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Non-photochemical quenching of chlorophyll fluorescence and operation of the xanthophyll cycle in estuarine microphytobenthos
Journal of Experimental Marine Biology and Ecology 326 (2005) 157 – 169 www.elsevier.com/locate/jembe
Non-photochemical quenching of chlorophyll fluorescence and operation of the xanthophyll cycle in estuarine microphytobenthos J. Seroˆdio a,*, S. Cruz a,b, S. Vieira a, V. Brotas b b
a Departamento de Biologia, Universidade de Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal Instituto de Oceanografia, Faculdade de Cieˆncias da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
Received 29 March 2005; received in revised form 6 May 2005; accepted 25 May 2005
Abstract The induction of non-photochemical quenching of chlorophyll fluorescence (NPQ) and its relationship with the operation of the xanthophyll cycle were studied in estuarine microphytobenthos assemblages. NPQ and xanthophyll cycle operation were characterised by quantifying the induction kinetics and light-response curves of NPQ formation and of the production of xanthophyll cyle pigments diadinoxanthin (DD) and diatoxanthin (DT), on suspensions of benthic microalgae collected during two spring-neap tidal cycles, in summer (July) and in winter (November). The NPQ light response was characterised by a large intraday variability and by the high NPQ values attained under high irradiances. Maximum daily NPQ often reached values above 4.0, and values above 5.0 were observed on 2 days during the November sampling period. Changes in NPQ were generally followed by proportional variations in the DT content, both during exposure to high light and upon transition from darkness to low light, leading to highly significant correlations between NPQ and the DT content or the degree of deepoxidation, DT / (DD + DT). Higher NPQ values in the dark than under low light were consistently observed. This was due to the generalised increase of F mV levels under low light to values higher than the F m measured in the dark, and was associated to the decrease of the DT content upon exposure to low light. Significant seasonal variability was found regarding the parameters related to NPQ induction kinetics, indicating a higher capacity for NPQ operation in November than in July, both in the dark and as a response to high light. This seasonal variation in the NPQ light response was found to be associated to substantial changes in the taxonomic composition of microphytobenthos assemblages, and was interpreted as resulting from changes in the potential photoprotective response associated to thermal acclimation to winter conditions. D 2005 Elsevier B.V. All rights reserved. Keywords: Chlorophyll-a fluorescence; Diatoms; Microphytobenthos; Non-photochemical quenching; Photoprotection; Xanthophyll cycle
1. Introduction * Corresponding author. Tel.: +351 234370787; fax: +351 234426408. E-mail address: [email protected] (J. Seroˆdio). 0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2005.05.011
Microphytobenthos assemblages inhabiting estuarine tidal flats are subjected to a highly variable and extreme light regime caused by rapid changes in
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water cover and by the direct exposure to sunlight for prolonged periods during diurnal low tides. In microalgae, the exposure to photosynthetically supersaturating light triggers the operation of processes that lower the yield of chlorophyll a fluorescence and are thus generally termed as non-photochemical quenching (NPQ) processes (Mu¨ller et al., 2001). In diatoms, these processes include rapidly reversible, photoprotective energy-dissipating mechanisms (denergy-dependent quenchingT, q E) and slowly reversible changes involving damages to the photosynthetic apparatus (dphotoinhibitory quenchingT, q I) (Quick and Horton, 1984; Mu¨ller et al., 2001). Photoprotective q E involves the operation of the xanthophyll cycle, through the rapid and reversible conversion of pigment diadinoxanthin (DD) into the energy-dissipating form diatoxanthin (DT) within the light-harvesting complexes. This leads to the dissipation of excessive energy to non-radiative pathways, decreasing the transfer of captured excitation energy to the photosystem II (PSII) reaction centers and thus limiting the amount of photodamage to the photosynthetic apparatus (Olaizola and Yamamoto, 1994). The photoprotective down-regulation of PSII through the operation of the xanthophyll cycle is well documented for diatoms, having received considerable attention in a number of recent studies (Owens, 1986; Ting and Owens, 1993; Arsalane et al., 1994; Olaizola and Yamamoto, 1994; CasperLindley and Bjo¨rkman, 1998; Jakob et al., 1999, 2001; Lavaud et al., 2002a,b, 2004; Ruban et al., 2004). However, as all these studies have been based on microalgae grown in culture, little is known regarding the operation of NPQ processes in natural diatom-dominated communities. In the particular case of microphytobenthos, no studies have yet been published regarding NPQ induction or the operation of the xanthophyll cycle. The interest on the study of NPQ functioning in microphytobenthos is also justified by the extreme light regime to which microalgae are subjected in their natural environment, and by the hypothesis that benthic microalgae may use their migratory ability to behaviourally regulate light exposure and avoid photodamage (Consalvey et al., 2004). Furthermore, it was recently shown that diatoms may develop an exceptionally high capacity for rapid and large NPQ induction under light stress (Lavaud et al.,
2002a; Ruban et al., 2004). In this study, the operation of NPQ in natural estuarine microphytobenthos assemblages was characterised regarding (i) NPQ induction kinetics under high irradiance and its relationship with the xanthophyll cycle, (ii) the formation of NPQ in the dark, and (iii) the seasonal variability of NPQ induction kinetics and light response.
2. Materials and methods 2.1. Sampling Benthic microalgae were collected on an intertidal mudflat located near Vista Alegre, Canal de ´Ilhavo, on the Ria de Aveiro, a mesotidal estuary located on central west coast of Portugal. Sampling site consists of fine muddy sediments (97% particles b 63 Am), where microphytobenthos assemblages are dominated throughout the year by diatoms of the genera Navicula, Nitzschia, and Gyrosigma. Sampling was carried out in May 2004 (NPQ and pigment content analysis, see below) and during two spring-neap tidal cycles, on July and November 2004 (NPQ induction kinetics and NPQ vs. E curves, see below). Microalgae were collected by placing two pieces of lens tissue on the surface of the sediment shortly after the beginning of diurnal low tide. After ca. 1 h, the upper piece of lens tissue was removed and microalgae were resuspended on filtered sea water. Replicated samples were used for determination of taxonomic composition. Microalgal suspensions were fixed in 1% v/v formaldehyde and viewed using bright-field microscopy for determination of the relative abundance of major taxonomic groups (diatoms, euglenophytes and cyanobacteria). Diatom identification was carried out on subsamples oxidized using saturated potassium permanganate solution and digested in concentrated HCl. 2.2. Chlorophyll fluorescence Variable fluorescence was measured using a Pulse Amplitude Modulation (PAM, Schreiber et al., 1986) fluorometer comprising a computer-operated PAMControl Unit (Walz, Effeltrich, Germany) and a
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WATER-EDF-Universal emitter-detector unit (Gademann Instruments GmbH, Wu¨rzburg, Germany). This fluorometer uses a modulated blue light (LED-lamp peaking at 450 nm, half-bandwidth of 20 nm) as source for measuring, actinic and saturating light, emitted at a frequency of 18 Hz when measuring F o (see notation, Table 1), or 20 kHz when measuring other fluorescence parameters. Fluorescence was measured using a 6-mm diameter Fluid Light Guide fiberoptics bundle or a 1.5-mm plastic fiberoptics, delivering the measuring, actinic and saturating light provided by the fluorometer, connected to a fluorescence cuvette (KS-101, Walz, Effeltrich, Germany). Sample temperature was maintained constant at 20 8C by connecting the fluorescence cuvette to a recirculating water bath (Frigiterm-10, Selecta, Spain). Maximum light levels attained were 920 (6 mm fiber) and 1700 (1.5 mm fiber) Amol quanta m 2 s 1. Due to the frequent occurrence of F mV values under low irradiances higher than the F m value measured after dark-adaptation, NPQ was calculated by adapting the non-photochemical quenching coefficient (Schreiber et al., 1994): NPQ ¼ ðFmV m FmV Þ=FmV
ð1Þ
where F mV m is the maximum F mV value, higher than F m, that is measured under low actinic irradiance. All measurements were carried out on the day of sampling.
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2.3. Pigment analysis Samples were centrifuged (3000 g, 10 min) and pigments were extracted from pellets using 3 ml of 95% cold buffered methanol (2% ammonium acetate). Samples were sonicated for 1 min and stored in the dark at 20 8C for 15 min. Extracts were filtered onto Millipore FluoroporeTM membrane filters (0.2 Am FG) immediately before HPLC analysis. Pigment extracts were analysed using a Shimadzu HPLC comprising a solvent delivery module (LC-10ADVP) with a system controller (SCL-10AVP), a photodiode array (SPD-M10AVP), and a fluorescence detector (RF10AXL). Chromatographic separation was carried out using a C18 column for reverse phase chromatography (Supelcosil, 25 mm long, 4.6 mm in diameter and 5 Am particles) and a 35 min long elution program. The solvents used were 0.5 M ammonium acetate in methanol and water (85:15, v/v), acetonitrile and water (90:10, v/v), and 100% ethyl acetate. The solvent gradient followed Kraay et al. (1992) adapted by Brotas and Plante-Cuny (2003) with a flow rate of 0.6 ml min 1 and an injection volume of 100 Al. Identification and calibration of HPLC peaks were done with chlorophyll-a from Sigma and h-carotene standards and chlorophyll c 2, chlorophyll c 3, fucoxanthin, neoxanthin, violoxanthin, diadinoxanthin, aloxanthin, diatoxanthin, lutein, zeaxanthin and chlorophyll b standards from DHI (Cartaxana and Brotas, 2003). Pigments were identified from absorption spectra and
Table 1 Notation Chl DD, DT DT /(DD + DT) DF / F mV E E NPQmin ETR V F s, F m F mV m F o, F m Fv / Fm k NPQ NPQ NPQ0, NPQm NPQ(E), NPQ(E,t) t
Chlorophyll (mg l 1) Diadinoxanthin and diatoxanthin [mol (100 mol Chl a) 1] Relative content of diatoxanthin [= DT / (DD + DT) 100] (%) Effective quantum yield of PSII (dimensionless) Spectrally averaged photon irradiance of PAR (400–700 nm) (Amol quanta m 2 s 1) Irradiance under which NPQ reaches a minimum value in a NPQ vs. E curve Relative electron transport rate (= E DF / F mV ) (dimensionless) Steady-state and maximum fluorescence emitted by a light-adapted sample (arbitrary units) Maximum F mV value measured under low irradiance (arbitrary units) Minimum and maximum fluorescence emitted by a dark-adapted sample (arbitrary units) Maximum quantum yield of PSII of a dark-adapted sample (dimensionless) Rate constant of NPQ induction (min 1) Non-photochemical quenching of fluorescence [= ( F mV F m) / F mV ] (dimensionless) NPQ at the start and after 6 min of exposure to high irradiance NPQ measured under irradiance E (NPQ vs. E curve), and after t minutes of exposure to irradiance E (NPQ induction experiments) Time of exposure to high irradiance during a NPQ induction experiment (min)
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retention times, and concentrations were calculated from the area of pigment peaks in the photodiode array. Diatoxanthin relative content was calculated as DT / (DD + DT) 100.
