Diel tuning of photosynthetic systems in ice algae at Saroma-ko Lagoon, Hokkaido, Japan

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Polar Science 3 (2009) 57e72 http://ees.elsevier.com/polar/

Diel tuning of photosynthetic systems in ice algae at Saroma-ko Lagoon, Hokkaido, Japan Shimpei Aikawa a, Hiroshi Hattori b, Yasushi Gomi c,1, Kentaro Watanabe c,d, Sakae Kudoh c,d, Yasuhiro Kashino a,*, Kazuhiko Satoh a a Department of Life Science, University of Hyogo, 3-2-1 Kohto, Kamigohri, Ako-gun, Hyogo 678-1297, Japan Department of Biological Science and Engineering, Tokai University, Minami-sawa, Minami-ku, Sapporo 005-8601, Japan c National Institute of Polar Research, Kaga 1-chome, Itabashi-ku, Tokyo 173-8515, Japan d Department of Polar Science, The Graduate University for Advanced Studies, Kaga 1-chome, Itabashi-ku, Tokyo 173-8515, Japan b

Received 28 August 2008; revised 23 October 2008; accepted 22 December 2008 Available online 4 February 2009

Abstract Ice algae are the major primary producers in seasonally ice-covered oceans during the cold season. Diurnal change in solar radiation is inevitable for ice algae, even beneath seasonal sea ice in lower-latitude regions. In this work, we focused on the photosynthetic response of ice algae under diurnally changing irradiance in Saroma-ko Lagoon, Japan. Photosynthetic properties were assessed by pulse-amplitude modulation (PAM) fluorometry. The species composition remained almost the same throughout the investigation. The maximum electron transport rate (rETRmax), which indicates the capacity of photosynthetic electron transport, increased from sunrise until around noon and decreased toward sunset, with no sign of the afternoon depression commonly observed in other photosynthetic organisms. The level of non-photochemical quenching, which indicates photoprotection activity by dissipating excess light energy via thermal processes, changed with diurnal variations in irradiance. The pigment composition appeared constant, except for xanthophyll cycle pigments, which changed irrespective of irradiance. These results indicate that ice algae tune their photosynthetic system harmonically to achieve efficient photosynthesis under diurnally changing irradiance, while avoiding damage to photosystems. This regulation system may be essential for productive photosynthesis in ice algae. Ó 2009 Elsevier B.V. and NIPR. All rights reserved. Keywords: Ice algae; Diurnal change; Photosynthesis; Non-photochemical quenching; Saroma-ko Lagoon

Abbreviations: Ek, light saturation index; ETR, electron transfer rate; F, measured fluorescence yield at a given time; Fm, maximal fluorescence yield of a dark-adapted sample; Fm0 , maximal fluorescence yield reached by a pulse of saturation light under actinic light; Fo, minimum fluorescence yield; Fv, variable fluorescence yield determined by Fm  Fo; Fv/Fm, maximum quantum yield of photosystem II; NPQ, non-photochemical quenching; NPQss, steady-state NPQ; PAR, photosynthetically active radiation; RLC, rapid light curve. * Corresponding author. Tel./fax: þ81 791 58 0185. E-mail address: [email protected] (Y. Kashino). 1 Present address: East China Sea Fisheries Oceanography Division, Seikai National Fisheries Research Institute, 1551-8 Taira-machi, Nagasaki 851-2213, Japan. 1873-9652/$ - see front matter Ó 2009 Elsevier B.V. and NIPR. All rights reserved. doi:10.1016/j.polar.2008.12.001

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S. Aikawa et al. / Polar Science 3 (2009) 57e72

1. Introduction Ice algal communities, dominated by diatom species (Garrison et al., 1986), are widely distributed throughout seasonally ice-covered polar seas in the Northern and Southern Hemispheres (Horner, 1976, 1985) and are important primary producers in highlatitude oceans (Nelson et al., 1995), and hence in the global oceans. Seasonal sea ice, which also develops in lower-latitude areas such as Saroma-ko Lagoon, Hokkaido, Japan, enables the development of ice algae underneath the sea ice (Hoshiai and Fukuchi, 1981). Saroma-ko (ca. 150 km2) is a seasonally ice-covered lagoon located on the northeast coast (ca. 44 N, 144 E) of Hokkaido. The lagoon is covered by sea ice (20e40 cm thickness) during winter, with covering of snow on the ice (Hoshiai and Fukuchi, 1981; Kishino, 1993). Under-ice irradiance is severely reduced because of reflection and absorption by the snow cover and the sea ice itself (Kishino, 1993). To grow beneath sea ice, ice algae possess the ability to efficiently utilize the low intensities of light received in this environment (Arrigo et al., 1993; Burkholder and Mandelli, 1965; Suzuki et al., 1995, 1997). In the ocean, the light environment fluctuates widely (Kirk, 1994); similarly, beneath sea ice the light environment fluctuates according to diurnal changes in the incident angle of the sun, weather conditions, and snow cover (Kishino, 1993). For example, it has been reported that during the period between 25 February and 9 March 1992, irradiance beneath sea ice in Saroma-ko Lagoon varied periodically between 0 and 35 mmol photons m2 s1 (Kudoh et al., 1997). During this period, the rate of carbon fixation in ice algae increased in proportion to irradiance, as the rate of carbon fixation in ice algae is limited by irradiance (Kudoh, 1993, 1995). Several studies have investigated Saroma-ko Lagoon and areas in Antarctica with the aim of assessing diurnal change in the electron transport rate in ice algae (Kudoh et al., 2003; McMinn et al., 2003). A previous study conducted in Saroma-ko Lagoon revealed that on 5 and 6 February 1998, the effective quantum yield of photosystem II, as measured under constant irradiance (165 mmol photons m2 s1), decreased continuously during the daytime, reached a minimum around sunset, and appeared to recover during the night, although this recovery was based on just a single measurement (Kudoh et al., 2003). A previous study conducted in Antarctica reported that the maximum relative electron transport rate (rETRmax) varied with diurnal change in irradiance; however, the

