The influence of photoacclimation on the effects of excessive photosynthetically active (PAR; 400-700 nm) and ultraviolet (UVR; 280-400 nm) radiation was assessed for the marine diatoms Thalassiosira weissflogii (Grunow) Fryxell and Hasle and Thalassiosira antarctica (Comber). Low and high PAR acclimated cultures were subjected to simulated surface irradiance (SSI) that mimicked irradiance around noon, including UVR. PSII efficiency, xanthophyll conversion, superoxide dismutase (SOD) activity, carbohydrate buildup, and lipid peroxidation were investigated after 30 min SSI and during 120 min recovery in low irradiance. Furthermore, viability loss was measured during 4 h SSI. Prior to SSI, the diadino-diatoxanthin pool was increased in high irradiance acclimated cells, compared with cells grown under low irradiance. Thirty-minutes SSI caused a pronounced decline in PSII efficiency. This coincided with de-epoxidation of diadinoxanthin in high irradiance acclimated cells, which was completely reversed during recovery in low irradiance. De-epoxidation was lower for low irradiance acclimated cells, whereas PSII efficiency and carbohydrate buildup were lower during the recovery phase. Furthermore, clear UVR effects on PSII efficiency were observed in low irradiance but not in high irradiance acclimated cells. Although 30 min SSI did not increase cellular SOD activity and lipid peroxidation, prolonged (4 h) SSI caused viability loss in low irradiance acclimated cells, which was enhanced by UVR. Therefore, PAR and UVR-induced PSII inactivation and viability loss were reduced by high irradiance-mediated changes in light harvesting and the xanthophyll pigments. In addition to photoacclimation-modulated differences, minor sensitivity differences were found between species.

The enzyme superoxide dismutase (SOD) holds a key position in the microalgal antioxidant network. The present research focused on oxidative stress responses in the Antarctic diatom Chaetoceros brevis F. Schütt during transition to excess (including ultraviolet radiation [UVR]) and limiting irradiance conditions. Over a 4 d period, cellular responses of thiobarbituric acid reactive substances (TBARS, a general oxidative stress indicator), SOD activity, photosynthetic and xanthophyll cycle pigments, PSII efficiency, and growth were determined. In addition, oxidative responses were measured during a daily cycle. Changing irradiance conditions significantly affected growth rates of C. brevis. PSII efficiency decreased significantly during periodic excess irradiance and increased under low irradiance conditions. Transition to excess irradiance increased the ratio of xanthophyll to light-harvesting pigments, whereas the opposite was observed for cultures transferred to low irradiance. This acclimation process was completed after 2 d in the new irradiance environment. SOD activity increased significantly after the first day regardless of the new irradiance environment but returned to preexposure values on the fourth day. We hypothesize that SOD activity may be temporarily elevated in C. brevis after irradiance shifts, thereby reducing oxidative stress when photoacclimation is in progress.

Antioxidant enzymes such as superoxide dismutase (SOD) play a key role in the removal of reactive oxygen species produced during visible and ultraviolet irradiance stress in microalgae and plants. However, little is known about the enzymatic antioxidative stress responses in ecologically important Antarctic marine microalgae. SOD in particular is difficult to analyze, possibly due to problems in obtaining sufficient quantities necessary for reliable and reproducible enzymatic assays. The aim of the present work was to create a sensitive, easy-to-use and reliable method for SOD determination in Antarctic microalgal material by comparing and optimizing existing protein extraction procedures and SOD assays in the marine Antarctic diatom Chaetoceros brevis. Optimization was achieved in cell disruption (sonication) and protein extraction procedures, extraction buffers, SOD assay methods (xanthine/xanthine oxidase and NBT/riboflavin photometric quantitative methods and native gel electrophoresis qualitative method) and the assay temperature. Protein extraction was optimal at low sonication amplitudes after a few pulses, irrespective of the type of buffer used. Extraction efficiency varied highly between the tested buffers; most protein was extracted in the presence of 1% of Triton X-100. SOD activity was best quantified using the NBT/riboflavin method in combination with a buffer containing potassium phosphate and Triton X-100. Moreover, the NBT/riboflavin method was demonstrated to be the most reliable and sensitive method at low temperatures (5 degrees C).

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