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UV-absorbing pigments, photosynthesis and UV exposure in Antarctica: comparison of terrestrial and marine algae
Aquatic Botany, 45 ( 1993 ) 231-243
231
Elsevier Science Publishers B.V., Amsterdam
UV-absorbing pigments, photosynthesis and UV exposure in Antarctica: comparison of terrestrial and marine algae Adele Post and A.W.D. Larkum Macleay Building A 12, School of Biological Sciences, University of Sydney, Sydney, N.S. W.. 2006, Australia (Accepted 22 December 1992)
ABSTRACT Post, A. and Larkum, A.W.D., 1993. UV-absorbing pigments, photosynthesis and UV exposure in Antarctica: comparison of terrestrial and marine algae. Aquat. Bot., 45:231-243. Since Antarctic plants experience a wide range of ultraviolet (UV) exposure, the pigment content of Antarctic algae (Palmaria decipiens (Reinsch) Ricker, Enteromorpha bulbosa (Suhr) Montagne, Prasiola crispa (Lightf.) Kiitz. sp. antarctica(Kiitzing) Knebel ) was monitored over a year. In summer the mature form of the marine rhodophyte Palmaria contains a range of UV-absorbing pigments in high concentration providing a broad absorbance with a maximum at 337 rim. Juvenile fronds develop through winter with smaller absorbance maxima at 322 nm, 309 nm and 295 nm. The terrestrial chlorophyte Prasiola crispa contains a single UV-absorbing pigment with a maximum at 325 nm. Compared with other green algae, including the marine Enteromorpha, the UV-absorbing pigment in Prasiola is present in high concentrations. Variations in the level of UV-absorbance relative to chlorophyll in Prasiola, appear to correspond with varying UV exposure. To test this, Prasiola was maintained with an enhanced ratio of UV-B to visible light to simulate the effects of stratospheric ozone depletion. After 4 weeks the chlorophyll content and photosynthetic rates were reduced in the presence of enhanced UV-B light, but the ratio of UV-absorbing pigments to chlorophyll was unchanged. This suggests that even for Antarctic algae, that contain high levels of UV-absorbing pigments, exposure to sunlight with an increased ratio of UV-B to visible light is stressful.
INTRODUCTION
Pigments absorbing ultraviolet ( U V ) light are found in all classes of algae (Sivalingam et al., 1974). They are thought to be mycosporine-like amino acids (Dunlap et al., 1986; Carreto et al., 1990; Karentz et al., 1991a) with absorbance maxima ranging from 310 to 360 nm. Karentz et al. ( 1991 a) surveyed marine organisms from Antarctica and found such UV-absorbing pigCorrespondence to: A. Post, Macleay Building A 12, School of Biological Sciences, University of Sydney, Sydney, N.S.W. 2006, Australia.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-3770/93/$06.00
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ments to be common. Some evidence suggests that the concentrations of algal UV-absorbing pigments depend on the degree of exposure to UV light (Scherer et al., 1988; Carreto et al., 1990; Marchant et al., 1991 ). The UV exposure in Antarctica varies greatly throughout the year, because of the low angle of the sun at high latitudes, variations in cloud cover, sea-ice formation, and snow and ice buildup on the surface of terrestrial plants. The effect of increasing UV exposure on Antarctic organisms is now an important consideration (EI-Sayad et al., 1990; Karentz et al., 1991b), as the level of UV light in spring has increased owing to stratospheric ozone depletion (Frederick and Snell, 1988 ). This study attempted to determine if the extreme seasonal differences in UV exposure in Antarctica caused a variation in UV-absorbing pigments. Over one year algae were collected in the vicinity of Casey (66 ° 17'S, 110 ° 32' E). A terrestrial chlorophyte alga which grows in perennial mats was chosen as it is fully exposed to sunlight in summer. It was compared with two marine algae, a green alga found in s u m m e r and a red alga that persisted over winter. The effects of artificially enhancing the ratio of UV to visible light on the photosynthesis and pigments of the terrestrial alga were also determined. MATERIALS AND METHODS
Sampling Algal samples were collected in the area of Casey, Wilke's Land, East Antarctica (66 ° 17'S, 110°32'E). The highest temperatures occur in January (mean monthly m a x i m u m of 2.6°C), the coldest month is August (mean monthly m a x i m u m of - 11.3 °C) and spring temperatures are low (October mean monthly m a x i m u m of - 7 . 5 ° C ) (Australian Meteorological Bureau Data, 1969-1988). Prasiola crispa (Lightf.) Kiitz. ssp. antarctica (Kiitzing) Knebel (in the text indicated as Prasiola) is a green foliose alga that frequently grows in areas near to penguin rookeries which provide conditions of low pH and high nutrients. When undisturbed, mats gradually form which are composed of crinkled Prasiola thalli. These mats experience a wide range of hydration as they can be under water due to melting snow and ice while, at other times, be very dry; most frequently during the growing season the mat acts as a sponge and the thalli are moist. Thalli are parenchymatous with apparently diffuse growth and vary in size up to 1 cm 2. To standardise field samples, collections were made from Shirley Island (66 ° 17'S, 110°30'E) and only sections of thick ( 15 ram) healthy mats were used. The tops of mats are often paler than the lower sections, so the Prasiola thalli were sorted into top, middle and base layers, approximately 5 m m each. Marine algae were collected from along the shoreline in the intertidal re-
ANTARCTIC ALGAE: UV EXPOSURE AND PHOTOSYNTHESIS
2 33
gion (down to 1 m depth). Owing to the availability of samples over winter,
Palmaria decipiens (Reinsch) Ricker (in the text indicated as Palmaria) was studied. In summer, Palmaria fronds are up to 40 cm in length and up to 3 cm in width and are oriented vertically with a holdfast attachment to rocks.
Palmaria is frequently found at this time of the year growing together with Enteromorpha bulbosa (Suhr) Montagne (in the text indicated as Enteromorpha). Seawater temperatures do not vary greatly over the year and do not go higher than - 1.8°C. Sea-ice forms over winter and m o v e m e n t of the ice scours the rocks at the shore so that most algae, including Enteromorpha are removed. Palmaria survives over winter as it has strong holdfasts with short fronds, from which side shoots develop over the year. In winter samples could be collected either by digging through the sea-ice or when the ice was blown out by intermittent blizzards.