diurnal cycle in rETRmax varied between observations because of changing weather conditions (McMinn et al., 2003). In the natural environment, irradiance changes are not only gradual: they can be sudden in response to factors such as changes in the weather. Excess incident irradiance is harmful to photosynthetic organisms because it destroys photosystems (Aro et al., 1993; Demmig-Adams, 1990). To prevent such damage, photosynthetic organisms have developed a protection mechanism that dissipates excess light energy as heat (the thermal dissipation mechanism). This energydissipating process is detected as a decrease (quenching) of chlorophyll fluorescence, known as non-photochemical quenching (NPQ) (DemmigAdams, 1990; Krause and Weis, 1991; Olaizola and Yamamoto, 1994). This mechanism is regulated according to irradiance, meaning that efficient photosynthesis continues under existing light conditions. It is known that diatoms possess a xanthophyll cycle (i.e., the diadinoxanthin cycle) as a thermal dissipation mechanism (Kashino and Kudoh, 2003; Olaizola and Yamamoto, 1994; Olaizola et al., 1994). The diadinoxanthin cycle consists of two types of xanthophylls that are converted to each other: diadinoxanthin and diatoxanthin (Olaizola et al., 1994). In studies of cultured mesophilic diatoms, the total amount of diadinoxanthin plus diatoxanthin increased with increasing growth irradiance (Arsalane et al., 1994; Ban et al., 2006; Kashino and Kudoh, 2003). Ice algae also possess the diadinoxanthin cycle (Kudoh et al., 2003). Furthermore, in ice algae the total amount of diadinoxanthin plus diatoxanthin increases with increasing irradiance (Ikeya et al., 2000; Kudoh et al., 2003). The change in thermal dissipation activity that accompanies variations in diurnal irradiance has been investigated in situ in many photosynthetic organisms, including higher plants, macroalgae, and dinoflagellates (Huang et al., 2006; Ralph et al., 2002; Warner and Berry-Lowe, 2006), as well as reef corals (Gorbunov et al., 2001; Lesser and Gorbunov, 2001; Levy et al., 2004; Winters et al., 2003). The xanthophyll cycle in dinoflagellates is a diadinoxanthin cycle, similar to that in diatoms. The xanthophyll cycle in higher plants and green algae is a violaxanthin cycle that consists of violaxanthin, antheraxanthin, and zeaxanthin (Demmig-Adams, 1990). A common factor of the above studies is that the level of NPQ increased from sunrise until around noon, when it showed a maximum, decreasing thereafter toward sunset. This trend shows an inverse correlation

S. Aikawa et al. / Polar Science 3 (2009) 57e72

with the effective quantum yield of photosystem II. In contrast to the large number of studies of mesophilic photosynthetic organisms, few reports have considered diurnal change in NPQ in ice algae. Kudoh et al. (2003) studied NPQ under constant irradiance (165 mmol photons m2 s1) in ice algae at Saroma-ko Lagoon. The level of NPQ failed to show any strong correlation with diurnal change in the effective quantum yield of photosystem II or the total amount of diadinoxanthin plus diatoxanthin; thus, an additional study is warranted. In the present study, we focused on the photosynthetic response of ice algae to changes in irradiance, and examined diurnal changes in photosynthesis in ice algae at Saroma-ko Lagoon. The lagoon is connected to the Sea of Okhotsk by two narrow channels. Water exchange between the lagoon and the sea is controlled by tidal forces. During the winter season, most of the lagoon is covered by sea ice, up to 45 cm in thickness (McMinn and Hattori, 2007). The photosynthetic properties of the ice algae were assessed using pulseamplitude modulation (PAM) fluorometry by determining (i) rETRmax, which indicates the ability of photosynthesis; (ii) the light saturation index (Ek), which indicates the photoacclimation status of microalgae; (iii) the maximum quantum yield of photosystem II defined as the ratio of variable and maximum fluorescence yield (Fv/Fm), which indicates the utilization efficiency of light energy; and (iv) NPQ, which indicates the level of dissipation of excess light energy. We also examined diurnal change in the total amounts of diadinoxanthin and diatoxanthin, which plays an important role in the diadinoxanthin cycle. 2. Materials and methods 2.1. Collection of ice algae Ice algae were collected between 2 and 4 March 2005 at a station (44 070 1100 N, 143 570 2100 E) located w2 km offshore from the Saroma Research Center for Aquaculture, which is located on the eastern shore of Saroma-ko Lagoon (Figure 1). Ice cores of ca. 40 cm length were cut from sea ice using an ice auger (internal diameter ca. 75 mm) after removal of the accumulated snow layer (ca. 13 cm thickness). The lowermost section of the sea ice (3e5 cm thick) was brown in color, and was immediately sliced horizontally using an ice saw, all within a black plastic bag to avoid exposure to sunlight. The sliced sea ice was kept in pre-cooled, filtered seawater prepared by filtering seawater collected at Saroma-ko Lagoon on 2 March

59

2005 using a GF/F filter (Whatman; Maidstone, UK). During transportation to the laboratory in the Saroma Research Center for Aquaculture, the ice core samples were kept at near-freezing temperatures and in the dark in a thermos box. Transportation took less than 20 min. The collected ice cores were crushed in pre-cooled, filtered seawater in the dark to release the ice algae. Ice algae suspended in the seawater were directly used for measurements of fluorescence or filtrated for pigment determination. Aliquots were kept for the assessment of species composition after the addition of 9% glutaraldehyde. Fluorescence measurements were performed 30e35 min after ice-coring; the measurements took less than 60 min. The ice algae were kept at nearfreezing temperatures in a thermos box before measurement. Filtration of ice algae for pigment determination was performed 50e105 min after sample collection, once the collected sea ice had melted completely, taking approximately 20 min. Ambient irradiance on the surface of sea ice at the sampling station was measured using a LI-COR 2 quantum sensor with a LI-1400 data logger (LI-COR Bioscience; Lincoln, Nebraska). 2.2. Assessment of the species composition of ice algae Ice algae were identified by microscopic observation using an Eclipse E-400 (Nikon; Tokyo, Japan) equipped with a DP70-WPCXP CCD camera (Olympus; Tokyo, Japan). The species composition was determined by counting the cell numbers of each species until the total number exceeded 1000. Biovolume estimates for each cell were made based on linear dimensions using a geometric formula (Hillebrand et al., 1999). The average biovolume of each species was calculated using the values of 35 cells. The amount of organic carbon attributable to each algal species was estimated based on the average volume and number of each species, using a conversion factor (Strathmann, 1967). 2.3. Measurement of fluorescence We used a PAM chlorophyll fluorometer, PhytoPAM (Heinz Walz; Effeltrich, Germany), along with accompanying control and analysis software, PhytoWin, for measurements of photosynthetic fluorescence in ice algae. The concentration of ice algae, as assessed by Phyto-PAM, was 117e280 mg chlorophyll.L1, sufficient to assess the photosynthesis of the algae (Ban et al., 2006). The wavelengths of measuring light,

