effect of ultraviolet radiation on thallus absorption and photosynthetic pigments in the red alga...

Journal of Photoc&mistry

ELSEVIER .I. Photochem. Photobiol. B: Biol. 48 (1999) 75-82

Effect of ultraviolet radiation on thallus absorption and photosynthetic pigments in the red alga Porphyra umbilicalis

Jo& Aguilera a, Carlos Jimkez aT*, Filix L. Figueroa a, Michael Lebert b, Donat-P. Hider b a Departamento de Ecologia, Facultad de Ciencias, Universidad de Mblaga, Campus Universitario de Teatinos s/n, E-29071 M&ga, Spain

hinstitutf% Botanik und Pharmazeutische Biologie der Friedrich-Alexander-Universitiit, StaudtstraJe 5. D-91058 Erlangen, Germany

Received 2 October 1998; accepted 20 January 1999

Abstract

The effect of ultraviolet (UV) radiation on thallus absorption, package effect, the concentration of photosynthetic pigments, photosynthetic oxygen production and effective quantum yield has been studied in the intertidal red macroalga Porphyru umbilicalis in the laboratory. High doses of UV-A and UV-B radiation result in a rapid decrease of thallus absorption and, after a 6 h exposure, total absorption is reduced to 25% of the initial value. Moreover, significant differences in the absorption peaks of the main pigments are found: while chlorophyll a (Chl a) and phycocyanin absorption peaks decrease by 65-67%, carotenoids and phycoerythrin (PE) peaks decrease by 75-82%. Uncoupling of the transfer of energy between PE and Chl a by UV is revealed by a gradual increase of fluorescence of PE up to 11 h of exposure, followed by a subsequent decrease of fluorescence of the PE, in parallel with the photobleaching of the pigments. Thalli with higher pigment concentration present a greater sensitivity to UV radiation, as revealed by a more pronounced decrease in total thallus absorption, oxygen production and effective quantum yield, and a less effective recovery under low irradiation. Exposure of the thalli to artificial UV radiation in an experimental chamber with spectra and doses more similar to those of the natural environment reveals that PAR + LJV-A radiation promotes a gradual increase of the total absorption over 24 h; in contrast, PAR + UV-A + UV-B induces a significant decrease of the thallus absorption. However, the concentration in vitro of Chl a, carotenoids and biliproteins does not change in any of these light treatments. The spectrally averaged in vivo absorption cross section normalized to Chl a (a*) increases after 24 h in PAR + LJV-A, but it does not change in PAR and PAR + UV- A+UV-B, indicating that the degree of packing of the pigments in the membranes of the thylakoids (package effect) is decreased by PAR + UV-A, but that the reverse is induced by PAR + UV-A + UV-B. It is proposed that UV-A radiation induces an enhancement of the efficiency of light capture mediated by a relaxation of pigment packing in the light-harvesting antennae of this intertidal macroalga, while the reverse is promoted by UV-B radiation. 0 1999 Elsevier Science S.A. All rights reserved.

Keywords: Intertidal system; UV radiation; Thallus absorption; Package effect: Photosynthesis; Photoinhibition; Porphyra

1. Introduction

The current thinning of the ozone layer is resulting in increased levels of ultraviolet ( W) radiation at the Earth’s surface. Consequently, at present there is great concern about the possible impact that increasing UV radiation may have on natural ecosystems, especially marine systems [ l-51. UV radiation has been found to affect aquatic organisms in many ways, in&ding a prominent decrease of photosynthetic activity of autotrophs. Phytoplankton are thought to be responsible for about half of the biomass production on our planet [ 61. Recent investigations have shown that many phytoplankton organisms are quite sensitive to enhanced UV-B radiation, suffering changes at the level of the photosynthetic

* Corresponding author. Tel. +34-95-213-2385; Fax: + 34-95-213-2000; E-mail: [email protected]

apparatus and pigments involved in light reception and in photosynthetic production, carbon and nitrogen metabolism [7,8] and growth rate. Orientation and motility have been

found to be drastically impaired in several species, even after short exposure to UV-B [ 9- 111.

In contrast to the bulk of research concerning the deleterious effects of ultraviolet radiation on microalgae, marine macroalgae have been largely overlooked. Benthic macroalgae, in contrast to phytoplankton, are fixed and restricted to their growth site and thus have no opportunity to avoid

high irradiances of visible light (photosynthetically active radiation, PAR; A = 400-700 nm) or UV radiation by vertical migration. This suggests that sublittoral macroalgae may

show a lower tolerance to environmental stress, particularly to high irradiances and to UV radiation, while supralittoral or intertidal algae should be more adapted to cope with higher

101 l-1344/99/$ - see front matter 0 1999 Elsevier Science S.A. All rights reserved. Prrs1011-1344(99)00015-9

16 J. Aguilera et al. /J. Photochem. Photobiol. B: Biol. 48 (1999) 75-82

UV levels at the surface. In the field, strong sunlight depresses the photosynthetic activity of marine macroalgae, causing dynamic photoinhibition. Photosynthetic activity follows a diurnal pattern so that the lowest levels usually occur between noon and afternoon [ 12,131. Recent investigations described higher reduction of photosynthetic activity in subtidal than in intertidal algae exposed to full sunlight [ 14-171. This depletion of the photosynthetic capacity may be followed by a decrease in the primary production, i.e., growth rate, increasing pigment photobleaching and tissue damage in some macroalgae from shaded and deep areas after exposure to sunlight [ 18,191.

the laboratory at 14 f 1 “C in aerated 5 1 beakers in Provasoli’ s enriched seawater. Algae were illuminated by daylight fluorescent lamps (Osram-L 18 W, Germany) at an irradiance of 15 W me2 in a light/dark regime of 12 h/12 h. Under these conditions, the pigment concentration of the thalli was similar to that in wild-type ones freshly harvested from the intertidal system.

