Dynamics of short-term acclimation to UV radiation in marine diatoms

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Journal of Photochemistry and Photobiology B: Biology 89 (2007) 1–8 www.elsevier.com/locate/jphotobiol

Dynamics of short-term acclimation to UV radiation in marine diatoms Manuela Fouqueray *, Jean-Luc Mouget, Annick Morant-Manceau, Ge´rard Tremblin Laboratoire de Physiologie et Biochimie Ve´ge´tales, EA 2663, Faculte´ des Sciences et Techniques, Universite´ du Maine, Avenue Olivier Messiaen, 72085 Le Mans cedex 9, France Received 11 May 2007; received in revised form 23 July 2007; accepted 29 July 2007 Available online 2 August 2007

Abstract In order to investigate the dynamics of the acclimation of marine diatoms to ultraviolet radiation (UVR), Amphora coffeaeformis, Odontella aurita and Skeletonema costatum were exposed for 5 h per day to a combination of UVA and UVB (UVBR/UVAR ratio 4.5%) with a total UVR daily dose of 110 kJ m2, which is equivalent to that observed in the natural environment. This treatment was applied in the middle of the photoperiod and was repeated on five successive days. During the UVR treatment, chlorophyll fluorescence parameters were monitored, damage and repair constants were calculated from effective quantum yield values (/PSII), and rapid light curves (electron transport rate versus irradiance curves using short light steps of different intensity) were plotted to determine the maximum relative electron transport rate (rETRmax) and maximum light use efficiency (a). In all species the growth rate was lower than control from day 1–3, but increased thereafter, except for S. costatum. The cellular chlorophyll a content increased significantly with repeated daily exposure to UVR for A. coffeaeformis only. In all species, the fluorescence parameters (Fm, the maximum fluorescence level measured in the dark, /PSII, rETRmax and a) decreased during UVR exposure, in contrast to F0 (the minimum fluorescence level measured in the dark). The response to UVR stress was species-specific. S. costatum was very sensitive, and failed to survive for more than three days, whereas A. coffeaeformis and O. aurita were able to acclimate to UVR stress. These two species used different strategies. In A. coffeaeformis, the repair constant was lower than the damage constant, but /PSII values returned to baseline values at the beginning of each experimental day, indicating that an effective active recovery process occurred after stress. In O. aurita, the repair processes took place during the stress, and could account for the UVR tolerance of this species.  2007 Elsevier B.V. All rights reserved. Keywords: Chlorophyll fluorescence; Diatoms; Exposure response curve; Rapid light curve; UVR acclimation

1. Introduction Phytoplankton in the oceans contributes to more than 30% of the total primary production [1], and inhibiting this productivity will affect marine ecosystems and the economic exploitation of these resources. Ultraviolet radiation (UVR) has deleterious effect on phytoplankton organisms: it reduces their growth rate and photosynthetic activity, damages their DNA, and degrades photosynthetic pigments and proteins (especially the D1 protein of the PSII complex) [2–4]. The sensitivity of phytoplankton species *

Corresponding author. Tel.: +33 2 43 83 27 01; fax: +33 2 43 83 39 17. E-mail address: [email protected] (M. Fouqueray). 1011-1344/$ - see front matter  2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2007.07.004

to UVR is highly variable due to species-specific responses [5]. Microalgae have active repair systems to deal with this harmful radiation, and these have been studied in the laboratory [6–8]. These studies have shown that exposure response curves (ERCs) to UVR usually have three phases: an initial time-dependent phase (usually linear), a transition phase, and a time-independent phase [7]. ERCs have been used to explain the inhibition of carbon fixation by UVR in the diatom Thalassiosira pseudonana [7], and the changes in chlorophyll fluorescence parameters observed in the green alga Dunaliella tertiolecta exposed to UVR [8]. Fast acquisition techniques designed to monitor photosynthetic performance continuously are required to investigate the short-term response of algae to UVR. Chlorophyll a fluorescence from PSII is a useful method that provides