Pigment extraction To quantify the UV-absorbing pigments in relation to chlorophyll, fresh samples were extracted in methanol (analytical grade low in UV-absorbance ) by grinding in a mortar and pestle with acid-washed sand. The extract was then centrifuged (3000 g for 10 m i n ) and the absorbance spectrum of the supernatant determined (200-700 n m ) , using an Hitachi U-3200 spectrophotometer. Concentrations of UV-absorbing pigments were expressed either as a ratio of the absorbance m a x i m u m in the UV compared with the chlorophyll absorbance m a x i m u m or as the absorbance at the UV peak maxima per unit weight of chlorophyll: for Prasiola, A32s (mg chlorophyll a+b)- l; for Palmaria, A337 (mg chlorophyll a ) -1. Chlorophyll and carotenoid concentrations were calculated (Lichtenthaler and Wellburn, 1983 ).
Culturing Prasiola under enhanced UV light Prasiola was collected in early November and the thalli from the middle and base layers of the mat rinsed and placed in shallow trays. Snow was collected from the sampling site, melted as required and added to the Prasiola to keep it moist. The trays were placed in an environment cabinet at 3 °C with a 12 h day/night cycle. Light was provided by GroLux tubes with UV-B enhancement from Westinghouse FS20 tubes which have peak emission at 313 nm. Samples were covered by sheets of mylar (to filter out UV-B and UV-C ) or by cellulose acetate (to filter out UV-C). Three replicate trays were used for each treatment. The UV-B radiation was measured in units of W m -2 using an International Light meter (IL- 1350 ) with a 313-nm filter on the sensor. Irradiance (400-700 n m ) was measured in units o f # m o l m -2 s-~ with a Li-Cor (Li-1000) light meter. Irradiance values (400-700 n m ) were converted to W m -2 using the conversion factors of Thimijan and Heins ( 1983 ): 4.57 for sunlight and 4.80 for GroLux tubes. Ambient levels of UV-B and
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A. POST AND A.W.D. LARKUM
visible light were measured at midday on the collection day, 13 November 1990: clear sky, visible light of 438 W m -2, UV-B light of 1.2 W m -2. The ratio of UV-B to visible light in the environment chamber was enhanced to double that in the ambient sunlight at midday. Incident irradiance on samples of Prasiola in the chamber was 29 W m -2 of visible light, with enhanced UVB radiation of 0.17 W m -2.
Photosynthetic measurements The photosynthetic oxygen-evolution rates of samples were measured with a Rank oxygen electrode. A known fresh weight (about 50 mg) of thallus was placed in the electrode chamber with the sample buffer and excess bicarbonate (20 m M ) . The sample buffer contained 20 m M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) buffer, pH 6.7 in deionised water for Prasiola, and pH 8.0 in seawater for Palmaria and Enteromorpha. Measurements were made at 4°C and 20°C for the marine algae. Prasiola in the field grows at low pH and experiences a range of temperatures from above ambient, owing to warming from sunlight (surface temperatures above 20 °C), to below freezing. Prasiola was routinely measured at 20 °C as photosynthetic rates are high at this temperature. After the sample had equilibrated to the required temperature, the oxygen evolution was measured at increasing irradiance. The sensor of the Li-Cor meter was positioned on the far side of the oxygen-electrode chamber in line with the projector lamp. The sample was then removed from the chamber and the chlorophyll content determined. RESULTS
Palmaria and Enteromorpha UV-absorbing pigments change as Palmaria undergoes its annual cycle of growth (Fig. 1 ). In summer (January) Palmaria has large fronds with the highest levels of UV-absorbing pigment. The spectrum of the pigment extract of the fronds (Fig. 1a) shows a broad UV-absorbance peak spanning from approximately 300 n m to 400 nm. The m a x i m u m absorbance was at 337 nm, with a concentration of A337 (mg chlorophyll a) -1 of 580 (Table 1 ). The ratio of UV-absorbance peak height to chlorophyll peak height was 7.8. In addition to the major peak at 337 n m in the methanol extract of Palmaria, collected in summer, the UV-absorbance had a shoulder at 360 nm. In winter Palmaria had small fronds and in the July sample (Fig. l c), the absorbance at 337 n m was reduced to a shoulder on a peak of 322 nm, with a value for the A337(mg chlorophyll a ) - 1 of only 95 (Table 1 ). With the drop in absorbance at 337 n m it is possible to resolve clearly the smaller absorbance peaks
235
ANTARCTIC ALGAE: UV EXPOSURE AND PHOTOSYNTHESIS
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Fig. 1. Seasonal variation in UV-absorbing pigments in Palmaria decipiens. Table 1 shows the absorbance values relative to the chlorophyll concentration. TABLE 1 Seasonal variations in the UV-absorbing pigment content of Palmaria decipiens, expressed per unit weight of chlorophyll a Sample date
January. April July October
Pigment absorbance (mg chlorophyll a) 309 nm
322 nm
337 nm
360 nm
* 215 182 183
* 290 187 288
580 296 95 305
287 147 * 161
*Indicates where values were not calculated owing to the absence of a clear peak or shoulder in the absorbance spectrum at that wavelength. Values are the mean of three samples.
at 322 nm, 309 nm and 295 nm. The extracts of samples collected in October, showed an increasing absorbance at 337 nm and 360 nm, corresponding with the regrowing of larger fronds. Comparison of oxygen evolution at increasing irradiances (Fig. 2a), of
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Fig. 2. Photosynthetic rates as a function of irradiance. (a) Palmaria decipiens collected in summer (solid lines), measured at 20°C (squares) and at 4°C (circles) and collected from under sea-ice in October (dashed line), measured at 20 °C. (b) Enteromorpha bulbosa collected in summer (solid lines) and measured at 20°C (squares) and at 4°C (circles). summer fronds of Palmaria and o f material sampled from under the sea-ice in October, showed that the mature fronds are not light-saturated, even at high irradiance at either 4 ° C or 20°C; however, the October samples were light-saturated above an irradiance o f 400/tmol m - 2 s - 1. Samples o f Enteromorpha collected in summer (Fig. 2b ) showed light-saturation of photosynthesis at both 4 ° C and 2 0 ° C above irradiances of 500 pmol m -2 s-1. Photosynthetic rates were higher for both algae at 20°C. The Enteromorpha had a low content o f UV-absorbing pigments, the methanol extract gave a ratio of 0.75 between the peak U V absorbance at 340 nm and the chlorophyll absorbance at 666 nm. No samples were present in winter collections o f algae from below the sea-ice.