2.2. Experimental design

The present work is a laboratory approach to describing the effects of different doses of UV-A and I-IV-B radiation on thallus absorption and pigmentation of the supralittoral red macroalga Porphyra umbilicalis. This alga is submitted to high irradiance in the natural environment, and protection mechanisms against UV radiation may be expected. Recently, it has been reported [ 201 that natural UV radiation caused a 28% depletion in the average quantum yield in P. leucosticta and that the accumulation of pigments was also affected, in the short term, by solar radiation. However, at present little information is available on the effects of UV radiation on photosynthetic pigments in macroalgae, in contrast to the effects of other light qualities, e.g., blue, red and far-red light [ 21,221. Porphyra acclimates to both light quality and quantity changes by altering the phycobiliprotein/chlorophyll a ratio [ 21,231, by rapid state transitions [ 241 and by an effi- cient mechanism of non-photochemical heat dissipation of excess energy absorbed by the photosynthetic apparatus

~251.

In a first set of experiments, discs 2 cm in diameter were cut from the thalli, transferred to Petri dishes filled with cul- ture medium, and exposed for 6 h to artificial UV radiation produced by a transilluminator (280-400 nm, peak at 3 12 nm; Bachhofer, Reutlingen, Germany; see Ref. [26] for details). High doses of UV-A + UV-B produced by the transilluminator (Table 1) may be useful for the study of the different types of photodamage at the level of pigment composition. The fluence rates produced by the unfiltered transilluminator (over 15 W m- *) are very high in comparison to solar UV-B, and will almost certainly produce a wide range of biological effects, including DNA damage, membrane damage and protein deformation, as well as pigmentation changes. Long-term exposure under high UV doses was not intended to simulate natural conditions, but it was useful for investigating the photobleaching kinetics of the pigments.

2. Materials and methods

2.1. Algal material and pretreatment

Thalli of Porphyra umbilicalis L. Kiitzing were collected from upper intertidal habitats at Helgoland, North Sea, and provided by the Biologische Anstalt Helgoland. Prior to the spectral treatments, algae were precultivated for 10 days in

In a second set of experiments, the possible photoprotective role of phycobiliproteins was tested. Two batches of thalli of P. umbilicalis were used, i.e., low-phycobiliprotein-content (LP) thalli and high-biliprotein-content (HP) thalli. The first batch consisted of thalli freshly harvested from the field and cultivated in the laboratory for 10 days in the above-men- tioned conditions; HP thalli were induced by Professor Klaus Liming at the Biologische Anstalt Helgoland of Hamburg after long cultivation of P.umbilicalis in the laboratory at high nitrate concentration and low irradiation ( 10 W mp2). Concentrations of phycoerythrin (PE) and phycocyanin (PC) were 8.2 and 3.8 mg g- ’ dry weight (DW), respectively, in the former group of thalli, and 32.0 and 10.7 mg g-i DW, respectively, in the second. These thalli were also exposed to the UV radiation produced by the transilluminator

Table 1 Irradiance of the different radiation treatments (W m-*) used in the two illumination systems: the transilluminator. which provides high UV doses, and the experimental chamber simulating the natural light field for UV radiation. A combination of visible and ultraviolet lamps provided the whole spectrum from

UV-C to PAR in the experimental chamber. Selective filters were used to cut off light below a determined wavelength

Light field PAR UV-A UV-B uv-c UV-B/UV-A UV-A/PAR UV-B/PAR (A=400-7OOnm) (h=315dOOnm) (h=280-315nm) (A<280nm)

Transilluminator 0 14.6 15.9 1.09

Experimental chamber Selective filters WG 396 25 0.7 0.0 0.03 WG 320 20 5.7 0.0 0.28 WG 295 25 12.0 1.3 0.11 0.48 0.052 No filter 22 15.7 4.3 0.06 0.27 0.71 0.195

J. Aguiiera et al. /J. Photochem. Photobid. 8: Bid. 48 (19991 75-82 77

for 90 min, after which photosynthetic oxygen production, effective quantum yield and thallus absorption were measured. Oxygen evolution was measured by means of a YSI 5775 microelectrode at a saturating irradiance of 350 p,mol quanta rnp2 s-l of PAR. The effective quantum yield was measured by means of a pulse amplitude modulated fluorom- eter (PAM-2000, Waltz, Effeltich, Germany) at a low irradiance of white light of 30 pm01 m - * s - ‘, and calculated as AFIF,‘, where AF is the difference between the respective maximal fluorescence (F,’ ) of a light-adapted plant and the fluorescence level in daylight (F,) [ 271. After irradiation, thalli were placed under white light of low irradiance, and recovery of photosynthetic O2 production and of effective quantum yield was followed for 24 h.

