effects of solar radiation on photosynthesis, uv-absorbing compounds and enzyme activities of the...

Journal of PhotociFmistry

Photobiology

ELSEVIER B:BiologT”

J. Photochem. Photohiol. B: Biol. 47 ( 199X) 46-57

Effects of solar radiation on photosynthesis, UV-absorbing compounds and enzyme activities of the green alga DasycZadus verrnicularis from

southern Spain

Received 26 July I 9%; accepted I h October 1998

Abstract

The effect of different wavebands of solar radiation (photosynthetically active radiation (PAR), ultraviolet A (UV-A) and ultraviolet B ( UV-H ) ) produced by use of cut-off filters on chlorophyll fluorescence of the green alga Dusyclud~ vrrmiculuris was assessed in summer- autumn 1996 at a shallow site in Cabo de Data-Nijar, southern Spain. Similar experiments were carried out under outdoor conditions at Milaga during summer and autumn 1997. In plants growing under in situ natural light conditions (2.5 m depth), the yield of variable chlorophyll fluorescence ( AF/ F,,,’ ) decreases with increasing sunlight. The full solar spectrum (PAR + UV-A + UV-6) has more accentuated, long- lasting effects on fluorescence than irradiation deprived of UV-B. In general, decreases in AFIF,,,’ do not exceed 30% in the three treatments. Under outdoor conditions, photoinhibition measured as a decrease in optimum quantum efficiency (F, /F,,,) varies between 40 and 7S% with no obvious differences between treatments; however, recovery of photosynthesis after shade exposure is fasterinplants treated withPAR + UV- A. Daily changes in nitrate reductase (NR) and carbonic anhydrase (CA] activities are antagonistic during the onset of natural radiation. The concentration of UV-absorbing compounds with maximum absorption at 348 and 332 nm is higher than that reported for other green algae. These compounds increase in plants exposed to the full solar spectrum ( PAR t UV-A + UV-B) and decrease under PAR alone and PAR + UV- A conditions at noon. which underlines a possible photoprotective mechaGm. Overall, data show that D. r~~rmic~luris is able to tolerate high solar radiation. Two physiological strategies seem to be basically active: dynamic photoinhibition at noon and an enhanced concentration of UV-screening substances. 0 1998 Elsevier Science S.A. All rights reserved.

1. Introduction

Since the detection of stratospheric ozone depletion, which has resulted in an increase in UV-B radiation [ 1,2], a con- siderable effort has been made to assess its deleterious effects on photosynthetic organisms, especially in terrestrial plants and phytoplankton [ 3-5 ] . In contrast, the effects of UV radiation on photosynthesis of benthic marine macroalgae have been less addressed [6-81, although these organisms are ecologically important in coastal primary production, serving as a food source for herbivores and detritivores, as well as a nursery area forjuvenile invertebrate and fishes. The majority of these experimental approaches in macroalgae have heen conducted under artificial irradiation [ 9-121, The difficulty

‘* C’orrcsponding author. Tel.: t 0471-481 I-334: Fax: + 0471-4X31- 13’).

E-muI: IgomerCn:awi-hremerhaven.dc

of accurately reproducing natural sunlight obviously restricts the significance of some responses and, hence, experimentation using natural solar radiation has become a priority. For example, studies in the kelp Ecklonia radium [ 131, the red alga Chondrus crispus [ 141 and several Arctic macroalgae [ 1.5,16] revealed changes in organic composition and photoinhibition of photosynthesis under current natural flux of UV radiation.

In southern Spain, photosynthesis under natural solar radiation of a variety of intertidal and upper sublittoral algae is characterized by dynamic photoinhibition at noon and recovery in the afternoon [ 17-20 1, whereas species from depths greater than IO m show chronic photoinhibition (or even photodamage). A delay in recovery becomes particularly evident when subtidal plants are exposed to surface irradiance levels [ 17,201. The intense photosynthetically active radia-

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I. G6mez et al. /J. Photochern. Photohid. B: Bid. 47 (I 998) 46-57 47

Water surface

f

I-‘-------/--._,- --\.,-- ----‘- .____.. ._ _ ./----,_.,-‘. ____ .- - ----. ~- . .-,. _,_,

Ultraphan (395 nm)

Stainless-steel sticks

Fig. I. Schematic drawing illustrating the cut-off filter design set at a shallow site in Cabo de Gata-Nijar during lield experiment on 20-24 Sept., 1996.

tion (PAR; 400-700 nm) commonly measured in this region has prompted attempts to evaluate whether natural PAR and UV radiation have differential effects on photosynthesis in these macroalgae. For some species. e.g., the endemic intertidal red alga Rissoellu verruculosa, levels of PAR close to 2000 p,mol m ’ s ‘, typical of a summer midday, exert a similar photoinhibitory effect on photosynthesis to the full solar spectrum including UV-A and UV-B radiation [ 2 11. In contrast, the upper intertidal red alga Porphyru leucosticftr is more photoinhibited by natural 1JV radiation than by PAR alone [ 191. Such findings emphasize the need for further field approaches to understand fully the photobiological strategies of algae adapted to enhanced solar radiation.

The present study expands such investigations and was designed to examine to what extent exposures to actual regimes of natural solar radiation affect photosynthesis and the concentration of UV-absorbing compounds in both field plants and specimens incubated under outdoor conditions. Basically, the work deals with the daily patterns of photoinhibition and recovery of photosynthesis as well as t,olerance capacity to short-term solar stress. To distinguish between the effects due to different solar spectrum components ( UV- A. UV-B and PAR), manipulations of specific wavelengths using cellulose acetate cut-off filters were carriedout. Finally, related processes such as changes in activities of key nutrient transport enzymes were also determined in order to give new insights into the whole photobiological strategy of the alga.

