PHOTOPHYSIOLOGICAL PROPERTIES OF THE MARINE PICOEUKARYOTE PICOCHLORUM RCC 237 (TREBOUXIOPHYCEAE, CHLOROPHYTA)1 Céline Dimier, Federico Corato, Giovanni Saviello and Christophe Brunet2 Stazione Zoologica "Anton Dohrn", Villa Comunale, 80121 Naples, Italy Running title: Photophysiology of Picochlorum 1Received Accepted 2Author for correspondence: email [email protected] 1 Key index words: Fluorescence quantum yield; Non-Photochemical Quenching of fluorescence; Photoacclimation; Picoplankton; Xanthophyll Cycle; Zeaxanthin. Abbreviations: Ax, antheraxanthin; chl a, chlorophyll a; DPS, de-epoxidation state [=(Ax+Zx)/(Vx+Ax+Zx)]; DTT, Dithiotreitol; ETR, electron transport rate; FALS, Forward Angle Light Scatter; Fq'/Fm', operating efficiency of PSII; Fv/Fm, maximum photochemical efficiency of PSII; HL, High Light; LHC, Light-Harvesting Complex; LL, Low Light; ML, Moderate Light; NF, Norflurazon; NPQ, Non-photochemical quenching; PAM, Pulse Amplitude Modulation; PFD, Photon flux density; PSII, Photosystem II; RALS, Right Angle Light Scatter; RLC, Rapid light curve; Vx, violaxanthin; Zx, zeaxanthin. 2 The photophysiological properties of strain RCC 237 belonging to the marine picoplanktonic Picochlorum (Henley et al.) genus were investigated under different photon flux densities (PFD, ranging from 40 to 400 µmol photons·m-2·s-1), mainly focusing on the development of the xanthophyll cycle and its relationship with the non-photochemical quenching of fluorescence (NPQ). The xanthophyll cycle functioning and its photoprotective role was investigated by applying a progressive increase of PFD and using Dithiotreitol and Norflurazon to block specific enzymatic reactions in order to deeply study the relationship between xanthophyll cycle and NPQ. These two processes were significantly related only during the gradually increasing light periods and not during stable light periods, where NPQ and zeaxanthin were decoupled. This result reveals that NPQ is a photoprotective process developed by algae only when cells are experiencing increasing PFD or in response to stressful light variations, for instance after a sudden light shift. Results showed that the photobiological properties of Picochlorum strain RCC 237 seem to be well related to the surface water characteristics, since it is able to maintain its photosynthetic characteristics under different PFDs and to quickly activate the xanthophyll cycle under high light. 3 INTRODUCTION In natural environments, phytoplankton cells continuously experience light variations due to the diel cycle and the vertical displacement of cells within the mixed layer due to hydrodynamics. Since light is crucial for survival and success of algae in aquatic ecosystems, they have to continuously photoacclimate, through biochemical, physiological and/or morphological variations. Photophysiological properties exhibited by algae seemed to be related to the environment where they grow (Stomp et al. 2004, Strzepek and Harrison 2004), converging toward the hypothesis that light is a relevant factor driving competition, exclusion and/or diversity in the algal community (Litchman and Klausmeir 2001, Floder and Burns 2005). An important trait of phytoplankton functional diversity is cell size that is in part responsible for the variety of biological and/or ecological behaviors with respect to the environment, e.g. sinking rate, nutrient consumption, light utilization and packaging effect (Fogg 1991, Raven 1998). Picoplankton (size < 3 µm) are known to be important contributors to autotroph communities in many coastal and oceanic ecosystems (e.g. Raven 1998, Not et al. 2005). The high level of taxonomic biodiversity in the picoeukaryotic fraction (Moon-van der Staay et al. 2001, Diez et al. 2004) reinforces the key role of this group in marine ecosystem structure and functioning. Nevertheless, very few data exist on their photophysiology (e.g. Wilhem et al. 1989; Timmermans et al. 2005), in contrast to prokaryotes (e.g. Bibby et al. 2003 and references therein, Stomp et al. 2004) and to the larger cell size eukaryotic community (e.g. Casper- Lindley and Bjorkman 1998, Lavaud et al. 2004). An important photoprotective mechanism active at short temporal scales is the dissipation of excess energy, through the non-photochemical quenching of chl a fluorescence that is catalyzed 4 by the xanthophyll cycle (Finazzi et al. 2006). This process involves enzymatic conversion from violaxanthin to antheraxanthin and zeaxanthin in plants and green algae (Gilmore et al. 1994, Masojidek et al. 1999) and from diadinoxanthin to diatoxanthin in chromophyte alge (Lavaud et al. 2002). Accumulation of zeaxanthin or diatoxanthin is triggered by the formation of a pH gradient across the thylakoid membrane (Demmig-Adams and Adams 2000). Much attention has been paid to the photoprotection mechanism in chromophyte algae (e.g Lavaud et al. 2004 and references therein, Harris et al. 2005), while few studies have dealt with marine green micro- algae (e.g. Gilmore and Yamamoto 2001, Garcia-Mendoza et al. 2002). The presumed minor contribution of green algae into