237
ANTARCTIC ALGAE: UV EXPOSURE AND PHOTOSYNTHESIS
Prasiola
Prasiola does not experience the extreme mechanical disturbance of the marine environment. In summer, mats are exposed and they can be disturbed by penguins, but in winter, even though they are most frequently covered by snow and ice, mats generally remain intact. Photosynthetic activity in Prasiola has been measured down to - 1 5 ° C (Becket, 1982). Prasiola thalli appear to grow steadily whenever conditions are favourable, without going through varying stages of development or maturity over the year. The spectrum of the methanol extract of Prasiola thalli shows a single broad UV-absorbance peak with a m a x i m u m absorbance at 325 n m (Fig. 3). The UVabsorbance covers the range from approximately 290 n m to 360 nm. To limit the variations in field material, Prasiola samples were collected from the same site and sorted routinely. Middle sections of mats collected in summer (December 1989 ) and winter (July 1990), showed a difference in the absorbance of the UV-absorbing pigment relative to chlorophyll (Fig. 3). The summer sample had a value for A325 (mg chlorophyll a + b ) - 1 of 186 compared with a value of 115 for the winter sample. No variation in the chlorophyll a/b ratio was found. No UV light was detected at the base of a 15-mm thick Prasiola mat when bright s u m m e r sunlight was shining directly on the surface, which means a gradient of UV light occurs through the thickness of the mat. The effect of 2.0
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A. P O S T A N D A . W . D . L A R K U M
this naturally occurring shading was analysed in summer by assaying the UVabsorbing pigment at differing levels in a mat (Table 2). The UV-absorbance relative to chlorophyll was greatest in the top layer which had a value for A325 (mg chlorophyll a + b) - ' of 215. Prasiola mats are frequently a paler green on the surface than at the base and analysis of the top layer showed it had the lowest chlorophyll content per unit weight, approximately 60% of the middle and base layers (Table 2). The chlorophyll a/b ratio was constant through the mat layers. The ratio of carotenoids to chlorophyll was higher in the top layer. Photosynthetic rates of thalli were also measured on these samples and expressed on a chlorophyll basis to make comparison easier, as this takes into account the variation in chlorophyll concentration (Fig. 4). The quantum efficiency was identical throughout the mat and light-saturated rates of oxygen evolution were similar. The photosynthetic rates and the light-saturating irradiances are compaTABLE 2 UV-absorbing pigment content, chlorophyll content and ratio of carotenoids to chlorophyll in sections through a Prasiola crispa mat collected in summer (n = 3, _+standard errors) Cross-section layer
Chl a/b
Chlorophyll content mgchl (gFW) -'
Carotenoid: chlorophyll (w:w)
A325 (mg chl ) - '
Top Middle Base
2.75+0.01 2.73+0.06 2.73_+0.04
1.7_+0.2 2.9+0.1 2.8_+0.1
0.324_+0.012 0.279_+0.008 0.259_+0.002
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Fig. 4. Photosynthetic rates as a function of irradiance for thalli sampled in sections through a naturally occurring mat of Prasiola crispa; top layer (dashed line), base layer (solid line). Photosynthetic rates are expressed on a chlorophyll basis, the chlorophyll content is shown in Table 2.
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ANTARCTIC ALGAE: UV EXPOSURE AND PHOTOSYNTHESIS
rable with those observed for Prasiola crispa in culture (Jacob et al., 1991 ) and for Prasiola stipitata Suhr ex Jessen (Raven and Johnston, 1991 ). The effect of increasing UV-B light exposure on photosynthesis and on the concentration of UV-absorbing pigment was tested in Prasiola. Material was collected soon after it had melted out after winter and the middle and base layers combined, rinsed and then divided into two sets of three. One set was used as a control and had no UV-B exposure. The other set was exposed to U V radiation using a ratio of UV-B to visible light which was enhanced to double that measured in ambient midday sunlight on a clear day at the time of collection. By the second week the photosynthetic rates in all replicates exposed to UV-B were reduced. But there was no change in the ratio of UVTABLE3 Pigment content ofPrasiola crispa at the start and after 4 weeks of culturing Chi a/b
Treatment
Initial
Chlorophyll content mgchl (g EVe) -1
Carotenoid: chlorophyll
A325 (mg chl) - t
2.71 _+0.01
2.9+0.3
0.273+0.015
187+21
2.46 _+0.03 2.28 + 0.03
2.4 + 0.1 1.7 + 0.1
0.283 + 0.007 0.304 + 0.006
184 + 6 188 + 11
(W:W)
4 Weeks minus UV-B plus UV-B
Control samples were covered with mylar to filter out UV-B radiation. Samples exposed to enhanced UV-B were covered by cellulose acetate to filter out UV-C radiation (initial sample: n = 3, 4 weeks sample: n = 12, + standard errors)
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Fig. 5. P h o t o s y n t h e t i c rates as a f u n c t i o n o f irradiance for Prasiola crispa grown u n d e r low light in a cabinet with n o ultraviolet light (solid line) a n d with e n h a n c e d ultraviolet light (dashed line). The inset shows the gross p h o t o s y n t h e t i c rates in the light limiting range for photosynthesis. Photosynthetic rates are expressed on a chlorophyll basis, the chlorophyll content is shown in Table 3.
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A. POST AND A.W.D. LARKUM
absorbance to chlorophyll at 325 nm from the initial value even after 4 weeks (Table 3 ). The photosynthetic rates measured at 4 weeks (Fig. 5 ) showed an increase in respiration in the Prasiola exposed to UV-B, but the light-saturated rate of oxygen evolution of the exposed samples were only 40% of the control rate. The photosynthetic rates corrected for dark respiration (Fig. 5, inset) clearly showed the reduction in the photon yield in the samples exposed to UV-B. In parallel with the drop in photosynthetic rates there was a drop in the chlorophyll content per unit weight in the exposed samples although the chlorophyll a/b ratio was similar (Table 3 ). DISCUSSION
The UV and visible light environment of Antarctic algae varies greatly over the year. At Casey in mid-winter the sun is above the horizon for less than 3 h and the sun reaches a maximum elevation angle of only 5 °. The snow and ice cover is thick with over 1 m of sea-ice on the edges of the shoreline. By contrast, in summer the days are long with skies frequently clear and the Prasiola mats are fully exposed to high levels of UV light. Biologically active wavelengths of UV can also penetrate Antarctic seawater to depths of 10 m (Karentz and Lutze, 1990). Antarctic seawater has a constant temperature of - 1.8 °C (Wiencke and tom Dieck, 1989; and personal observation). Terrestrial plants experience a range of temperatures. On clear summer days the surface temperature of Prasiola can be above 20 oC, while freezing temperatures occur overnight (Becker, 1982; and personal observation). Prasiola can also tolerate a wide range of salinity, possibly assisted by the thick cell walls (Jacob et al., 1991 ). Although marine temperatures are stable, sea-ice scours the shoreline and the larger fronds which are enclosed in the ice are torn away. The Palmaria plants which survive generally consist of the holdfast with a short, sometimes broken, frond. Later in the year lateral fronds also develop from this. The development of lateral shoots has been observed in Palmaria fronds in culture and is dependent on the seasonal changes in day-length (Wiencke, 1990). Palmaria contains a range of UV-absorbing pigments. The greatest absorbance is in summer and is maximum at 337 nm with a shoulder at 360 nm. Over the year other peaks were observed at 322 nm and 309 nm. Palmaria collected from Palmer Station (64°46'S, 64°03'W) in early summer (Karentz et al., 1991 a), contained mycosporine-glycine (310 nm), palythine (320 nm ), palythene (360 nm) and four pigments absorbing in the range 330-334 nm. The Palmaria collected at Casey over the winter, with juvenile lateral fronds, had reduced UV-absorbing pigment content compared with the summer (Table 1 ). In addition, the absorbance peaks at 309 nm and 322 nm suggest the pigments are mycosporine-glycine and palythine which have the simplest chemical structures.
