Localization of lipoxygenase activity on the oil bodies and in protoplasts using a novel fluorescence imaging method

Lipoxygenase (linoleate:oxygen oxidoreductase; EC 1.13.11.12; LOX) catalyzes oxygenation of polyenoic fatty acids, which precedes the degradation of storage lipids during seed germination in sunflower. In the present work, it has been confirmed that 2′,7′-dichlorodihydrofluorescein diacetate (H 2DCFDA) produces fluorescence in presence of lipid hydroperoxides (LOX reaction products). This work provides new information on spatial localization of transiently enhanced LOX activity in protoplasts from 5 d old seedling cotyledons of sunflower ( Helianthus annuus L. cv. Morden) by exploiting H 2DCFDA as a probe for fluorescence detection from LOX activity sites. Use of LOX inhibitors [nordihydroguaiaretic acid (NDGA) and propyl gallate (PG)] confirms oil bodies as LOX activity sites. Oil body surface has been shown to possess LOX activity in 5 d old seedling cotyledons. ► LOX localization by fluorescence microscopy and CLSM. ► H 2DCFDA oxidation by LOX. ► Oxidation product (2′,7′-dichlorofluorescein) is fluorescent. ► 2′,7′-dichlorofluorescein formation inhibited by LOX inhibitors. ► Enhanced LOX activity detected in seedling protoplasts and oil bodies.