Another set of discs was placed in Petri dishes and exposed for 24 h to different spectral ranges of UV radiation with doses more similar to natural solar radiation. We shall refer to this system as the experimental chamber. A combination of nine fluorescent lamps (2 X Radium NL 36 W/25; 2 X Q- Panel UVA-340; 2 X Osram-L 36 W/32; 2 X Philips TL 40 W/ 12 UVB; 1 X Q-Panel UVA-35 1) provided UV + PAR radiation from 260 to 700 nm. Radiation was cut off with selective filters (WG396; WG320; WG295; Schott, Mainz, Germany) to provide four different spectral qualities with different doses and ratios (Table 1) . Appropriate ordering of the lamps and placing the samples 60 cm away provided a homogeneous light field for the plants in the different treatments. Spectra were measured using a double monochro- mator spectroradiometer (Optronic Instruments, model 752, Orlando, FL, USA) with an Ulbricht sphere and a quartz cable.

2.3. Fluorescence emission spectra

In order to analyse the fluorescence of phycobiliproteins, another set of thalli of P. umbilicalis was exposed to the UV radiation produced by the transilluminator and their fluorescence emission spectra measured at discrete time intervals with a Shimadzu RF5000 spectrofluorometer (Shimadzu, Kyoto, Japan). The excitation wavelength was fixed at 520 MI (maximum of PE absorption). For the fluorescence measurements thalli of P. umbilicalis were placed in 1 mm path length quartz cuvettes (Hellma, Miillheim, Germany).

2.4. Absorption spectroscopy

Absorption spectra of thalli of P. umbilicalis were measured during exposure to the transilluminator and in the experimental chamber. Discs were temporarily removed, mounted between two slides and placed next to the detector, perpen- dicular to the light beam, of a single-beam spectrophotometer (Beckman DU-70, Palo Alto, CA, USA). The total absorption of the thalli was calculated by integration of the absorption spectra in the range 400-700 nm, after correction for scattering.

2.5. Photosynthetic pigments

Pigment concentration was analysed initially and after 24 h of exposure in the experimental chamber. Pigments were extracted from frozen samples kept in liquid nitrogen. Chlo- rophyll a (Chl a) was extracted in acetone (90%) neutralized with MgC03. Extracts were centrifuged at 10 OOOg for 10 min, and the Chl a concentration in the supernatant was determined spectrophotometrically [ 281. Total carotenoidcontent was estimated by multiplying the optical density at 480 nm by a factor of 4.4. Biliproteins were extracted in phosphate buffer 0.1 M, pH 6.5 at 4°C containing 10 mM Na,-EDTA and 4 mM phenylmethyl-sulphonyltluoride (PMSF) . The extracts were centrifuged at 15 OOOg for 15 min and the supernatant used for PE and PC determination. Biliproteinconcen- tration was determined spectrophotometrically [ 291.

From these data, the spectrally averaged in vivo absorption cross section normalized to Chl a (a*) was calculated, which is related to the efficiency of light capture; a* within the PAR range (400-700 nm) is taken as the ratio between the average thallus absorption and total Chl a concentration (mg m - 2). The optical cross sections of Chl a, total carotenoids, PE and PC were obtained as the ratio between the absorption of the thalli at 678, 480, 565 and 624 nm, respectively, and the specific pigment concentration, estimated as above. For more details, see Ref. [ 301. Variations in a* have been attributed to changes in the degree of packing (package effect) of the pigments in the membranes of the thylakoids [ 30,3 11. It has to be stated that in vivo absorption at 480 nm is not only due to carotenoids. Phycoerythrin also has a considerable absorption cross section at this wavelength.

2.6. Statistics

Results have been expressed as the mean values f standard deviation (S.D.) of four replicates. Statistical significance of the means was tested with a one-way ANOVA followed by Fisher’s protected least significance difference (LSD) mul- tirange test at a significance level of p < 0.05 [ 321.

3. Results

3.1. High doses of UV radiation

Fig. 1 shows the absorption spectra of P. umbilicalis during exposure to the unfiltered radiation of the transilluminator. High doses of UV-A and UV-B promoted a rapid decrease of total absorption of the thalli of P. umbilicalis. After 2 h, total absorption of the thalli decreased by approximately30%, but no significant differences appeared in the rate of bleaching between the pigment peaks during the first 2 h of exposure (Table 2). After 6 h of exposure, the total absorption of the thalli was only 25% of the initial value. However, the extent of the decrease significantly differed between the pigment peaks (Table 2). While Chl a and PC decreased by 65 and

78 J. Aguilera et al. /J. Photochem. Photobid. B: Biol. 48 (1999) 75-82

350 400 450 500 550 600 650 700 750

Wavelength [nm]

Fig. 1. Absorption spectra of Porphyrrr umbilicalis measured at increasing times of exposure to high doses of UV-A+UV-B produced by a transilluminator.

67%, respectively, carotenoids and PE peaks decreased by 75 and 82%, respectively. An increase of PE fluorescence at 580 nm was detected after the first 4 h of exposure (Fig. 2) ; maximal fluorescence of PE was found after around 11 h of exposure; this could be related to the uncoupling between PE and Chl a; afterwards the fluorescence decreased, probably due to photobleaching of the pigment-protein complex. This increase and further decrease of the PE fluorescence induced by UV radiation was accompanied by a gradual shift of 8 nm to shorter wavelengths.