2. Materials and methods

2.1. Field experiments

The effect of in situ solar radiation on photosynthetic performance of Dasycladus vermicularis was assessed in plants growing on a shallow subtidal sandy platform at Cabo de Gata-Nijar (36” 52’ N, 2” 12’ W, Almeria) during September 1996. Thalli attached to stones at a depth of 2.5 m were covered with three different acetate cut-off filters using PVC-

stainless steel frames tied to the bottom (Fig. 1). Thus a combination of three radiation treatments was set up: 1. PAR + UV-A + UV-B, by use of Ultraphan 295 (Digefra

GmbH, Munich, Germany). 2. PAR + UV-A, by use of Folex 320 (Folex GmbH,

Dreieich, Germany). 3. PAR alone, by use of Ultraphan 395 (Digefra GmbH,

Munich, Germany). To evaluate the degree of acclimatization of algae, the

system was maintained for four days under these conditions and daily cycles from 0900 to 1800 h of pulse amplitude modulated (PAM) fluorescence were carried out on the first day and after four days’ exposure. Every two hours, plants were removed from the bottom by SCUBA diving and immediately measured for chlorophyll fluorescence at a site close to the experimental system.

2.2. Outdoor system

Algal material was collected in July 1997 and the experiments were carried out using a tank system placed on a roof at the campus of Universidad de Maaga, Mrilaga (36”47’ N, 0.4”19’ W). After sampling, algae were transported to the laboratory in an ice-chest, cleaned from epibionts and maintained under artificial illumination (80 p,mol photon m 2 s-l; daylight lamps, Osram L2OW/ 10s; 12: 12 h 1ight:darkness regime) for five days in 5 1 Plexiglas beakers containing filtered seawater at 16°C. The seawater was renewed every two days. Forphysiological experiments, algal material of approximately 50 g fresh weight (FW, 70-80 individual thalli) was transferred to the outdoor conditions consisting of three 0.5 1 cuvettes each supplied with constant bubbling of air. The cuvettes were placed in a thermostatically controlled water bath ( 16-20”(Z). Similarly to the in situ experiments, the algae in the cuvettes were covered with the cut-off filters previously described (Fig. 2). Measurements of Chl fluorescence and activities of key nutrient transport enzymes ( nitrate reductase and carbonic anhydrase) and UV- absorbing compounds were performed from 0830 to 1900 h at intervals of 2 h on 4 July, 1997. In a second experiment

I. G&m et al. /.I, Photochenz. Photohid B: Bid. 47 (1998) 46-57

Folex 320 nm

(PAR+,UVA) , (PAR+UVA+UVB)

I. .___^ ̂ ^.... I

Fig. 2. Outdoor system placed on the roof of the Science Faculty of Univer-

sity of Maaga during two daily cycles on 4 June and 23-24 Oct. 1097. Before physiological measurements, algae were pre-incubated under artificial white light (70-80 pmol m-‘s ’ ) for several days and then maintained

for some hours under a light-shading screen.

carried out on 23-24 Oct., 1997, the photoinhibition and the capacity of recovery of photosynthesis were analysed in plants exposed for 2 h to solar radiation at noon and then shaded for 24 h. To induce recovery of photosynthesis, plants were covered with a light-shading screen and measured at different time intervals (2,4, 20 and 24 h) .

2.3. Light measurements

The in situ incident irradiance at Cabo de Gata-Nijar was monitored over the entire day with a PUV 5 IO radiometer (Biospherical Instruments Inc., USA) positioned adjacent to the experimental site. This device recorded instantaneous irradiance at four UV bands (305,320,340 and 380 nm) and PAR (400-700 nm) . The incident irradiance at 2.5 m depth was estimated using light attenuation coefficients (&) cal- culated from light profiles (twice during the day) made using an underwater PUV 500 radiometer. It must be emphasized that the coast of Cabo de Gata-Nijar is characterized by very clear waters (Type I, Jerlov classification) and good weather conditions throughout the year. Therefore, marked variations in the light attenuation patterns during the day would be unlikely. Daily doses of quantum solar energy (kJ m ‘1 on each day were determined by integrating the instantaneous irradiances from dawn to sunset. Because the PUV 5 10 instrument does not provides broad-UV records, an estimation of UV-A (320-400 nm) and W-B (280-320 nm) using two linear equations described by Orce and Helbling 1221 was necessary:

where I 34,J, 1380, 1,,,,, lq2,, are the readings from the PUV 5 10 radiometer. Daily doses of PAR were integrated directly from the PUV 5 10 data.

In the outdoor experiments, the incident solar radiation received by the plants over the day and during the short-term exposures was recorded with an ELDONET dosimeter (Real Time Computer, Erlangen, Germany) at three different bands,280-315nm(UV-B),315-400nm(UV-A) and400- 700 (PAR). PAR readings in W m- ’ were converted to quantum irradiance (1 pmol photon m -’ s- ’ ) using the conversion factor 4.6. The light-shading screen used forrecovery lowered the incident PAR received by plants to values lower than 100 kmol photon m-’ s- ‘, whereas UV radiation was completely excluded.