the phytoplankton community, as well as the similarity of their xanthophyll cycle to the well-documented one found in terrestrial plants, (e.g. Havaux and Niyogi 1999, Demmig-Adams and Adams 2000) make the study of photoregulation in this group of low ecological interest. Recently, it has been shown that these algae are among the main picoeukaryote contributors in many ecosystems (Diez et al. 2004, Not et al. 2005), highlighting their capacity to adapt to different ecosystem characteristics and their probably relevant role in ecosystem structure and functioning. This study investigates the photophysiological properties of the strain RCC 237 belonging to the picoeukaryotic genus Picochlorum (Chlorophyta, Trebouxiophyceae). The short-term photoacclimative process is dissected in order to test the presence of an efficient acclimation to high photon flux density, through rapid xanthophyll cycle activation. Our assumption is based on the biological peculiarities of picoeukaryotes, e.g. low sinking rate and packaging effect (Raven 1998), that may lead this group to be more sensitive to light than large cells. This could be the case of the strain RCC 237 that was isolated from Mediterranean surface waters. Pigments, quenching of fluorescence and PSII efficiency were used as photoacclimative 5 indices during different light treatments and in presence or absence of enzymatic inhibitors linked to xanthophyll cycle activity. MATERIALS AND METHODS Algal model and culture conditions. The strain RCC 237 of Picochlorum (Chlorophyta, Trebouxiophyceae), isolated by Dr. D. Vaulot from the surface layer (20 m depth) of the Mediterranean Sea, was provided by the Roscoff Culture Collection (France; Vaulot et al. 2004). This small non-flagellated coccoid strain (diameter 1.5 µm) was cultivated non-axenically at 20° -2 -1 C under 40 µmol photons·m ·s (measured by a 4 π QSL-2100, Biospherical instruments INC, San Diego, USA) provided by one lamp (OSRAM, Decostar 51, 50 W; Munich, Germany) with a 12:12 light:dark photoperiod. Algae were grown in Keller medium (Keller et al. 1987) in 3-L glass cylinders and the cultures were continuously aerated and maintained in exponential phase by daily dilution of half of the culture with fresh medium during more than 10 days before the experiments. The pH and temperature were estimated daily with a HI- 9214-Stick pHmeter (Hanna Instruments, Woonsocket, USA) while cell concentration was controlled by flow cytometry (see below). Experimental design and sampling. The four experiments were conducted in triplicate aerated cultures at 20° C. Each culture flask was illuminated by one lamp (OSRAM, Decostar 51, 50 W; Munich, Germany), and the three lamps were mounted on a prototype device, called “PLIS” (“Progressive Light Increase System”) allowing gradual changes of PFD on the culture flasks, controlled by a BASIC program. A 3-L glass culture flask was maintained under the 6 initial conditions as an experimental control. During the four experiments, 30 mL of culture was sampled (see below) for HPLC-analyzed pigments, absorption spectrum on a filter by a spectrophotometer, cellular parameters by flow cytometry, quantum yield of fluorescence and rapid light curves (RLCs) by Phyto-Pam. Low light to high light experiment. The “PLIS” was setup as follows: after 30 minutes at 40 µmol photons·m-2·s-1, light was gradually increased to 200 µmol photons·m-2·s-1, over 60 min. -2 -1 For the following three hours, light remained constant at 200 µmol photons·m ·s and then -2 -1 increased to 400 µmol photons·m ·s over 30 min, remaining constant at this high light value for 2 hours. Frequency of sampling was high, especially during the two increasing light periods, with a total of 23 sampling points (Fig. 1a) High light to low light experiment. This experiment was conducted on cells acclimated at -2 -1 400 µmol photons·m ·s during 6 hours (i.e. the duration of the previous experiment) in order to study the relaxation of the photosynthetic apparatus under low light (40 µmol photons·m-2·s-1) during 3 hours. DTT addition experiment. After one hour of illumination at 40 µmol photons·m-2·s-1, the -1 DTT was injected in aqueous solution into the culture to a final concentration of 500 µmol·L (Lohr and Wilhelm 2001). Cells were incubated for 10 minutes with the inhibitor before the shift to high light (400 µmol photons·m-2·s-1). The control culture with no-addition of DTT followed the same light change as the three other flasks. 7 NF addition experiment. A methanolic solution of NF was injected into the culture flask -1 to a final concentration of 10 µmol·L methanol (2.5% v/v methanol; Garcia-Plazaola et al. 2002). Cells were incubated for 10 minutes with the inhibitor before being shifted to 400 µmol photons·m-2·s-1. It was previously verified that methanol (without NF) at this concentration has no effect on the algal photophysiology (data not shown). Pigment analysis. Samples of 10 mL were filtered onto GF/F glass-fiber filters (Whatman, Maidstone, UK) and immediately stored in liquid nitrogen until analysis. Using the method outlined in Casotti et al.