assuming a linear relationship between DT content and NPQ, which has been verified for diatoms (Arsalane et al., 1994; Olaizola and Yamamoto, 1994; Lavaud et al., 2002a; Ruban et al., 2004):
2.4. NPQ induction and relaxation
NPQðt Þ ¼ NPQm þ ðNPQ0 NPQm ÞekNPQ t
Microphytobenthos suspensions were concentrated by centrifugation (3000 g, 10 min) and resuspended in filtered sampling site water to a final concentration always higher than 50 Ag Chl a ml 1. Before measuring, samples were supplemented with NaHCO3 (4 mM final concentration) and dark-adapted for 30 min. F o and F m were measured, and the maximum quantum yield, F v/F m, was determined by applying saturating pulses each 2 min during 10 min. Samples were then exposed to low actinic irradiance (44 Amol quanta m 2 s 1) for 15 min, and to 1700 Amol quanta m 2 s 1 for 30 or 60 min. During light exposure, the effective quantum yield of PSII, DF / F mV = [( F mV F s) / F mV] (Genty et al., 1989), was determined each 90 s and the relative electron transport rate was obtained from the delivered actinic irradiance, E, by calculating ETR = E DF / F mV . Actinic light was then switched off and saturating pulses were applied each 2 min during 30 min. NPQ induction experiments were carried out on 5 days along each spring-neap tidal cycle, 2–3 times each day. For comparing the evolution of NPQ and of xanthophyll cycle pigments DD and DT content during exposure to high light, samples were dark-adapted and exposed to low light as described above, after which the actinic light was increased to 920 Amol quanta m 2 s 1. After 1, 2, 3, 5, 10, 15, 30 and 60 min of light exposure, the sample was rapidly (b 10 s) frozen in liquid nitrogen for HPLC analysis, assuring that the xanthophyll cycle was stopped virtually immediately after light exposure. Samples were also collected for HPLC analysis at the end of the dark-adaptation period and immediately after exposure to low light. NPQ was measured immediately before the collection for HPLC analysis, and was calculated by applying Eq. (1) using the highest F mV value measured under 44 Amol quanta m 2 s 1 as F mV m. The NPQ increase following exposure to high light was described by a first-order kinetics model, derived from the model of Olaizola and Yamamoto (1994) for the variation of DT concentration under high light by
ð2Þ
where NPQ0 and NPQm are the values measured immediately before the start of the high-light period and after the complete de-epoxidation of the DD pool to DT following a first-order kinetics, respectively, and k NPQ is the rate constant of NPQ increase. k NPQ was estimated by fitting Eq. (2) to the first 6 min of the time series of NPQ measurements under high light. The model was fitted iteratively using MS Excel Solver. 2.5. NPQ vs. E curves The light response of NPQ was characterised by constructing NPQ vs. E curves, by measuring NPQ on microalgal suspensions exposed to increasing irradiance levels. After 30 min of dark-adaptation, F o and F m were measured, and the sample was subsequently exposed to 12 increasing irradiance levels, from 21 to 1700 Amol quanta m 2 s 1. Under each irradiance level, F s and F mV were measured each 90 s, until a steady-state in DF/F mV was reached (minimum 7.5 min). NPQ was calculated by applying Eq. (1) using the highest F mV value measured as F mV m. NPQ vs. E curves were constructed 2–3 times each day, on the days when NPQ induction experiments were carried out.
3. Results 3.1. NPQ induction and relaxation Fig. 1 illustrates the pattern of variation of NPQ and DF / F mV during the sequential exposure to low and high irradiance of dark-adapted microphytobenthos samples. Upon the transition to low light, a decrease in NPQ of dark-adapted samples was often observed, usually leading a steady-state within 15–20 min. During this period, DF / F mV decreased immediately after transition to low light, but in many cases it recovered to values close or above the F v / F m value measured at the end of the dark period (Fig. 1B). Following an
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Fig. 2. NPQ induction kinetics in microphytobenthos suspensions exposed to 1700 Amol quanta m 2 s 1, and fit of Eq. (2) for the estimation of the rate constant of NPQ induction (k NPQ). Same NPQ data as displayed in Fig. 1.
Fig. 1. Typical changes in F v / F m, DF / F m V and NPQ induction kinetics in dark-adapted microphytobenthos suspensions exposed to low (44 Amol quanta m 2 s 1; gray bar) and high (1700 Amol quanta m 2 s 1; white bar) irradiance and subsequent recovery and relaxation in the dark (black bars). Samples collected in 24 July (A) and 15 November 2004 (B).
initial large decrease with the start of the high-light exposure (on average, 74.3%), DF / F mV recovered gradually, usually reaching a steady-state within 30 min. This variation in DF / F mV was paralleled by a rise in NPQ that comprised an initial rapid and large change followed by a slower increase, usually leading to a steady-state within 20–30 min of high-light exposure. The mean values of the NPQ measured after
30 min of high-light exposure, NPQ(1700,30V), calculated for each sampling period ranged from 1.97 in July to 3.11 in November (Table 2). NPQ(1700,30V) attained a maximum value of 5.36 in the November sampling period. In both sampling periods, F o and F m levels were lower after returning to darkness than before the light stress, and F v / F m recovered rapidly and almost completely to the initial values. Considering the two sampling periods, the recovery of F v / F m reached, on average, 87.2% after 10 min and 92.5% after 20 min. The fit of Eq. (2) to the first 6 min of the NPQ time series was in all cases very good, with the model explaining over 97% of the variability (Fig. 2). Model fit decreased for longer NPQ time series, indicating that the NPQ increase after 6 min was due to processes not described by a first-order kinetics. k NPQ
Table 2 Mean values of the parameters used to characterise the NPQ induction kinetics and the NPQ vs. E curves, measured in the sampling periods of July (n = 13) and November (n = 12) 2004 NPQ induction
July November a
NPQ vs. E curves
F v / F ma
DF / F mV (44)
DF / F mV (1700)
F v / F mb
k NPQ
NPQ (1700,30 V)
NPQ (0)
NPQ (1700)
E NPQmin
0.614 0.553 *
0.591 0.550 ns
0.171 0.130 ns
0.580 0.509 ns
0.52 0.74 *
1.97 3.11 **
0.45 0.80 *
1.50 3.43 ***
178.5 125.7 ns
Measured after 30 min of dark-adaptation. Measured after 20 min recovery in the dark. Numbers in the table header are irradiances in Amol quanta m2 s1 under which DF / F mV and NPQ were measured. Levels of statistical significance of Student’s t-test used to compare mean values. b
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ranged from 0.16 to 1.18 min 1, attaining mean values of 0.52 and 0.74 min 1 for July and November, respectively (Table 2). Although the described patterns of NPQ induction and relaxation were consistently observed throughout the studied periods, large intraday variability in the NPQ light response was frequently found. Considering both sampling periods, intraday variability (c.v.) in k NPQ and NPQ(1700,30V) ranged from 3.9% to 61.6%, and from 8.2% to 67.1%, respectively. 3.2. NPQ and DT The variations in NPQ observed upon changes in irradiance were generally followed by proportional variations in DT content. This was observed under exposure to high light, during which the build-up of NPQ was closely paralleled by a proportional increase in DT concentration (Fig. 3). Parallel variations in NPQ and DT were also observed during exposure to low light following dark-adaptation, both decreasing from the high values measured at the end of the dark period (Fig. 3), and leading to highly significant correlations between NPQ and DT / (DD + DT) or DT content (r 2 = 0.873, P b 0.001 and r 2 = 0.751, P b 0.001, respectively; Fig. 4). The decrease in NPQ under low light was due to the steady increase of F mV to values higher than the F m value measured after dark-adaptation (Fig. 3). In all cases, the DT
Fig. 4. Linear relationship between NPQ and DT / (DD + DT) during exposure to high irradiance (920 Amol quanta m 2 s 1).
content in dark-adapted samples was higher (averaging 0.64 F 0.075 mol DT (100 mol Chl a) 1) than after exposure to low light (0.59 F 0.028 mol DT (100 mol Chl a) 1). The DD pool size in dark-adapted samples averaged 9.64 mol DD (100 mol Chl a) 1. 3.3. NPQ vs. E curves Higher NPQ values in the dark, NPQ(0), than under low light were also consistently observed in the NPQ vs. E curves constructed during the two
Fig. 3. Decrease of NPQ and DT, and increase of F mV above F m upon transition from darkness (black bar) to low irradiance (44 Amol quanta m 2 s1; gray bar) of a dark-adapted microphytobenthos sample, and subsequent NPQ induction and DT production upon exposure to high irradiance (920 Amol quanta m 2 s 1; white bar) for 60 min.