It must be mentioned that the use of different instrumen- tation for light measuring resulted in differences, particularly of UV irradiance values. A key factor is the fact that the integrated readings from the solar spectrum vary significantly near the upper limit of the UV-B region (3 10 and 320 nm) . For example, the UV-B integral from the ELDONET dosimeter was 70% of the value estimated using the conversion described by Orce and Helbling [22] and 80% when data were compared to the integral between 300 and 3 15 nm from an Li-1800 spectra-radiometer (Licor Inc., Lincoln, USA). Therefore, inter-calibrations with this latter instrument were used to check our light data.

2.4. Determination oj’ W-absorbing compounds

The absorbance of UV-absorbing compounds was monitored in thalli incubated under natural solar radiation (outdoor) throughout an entire day. Additionally, seasonal changes in these compounds were assessed in samples ( lO- 20 mg dry weight, DW) collected at Cabo de Gata-Nijar during 1996 and 1997 and at two sites in Mexico and the Canary Islands (see below).

The absorbance of Fhe UV-absorbing compounds was first determined by high-pressure liquid chromatography (HPLC) after extraction for 1.5-2 h in screw-capped centri- fuge vials with 1 ml 25% (vol./vol.) aqueous methanol in a water bath at 45°C. Vacuum-dried extracts (Speed Vat Con- centrator SVC IOOH) were re-dissolved in 800 pJ methanol ( 100%) and then filtered through a 0.2 km membrane. Sam- ples were then measured using a water HPLC method modified from Ref. [23]. Basically, the W-absorbing compounds were first separated on a stainless steel Knauer Sperisorb RP-8 column (5 pm, 250X4 mm id.) protected with an RP-8 guard cartridge C 20 X 4 mm id.). The mobile phase containing 25% aqueous methanol (voI./vol.) and 0.1% acetic acid (vol./vol.) in water was then run isocrati- tally at a flux rate of 0.7 ml min -- ‘. The W-absorbing compounds were detected at 331) nm and absorption spectra obtained by scanning (290-400 nm) directly on the HPLC- separated peaks (stainless steel Knauer Spherisorb RP-8 column).

2.5. Pulse amplitude modulated fluorescence

In vivo analysis of Chl fluorescence was made in each case using a portable modulation fluorometer (PAM 2000, Walz,

I. Gdmez et al. /J. Photochem. Photohid. 8: Bid. 47 (1998) 4657 49

Effeltrich, Germany) according to Schreiber et al. [ 241. In plants from in situ treatments, measurements of the effective quantum yield ( AhFIF,,‘) [ 251 were performed. Plants were taken away and measured immediately under a light-shading screen ( 20% of the sunlight irradiance) to avoid an excessive decrease of the fluorescence due to energy-dependent quenching (@) [ 261. In general, no obvious differences in the fluorescence signal were observed during the lapse of 10 min. The AFIF,’ parameter gives insights into the overall quantum yield of PSI1 photochemistry and represents the difference between the maximal fluorescence of a light- adapted sample (F,,,‘) and the current steady-state fluorescence (F,,). For plants incubated under outdoor conditions, the optimal quantum yield (F,,/F,,,) was also measured, which indicates the quantum efficiency of plants incubated in darkness for 15 min. The variable fluorescence (F,) is defined as the difference between a basal state of fluorescence (Fo), i.e., all PSI1 centres are oxidized, and the maximal fluorescence emission (F,) when all centres arereduced ( see Ref. [ 271 for considerations on the use of this methodology).

2.6. Determination of enzyme activities

Nitrate reductase (NR, E.C. 1.6.6.1) activity was assayed as described by Corzo and Niell [ 281. Samples of approximately 0.15 g FW were incubated for 30 min at 30°C in a solution containing 0.3 M KNOX and 0.5 mM glucose. Fol- lowing incubation, the assay medium without sample was frozen in liquid nitrogen and re-thawed at ambient temperature. The concentration of NO, was measured colorimetri,- tally after dilution of 1 ml of extract with 1 ml of N,N-( I, naphthyl)ethylenediamine and 1 ml of sulfanilamide [29].

Total carbonic anhydrase (CA, E.C. 4.2.1.1) activity was determined in samples previously stored in liquid nitrogen according to the methodology described by Haglund et al. f30.1 by determining the time taken for a linear drop of pH from 8.5 to 7.5 at 0-2°C. First, samples were thawed at room temperature, ground in a mortar and dissolved in 3 ml of a buffer solution prepared with 50 mM Tris, 25 mM isoascorbic acid and 5 mM EDTA at pH 9. The reaction was started by adding 1 ml of ice-cold CO,-saturated distilled water. The relative enzyme activity (REA) was then estimated using the following formula:

REA=(T,,/?;:)-1

where T,) and T, are the times taken for pH change in the non- catalysed (buffer only) and catalysed (extract) reactions, respectively.

2.7. Statistical analysis

Differences between UV-filter treatments ( F,,Tnd) and time of day ( Fhoor) were assessedusing two-way ANOVA (Model I). When a significant effect was detected, one-way ANOVA and the LSD Fisher test were used for comparisons of means according to Ref. [ 3 11. To meet normality and avoid corre-

lation between means and variances, data were log trans- formed. Percentage and ratio data were adjusted to a normal distribution by applying an arcsin (square root) correction.

