Involvement of the oxidative burst in phytoalexin accumulation and the hypersensitive reaction

The role of the oxidative burst, transient production of activated oxygen species such as H2O2 and superoxide (O2-) in elicitation of phytoalexins and the hypersensitive reaction (HR) was investigated in white clover (Trifolium repens L.) and tobacco (Nicotiana tabacum L.). H2O2 and O2- production was measured as chemiluminescence (CL) mediated by luminol, which was added to suspension-cultured white clover just before measurement in an out-of-coincidence mode scintillation counter. Maximum CL occurred between 10 and 20 min after addition of 0.4 X 10(8) colony-forming units/mL of incompatible Pseudomonas corrugata or 158 micromolar HgCl2. Autoclaved P. corrugata produced a slightly higher response. Elicitation of cells with 25 micromolar HgCl2 did not produce CL. Preincubation of plant cells in superoxide dismutase, which converts O2- to H2O2, for 2 min before addition of bacteria did not significantly increase maximum CL levels (P less than or equal to 0.05). Preincubation of plant cells with catalase for 2 min before addition of bacteria prevented the increase in CL, confirming that H2O2 is the substrate for the luminol reaction. Addition of live bacteria or HgCl2 (25 and 158 micromolar) to white clover increased levels of the phytoalexin medicarpin during a 24-h period, but addition of autoclaved bacteria did not elicit formation of medicarpin. Preincubation of plant cells with catalase, which quenched the bacteria-induced oxidative burst, did not decrease phytoalexin accumulation. Live bacteria infiltrated into Havana 44 tobacco leaf panels induced development of the HR, but autoclaved bacteria did not. Incubation of live bacteria with superoxide dismutase and catalase before infiltration into tobacco leaves did not interfere with development of the HR. Tobacco leaf panels infiltrated with up to 158 micromolar HgCl2 did not develop an HR. These results suggest that an oxidative burst consisting of and O2- does occur during these two plant defense responses.