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Fig. 5. Typical light responses of F mV (normalised to F m) and NPQ in microphytobenthos suspensions, showing a clear increase in F mV above F m and correspondent NPQ decrease for E b E NPQmin, and an increase for E N E NPQmin.
spring-neap tidal cycles (Fig. 5). The decrease of NPQ upon illumination with low irradiances was due to a marked increase in F mV to levels above F m. Except for one case, all curves showed high NPQ(0) values that decreased steeply with irradiance until a minimum was reached. The irradiance at which NPQ was minimum, E NPQmin, averaged 178.5 and 125.7 Amol quanta m 2 s 1 in July and November, respectively (Table 2). On average, NPQ(0) values reached 0.45 in July and 0.80 in November. For irradiances above E NPQmin, NPQ increased virtually linearly with irradiance, reaching maximum values at 1700 Amol quanta m 2 s 1 that were typically 2–4 times higher than those observed in the dark. Average NPQ(1700) values for each tidal cycle were similar to those measured in the NPQ induction experiments. 3.4. Seasonal variability Significant differences were found between the July and November spring-neap tidal cycles regarding the parameters related to NPQ operation (Table 2). In general, these results indicate a higher capacity for NPQ induction in November than in July, both in the dark and as a response to high light. F v / F m was significantly lower in November than in July, probably related to a higher formation of NPQ in the dark and
not to a lower potential efficiency of the photosynthetic apparatus as its performance under actinic light (DF / F mV at 44 and at 1700 Amol quanta m 2 s 1) and F v / F m recovery were not significantly different in both periods (Table 2). Accordingly, NPQ in the dark was also significantly higher in November (Table 2), this difference becoming higher when considering NPQ under low irradiances such as 21 Amol quanta m 2 s 1. Considering the response to high irradiance, significant differences were found both regarding the initial rate of NPQ induction (k NPQ) and the maximum NPQ attained (both in the NPQ induction experiments and in the NPQ vs. E curves), all significantly higher in November (Table 2). Regarding E NPQmin, no significant differences were found between the two periods, although average values were higher in July. This large seasonal variation in the NPQ light response was associated to substantial changes in the taxonomic composition of the microphytobenthos assemblages regarding the dominant species of diatoms. In both sampling periods, the diatom species composition was relatively constant among the different days, with maximum variation in the relative abundance of individual species remaining below 5%. In July, five diatom species accounted, on average, for 87.0% of total species relative abundance (Table 3): Nitzschia frustulum (Ku¨tzing) Grunow,
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Nitzschia perspicua Cholnoky, Parlibellus crucicula (W. Smith) Witkowski, Lange-Bertalot & Metzeltim, Navicula cf. gregaria Donkin and Nitzschia draveillensis Coste and Ricard. The relative abundance of the remaining diatom species was in all cases less than 2%. In November, the number of species with relative abundances above 2% increased to 10, of which four also had abundances above 2% in the July sampling period: N. cf. gregaria, P. crucicula, N. frustulum and N. perspicua. During this period, the microphytobenthos assemblages were largely dominated by Gyrosigma fasciola (Ehrenberg) Griffth and Henfrey, with relative abundances ranging from 35.6% to 49.8%, and averaging 41.5% of total cell counts (Table 3). In July, the maximum abundance of this species reached only 1.9%. Remaining species with abundances above 2% were Nitzschia acuminata W. Smith, Entomoneis alata (Ehrenberg) Ehrenberg, Tryblionella apiculata Gregory, Navicula phyllepta Ku¨tzing, and Entomoneis paludosa (W.Smith) Reimer, but in all cases abundances were less than 7%. In both sampling periods, euglenophytes (Euglena sp.) and cyanobacteria (Merismopedia sp., Oscillatoria spp.) accounted for less than 1% of cell counts.
Table 3 Mean relative abundance (%) of the main taxa found in microphytobenthos suspensions collected in the sampling periods of July and November 2004 Taxa Diatoms Nitzschia frustulum (Ku¨tzing) Grunow N. perspicua Cholnoky N. draveillensis Coste and Ricard N. acuminata W. Smith Parlibellus crucicula (W. Smith) Witkowski, Lange-Bertalot and Metzeltim Navicula cf. gregaria Donkin N. phyllepta Ku¨tzing Gyrosigma fasciola (Ehrenberg) Griffth and Henfrey Tryblionella apiculata Gregory Entomoneis alata (Ehrenberg) Ehrenberg E. paludosa (W. Smith) Reimer Other diatoms Euglenophytes Cyanobacteria
July
November
30.3 25.2 5.7 – 18.7
5.4 3.5 – 6.6 5.9
6.9 – –
6.6 3.5 41.4
– – – 12.3 0.4 0.3
4.6 5.1 2.8 14.1 0.3 0.1
Mean values obtained by averaging the relative abundances determined for each sampling date in each spring-neap cycle. For diatoms, only relative abundances N 2% are presented.
4. Discussion 4.1. NPQ induction kinetics The results obtained on the kinetics of NPQ induction by microphytobenthos assemblages revealed a comparatively high capacity for the rapid development of large NPQ under exposure to supersaturating irradiances. Despite the large intraday variability observed in the NPQ light response, daily maxima of k NPQ and NPQ(1700,30V) were in many cases higher than the values published for diatoms exposed to 2000 Amol quanta m 2 s 1 (Table 4). This was particularly true in the case of NPQ(1700,30’) in the November sampling period, when 50% of the values were higher than 3.0, and 25% higher than 4.0. These values are higher than the highest NPQ values measured in microalgae or plants, for which NPQ = 4.0 is considered as the maximum ordinarily reached (Rohae`ek, 2002; Ruban et al., 2004). NPQ values significantly higher than 3.0 have been reported for diatoms, but only for Phaeodactylum tricornutum grown under an intermittent light regime (Lavaud et al., 2002a; Table 4). It is conceivable that even higher NPQ values could have been measured in microphytobenthos since, as some results suggest (NPQ continuing to decrease at the end of the low-light exposure; Fig. 1B), F mV could have increased to higher levels if the exposure to low light following prolonged darkness had been longer. The continuous increase of NPQ during the 30 min of light stress only partially resulted from the deepoxidation of the cellular DD pool, as shown by the decrease of the fit of the first-order kinetics model (Eq. (2)) for NPQ time series longer than the first 6 min of high-light exposure (Fig. 2). The slower increase in NPQ after this initial period could be due to de novo synthesis of DD or to damages caused to the photosynthetic apparatus ( q I), unrelated to DT production. However, under the experimental conditions applied, the NPQ developed by microphytobenthos appears to result mostly from the operation of photoprotective energy-quenching processes ( q E). This is supported by the rapid recovery of F v / F m after the light stress period, which has been taken as an indication of the occurrence of rapidly-reversible down-regulation of PSII rather than the onset of chronic photoinhibition (Schofield et al., 1998;
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Table 4 NPQ induction kinetics (k NPQ, NPQ(E,30V)) measured in this study and published results for diatoms Source
Microalgae
Growth conditions
E
k NPQ
NPQ(E,30 V)
Arsalane et al. (1994) Casper-Lindley and Bjo¨rkman (1998) Lavaud et al. (2002a)
P. tricornutum P. tricornutum P. tricornutum
2000 1200 2000
Lavaud et al. (2004)
P. tricornutum Skeletonema costatum Microphytobenthos
100, 12/12 h 100, 15/9 h 40, 16/8 h 40, 5/55 min 40, 16/8 h July November
1700
0.40 0.71 0.46 0.72 0.68 1.11 0.46–0.79 0.54–1.18
2.05 1.75 3.12 10.23 2.60 0.92 2.05–2.75 1.72–5.36
This study
2000
Growth conditions: irradiance (Amol quanta m2 s1 ) and photoperiod (light/dark period), or sampling period. k NPQ was estimated by fitting Eq. (2) to published values (goodness of fit in all cases N0.95).