Short exposure (90 min) of LP and HP thalli of P. umbilicalis to the UV radiation produced by the transilluminator induced a significant decrease of photosynthetic O2 production, effective quantum yield and total absorption of the thalli. Fig. 3 (a) shows that the capacity for O2 production decreased by about 25% after exposure in LP thalli and by about 50% in HP ones. LP thalli were less affected by the deleterious effects of UV radiation, and their capacity for recovery was higher; O2 production of LP thalli was around 95-100% after 3 h under white light of low intensity. However, HP thalli showed a much slower recovery after UV radiation treatment, and O2 production reached only 65% of the initial value after 24 h under low light. Similar results were obtained for the response of the effective quantum yield (Fig. 3(b) ) . Both LP and HP thalli showed similar quantum yields before the irradiation treatment; it decreased by about 60% after UV treatment in HP thalli, and it did not show any significant recovery during the next 24 h under low light. LP thalli were

Oh - I

540 560 5L

Wavelength [nm]

660

Fig. 2. Fluorescence emission spectra of phycoerythrin (excitation at 520 nm) after different times of exposure to enhanced UV radiation produced

by a transilluminator.

less affected by UV radiation, decreasing their quantum yield by only 25% after 90 min of exposure; these thalli also showed a high capacity for recovery, and after 24 h under low light their quantum yield had increased to 95% of the initial value. This differential response of the capacity for O2 production and of effective quantum yield of LP and HP thalli of P. umbilicalis after a short exposure to high levels of UV radiation was also evident for the variation of the total absorption of the thalli (Fig. 3 (c) ) . The total absorption of HP thalli decreased by about 30% after 90 min of exposure, while the decrease in LP thalli was about 10%. In both cases the total absorption continued to decrease for at least two more hours after transfer of the thalli to low light, leading to a final 42% decrease in HP and 20% in LP. After 24 h of recovery under low light, the total absorption of the thalli was lo-12% lower than the initial value in both cases.

3.2. Simulation of natural UVjeld

Fig. 4 shows the change of the total absorption of the thalli of P. umbiEicaZis under different UV treatments in the experimental chamber. Under PAR, there was a transient increase of total absorption by about 20% after 3 h, returning to the initial values after 24 h. PAR + UV-A promoted an increase of the total absorption, which took place in two phases: a rapid increase in the first 1.5 h, and a second one after 6 h. Thereafter the total absorption of the thalli remained constant for the rest of the experiments. The final absorption of the

Table 2 Absorption changes in Porphyru umbilicalis in PAR (400-700 nm) and at several key wavelengths representative for the main pigments after different exposure times in the hansilluminator. The values measured before exposure (initial) were arbitrarily set to 100%. Values represent means of four replicates + S.D.

Means with different superscripts after 6 h of exposure were significantly different at p < 0.05.

Exposure time PAR (h=400-700nm)

Chl a (A=678nm)

Carotenoids (A=480nm)

Phycoerythrin

(A=565 nm) Phycocyanin (h=624nm)

Initial 0% 0% 0% 0% 0% 30 min -1.5+1.0 -4.5&Z -1kO.O -5.5kl.O -7+ 1.4 2h -2i3.0+3.3 -31.0+0 - 28 i 7.0 - 24.0 + 7.0 -33*0.0 6h -75.0+0.4” - 65.0 + Oh -75jy1.36 -s2.0+ 1.7’ - 67 f 3.0h

J. Aguilera et al. /J. Photochem. Photobiol. B: Biol. 48 (1999) 75-82 79

1

3

% 0.6

7 ", 0.4

i om8 t

0.2

0 3 4 9

0.7

2 .z 0.6

> B 0.5

s 0.4

6 f 'a

8 0.1 ~

0.3

0.2

m HP q ILP (4

(b)

J-7

Fig. 3. Influence of exposure to UV radiation produced by a transilluminator

(1.5 h) and recovery for 24 h on (a) Oz production, (b) effective quantum yield and (c) total absorption of high- and low-phycobiliprotein-content thalli of Porphyra umbilicalis. Initial values in (c) have arbitrarily been set

to 100%. HP, high-phycobiliprotein-content thalli; LP, low-phycobiliprotein-content thalli.

thalli was 40% higher than the initial value. PAR+ UV- A + UV-B induced an increase of thallus absorption, with a maximum after 3 h. A continuous decrease was detected afterwards, and the total absorption decreased by about 25% after 24 h. When light was not filtered (PAR + UV-A + UV- B + UV-C) , an immediate decrease of thallus absorption was detected, decreasing by 45% after 24 h.

Fig. 5 summarizes the concentration of the main photosynthetic pigments in the thalli of P. umbilicdis at the initial

-+- PAR - PARtUV-A -o- PARtUV-tA+B)

T i -t- PARtW-(AtBtC)

1

125

501 I I I I I I I I 0 3 6 9 12 15 18 21 24

Exposure time (h)

Fig. 4. Influence of exposure to four different spectral qualities of PAR + UV radiation in the experimental chamber on thallus absorption in Porphyra umbilicalis.

I Initial I PAR ei PARttJV-A

10 u PARtUWAtB) q PARtUV-(AtRtC)

" Chla Car PE PC Fig. 5. Influence of exposure to four different spectral qualities of PAR + UV

radiation in the experimental chamber on pigment content in Porphyra umbilicalis.

time and after 24 h of exposure in the experimental chamber. The concentrations of Chl a, total carotenoids and phycobiliproteins (PE and PC) did not change with respect to the initial ones after 24 h under PAR, PAR+UV-A and PAR + UV-A + UV-B. When plants were exposed to the full spectrum (PAR + UV-A + UV-B + UV-C), the concentrations of all pigments decreased. Both PE and PC suffered a drastic decrease by about M-60% after exposure to the full spectrum.

