Protection of photosystem II against UV-A and UV-B radiation in the cyanobacterium Plectonema boryanum: The role of growth temperature and growth irradiance

Photochemistry and Photobiology, Dec 2000 by Ivanov, Alexander G, Miskiewicz, Ewa, Clarke, Adrian K, Greenberg, Bruce M, Huner, Norman P A

Modulated Chl Jiuorescence. Chl a fluorescence of a dark adapted (30 min) control and UV-treated P. boryanum cell suspensions was measured using a PAM 101 Chl fluorescence measuring system (Heinz Walz GmbH, Effeltrich, Germany) equipped with an emitterdetector cuvette assembly unit ED-101US/D as described in Schreiber (16). Instantaneous (dark) chlorophyll fluorescence at open PSII centers (Fo) was excited by a nonactinic modulated measuring beam (650 nm, 0.12 wmol m-2 s-1) at 1,6 kHz in the dark and 100 kHz in the light. Maximum fluorescence at closed PSII centers (Fm) was induced by saturating white light pulses (800 ms, 2800 lmol m-2 s-1) provided by a Schott lamp (KL 1500, Schott Glaswerke, Mainz, Germany) and controlled from a Walz PAM 103 Trigger Control Unit. The actinic light corresponded to the growth irradiance of either 6 or 150 ol m-2 s^sup -1^ PPFD. All measurements were performed at the corresponding growth temperature. The photochemical (qP) and nonphotochemical (qN) fluorescence quenching parameters were evaluated under steady-state photosynthesis and calculated according to Schreiber (16).

Extraction of pigments. For pigment extraction, cyanobacterial cells were harvested by centrifugation and concentrated under vacuum to remove the remaining water. Cells were broken quickly using 0.1 mm zirconia/silica beads (3 X 20 s cycle) and a MINIBEADBEATER`' (Biospec Products, Inc., Bertlesville, OK). Carotenoids and Scy were extracted with 100% (vol/vol) acetone at 4'C and dim light. For extraction of OS-MAA, samples were extracted in 30% (vol/vol) methanol for 30 min at 50 deg C in darkness as described by Bohm et al. (12). Extracts were clarified by centrifugation. The supernatant was filtered through a 0.22 Jim syringe filter and samples were stored at -80 deg C until analysis.

Pigment analysis. Carotenoids were separated and quantified by reversed-phase high-performance liquid chromatography (HPLC) as described previously (17) with some modifications. The system contained a Beckman System Gold programmable solvent module 126, diode array detector module 168 (Beckman Instruments, San Ramon, CA), CSC-Spherisorb ODS-I reverse-phase column (5 Rm particle size, 25 X 0.46 cm ID) with an Upchurch Perisorb A guard column (both columns from Chromatographic Specialties Inc., Concord, Ontario, Canada). Samples were injected using a Beckman 210A sample injection valve with a 20 liL sample loop. Pigments were eluted isocratically for 6 min with a solvent system of acetonitrile:methanol:0.1 M Tris-HCl (pH 8.0), (72:8:3.5, vol/vol/vol), followed by a 2 min linear gradient to 100% methanol:hexane (75: 25, vol/vol) which continued isocratically for 4 min. Total run time was 12 min. Flow rate was 2 cm3 min-'. Absorbance was detected at 440 nm and peak areas were integrated by Beckman System Gold software. Retention times and response factors of Chl a and beta-carotene were determined by injection of known amounts of pure standards purchased from Sigma (St. Louis, MO). The retention times of zeaxanthin and a carotenoid, tentatively identified spectroscopically as myxoxanthophyll, were determined by using pigments purified by thin-layer chromatography as described in Diaz et al. (18). To determine the concentration of the putative myxoxanthophyll, the response factor was calculated using a specific extinction coefficient of 2160 Lg-t cm-tI at 478 nm (19).