S. Aikawa et al. / Polar Science 3 (2009) 57e72

60 143o30’

143o45’

44o15’

144o00’

144o15’

km

Saroma-ko Lagoon 0

5

10

A

B 44o00’ 46oN

Sea of Okhotsk

44oN

Hokkaido

42oN

140oE

142oE

144oE

146oE

Fig. 1. Location maps of the study area showing the sampling station (A) and Abashiri Meteorological Observatory (B).

actinic light, and saturating pulse light were 470, 655, and 655 nm, respectively, and the intensity of the measuring light was 1 mmol photons m2 s1. A saturating pulse was supplied at the maximum level specified in Phyto-PAM (intensity level 10 and duration of 0.5 s). Before the start of each measurement, ice algae were kept in the dark for more than 30 min. Fv/Fm is defined as Fv =Fm ¼ ½Fm  Fo =Fm ;

ð1Þ

where Fm is the maximum fluorescence achieved by exposing the dark-adapted ice algae to a saturating illumination pulse, and Fo is the minimum fluorescence determined by exposing the ice algae to measuring light. The relative electron transport rate (rETR) under a given irradiance (photosynthetically active radiation; PAR) can be calculated by rETR ¼ PAR  ð½Fm0  F=Fm0 Þ ¼ PAR  ðDF=Fm0 Þ; ð2Þ

where F and Fm0 are the transient and maximum fluorescence levels under actinic light at a given time, respectively. rETR was determined by illuminating ice algae at 64, 90, 120, 180, or 350 mmol photons m2 s1 for 300 s, during which time a train of saturation light pulses was applied at 20 s intervals. DF/Fm0 at a stationary level measured at 285 s after the onset of actinic light was used to calculate rETR under the respective PAR. Light response curves were obtained by plotting rETR at a stationary level versus PAR, and fitted to the following equation (Eilers and Peeters, 1988; Zonneveld, 1998) using a Lavenberg Marquardt regression algorithm included in KaleidaGraph ver 4.0 for Macintosh (Synergy Software; Reading, Pennsylvania, USA):   ð3Þ rETR ¼ PAR= a  PAR2 þ b  PAR þ c ; where a, b, and c are the regression coefficients used to fit to the curve. Using these regression coefficients, the maximum value of rETR (rETRmax) and Ek were calculated as

S. Aikawa et al. / Polar Science 3 (2009) 57e72

h i1 rETRmax ¼ b þ 2ðacÞ0:5 and

ð4Þ

Ek ¼ rETRmax =a ¼ rETRmax  c;

ð5Þ

suctioning at 20e25 kPa. The filtrated ice algae on the filters were stored at e196  C in Dry Shipper SC4/3V (My Science; Tokyo, Japan). Photosynthetic pigments were extracted using 90% N,N-dimethylformamide (Furuya et al., 1998), and were analyzed by HPLC as described in Kashino and Kudoh (2003).

where a is the initial slope of the light response curve. To determine the parameter NPQ using Phyto-PAM, Fm and Fm0 were determined by a train of saturating flashes at 20 s intervals during actinic light illumination for 300 s and subsequent darkness for >80 s. The intensities of actinic light were 64, 120, 240, and 350 mmol photons m2 s1. The value of NPQ was calculated according to the following equation: NPQ ¼ ½Fm  Fm0 =Fm0 :

3. Results 3.1. Light and weather conditions The weather was calm and fine during the study period of 2e4 March 2005. The solar radiation on 2 March 2005, as observed at Abashiri Meteorological Observatory (44 000 5600 N, 144 170 0100 E; Figure 1), located 28 km east-southeast of the sampling station, increased after sunrise, reached a maximum around noon, and then decreased toward sunset (Figure 2, top panel). The course of change and intensity of sunlight were almost identical during each of the 3 days of our investigation. Similarly, PAR on the surface of sea ice at the sampling station showed a bell-shaped temporal trend on 4 March 2005 (Figure 2, bottom panel). The

ð6Þ

The steady-state value of NPQ (NPQss) was calculated by averaging the last three values during the illumination period. 2.4. Pigment determination Melted sea ice containing ice algae (85e120 mL) was filtered onto a GF/F glass fiber filter (Whatman) by

2.5

Solar radiation (MJ/m2)

61

2 March 2005 3 March 2005

2

4 March 2005

1.5 1 0.5

Photosynthetically active radiation (μmol photons·m-2·s-1)

0

1500 2 March 2005 3 March 2005 1000

4 March 2005

500

0 6:00

8:00

10:00

12:00

14:00

16:00

18:00

Time of day Fig. 2. Diurnal change in the environmental light conditions from 2 through 4 March 2005. Top panel, solar radiation measured at Abashiri Meteorological Observatory. Bottom panel, irradiance on the surface of sea ice at the sampling station.