The in vivo absorption cross section normalized to Chl a (a”) in the PAR region of the spectrum increased after 24 h of exposure under PAR + UV-A (Table 3), but it did not change when the thalli were exposed to PAR or PAR + UV- A + UV-B. A major increase of a” under PAR + UV-A was detected at the level of the Chl a and carotenoids absorption peaks, indicating a decrease of the degree of packing of those pigments after exposure to W-A. This result is confirmed by the increase of the absorption of the thalli of P. umbilicalis under UV-A radiation (Fig. 4). A different behaviour was found in plants exposed to PAR + W-A + UV-B; here the concentration of Chl a also remained constant after 24 h (Fig. 5), but the total absorption of the thalli decreased (Fig. 4);

80 .I. Aguilera et al. /J. Photochem. Photobiol. B: Biol. 48 (1999) 75-82

Table 3 Spectrally averaged in vivo absorption cross section in the PAR region normalized to Chl a (a”), and at selected wavelengths corresponding to the maximum absorption peaks of the main photosynthetic pigments related to the pigment concentration at initial time and after 24 h of exposure in the experimental chamber. Values of a* are measured in m2 mg- ’ pigment ( X lo- ‘). Values are the mean of four replicates f S.D. Different superscripts indicate significant differences

between treatments at p < 0.05

Light quality PAR

(A=400-700nm)

Chl a

(A=678 nm)

Carotenoids

(A=480nm)

Phycoerythrin

(A=565nm)

Phycocyanin

(A=624 nm)

Initial 24 h PAR 24 h PAR + UV-A

24 h PAR + UV-A + W-B 24 h PAR + UV-A + W-B + W-C

3.5 +0.12 d 3.65 f 0.80 a 14+ 1.75 ‘% 2.7*0.64” 3.9 *0.40 d 3.3 kO.66 a 3.91 f 0.78 a 12k1.19” 3.0*0.14” 4.0 f 0.26 ’

4.0f0.39 h 4.40 f 0.45 b 16+0.58 h 3.2kO.12 a 4.6 f 0.59 ’ 3.1+0.68 a 3.7OkO.70 a lOkO.55 c 2.0 * 0.50 h 3.4 f 0.40 b

2.5 kO.52 ’ 2.10+0.36’ 9.6*0,2’ 2.3 f 0.24 h 4.5*0.19”

however, a* in the PAR region and at the absorption peak of Chl a (678 nm) did not change. Under this treatment, significant changes occurred at the level of carotenoids and phycobiliproteins, where DthallDsol (absorption of the thalli in viva/absorption of the pigment extracts) significantly decreased without a significant change of their total concentrations in the thalli (Fig. 5). Thus, an increase of the packing of carotenoids may be expected under UV-B radiation; the more pronounced decrease of the absorption of the thalli under UV-A+UV-B occurred in the blue region (not shown). Moreover, a* significantly decreased under unfiltered radiation, except at the phycocyanin absorption peak. Under unfiltered radiation, the decrease of the absorption of the thalli (Fig. 4) was due both to a decrease of the pigment concentrations (Fig. 5) and to an increase of the package effect (decrease of a* for Chl a, carotenoids and PE in Table

3).

4. Discussion

Data obtained in this study reveal that the extent of the damaging effects of UV radiation depends on the duration of the exposure as well as on its spectral composition, apart from its fluence rate. UV radiation of different spectral composi- tions promotes different responses in the pigmentation pattern of P. umbilicalis. Exposure of the thalli to high levels of UV- A + UV-B radiation emitted by a transilluminator induces a significant decrease of the maximal absorption peaks of all pigments. Nevertheless, the depletion of carotenoids andphy- coerythrin absorption peaks is more drastic than that of Chl a and phycocyanin. It has previously been demonstrated that strong UV radiation can photo-oxidize, and thereby bleach, all types of photosynthetic pigments [ 331. The differential pigment bleaching in these experiments confirms previous observations in different species of phytoplankton using artificial UV radiation in which the accessory pigment PE was bleached first, followed by the protective carotenoids, while the chlorophylls were more resistant [ 34,351. This pattern of inhibition agrees with the spatial distribution of these pigments in the chloroplast. On the other hand, PE is located in the peripheral part of the hexameric discs of the phycobili-

somes [ 361. This pigment has rapid binding and dissociation capability, favouring rapid phycobilisome acclimation to changing irradiances [ 371. PE has also been found to form free aggregates, not attached to the phycobilisomes [ 381. According to these ultrastructural evidences, these pigments may reduce the amount of light available for absorption by the light-harvesting pigment-protein complexes, thus poten- tially limiting photoinhibition and photodamage caused by high it-radiance or UV radiation. Nevertheless, other authors [ 391 reported that UV-B radiation induces a rapid increase in intracellular carotenoids and a marked decline in Chl a in the unicellular marine prasinophyte Tetraselmis.

