ or ATP was removed, no in vitro activation of NADPH-dependent O 2 - generating reaction occurred after treatment with HWC-elicitor or digitonin. These results indicate that a novel O 2 - generating NADPH oxidace may be activated in the plasma membrane of potato cells by inoculation with an incompatible race of P. infestans or treatment with the fungal elicitor and that Ca 2+, cytosolic protein components and ATP may be involved in the activation process.