Ralph et al., 2002). Furthermore, the parallel decrease of F m and F o observed after the exposure to supersaturating irradiance suggests the operation of photoprotective mechanisms, because damage to the photosynthetic apparatus normally results in the decrease of F m and in the increase of F o (Gilmore et al., 1996; Schofield et al., 1998; Mu¨ller et al., 2001; Ralph et al., 2002). The correlation found between NPQ and the degree of de-epoxidation, DT / (DD + DT), during the 60 min of high-light exposure indicates that NPQ operation under the tested conditions is based on the formation of DT. This further confirms the minor effects of photoinhibition, which would have lead to a larger increase in NPQ than in DT / (DD + DT) (Arsalane et al., 1994). This result supports the use of k NPQ as an estimator of the initial rate of xanthophyll de-epoxidation. 4.2. NPQ induction in the dark A consistent result regarding the operation of NPQ in microphytobenthos was the generalised occurrence of high NPQ values in dark-adapted samples and its gradual decrease under low light, due to the increase of F mV to values higher than the F m measured in the dark. The relaxation of NPQ upon exposure to low light was associated to the decrease of the cellular DT content and of the degree of xanthophyll de-epoxidation, with considerably high levels of de-epoxidation (DT / (DD + DT) N 5%) being found in dark-adapted samples. Relatively high degrees of de-epoxidation were also measured after exposure to low irradiance (Fig. 3), causing the observation of high DT / (DD + DT) values while NPQ = 0 (i.e., negative intercept of the linear regression line in Fig. 4). This was
probably due to the fact that the exposure to low light was insufficient to completely epoxidate the DT formed in the dark, and that NPQ was calculated on the basis of the maximum F mV level. The relationship between NPQ and DT / (DD + DT) may be affected by the presence of Euglenophytes, microalgae that contain DD but do not produce DT as a response to high light (Casper-Lindley and Bjo¨rkman, 1998). A significant presence of Euglenophytes, however, should have contributed to lower the measured degree of de-epoxidation (for the same NPQ level) and could not explain the high levels measured in the dark and under low light. The formation of NPQ in the dark ( F mV N F m) has been reported for diatoms (Falkowski et al., 1986; Demers et al., 1991; Ting and Owens, 1993; Geel et al., 1997; Casper-Lindley and Bjo¨rkman, 1998; Jakob et al., 1999; Mouget and Tremblin, 2002) and microphytobenthos assemblages (Seroˆdio, 2004). Nevertheless, the formation of NPQ or DT in the dark was not detected in a number of studies on the operation of the xanthophyll cycle in diatoms (Arsalane et al., 1994; Olaizola and Yamamoto, 1994; Dijkman and Kroon, 2002; Lavaud et al., 2002a, 2004). The observation of F mV levels higher than F m was shown to result from the activation of the DD de-epoxidase, and consequent de-epoxidation of DD to DT, as induced by the formation of a transthylakoidal proton gradient, as when responding to supersaturating irradiances (Ting and Owens, 1993; Jakob et al., 1999; Lavaud et al., 2002b). As a result of the accumulation of DT in the dark, absorbed light energy is diverted from the PSII reaction centers, lowering the F m emission. Under low light levels, the activation of ATP synthase and carbon metabolism dissipate the proton gradient
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at a higher rate than the proton accumulation caused by PSII activity, leading to the gradual epoxidation of DT to DD, the relaxation of NPQ, and the increase of F mV levels above F m. This explanation is supported by the observation of the simultaneous decrease of NPQ and DT content and the increase of F v / F m upon exposure to low irradiance after prolonged darkness (Jakob et al., 1999). In diatoms, the formation of a pH-gradient in the dark has been generally attributed to chlororespiration (Ting and Owens, 1993; Olaizola and Yamamoto, 1994; Jakob et al., 1999), although other physiological processes, such as CO2-concentration mechanisms and acetate accumulation during lipid degradation, may also contribute to lower the thylakoid lumen pH and induce the xanthophyll deepoxidation, decreasing F m (Jakob et al., 2001). The high NPQ levels in the dark consistently observed in natural samples of microphytobenthos may result from an increased DD cellular pool, allowing high levels of DT production and accumulation. However, the capacity for the formation of NPQ in the dark appear not to be related with a large DD pool, as diatoms containing an artificially-induced large DD pool did not show any DT accumulation in the dark (Lavaud et al., 2002a). NPQ induction in the dark could also result from an increased content or activity of DD de-epoxidase, enabling higher degrees of deepoxidation of a small DD pool. DD de-epoxidase activity could be increased either by requiring a lower transthylakoidal proton gradient for activation, or by an increased activity of physiological processes causing the intrathylakoidal proton accumulation (Jakob et al., 2001). The capacity to develop NPQ in the dark has been considered an adaptive advantage, representing as a way to prevent degradation of xanthophyll cycle pigments during prolonged periods of darkness, thus providing readily functional photoprotection upon reillumination by otherwise photodamaging light levels (Hoefnagel et al., 1998; Jakob et al., 1999). In the case of microphytobenthos inhabiting estuarine intertidal flats, benthic microalgae are frequently subjected to rapid changes in light exposure, including the sudden exposure to high levels of sunlight after withstanding long periods in the dark. This occurs frequently, when tidal ebb takes place during the day, exposing microalgal biofilms to full sunlight after a continuous period of several hours in dark-
ness during night or high tide. Exposure to solar light after long periods of darkness may also take place when microalgae buried in aphotic layers of the sediment are resuspended and brought to the surface. The maintenance of functional xanthophyll cycle pigments by benthic diatoms can thus be considered as advantageous and may explain the high levels of NPQ in the dark exhibited by microphytobenthos samples. 4.3. Seasonal variability: NPQ light response and acclimation Significant seasonal variability was found regarding the fluorescence indices related to maximum NPQ capacity (NPQ(1700), NPQ(1700,30V)), NPQ in the dark ( F v / F m, NPQ(0)), and NPQ induction kinetics (k NPQ). Higher values were observed in November, suggesting important changes in the potential photoprotective response of the assemblages associated to acclimation to winter conditions. Seasonal changes in NPQ operation may be expected from the variation in the pool of xanthophyll cycle pigments, since it has been shown that the xanthophyll pool size increases as a response to lower growth temperatures, both in diatoms (Anning et al., 2001) and in plants (Mu¨ller et al., 2001). The enhanced NPQ operation observed in November could thus result from an increase of the DD pool size (and other factors affecting DT production, see above) as a result of acclimation to lower temperature. The exposure to direct sunlight under low temperatures can be particularly damaging to the photosynthetic apparatus, as the operation of the xanthophyll cycle is an enzyme-mediated process, and low temperature may thus be expected to slow down the photoprotective response under high light. An increase in the content of the xanthophyll cycle pigments in winter would thus be advantageous for microphytobenthos cells, enhancing photoprotection against photodamages by allowing to attain higher degrees of de-epoxidation at low temperatures. The increase in the NPQ in the dark in November (and consequent lower dark-adapted F v / F m) would thus represent a side effect of the increase in the DD pool size and of the capacity for NPQ operation. Because the DD content has been shown to increase with growth irradiance in various microalgae
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(Casper-Lindley and Bjo¨rkman, 1998; Moisan et al., 1998; Meyer et al., 2000; Rijstenbil, 2003), a decrease in the DD pool size could be expected in November considering the lower irradiance levels, thus counterbalancing the effect of lower temperatures. However, due to the high water turbidity in estuaries (Seroˆdio and Catarino, 1999) and to the migratory behaviour of benthic microalgae (Consalvey et al., 2004), microphytobenthos biofilms are exposed to light almost only during diurnal low tides. During these periods, microalgae are exposed to direct sunlight which, even during winter months, reaches considerably high levels (N1000 Amol quanta m 2 s 1), justifying the maintenance of high levels of photoprotection. Considering the substantial changes in taxonomic composition that were found between July and November, the seasonal increase in the NPQ capacity may be interpreted as resulting from the diatom species succession, associated to a potential competitive advantage of populations with the ability of developing a higher photoprotection capacity. In phytoplankton, the magnitude of xanthophyll cycling has been considered as a process for species succession (Meyer et al., 2000). However, the community composition might have changed due to causes unrelated to photoprotection, and the increase in NPQ capacity may be the result of the ability of most species to physiologically acclimate to changing light and temperature conditions on a seasonal time scale. The pattern of the NPQ vs. E curves found for microphytobenthos is similar to the NPQ light re¨ quist, sponse of cyanobacteria (Campbell and O 1996; Green and Oliver, 2003). Although the variation of NPQ under low light observed in cyanobacteria has been attributed to state transitions, a physiological process that does not occur in diatoms (Owens, 1986; Ting and Owens, 1993; Lavaud et al., 2002a), in both cases NPQ attains a minimum value at relatively moderated irradiances (E NPQmin). In cyanobacteria, a relationship was found between E NPQmin and the photoacclimation status of the microalgae (Camp¨ quist, 1996). The similarity between the bell and O NPQ vs. E curves in the two groups of microalgae and the fact that E NPQmin is determined by the magnitude of NPQ in the dark, its relaxation under low light, and its onset under high light, factors shown to be associated to thermal acclimation or photoacclimation in diatom-dominated microphytobenthos, suggest the
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possibility that a similar relationship may also exist for diatoms. As with cyanobacteria, this would allow to determine the integrated light regime to which natural assemblages of microalgae are acclimated (Green and Oliver, 2003). Although no significant seasonal variability was found regarding E NPQmin in this study, the higher mean value observed in July concurs with what would be expected in the case of photoacclimation to higher irradiances. 4.4. Measuring NPQ in undisturbed microphytobenthos While the study of benthic microalgae in suspension may provide valuable information on their potential photoprotection capacity, the obtained results may not represent the physiological response of undisturbed microphytobenthos assemblages under natural conditions. Because much of the particular physico-chemical conditions of the microenvironment of benthic biofilms are lost when the microalgae are resuspended, the characterization of the photoprotective response of intact microphytobenthos assemblages can only be adequately assessed through the measurement of NPQ in undisturbed sediment samples. This, however, is strongly hampered by two types of factors. First, the vertical migratory response of benthic microalgae to changes in light conditions, including darkening, and its large impact on fluorescence parameters (Seroˆdio et al., 1997) makes it virtually impossible to apply NPQ indices based on the F m level. Second, light and fluorescence attenuation in the upper layers of the sediment and the depth-integration of fluorescence signals may cause the light-dependent underestimation of NPQ indices (Seroˆdio, 2004). This study has shown that the application of fluorescence quenching analysis to benthic microalgae is further complicated by the frequent formation of NPQ in the dark and resulting F mV values higher than F m, as it leads to negative NPQ values (Ting and Owens, 1993; Geel et al., 1997; Mouget and Tremblin, 2002). In conclusion, the study of NPQ in intact microphytobenthos assemblages presents problems that cannot be adequately overcame with the methods currently available. Alternative approaches must be developed that allow for the non-invasive monitoring of NPQ indices independently of F m quenching in the dark and of changes
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in vertical distribution of microalgae within the photic layers of the sediment.
Acknowledgements We wish to thank Carlos Rafael Mendes for help in the HPLC analysis, Helena Coelho for help in field and laboratory work, and Jorge Marques da Silva for critical comments on the manuscript. This work was supported by project POCTI/MAR/15318/99, funded by Fundac¸a˜o para a Cieˆncia e a Tecnologia, and by Programme PRODEP III. We thank one anonymous reviewer for critical comments on the manuscript. [SS]
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Sargassum natans (Phaeophyceae): responses under natural sunlight. J. Phycol. 34, 104 – 112. Schreiber, U., Schliwa, U., Bilger, W., 1986. Continuous recording of photochemical and nonphotochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth. Res. 10, 51 – 62. Schreiber, U., Bilger, W., Neubauer, C., 1994. Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. In: Shulze, E.D., Caldwell, M.M. (Eds.), Ecophysiology of Photosynthesis. Springer-Verlag, Berlin, pp. 49 – 70. Seroˆdio, J., 2004. Analysis of variable chlorophyll fluorescence in microphytobenthos assemblages: implications of the use of depth-integrated measurements. Aquat. Microb. Ecol. 36, 137 – 152. Seroˆdio, J., Catarino, F., 1999. Fortnightly light and temperature variability on estuarine intertidal sediments and implications for microphytobenthos primary productivity. Aquat. Ecol. 33, 235 – 241. Seroˆdio, J., Marques da Silva, J., Catarino, F., 1997. Nondestructive tracing of migratory rhythms of intertidal benthic microalgae using in vivo chlorophyll a fluorescence. J. Phycol. 33, 542 – 553. Ting, C.S., Owens, T.G., 1993. Photochemical and nonphotochemical fluorescence quenching processes in the diatom Phaeodactylum tricornutum. Plant Physiol. 101, 1323 – 1330.