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rapid, sensitive and non-intrusive measurements of photosynthesis. An increasing number of studies [8–11] show that fluorimetric methods can be used to develop ERCs and RLCs. Thus, Heraud and Beardall [8] show that modulated fluorimetry can satisfactorily be used in place of oximetry or labelled carbon fixation to produce ERCs. Measurements of the fluorescence parameters (maximum quantum yield and effective quantum yield) during UVR exposure allow to quantify damage and repair constants in different species in order to compare their sensitivity and capacity to acclimate to UVR. The RCLs are a technique recently applied to the determination of electron transport rate (ETR) versus irradiance curves. They use short light steps (tens of seconds) of different intensity, and have been applied to seagrasses, microphytobenthos and diatoms [9–11]. The RLCs do not achieve steady-state conditions at each light level, and have been used to limit the effect of light acclimation [12]. In a natural environment, such as the open ocean, changes in the position of microalgae in the water column provide protection against UVR. In artificial aquaculture ecosystems, such as oyster-ponds, the shallow depth and low water turbidity offer no protection against UV radiation. Rech et al. [13] show that some of the diatoms, commonly found in oyster-ponds where they constitute a major food source [14,15], are able to acclimate to long-term UVR, but some highly sensitive species fail to do so. The aim of this study was to characterize the dynamics of short-term acclimation to UVR in three marine diatoms: one species has been classified as tolerant (Amphora coffeaeformis) and another as sensitive (Skeletonema costatum) [13]. The UVR acclimation of Odontella aurita is still unknown and it was included in this study, because this species is of considerable economic importance. It is cultured at an industrial scale for the production of eicosapentaenoic acid [16]. Algae were exposed to UVR stress for five days in order to determine the time required for UVR resistance mechanisms to develop at the photosynthetic level. The ERCs were used to determine the damage and repair constants, and RLCs were used to estimate the maximum relative electron transport rate (rETRmax) and the maximum light use efficiency (a) during the repeated exposures to UVR. 2. Materials and methods 2.1. Culture conditions The Bacillariophyceae A. coffeaeformis (Agardh) Ku¨tzing, O. aurita (Lyngbye) Agardh and S. costatum (Greville) Cleve were obtained from the microalgal culture collection of the ‘‘Laboratoire de Biologie Marine’’ of the University of Nantes (France). Stock and control cultures were grown in semi-continuous mode in artificial seawater (Harrison et al. [17] modified according to Perkins et al. [11]) in 500 mL Erlenmeyer flasks containing 250 mL of culture in a thermo-regulated culture chamber (15 ± 1 C). A. coffeaeformis was grown in the same medium,

but with the concentration of CaCl2 reduced from 9.4 to 0.25 mM to limit cell adhesion [18]. Photosynthetically active radiation (PAR) was provided for 14 h d1 from a high-intensity discharge lamp (Osram HQI-BT, 400W, Munich, Germany). Irradiance (100 lmol photons m2 s1) was measured at the bottom of the Erlenmeyer, where the algae settled, using a Walz US-SQS 4p waterproof light probe (Walz GmBH, Effeltrich, Germany) connected to a Li-Cor 189 quantameter (Li-Cor Instruments, Lincoln, USA). Before the UVR treatment, cell density was estimated using Nageotte (for O. aurita) or Neubauer haematocytometers, and the specific growth rate (d1) was calculated as described in Rech et al. [13], between days 1 and 3, and between days 3 and 5. The chlorophyll a (Chl a) content was determined after UVR treatment (for exposed and control samples), using 30 mL aliquots of culture filtered over Whatman GF/C. Pigments were extracted in dimethylformamide, and total Chl a was determined by spectrophotometry [19]. 2.2. UVR treatment All the experiments (control and UVR) were performed in a custom-made frame described by Rech et al. [13], in which cells received PAR from below and UVR from above. The UVR source consisted of a Vilber-Lourmat VL315 BLB (Torcy, France), fitted with three Sylvania F15W-BLB T8 (SLI, Raunheim, Germany) auto-filtrating fluorescent tubes to provide the UVAR (peak emission at 365 nm), and a Vilber-Lourmat VL115M fitted with two Vilber-Lourmat 15 M tubes, and an additional filter to provide the UVBR (peak emission at 315 nm). UVR irradiance was measured using an HD 9021 UVR radiometer (DeltaOhm, Padova, Italy) fitted with an LP 9021 UVAR probe (spectrum range: 315–400 nm) for UVAR, and an LP 9021 UVBR probe (spectrum range: 280–315 nm) for UVBR. The UVR irradiance was adjusted by inserting neutral density filters between the UVR sources and the culture flasks. Before the exposure to UVR, algae in the exponential growth phase were transferred into 500 mL glass beakers covered with a thin polyethylene film to prevent evaporation of the medium. The polyethylene film was replaced before each UVR exposure to prevent any modification of the UVR treatment, and it did not significantly alter the UVR sprectrum [13]. The cultures were not diluted during the UVR treatment. Before the first exposure, when algae settled to the bottom of the flask, part of the supernatant medium was renewed. The light treatment consisted of 100 lmol photons m2 s1 of PAR, 5.84 W m2 of UVAR and 0.28 W m2 of UVBR (0.61 W m2 UVAR and 0.23 W m2 UVBR, weighted according to Cullen et al. [20]). The UVBR/UVAR ratio (4.5%), and the total daily dose of UVR (110 kJ m2) were equivalent to those measured in June at the same latitude as the oyster-ponds (4801 0 N, 0009 0 E). The UVR treatment was applied for 5 h in the middle of the photoperiod and was repeated on five consecutive days.