3. Results

3.1. Field experiments: underwater quantum irradiance and photosynthetic responses

In situ quantum irradiance at Cabo de Gata-Nijar during the experiments was highest between 1200 and 1600 h, with values sometimes exceeding 2000 p,rnol photon m ~’ s ’ of PAR, especially on 20 Sept., 1996 (Fig. 3). UV-A and W- B radiation followed a similar daily cycle, with maxima of UV-B radiation being relatively higher on 24 Sept., 1996 than on 20 Sept., 1996. The integrated daily doses for the whole UV-B (280-320 nm) and W-A (320-400 nm) ranges measured at the surface on 20 Sept. reached values close to 39 and 880 kJ rn~-‘, respectively. Due to light attenuation ( K,, > 0.3 m ’ ) , these values decreased approximately 58% at 2.5 m depth (Table 1) . Similar UV-B radiation levels were measured on 24 Sept. Light attenuation for PAR was, however, higher on 20 Sept. compared to 24 Sept., which resulted in 28% lower irradiance at 2.5 m during this period.

8:OO iO:OO i2:OO 14:00 16:OO 18:OO 20 00

Time of day (h)

Fig. 3. Daily changes in solar radiation at Cabo de Gata-Nijar during the field experiments measured using a PUV 510 radiometer. Ultraviolet A and B (UV-A and UV-B) were estimated from instantaneous PUV 5 10 readings

at wavelength bands of 305, 320, 340 and 380 nm using attenuation coefficient (&) values from an underwater PUV 500 radiometer. See text for details of calculations.

50

Table I

1. Gdmez et al. / .I. Photochem. Photohiol. B: Biol. 47 (1998) 46-57

Optical characteristics and light availability at the experimental site (surface and 2.5 m depth) in Cabo de Gata-Nijar during field exposure of Dusycladus

oermiculuris to three different irradiation treatments. Detaila of calculations of rates and doses of k-radiance are given in text

Irradiation Attenuation coefficient

(K,,, In ‘1

Maximum quantum irradiance rate at noon ~1300-1400 h) (UV, Wm ‘:PAR,pmolm ‘s ‘)

\%Ufd‘X 2.5 m

Daily dose

(kJ rn..“)

Surface 2.5 m

20 Sept., 1996 UV-B (28&320 nm) UV-A (320-400 nm) PAR (400-700 nm)

24 Sept., 1996 UV-B (280-320 nm)

UV-A (320-400 mn) PAR (40&700 nm)

0.348 2.44 0.229 45.49 0.170 2344.96

0.327 1.83

0.223 3Y.87

0.097 1782.94

Under these light conditions, the effective quantum yield of fluorescence ( AF/ F,,’ ) showed daily changes during the two series of measurements (Fig. 4). During the first day of exposure, a significant effect of daily changes in solar radiation and type of incident radiation was found (two-way ANOVA, p < 0.05). In general, after a high value of AF/F,,,’ close to 0.68 at dawn, a decrease was observed, which was significant in plants filtered from the whole UV region (PAR alone) and UV-B (PAR + UV-A) (p < 0.01, Fisher LSD). However, no marked decline at noon was measured in these plants; rather AFl F,,,’ values increased (p < 0.05; Fisher LSD). In contrast, in plants exposed to PAR + UV-A + UV-B conditions, fluorescence continued to decrease to minimum values of 0.5 in the afternoon, thereby differing significantly from the other treatments (p < 0.05). Between 1600and 1830

I- -’ ___--

CV- 1

0800 1000 1200 1400 1600 1800 2000

Time of day (h) Fig. 4. Daily changes in effective quantum yield of fluorescence (&F/F,,,’ )

of Dnsycladur vermiculntis growing in situ at Cabo de Gala-Nijar. Effects of three different UV cut-off filter treatments during two daily periods in Sept. 1996. Data are means + S.D. of eight plank

-

1.04 39.07 16.37 27.64 881.16 496.33 1924.05 9279.88 5489.56

0.82 37.63 16.58 22.52 944.02 523.79

1405.46 9121.51 7144.80

h, a recovery in fluorescence emission was measured in all treatments, which was especially accentuated in plants exposed to PAR alone, with values comparable to that measured at dawn (p > 0.05). After four days under the three experimental treatments, the plants exposed to UV radiation (PAR + UV-A + UV-B and PAR + UV-A) were more strongly photoinhibited than those irradiated solely with PAR (p < 0.01, two-way ANOVA; Fig. 4, bottom). In these treatments, AFIF,,,’ between 24 and 32% in relation to initial values measured at 0815 h (p CO.05). In plants receiving only PAR, no depletion of fluorescence at noon was detected over the measuring period (p > 0.05).

3.2. Daily changes in solar radiation and photosynthetic pe$ormance of plants under outdoor conditions

As in the field experiments, irradiation was high during exposure of plants to outdoor conditions on 4 June, 1997 (Fig. 5 ( a) ) . Maximum values of PAR ( 1900 pmol photon m -2 so ‘),UV-B(0.7Wmm-‘)andUV-A(56Wm-~‘)were measured between 1300 and 1500 h. The integrated daily doses for UV-A and UV-B were 1411 and 16.2 kJ m -*, respectively, while PAR was above I I 000 k.J m ‘.

