Ultrastructure and photosynthesis in the supralittoral ... - Phycologia

Phycologia (2006) Volume 45 (2), 168–177

Published 23 February 2006

Ultrastructure and photosynthesis in the supralittoral green macroalga Prasiola crispa from Spitsbergen (Norway) under UV exposure ANDREAS HOLZINGER1*, ULF KARSTEN2, CORNELIUS LU¨TZ1

AND

CHRISTIAN WIENCKE3

1

University of Innsbruck, Institute of Botany, Department of Physiology and Cell Physiology of Alpine Plants, Sternwartestrasse 15, A-6020 Innsbruck, Austria 2 University of Rostock, Department of Biological Sciences, Applied Ecology, Albert-Einstein-Strasse 3, D-18051 Rostock, Germany 3 Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany A. HOLZINGER, U. KARSTEN, C. LU¨TZ AND C. WIENCKE. 2006. Ultrastructure and photosynthesis in the supralittoral green macroalga Prasiola crispa from Spitsbergen (Norway) under UV exposure. Phycologia 45: 168–177. DOI: 10.2216/05-20.1 The effects of mild artificial UV conditions (4.7 W m22 UV-A, 320–400 nm and 0.20 W m22 UV-B, 280–320 nm) plus PAR (25–30 mmol photons m22 s21) at 4–68C on photosynthesis and ultrastructure of the aeroterrestrial green macroalga Prasiola crispa (Lightfoot) Ku¨tzing from the Arctic were investigated. Specimens were collected from an aeroterrestrial ˚ lesund, Spitsbergen, Norway, 78855.59N, 11856.09E). Exposure for 6 h and 24 h to PAR rocky cliff in Kongsfjorden (Ny-A 1 UV-A 1 UV-B led to a decrease in the optimum quantum yield (PS II efficiency, Fv/Fm) to 73.7% and 42.0% of the control value, respectively. In addition, the relative electron transport rate as a function of the photon fluence rate (PI curve) was determined. This showed that the a value declined in concert with Fv/Fm in UV-exposed samples. In parallel to the fluorescence measurements, tissue samples were prepared for transmission electron microscopy. Whereas 6 h of UV treatment did not lead to significant alterations in the ultrastructure compared to control samples, 24 h of UV treatment caused slight defects. Occasionally, thylakoid membranes showed dilatations and the number of plastoglobuli appeared reduced. Formation of a large number of cytoplasmic globules with a diameter in the range of 0.4–0.8 mm and a medium electron-dense contrast was observed. The data presented demonstrate that P. crispa is rather insensitive to experimental UV treatment and thus seems to be well adapted to cope with increased UV irradiation expected to occur as a consequence of decreased stratospheric ozone concentrations in the northern hemisphere. KEY WORDS: chloroplast, green alga, thylakoid, ultrastructure, UV

INTRODUCTION Since the first records of stratospheric ozone losses above the Arctic, serious concerns have arisen on the impact of enhanced ultraviolet B radiation (UV-B, 280–315 nm) on the northern biosphere (Hessen 2001). The Arctic Kongsfjorden (Spitsbergen, Norway) is a marine coastal ecosystem that has been intensively studied over the last years as a model for global change (Hop et al. 2002, and references therein). A typical feature of the fjord is a well structured macroalgal community down to a depth of almost 40 m consisting of about 60 species (Hop et al. 2002). Macroalgae play an important role in primary production as a food source for herbivores and detrivores, as well as a nursery area and habitat for fish and invertebrates (Lippert et al. 2001). Macroalgal vegetation that grows close to the water surface experiences a seasonal fluctuation of environmental factors including UV-B (Hanelt et al. 2001; Hop et al. 2002). There are many negative effects of UV-B on organisms, particularly when absorbed by nucleic acids and proteins, which may cause photodamage and conformational changes that finally result in various disturbed vital metabolic functions such as transcription, DNA replication and translation (Lao & Glazer * Corresponding author ([email protected]).