M. Fouqueray et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 1–8

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2.3. Fluorescence measurement

2.6. Statistical analyses

On day 1 and on alternate days thereafter, the in-vivo Chl a fluorescence was measured on 2 mL algal samples (maintained at 15 C) with a FMS 1 modulated fluorimeter (Hansatech Ltd., Cambridge, UK), modified for use at low Chl a concentrations [20]. Fluorescence measurements were made every 30 or 60 min during the UVR treatment (5 h) for the ERCs and RLCs respectively. Samples were darkadapted for 5 min, to limit their dark recovery (there was no significant difference after 5, 15 or 30 min of dark-adaptation, data not shown) before measuring the fluorescence parameters. Following dark-adaptation, the minimum fluorescence level (F0) was measured, and the maximum fluorescence level (Fm) was obtained using a saturating flash (4300 lmol photons m2 s1 during 0.7 s). These parameters made it possible to calculate the variable fluorescence Fv = Fm  F0, and the maximum quantum efficiency of PSII (Fv/Fm). The effective quantum yield efficiency of PSII (/PSII) was obtained using a different sample, in order to avoid any influence of the light exposure history. Fs (the steady-state fluorescence) was measured after 20 s of exposure to actinic light similar to the growth irradiance (100 lmol photons m2 s1), and F 0m (the maximum fluorescence level of the light acclimated sample) was acquired after a saturating flash. The /PSII parameter was calculated from /PSII ¼ ðF 0m  F s Þ=F 0m .

All measurements were made on three or four replicates (from different cultures), and the results were expressed as means and standard errors. Significant variations were determined by analysis of variance (ANOVA), and differences between treatments were tested with Tukey’s test, using Sigma Stat 3.1. All conclusions are based on a level of significance of at least 5% (P < 0.05).

2.4. Determination of the damage and repair constants Fluorescence parameters were used to achieve ERCs. Values of /PSII were fitted (using SigmaPlot 9.0) to the equation of Neale [22]: P/Pinitial = (r/(k + r)) + (k/ (k + r))e(k+r)t, where P is /PSII, Pinitial is the value of /PSII at the start of the treatment, t is the time in minutes, k (min1) is a constant related to damage to the photosynthetic performance and r (min1) is a constant related to the repair process during UVR exposure. These constants were quantified on each experimental day. 2.5. Rapid light curves RLCs were plotted using samples different from those used for the ERCs. Samples were dark-adapted for 5 min, then RLCs were obtained by exposing algae to nine successive decreasing irradiance levels (from 800 to 0 lmol photons m2 s1). Each irradiance lasted 20 s, and after this the effective quantum yield efficiency of PSII was measured. The light levels and the decreasing order of the light steps were assessed as recommended by Perkins et al. [11] (data not shown). The relative electron transport rate (rETR), uncorrected for the cellular absorptance, was calculated from the equation rETR (arbitrary unit) = /PSII · E, where E represents the irradiance. Data from RLCs were fitted using the Eilers and Peeters model [23]. Consequently, the maximum rETR (rETRmax) and the maximum light use efficiency (a) are dimensionless.