Independently of the size of the phycobilisomes (i.e., the degree of bleaching), the energy transfer to Chl a may or may not be affected when the thalli are exposed to UV radiation. In these cases, it would be possible to obtain a higher or lower fluorescence yield, respectively. It seems to be clear that when the photosynthetic electron transport chain is intact (before major UV damage), the phycobilin fluorescence should be very low, since most of the energy flows to Chl a. During UV irradiation we have found a significant increase in fluorescence of PE and a shift in the maximum of the emission spectrum to lower wavelengths. This means that energy transfer to Chl a is no longer possible, and energy is dissipated as fluorescence. The increase of fluorescence emission and the shift in the maximum related to the dissociation of the phycobilisomes have been previously described as consequences of the uncoupling of the energy transfer path- way along the different phycobiliproteins within the phycobilisomes [40-42]. More recently, several authors [35,43] supported this hypothesis with the observation of UV effects on the breakdown of the high-molecular-mass aggregates into hexamers, which further disintegrate into trimers and finally into monomers, with parallel changes in the absorption and fluorescence properties of biliproteins, both in flagellates and cyanobacteria.

Thalli of P. umbilicalis irradiated with UV of different spectral composition and with doses of UV-A and UV-B close to the levels of natural radiation show a differential pigment response. First, PAR promotes an increase of thallus absorption that returns to the initial value after 24 h. When plants are illuminated with PAR + UV-A, thallus absorption

J. Aguilera et al. /J. Photochem. Photobiol. B: Biol. 48 (1999) 75-82 81

increases, while PAR + UV-A + UV-B promotes a decrease of pigment peak absorption. It has been found that pigment concentration does not significantly vary under those treatments, thus rearrangement of the pigments in the thylakoids of P. umbilicalis may be expected. UV-A induces a relaxation of pigment packing in the light-harvesting antennae, while the reverse is promoted by W-B radiation. A deleterious effect of UV-A or UV-B radiation on the pigment content of P. umbilicalis has not been detected. Moreover, PAR, PAR + UV-A and PAR + UV-A + UV-B result in differential thalli absorption and package effect. UV-A induces a clear increase of total absorption, with a significant decrease of the packing of Chl a and carotenoids. Nevertheless, under UV- A+UV-B radiation thallus absorption decreases, but this decrease is not due to photodestruction of the pigments but to an increasing package effect. This response under natural UV-B levels may be considered as being photoprotective.

On the other hand, it has also been shown in this work that low-phycobiliprotein-content thalli (concentrations similar to those found in wild-type algae) of P. umbilicalis are less affected by UV radiation than high-phycobiliprotein-content ones. This result can be explained by the fact that in HP thalli the density of absorbing molecules within the light path is higher than in LP ones, thus in the former the probability of a photon of UV radiation passing through the thylakoids without being absorbed is small compared to that in the LP ones. HP thalli would absorb more UV radiation than LP ones and thus its negative effects on physiological and metabolic performance of tbe thalli should be more pronounced. How- ever, one could argue that the HP thalli should be less sensitive than the LP thalli. High concentrations of phycobiliproteins, many of which may anyway be uncoupled from light harvesting, would shade the more vulnerable reaction centres. This has been shown not to be the case in P. umbil-

icalis. UV radiation uncouples the transfer of energy from phycobiliproteins to Chl a, as shown by the increase of the fluorescence emission of PE with further breakdown of the phycobilisomes. Reversible uncoupling of energy transfer resulting in considerable fluorescence emission of PE has been suggested as a protective mechanisms in picoplankton [ 441. The presence of UV-absorbing mycosporine-like amino acid compounds (e.g., Porphyra-334) has been reported in Porphyra [45,46]. These molecules could be responsible for the better adaptation of this intertidal macrophyte to cope with high natural levels of UV radiation during the emersion periods.

In conclusion, UV radiation induces significant changes in the spectroscopic features of P. umbilicalis, with low-phycobiliprotein-content thalli being less sensitive to its deleterious effects. UV radiation also seems to promote a rearrangement of the photosynthetic pigments of the antennae of this intertidal macrophyte, UV-A radiation inducing a relaxation of the packing of the pigments, while UV-B induces an increase of their degree of packing as a possible protective mechanism.

Acknowledgements

This work was supported by a grant from the Ministry of Education and Science of Spain (Project CICYT AMB97- 102 1 -CO2-0 1) to F.L.F., by the European Union (Environ- ment Programme, EV5V-CT94-0425; DG XII to D.-P.H. and ENV4-CT-96-0188 to F.L.F.), and by the Accibn Integrada Hispano-Alemana No. 133-B, DAAD (322-AI-e&). J.A. received a doctoral fellowship from the Junta de Andalucia. The authors thank Mrs Andrea Schumacher for technical help.

References

[ 11 R.C. Worrest, Review of literature concerning the impact of UV-B radiation upon marine organisms, in: J. Calkins (Ed.), The Role of

Solar Ultraviolet Radiation in Marine Ecosystems, Plenum, New York, 1982, pp. 429-457.

[2] R.C. Smith, B.B. F’rezelin, K.S. Baker, R.R. Bidigare, N.P. Boucher, T. Coley, D. Karentz, S. MacIntyre, H.A. Matlick, D. Menzies, M. Ondrusek, Z. Wan, K.S. Waters, Ozone depletion: ultraviolet radiation and phytoplankton biology in antarctic waters, Science 255 ( 1992)

952-959. [3] CR. Booth, J.H. Morrow, T.P. Coohill, J.J. Cullen, J.E. Frederick,

D.-P. Hader, 0. Holm-Hansen, W.H. Jeffrey, D.L. Mitchell, P.J.