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1996; Buma et al. 2003). In addition, cell biology and physiology may also be affected, leading to increasing mortality (Franklin & Forster 1997). Photosynthesis is one of the key processes to be potentially impaired after UV-B exposure due to damage to the D1 protein (Vass 1997) or to Rubisco (Bischof et al. 2000) or loss of pigments (Bischof et al. 2000), as well as reduced gene expression (Mackerness et al. 1999). While the underlying biological mechanisms are quite well understood, much less is known about UV-induced ultrastructural changes in algal cells (Meindl & Lu¨tz 1996, Lu¨tz et al. 1997, Holzinger et al. 2004). So far, the effects of UV radiation on photosynthesis and the ultrastructure of two sublittoral red algal species from the Arctic have been investigated (Holzinger et al. 2004). Photochemical efficiency of PS II in Palmaria palmata and Odonthalia dentata strongly decreased to about one-third of the initial value under UV. Concomitantly, the fine structure of the photosynthetic apparatus was severely influenced by UV. Thylacoid membranes appeared wrinkled, lumen dilatations occurred and the outer membranes were altered. Moreover, mitochondria were damaged, and formation of numerous plasma vesicles was observed. The data clearly demonstrated a high UV sensitivity for both red algal species as well as a strong correlation between the physiological and ultrastructural levels (Holzinger et al. 2004).

Holzinger et al.: UV effects in Prasiola To gain insight into whether UV induces similar changes in photosynthesis and ultrastructure in green macroalgae, the supralittoral alga Prasiola crispa was collected from a rocky cliff in the inner part of the Arctic Kongsfjorden (Spitsbergen, Norway). Prasiola is an ecologically interesting genus due to its ability to grow subaerially on various hard substrata, often several meters above sea level. As a consequence of living under almost terrestrial conditions, Prasiola experiences strongest amplitudes of all prevailing environmental parameters including UV, and hence species of the genus have developed a range of morphological, physiological and biochemical protective mechanisms such as thick cell walls as a measure against dehydration (Jacob et al. 1992a, b), or the formation of UV sunscreens to cope with UV radiation (Jackson and Seppelt 1997, Hoyer et al. 2001, Karsten et al. 2005). However, in P. crispa ssp. antarctica, DNA damage has been reported due to UV-B radiation (Lud et al. 2001). In the present study the simultaneous investigation of photosynthesis and ultrastructure in P. crispa in response to experimental UV exposure was undertaken to obtain more information on survival strategies of this species.

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As the radiation source, a combination of daylight fluorescence tubes (Osram L85/W19) and Q-Panel UV-A-340 fluorescence tubes (Q-Panel Company, Cleveland, Ohio, USA) were used. Radiation measurements were carried out with a Li-Cor LI-190-SB cosine corrected sensor connected to a LiCor LI-1000 data logger (Lambda Instruments, Lincoln, Nebraska, USA) for PAR, and with a PMA 2100 broad-band UV radiometer (Solar Light Co., Philadelphia, Pennsylvania, USA) equipped with a PMA 2101 UVB detector and a PMA 2100 UVA detector. Although the C.I.E. definition for UV-B is 280–315 nm, we used the broader waveband (280–320 nm) because our UV radiometer was calibrated to these spectral wavelengths. While half of the containers were exposed to the full radiation spectrum, the other half were kept under a specific filter foil to cut off UV-A and UV-B (PAR treatment) (400 nm cutoff; Folex PR, Folex, Dreieich, Germany). Scattering effects were neglected. After 6 h and 24 h of PAR and PAR 1 UV-A 1 UV-B treatment samples were taken for ultrastructural examination and for investigation of photosynthetic performance. Photosynthetic performance

MATERIAL AND METHODS Algal material and study site Specimens of the green macroalga P. crispa (Lightfoot) Ku¨tzing were collected during the summer season 2002 from an aeroterrestrial rocky cliff. The collection site was located 100 m from the shore and about 2–3 m above sea level underneath a seagull colony in the inner part of the Kongsfjorden (Ny˚ lesund, Spitsbergen, Norway; 78855.59N, 11856.09E). AlA though at this location in summer on a sunny day at noon about 1.2 W m22 UV-B (280–320 nm) and 25 W m22 UV-A (320–400 nm) can be measured, the plants occur partly in the shade or may be covered by a small layer of snow until late spring. Algal samples were collected from the rocks with tweezers and placed in black bags to avoid exposure to high irradiance during transportation. These samples are considered as ‘freshly harvested’ material and were, for control reasons, directly fixed for transmission electron microscopy (TEM) (see below). After collection, samples were cleaned from visible contaminants such as faeces of birds, maintained for a maximum of 24 h in the laboratory under dim light conditions and in running seawater at 3–48C pumped directly from the fjord before the UV-exposure experiments.