3. Results 3.1. Growth rate and chlorophyll a content Growth rates and Chl a contents measured on the experimental days (day 1, day 3 and day 5) are shown in Tables 1 and 2, respectively. Between days 1 and 3, the growth rate of the algae exposed to UVR was lower than that of the controls. Between days 3 and 5, the growth rate increased in A. coffeaeformis and O. aurita, but not in S. costatum. The Chl a content was not different from the control on day 1, and increased significantly on day 5 only in A. coffeaeformis. 3.2. Fluorescence parameters For all three species, UVR treatment induced a reduction in Fm, and an increase in F0 during the stress (Fig. 1). In A. coffeaeformis, on the first day of UVR exposure, Fm values fell during the first 120 min to reach 50% of the initial value; and they then remained constant until the end of the treatment. Fm fell less on days 3 and 5, and remained constant after 60 min of UVR exposure. O. aurita shows a slight increase in F0 on the first day of exposure to UVR, and there was no change on days 3 and 5. The Fm values fell during the first 60 min on each day, and then remained constant. In S. costatum, F0 and Fm fell continuously during the 5 h of the UVR treatment of the first day. On the day 3, 30 min after the start of the UVR treatment, Fm was not significantly different from F0, and this species failed to survive. On the experimental days (1, 3 and 5), F0 like the Fm values measured at the start of the exposure,

Table 1 Growth rates (d1) of Amphora coffeaeformis, Odontella aurita and Skeletonema costatum grown under 100 lmol photons m2 s1 without exposure to UVR (control) and between days 1–3, and 3–5, of UVR exposure, (means ± SE, n = 4)

Amphora coffeaeformis

Control

Day 1–3

0.35 ± 0.06a

0.12 ± 0.03b

0.27 ± 0.09a

a

b

0.13 ± 0.03

0.23 ± 0.05c

n.g.

n.d.

Odontella aurita

0.33 ± 0.02

Skeletonema costatum

0.23 ± 0.12

Day 3–5

n.g.: no growth; n.d.: not determined. For each species, non-significantly different data share the same superscript letter (Tukey’s test, p 6 0.05).

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Table 2 Chlorophyll a (lg Chl a 106 cells) content of Amphora coffeaeformis, Odontella aurita and Skeletonema costatum before and after UVR exposure on each experimental day, (means ± S.E., n = 3) Control

Day 1

Day 3

Day 5

Amphora coffeaeformis

2.20 ± 0.31a

2.23 ± 0.28a

3.52 ± 0.28a

3.69 ± 0.44b

Odontella aurita

4.96 ± 1.05a

5.78 ± 1.29a

6.57 ± 1.77a

7.21 ± 1.68a

Skeletonema costatum

0.73 ± 0.23a

0.67 ± 0.24a

1.54 ± 0.62a

n.d.

n.d.: not determined. For each species, non-significantly different data share the same superscript letter (Tukey’s test, p 6 0.05).

Amphora coffeaeformis

1600

day 1

800

F0 , Fm (a.u.)

800 1200

600

600 800

400

400

400

200

200

0

0

0

day 3

day 3

day 3

1600

Skeletonema costatum day 1

day 1

Odontella aurita

800 800 600

F0 , Fm (a.u.)

1200 600

400

800

400 200

400

F0 Fm

200 F0 Fm

0

0 0

0

1600

60

day 5

day 5

120

180

240

300

Time (min)

F0 ,Fm (a.u.)

800 1200 600 800

400

400

200 F0 Fm

F0 Fm

0

0 0

60

120

180

240

300

0

60

Time min

120

180

240

300

Time min

Fig. 1. Changes in the minimum (F0) and maximum (Fm) fluorescence levels with UVR time exposure (min) on different experimental days (days 1, 3 and 5) in Amphora coffeaeformis, Odontella aurita and Skeletonema costatum. (Symbols are means ± SE, n = 3); a.u.: arbitrary unit.

were not significantly different in A. coffeaeformis and O. aurita. 3.3. Effective quantum yield UVR exposure induced a variable reduction in the effective quantum yield (/PSII) of all three species during the five experimental days (Fig. 2). In A. coffeaeformis, /PSII decreased to a similar extent during the successive exposures, reaching only 40% of the initial value at the end of the daily UVR. In O. aurita, the decrease in /PSII ranged from 42% (day 1) to 31% (day 5). For S. costatum, the decrease in /PSII was linear on day 1. This species failed to survive, as demonstrated by its low /PSII on day 3, and values closed to zero on day 5 (data not shown).

