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Studies of in vivo chlorophyll a (chl a ) fluorescence per cell, measured with a flow cytometer, and the pigment composition of cultured phytoplanktonic cells suggested that the xanthophyll cycle plays a role in the prevention of photoinhibitory damage to the photosynthetic apparatus. Two species were tested: Alexandrium excavatum (Dinophyceae) and Thalassiosira pseudonana (Bacillariophyceae). Cells were grown under 3 acclimation light conditions: continuous low light (50 @m01 photons m-' S-'), continuous high light (500 @m01 photons m-' S-') and fluctuating light (50, 1000, and 50 pm01 photons m-' S-'; cycle frequency = 1 h-'). The time course of in vivo fluorescence per cell signal showed rapid decreases in fluorescence when cells were suddenly submitted to a continuous high light intensity (1000 ~tmol photons m-' S-') for short periods of time (30 to 60 min). This decrease was ca 50 to 65 O/O of the initial fluorescence level. When the experimental light was turned off, fluorescence returned to a level generally close to its initial value. Concurrent with these rapid fluorescence decreases, the concentration of the epoxy-free pigment diatoxanthin increased at the expense of its parent compound, diadinoxanthin, upon exposure to high light. The process reversed when the light was turned off. Comparison among the 3 acclimation conditions suggests that fluorescence quenching is related to the concentration of diatoxanthin per unit chl a in the same way for h ~ g h lightand low-light-acclimated cells. In addition to the fast, reversible reaction in pigments, the highlight-acclimated cells maintain a positive concentration of diatoxanthin even when kept in darkness for 1 to 2 h. Low-light-acclimated cells lose their diatoxanthin fraction when put in the dark. Thus acclimation controls the long-term accumulation of the xanthophyll-cycle pigments in cells but it seems independent of the rapid reactions of the xanthophyll cycle that are related to fluorescence quenching. For both species, but particularly for A. excavatum, fluorescence quenching, resulting from light exposure and accompanied by xanthophyll pigment changes, suggests that non-radiative energy dissipation is taking place in the pigment bed, as in higher plants possessing an active xanthophyll cycle. This mechanism potentially allows algae to accomodate to rapid changes in the llght field and permits cells to build up longer-term mechanisms of photoprotection such as regulation of pigment content and composition. INTRODUCTION between the growth rate of the cell and the fluctuations of the external environment. These physiological Phytoplankton grow in very dynamic environments. responses to light fluctuations involve several mechanAs a result of hydrodynamic processes, they are isms, observed as changes in the fluorescence yield exposed to large fluctuations in light intensity, over a (Kiefer 1973, Heaney 1978, Vincent 1979, 1980, Harris wide range of time scales from seconds to hours (Dera 1980a), the size and density of photosynthetic units and & Olszewslu 1967, Legendre 1981, Demers et al. 1986). the composition and distribution of pigments (HaLldal The physiological responses of algae serve as a buffer 1970, Chan 1978, Prezelin & Sweeney 1978, Riper et al. O Inter-Research/Printed in Germany 0171-8630/91/0076/0185/$ 03.00 186 Mar Ecol. Prog. S 1979, Falkowslu & Owens 1980, Perry et al. 1981, Sukenik et al. 1987), and the chemical composition as well as the activity level of photosynthetic enzymes (Beardall & Morris 1976, Gibson 1978, Laws & Bannister 1980, Falkowski & Wirick 1981). These mechanisms do not all respond at the same rate (Harris 1980b). According to the hierarchical model of Harris (1980b), fluorescence is recognized to respond at very rapid time scales (10 to ca 1000 S), whereas pigment synthesis generally responds at time scales varying between hours and days. Sakshaug et al. (1987), using flow cytometry, demonstrated rapid (on the order of minutes) and reversible changes in the in vivo fluorescence per cell when diatom cells were transferred from low to high light intensities. They interpreted their data as a manifestation of a protective mechanism against strong light intensity. We show here that these rapid changes in the in vivo fluorescence per cell are associated with rapid and reversible changes in the concentration of particular carotenoid pigments taking part in the xanthophyll cycle. The cycle involves a light-driven reaction which transforms epoxy-containing xanthophylls (oxy-derivatives of carotenes) into epoxy-free pigments (Hager 1975). In higher plants and chlorophytes, the pigments involved in this cycle are violaxanthin, antheraxanthin and zeaxanthin (Hager 