In vivo chlorophyll fluorescence was measured using a portable pulse amplitude modulated fluorometer (DIVING PAM, Walz, Effeltrich, Germany). The maximum quantum yield (Fv/ Fm) of photochemistry was determined in six individual samples, which were placed in a cooled seawater cuvette during the measurement. Determination of Fv/Fm in P. crispa was conducted as described by Hanelt (1998). After exposure for 10–15 min to darkness, minimal fluorescence (F0) was measured with a pulsed measuring beam (approximately 0.3 mmol photons m22 s21, 650 nm), followed by short pulses of saturating white light (0.4–0.8 s, 1000–5000 mmol photons m22 s21) to record Fm (Fv 5 Fm 2 F0). Additionally, photosynthesis vs irradiance curves (PI curves) were monitored by means of the DIVING-PAM according to the method described by Karsten et al. (2001) and Holzinger et al. (2004), to calculate maximum photosynthetic electron transport rates (ETRmax). Respective relative electron transport rates (ETRs) were determined by multiplying the effective quantum yield DF/Fm9 (Genty et al. 1989) with the respective actinic photon fluence rate emitted by the fluorometer (Schreiber et al. 1994). The data were fitted with two photosynthesis models (Platt et al. 1980; Walsby 1997) that differ in taking into account photoinhibition. Light microscopy and TEM

Radiation experiments Thalli of P. crispa (diameter, 1–1.5 cm) were exposed to PAR and PAR 1 UV for a period of 24 h. For each sampling (6 h PAR, 6 h PAR 1 UV, 24 h PAR, 24 h PAR 1 UV), two duplicate containers (i.e. altogether eight containers each filled with four to five individual plantlets), were used always. From each container three individual thalli were randomly selected for photosynthesis measurements. This experimental design had to be chosen to guarantee homogeneous radiation of all vessels under the technical limitations at the field station. The containers were kept in ice/water at 4–68C, and were irradiated from the top with 25–30 mmol PAR m22 s21, 4.7 W m22 UV-A (320–400 nm) and 0.20 W m22 UV-B (280–320 nm).

Approximately 2 mm long pieces from the tips of the algae were cut with a razor blade and fixed with 2.5% glutaraldeyde for 2 h in 50 mM sodium cacodylate buffer (pH 7.0), rinsed, postfixed in 1% OsO4 in the same buffer for 4 h, rinsed, dehydrated in increasing concentrations of ethanol (each 15 min), infiltrated with Spurr embedding resin (Serva, Heidelberg, Germany) and polymerized for 8 h at 708C. Semithin sections of the embedded material were investigated with a Zeiss Axiovert 200 M microscope using a 63 3 1.4 NA oil lens; images were collected with a Zeiss Axiocam MCR5. Ultrathin sections were counterstained in aqueous uranyl acetate and Reynolds’ lead citrate and examined with a Zeiss EM 902 transmission electron microscope at 80 kV. Images

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Fig. 1. Changes in PS II efficiency (Fv/Fm) of Prasiola crispa after 6 h and 24 h exposure to PAR 1 UV-A 1 UV-B. The mean values of Fv/Fm 6 SD (n 5 6) are given. Significant differences between 6 h PAR and 6 h UV treatment, as well as between 24 h PAR and 24 h UV treatment, were recorded (t test, P , 0.001).

were captured on Kodak negative material, digitalized and further processed with Adobe Photoshop software. Statistics Mean values of photosynthetic parameters and standard deviations were calculated from six replicates per treatment. For the PS II efficiency measurements, t test comparisons were undertaken between each PAR and PAR 1 UV data pair using the commercial software InStat (GraphPad Inc, San Diego, California, USA). Statistical significance of PI curve means was tested with a model 1 one-way ANOVA followed by a Tukey multiple comparison test (Sokal & Rohlf 1995). RESULTS Photosynthesis The optimum quantum yield of P. crispa collected in the field and kept for 24 h in the laboratory under dim light conditions