Non-photochemical quenching of chlorophyll fluorescence and operation of the xanthophyll cycle in estuarine microphytobenthos J. Seroˆdio a,*, S. Cruz a,b, S. Vieira a, V. Brotas b b
a Departamento de Biologia, Universidade de Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal Instituto de Oceanografia, Faculdade de Cieˆncias da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
Received 29 March 2005; received in revised form 6 May 2005; accepted 25 May 2005
Abstract The induction of non-photochemical quenching of chlorophyll fluorescence (NPQ) and its relationship with the operation of the xanthophyll cycle were studied in estuarine microphytobenthos assemblages. NPQ and xanthophyll cycle operation were characterised by quantifying the induction kinetics and light-response curves of NPQ formation and of the production of xanthophyll cyle pigments diadinoxanthin (DD) and diatoxanthin (DT), on suspensions of benthic microalgae collected during two spring-neap tidal cycles, in summer (July) and in winter (November). The NPQ light response was characterised by a large intraday variability and by the high NPQ values attained under high irradiances. Maximum daily NPQ often reached values above 4.0, and values above 5.0 were observed on 2 days during the November sampling period. Changes in NPQ were generally followed by proportional variations in the DT content, both during exposure to high light and upon transition from darkness to low light, leading to highly significant correlations between NPQ and the DT content or the degree of deepoxidation, DT / (DD + DT). Higher NPQ values in the dark than under low light were consistently observed. This was due to the generalised increase of F mV levels under low light to values higher than the F m measured in the dark, and was associated to the decrease of the DT content upon exposure to low light. Significant seasonal variability was found regarding the parameters related to NPQ induction kinetics, indicating a higher capacity for NPQ operation in November than in July, both in the dark and as a response to high light. This seasonal variation in the NPQ light response was found to be associated to substantial changes in the taxonomic composition of microphytobenthos assemblages, and was interpreted as resulting from changes in the potential photoprotective response associated to thermal acclimation to winter conditions. D 2005 Elsevier B.V. All rights reserved. Keywords: Chlorophyll-a fluorescence; Diatoms; Microphytobenthos; Non-photochemical quenching; Photoprotection; Xanthophyll cycle
1. Introduction * Corresponding author. Tel.: +351 234370787; fax: +351 234426408. E-mail address: [email protected] (J. Seroˆdio). 0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2005.05.011
Microphytobenthos assemblages inhabiting estuarine tidal flats are subjected to a highly variable and extreme light regime caused by rapid changes in
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water cover and by the direct exposure to sunlight for prolonged periods during diurnal low tides. In microalgae, the exposure to photosynthetically supersaturating light triggers the operation of processes that lower the yield of chlorophyll a fluorescence and are thus generally termed as non-photochemical quenching (NPQ) processes (Mu¨ller et al., 2001). In diatoms, these processes include rapidly reversible, photoprotective energy-dissipating mechanisms (denergy-dependent quenchingT, q E) and slowly reversible changes involving damages to the photosynthetic apparatus (dphotoinhibitory quenchingT, q I) (Quick and Horton, 1984; Mu¨ller et al., 2001). Photoprotective q E involves the operation of the xanthophyll cycle, through the rapid and reversible conversion of pigment diadinoxanthin (DD) into the energy-dissipating form diatoxanthin (DT) within the light-harvesting complexes. This leads to the dissipation of excessive energy to non-radiative pathways, decreasing the transfer of captured excitation energy to the photosystem II (PSII) reaction centers and thus limiting the amount of photodamage to the photosynthetic apparatus (Olaizola and Yamamoto, 1994). The photoprotective down-regulation of PSII through the operation of the xanthophyll cycle is well documented for diatoms, having received considerable attention in a number of recent studies (Owens, 1986; Ting and Owens, 1993; Arsalane et al., 1994; Olaizola and Yamamoto, 1994; CasperLindley and Bjo¨rkman, 1998; Jakob et al., 1999, 2001; Lavaud et al., 2002a,b, 2004; Ruban et al., 2004). However, as all these studies have been based on microalgae grown in culture, little is known regarding the operation of NPQ processes in natural diatom-dominated communities. In the particular case of microphytobenthos, no studies have yet been published regarding NPQ induction or the operation of the xanthophyll cycle. The interest on the study of NPQ functioning in microphytobenthos is also justified by the extreme light regime to which microalgae are subjected in their natural environment, and by the hypothesis that benthic microalgae may use their migratory ability to behaviourally regulate light exposure and avoid photodamage (Consalvey et al., 2004). Furthermore, it was recently shown that diatoms may develop an exceptionally high capacity for rapid and large NPQ induction under light stress (Lavaud et al.,
2002a; Ruban et al., 2004). In this study, the operation of NPQ in natural estuarine microphytobenthos assemblages was characterised regarding (i) NPQ induction kinetics under high irradiance and its relationship with the xanthophyll cycle, (ii) the formation of NPQ in the dark, and (iii) the seasonal variability of NPQ induction kinetics and light response.
2. Materials and methods 2.1. Sampling Benthic microalgae were collected on an intertidal mudflat located near Vista Alegre, Canal de ´Ilhavo, on the Ria de Aveiro, a mesotidal estuary located on central west coast of Portugal. Sampling site consists of fine muddy sediments (97% particles b 63 Am), where microphytobenthos assemblages are dominated throughout the year by diatoms of the genera Navicula, Nitzschia, and Gyrosigma. Sampling was carried out in May 2004 (NPQ and pigment content analysis, see below) and during two spring-neap tidal cycles, on July and November 2004 (NPQ induction kinetics and NPQ vs. E curves, see below). Microalgae were collected by placing two pieces of lens tissue on the surface of the sediment shortly after the beginning of diurnal low tide. After ca. 1 h, the upper piece of lens tissue was removed and microalgae were resuspended on filtered sea water. Replicated samples were used for determination of taxonomic composition. Microalgal suspensions were fixed in 1% v/v formaldehyde and viewed using bright-field microscopy for determination of the relative abundance of major taxonomic groups (diatoms, euglenophytes and cyanobacteria). Diatom identification was carried out on subsamples oxidized using saturated potassium permanganate solution and digested in concentrated HCl. 2.2. Chlorophyll fluorescence Variable fluorescence was measured using a Pulse Amplitude Modulation (PAM, Schreiber et al., 1986) fluorometer comprising a computer-operated PAMControl Unit (Walz, Effeltrich, Germany) and a
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WATER-EDF-Universal emitter-detector unit (Gademann Instruments GmbH, Wu¨rzburg, Germany). This fluorometer uses a modulated blue light (LED-lamp peaking at 450 nm, half-bandwidth of 20 nm) as source for measuring, actinic and saturating light, emitted at a frequency of 18 Hz when measuring F o (see notation, Table 1), or 20 kHz when measuring other fluorescence parameters. Fluorescence was measured using a 6-mm diameter Fluid Light Guide fiberoptics bundle or a 1.5-mm plastic fiberoptics, delivering the measuring, actinic and saturating light provided by the fluorometer, connected to a fluorescence cuvette (KS-101, Walz, Effeltrich, Germany). Sample temperature was maintained constant at 20 8C by connecting the fluorescence cuvette to a recirculating water bath (Frigiterm-10, Selecta, Spain). Maximum light levels attained were 920 (6 mm fiber) and 1700 (1.5 mm fiber) Amol quanta m 2 s 1. Due to the frequent occurrence of F mV values under low irradiances higher than the F m value measured after dark-adaptation, NPQ was calculated by adapting the non-photochemical quenching coefficient (Schreiber et al., 1994): NPQ ¼ ðFmV m FmV Þ=FmV
ð1Þ
where F mV m is the maximum F mV value, higher than F m, that is measured under low actinic irradiance. All measurements were carried out on the day of sampling.