ANTARCTIC ALGAE: UV EXPOSURE AND PHOTOSYNTHESIS
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Mature Palmaria fronds collected in January showed higher rates of photosynthesis at 20°C than at lower temperatures, but the oxygen evolution is not light-saturated at either temperature under these conditions. Palmaria collected from below the sea-ice was light-saturated at lower irradiance with saturation occurring above 400/zmol m -2 s-~, even when measured at 20°C. The increase in photosynthetic capacity in summer corresponds with the time when Palmaria has the maximum content of UV-absorbing pigments. The chlorophyte, Prasiola contains a single UV-absorbing pigment with absorbance maximum at 325 rim. Enteromorpha, in the Ulvophyceae is closely related to Prasiola (Raven and Johnston, 1991 ), but Enteromorpha had low levels of UV-absorbing pigment. These differences in the content of UV-absorbing pigments correlate with the varying light environment of the two algae. Prasiola is fully exposed to sunlight while the marine Enteromorpha is largely shaded by the taller Palmaria fronds in summer. Prasiolajaponica Yatabe collected in Japan (Sivalingam et al., 1974) contained a pigment also absorbing at 325 nm, but the ratio of the absorbance of the UV-absorbing pigment to the absorbance of chlorophyll at 663 nm was only 0.8. This is much lower than typical values found in summer samples of Prasiola, such as the middle-layer thaUi (Fig. 3) which have a ratio of the UV-absorbing pigment to chlorophyll absorbance of 2.7; the surface layer thalli have an even higher value of 3.4. While it has been suggested that green algae commonly contain low levels of the UV-absorbing pigments (Karentz et al., 1991 a), this is clearly not the case for Prasiola growing in Antarctica. The results suggest a seasonal variation occurs in the level of UV-absorbance relative to chlorophyll in Prasiola, with the highest levels found in the top layers of mats in summer. Under ambient conditions, regulation of the UV-absorbing pigments relative to chlorophyll appears to occur up to this maximum level and decreases with shading. Other studies have found regulation of UV-absorbing pigments in algae is related to UV exposure (Scherer et al., 1988; Carreto et al., 1990; Marchant et al., 1991 ). The variations in UV-absorbing pigments found over the year for Palmaria and Prasiola and the variations found in the layers of the Prasiola mat tend to support that conclusion. However, when Prasiola was exposed to an enhanced ratio of UV-B to visible light, the concentration of UV-absorbing pigments relative to chlorophyll remained constant (Table 3). Compared with sunlight the total UV-B exposure in the cabinet was low, because the level of visible light was restricted and this may have limited the synthesis of UV-absorbing pigments. Yet there were some similarities between the effects of UV in the environmental cabinet and bright sunlight on the surface layer of Prasiola in the field, as shown with the decrease in chlorophyll per unit of weight with no change in the chlorophyll a/b ratio (Table 3 ). The enhanced ratio of UV-B light resulted in a reduction in oxygen evolution under both light-saturating and limiting con-
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ditions when photosynthetic rates were expressed on a chlorophyll basis (Fig. 5 ) to allow for the change in chlorophyll concentration. These Antarctic algae contain a range of UV-absorbing pigments. Measurements of the pigments in the marine and terrestrial algae indicate that the maximum levels of UV-absorbing pigments occur in summer. The concentration of the pigments in these species can change with the stage of development and with variations in exposure to sunlight. Very high concentrations of UVabsorbing pigments occur in the mature fronds of Palmaria in summer. By contrast the concentrations in Prasiola are lower, but even these are high by comparison with Enteromorpha, a shade plant, and with temperate species (Sivalingam et al., 1974). The concentration of the UV-absorbing pigment found in Prasiola in the summer was not increased any further by exposure to an enhanced ratio of UV-B radiation to visible light, although the chlorophyll concentration and photosynthetic rates decreased, and this may have important and general implications. The main effect of stratospheric ozone depletion over Antarctica has been to increase the ratio of UV-B to visible light in spring (Frederick and Snell, 1988; Karentz, 1991 ). Our simulation of this type of change, albeit at a low level of total irradiance, suggests that such changes in the ratio of UV-B to visible light may lead to reduced productivity even in those Antarctic plants that already contain high levels of UV-absorbing pigments. ACKNOWLEDGEMENTS
This study depended on the support of the Australian Antarctic Division, the Antarctic Science Advisory Committee and the Australian Research Council.
