INTRODUCTION MATERIALS AND METHODS. Alexandrium tamarense (strain ATKR ) used in the present study was isolated in Kure Bay,

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1 Nitrate Availability for the Accumulation of Shinorine, Palythine, and Porphyra-334 (Mycosporine-like Amino Acids) in Dinoflagellate Alexandrium tamarense Nobuyuki Kobashi 1, Ai Murata 1, Hitomi Taira 2, and Taguchi, Satoru 1 1 Department of Environmental Engineering for Symbiosis, Faculty of Engineering, Soka University, 2 Soka Women College, Tangi Cho, Hachioji City, Tokyo , Japan. INTRODUCTION Dinoflagellates may be one of the most evolved groups among phytoplankton to adapt to the environmental stress. One of the most significant adaptation is to evolve a development of flagella (Goldstein 1992) to perform a diel vertical migration (Eppley et al. 1968). Some of them are exposed to a wide range of irradiance in a water column. They may be damaged by a high irradiance and ultraviolet radiation (UVR) at the surface layer although they have a tendency to avoid the top 0.5m of the water column (Halse 1950). Some of them may produce the UVR absorbing compounds such as mycosporine-like amino acids (MAAs) to protect themselves from UVR damage (Dunlap and Schick 1998). Since photosynthetic dinoflagellates migrate daily in a water column (Graham and Wilcox 2000) and nitrogen depletion in the surface layer may cause them to migrate downward from high light intensity (Heaney and Epply 1981), they take advantage to harvest nitrogen such as nitrate (NO 3 ) accumulated in a high concentration in a deep layer during the night (Cullen and Horrigan 1981, Tyler and Seliger 1981). Availability of nitrogen to the accumulation of MAAs in relation to photosynthesis has been studied on the different species of red alga Porphyra (Phodophyta) (Korbee et al. 2005). The most and least accumulated MAA under ammonium was porphyra-334 (P334) and shinorine (SHIN), respectively. However, the effect of NO 3 concentration has not been well known whether the NO 3 concentration control the accumulation of MAAs in unicellular algae. Some of major MAAs known for dinoflagellates such as Alexandrium tamarense are palythine (PAL), P334, and SHIN (Carreto et al. 2005). The response of individual MAA to NO 3 concentration was less studied in A. tamarense. In the present study, nitrate availability was hypothesized to affect on the concentrations of three MAAs in relation to chlorophyll a (Chl a) in dinoflagellate Alexandrium tamarense. Consequently this effect also induce the compositional change of MAAs. Adaptation to nitrate availability can explain the surviving strategy with nitrate-induced accumulation and composition of MAAs of photosynthetic dinoflagellate in a water column. MATERIALS AND METHODS Alexandrium tamarense (strain ATKR ) used in the present study was isolated in Kure Bay,

2 Japan. Cells were maintained at 17 С and 33PSU salinity under 350 µmol m -2 s -1 which was provided by cool white fluorescence tubes under a 12 hr light and 12 hr dark cycle (Hamasaki et al. 2001). Exponentially growing cells were inoculated into a modified f/2 medium without silicate using aged seawater (Guillard and Ryther 1962). All treatments with four different nitrate concentrations (12, 25, 50, and 100 µm) were conducted in triplicate in 2L sterilized screw-top polycarbonate bottles. At day 6 during the exponential growth phase, duplicate subsamples from each experimental bottles were taken for the measurement of concentration of Chl a, PAL, P334, and SHIN. Subsamples for Chl a analysis were collected on GF/F glass fiber filters. The subsamples was homogenized in 90% acetone into 15ml centrifuge tube on ice with an ultrasonic homogenizer and was extracted in the dark at -20 С for 24 h. An extracted subsumple was analyzed on a high performance liquid chromatography (HPLC) (Beckman, 168 Diode Array Detector, C18 reversed-phase Ultrasphere 3µm column) using solvent gradient system (Head and Horne 1993). Subsumples for the analysis of PAL, P334, and SHIN were collected on GF/A glass fiber filters. Cell materials on the filter were extracted using the method of Klisch and Häder (2000). An extracted subsample was analyzed on HPLC (Beckman, 168 Diode Array Detector) using a Shiseido SG 120 guard column, 5 µm column (250 mm 4.6 mm I.D.) (Adams and Shick 1996). Concentrations of MAAs were determined from peak areas detected at 334 nm using peak areas for particular MAA standards (Taira et al. 2004), and then the Chl a specific MAA concentration was calculated by dividing the MAA concentration with Chl a concentration. The reconstruction of MAA specific absorption coefficient was conducted with the Chl a concentration, the individual MAA concentration of the given sample, and the spectral distribution of the standard of the individual MAA (Taira et al. 2004). RESULTS Concentration of Chl a was a function of nitrate concentration as indicated by the equation: Chl a = 0.34 NO , r 2 = 0.97, p<0.01. It was increased by 4.6 fold from the lowest to the highest concentration of NO 3 (Table 1). The most abundant MAA was PAL while the least abundant MAA was P334 except for those at 100 µm NO 3 concentration. The most increase of 74 fold between the lowest and highest NO 3 concentration was observed for P334 while the least increase of 11 fold was observed for PAL (Table 1). All three MAAs indicated the increase of MAA concentration per Chl a concentration with NO 3 concentration to 50 µm and the decrease with higher NO 3 concentration (Fig.1). The most enhancement of 20 fold increase was observed for P334 while the least enhancement of 5 fold increase was observed for PAL between 12 and 50 µm NO 3 concentrations. As a sum of three MAAs, it indicated 7 fold increase between 12 and 50 µm NO 3 concentrations. Even at higher 100 µm NO 3 2