S. Aikawa et al. / Polar Science 3 (2009) 57e72

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Table 1 Number of cells collected on 3 and 4 March 2005. Numbers in parentheses represent the proportion (%) of the total cells in the sample. Species

Number of Cell 3 March 2005

4 March 2005

9:00

11:00

14:00

17:00

9:00

11:00

14:00

17:00

Detonula confervacea Melosira arctica Thalassiosira spp. Navicula transitans Nitzschia frigida Achnanthes taeniata Odontella spp. Fragilariopsis spp. others

783 (74.5) 145 (13.8) 34 (3.24) 10 (0.951) 31 (2.95) 41 (3.90) 0 (0.00) 0 (0.00) 7 (0.666)

810 (76.8) 100 (9.48) 105 (9.95) 16 (1.52) 6 (0.569) 13 (1.23) 0 (0.00) 5 (0.474) 0 (0.00)

723 (68.1) 45 (4.24) 78 (7.34) 16 (1.51) 156 (14.7) 24 (2.26) 0 (0.00) 11 (1.04) 9 (0.847)

616 (59.8) 185 (18.0) 86 (8.35) 18 (1.75) 32 (3.11) 69 (6.70) 5 (0.485) 6 (0.583) 13 (1.26)

738 (65.1) 105 (9.26) 86 (7.58) 92 (8.11) 32 (2.82) 72 (6.35) 7 (0.617) 0 (0.00) 2 (0.176)

727 (65.9) 168 (15.2) 64 (5.80) 34 (3.08) 15 (1.36) 59 (5.34) 0 (0.00) 0 (0.00) 37 (3.35)

710 (66.6) 144 (13.5) 64 (6.00) 27 (2.53) 12 (1.13) 102 (9.57) 0 (0.00) 0 (0.00) 7 (0.657)

722 (69.8) 127 (12.3) 50 (4.83) 47 (4.54) 12 (1.16) 67 (6.47) 0 (0.00) 0 (0.00) 10 (0.966)

Total

1051

1055

1062

1030

1134

1104

1066

1035

diurnal change in PAR is comparable to that of solar radiation recorded at Abashiri Meteorological Observatory (Figure 2, top panel). 3.2. Species composition of ice algae Species composition was examined by counting cell numbers under microscopic observation (Table 1) to exclude the possibility of species-dependent differences in photosynthetic properties. All of the observed ice algal species belonged to the diatoms: no dinoflagellates were found. On 3 March 2005, the most dominant species in all four specimens collected at different time stamps was Detonula confervacea, although the abundance of this species as a percentage of the total algal cell numbers varied between 60% (17:00; all times are expressed in local time) and 77% (11:00). The second most dominant species in the specimens collected at 9:00 and 17:00 was Melosira arctica, and that in the samples collected at 11:00 and 14:00 was Thalassiosira spp. We also found small numbers of Navicula transitans, Nitzschia frigida, Achnanthes taeniata, Odontella spp., and Fragilariopsis spp. The three main species of centric diatoms made up more than 80% of the total observed ice algal cells. A similar species composition was observed for samples collected on 4 March 2005 (Table 1). Measurements of chlorophyll fluorescence depend on the amount of chlorophyll a present in ice algal cells, which is influenced by the amount of organic carbon (Miyai et al., 1988). In turn, the amount of organic carbon depends on the cell volume (Strathmann, 1967), which varies significantly between species (Hillebrand et al., 1999). Accordingly, we estimated the amount of organic carbon in each species (Table 2). The proportion of organic carbon

contributed by Thalassiosira spp. relative to the total organic carbon in the sample (Table 2) was slightly higher than the proportion of the number of cells of Thalassiosira spp. relative to the total number of cells in the sample (Table 1), reflecting its large cell volume. Reflecting this increase, the ratios of organic carbon contributed by D. confervacea and M. arctica showed slight reductions. Nonetheless, the majority (> w80%) of the organic carbon contained in the samples belonged to the three major species, with D. confervacea being the most abundant. The origin of chlorophyll a fluorescence could therefore be ascribed to these three major centric diatoms. 3.3. Light response curves Over the past decade, the protocol for PAM analysis has been improved to the degree that a light response curve could be measured within just 3 min (rapid light curve; RLC) (Ralph and Gademann, 2005; Schreiber et al., 1997; White and Critchley, 1999). An RLC is obtained by plotting rETR against irradiance (PAR) (Figure 3, dashed line). rETR is determined as the product of PAR and effective quantum yield (DF/Fm0 ) at each respective PAR. The effective quantum yield is automatically determined by the built-in protocol (Light Curve in Phyto-Win) at the end of each illumination step of 10e30 s, usually comprising 11 consecutive steps, and the irradiance increases stepwise as the protocol progresses. The condition that differs between the RLC and the well-known light response curve (generally referred to as the PeI curve, determined by gas exchange or 14C-fixation measurements) is the illumination period: the illumination period at each step for RLC is at most 30 s, insufficient to enable complete equilibrium.

334 76.7 77.0 66.6 6.63 25.8 0.000 0.000

17:00

(54.9) (14.5) (16.5) (6.39) (1.11) (6.57) (0.00) (0.00) 329 86.9 98.6 38.2 6.63 39.3 0.000 0.000

14:00

(54.7) (16.5) (16.0) (7.82) (1.35) (3.69) (0.00) (0.00) 336 101 98.6 48.1 8.29 22.7 0.000 0.000

11:00

(47.2) (8.76) (18.3) (18.0) (2.45) (3.83) (1.44) (0.00) 9:00

342 63.4 132 130 17.7 27.7 10.5 0.000 (46.6) (18.2) (21.6) (4.17) (2.89) (4.34) (1.22) (0.909) 285 112 132 25.5 17.7 26.6 7.47 5.56

14:00

335 27.2 120 22.7 86.3 9.24 0.000 10.2 375 60.4 162 22.7 3.31 5.00 0.000 4.64 362 87.5 52.4 14.2 17.1 15.8 0.000 0.000 Detonula confervacea Melosira arctica Thalassiosira spp. Navicula transitans Nitzschia frigida Achnanthes taeniata Odontella spp. Fragilariopsis spp.

9:00

(66.0) (15.9) (9.53) (2.58) (3.12) (2.87) (0.00) (0.00)

(59.3) (9.54) (25.6) (3.58) (0.523) (0.791) (0.00) (0.734) 11:00

(54.8) (4.45) (19.7) (3.71) (14.1) (1.51) (0.00) (1.67)

17:00

4 March 2005 3 March 2005

Organic carbon (ng) Species

Table 2 Amounts of organic carbon (ng) in ice algal cells collected on 3 and 4 March 2005. Numbers in parentheses represent the proportion (%) of the total organic carbon in the sample.