Fig. 2. PI curve of Prasiola crispa under control (PAR) conditions (open white circles) and after 24 h exposure to PAR 1 UV-A 1 UVB (black squares). Given is the relative electron transport rate (relative ETR). Both symbols represent the mean value of six replicates (SD , 19%). The black and dotted lines each represent a fitted curve of the respective data measured based on the Walsby (1997) equation: P 5 Pm (1 2 e2aI/Pm) 1 bI, Pm: maximum photosynthesis (where a is the positive slope at limiting photon fluence rates; b is the negative slope at inhibiting photon fluence rates; I is irradiance).

(5 initial value) of 0.620 6 0.020 (n 5 6) (data not shown). This Fv/Fm value remained absolutely unchanged over a period of at least 7 d, as well as under the 6 h (Fv/Fm 5 0.619 6 0.016) and 24 h PAR (Fv/Fm 5 0.628 6 0.031) treatments in the radiation experiment. After 6 h of PAR 1 UV-A 1 UVB treatment, the optimum quantum yield of P. crispa thalli exhibited a significant decline to 73.7% of the 6 h PAR control (P , 0.001; Fig. 1). After 24 h of UV exposure, a stronger decrease in the optimum quantum yield to 42.0% of the 24 h PAR control was observed (P , 0.001; Fig. 1). The relative ETR as a function of the photon fluence rate (PI curve) was recorded and the data points were fitted using two models (Platt et al. 1980; Walsby 1997; Fig. 2). Both mathematical treatments gave almost identical results (Table 1). Control plants (PAR only) of P. crispa exhibited an a value of 0.086–0.088, a Pmax of 2.043–2.045 and an Ik value of 23–24 (Fig. 2, Table 1). Exposure to 24 h of PAR 1 UVA 1 UV-B led to a strong decrease in a (0.046) (P , 0.001) and Pmax (1.243) (P , 0.001), while Ik remained almost unchanged at 27 mmol photons m22 s21 (P . 0.05) (Fig. 2, Table

Table 1. Photosynthesis-irradiance (PI) curve parameters of Prasiola crispa treated with artificial ultraviolet radiation for 6 h and 24 h.1 PI curve parameters2 a

Treatment Control 6h UV 24h UV

0.086 0.088 0.068 0.068 0.046 0.046

6 6 6 6 6 6

a

0.010 0.008 0.007b 0.009 0.011 0.012

b

Pmax

20.002 20.001 n.d. n.d. n.d. n.d.

6 6 6 6 6 6

2.043 2.045 2.306 2.307 1.243 1.244

Ik a

0.188 0.179 0.267a 0.272 0.113b 0.118

24 23 34 34 27 27

6 6 6 6 6 6

Model a

4 3 5b 4 3a 3

Platt et al. (1980) Walsby (1997) Platt et al. (1980) Walsby (1997) Platt et al. (1980) Walsby (1997)

1 Data were recorded as relative electron transport rates (relative ETR) and fitted with the PI model equation of Walsby (1997) and Platt et al. (1980). Given are the mean values 6 SD (n 5 6). 2 Analysis of variance showed significant differences between respective controls and radiation treatments (a Platt et al. model: F 2,15 5 27.756, p , 0.0001, Tukey post hoc results indicated by lower case letters; a Walsby model: F2,15 5 27.488, p , 0.0001; Pmax Platt et al. model: F2,15 5 46.209, p , 0.0001, Tukey post hoc results indicated by lower case letters; Pmax Walsby model: F2,15 5 46.025, p , 0.0001; Ij, Platt et al. model: F2,15 5 9.48, p , 0.01, Tukey post hock results indicated by lower case letters: Ik Walsby model: F2,15 5 16.412, p , 0.001), a; positive slope at limiting photon fluence rates (mmol electrons mmol21 photons); b: negative slope at inhabiting photon fluence rates (mmol electrons mmol21 photons); Pmax: maximum light-saturated photosynthetic rate (rel. ETR); Ik: initial value of light-saturated photosynthetic rate (mmol photons m22 s21); n.d.: not detected (no photoinhibition detectable).