1975); the formation of zeaxanthin helps in the process of nonradiative dissipation of excess light energy (Demmlg et al. 1987). In phytoplankton, equivalent reactions are found with the pigments diadinoxanthin and diatoxanthin (Hager 1975, Cunningham & Schiff 1986). These latter pigments are present in the major groups of marine phytoplankton, diatoms, dinoflagellates and prymnesiophytes (Liaaen-Jensen 1978). We also show that the xanthophyll cycle is particularly active in 1 of the 2 species tested, Alexandrium excavatum, under light intensity conditions representative of the ocean euphotic zone, and that, in this species, photo-acclimation to low, high or fluctuating light regimes is reflected in the accumulation of the xanthophyll-cycle pigments and in the fluorescence variations upon exposure to excessive light. MATERIALS AND METHODS Alexandrium excavatum and Thalassiosira pseudonana, clone 3H (obtained from the Center for Culture of Marine Phytoplankton, Bigelow Laboratory, Boothbay Harbour, ME, USA) were grown at 18°C in batch cultures made from St. Lawrence estuary seawater (28 %O salinity) collected at the Maurice-Lamontagne Institute pumping system. Growth medium was enriched according to the 'f/2' recipe of Guillard & Ryther (1962). Cultures were grown under 3 light conditions (250 W Super Metal Halide arc lamp Optimarc TM, Duro-Test Electric Ltd, NJ, USA), referred to, in this text, as the acclimation conditions: continuous low light [photosynthetically active photon flux density (PPFD) = 50 pm01 photons m-' S-'], continuous high light (PPFD = 500 11mol photons m-' S-') and fluctuating light (light gradually increased within 30 min from 50 to 1000 pm01 photons mP2 S-' and then gradually decreased to 50 ~imol photons m-' S-' over the next 30 mini cycle frequency = 1 h-'). These conditions were chosen to resemble those for bottomand top-euphoticzone cells and cells growing in dynamic environments, respectively, with changes in light conditions shorter than generation times. After 3 to 5 d of acclimation to the light conditions, experiments were carried out in the laboratory. During the experiments, the cultures were submitted to two or three 30 to 60 min cycles of exposure to high light followed by darkness. The cells were pre-darkened for 30 min before beginning the experiments to homogenize starting conditions among the cultures and to maximize fluorescence yield. A 300 W projection bulb (Sylvania) was used as the experimental light; light intensity was generally 1000 ymol photons m-' S-' except for one experiment where hght was increased stepwise by changing voltage (no spectral change) from 50 to 1000 pm01 photons m-2 s-' in 100 min. Temperature was regulated using a water bath into which the culture vessels were plunged. Approximately every 5 min, per cell in vlvo fluorescence (excitation light: 424/44 nm band of a mercury lamp; collected emission: > 650 nm) and Coulter cell volume were determined using a FACS Analyzer flow cytometer (Beckton-Dickinson, Mountain View, California). At least 2000 counts of fluorescent particles were used to generate the data set. The acquisition time was ca 30 s and the first measurement (to) was done before the projection lamp was turned on. At the beginning of the experiment (i.e. just before turning the experimental light on) and during the stable phases of the fluorescence signal, 200 to 400 m1 of the experimental culture were filtered onto Whatman GF/F glass-fiber filters and the pigments were extracted immediately with successive 1 to 2 m1 volumes of 100% acetone, HPLC-grade, until a further 1 m1 extraction of the residues showed undetectable concentration of pigments. Pigment composition and concentration were then analyzed by high performance liquid chromatography (HPLC), using the method developed by Mantoura & Llewellyn (1983) A PerlunElmer Pecosphere RP 18, 3 pm column, was used for separation. An 8 min gradient, starting with an 80: 10: 10 solution of methano1:water:ion-pairing solution (Mantoura & Llewellyn 1983) and ending with an 80:20 solution of methanol:acetone, was followed by an isocratic hold until 13 min. A Spectra Physics 8800 Demers et al.: Rapid light-induced changes in phytoplankton 187 pump controlled flow rate through the column (1.5 m1 min-l). Detection was done at 440 nm using a Spectra Physics SP 8490 UV-VIS detector. Identification and quantification were achieved using external pigment standards, TLC-prepared from Alexandrjum excavatum or obtained commercially from Sigma (chlorophyll a , (3-carotene).

marine ecology. progress series

Rapid light-induced changes in cell fluorescence and in xanthophyll-cycle pigments of Alexandrium excavatum (Dinophyceae) and Thalassiosira pseudonana (Bacillario-phyceae): a photo-protection mechanism