3.4. Damage and repair constants Changes in fluorescence parameters with time fitted well with the Neale [21] equation (correlation coefficients usually >0.99), making it possible to estimate the damage (k) and repair (r) constants (Table 3). In A. coffeaeformis, the k values were greater than the r values, and they both increased with the UVR exposure. In O. aurita, r values higher than the k values were observed on each experimental day. S. costatum shows no change in the k values, but the repair constant was equal to zero on day 1, and had increased on day 3. The r/k values greater than 1 were only observed for O. aurita. This ratio increased with the number of exposures to UVR in all three species, but the changes were not significant.

M. Fouqueray et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 1–8 0.8

Amphora coffeaeformis

day 1 day 3 day 5

Amphora coffeaeformis

5

80

rETR (a.u.)

φ PSII

0.6

0.4

60

40

20

0.2

0

0.0

Odontella aurita

80

Odontella aurita

rETR (a.u.)

φ PSII

0.6

0.4

60

40 t0 t60 t180 t300

20

0.2

0

0.0

Skeletonema costatum

Skeletonema costatum

80

rETR (a.u)

0.4

φ

PSII

0.6 60

40

20

0.2

0 0.0

0 0

60

120

180

240

200

300

400

600

800

Irradiance (µmol photons m-2 s-1 )

Time (min)

Fig. 3. Rapid light curve obtained during day 1 of UVR exposure: at the start (t0), and after 60 min (t60), 180 min (t180) and 300 min (t300) of exposure to UVR. (Symbols are means ± SE, n = 3); a.u.: arbitrary unit.

Fig. 2. Change in the effective quantum yield (/PSII) with UVR exposure time (min) on days 1 (d), 3 (s) and 5 (.) in Amphora coffeaeformis, Odontella aurita and Skeletonema costatum. (Symbols are means ± SE, n = 3).

tively, after the exposure started. In A. coffeaeformis, a was significantly lower on day 5 than on days 1 and 3 at the beginning of the UVR treatment. The parameter rETRmax decreased by ca. 40% in A. coffeaeformis, with a significant reduction from 240 min of UVR exposure onward on each experimental day. In O. aurita, this reduction reached 25%, but was not significant during the stress, and rETRmax was significantly lower at the start of day 5 than on day 1 or 3. In S. costatum, UVR exposure induced a significant decrease in a and rETRmax during the treatment. There was significant difference of a values between the start and 60 min of UVR exposure on day 1, and the reduction of rETRmax became significant from 240 min onward.

3.5. Rapid light curves RLCs were recorded on each experimental day after 0, 60, 120, 180, 240 and 300 min of UVR exposure. By way of an example, RLCs obtained for the three species on day 1 before and after 60, 180 and 300 min of exposure to UVR are shown in Fig. 3. The photosynthetic parameters calculated from RLCs are shown in Fig. 4. During UVR stress, a decreased for all species and on each experimental day. The reduction in a reached 45% in O. aurita, and 70% in A. coffeaeformis. The reduction of a in A. coffeaeformis and O. aurita was significant from 60 and 120 min, respec-

Table 3 Damage (k) and repair (r) constants (103 min) in Amphora coffeaeformis, Odontella aurita and Skeletonema costatum calculated, using the equation of Neale [21], from the /PSII values, (means ± SE, n = 3) Amphora coffeaeformis k

Odontella aurita

r a

r/k a

Day 1

1.80 ± 0.37

0.40 ± 0.31

0.17 ± 0.12

Day 3

2.54 ± 0.46a

0.85 ± 0.50a

Day 5

2.26 ± 0.07a

1.12 ± 0.28a

k a

Skeletonema costatum r

a

r/k a

k a

2.96 ± 0.12

3.79 ± 0.46

1.31 ± 0.17

0.29 ± 0.18a

0.66 ± 0.14b

0.87 ± 0.48a

1.1 ± 0.58a

0.49 ± 0.13a

2.17 ± 0.52a

4.37 ± 1.39a

1.99 ± 0.57a

r a

r/k a

1.90 ± 0.37

0±0

0 ± 0a

2.06 ± 0.37a

0.23 ± 0.14a

0.09 ± 0.05a

n.d.

n.d.

n.d.

n.d.: not determined. For each species, and each constant, non-significantly different data share the same superscript letter (Tukey’s test, p 6 0.05).

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M. Fouqueray et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 1–8 80

Amphora coffeaeformis

0.6

α (a.u.)

Amphora coffeaeformis

day 1 day 3 day 5

rETR max (a.u.)

0.8

0.4

0.2

Odontella aurita

40

20

Odontella aurita

80

rETR max (a.u.)