PAM fluorescence parameters (Fig. 5(b), (c) ) varied as a function of both light treatments and time of day (FJF,,,,

p < 0.05; AFIF,,,‘, p < 0.01, two-way ANOVA). After a 2 h exposure to PAR + UV-A, F, IF,,, decreased approximately 30%, whereas under PAR+ UV-A+ UV-B and FAR, the decline in quantum efficiency was close to 54% of the initial value (p < 0.05). At noon, a slight increase in F,,IF,,, in plants exposed to PAR + UV-A and PAR was measured, but values did not increase in the afternoon. A similar pattern was observed for the variations in AFIF,,,‘; however, the differences between PAR + UV-A + UV-B-treated plants and those exposed to PAR + UV-A and PAR alone were highly significant (p < 0.001 ) at noon, indicating a marked effect of UV-B on the photoinhibition of photosynthesis during the onset of solar radiation. The absence of a marked decline in

0500 0800 1200 1600 2000

Light shading screen Solar radiation

6 s

0.8

‘G E al 0.6

iq 2x0.4 =lL iI-

g 0.2

E .- Is 0 0, l----I---r--r-~i

% 0.8

'5. (c)l

E 0.6

z? fE

!% o'4 .z

5 0.2

5 0 , , , , , , / 4 0500 0800 1200 1600 2000

Time of day (h)

Fig. 5. Effects ofdaily changes in solar radiation on chlorophyll Auorescence

of Dusyladus vennicuknir measured in an outdoor system at Malaga on 4 June 1997. (a) Daily course of sunlight for three different wavelength bands (PAR, UV-A and W-B) measured with an ELDONBT instrument. (b)

Changes in optimal quantum efficiency (F, /fi’,,,) and (c) effective quantum yield (M/F,,,’ ) measured in plants exposed to three different IJV cut-off filter treatments. Data are means It S.D. of eight plant\.

Solar radiation (2 h)

fluorescence at noon in this experiment constitutes the main difference in relation to the data recorded in September 1996.

3.3. Photoinhibition and recovery qfphotosynthesis following short-term exposure to solar radiation

Fig. 6 shows the daily variation in solar radiation and total UV doses measured during the experiments and the sequence of exposure and recovery. During these experiments in Octo- ber 1997, daily irradiances for PAR and UV-A close to 4865.8 and 778.0 kJ m I, respectively, were significantly lowerthan that measured at the surface in September 1996 or July 1997, but comparable to the doses reaching 2 m depth at Cabo de Gata-Nijar in September 1996. The UV-B dose, close to 9.8 kJ m ‘, was, however, significantly lower than that measured during the other seasons. During the sunlight exposure period, plants received a PAR dose close to 1600 kJ mm ‘, whereas UV-A and UV-B reached 260 and 4 kJ I~-?, respectively.

Two hours under these light conditions caused considera- ble photoinhibition of photosynthesis (p < 0.05, Fig. 7). In both F,,IF,,, and AFIF,,,’ a significant differential effect of treatments was observed (p < 0.05, two-way ANOVA) and in all cases 24 h under shade conditions allowed the plants to recover initial fluorescence values completely. In general, F, / F,,, values in plants exposed to PAR + UV-A were less strongly inhibited than thalli maintained under PAR + UV- A + UV-B and PAR alone (p < 0.05). Similarly, a marked recovery in fluorescence was observed already after 2 h under shading in this treatment, while a large time period was required to complete recovery under PAR + UV-A + UV-B and PAR. On the other band, the ANOVA and Fisher LSD mean comparison test did not detect differences between PAR + UV-A +UV-B and PAR treatments. Results from AF/F,,,’ indicated a lower, but still significant, decrease (17 < 0.05). Here, PAR + UV-A + UV-B treatment causedthe

Time of day (h) Fig. 6. Daily variations in natural solar radiation (PAR, UV-A and UV-B) during experiments on exposure and recovery carried out m an outdoor system at Malaga on 23-24 Oct., 1997. Arrows denote the sequence of experimentation and times when samples were taken for measurements. Shaded areas indicate

periods for which plants were covered with a light-shading screen for recovery.

52 1. Gdmrz et al. /.I. Photochem. Photobiol. B: Biol. 47 (1998) 46-57

c 0.8 -r -- ---.------ ---

75 E ,-

E 0

1 __.____.,_. ---- .--.-.. . PAR+UVA+UVB:! . PAR+UVA

lnttlal +-+ 2 h 4 h 20 h 24 h 2h --

Solar Recovery Radiation

Fig. 7. Changes in PAM chlorophyll fluorescence of Dusycladus vrm?icu~ hr;s measured throughout a day in an outdoor system at Mlilaga on 23-24 Oct., 1997: (a) optimal quantum efficiency (F, /F,,,) and (b) effective

quantum yield ( AFIF,‘) of plants exposed for 2 h to three different IJV cut-off filter treatments and subsequent recovery under shade conditions. Data are means f S.D. of eight plants.

most effective decline in fluorescence compared to PAR + UV-A and PAR alone. Similarly to FJF,,,, the effective quantum yield recovered rapidly in plants exposed to PAR + UV-A (p < 0.05).

3.4. Changes in enzyme activities

Activities of the key nutrient uptake enzymes nitrate reductase (NR) and carbonic anhydrase (CA) measured throughout the day are shown in Fig. 8. NR activity was high (0.7 pmol NO? g ’ FW min ’ ) in plants incubated for five days under < 100 f*mol photon m ~~’ s ’ white light; however, after 4 h under PAR + UV-A + UV-B, a significant decrease (p < 0.05) was recorded. In plants receiving PAR + UV-A and PAR alone, this decline was lower. After a significant increase at 1430 h (p<O.O5) in the three treatments, NR activity again decreased in the afternoon, which was more marked in PAR + UV-A and PAR treatments (p < 0.05).

The CA activity measured in plants exposed to UV radiation had a peak (p < 0.05) at 1230, coinciding with a minimum of NR activity. Plants maintained under PAR + UV-A treatment showed the strongest decreases in CA activity. The exposure to natural PAR affected CA activity only at the beginning of the measuring period (p < 0.05)) but no further changes were observed.