(56.9) (13.1) (13.1) (11.3) (11.3) (4.40) (0.00) (0.00)

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Figure 4 shows the kinetics of the effective quantum yield of photosystem II (DF/Fm0 ) during illumination at 90 mmol photons m2 s1 for 300 s followed by 200 s of darkness. The value of DF/Fm0 was far from an equilibrium at 10 or 30 s when the built-in protocol determines DF/Fm0 for RLC. In some experiments, measurements taken at a non-equilibrium status could be meaningful; however, we found that the kinetics differed markedly between ice algal samples (data not shown). Our intention was to compare the photosynthetic properties between ice algae collected at different times of the day; accordingly, we manipulated the method used in assessing the light response curve in order to compare the values of photosynthetic parameters obtained for ice algae collected at different times. Taking the kinetics of DF/Fm0 (see Figure 4) into account, we measured DF/Fm0 after 285 s illumination at 64, 90, 120, 180, or 350 mmol photons m2 s1. The light response curve obtained using this procedure was compared with RLC determined using a built-in program (Light Curve in Phyto-Win), using ice algae collected on 2 March 2005 (Figure 3). The values of rETR calculated using our method (solid line in Figure 3) are about twice as high as those of the RLC (dashed line) over the selected irradiance range. This disparity is derived from the different illumination times used to measure DF/Fm0 , as DF/Fm0 increases gradually and reaches a quasi-steady state after w100 s under actinic light (Figure 4). The value of DF/Fm0 is relatively small at 10 s, when the built-in program determines the value of DF/Fm0 for RLC. Use of the built-in algorithm does not allow for extension of the time duration beyond 30 s; accordingly, we manually measured the value of DF/Fm0 using the method described above. 3.4. Diurnal change in the maximum electron transport rate, light saturation index, and maximum quantum yield of photosystem II Using our method developed for the light response curve, we obtained rETRmax for ice algal samples collected at different times of the day. Figure 5 shows diurnal change in rETRmax for ice algae collected on 3 and 4 March 2005. The value of rETRmax increased from sunrise, reached a maximum around noon, and subsequently decreased toward sunset. This simple bell-shape kinetics is observed on both 3 and 4 March 2005, and the shape of the trend line is similar to the diurnal trend of sunlight (Figure 2).

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64 25

20

rETR

15

10

5

0 0

100

200

300

400

500

600

Irradiance (μmol photons·m-2·s-1) Fig. 3. Comparison of light response curves measured using the in-built method in Phyto-PAM (dashed line) and the method applied in this study (solid line). Ice algal samples were collected on 2 March 2005. In some data points, error bars in RLC obtained by three measurements are smaller than symbols.

by >80 s darkness. We used actinic light intensities of 64, 120, 240, or 350 mmol photons m2 s1. The kinetics shown in Figure 8 changed over the course of the day. In the morning (9:00) and evening (17:00), when the environmental irradiance was relatively low, the kinetics of NPQ were almost identical for all irradiances (Figure 8). At noon, the response to each irradiance level showed a marked change: the levels of NPQ became higher at higher irradiance levels. For example, the level of steady-state NPQ (NPQss) attained after 300 s illumination under 350 mmol photons m2 s1 was twice that under 120 mmol photons m2 s1. Despite this difference observed between ice algal samples collected at different times of the day, it

Figure 6 shows the diurnal change in Ek. The trace of Ek during the day is similar to that of rETRmax (Figure 5) on both days, with a maximum obtained at noon. Fv/Fm showed the opposite trend (i.e., a concave trend; Figure 7) on both days, with low values around midday followed by an increase during the evening to levels similar to those recorded during the morning. 3.5. Diurnal change in non-photochemical quenching The kinetics of NPQ were recorded using darkadapted (>35 min) ice algae collected at different times of the day on 3 March 2005 (Figure 8) and 4 March 2005 under 300 s actinic illumination followed 0.7

∇

Effective quantum yield ( F/Fm’)

0.6 0.5 0.4 0.3 0.2 0.1 0

OFF

ON 0

100

200

300

400

500

Time (sec) Fig. 4. Kinetics of effective quantum yield (DF/Fm0 ) in ice algae collected on 2 March 2005 under 90 mmol photons m2 s1. Arrows indicate the time point when the actinic light was turned on and off. Saturation light pulses were applied at 20 s intervals.

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65

30 25

rETRmax

20 15 10

3 March 2005 4 March 2005

5 0 8:00

10:00

12:00

14:00

16:00

18:00

Time of day Fig. 5. Diurnal change in rETRmax in ice algae collected on 3 March 2005 (solid line) and 4 March 2005 (dashed line). Ice algae were collected at 9:00, 11:00, 14:00, and 17:00 on 3 and 4 March 2005. rETRmax was obtained from the light response curves using DF/Fm0 during 300 s illumination at each irradiance, as shown in Figure 3 (solid line).

appears that 300 s is sufficient for NPQ to reach a steady-state, and NPQ decreased rapidly upon darkness after 300 s illumination, irrespective of the intensity of actinic light and the time when the sample was collected (Figure 8). The rapid decrease in NPQ upon darkness (Figure 8), as well as the reversible decrease in Fv/Fm (Figure 7), enables us to assume that photoinhibition made a negligible contribution to NPQ. Therefore, we can evaluate NPQ in all of the ice algal samples without any influence of photoinhibition. Similar NPQ patterns were also obtained on 4 March 2005 (data not shown). As described above, NPQ reached a steady-state after 300 s illumination (Figure 8). Figure 9 shows NPQss in ice

algal samples collected at different times of the day under different irradiance levels. For samples collected on 3 March 2005, the value of NPQss changed according to the time of day. Under the lowest irradiance tested (64 mmol photons m2 s1), NPQss showed a slight decrease around noon. Under 120 mmol photons m2 s1, NPQss was almost constant during the morning and around noon, but increased during the evening. Under 240 mmol photons m2 s1, NPQss on 3 March 2005 increased around noon. Under the highest irradiance tested (350 mmol photons m2 s1), NPQss showed an even more prominent increase around noon. The trends in NPQss on 4 March 2005 are similar to those on 3 March 2005 (Figure 9).

