A method for the assessment of light-induced oxidative stress in embryos of fucoid algae via confocal laserscan microscopy

A method was developed for measurement of active oxygen production in embry onic stages of the brown seaweed Fucus spiralis. using the label CM-DCFH-DA . Active oxygen species convert the label into the green fluorescent CM-DCF (exc/em 488/530 nm) that is detected via confocal laserscan microscopy and quantitative image analysis. Loading of the label did not harm the embryos ; loading efficiency was age-independent, and the esterases needed for conv ersion to CM-DCFH were not inhibited by the effective UV dose (2 W m(-2)) a pplied in the experiments. After correction for daily valiation of the lase r power, and calibration with DCF standard solutions, this automated analys is of confocal images rendered active oxygen concentrations in fucoid embry os (muM DCF). An experiment was designed for the assessment of active oxyge n production following irradiance stress in the light-sensitive embryos. Di m-light-acclimated, 1-, 2- and 3-day-old embryos were transferred for 60 mi n to conditions (4 pi -irradiance 300 mu mol photons m(-2) s(-1)), optional ly without UV radiation, including UVA, or including UVA plus UVB. PSII yie ld measurements (PAM fluorometer) were carried out in order to assess the d egree of photoinhibition under these light conditions. The imposed light st ress initially caused a rapid decrease of the PSII yields (Phi (P)). With i ncreasing embryo age, minimum Phi (P) values attained under light stress re mained higher. Consequently, electron transport rates (ETR) would increase with embryo age, i.e., with the development of their photosynthetic apparat us. Active oxygen production increased with ETR, and when WE was included, relatively greater amounts of active oxygen were produced. A slow, second-p hase decrease of Phi (P) under light stress that was proportional to active oxygen production indicated that some photooxidative damage was caused, in particular during UVB exposure. Recovery from light stress was a rapid pro cess in the absence of UVB; in such cases Phi (P) was almost restored to th e initial values within 60 min. The relative state of recovery of Phi (P) w as correlated with both the effective UV dose and active oxygen production rate (DCF). Recovery was slowest in embryos exposed for 60 min to an experi mental UVB dose, which was representative of a situation at low tide, on a sunny day. The results suggest that active oxygen may cause an in situ inhi bition of growth of the earliest life stages of F. spiralis.