Generation of chemiluminescence during enzymatic isolation of protoplasts from leaves of Oryza sativa

The isolation of protoplasts from basal leaf segments of rice (Oryza sativa) was associated with a dramatic increase in chemiluminescence, which may be interpreted as a measure of cellular active oxygen. This chemiluminescence emission was sensitive to inhibition by catalase (EC 1.11.1.6), hydroquinone, ascorbic acid and azide but: not by superoxide dismutase (EC 1.15.1.1) or 1,4,diazabicyclo [2.2.2.] octane. Peroxidase (EC 1.11.1.7) activity increased in the protoplasting solution during tissue digestion. The incubation of isolated cell walls in protoplasting solution also resulted in a stimulation of chemiluminescence which was again associated with elevated levels of peroxidase and hydrogen peroxide. We conclude that protoplast isolation in rice is associated with an increase in active oxygen species as measured by the increased chemiluminescence emission which results, in part, from the action of a peroxidase/hydrogen peroxide system.