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Figs 3, 4. Details of Prasiola crispa thalli. Semithin sections viewed with a light microscope. Control samples with intact chloroplast (Chl), cell wall (CW), and nucleus (N). Fig. 3. Transverse section. Fig. 4. Longitudinal section. Bars 5 10 mm.

1), and after 6 h of UV treatment a showed an intermediate value of 0.068; Ik and Pmax were slightly higher with 34 and 2.307, respectively (Table 1). Under control conditions, P. crispa exhibited a decrease in relative ETR at photon fluence rates above 300–400 mmol photons m22 s21, which indicates some photoinhibition (Fig. 2). However, after treatment with 24 h of UV, no photoinhibition could be measured any more (Fig. 2), although the maximum ETR rate dropped to 63% of the control. Light microscopy and TEM Light microsocopy of semithin section revealed the known cell architecture of P. crispa (cf. Knebel 1935; Jacob et al. 1992a). The thalli consist of monostromatic blades (cell monolayers) measuring 16.1 6 1.1 mm in diameter (Figs 3, 4). At transverse sections, the diameter of the single protoplasts was determined at 4.4 6 0.6 mm, and cells are conspicuously arranged into packets forming rectangles (Figs 5– 9). The single chloroplast in each cell is stellate and axial. Control samples (freshly harvested material and samples irradiated for 24 h with PAR only) of P. crispa exhibited a good preservation of the ultrastructure (Figs 5–9). Details such as the stellate chloroplast with numerous plastoglobuli, pyrenoids, nucleus, small vacuoles, cytoplasmic globules and the cell wall are visible (Figs 5–7). It can be regarded as a sign of quality that the fixation protocol preserved microtubules (Figs 7, 9); also, the preservation of mitochondria is good (Fig. 8).

Exposing P. crispa to PAR 1 UV-A 1 UV-B for 6 h did not lead to significant alterations in the ultrastructure compared with that of control samples (Figs 10, 11). The arrangement of thylakoids (Fig. 10), the mitochondria with cristae (Fig. 11) and the nucleus remained unaffected. In contrast, PAR 1 UV-A 1 UV-B treatment for 24 h (Figs 12–15) caused slight alterations at the chloroplasts and the number of plastoglobuli was reduced. The low magnification appearance of the thallus (Fig. 12) cannot resolve differences to the control samples; however, larger magnifications occasionally reveal dilatations of the thylakoids (Fig. 15, arrow). The number of globules with a diameter in a range of 0.4–0.8 mm (Fig. 14) increased by at least 3- to 4-fold compared with that of control samples (Fig. 5) and samples exposed to 6 h of UV irradiation (Fig. 11). These globules, only sparsely seen in control samples, appeared to be very abundant in 24 h UV-irradiated cells (Fig. 14) and exhibited a medium electron-dense contrast. Moreover, some mitochondria showed slight damage (Fig. 15).

DISCUSSION In the present study, the green macroalga P. crispa was investigated for its ability to cope with mild, artificial UV radiation. Whereas UV treatment for 6 h caused mild effects on optimum quantum yield, exposure of the thalli for 24 h caused

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Figs 5–9. Details of the ultrastructure of Prasiola crispa control samples.

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Figs 10, 11. Details of the ultrastructure of Prasiola crispa thalli exposed for 6 h to PAR 1 UV-A 1 UV-B. Fig. 10. Details of a chloroplast with plastogolbuli (arrow) and starch grain (S). Fig. 11. Mitochondrium (arrow) in the vicinity of chloroplast lobes, vacuoles (V) and globules (G). Bars 5 1 mm.

