IN-VIVO MEASUREMENT OF ACTIVE OXYGEN PRODUCTION IN THE BROWN ALGA FUCUS-EVANESCENS USING 2',7'-DICHLOROHYDROFLUORESCEIN DIACETATE

Intracellular production of active oxygen in the brown alga Fucus evan escens C. Ag: was studied by measuring the capacity for in vivo conver sion of 2',7'-dichlorohydrofluorescein diacetate (DCFH-DA) to the fluo rescent dye 2',7'-dichlorofluorescein (DCF), both in emersed and immer sed seaweeds. Algae were incubated in seawater containing DCFH-DA unde r a range of conditions, and it was also possible to load algae with D CFH-DA and then follow subsequent DCF production. in emersed tissue. D CF formation was linear for at leash 2 h in both darkness and light, w ith the rate of formation increasing with the light level. DCF formati on was temperature dependent. It also increased when algae were treate d with H2O2 or methyl viologen (paraquat), which disrupts photosystem 1 electron transport and increases O-2(-) production. Exogenous catala se reduced in vivo DCF production, presumably by lowering cellular con centrations of H2O2. Hydrogen peroxide was released into the seawater by illuminated algae resulting in external dye conversion to DCF. Howe ver, this does not interfere with in vivo measurement of DCF by loaded , washed algae because DCF leakage appeared to be negligible. Internal DCF did not affect photosynthetic oxygen production relative to untre ated controls. Overall, our data suggest that DCFH-DA is a potentially very useful probe for studying active oxygen metabolism in seaweeds s ubjected to environmental stresses.