REFERENCES Becker, E.W., 1982. Physiological Studies on Antarctic Prasiola crispa and Nostoc commune at low temperatures. Polar Biol., 1: 99-104. Carreto, J.I., Carignan, M.O., Daleo, G. and De Marco, S.G., 1990. Occurrence of mycosporinelike amino acids in the red-tide dinoflagellate Alexandrium excavatum: UV photoprotective compounds? J. Plankton Res., 12:909-921. Dunlap, W.C., Chalker, B.E. and Oliver, J.K., 1986. Bathyrythmic adaptations of reef building corals at Davies Reef, Australia. III UV-B absorbing compounds. J. Exp. Mar. Biol. Ecol., 104: 239-248. EI-Sayad, S.Z., Stephens, F.C., Bidigare, R.R. and Ondrusek, M.E., 1990. Effect of ultraviolet radiation on Antarctic marine phytoplankton. In: K.R. Kerry and G. Hempel (Editors), Antarctic Ecosystems. Ecological Change and Conservation, Springer, Berlin, pp. 379-385. Frederick, J.E. and Snell, H.E., 1988. Ultraviolet radiation levels during the Antarctic spring. Science, 241: 438-440. Jacob, A., Kirst, G.O., Wiencke, C. and Lehmann, H., 1991. Physiological responses of the
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Antarctic green alga Prasiola crispa spp. antarctica to salinity stress. J. Plant Physiol., 139: 57-62. Karentz, D., 1991. Ecological considerations of Antarctic ozone depletion. Antarct. Sci., 3: 311. Karentz, D. and Lutze, L.H., 1990. Evaluation of biologically harmful ultraviolet radiation in Antarctica with a biological dosimeter designed for aquatic environments. Limnol. Oceanogr., 35: 549-561. Karentz, D., McEuen, F.S., Land, M.C. and Dunlap, W.C., 199 la. Survey of mycosporine-like amino acid compounds in Antarctic marine organisms: potential protection from ultraviolet exposure. Mar. Biol., 108:157-166. Karentz, D., Cleaver, J.E. and Mitchell, D.L., 1991 b. Cell survival characteristics and molecular responses of Antarctic phytoplankton to ultraviolet-B radiation. J. Phycol., 27: 326-341. Lichtenthaler, H.K. and Wellburn, A.R., 1983. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans., 4:591-592. Marchant, H.J., Davidson, A.T. and Kelly, G.J., 1991. UV-B protecting compounds in the marine alga Phaeocystis pouchetii from Antarctica. Mar. Biol., 109:391-395. Raven, J.A. and Johnston, A.M., 1991. Photosynthetic inorganic carbon assimilation by Prasiola stipitata (Prasiolales, Chlorophyta) under emersed and submersed conditions: relationship to the taxonomy ofPrasiola. Br. Phycol. J., 26: 247-257. Scherer, S., Chen, T.W., Boger, P., 1988. A new UV-A/B protecting pigment in the terrestrial cyanobacterium Nostoc commune. Plant Physiol., 88: 1055-1057. Sivalingam, P.M., Ikawa, T., Yokohama, Y. and Nisizawa, K., 1974. Distribution of a 334 UVabsorbing-substance in algae, with special regard of its possible physiological roles. Bot. Mar., 17: 23-29. Thimijan, R.W. and Heins, R.D., 1983. Photometric, radiometric, and quantum light units of measure: a review of procedures for interconversion. Hortic. Sci., 18:818-822. Wiencke, C., 1990. Seasonality of red and green macroalgae from Antarctica--a long-term culture study under fluctuating Antarctic daylengths. Polar Biol., 10: 601-607. Wiencke, C. and tom Dieck, I., 1989. Temperature requirements for growth and temperature tolerance of macroalgae endemic to the Antarctic region. Mar. Ecol. Prog. Ser., 54:189-197. Worrest, R.C., van Dyke, H. and Thomson, B.E., 1978. Impact of enhanced simulated solar ultraviolet radiation upon a marine community. Photochem. Photobiol., 27: 471-478.
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UV-absorbing pigments, photosynthesis and UV exposure in Antarctica: comparison of terrestrial and marine algae Adele Post and A.W.D. Larkum Macleay Building A 12, School of Biological Sciences, University of Sydney, Sydney, N.S. W.. 2006, Australia (Accepted 22 December 1992)
ABSTRACT Post, A. and Larkum, A.W.D., 1993. UV-absorbing pigments, photosynthesis and UV exposure in Antarctica: comparison of terrestrial and marine algae. Aquat. Bot., 45:231-243. Since Antarctic plants experience a wide range of ultraviolet (UV) exposure, the pigment content of Antarctic algae (Palmaria decipiens (Reinsch) Ricker, Enteromorpha bulbosa (Suhr) Montagne, Prasiola crispa (Lightf.) Kiitz. sp. antarctica(Kiitzing) Knebel ) was monitored over a year. In summer the mature form of the marine rhodophyte Palmaria contains a range of UV-absorbing pigments in high concentration providing a broad absorbance with a maximum at 337 rim. Juvenile fronds develop through winter with smaller absorbance maxima at 322 nm, 309 nm and 295 nm. The terrestrial chlorophyte Prasiola crispa contains a single UV-absorbing pigment with a maximum at 325 nm. Compared with other green algae, including the marine Enteromorpha, the UV-absorbing pigment in Prasiola is present in high concentrations. Variations in the level of UV-absorbance relative to chlorophyll in Prasiola, appear to correspond with varying UV exposure. To test this, Prasiola was maintained with an enhanced ratio of UV-B to visible light to simulate the effects of stratospheric ozone depletion. After 4 weeks the chlorophyll content and photosynthetic rates were reduced in the presence of enhanced UV-B light, but the ratio of UV-absorbing pigments to chlorophyll was unchanged. This suggests that even for Antarctic algae, that contain high levels of UV-absorbing pigments, exposure to sunlight with an increased ratio of UV-B to visible light is stressful.
INTRODUCTION
Pigments absorbing ultraviolet ( U V ) light are found in all classes of algae (Sivalingam et al., 1974). They are thought to be mycosporine-like amino acids (Dunlap et al., 1986; Carreto et al., 1990; Karentz et al., 1991a) with absorbance maxima ranging from 310 to 360 nm. Karentz et al. ( 1991 a) surveyed marine organisms from Antarctica and found such UV-absorbing pigCorrespondence to: A. Post, Macleay Building A 12, School of Biological Sciences, University of Sydney, Sydney, N.S.W. 2006, Australia.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-3770/93/$06.00
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ments to be common. Some evidence suggests that the concentrations of algal UV-absorbing pigments depend on the degree of exposure to UV light (Scherer et al., 1988; Carreto et al., 1990; Marchant et al., 1991 ). The UV exposure in Antarctica varies greatly throughout the year, because of the low angle of the sun at high latitudes, variations in cloud cover, sea-ice formation, and snow and ice buildup on the surface of terrestrial plants. The effect of increasing UV exposure on Antarctic organisms is now an important consideration (EI-Sayad et al., 1990; Karentz et al., 1991b), as the level of UV light in spring has increased owing to stratospheric ozone depletion (Frederick and Snell, 1988 ). This study attempted to determine if the extreme seasonal differences in UV exposure in Antarctica caused a variation in UV-absorbing pigments. Over one year algae were collected in the vicinity of Casey (66 ° 17'S, 110 ° 32' E). A terrestrial chlorophyte alga which grows in perennial mats was chosen as it is fully exposed to sunlight in summer. It was compared with two marine algae, a green alga found in s u m m e r and a red alga that persisted over winter. The effects of artificially enhancing the ratio of UV to visible light on the photosynthesis and pigments of the terrestrial alga were also determined. MATERIALS AND METHODS
Sampling Algal samples were collected in the area of Casey, Wilke's Land, East Antarctica (66 ° 17'S, 110°32'E). The highest temperatures occur in January (mean monthly m a x i m u m of 2.6°C), the coldest month is August (mean monthly m a x i m u m of - 11.3 °C) and spring temperatures are low (October mean monthly m a x i m u m of - 7 . 5 ° C ) (Australian Meteorological Bureau Data, 1969-1988). Prasiola crispa (Lightf.) Kiitz. ssp. antarctica (Kiitzing) Knebel (in the text indicated as Prasiola) is a green foliose alga that frequently grows in areas near to penguin rookeries which provide conditions of low pH and high nutrients. When undisturbed, mats gradually form which are composed of crinkled Prasiola thalli. These mats experience a wide range of hydration as they can be under water due to melting snow and ice while, at other times, be very dry; most frequently during the growing season the mat acts as a sponge and the thalli are moist. Thalli are parenchymatous with apparently diffuse growth and vary in size up to 1 cm 2. To standardise field samples, collections were made from Shirley Island (66 ° 17'S, 110°30'E) and only sections of thick ( 15 ram) healthy mats were used. The tops of mats are often paler than the lower sections, so the Prasiola thalli were sorted into top, middle and base layers, approximately 5 m m each. Marine algae were collected from along the shoreline in the intertidal re-
ANTARCTIC ALGAE: UV EXPOSURE AND PHOTOSYNTHESIS
2 33
gion (down to 1 m depth). Owing to the availability of samples over winter,
Palmaria decipiens (Reinsch) Ricker (in the text indicated as Palmaria) was studied. In summer, Palmaria fronds are up to 40 cm in length and up to 3 cm in width and are oriented vertically with a holdfast attachment to rocks.