3.5. Changes in W-absorbing compounds

HPLC measurements using aqueous methanol as solvent for plants exposed throughout a day to solar radiation on 4

June, 1997 revealed two major UV-absorbing compounds with maximum absorptions at 348 and 332 nm. These substances, probably a mixture of compounds, showed high optical densities ( > 2 O.D. per 100 mg DW) throughout the day. In Fig. 9, the results of the compound absorbing at 348 nm are shown as an example. At noon, values significantly decreased in plants exposed to PAR + UV-A and PAR alone, whereas they remained high in plants exposed to PAR + UV- A + UV-B (p < 0.05 1. During the first hours of the day and in the afternoon, no significant differences between cut-off filter treatments were observed. Interestingly, a high absorb-

Light shading screen Solar radiation

.__-

I? u

Time of day (h) Fig. 8. Effects of daily changes in solar radiation on enzyme activities of

nitrate reductase (NR) and carbonic anhydmse (CA) determined in Dusy- claduus vrm~icuhri~s exposed to three different UV cut-off filter treatments under outdoor conditions on 4 June, 1997. Data are means + S.D. of three plants.

tight shading sc*eeri

t Solar radiation

o&o lOi 12’30 16iO

m Time of day (h)

~ n PAR+UVA+UVB i” PARcUVA I ! PAR

Fig. 9. Effects of daily changes in solar radiation on the concentration of [JV-absorhing compounds of Dasyckuius wmiiculark. Data represent the O.D. per 100 mg DW of a major compound with absorption maximum at

348 nm and were determined throughout a daily cycle under outdoor conditions at Milaga on 4 June, 1997. Data are means f S.D. of three plants.

I. G&e7 et ul. /J. Photochrrn. Photohiol. B: Bid. 47 (199X) 46-57 5.1

ante close to 8 O.D. per 100 mg DW was measured in plants previously acclimatized to white light (initial).

4. Discussion

4. I. Efjcects qf UV radiation in the natural habitat

Before Wood’s work on the brown alga Ecklonia radiata [ 131, information on the effects of UV radiation on the biol- ogy of macroalgae was scarce or simply not available. Using held and tank experiments under natural light conditions, this author found that incident sunlight at 5 m depth ( 30%Zo UV radiation) during summer was enough to cause tissue dam- age, reduction in growth, pigment degradation and decrease of survival of young sporophytes after removal of adult kelp canopy [ 131. These findings identifying UV radiation rather than high PAR as the major deleterious factor on these bio- logical parameters raised important questions about the role of natural UV radiation on algae exposed normally to very high quantum irradiances.

In general, Mediterranean macroalgae receive during summer unweighted daily UV doses higher than 1000 kJ m ’ and data obtained through PAM fluorescence over the course of the day in the field indicate that photosynthesis at noon is inhibited in the presence of UV, especially UV-B [ 17-201. Fluorescence quenching parameters and AFIF,,,’ have been used to characterize the light responses in macroalgae exposed to high sunlight. For example, in Porphyru leucosticta, simultaneous measurements of AFIF,,,’ and F, / F,,, indicate similar patterns of variation during the day, i.e., a decline at noon and a recovery in the afternoon [ 19 1. Such a response represents a regulative process to cope with extreme radiant energy, the expression of which basically depends on the prevailing light conditions and zonation patterns of algae [ 6,7,9,15,16,26]. It has been demonstrated that a reduction in the photochemical yield of PSI1 (AFIP,,,‘) is closely related to a decline in electron transport rates and is the result of adjustments in both the concentration (photochemical quenching) and the photon absorption efficiency ( F, IF,,,) of the PSI1 reaction centres [ 24,25 1.

The decreases in AFIF,,’ measured in D. vermicularis growing in situ ( < 30%) are in general lower than that measured in Porphyra leucosticta [ 19 ] and other red algae from the same geographic area [ 321, which could indicate a differential ability of D. vermicularis to tolerate current levels of UV radiation. The high solar radiation, the high light pen- etration and low scattering measured in Cabo de Gata-Nijar allow us to argue that this species has effective adaptations to cope with enhanced UV radiation. Our data are consistent with previous findings reported by Perez-Rodriguez et al. [ 121 using artificial UV dosage. The authors concluded that a weighted UV dose as high as 1000 kJ m ~’ is required to cause 25% photoinhibition of F,/F,,, [ 121. In this context, differences in structure and organization of the light-harvest- ing system, thallus morphology and morpho-functional char-

acteristics, which in turn determine the photobiological strategies in macroalgae [ 9,33-351, may be invoked as determining factors explaining inter-specific differences. Undoubtedly, measurements of in situ quantum yield in marine macroalgae involve practical difficulties, mainly related to possible changes in n-radiance conditions at the time of measurements [6], particularly when experiments include transfer of samples from the growth site to the surface [ IX]. In any case, the light-dependent adjustments of photochemical energy-conversion mechanisms, including down- regulation of the PSI1 activity (photoinhibition), may have consequences for primary production [ 34,361, which may be important in a scenario of enhanced solar UV radiation I 141.