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Time of day Fig. 7. Diurnal change in Fv/Fm in ice algae collected on 3 March 2005 (solid line) and 4 March 2005 (dashed line). Ice algae were collected at the same time as those in Figure 5 and were adapted to the dark for 30e35 min.

3.6. Diurnal change in the composition of photosynthetic pigment Figure 10 shows the composition of photosynthetic pigment in ice algae collected at different times of the day on 3 and 4 March 2005. The proportions of chlorophyll a, fucoxanthin, chlorophyll c1 þ c2, and ßcarotene relative to the total amount of major photosynthetic pigments were 50%, 30%, 15%, and 0.5%, respectively. We observed no significant changes in the relative amounts of these pigments over the 2 days, except at 11:00 on 3 March 2005. In contrast, the amount of diadinoxanthin plus diatoxanthin (DD þ DT), which controls NPQ via the xanthophyll cycle in diatoms, increased with increasing integrated irradiance (Figure 10, bottom panel). 4. Discussion Because the measurement of photosynthesis using PAM fluorometry is rapid and reliable, it is currently applied to assess the photosynthesis of marine and fresh water phytoplankton and ice algae in situ (e.g., Ban et al., 2006; Kashino et al., 2002; Kudoh et al., 2003; Lesser and Gorbunov, 2001; McMinn et al., 2003; Warner and Berry-Lowe, 2006). Recently, a convenient built-in protocol was added to Phyto-Win to enable the user to obtain a light response curve (rapid light curve, RLC) for the assessment of the state

of photosynthetic acclimation (Ralph and Gademann, 2005; Schreiber et al., 1997; White and Critchley, 1999). This method is frequently applied to in situ measurements for phytoplankton, including psychrophilic phytoplankton (e.g., McMinn et al., 2005; Ralph et al., 1999). In the present study, we evaluated RLC by applying this built-in protocol to ice algal samples (Figure 3). We confirmed that the assessment of DF/Fm0 during the early part of the measurement (Figure 4) using the built-in protocol results in a small DF/Fm0 value, yielding a small rETR. Furthermore, the kinetics of DF/Fm0 varied between ice algal samples collected at different times of the day, especially during the early part of the measurement upon illumination. It is therefore inappropriate to compare photosynthetic parameters obtained by the built-in protocol between different ice algal samples. Accordingly, we determined a light response curve by measuring steady-state values of DF/Fm0 (Figure 3), which enabled us to compare photosynthetic parameters derived from the light response curves obtained for ice algae collected at different times of the day. Seroˆdio et al. (2006) reported in detail on the photosynthetic properties obtained from RLC compared with those obtained from steady-state light response curves. The authors observed a linear relationship between ETRmax values calculated using the two methods, although the slope of the regression

Fig. 8. Diurnal change in non-photochemical quenching (NPQ) in ice algae collected on 3 March 2005. NPQð½Fm  F0m =F0m Þ was recorded under actinic light (64 (circles), 120 (triangles), 240 (squares), and 350 (diamonds) mmol photons m2 s1) for 300 s followed by >80 s of darkness. The times of day when ice algae were collected are shown on the panels.

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Time of day Fig. 9. Diurnal change in steady-state levels of NPQ (NPQss) determined on 3 March 2005 (top panel) and 4 March 2005 (bottom panel). The intensities of actinic light were 64 (circles), 120 (triangles), 240 (squares), and 350 (diamonds) mmol photons m2 s1. The magnitude of NPQss was calculated by averaging the last three values of NPQ during illumination by actinic light (see Figure 8).

equations of ETRmax by RLC on ETRmax by light response curve varied between 0.64 and 0.97 for ambient irradiances of 261 and 1700 mmol photons m2 s1, respectively. One of their conclusions (in addition to the general usefulness of RLC) is that RLC may be particularly useful for obtaining a series of light response curves on the same samples without significantly altering their photoacclimation status. Therefore, it is appropriate to use the method applied in the present study to assess the photosynthetic properties of ice algae with different acclimation status beneath the sea ice. The method of PAM analysis was originally developed using higher plants and cyanobacteria (e.g., Genty et al., 1989; Krause and Weis, 1991; Schreiber et al., 1994). The method is now applied to phytoplankton, including chlorophyll c-containing algae; however, there exists insufficient physiological research on photosynthesis in such chlorophyll c-containing algae to implicitly apply the PAM method to

these algae. Nevertheless, the method is rapid and reliable, although we should remain aware of the issues outlined above. Using the method applied in this work for the light response curve, we obtained rETRmax values for ice algal samples collected at different times of the day (Figure 5). The value of rETRmax varied (Figure 5) with environmental irradiation (Figure 2). There exists a clear and reproducible agreement between the trends in rETRmax and environmental light intensity. It is naturally expected that rETR, and accordingly the rate of photosynthesis, would increase to some extent with increasing irradiance, as shown in the light response curve (Figure 3). An increase in net photosynthesis with increasing irradiance during the day is frequently reported for other photosynthetic organisms such as soybean (Huang et al., 2006; Huck et al., 1983) and coral symbionts (Lesser and Gorbunov, 2001; Levy et al., 2004), although an afternoon depression is observed in many cases.

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0.5 Chl a 0.4 Fucoxanthin

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Time of day Fig. 10. Diurnal change in pigment composition in ice algae collected on 3 March 2005 (solid lines) and 4 March 2005 (dashed lines). Ice algae were collected at the same times as those in Figure 5, and the extracted pigments were assessed by HPLC. The relative amount of pigment is expressed as a molar ratio against all the major pigments shown in the figure (chlorophyll a (Chl a; open circles), fucoxanthin (triangles), chlorophyll c1 þ c2 (Chl c; squares), diadinoxanthin þ diatoxanthin (DD þ DT; diamonds), ß-carotene (closed circles)). Top panel, all major photosynthetic pigments. Bottom panel, enlargement showing diadinoxanthin þ diatoxanthin and ß-carotene.