a stronger decrease in Fv/Fm and alterations at the ultrastructural level. In polar macroalgae, enhanced UV radiation can exert deleterious effects on photosynthesis and other physiological processes (Hanelt et al. 1997; Aguilera et al. 1999). In many species, strong solar or artificial radiation may depress algal photosynthesis due to PAR or UV impacts (or both) (Ha¨der 1993; Ha¨der & Figueroa 1997; Hanelt 1998). Previous studies have shown that for the evaluation of PAR- and UV-induced inhibition of photosynthesis in macroalgae, the in vivo chlorophyll fluorescence of PS II, as used in the present investigation, is a suitable method (Hanelt et al. 1997; Bischof et al. 1998; Hanelt 1998). The optimum quantum yield (Fv/Fm) is a sensitive parameter for detecting UV stress in macroalgae (Cordi et al. 1997). At the surface of Kongsfjorden during summer at noon and without clouds, photon fluence rates (PAR) of 1300 mmol m22 s21, in parallel with 19 W m22 UVA and 1.1 W m22 UV-B can be measured (Bischof et al. 1998). We used a combination of artificial lamps that emitted 25–30 mmol m22 s21 PAR, 4.7 W m22 UV-A and 0.2 W m22 UV-B, a regime that can be characterized as mild, and which equals more cloudy and foggy conditions typical of many summer days in the Kongsfjorden region. In addition, due to a previous study on UV effects on photosynthesis and ultrastructure of two red algal species from the upper and lower

sublittoral (Holzinger et al. 2004), the same experimental design was chosen for better comparison of the data. In the field, P. crispa will often experience the UV-B and UV-A irradiances applied in this study, combined with higher PAR. The data on the relative ETRs as a function of increasing photon fluence densities in P. crispa indicate a negative UV effect on the a values of these PI curves. Since the Ik values represent the quotient Pmax/a, decreasing a leads to an almost unchanged Ik if Pmax decreases, as was the case after 24 h of UV exposure. The a value is a measure of the efficiency of the photosynthetic apparatus to absorb photons in the irradiance-limited range of the PI curve. Consequently, UV-induced changes in a point to a disturbance of the light-harvesting systems. In contrast to the relatively small changes in the a values of the PI curves for P. crispa, the red macroalgae P. palmata and O. dentata exhibited a particularly strong UV effect on the a values (Holzinger et al. 2004). Thus, the effect of UV radiation on the photosynthesis in Prasiola is much smaller compared with that of both red algal species that were exposed to the same experimental design. This is in good agreement with the observation that even 2.0 W m22 UV-B did not significantly alter the photosynthetic performance of the closely related P. crispa ssp. antarctica (Lud et al. 2001). On the other hand, these authors reported DNA damage under the UV treatment applied. Consequently, the interpretation of

← Fig. 5. Longitudinal section revealing the axial chloroplast (Chl) with numerous plastoglobuli; within the cytoplasm several vacuoles (V), occasional granules (G) and the nucleus (N) are visible. Fig. 6. Section in an area showing the nucleus (N) surrounded by the chloroplast containing a pyrenoid (Py). Fig. 7. Nucleus (N), chloroplast with pyrenoid (Py) and microtubules (arrows). Fig. 8. Detail with mitochondria (arrows) close to the chloroplast. Fig. 9. Microtubules (arrow) in close vicinity to the nucleus (N). Bars 5 1 mm.

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Figs 12–15. Details of the ultrastructure of Prasiola crispa thalli exposed for 24 h to PAR 1 UV-A 1 UV-B. Fig. 12. Overview. Fig. 13. Detail with nucleus (N), pyrenoid (Py) and starch grain (s) within the chloroplast. Fig. 14. Large number of globules (G) and vacuoles (V). Fig. 15. Beginning dilatation of the thylakiod membrane (arrow), starch grain (S) and slightly damaged mitochondrion (M) in vicinity of the chloroplast. Bars 5 1 mm.