3 Table 1. Concentrations (nmol L -1 ) of Chl a, PAL, P334, and SHIN at four different concentrations of NO 3 (µm). Numbers in parenthesis indicate percentage calculated with the summed three MAAs. concentration, the accumulation of all MAAs in relative to Chl a concentration was not maintained at the similar level to 50 µm NO 3 concentration. Fig. 1. Relationship between NO 3 concentration and MAA concentration per Chl a concentration. The most dominant MAA was PAL with a descending order of SHIN and P334 at the lowest 12 µm NO 3 concentration (Fig. 2). The dominant MAA was SHIN with a descending order of P334 and PAL at the highest 100 µm NO 3 concentration. Maximum MAA specific absorption coefficient per Chl a concentration at 100 µm NO 3 concentration was 4.6 times higher than one at 12 µm NO 3 concentration. Although maximum MAA specific absorption coefficient per Chl a concentration was varied with NO 3 concentration, the wavelength at maximum MAA specific absorption coefficient per Chl a concentration did not show a significant shift, i.e., from 327 to nm. All three maximum MAA specific light absorption coefficients per Chl a concentration were enhanced by 10 fold and saturated at the approximate value of ~0.2 m 2 (µmol Chl a) -1 for all MAAs as the relative contents of MAA per Chl a concentration were increased to 12 µmol MAAs per µmol Chl a (Fig. 3). However there was some difference in their response. The most sensitive MAA to the relative concentration of MAA to Chl a concentration was P334 while the least sensitive MAA was PAL. 3

4 Fig. 2. MAA specific absorption coefficient per Chl a concentration at (A) 12 and (B) 100 µm NO 3 concentrations. Fig. 3. Relationship between Chl a specific MAA concentration and the maximum Chl a specific absorption coefficient of each MAA. DISCUSSION The present study may suggest that Alexandrium tamarense take advantage to migrate either to a deep layer of the water column, where NO 3 availability is usually high, in order to enhance their sun screen capability (Garcia-Pichel 1994) or to a surface layer, where light is available for photosynthesis, under a protection with multiple MAAs in their cells (Carreto et al. 2005) for the possible harmful UVR. Enhancement of both Chl a and MAAs with NO 3 concentration is also observed for macroalgae (e.g., Korbee et al. 2005) as long as light is available as indicated in the present study. The most accumulated MAA in the present study is also confirmed for red algae (Korbee et al. 2005). Nitrate is also assimilated by Alexandrium tamarense even in a dark condition (Murata 2008). However, the 4

5 saturation of UVR absorption of MAAs with accumulation of MAAs in the cell may suggest that package effect similar to one for chlorophyll pigments (Berner et al. 1989) is possibly occurred for MAAs. Despite of this unexpected phenomena, these observations may suggest that A. tamarense take advantage of high NO 3 availability in a deep layer of water column to enhance the concentration with changing the relative composition of MAAs as the surviving strategy even though light is not sufficiently available for their photosynthesis. The high accumulation and the shift of relative abundance of MAAs toward to longer wavelengths under high availability of NO 3, which were observed in the present study, can be considered as the surviving strategy for Alexandrium tamarense. The present study suggests that Alexandrium tamarense may prepare for a possibly stressful light environment prior to the emigration to the surface layer. ACKNOWLEDGEMENTS We thank Dr. K. Yabe for providing standard of MAAs. REFERENCE Adams, N. L. and M. J. Shick (1996). Photochem. Photobiol. 64, Berner, T., Z. Dubinsky, K. Wyman, and P. G. Fallows (1989). J. Phycol. 25, Carreto, J. I., M. O. Carignan and N. G. Montoya (2005). Mar. Biol. 146, Cullen, J. J. and S. G. Horrigan (1981). Mar. Biol. 62, Dunlap, W.C. and J. M. Schick (1998). J. Phycol. 34, Eppley, R. W., O. Holm-Hansen, and J. D. H. Strickland (1968). J. Phycol. 4, Garcia-Pichel, F. (1994). Limnol. Oceanogr. 39, Goldstein, S. F. (1992), In, Algal Cell Motility, (ed.) by M. Melkonian, Chapman & Hall, London, UK. pp Graham, L. E. and L. W. Wilcox (2000). Algae, Prentice Hall, HJ. Guillard, R. R. L. and J. H. Ryther (1962). Can. J. Microbiol. 8, Hamasaki, K., M. Horie, S. Tokimitsu, T. Toda and S. Taguchi (2001). J. Plankt. Res. 23, Halse, G. R. (1950). Oikos, 2, Head, E. J. H. and Horne, E. P. W. (1993). Deep-Sea Res II, 40, Heaney, S. I. and R. W. Eppley (1981). J. Plankt. Res. 3, Klish, M. and D. P. Häder (2000). J. Photochem. Photobiol. B: Biol. 55, Korbee, N., P. Huovinen, F. L. Figuero, J. Aguilera and U. Karsten (2005). Mar. Biol. 146, Murata, A. (2008). Ph. D. dissertation, Soka University, Tokyo. Taira, H., J. I. Goes, H. R. Gomes, K. Yabe, and S. Taguchi (2004). Plankt. Biol. Ecol. 51, Tyler, M. A. and H. H. Seliger (1981). Limnol. Oceanogr. 26,