4.2. Solar stress and recovery capacity

As was outlined above, down regulation of the PSI1 during high irradiances at noon is a reversible process, and if no chronic photoinhibition (alterations in the Dl protein tum- over rates) or total loss of photosynthetic activity due to photodamage occurs, photosynthesis recovers to non-inhibited values when solar stress is absent [6,35,37]. Thus, the recovery kinetics following short-term exposure to solar radiation are a good indicator of the light-stress tolerancecapacity of the photosynthetic apparatus andconsequently, differential effects of PAR, UV-A and UV-B can be outlined. This type of experiment has been carried out in several macroalgal assemblages from temperate regions and reveals that sun- adapted algae (intertidal and upper sublittoral species) require a shorter time under shade conditions to recover photosynthetic performance fully than shade-adapted algae growing at subtidal locations [l&,20,35]. In D. verrnicularis, exposure for two hours to UV doses close to 260 kJ m -’ with a PAR background of 1600 kJ rn~ 2 was enough to cause a 65% decrease in F,IF,,, and a 77% decrease in AFIF,,’ in plants previously acclimatized to low white-light irradiances. After 24 h under shade conditions, complete recovery was observed in all treatments, which clearly indicated that plants exhibited dynamic photoinhibition. These results resemble recovery patterns described for two intertidal ‘sun-type’ red algae, Asparagopsis armata 1201 and Gelidium lut~folium [ 321 from Gibraltar strait, southern Spain. Interestingly, the recovery in D. vermiculwis was faster in algae exposed to PAR + W-A, with full recovery of photosynthesis already after 2 h. In plants maintained under PAR + UV-A + UV-B and PAR alone, a complete recovery was reached after 24 h. When data from AFl F,,,’ are analysed, a slightly greater effect of UV-B could be demonstrated, agreeing with data reported previously by Perez-Rodriguez et al. [ 121, who did not find any deleterious effect of UV-A on F, I F,,, in algae irradiated with a combination of PAR and UV lamps.

The rapid recovery observed in plants exposed to UV radiation deprived of UV-B (PAR + UV-A) is consistent with the increase of fluorescence observed under filters cutting off UV-B radiation during the experiment performed in summer (4 June, 1997). Here, after a photoinhibition phase during

54 1. Gdmez et al. /J. Photorhem. Photohid. B: Bid. 47 (1998) 46-57

the first few hours, both F,l F,,, and hFl F,’ increased during the onset of irradiation. However, the subsequent decline with decreasing irradiance in the afternoon is difficult to interpret. Probably, algae were not able to maintain a high photosynthetic performance for a long time, and due to the extreme irradiation to which they were exposed, chronic photoinhibition occurred (Fig. 5 (a) ) . The idea that any UV-A quanta could be effectively used for photosynthesis in D. vennicu- &is, as has been postulated for some species of macroalgae growing under limited PAR irradiance [ 38 1, is unlikely, since this species clearly does not suffer limitation by PAR. Although light requirements for oxygen evolution in D. vermicularis se high (irradiances for saturation of photosyn,- thesis ranging between 250 and 500 bmol photon m ’ s ’ [ 121) , an estimation of the daily period at which algae are light saturated (H,;,,) results in values close to l& 13 h per day. Apparently the response of D. vermiculuris to a solar spectrum deprived of UV-B (PAR + UV-A) is a conse- quence of acclimatization processes: for example, plants exposed to PAR + W-A for a day under field conditions in autumn and under outdoor treatments in summer did not suffer photoinhibition at noon. However, after four days, field plants were inhibited in a similar way as in PAR +UV- A + UV-B treatments. A first explanation could be that the inhibitory effect of UV-A in the absence of UV-B on photosynthetic performance required a longer time to become evident. Alternatively, the absence of UV-B for a scale of days, depriving plants of an environmental trigger inducing photoprotective mechanisms, e.g., synthesis and accumulation of UV-screening substances (see below), could be an additional explanation.

4.3. lnj’luence of solar radiation on activities of nutrient transport enzymes

The reduction of nitrate to nitrite by NR is the first step in the nitrate assimilation pathway in algae [ 39,401. On the other hand, external CA is involved in an active carbon- concentrating mechanism catalysing the dehydration of HCO? ~- to form CO, and has been described in a variety of micro- and macroalgae [ 41,421. Despite the importance of these enzymes and the fact that they are strongly regulated by environmental factors, few efforts have been made in order to assess the impact of enhanced UV radiation. Only recently has it been demonstrated that UV-B radiation inhibits the activity of certain enzymes related to inorganic nitrogen metabolism in algae [ 43-45 1.

In the present study, variations in NR and CA activities do not conclusively add evidence for a marked effect of UV radiation. However, some trends may be outlined. During the first few hours (0800 to 1430). changes in NR and CA were antagonistic. On the other hand, for the plants exposed to the three treatments NR activity tended to increase after a 6 h exposure to sunlight, agreeing with some diurnal rhythms reported for other macroalgae such as CJlva,fene.stratu [ 461 and Gracilaria tenuistipitata 147 1. Changes in enzyme activ-

ity have also been reported in the endemic intertidal Medi- terranean red alga Rissoellll verruculosa [ 211. Such variations, however, only partially match daily variations in photosynthesis, which suggests that both processes, energy quenching and down regulation of the PSI1 and nutrient transport, could be differentially affected in the presence of enhanced solar radiation. Therefore, the question whethel short wavelengths act as triggers for the activation or deac- tivation of carbon and nitrogen transport mechanisms, as in processes dealing with energy dissipation in PSII, remains open.