Neale, I. Sobolev, J. van der Leun, R.C. Worrest, Impacts of solar UVR on aquatic microorganisms, Photochem. Photobiol. 65 (1997) 252-269.

[ 41 R. Santas, Solar UV effects in algal assemblages of the Caribbean and the Mediterranean Seas, in: C.S. Zerefos, A.F. Bais (Eds.), Nato AS1 Series I, 52, Solar Ultraviolet Radiation Modelling, Measurements

and Effects, Springer, Berlin, 1997, pp. 233-250. [5] D.-P. Hider, F.L. Figueroa, Photoecophysiology of marine macroal-

gae, Photochem. Photobiol. 66 (1997) 1-14. [6] R.A. Houghton, G.M. Woodwell, Global climatic change, Sci. Amer.

260 (1989) 18-26. [ 7 ] G. DGhler, M.R. Ah, Assimilation of 15N-ammonia during irradiance

with ultraviolet-B and monochromatic light in Thalassiosira rotula, CR. Acad. Sci. Paris, Serie D 308 (1989) 513-518.

[8] R.P. Sinha, H.D. Kumar, A. Kumar, D.-P. Hider, Effects of UV

irradiation on growth, survival, pigmentation and nitrogen metabolism enzymes in cyanobacteria, Acta Protozool. 34 (1995) 187-192.

[9] D.-P. Hider, M. Hiider, Ultraviolet B inhibition of motility in green

and dark bleached Euglena gracilis, Curr. Microbial. 17 ( 1988) 215- 220.

101 S. Gerber, D.-P. Hider, Effect of enhanced solar radiation on chlo-

rophyll fluorescence and photosynthetic oxygen production of five

species of phytoplankton, FEMS Microbial. Ecol. 16 (1995) 3342. 111 C. Jiminez, F.L. Figueroa, J. Aguilera, M. Lebert, D.P. Hider, Pho-

totaxis and gravitaxis in Dunaliella bardawil: influence of UV radiation, Acta Protozool. 35 (1996) 287-295.

[ 121 W.J. Henley, S.T. Lindley, G. Levavasseur, C.B. Osmond, J. Ramus,

Photosynthetic response of Vlva rotundata to light and temperature during emersion on an intertidal sand flat, Oecologia 89 (1992) 516- 523.

[ 131 D. Hanelt, J. Li, W. Nultsch, Tidal dependence of photoinhibition in marine macrophytes of the South China Sea, Bot. Acta 107 ( 1994) 6672.

[14] M. Maegawa, M. Kunieda, W. Kida, The influence of ultraviolet radiation on the photosynthetic activity of several algae from different depths, Jpn. J. Phycol. 41 (1993) 207-214.

[ 151 L.A. Franklin, R.M. Forster, The changing irradiance environment: consequences for marine macrophyte physiology, productivity and ecology, Eur. J. Phycol. 32 (1997) 207-232.

82 J. Aguilera et al. /J. Photochem. Photobiol. B: Biol. 48 (1999) 75-82

[25] S.K. Herbert, Photoinhibition resistance in the red alga Porphyra perforata. The role of photoinhibition repair, Plant Physiol. 92 ( 1990) 514-519.

[26] S. Gerber, D.-P. Hider, Effects of artificial UV-B and simulated solar radiation on the flagellate Euglena gracilis: physiological, spectro- scopical and biochemical investigations, Acta Protozool. 34 ( 1995)

13-20.

~27 ] B. Genty, J.-M. Briantais, N.R. Baker, The relationship between the quantum yield of photosynthetic electron transport and quenching of

chlorophyll fluorescence, Biochim. Biophys. Acta 990 ( 1989) 87- 92.

128 ] S.W. Jeffrey, G.F. Humphrey, New spectrophotometric equations for determining chlorophylls a, b, cl and c2 in higher plants, algae and natural phytoplankton, Biochem. Physiol. Pllan. 167 (1975) 191- 194.

[ 291 S. Beer, A. Eshel, Determinining phycoerythrin and phycocyanin concentrations in aqueous crude extracts of red algae, Aust. J. Mar. Freshw. Res. 36 (1985) 785-792.

[ 161 S. Sagert, R.M. Forster, P. Feuerpfeil, H. Schubert, Daily course of photosynthesis and photoinhibition in Chondrus crispus (Rhodo- phyta) from different shore levels, Eur. J. Phycol. 32 ( 1997) 363-

371. [ 17 J D. Hanelt, Capability of dynamic photoinhibition in Arctic macroal-

gae is related to their depth distribution, Mar. Biol. 131 ( 1998) 361-

369. [ 18 J J.M. Kain, Seasonal growth and photoinhibition in Plocamium carti-

lagineum (Rhodophyta) of the Isle of Man, Phycologia 26 ( 1987)

88-99. [ 191 C.W. Grobe, T.M. Murphy, Inhibition of growth of Ulva expansa

(Chlorophyta) by ultraviolet-B radiation, J. Phycol. 30 ( 1994) 783-

790. [20] F.L. Figueroa, S. Salles, J. Aguilera, C. Jimenez, J. Mercado, B.

Viiiegla, A. Flares-Moya, M. Altamirano, Effects of solar radiation

on photoinhibition and pigmentation in the red alga Porphyra leucosticta, Mar. Ecol. Prog. Ser. 151 (1997) 81-90.