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2.3. Pigment analysis Samples were centrifuged (3000 g, 10 min) and pigments were extracted from pellets using 3 ml of 95% cold buffered methanol (2% ammonium acetate). Samples were sonicated for 1 min and stored in the dark at 20 8C for 15 min. Extracts were filtered onto Millipore FluoroporeTM membrane filters (0.2 Am FG) immediately before HPLC analysis. Pigment extracts were analysed using a Shimadzu HPLC comprising a solvent delivery module (LC-10ADVP) with a system controller (SCL-10AVP), a photodiode array (SPD-M10AVP), and a fluorescence detector (RF10AXL). Chromatographic separation was carried out using a C18 column for reverse phase chromatography (Supelcosil, 25 mm long, 4.6 mm in diameter and 5 Am particles) and a 35 min long elution program. The solvents used were 0.5 M ammonium acetate in methanol and water (85:15, v/v), acetonitrile and water (90:10, v/v), and 100% ethyl acetate. The solvent gradient followed Kraay et al. (1992) adapted by Brotas and Plante-Cuny (2003) with a flow rate of 0.6 ml min 1 and an injection volume of 100 Al. Identification and calibration of HPLC peaks were done with chlorophyll-a from Sigma and h-carotene standards and chlorophyll c 2, chlorophyll c 3, fucoxanthin, neoxanthin, violoxanthin, diadinoxanthin, aloxanthin, diatoxanthin, lutein, zeaxanthin and chlorophyll b standards from DHI (Cartaxana and Brotas, 2003). Pigments were identified from absorption spectra and
Table 1 Notation Chl DD, DT DT /(DD + DT) DF / F mV E E NPQmin ETR V F s, F m F mV m F o, F m Fv / Fm k NPQ NPQ NPQ0, NPQm NPQ(E), NPQ(E,t) t
Chlorophyll (mg l 1) Diadinoxanthin and diatoxanthin [mol (100 mol Chl a) 1] Relative content of diatoxanthin [= DT / (DD + DT) 100] (%) Effective quantum yield of PSII (dimensionless) Spectrally averaged photon irradiance of PAR (400–700 nm) (Amol quanta m 2 s 1) Irradiance under which NPQ reaches a minimum value in a NPQ vs. E curve Relative electron transport rate (= E DF / F mV ) (dimensionless) Steady-state and maximum fluorescence emitted by a light-adapted sample (arbitrary units) Maximum F mV value measured under low irradiance (arbitrary units) Minimum and maximum fluorescence emitted by a dark-adapted sample (arbitrary units) Maximum quantum yield of PSII of a dark-adapted sample (dimensionless) Rate constant of NPQ induction (min 1) Non-photochemical quenching of fluorescence [= ( F mV F m) / F mV ] (dimensionless) NPQ at the start and after 6 min of exposure to high irradiance NPQ measured under irradiance E (NPQ vs. E curve), and after t minutes of exposure to irradiance E (NPQ induction experiments) Time of exposure to high irradiance during a NPQ induction experiment (min)
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retention times, and concentrations were calculated from the area of pigment peaks in the photodiode array. Diatoxanthin relative content was calculated as DT / (DD + DT) 100.
assuming a linear relationship between DT content and NPQ, which has been verified for diatoms (Arsalane et al., 1994; Olaizola and Yamamoto, 1994; Lavaud et al., 2002a; Ruban et al., 2004):
2.4. NPQ induction and relaxation
NPQðt Þ ¼ NPQm þ ðNPQ0 NPQm ÞekNPQ t
Microphytobenthos suspensions were concentrated by centrifugation (3000 g, 10 min) and resuspended in filtered sampling site water to a final concentration always higher than 50 Ag Chl a ml 1. Before measuring, samples were supplemented with NaHCO3 (4 mM final concentration) and dark-adapted for 30 min. F o and F m were measured, and the maximum quantum yield, F v/F m, was determined by applying saturating pulses each 2 min during 10 min. Samples were then exposed to low actinic irradiance (44 Amol quanta m 2 s 1) for 15 min, and to 1700 Amol quanta m 2 s 1 for 30 or 60 min. During light exposure, the effective quantum yield of PSII, DF / F mV = [( F mV F s) / F mV] (Genty et al., 1989), was determined each 90 s and the relative electron transport rate was obtained from the delivered actinic irradiance, E, by calculating ETR = E DF / F mV . Actinic light was then switched off and saturating pulses were applied each 2 min during 30 min. NPQ induction experiments were carried out on 5 days along each spring-neap tidal cycle, 2–3 times each day. For comparing the evolution of NPQ and of xanthophyll cycle pigments DD and DT content during exposure to high light, samples were dark-adapted and exposed to low light as described above, after which the actinic light was increased to 920 Amol quanta m 2 s 1. After 1, 2, 3, 5, 10, 15, 30 and 60 min of light exposure, the sample was rapidly (b 10 s) frozen in liquid nitrogen for HPLC analysis, assuring that the xanthophyll cycle was stopped virtually immediately after light exposure. Samples were also collected for HPLC analysis at the end of the dark-adaptation period and immediately after exposure to low light. NPQ was measured immediately before the collection for HPLC analysis, and was calculated by applying Eq. (1) using the highest F mV value measured under 44 Amol quanta m 2 s 1 as F mV m. The NPQ increase following exposure to high light was described by a first-order kinetics model, derived from the model of Olaizola and Yamamoto (1994) for the variation of DT concentration under high light by
ð2Þ
where NPQ0 and NPQm are the values measured immediately before the start of the high-light period and after the complete de-epoxidation of the DD pool to DT following a first-order kinetics, respectively, and k NPQ is the rate constant of NPQ increase. k NPQ was estimated by fitting Eq. (2) to the first 6 min of the time series of NPQ measurements under high light. The model was fitted iteratively using MS Excel Solver. 2.5. NPQ vs. E curves The light response of NPQ was characterised by constructing NPQ vs. E curves, by measuring NPQ on microalgal suspensions exposed to increasing irradiance levels. After 30 min of dark-adaptation, F o and F m were measured, and the sample was subsequently exposed to 12 increasing irradiance levels, from 21 to 1700 Amol quanta m 2 s 1. Under each irradiance level, F s and F mV were measured each 90 s, until a steady-state in DF/F mV was reached (minimum 7.5 min). NPQ was calculated by applying Eq. (1) using the highest F mV value measured as F mV m. NPQ vs. E curves were constructed 2–3 times each day, on the days when NPQ induction experiments were carried out.
3. Results 3.1. NPQ induction and relaxation Fig. 1 illustrates the pattern of variation of NPQ and DF / F mV during the sequential exposure to low and high irradiance of dark-adapted microphytobenthos samples. Upon the transition to low light, a decrease in NPQ of dark-adapted samples was often observed, usually leading a steady-state within 15–20 min. During this period, DF / F mV decreased immediately after transition to low light, but in many cases it recovered to values close or above the F v / F m value measured at the end of the dark period (Fig. 1B). Following an
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Fig. 2. NPQ induction kinetics in microphytobenthos suspensions exposed to 1700 Amol quanta m 2 s 1, and fit of Eq. (2) for the estimation of the rate constant of NPQ induction (k NPQ). Same NPQ data as displayed in Fig. 1.
Fig. 1. Typical changes in F v / F m, DF / F m V and NPQ induction kinetics in dark-adapted microphytobenthos suspensions exposed to low (44 Amol quanta m 2 s 1; gray bar) and high (1700 Amol quanta m 2 s 1; white bar) irradiance and subsequent recovery and relaxation in the dark (black bars). Samples collected in 24 July (A) and 15 November 2004 (B).
initial large decrease with the start of the high-light exposure (on average, 74.3%), DF / F mV recovered gradually, usually reaching a steady-state within 30 min. This variation in DF / F mV was paralleled by a rise in NPQ that comprised an initial rapid and large change followed by a slower increase, usually leading to a steady-state within 20–30 min of high-light exposure. The mean values of the NPQ measured after
30 min of high-light exposure, NPQ(1700,30V), calculated for each sampling period ranged from 1.97 in July to 3.11 in November (Table 2). NPQ(1700,30V) attained a maximum value of 5.36 in the November sampling period. In both sampling periods, F o and F m levels were lower after returning to darkness than before the light stress, and F v / F m recovered rapidly and almost completely to the initial values. Considering the two sampling periods, the recovery of F v / F m reached, on average, 87.2% after 10 min and 92.5% after 20 min. The fit of Eq. (2) to the first 6 min of the NPQ time series was in all cases very good, with the model explaining over 97% of the variability (Fig. 2). Model fit decreased for longer NPQ time series, indicating that the NPQ increase after 6 min was due to processes not described by a first-order kinetics. k NPQ
Table 2 Mean values of the parameters used to characterise the NPQ induction kinetics and the NPQ vs. E curves, measured in the sampling periods of July (n = 13) and November (n = 12) 2004 NPQ induction
July November a
NPQ vs. E curves
F v / F ma
DF / F mV (44)
DF / F mV (1700)
F v / F mb
k NPQ
NPQ (1700,30 V)
NPQ (0)
NPQ (1700)
E NPQmin
0.614 0.553 *
0.591 0.550 ns
0.171 0.130 ns
0.580 0.509 ns
0.52 0.74 *
1.97 3.11 **
0.45 0.80 *
1.50 3.43 ***
178.5 125.7 ns
Measured after 30 min of dark-adaptation. Measured after 20 min recovery in the dark. Numbers in the table header are irradiances in Amol quanta m2 s1 under which DF / F mV and NPQ were measured. Levels of statistical significance of Student’s t-test used to compare mean values. b
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ranged from 0.16 to 1.18 min 1, attaining mean values of 0.52 and 0.74 min 1 for July and November, respectively (Table 2). Although the described patterns of NPQ induction and relaxation were consistently observed throughout the studied periods, large intraday variability in the NPQ light response was frequently found. Considering both sampling periods, intraday variability (c.v.) in k NPQ and NPQ(1700,30V) ranged from 3.9% to 61.6%, and from 8.2% to 67.1%, respectively. 3.2. NPQ and DT The variations in NPQ observed upon changes in irradiance were generally followed by proportional variations in DT content. This was observed under exposure to high light, during which the build-up of NPQ was closely paralleled by a proportional increase in DT concentration (Fig. 3). Parallel variations in NPQ and DT were also observed during exposure to low light following dark-adaptation, both decreasing from the high values measured at the end of the dark period (Fig. 3), and leading to highly significant correlations between NPQ and DT / (DD + DT) or DT content (r 2 = 0.873, P b 0.001 and r 2 = 0.751, P b 0.001, respectively; Fig. 4). The decrease in NPQ under low light was due to the steady increase of F mV to values higher than the F m value measured after dark-adaptation (Fig. 3). In all cases, the DT
Fig. 4. Linear relationship between NPQ and DT / (DD + DT) during exposure to high irradiance (920 Amol quanta m 2 s 1).
content in dark-adapted samples was higher (averaging 0.64 F 0.075 mol DT (100 mol Chl a) 1) than after exposure to low light (0.59 F 0.028 mol DT (100 mol Chl a) 1). The DD pool size in dark-adapted samples averaged 9.64 mol DD (100 mol Chl a) 1. 3.3. NPQ vs. E curves Higher NPQ values in the dark, NPQ(0), than under low light were also consistently observed in the NPQ vs. E curves constructed during the two
Fig. 3. Decrease of NPQ and DT, and increase of F mV above F m upon transition from darkness (black bar) to low irradiance (44 Amol quanta m 2 s1; gray bar) of a dark-adapted microphytobenthos sample, and subsequent NPQ induction and DT production upon exposure to high irradiance (920 Amol quanta m 2 s 1; white bar) for 60 min.