In this study, we want to emphasize that rETRmax, which corresponds to the capacity of photosynthesis, as does rETR (data not shown), changed according to diurnal changes in light conditions, thereby achieving efficient photosynthesis in ice algae. It is logical to expect that the photosynthetic system would be regulated according to the light environment, which changes diurnally. Similar diurnal changes have been reported for reef corals inhabiting tropical oceans (Gorbunov et al., 2001; Lesser and Gorbunov, 2001; Levy et al., 2004; Winters et al., 2003). The precise molecular mechanisms responsible for the observed diurnal changes in photosynthetic capacity remain to be determined in future research. Another interesting feature observed in this study is the diurnal change in Ek, which is commonly used as a parameter to characterize the photoacclimation status of microalgae (e.g., Behrenfeld et al., 2004,

2008; Falkowski, 1984). The pattern of Ek change (Figure 6) is in good agreement with that of rETRmax (Figure 5), and therefore, with that of environmental irradiance (Figure 2). Ek was larger at noon, when the level of solar radiation was at its highest. This finding is consistent with our previous finding that Ek values are higher in diatoms grown under higher irradiance (Ban et al., 2006), thereby clearly demonstrating that ice algae beneath sea ice delicately adjust its photoacclimation status according to diurnal changes in solar radiation, even over the course of a single day. In the present study, the kinetics of NPQ under illumination changed according to the time of day when the ice algae were collected. At around noon, NPQ increased more rapidly than in the morning and evening (Figure 8). In addition to this acceleration in NPQ increase, the magnitude of NPQss achieved after 300 s illumination

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was elevated under high irradiance (Figure 8). Reflecting these changes in NPQ, NPQss also changed with changing sunlight intensity (Figure 9). NPQss under low irradiance (64 and 120 mmol photons m2 s1) was lower at noon than during the morning and evening. This pattern is similar to (although more pronounced and reproducible than) that reported by Kudoh et al. (2003) for ice algae in Saroma-ko Lagoon, as assessed under 165 mmol photons m2 s1. At noon, the photosynthetic system is regulated to operate at a high rate of electron transport (Figure 5), thereby reducing the probability of photodamage under low irradiance beneath sea ice; in addition, the necessity of NPQ may be reduced under such low irradiance. In contrast, NPQss under high irradiance was higher at noon than during the morning and evening (Figure 9). It is possible that the energy-dissipating system is more actively regulated in a high-light environment. This proposal is supported by the data presented in Figure 8, in which the NPQ kinetics are almost identical irrespective of the given irradiance in the morning and evening, and show marked changes with irradiance at noon. It is widely accepted that NPQ is caused by the xanthophyll cycle performed by interconversion between diadinoxanthin and diatoxanthin when photoinhibition is negligible. The contribution of photoinhibition to NPQ can be excluded in the present measurements (Figure 8). The amounts of these pigments increased over the course of the day relative to the other major photosynthetic pigments, which remained relatively constant (Figure 10); however, changes in the amount of diadinoxanthin plus diatoxanthin do not appear to be solely related to changes in NPQ and NPQss. It is possible that other effective energy-dissipating system(s) (in addition to the xanthophyll cycle) operate in ice algae, which inhabit low-light environments beneath sea ice. In any case, energy-dissipating systems are possibly regulated to more effectively protect photosystems under high-light intensities (at noon) than under low-light intensities (in the morning and evening). A high capacity to dissipate excess light energy under extremely high irradiances (e.g., the value of 350 mmol photons m2 s1 used in this study) compared with the irradiance experienced in the field might assure effective protection mechanisms when the sea ice melts in spring. Fv/Fm is known to be an indicator of photoinhibition (Rintama¨ki et al., 1995) which is an irreversible process. In the present study, Fv/Fm values obtained for ice algae were low around noon, but recovered in the evening (Figure 7). The same trend was also reported by Kudoh et al. (2003) for ice

algae in the Saroma-ko Lagoon, meaning that we have confirmed the reproducibility of this earlier finding. The decrease in Fv/Fm observed around noon could be attributed to the downregulation of photosystem II, which is largely influenced by NPQ, as described above. Similar diurnal changes have been reported for other mesophilic photosynthetic organisms such as soybean (Huang et al., 2006) and symbiotic dinoflagellates from reef-building corals (Hoegh-Guldberg and Jones, 1999). The course of change observed in rETRmax (Figure 5) is the opposite of that observed in Fv/Fm (Figure 7), which initially appears incongruous. The entire chain of photosynthesis is regulated by the light-dependent modulation of RubisCO activity (Geiger and Servaites, 1994). The value of rETRmax is obtained from DF/Fm0 measured at quasi-steadystate condition (Figures 3 and 4), while Fv/Fm is measured under a dark-adapted status, independent of the CalvineBenson cycle. The lower rETRmax observed in the morning and evening is possibly related to the lower activity of RubisCO, while the higher rETRmax observed at noon may be the result of light acclimation to achieve higher RubisCO activity. An afternoon depression in photosynthesis activity is commonly observed in phytoplankton, macroalgae, and higher plants (Huang et al., 2006; Huck et al., 1983; Lesser and Gorbunov, 2001; Levy et al., 2004; Quick et al., 2006; Ralph et al., 1999). Although the intervals between measurements (sampling times) were somewhat long in the present work, we did not observe any depression in rETRmax (Figure 5). In higher plants, stomatal closure could be a factor explaining the observed afternoon depression (Quick et al., 2006), but this is clearly not a factor in the case of phytoplankton. Nonetheless, an afternoon depression has been observed in coral symbionts (Levy et al., 2004), even for rETRmax (Ralph et al., 1999). There must exist a factor that causes an afternoon depression in phytoplankton, such as photorespiration (Osmond, 1981). Ice algae may possess more efficient mechanisms to avoid the afternoon depression, such as high NPQ activity, as discussed above. 5. Concluding remarks The availability of light beneath the sea ice changes according to the movement of the sun in low-latitude areas such as Saroma-ko Lagoon, as is evident from Figure 2. We observed that ice algae in Saroma-ko Lagoon changed their photosynthetic capacity