4.4. The role of UV-ahsorbing compounds

Our data show that the contents of UV-absorbing compounds (maximum absorption at 332 and 348 nm) were high throughout the day, but only during the onset of solar radiation at noon could UV-dependent changes beobserved. Plants exposed to UV-B-filtered radiation (PAR alone and PAR + UV-A ) showed a significantly lower absorbance (O.D.) than plants exposed to full solar radiation, which had constant values along the day. Although most of these compounds absorb mainly within the UV-A region (31 O-360 nm) and, hence, would protect more efficiently against UV- A radiation, a stimulation in the concentration of these substances by increasing PAR has been reported for the red alga C’hondrus crispus I 48 ] and the dinoflagellate Alexandrium cxc~~vc1ru1~~ [ 491. On the other hand, our results agree in that W-B can also induce synthesis, which has also been shown in a variety of algae f S&53]. The high O.D. values found in plants acclimatized for several days to white light do not agree with a possible UV-photoprotective role in D.vermiwlaris. However, it must be emphasized that under artificial lamps with a low PAR background ( 32 p,mol m -’ s ’ ), a marked increased in these UV-absorbing compounds was also seen

1181. As was pointed out by P&ez-Rodriguez et al. [ 1X], the

HPLC patterns and absorption spectra of these compounds in Dasycludus differ from those of other well-known mycosporine-like amino acids (MAAs, e.g., shinorine, asterina- 330 or porphyra-334) extracted mainly from red algae [ 54- 56]. The accumulation of [IV-absorbing compounds in 11. vermkuluris constitutes a clear exception within the green algae, which were found to have very low amounts [ 541. This may be related to the relatively low photoinhibition of photosynthesis found in field plants (see above), despite the high natural irradiance to which they were exposed. The unusually high background of these compounds in plants incubated under full solar radiation ( PAR + UV-A + UV-B), which was relatively constant throughout the day, could attenuate daily changes in photosynthesis. Unfortunately, the chemical structure and induction mechanisms of these compounds are up to now unknown, but some evidence suggests a photoprotective role, similar to that proposed by MAAs: the recently documented excretion to the medium of these compounds in the form of a ‘yellow-brown’ mixture by D.

I. G&vez et al. / .I. Photachem. Photobid B: Bid. 47 (1998) 46-57 55

Table 2 Seasonal variations in UV-absorbing compounds 0fDasycladu.s mvmicuinris collected during 1996-1997 at Cabo de Gata-Nijar. The mean daily doses of solar radiation for each month measured with an ELDONET dosimeter at Milaga are also given. Data from algae collected at the east coast of Mexico (Quintana Roo) and two sites at the Canary Islands (Fuerteventura and Gran Canaria) are also shown for comparison. Data are means (S.D.) of four replicates. ND:

data not available

Location/month

Cabo de Gata-Nijar: Sept. 1996

Feb. 1997 April 1997 June 1997

Qintana Roo:

Feb. 19%

Fuerteventura: Jan. 1998

Gran Canaria: Jan. 1998

Comp-332 Comp-348 Average daily dose (kJ m ‘)

(O.D. per 100 mg DW) PAR IJV-A UV-B

4.1 (0.9) 3.1 (0.4) 7509.55 (1796.5) 330.59 ( 208.2) 15.14 (3.3)

3.3 (0.8) 2.0 (0.2) 4751.17 (1218.1) ND ND

I.1 (0.6) 0.9 (0.4) 7830.19 (2066.0) 835.42 (224.5) 12.16 (4.3) 3.5 (0.2) 2.5 (0.9) 9648.15 (2901.4) I 171.28 (350.3) 13.78 (4.1)

0.6 (0.9) 1.3 (0.8)

6.9 (0.9) 4.6 (0.2)

5.1 (1.7) 3.4 (0.1)

vermiculuris [ 181 may correspond to the series of coloured algal exudates (especially from brown algae) absorbing in the UV region described a long time ago [ 57-591.

The question whether these compounds are excreted as a response to a long-term environmental light control was addressed by collecting plants in different seasons and by comparing data with samples from the Atlantic (east coast of Mexico and Canary Islands, Table 2). On a seasonal basis, the UV-absorbing compounds of D. vermicularis reach maximum values in September (late summer-autumn), i.e., after plants had been exposed for several months to very high sunlight, including high UV-A and UV-B doses. In winter material (January 1998) from the Canary Islands the values are constitutively high (3.4-6.9 O.D. per 100 mg DW) and comparable to maxima measured in samples from Cabo de Gata-N[jar in October 1997 (Fig. 9), while in plants collected in Mexico at 4-5 m depth, the concentrations of UV-absorbing compounds were lower. These data are clearly novel and suggest that abundant accumulation of UV-absorbing compounds is an inherent chemical attribute of this species. Undoubtedly, the perspectives of research on this topic are promising and more detailed screenings as well as the chemical structure are fundamental to elucidate the relationships between enhanced UV-solar radiation and UV-absorbing compounds in macroalgae.

Acknowledgements

This study was supported by grants from the European Union (Environmental and Climate Programme, CT96 ENV4-0188) and the Ministerio de Educacibn y Cultura, Spain (CICYT, AMB97-1021-C02-01). Field studies were carried in the framework of the Action Integrada Hispano-

Alemana (DAAD, 133-B, 322-AI-E-DR). U.K. acknowl- edges a grant from the Deutsche Forschungsgemeinschaft, Germany (KA 899/3- 1) . The Agencia de Medio Ambiente, Junta de Andalucia, provided facilities and logistic support at the Natural Park Cabo de Gata-Nijar. The authors are also indebted to C. Jimhnez, D.-P. Hider and M. Lebert for val- uable help during field experiments and C. Maestre for skillful technical assistance. R. Haroun and J. Espinoza-Avalos kindly collected samples for extraction of UV-absorbing compounds in the Canary Islands and in QuintanaRoo (Mex- ice), respectively. This is publication No. 1533 of the Alfred- Wegener-Institut fiir Polar- und Meeresforschung.

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