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Fig. 5. Typical light responses of F mV (normalised to F m) and NPQ in microphytobenthos suspensions, showing a clear increase in F mV above F m and correspondent NPQ decrease for E b E NPQmin, and an increase for E N E NPQmin.
spring-neap tidal cycles (Fig. 5). The decrease of NPQ upon illumination with low irradiances was due to a marked increase in F mV to levels above F m. Except for one case, all curves showed high NPQ(0) values that decreased steeply with irradiance until a minimum was reached. The irradiance at which NPQ was minimum, E NPQmin, averaged 178.5 and 125.7 Amol quanta m 2 s 1 in July and November, respectively (Table 2). On average, NPQ(0) values reached 0.45 in July and 0.80 in November. For irradiances above E NPQmin, NPQ increased virtually linearly with irradiance, reaching maximum values at 1700 Amol quanta m 2 s 1 that were typically 2–4 times higher than those observed in the dark. Average NPQ(1700) values for each tidal cycle were similar to those measured in the NPQ induction experiments. 3.4. Seasonal variability Significant differences were found between the July and November spring-neap tidal cycles regarding the parameters related to NPQ operation (Table 2). In general, these results indicate a higher capacity for NPQ induction in November than in July, both in the dark and as a response to high light. F v / F m was significantly lower in November than in July, probably related to a higher formation of NPQ in the dark and
not to a lower potential efficiency of the photosynthetic apparatus as its performance under actinic light (DF / F mV at 44 and at 1700 Amol quanta m 2 s 1) and F v / F m recovery were not significantly different in both periods (Table 2). Accordingly, NPQ in the dark was also significantly higher in November (Table 2), this difference becoming higher when considering NPQ under low irradiances such as 21 Amol quanta m 2 s 1. Considering the response to high irradiance, significant differences were found both regarding the initial rate of NPQ induction (k NPQ) and the maximum NPQ attained (both in the NPQ induction experiments and in the NPQ vs. E curves), all significantly higher in November (Table 2). Regarding E NPQmin, no significant differences were found between the two periods, although average values were higher in July. This large seasonal variation in the NPQ light response was associated to substantial changes in the taxonomic composition of the microphytobenthos assemblages regarding the dominant species of diatoms. In both sampling periods, the diatom species composition was relatively constant among the different days, with maximum variation in the relative abundance of individual species remaining below 5%. In July, five diatom species accounted, on average, for 87.0% of total species relative abundance (Table 3): Nitzschia frustulum (Ku¨tzing) Grunow,
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Nitzschia perspicua Cholnoky, Parlibellus crucicula (W. Smith) Witkowski, Lange-Bertalot & Metzeltim, Navicula cf. gregaria Donkin and Nitzschia draveillensis Coste and Ricard. The relative abundance of the remaining diatom species was in all cases less than 2%. In November, the number of species with relative abundances above 2% increased to 10, of which four also had abundances above 2% in the July sampling period: N. cf. gregaria, P. crucicula, N. frustulum and N. perspicua. During this period, the microphytobenthos assemblages were largely dominated by Gyrosigma fasciola (Ehrenberg) Griffth and Henfrey, with relative abundances ranging from 35.6% to 49.8%, and averaging 41.5% of total cell counts (Table 3). In July, the maximum abundance of this species reached only 1.9%. Remaining species with abundances above 2% were Nitzschia acuminata W. Smith, Entomoneis alata (Ehrenberg) Ehrenberg, Tryblionella apiculata Gregory, Navicula phyllepta Ku¨tzing, and Entomoneis paludosa (W.Smith) Reimer, but in all cases abundances were less than 7%. In both sampling periods, euglenophytes (Euglena sp.) and cyanobacteria (Merismopedia sp., Oscillatoria spp.) accounted for less than 1% of cell counts.
Table 3 Mean relative abundance (%) of the main taxa found in microphytobenthos suspensions collected in the sampling periods of July and November 2004 Taxa Diatoms Nitzschia frustulum (Ku¨tzing) Grunow N. perspicua Cholnoky N. draveillensis Coste and Ricard N. acuminata W. Smith Parlibellus crucicula (W. Smith) Witkowski, Lange-Bertalot and Metzeltim Navicula cf. gregaria Donkin N. phyllepta Ku¨tzing Gyrosigma fasciola (Ehrenberg) Griffth and Henfrey Tryblionella apiculata Gregory Entomoneis alata (Ehrenberg) Ehrenberg E. paludosa (W. Smith) Reimer Other diatoms Euglenophytes Cyanobacteria
July
November
30.3 25.2 5.7 – 18.7
5.4 3.5 – 6.6 5.9
6.9 – –
6.6 3.5 41.4
– – – 12.3 0.4 0.3
4.6 5.1 2.8 14.1 0.3 0.1
Mean values obtained by averaging the relative abundances determined for each sampling date in each spring-neap cycle. For diatoms, only relative abundances N 2% are presented.
4. Discussion 4.1. NPQ induction kinetics The results obtained on the kinetics of NPQ induction by microphytobenthos assemblages revealed a comparatively high capacity for the rapid development of large NPQ under exposure to supersaturating irradiances. Despite the large intraday variability observed in the NPQ light response, daily maxima of k NPQ and NPQ(1700,30V) were in many cases higher than the values published for diatoms exposed to 2000 Amol quanta m 2 s 1 (Table 4). This was particularly true in the case of NPQ(1700,30’) in the November sampling period, when 50% of the values were higher than 3.0, and 25% higher than 4.0. These values are higher than the highest NPQ values measured in microalgae or plants, for which NPQ = 4.0 is considered as the maximum ordinarily reached (Rohae`ek, 2002; Ruban et al., 2004). NPQ values significantly higher than 3.0 have been reported for diatoms, but only for Phaeodactylum tricornutum grown under an intermittent light regime (Lavaud et al., 2002a; Table 4). It is conceivable that even higher NPQ values could have been measured in microphytobenthos since, as some results suggest (NPQ continuing to decrease at the end of the low-light exposure; Fig. 1B), F mV could have increased to higher levels if the exposure to low light following prolonged darkness had been longer. The continuous increase of NPQ during the 30 min of light stress only partially resulted from the deepoxidation of the cellular DD pool, as shown by the decrease of the fit of the first-order kinetics model (Eq. (2)) for NPQ time series longer than the first 6 min of high-light exposure (Fig. 2). The slower increase in NPQ after this initial period could be due to de novo synthesis of DD or to damages caused to the photosynthetic apparatus ( q I), unrelated to DT production. However, under the experimental conditions applied, the NPQ developed by microphytobenthos appears to result mostly from the operation of photoprotective energy-quenching processes ( q E). This is supported by the rapid recovery of F v / F m after the light stress period, which has been taken as an indication of the occurrence of rapidly-reversible down-regulation of PSII rather than the onset of chronic photoinhibition (Schofield et al., 1998;
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Table 4 NPQ induction kinetics (k NPQ, NPQ(E,30V)) measured in this study and published results for diatoms Source
Microalgae
Growth conditions
E
k NPQ
NPQ(E,30 V)
Arsalane et al. (1994) Casper-Lindley and Bjo¨rkman (1998) Lavaud et al. (2002a)
P. tricornutum P. tricornutum P. tricornutum
2000 1200 2000
Lavaud et al. (2004)
P. tricornutum Skeletonema costatum Microphytobenthos
100, 12/12 h 100, 15/9 h 40, 16/8 h 40, 5/55 min 40, 16/8 h July November
1700
0.40 0.71 0.46 0.72 0.68 1.11 0.46–0.79 0.54–1.18
2.05 1.75 3.12 10.23 2.60 0.92 2.05–2.75 1.72–5.36
This study
2000
Growth conditions: irradiance (Amol quanta m2 s1 ) and photoperiod (light/dark period), or sampling period. k NPQ was estimated by fitting Eq. (2) to published values (goodness of fit in all cases N0.95).