Palmaria is frequently found at this time of the year growing together with Enteromorpha bulbosa (Suhr) Montagne (in the text indicated as Enteromorpha). Seawater temperatures do not vary greatly over the year and do not go higher than - 1.8°C. Sea-ice forms over winter and m o v e m e n t of the ice scours the rocks at the shore so that most algae, including Enteromorpha are removed. Palmaria survives over winter as it has strong holdfasts with short fronds, from which side shoots develop over the year. In winter samples could be collected either by digging through the sea-ice or when the ice was blown out by intermittent blizzards.
Pigment extraction To quantify the UV-absorbing pigments in relation to chlorophyll, fresh samples were extracted in methanol (analytical grade low in UV-absorbance ) by grinding in a mortar and pestle with acid-washed sand. The extract was then centrifuged (3000 g for 10 m i n ) and the absorbance spectrum of the supernatant determined (200-700 n m ) , using an Hitachi U-3200 spectrophotometer. Concentrations of UV-absorbing pigments were expressed either as a ratio of the absorbance m a x i m u m in the UV compared with the chlorophyll absorbance m a x i m u m or as the absorbance at the UV peak maxima per unit weight of chlorophyll: for Prasiola, A32s (mg chlorophyll a+b)- l; for Palmaria, A337 (mg chlorophyll a ) -1. Chlorophyll and carotenoid concentrations were calculated (Lichtenthaler and Wellburn, 1983 ).
Culturing Prasiola under enhanced UV light Prasiola was collected in early November and the thalli from the middle and base layers of the mat rinsed and placed in shallow trays. Snow was collected from the sampling site, melted as required and added to the Prasiola to keep it moist. The trays were placed in an environment cabinet at 3 °C with a 12 h day/night cycle. Light was provided by GroLux tubes with UV-B enhancement from Westinghouse FS20 tubes which have peak emission at 313 nm. Samples were covered by sheets of mylar (to filter out UV-B and UV-C ) or by cellulose acetate (to filter out UV-C). Three replicate trays were used for each treatment. The UV-B radiation was measured in units of W m -2 using an International Light meter (IL- 1350 ) with a 313-nm filter on the sensor. Irradiance (400-700 n m ) was measured in units o f # m o l m -2 s-~ with a Li-Cor (Li-1000) light meter. Irradiance values (400-700 n m ) were converted to W m -2 using the conversion factors of Thimijan and Heins ( 1983 ): 4.57 for sunlight and 4.80 for GroLux tubes. Ambient levels of UV-B and
234
A. POST AND A.W.D. LARKUM
visible light were measured at midday on the collection day, 13 November 1990: clear sky, visible light of 438 W m -2, UV-B light of 1.2 W m -2. The ratio of UV-B to visible light in the environment chamber was enhanced to double that in the ambient sunlight at midday. Incident irradiance on samples of Prasiola in the chamber was 29 W m -2 of visible light, with enhanced UVB radiation of 0.17 W m -2.
Photosynthetic measurements The photosynthetic oxygen-evolution rates of samples were measured with a Rank oxygen electrode. A known fresh weight (about 50 mg) of thallus was placed in the electrode chamber with the sample buffer and excess bicarbonate (20 m M ) . The sample buffer contained 20 m M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) buffer, pH 6.7 in deionised water for Prasiola, and pH 8.0 in seawater for Palmaria and Enteromorpha. Measurements were made at 4°C and 20°C for the marine algae. Prasiola in the field grows at low pH and experiences a range of temperatures from above ambient, owing to warming from sunlight (surface temperatures above 20 °C), to below freezing. Prasiola was routinely measured at 20 °C as photosynthetic rates are high at this temperature. After the sample had equilibrated to the required temperature, the oxygen evolution was measured at increasing irradiance. The sensor of the Li-Cor meter was positioned on the far side of the oxygen-electrode chamber in line with the projector lamp. The sample was then removed from the chamber and the chlorophyll content determined. RESULTS
Palmaria and Enteromorpha UV-absorbing pigments change as Palmaria undergoes its annual cycle of growth (Fig. 1 ). In summer (January) Palmaria has large fronds with the highest levels of UV-absorbing pigment. The spectrum of the pigment extract of the fronds (Fig. 1a) shows a broad UV-absorbance peak spanning from approximately 300 n m to 400 nm. The m a x i m u m absorbance was at 337 nm, with a concentration of A337 (mg chlorophyll a) -1 of 580 (Table 1 ). The ratio of UV-absorbance peak height to chlorophyll peak height was 7.8. In addition to the major peak at 337 n m in the methanol extract of Palmaria, collected in summer, the UV-absorbance had a shoulder at 360 nm. In winter Palmaria had small fronds and in the July sample (Fig. l c), the absorbance at 337 n m was reduced to a shoulder on a peak of 322 nm, with a value for the A337(mg chlorophyll a ) - 1 of only 95 (Table 1 ). With the drop in absorbance at 337 n m it is possible to resolve clearly the smaller absorbance peaks
235
ANTARCTIC ALGAE: UV EXPOSURE AND PHOTOSYNTHESIS
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Fig. 1. Seasonal variation in UV-absorbing pigments in Palmaria decipiens. Table 1 shows the absorbance values relative to the chlorophyll concentration. TABLE 1 Seasonal variations in the UV-absorbing pigment content of Palmaria decipiens, expressed per unit weight of chlorophyll a Sample date
January. April July October
Pigment absorbance (mg chlorophyll a) 309 nm
322 nm
337 nm
360 nm
* 215 182 183
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at 322 nm, 309 nm and 295 nm. The extracts of samples collected in October, showed an increasing absorbance at 337 nm and 360 nm, corresponding with the regrowing of larger fronds. Comparison of oxygen evolution at increasing irradiances (Fig. 2a), of
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Fig. 2. Photosynthetic rates as a function of irradiance. (a) Palmaria decipiens collected in summer (solid lines), measured at 20°C (squares) and at 4°C (circles) and collected from under sea-ice in October (dashed line), measured at 20 °C. (b) Enteromorpha bulbosa collected in summer (solid lines) and measured at 20°C (squares) and at 4°C (circles). summer fronds of Palmaria and o f material sampled from under the sea-ice in October, showed that the mature fronds are not light-saturated, even at high irradiance at either 4 ° C or 20°C; however, the October samples were light-saturated above an irradiance o f 400/tmol m - 2 s - 1. Samples o f Enteromorpha collected in summer (Fig. 2b ) showed light-saturation of photosynthesis at both 4 ° C and 2 0 ° C above irradiances of 500 pmol m -2 s-1. Photosynthetic rates were higher for both algae at 20°C. The Enteromorpha had a low content o f UV-absorbing pigments, the methanol extract gave a ratio of 0.75 between the peak U V absorbance at 340 nm and the chlorophyll absorbance at 666 nm. No samples were present in winter collections o f algae from below the sea-ice.