0.8

0.6

α (a.u)

60

0

0.0

0.4

60

40

20

0.2

0

0.0

Skeletonema costatum

Skeletonema costatum

80

rETR max (a.u.)

0.8

0.6

α (a.u.)

day 1 day 3 day 5

0.4

60

40

20

0.2

0

0.0 0

60

120

180

240

300

Time min

0

60

120

180

240

300

Time (min)

Fig. 4. Change in the maximum light use efficiency (a) and the maximum rETR (rETRmax), with UVR time exposure (min) estimated from the RLCs on days 1(d), 3 (s) and 5 (.) in Amphora coffeaeformis, Odontella aurita and Skeletonema costatum. (Symbols are means ± SE, n = 3). a.u.: arbitrary unit.

These parameters decreased significantly between days 1 and 3. 4. Discussion Previous work on long-term acclimation to the same UVR treatment in marine diatoms shown that A. coffeaeformis was a tolerant species, whereas S. costatum was sensitive. Over a minimum period of two weeks of UVR exposure A. coffeaeformis maintained a growth rate and Chl a content similar to those of the control, whereas S. costatum failed to acclimate [13]. This study focuses on the short-term response and describes changes in chlorophyll fluorescence parameters in A. coffeaeformis, O. aurita and S. costatum exposed for five consecutive days of UVR treatment. ERCs and RLCs were used to estimate the degree of inhibition of photosynthetic activity during the treatment. The UVR exposure affected diatom species differently: A. coffeaeformis and O. aurita appeared to be more resistant than S. costatum. Algae exposed to UVR had a lower growth rate than control between days 1 and 3 in all species, illustrating the rapidity of the damaging impact of the harmful radiations. Between days 3 and 5, growth rate recovery in A. coffeaeformis and O. aurita indicated that these species had

begun to acclimate to UVR exposure. In Rech et al. [13], the growth rate of A. coffeaeformis was reduced by ca. 18% by the various long-term UVR treatments. In the present study, between days 3 and 5, the growth rate was 20% lower than control, indicating that this diatom had come close to being acclimated to the UVR. In S. costatum, the growth rate was inhibited by UVR exposure, illustrating the sensitivity of this diatom species to the UVR treatment used in this experiment. In the literature, a decrease in algal Chl a content in response to UVR treatment has often been reported [4,7]. However, we observed in O. aurita no significant change and in A. coffeaeformis an increase in the Chl a content after repeated exposure, as did Litchamn and Neale [24], who used a similar UVR treatment. The change of F0 and Fm with time and on different experimental days followed the same trend in all three species. The increase in F0 under UVR stress observed in these experiments is the result of photoinactivation or damage at the PSII reaction center, and the decrease in Fm indicates non-photochemical quenching processes [24–26]. The range of these differences became less pronounced as the number of UVR exposures increased in A. coffeaeformis and O. aurita, reflecting the ability of these species to acclimate to UVR. The effective quantum yield (/PSII) decreased

M. Fouqueray et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 1–8

during the UVR exposure, as observed in various microalgae both in the laboratory and in the field [27–30]. Overall, /PSII decreased during UVR treatment in A. coffeaeformis and O. aurita on all experimental days, but the photosynthetic apparatus of these diatoms recovered overnight. In S. costatum, there was no recovery, and the impact of UVR on /PSII was such that it induced cell death. Cell size could be one factor accounting for the differences in UVR tolerance between species. Karenzt [31] found that small species were more sensitive, although Waring [27] observed the contrary. In the present study, the smallest species was S. costatum (15–25 lm), and it was more sensitive than both A. coffeaeformis (30 lm) and O. aurita (40–50 lm). The changes in the chlorophyll fluorescence parameters (Fv/Fm, /PSII) were accurately described by the equation of Neale [21] in green microalgae species [8], which indicates that dynamic interactions occur between the processes that damage the photosynthetic apparatus and those involved in repair. The use of /PSII with the Neale’s equation allowed us to quantify damage and repair processes in diatoms in order to compare their sensitivity to UVR exposure. The damage (k) and repair (r) constants were lower than those published for Dunaliella tertiolecta by Heraud and Beardall [8] exposed to UVBR only. However, in the present study, the UVR treatment was a combination of UVAR and UVBR. Damage and repair constants illustrate species-specific sensitivity. Thus O. aurita was able to compensate for damage during UVR exposure by means of efficient repair mechanisms, which contrasted with A. coffeaeformis, which had greater recovery capacity after the exposure. Furthermore, successive UVR exposures induced an increase in the r/k ratio, indicating a reduction of the sensitivity of these diatoms to UVR. On the other hand S. costatum was not able to offset damage during or after the UVR stress. RLCs (Fig. 3) quickly provide information about the light saturation characteristics of rETR. As for traditional P/E curves, the photosynthetic parameters a and rETRmax could be used to compare the photosynthetic performances of different species. The decrease in a and rETRmax, as observed in Phaeodactylum tricornutum by Liang et al [32], indicates inhibition and/or down regulation of photosynthesis. Moreover, these parameters highlight speciesspecific sensitivity to UVR stress: a and rETRmax were more affected in A. coffeaeformis and S. costatum, but show greater change during stress in O. aurita. In A. coffeaeformis, the decrease in rETRmax may have been due to an inhibition during the stress itself, illustrated by the decrease in fluorescence parameters (Fm and /PSII). In O. aurita rETRmax fell at the beginning of day 5, but remained constant during the UVR stress, indicating down regulation of this parameter. The acclimation of the photosynthetic apparatus in response to repeated UVR exposure was different in the two UVR tolerant species; the efficiency of light capture being markedly reduced in A. coffeaeformis, but not in O. aurita.