Ralph et al., 2002). Furthermore, the parallel decrease of F m and F o observed after the exposure to supersaturating irradiance suggests the operation of photoprotective mechanisms, because damage to the photosynthetic apparatus normally results in the decrease of F m and in the increase of F o (Gilmore et al., 1996; Schofield et al., 1998; Mu¨ller et al., 2001; Ralph et al., 2002). The correlation found between NPQ and the degree of de-epoxidation, DT / (DD + DT), during the 60 min of high-light exposure indicates that NPQ operation under the tested conditions is based on the formation of DT. This further confirms the minor effects of photoinhibition, which would have lead to a larger increase in NPQ than in DT / (DD + DT) (Arsalane et al., 1994). This result supports the use of k NPQ as an estimator of the initial rate of xanthophyll de-epoxidation. 4.2. NPQ induction in the dark A consistent result regarding the operation of NPQ in microphytobenthos was the generalised occurrence of high NPQ values in dark-adapted samples and its gradual decrease under low light, due to the increase of F mV to values higher than the F m measured in the dark. The relaxation of NPQ upon exposure to low light was associated to the decrease of the cellular DT content and of the degree of xanthophyll de-epoxidation, with considerably high levels of de-epoxidation (DT / (DD + DT) N 5%) being found in dark-adapted samples. Relatively high degrees of de-epoxidation were also measured after exposure to low irradiance (Fig. 3), causing the observation of high DT / (DD + DT) values while NPQ = 0 (i.e., negative intercept of the linear regression line in Fig. 4). This was
probably due to the fact that the exposure to low light was insufficient to completely epoxidate the DT formed in the dark, and that NPQ was calculated on the basis of the maximum F mV level. The relationship between NPQ and DT / (DD + DT) may be affected by the presence of Euglenophytes, microalgae that contain DD but do not produce DT as a response to high light (Casper-Lindley and Bjo¨rkman, 1998). A significant presence of Euglenophytes, however, should have contributed to lower the measured degree of de-epoxidation (for the same NPQ level) and could not explain the high levels measured in the dark and under low light. The formation of NPQ in the dark ( F mV N F m) has been reported for diatoms (Falkowski et al., 1986; Demers et al., 1991; Ting and Owens, 1993; Geel et al., 1997; Casper-Lindley and Bjo¨rkman, 1998; Jakob et al., 1999; Mouget and Tremblin, 2002) and microphytobenthos assemblages (Seroˆdio, 2004). Nevertheless, the formation of NPQ or DT in the dark was not detected in a number of studies on the operation of the xanthophyll cycle in diatoms (Arsalane et al., 1994; Olaizola and Yamamoto, 1994; Dijkman and Kroon, 2002; Lavaud et al., 2002a, 2004). The observation of F mV levels higher than F m was shown to result from the activation of the DD de-epoxidase, and consequent de-epoxidation of DD to DT, as induced by the formation of a transthylakoidal proton gradient, as when responding to supersaturating irradiances (Ting and Owens, 1993; Jakob et al., 1999; Lavaud et al., 2002b). As a result of the accumulation of DT in the dark, absorbed light energy is diverted from the PSII reaction centers, lowering the F m emission. Under low light levels, the activation of ATP synthase and carbon metabolism dissipate the proton gradient
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at a higher rate than the proton accumulation caused by PSII activity, leading to the gradual epoxidation of DT to DD, the relaxation of NPQ, and the increase of F mV levels above F m. This explanation is supported by the observation of the simultaneous decrease of NPQ and DT content and the increase of F v / F m upon exposure to low irradiance after prolonged darkness (Jakob et al., 1999). In diatoms, the formation of a pH-gradient in the dark has been generally attributed to chlororespiration (Ting and Owens, 1993; Olaizola and Yamamoto, 1994; Jakob et al., 1999), although other physiological processes, such as CO2-concentration mechanisms and acetate accumulation during lipid degradation, may also contribute to lower the thylakoid lumen pH and induce the xanthophyll deepoxidation, decreasing F m (Jakob et al., 2001). The high NPQ levels in the dark consistently observed in natural samples of microphytobenthos may result from an increased DD cellular pool, allowing high levels of DT production and accumulation. However, the capacity for the formation of NPQ in the dark appear not to be related with a large DD pool, as diatoms containing an artificially-induced large DD pool did not show any DT accumulation in the dark (Lavaud et al., 2002a). NPQ induction in the dark could also result from an increased content or activity of DD de-epoxidase, enabling higher degrees of deepoxidation of a small DD pool. DD de-epoxidase activity could be increased either by requiring a lower transthylakoidal proton gradient for activation, or by an increased activity of physiological processes causing the intrathylakoidal proton accumulation (Jakob et al., 2001). The capacity to develop NPQ in the dark has been considered an adaptive advantage, representing as a way to prevent degradation of xanthophyll cycle pigments during prolonged periods of darkness, thus providing readily functional photoprotection upon reillumination by otherwise photodamaging light levels (Hoefnagel et al., 1998; Jakob et al., 1999). In the case of microphytobenthos inhabiting estuarine intertidal flats, benthic microalgae are frequently subjected to rapid changes in light exposure, including the sudden exposure to high levels of sunlight after withstanding long periods in the dark. This occurs frequently, when tidal ebb takes place during the day, exposing microalgal biofilms to full sunlight after a continuous period of several hours in dark-
ness during night or high tide. Exposure to solar light after long periods of darkness may also take place when microalgae buried in aphotic layers of the sediment are resuspended and brought to the surface. The maintenance of functional xanthophyll cycle pigments by benthic diatoms can thus be considered as advantageous and may explain the high levels of NPQ in the dark exhibited by microphytobenthos samples. 4.3. Seasonal variability: NPQ light response and acclimation Significant seasonal variability was found regarding the fluorescence indices related to maximum NPQ capacity (NPQ(1700), NPQ(1700,30V)), NPQ in the dark ( F v / F m, NPQ(0)), and NPQ induction kinetics (k NPQ). Higher values were observed in November, suggesting important changes in the potential photoprotective response of the assemblages associated to acclimation to winter conditions. Seasonal changes in NPQ operation may be expected from the variation in the pool of xanthophyll cycle pigments, since it has been shown that the xanthophyll pool size increases as a response to lower growth temperatures, both in diatoms (Anning et al., 2001) and in plants (Mu¨ller et al., 2001). The enhanced NPQ operation observed in November could thus result from an increase of the DD pool size (and other factors affecting DT production, see above) as a result of acclimation to lower temperature. The exposure to direct sunlight under low temperatures can be particularly damaging to the photosynthetic apparatus, as the operation of the xanthophyll cycle is an enzyme-mediated process, and low temperature may thus be expected to slow down the photoprotective response under high light. An increase in the content of the xanthophyll cycle pigments in winter would thus be advantageous for microphytobenthos cells, enhancing photoprotection against photodamages by allowing to attain higher degrees of de-epoxidation at low temperatures. The increase in the NPQ in the dark in November (and consequent lower dark-adapted F v / F m) would thus represent a side effect of the increase in the DD pool size and of the capacity for NPQ operation. Because the DD content has been shown to increase with growth irradiance in various microalgae
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(Casper-Lindley and Bjo¨rkman, 1998; Moisan et al., 1998; Meyer et al., 2000; Rijstenbil, 2003), a decrease in the DD pool size could be expected in November considering the lower irradiance levels, thus counterbalancing the effect of lower temperatures. However, due to the high water turbidity in estuaries (Seroˆdio and Catarino, 1999) and to the migratory behaviour of benthic microalgae (Consalvey et al., 2004), microphytobenthos biofilms are exposed to light almost only during diurnal low tides. During these periods, microalgae are exposed to direct sunlight which, even during winter months, reaches considerably high levels (N1000 Amol quanta m 2 s 1), justifying the maintenance of high levels of photoprotection. Considering the substantial changes in taxonomic composition that were found between July and November, the seasonal increase in the NPQ capacity may be interpreted as resulting from the diatom species succession, associated to a potential competitive advantage of populations with the ability of developing a higher photoprotection capacity. In phytoplankton, the magnitude of xanthophyll cycling has been considered as a process for species succession (Meyer et al., 2000). However, the community composition might have changed due to causes unrelated to photoprotection, and the increase in NPQ capacity may be the result of the ability of most species to physiologically acclimate to changing light and temperature conditions on a seasonal time scale. The pattern of the NPQ vs. E curves found for microphytobenthos is similar to the NPQ light re¨ quist, sponse of cyanobacteria (Campbell and O 1996; Green and Oliver, 2003). Although the variation of NPQ under low light observed in cyanobacteria has been attributed to state transitions, a physiological process that does not occur in diatoms (Owens, 1986; Ting and Owens, 1993; Lavaud et al., 2002a), in both cases NPQ attains a minimum value at relatively moderated irradiances (E NPQmin). In cyanobacteria, a relationship was found between E NPQmin and the photoacclimation status of the microalgae (Camp¨ quist, 1996). The similarity between the bell and O NPQ vs. E curves in the two groups of microalgae and the fact that E NPQmin is determined by the magnitude of NPQ in the dark, its relaxation under low light, and its onset under high light, factors shown to be associated to thermal acclimation or photoacclimation in diatom-dominated microphytobenthos, suggest the
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possibility that a similar relationship may also exist for diatoms. As with cyanobacteria, this would allow to determine the integrated light regime to which natural assemblages of microalgae are acclimated (Green and Oliver, 2003). Although no significant seasonal variability was found regarding E NPQmin in this study, the higher mean value observed in July concurs with what would be expected in the case of photoacclimation to higher irradiances. 4.4. Measuring NPQ in undisturbed microphytobenthos While the study of benthic microalgae in suspension may provide valuable information on their potential photoprotection capacity, the obtained results may not represent the physiological response of undisturbed microphytobenthos assemblages under natural conditions. Because much of the particular physico-chemical conditions of the microenvironment of benthic biofilms are lost when the microalgae are resuspended, the characterization of the photoprotective response of intact microphytobenthos assemblages can only be adequately assessed through the measurement of NPQ in undisturbed sediment samples. This, however, is strongly hampered by two types of factors. First, the vertical migratory response of benthic microalgae to changes in light conditions, including darkening, and its large impact on fluorescence parameters (Seroˆdio et al., 1997) makes it virtually impossible to apply NPQ indices based on the F m level. Second, light and fluorescence attenuation in the upper layers of the sediment and the depth-integration of fluorescence signals may cause the light-dependent underestimation of NPQ indices (Seroˆdio, 2004). This study has shown that the application of fluorescence quenching analysis to benthic microalgae is further complicated by the frequent formation of NPQ in the dark and resulting F mV values higher than F m, as it leads to negative NPQ values (Ting and Owens, 1993; Geel et al., 1997; Mouget and Tremblin, 2002). In conclusion, the study of NPQ in intact microphytobenthos assemblages presents problems that cannot be adequately overcame with the methods currently available. Alternative approaches must be developed that allow for the non-invasive monitoring of NPQ indices independently of F m quenching in the dark and of changes
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in vertical distribution of microalgae within the photic layers of the sediment.
Acknowledgements We wish to thank Carlos Rafael Mendes for help in the HPLC analysis, Helena Coelho for help in field and laboratory work, and Jorge Marques da Silva for critical comments on the manuscript. This work was supported by project POCTI/MAR/15318/99, funded by Fundac¸a˜o para a Cieˆncia e a Tecnologia, and by Programme PRODEP III. We thank one anonymous reviewer for critical comments on the manuscript. [SS]
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