Holzinger et al.: UV effects in Prasiola physiological data must always be performed with great care, since cellular alterations may already occur in the algae, which are not reflected by the photosynthetic performance. Prasiola crispa showed only a minor degree of photoinhibition under the highest photon fluence rates tested. In contrast, in the deep-water taxon O. dentata strong photoinhibition has been reported (Holzinger et al. 2004). Although ecophysiological data on the latter species are lacking, it has been documented that among other macroalgae from polar habitats, the degree of photoinhibition is normally a function of light availability corresponding to the collecting depth (i.e. shallowwater isolates such as P. palmata are more PAR/UV resistant than plants from deeper waters) (Bischof et al. 1998). This has also been confirmed for other macroalgae from Kongsfjorden. While the deepwater red alga Phycodrys rubens exhibited strong photobleaching after exposure to enhanced PAR/UV (Karsten et al. 2001), the supralittoral brown alga Fucus distichus was almost insensitive to UV radiation (Hanelt et al. 1997). The high sensitivity of photosynthesis under UV in O. dentata and P. rubens can be related to the lack of capability to form mycosporine-like amino acids (MAAs) as photoprotectants (Karsten et al. 1998). Both P. crispa and P. palmata synthesize and accumulate species-specific MAAs under UV stress (Karsten & Wiencke 1999; Hoyer et al. 2001), indicating a photoprotective function. In addition, the minor effects measured in photosynthetic performance in P. crispa are in agreement with the ultrastructure data, in which after 6 h of UV treatment no visible changes were observed, but the 24 h treatment led to some alterations in the ultrastructure. It has been described that geographic and environmental factors (Friedmann 1969) or osmolarity (Jacob et al. 1991; Jacob 1992) can be critical for the morphology and structure of Prasiola ssp. The data obtained in the present study on the thallus and cell dimension correlate well with those on P. crispa ssp. antarctica (Jacob et al. 1992a). Concerning a general characterization of the ultrastructure in Prasiola ssp., some early reports on different species are presented first. While Manton & Friedmann (1959) investigated the ultrastructure of gametes and zygotes in Prasiola stipitata, the vegetative thallus of Prasiola velutina was described in detail by Lokhorst & Star (1988). The latter authors presented images of vegetative cells that appeared to be very similar to those of the untreated control samples of P. crispa studied. Each cell contained a stellate chloroplast, starch granules and globular structures. A strong structural similarity also exists between P. crispa ssp. antarctica (Jacob et al. 1992a) and P. crispa from Spitsbergen studied here. Apparently the fixation protocol used in the present investigation (subjecting the fixatives in cacodylate buffer and alcohol dehydration), different from the methods described by Jacob et al. (1992a), who used HEPES-buffered seawater and an acetone dehydration, is well suited to study the ultrastructure of P. crispa. After 24 h of UV radiation, it is primarily the chloroplasts and mitochondria that are targeted. Moreover, an unusually high number of globules with medium electron-dense contrast were observed. The globules are comparable to those described by Jacob (1992), who reported a varying contrast of these compartments after different fixation protocols. The contents of these globules remains unclear; most likely they represent storage compounds, as they have the same appearance as storage compounds known from leaves and seeds (Gunning

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& Steer 1996). It must be emphasized that this type of UV treatment does not affect membranes in general, as judged from an intact nuclear envelope and membranes surrounding globules, vesicles and vacuoles. The UV-induced effects on the ultrastructure observed in P. crispa, especially concerning alterations of the chloroplast, are not as tremendous as those of macroalgae from other classes such as the red algal genus Palmaria (Poppe et al. 2003; Holzinger et al. 2004) or O. dendata, in which the chloroplast structures were severely damaged (Holzinger et al. 2004). In addition, in these red algae the effects were already clearly visible after 6 h of UV radiation with the same experimental setup. In another red macroalga, Phycodrys austrogeorgica, the chloroplast envelope and the thylakoid membranes became damaged and the phycobilisomes were detached from the thylakoids after 12 h of UV treatment (Poppe et al. 2003). In the same species, protein crystals were described in the cytoplasm, degraded after UV exposure. The supralittoral red alga Bangia atropurpurea exhibited similar alterations under UV, such as vesiculation of thylakoids (Poppe et al. 2003). In contrast, green algae from other groups apparently can cope very well with UV irradiation. For example, in the unicellular freshwater green alga Micrasterias denticulata, only wavelengths lower than 287 nm (filter cutoff, but in combination with drastically higher values of PAR as used in the present study) were harmful to the cells at the ultrastructural level (Meindl & Lu¨tz 1996; Lu¨tz et al. 1997). At 280 nm (filter cutoff) irradiation, stroma and grana lamellae appeared less clear, grana tended to disintegrate and the netlike arrangement of the thylakoids was lost (Lu¨tz et al. 1997). In addition, some of the dictyosomal cisternae were bent, the number of cisternae reduced and endoplasmic reticular cisternae with ribosomes were present in large sheets (Meindl & Lu¨tz 1996). Despite the fact that especially high pressure freeze fixation/freeze substitution results in good preservation of the ultrastructure in a variety of different algae and higher plants (e.g. Meindl et al. 1992; Holzinger 2000; Poppe et al. 2002; Walther & Ziegler 2002), classical chemical fixation still provides useful information. In fact, the preservation of membranous structures of the chloroplasts and various plasmatic components was excellent when using this fixation procedure. The advantages to performing the TEM preparation in the field, which is not possible with the high-pressure freezing method that requires a sophisticated apparatus, including a long range transport of the samples, exculpate the limitations of a chemical fixation protocol. Based on the obvious differences between the red macroalgae mentioned above and P. crispa, the question arises, ‘What makes the latter species so much more insensitive against the UV regime applied in the laboratory’? One possible answer might be the presence of MAAs. Prasiola crispa ssp. antarctica contains high concentrations of a unique UV-absorbing compound with an absorption maximum at 324 nm, which was characterized as a putative MAA due to chromatographic properties (Hoyer et al. 2001). The occurrence of this compound has also been confirmed in the closely related P. stipitata (Gro¨niger & Ha¨der 2002). Most interestingly, Karsten et al. (2005) showed the presence of MAAs not only in Prasiola species, but also in other morphologically diverse, closely related and in many cases aeroterrestrial green algae from the family Trebouxiophyceae. Only a few macroscopic green al-