237
ANTARCTIC ALGAE: UV EXPOSURE AND PHOTOSYNTHESIS
Prasiola
Prasiola does not experience the extreme mechanical disturbance of the marine environment. In summer, mats are exposed and they can be disturbed by penguins, but in winter, even though they are most frequently covered by snow and ice, mats generally remain intact. Photosynthetic activity in Prasiola has been measured down to - 1 5 ° C (Becket, 1982). Prasiola thalli appear to grow steadily whenever conditions are favourable, without going through varying stages of development or maturity over the year. The spectrum of the methanol extract of Prasiola thalli shows a single broad UV-absorbance peak with a m a x i m u m absorbance at 325 n m (Fig. 3). The UVabsorbance covers the range from approximately 290 n m to 360 nm. To limit the variations in field material, Prasiola samples were collected from the same site and sorted routinely. Middle sections of mats collected in summer (December 1989 ) and winter (July 1990), showed a difference in the absorbance of the UV-absorbing pigment relative to chlorophyll (Fig. 3). The summer sample had a value for A325 (mg chlorophyll a + b ) - 1 of 186 compared with a value of 115 for the winter sample. No variation in the chlorophyll a/b ratio was found. No UV light was detected at the base of a 15-mm thick Prasiola mat when bright s u m m e r sunlight was shining directly on the surface, which means a gradient of UV light occurs through the thickness of the mat. The effect of 2.0
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A. P O S T A N D A . W . D . L A R K U M
this naturally occurring shading was analysed in summer by assaying the UVabsorbing pigment at differing levels in a mat (Table 2). The UV-absorbance relative to chlorophyll was greatest in the top layer which had a value for A325 (mg chlorophyll a + b) - ' of 215. Prasiola mats are frequently a paler green on the surface than at the base and analysis of the top layer showed it had the lowest chlorophyll content per unit weight, approximately 60% of the middle and base layers (Table 2). The chlorophyll a/b ratio was constant through the mat layers. The ratio of carotenoids to chlorophyll was higher in the top layer. Photosynthetic rates of thalli were also measured on these samples and expressed on a chlorophyll basis to make comparison easier, as this takes into account the variation in chlorophyll concentration (Fig. 4). The quantum efficiency was identical throughout the mat and light-saturated rates of oxygen evolution were similar. The photosynthetic rates and the light-saturating irradiances are compaTABLE 2 UV-absorbing pigment content, chlorophyll content and ratio of carotenoids to chlorophyll in sections through a Prasiola crispa mat collected in summer (n = 3, _+standard errors) Cross-section layer
Chl a/b
Chlorophyll content mgchl (gFW) -'
Carotenoid: chlorophyll (w:w)
A325 (mg chl ) - '
Top Middle Base
2.75+0.01 2.73+0.06 2.73_+0.04
1.7_+0.2 2.9+0.1 2.8_+0.1
0.324_+0.012 0.279_+0.008 0.259_+0.002
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Fig. 4. Photosynthetic rates as a function of irradiance for thalli sampled in sections through a naturally occurring mat of Prasiola crispa; top layer (dashed line), base layer (solid line). Photosynthetic rates are expressed on a chlorophyll basis, the chlorophyll content is shown in Table 2.
239
ANTARCTIC ALGAE: UV EXPOSURE AND PHOTOSYNTHESIS
rable with those observed for Prasiola crispa in culture (Jacob et al., 1991 ) and for Prasiola stipitata Suhr ex Jessen (Raven and Johnston, 1991 ). The effect of increasing UV-B light exposure on photosynthesis and on the concentration of UV-absorbing pigment was tested in Prasiola. Material was collected soon after it had melted out after winter and the middle and base layers combined, rinsed and then divided into two sets of three. One set was used as a control and had no UV-B exposure. The other set was exposed to U V radiation using a ratio of UV-B to visible light which was enhanced to double that measured in ambient midday sunlight on a clear day at the time of collection. By the second week the photosynthetic rates in all replicates exposed to UV-B were reduced. But there was no change in the ratio of UVTABLE3 Pigment content ofPrasiola crispa at the start and after 4 weeks of culturing Chi a/b
Treatment
Initial
Chlorophyll content mgchl (g EVe) -1
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2.71 _+0.01
2.9+0.3
0.273+0.015
187+21
2.46 _+0.03 2.28 + 0.03
2.4 + 0.1 1.7 + 0.1
0.283 + 0.007 0.304 + 0.006
184 + 6 188 + 11
(W:W)
4 Weeks minus UV-B plus UV-B
Control samples were covered with mylar to filter out UV-B radiation. Samples exposed to enhanced UV-B were covered by cellulose acetate to filter out UV-C radiation (initial sample: n = 3, 4 weeks sample: n = 12, + standard errors)
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Fig. 5. P h o t o s y n t h e t i c rates as a f u n c t i o n o f irradiance for Prasiola crispa grown u n d e r low light in a cabinet with n o ultraviolet light (solid line) a n d with e n h a n c e d ultraviolet light (dashed line). The inset shows the gross p h o t o s y n t h e t i c rates in the light limiting range for photosynthesis. Photosynthetic rates are expressed on a chlorophyll basis, the chlorophyll content is shown in Table 3.