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Two of the three species studied here (O. aurita and S. costatum) are produced commercially outdoors in tanks or race-ways. Under natural conditions, these organisms are subjected to UVR, and sensitivity to UVR could be a key factor in selecting species (or clones) for industrial production. Our findings show that ERCs were more efficient than RLCs to discriminate physiological diatom responses to UVR stress. Damage and repair constants calculated from ERCs provided an easy way to quantify and compare species-specific sensitivity to UVR stress. These constants also provide information about the UVR tolerance of the diatoms from the first exposure. The dynamics of diatom resistance to UVR stress shown that their resistance increased with increasing numbers of exposures. The differences in sensitivity observed in these diatom species could therefore be related to defence mechanisms. In accordance with a previous study [13], A. coffeaeformis was shown to tolerate and be able to acclimate to this treatment. This tolerance seems to correlate with the recovery of the photosynthetic parameters. In O. aurita, damage was compensated from the first UVR exposure, indicating the rapid development of protection mechanisms, which could consist of an efficient turn-over of the D1 protein of PSII [33] or the activation of antioxidant enzymes [34]. We are currently exploring the biochemical aspects of UVR acclimation in marine diatoms. Acknowledgements The authors thank Pierre Gaudin from the University of Nantes (France) for providing diatom species and to Monika Ghosh for correcting the manuscript. We thank the anonymous reviewers for their useful comments. References [1] P.G. Falkowski, J.A. Raven, Aquatic Photosynthesis, Blackwell Science, Oxford, 1997. [2] J. Nilawati, B.M. Greenberg, R.E.H. Smith, Influence of ultraviolet radiation on growth and photosynthesis of two cold ocean diatoms, J. Phycol. 33 (1997) 215–224. [3] A.T. Davidson, The impact of UVB radiation on marine phytoplankton, Mutat. Res. 422 (1998) 119–129. [4] R.P. Sinha, D.-P. Ha¨der, Life under solar UV radiation in aquatic organisms, Adv. Space Res. 30 (2002) 1547–1556. [5] F.S. Xiong, J. Komenda, J. Kopecky, L. Nedbal, Strategies of ultraviolet-B protection in microscopic algae, Physiol. Plant 100 (1997) 378–388. [6] J.J. Cullen, M.P. Lesser, Inhibition of photosynthesis by ultraviolet radiation as a function of dose and dosage rate: result for a marine diatom, Mar. Biol. 111 (1991) 183–190. [7] M.P. Lesser, J.J. Cullen, P.J. Neale, Carbon uptake in a marine diatom during acute exposure to ultraviolet B radiation: relative importance of damage and repair, J. Phycol. 30 (1994) 183–192. [8] P. Heraud, J. Beardall, Changes in chlorophyll fluorescence during exposure of Dunaliella tertiolecta to UV radiation indicate a dynamic interaction between damage and repair processes, Photosynth. Res. 63 (2000) 123–134. [9] P.J. Ralph, R. Gademann, Rapid light curves: a powerful tool to assess photosynthetic activity, Aqua. Bot. 82 (2005) 222–237.

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