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gae investigated so far contain UV-absorbing compounds such as MAAs or coumarins (Pe´rez-Rodrı´guez et al. 1998; Jeffrey et al. 1999; Xiong et al. 1999). The function of MAAs is related to their ability to reduce UV-induced inhibition of photosynthesis (Neale et al. 1998). However, MAAs might not be the only key in understanding the higher UV resistance of P. crispa compared to other macroalgae. The Antarctic chlorophyte Scenedesmus sp. copes with UV by enhanced replacing of the D1 protein or Rubisco and repair of DNA damage (Lesser et al. 2002). Molecular responses are also observed in Antarctic phytoplankton as a consequence of UV irradiation (Karentz et al. 1991). For Prasiola, another factor might be of relevance: The samples used for this study were collected underneath a seagull colony. Data from the literature indicate that Prasiola species are extremely nitrophilous and prefer habitats rich in mixed excreta and faeces of birds (Rindi et al. 1999; Raven & Taylor 2003) and, hence, deal very well with eutrophic conditions. Considering a relation between the MAA concentration and nitrogen availability in different species of the red alga Porphyra (Korbee et al. 2005), as well as a nitrogen-dependency of photoacclimation in Ulva rotundata (Henley et al. 1991), it becomes obvious that nitrogen might be a critical factor for the good physiological performance of Prasiola under subaerial conditions. In summary, the data presented demonstrate that P. crispa from the Arctic Kongsfjorden is rather insensitive to applied UV radiation. Only 24 h of UV treatment caused effects at the physiological and ultrastructural levels, which, however, are less severe compared to those of other macroalgae treated with the same experimental design. Therefore, the supralittoral green macroalga P. crispa, which also occurs in habitats at lower latitudes with higher initial UV irradiation, seems to be adapted to cope with increasing UV, which has been reported as a consequence of further reduction of stratospheric ozone in the northern hemisphere (e.g. Heese 1996; Wa¨ngberg et al. 1996; Shindell 2003).

ACKNOWLEDGEMENTS The authors thank Claudia Daniel for collecting field samples ˚ lesund International Research and Monitoring and the Ny-A Facility for its support. Thanks also to Francesco Gordillo for providing and helping with the DIVING-PAM, as well as Beatrix Defregger and Lavinia Di Piazza for expert technical assistance with the studies of algal ultrastructure. We are grateful to Prof. Reinhard Rieger, Institute of Zoology, University of Innsbruck, for providing access to his department’s transmission electron microscope. Financial support by the Norsk Polar Institute and the LSF for C.L. and A.H. is kindly acknowledged.

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Received 14 April 2005; accepted 31 August 2005 Communicating editor: C. Amsler