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A. POST AND A.W.D. LARKUM
absorbance to chlorophyll at 325 nm from the initial value even after 4 weeks (Table 3 ). The photosynthetic rates measured at 4 weeks (Fig. 5 ) showed an increase in respiration in the Prasiola exposed to UV-B, but the light-saturated rate of oxygen evolution of the exposed samples were only 40% of the control rate. The photosynthetic rates corrected for dark respiration (Fig. 5, inset) clearly showed the reduction in the photon yield in the samples exposed to UV-B. In parallel with the drop in photosynthetic rates there was a drop in the chlorophyll content per unit weight in the exposed samples although the chlorophyll a/b ratio was similar (Table 3 ). DISCUSSION
The UV and visible light environment of Antarctic algae varies greatly over the year. At Casey in mid-winter the sun is above the horizon for less than 3 h and the sun reaches a maximum elevation angle of only 5 °. The snow and ice cover is thick with over 1 m of sea-ice on the edges of the shoreline. By contrast, in summer the days are long with skies frequently clear and the Prasiola mats are fully exposed to high levels of UV light. Biologically active wavelengths of UV can also penetrate Antarctic seawater to depths of 10 m (Karentz and Lutze, 1990). Antarctic seawater has a constant temperature of - 1.8 °C (Wiencke and tom Dieck, 1989; and personal observation). Terrestrial plants experience a range of temperatures. On clear summer days the surface temperature of Prasiola can be above 20 oC, while freezing temperatures occur overnight (Becker, 1982; and personal observation). Prasiola can also tolerate a wide range of salinity, possibly assisted by the thick cell walls (Jacob et al., 1991 ). Although marine temperatures are stable, sea-ice scours the shoreline and the larger fronds which are enclosed in the ice are torn away. The Palmaria plants which survive generally consist of the holdfast with a short, sometimes broken, frond. Later in the year lateral fronds also develop from this. The development of lateral shoots has been observed in Palmaria fronds in culture and is dependent on the seasonal changes in day-length (Wiencke, 1990). Palmaria contains a range of UV-absorbing pigments. The greatest absorbance is in summer and is maximum at 337 nm with a shoulder at 360 nm. Over the year other peaks were observed at 322 nm and 309 nm. Palmaria collected from Palmer Station (64°46'S, 64°03'W) in early summer (Karentz et al., 1991 a), contained mycosporine-glycine (310 nm), palythine (320 nm ), palythene (360 nm) and four pigments absorbing in the range 330-334 nm. The Palmaria collected at Casey over the winter, with juvenile lateral fronds, had reduced UV-absorbing pigment content compared with the summer (Table 1 ). In addition, the absorbance peaks at 309 nm and 322 nm suggest the pigments are mycosporine-glycine and palythine which have the simplest chemical structures.
ANTARCTIC ALGAE: UV EXPOSURE AND PHOTOSYNTHESIS
241
Mature Palmaria fronds collected in January showed higher rates of photosynthesis at 20°C than at lower temperatures, but the oxygen evolution is not light-saturated at either temperature under these conditions. Palmaria collected from below the sea-ice was light-saturated at lower irradiance with saturation occurring above 400/zmol m -2 s-~, even when measured at 20°C. The increase in photosynthetic capacity in summer corresponds with the time when Palmaria has the maximum content of UV-absorbing pigments. The chlorophyte, Prasiola contains a single UV-absorbing pigment with absorbance maximum at 325 rim. Enteromorpha, in the Ulvophyceae is closely related to Prasiola (Raven and Johnston, 1991 ), but Enteromorpha had low levels of UV-absorbing pigment. These differences in the content of UV-absorbing pigments correlate with the varying light environment of the two algae. Prasiola is fully exposed to sunlight while the marine Enteromorpha is largely shaded by the taller Palmaria fronds in summer. Prasiolajaponica Yatabe collected in Japan (Sivalingam et al., 1974) contained a pigment also absorbing at 325 nm, but the ratio of the absorbance of the UV-absorbing pigment to the absorbance of chlorophyll at 663 nm was only 0.8. This is much lower than typical values found in summer samples of Prasiola, such as the middle-layer thaUi (Fig. 3) which have a ratio of the UV-absorbing pigment to chlorophyll absorbance of 2.7; the surface layer thalli have an even higher value of 3.4. While it has been suggested that green algae commonly contain low levels of the UV-absorbing pigments (Karentz et al., 1991 a), this is clearly not the case for Prasiola growing in Antarctica. The results suggest a seasonal variation occurs in the level of UV-absorbance relative to chlorophyll in Prasiola, with the highest levels found in the top layers of mats in summer. Under ambient conditions, regulation of the UV-absorbing pigments relative to chlorophyll appears to occur up to this maximum level and decreases with shading. Other studies have found regulation of UV-absorbing pigments in algae is related to UV exposure (Scherer et al., 1988; Carreto et al., 1990; Marchant et al., 1991 ). The variations in UV-absorbing pigments found over the year for Palmaria and Prasiola and the variations found in the layers of the Prasiola mat tend to support that conclusion. However, when Prasiola was exposed to an enhanced ratio of UV-B to visible light, the concentration of UV-absorbing pigments relative to chlorophyll remained constant (Table 3). Compared with sunlight the total UV-B exposure in the cabinet was low, because the level of visible light was restricted and this may have limited the synthesis of UV-absorbing pigments. Yet there were some similarities between the effects of UV in the environmental cabinet and bright sunlight on the surface layer of Prasiola in the field, as shown with the decrease in chlorophyll per unit of weight with no change in the chlorophyll a/b ratio (Table 3 ). The enhanced ratio of UV-B light resulted in a reduction in oxygen evolution under both light-saturating and limiting con-
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ditions when photosynthetic rates were expressed on a chlorophyll basis (Fig. 5 ) to allow for the change in chlorophyll concentration. These Antarctic algae contain a range of UV-absorbing pigments. Measurements of the pigments in the marine and terrestrial algae indicate that the maximum levels of UV-absorbing pigments occur in summer. The concentration of the pigments in these species can change with the stage of development and with variations in exposure to sunlight. Very high concentrations of UVabsorbing pigments occur in the mature fronds of Palmaria in summer. By contrast the concentrations in Prasiola are lower, but even these are high by comparison with Enteromorpha, a shade plant, and with temperate species (Sivalingam et al., 1974). The concentration of the UV-absorbing pigment found in Prasiola in the summer was not increased any further by exposure to an enhanced ratio of UV-B radiation to visible light, although the chlorophyll concentration and photosynthetic rates decreased, and this may have important and general implications. The main effect of stratospheric ozone depletion over Antarctica has been to increase the ratio of UV-B to visible light in spring (Frederick and Snell, 1988; Karentz, 1991 ). Our simulation of this type of change, albeit at a low level of total irradiance, suggests that such changes in the ratio of UV-B to visible light may lead to reduced productivity even in those Antarctic plants that already contain high levels of UV-absorbing pigments. ACKNOWLEDGEMENTS
This study depended on the support of the Australian Antarctic Division, the Antarctic Science Advisory Committee and the Australian Research Council.
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