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according to diurnal change in their light environment over two consecutive days. The energy-dissipating performance of the algae was also adjusted diurnally. We can reasonably assume that ice algae are able to harmonically tune their total photosynthetic system to conduct effective photosynthesis in a naturally changing environment. The absence of an afternoon depression in photosynthesis in ice algae is a remarkable adaptation to achieve high productivity beneath the sea ice. Acknowledgments We are grateful to Prof. Satoru Taguchi and Dr. Sandric C.Y. Leong of Soka University and students at Tokai University and Soka University for their technical support at Saroma-ko Lagoon, and Abashiri Meteorological Observatory for providing solar radiation data. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) (18054028 for YK & 17053024 for KS), and grants from the Global COE (Centers of Excellence) Program from MEXT (KS), NIPR (10-27 for YK), the Hyogo Science and Technology Association (20I107 for YK), and a Sasakawa Scientific Research Grant from the Japan Science Society (18-225 for SA). The map in Figure 1 was created using Online Map Creation (http:// www.aquarius.geomar.de/omc/), which is supported by the Generic Mapping Tools (GMT). References Aro, E.M., Virgin, I., Andersson, B., 1993. Photoinhibition of photosystem II. Inactivation, protein damage and turnover. Biochim. Biophys. Acta 1143, 113e134. Arrigo, K.R., Robinson, D.H., Sullivan, C.W., 1993. A high resolution study of the platelet ice ecosystem in McMurdo Sound, Antarctica: photosynthetic and bio-optical characteristics of a dense microalgal bloom. Mar. Ecol. Prog. Ser. 98, 173e185. Arsalane, W., Rousseau, B., Duval, J.-C., 1994. Influence of the pool size of the xanthophyll cycle on the effects of light stress in a diatom: competition between photoprotection and photoinhibition. Photochem. Photobiol. 60, 237e243. Ban, A., Aikawa, S., Hattori, H., Sasaki, H., Sampei, M., Kudoh, S., Fukuchi, M., Satoh, K., Kashino, Y., 2006. Comparative analysis of photosynthetic properties in ice algae and phytoplankton inhabiting Franklin Bay, the Canadian Arctic, with those in mesophilic diatoms during CASES 03e04. Polar Biosci. 19, 11e28. Behrenfeld, M.J., Halsey, K.H., Milligan, A.J., 2008. Evolved physiological responses of phytoplankton to their integrated growth environment. Phil. Trans. R. Soc. B 363, 2687e2703. Behrenfeld, M.J., Prasil, O., Babin, M., Bruyant, F., 2004. In search a physiological basis for covariations in light-limited and lightsaturated photosynthesis. J. Phycol. 40, 4e25.

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Leegood, R.C., Stitt, M., 2006. The effect of water stress on photosynthetic carbon metabolism in four species grown under field conditions. Plant Cell Environ. 15, 25e35. Ralph, P.J., Gademann, R., 2005. Rapid Light Curves: a powerful tool for the assessment of photosynthetic activity. Aquat. Bot. 82, 222e237. Ralph, P.J., Gademann, R., Larkum, A.W.D., Schreiber, U., 1999. In situ underwater measurements of photosynthetic activity of coral zooxanthellae and other reef-dwelling dinoflagellate endosymbionts. Mar. Ecol. Prog. Ser. 180, 139e147. Ralph, P.J., Polk, S., Moore, K.A., Orth, R.J., Smith Jr., W.A., 2002. Operation of the xanthophyll cycle in the seagrass Zostera marina in response to variable irradiance. J. Exp. Mar. Biol. Ecol. 271, 189e207. Rintama¨ki, E., Salo, R., Lehtonen, E., Aro, E.-M., 1995. Regulation of D1-protein degradation during photoinhibition of photosystem II in vivo: phosphorylation of the D1 protein in various plant groups. Planta 195, 379e386. Schreiber, U., Bilger, W., Neubauer, C., 1994. Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. Ecol. Studies 100, 49e70. Schreiber, U., Gademann, R., Ralph, P.J., Larkum, A.W.D., 1997. Assessment of photosynthetic performance of Prochloron in Lissoclinum patella in hospite by chlorophyll fluorescence measurements. Plant Cell Physiol. 38, 945e951. Seroˆdio, J., Vieira, S., Cruz, S., Coelho, H., 2006. Rapid lightresponse curves of chlorophyll fluorescence in microalgae: relationship to steady-state light curves and non-photochemical quenching in benthic diatom-dominated assemblages. Photosynth. Res. 90, 29e43. Strathmann, R.R., 1967. Estimating the organic carbon content of phytoplankton from cell volume or plasma volume. Limnol. Oceanogr. 12, 411e441. Suzuki, Y., Kudoh, S., Takahashi, M., 1995. Photosynthetic characteristics of ice algae with special emphases on temperature and light conditions. Proc. NIPR Symp. Polar Biol. 8, 57e58. Suzuki, Y., Kudoh, S., Takahashi, M., 1997. Photosynthetic and respiratory characteristics of an Arctic ice algal community living in low light and low temperature conditions. J. Mar. Syst. 11, 111e121. Warner, M.E., Berry-Lowe, S., 2006. Differential xanthophyll cycling and photochemical activity in symbiotic dinoflagellates in multiple locations of three species of Caribbean coral. J. Exp. Mar. Biol. Ecol. 339, 86e95. White, A.J., Critchley, C., 1999. Rapid light curves: A new fluorescence method to assess the state of the photosynthetic apparatus. Photosynth. Res. 59, 63e72. Winters, G., Loya, Y., Ro¨ttgers, R., Beer, S., 2003. Photoinhibition in shallow-water colonies of the coral Stylophora pistillata as measured in situ. Limnol. Oceanogr. 48, 1388e1393. Zonneveld, C., 1998. Photoinhibition as affected by photoacclimation in phytoplankton: a model approach. J. Theor. Biol. 193, 115e123.