[ 211 F. Lopez-Figueroa, F.X. Niell, Red-light and blue-light photoreceptor controlling chlorophyll a synthesis in the red alga Porphyra umbilicalis and in the green alga Ulva rigida, Physiol. Plant 76 ( 1989) 391- 397.

[ 221 F.L. Figueroa, J. Aguilera, F.X. Niell, Red and blue light regulation of growth and photosynthetic metabolism in Porphyra umbilicalis (L.) Kiitzing (Bangiales Rhodophyta), Eur. J. Phycol. 30 (1995)

11-18. [23] F. Lopez-Figueroa, Diurnal variations in pigment contents in Por-

phyra laciniata and Chondrus crispus and its relation to the diurnal changes of underwater light quality and quantity, Mar. Ecol. 13 (1992) 285-305.

[ 241 K. Satoh, D.C. Fork, A new mechanism for adaptation to changes in light intensity and quality in the red alga Potphyra perforata. II Char- acteristics of state II-state III transitions, Photosynth. Res. 4 (1983) 61-70.

[ 301 J.M. Mercado, C. Jimenez, F.X. Niell, F.L. Figueroa, Comparison of methods for measuring light absorption by algae and their application to the estimation of me package effect, in: F.L. Figueroa, C. Jimdnez,

J.L. Perez-Llorens, F.X. Niell (Eds.), Scient. Mar. 60 (Suppl. 1). Underwater Light and Algal Photobiology, 1996, pp. 39-45.

[31] T. Bemer, Z. Dubinsky, K. Wyman, P. Falkowski, Photoadaptation

and the ‘package’ effect in DunalieZla tertiolecta (Chlorophyceae) , J. Phycol. 25 (1989) 70-78.

[32] P.R.Sokal,F.J.Rohlf,Biometry, W.H.Freeman,SanFrancisco, 1981.

[ 331 D.-P. Hider, R.C. Worrest, Effects of enhanced solar-ultraviolet radiation on aquatic ecosystems, Photochem. Photobiol. 53 ( 1991) 717-

725. [34] S. Gerber, D.-P. Hider, Effect of solar irradiation on motility and

pigmentation of three species of phytoplankton, Environ. Exper. Bot.

33(1993)515-521.

[ 351 R.P. Sinha, M. Lebert, A. Kumar, H.D. Kumar, D.-P. Hader, Spectro-

scopic and biochemical analyses of UV effects on phycobiliproteins of Anabaena sp. and Nostoc carmium, Bot. Acta 108 (1995) 87-92.

[ 361 L. Talarico, Phycobiliproteins and phycobilisomes in the red algae:

adaptive responses to light, in: F.L. Figueroa, C. Jiminez, J.L. Perez- Llorens, F.X. Niell (Eds.), Scient. Mar. 60 (Suppl. l), Underwater Light and Algal Photobiology, 1996, pp. 205-222.

[ 371 P. Algarra, W. Riidiger, Acclimatation processes in the light harvest-

ing complex of the red alga Porphyridium purpureum (Bory) Drew et Ross, according to irradiance and nutrient availability, Plant Cell

Environ. 16 (1993) 149-159. [38] L. Talarico, R-phycoerythrin from Audouinella saviana (Nemaliales,

Rhodophyta). Ultrastructural and biochemical analysis of aggregates and subunits, Phycologia 29 (1990) 292-302.

[39] J.I. Goes, N. Handa, S. Taguchi. T. Hama. Effect of UV-B radiation

on the fatty acid composition of the marine phytoplankter Tetraselmis sp.: relationship tocellularpigments,Mar. Ecol. Prog. Ser. 114 ( 1994) 259-274.

[40] E. Gantt, C.A. Lipschultz, B. Zilinskas, Further evidence for a phycobilisome model from selective dissociation, fluorescence emission, immunoprecipitation and electron microscopy, Biochim. Biophys.

Acta. 430 ( 1976) 375-388. [41] E. Morschel, K.-P. Koller, W. Wehrmeyer, Biliprotein assembly in

the disc-shaped phycobilisomes of Rhodella violacea. Electron micro-

scopical and biochemical analysis of C-phycocyanin and allophyco- cyanin aggregates, Arch. Microbial. 144 (1980) 268-271.

[42] A.N. Glazer, Directional energy transfer in a photosynthetic antenna, J. Biol. Chem. 264 (1989) l-4.

[43] M. Fischer, D.-P. I-Eider, UV effects on photosynthesis and phycobi-

liprotein composition in the flagellate Cyanophora paradoxa, FEMS Microbial. Ecol. 101 (1992) 121-131.

[44] P. Heathcote, M. Wyman, N.G. Carr, G.S. Be&lard, Partialuncoupling of energy transfer from phycoerythrin in the marine cyanobacterium Synechococcus sp. WH 7803, B&him. Biophys. Acta 1099 (1992)

267-270. [45] D. Karentz, F.S. McEuen, K.M. Land, WC. Dunlap, A survey of

mycosporine-like amino acid compounds in Antarctic marine organ-

isms: potential protection from ultraviolet exposure, Mar. Biol. 108 (1991) 157-166.

[46] D. Karentz, Ultraviolet tolerance mechanisms in Antractic marine

organisms, in: Ultraviolet Radiation in Antarctica: Measurements and Biological Effects, Antarctic Res. Ser. 62 (1994) 93-l 10.

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