Plant Ecology 154: 219–236, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

Section 5: Aquatic Plants and Aquatic Ecosystems

Cyanobacteria in the water of a rice field in India. (Photograph by R.P. Sinha)

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Plant Ecology 154: 221–236, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Responses of aquatic algae and cyanobacteria to solar UV-B Rajeshwar P. Sinha, Manfred Klisch, Almut Gröniger & Donat-P. Häder∗ Institut für Botanik und Pharmazeutische Biologie, Friedrich-Alexander-Universität, Staudtstr. 5, D-91058 Erlangen, Germany (∗ Author for correspondence, E-mail: [email protected])

Key words: Cyanobacteria, Macroalgae, Mycosporine-like amino acids (MAAs), Phytoplankton, Scytonemin, Ultraviolet-B radiation Abstract Continuous depletion of the stratospheric ozone layer has resulted in an increase in solar ultraviolet-B (UV-B; 280–315 nm) radiation reaching the Earth’s surface. The consequences for aquatic phototrophic organisms of this small change in the solar spectrum are currently uncertain. UV radiation has been shown to adversely affect a number of photochemical and photobiological processes in a wide variety of aquatic organisms, such as cyanobacteria, phytoplankton and macroalgae. However, a number of photosynthetic organisms counteract the damaging effects of UV-B by synthesizing UV protective compounds such as mycosporine-like amino acids (MAAs) and the cyanobacterial sheath pigment, scytonemin. The aim of this contribution is to discuss the responses of algae and cyanobacteria to solar UV-B radiation and the role of photoprotective compounds in mitigating UV-B damage.

Introduction Continued depletion of the stratospheric ozone layer, mainly due to anthropogenically released atmospheric pollutants such as chlorofluorocarbons (CFCs) is responsible for the increase in solar ultraviolet-B (UV-B; 280–315 nm) radiation reaching the Earth’s surface (Blumthaler & Ambach 1990; Crutzen 1992; Kerr & McElroy 1993; Lubin & Jensen 1995). In addition to the Antarctic ozone hole, ozone depletion has also been reported in the north polar region (Hoffman & Deshler 1991). Current estimates predict that ozone losses will continue throughout the next century if we fail to adhere to the 1992 Copenhagen Amendment (Slaper et al. 1996). Biologically effective doses of UV radiation penetrate deep into the water column (Smith & Baker 1979); they have been detected down to a depth of 70 m (Smith et al. 1992) and may thus affect the aquatic ecosystems (Häder et al. 1998). The penetration of water by UV-B depends on the optical properties of the water column. The depth of water required to remove 90% of the solar radiation at 310 nm varies from about 20 m in the clearest ocean water to a

few centimeters in brown humic lakes and rivers (Kirk, 1994). All aquatic organisms appear to be susceptible to UV-B, but to a highly variable extent. UV-B is a small (less than 1% of total energy) but highly active component of the solar spectrum which has the potential to cause wide ranging effects, including alteration in the structure of proteins, DNA and other biologically relevant molecules, chronic depression of key physiological processes and acute physiological stress. As a result, the productivity of ecosystems may be affected (Karentz et al. 1991a; Vincent & Roy 1993; Bothwell et al. 1994; Williamson 1995; Sinha & Häder 1996a). However, certain photosynthetic organisms which are simultaneously exposed to visible and UV radiation in their natural habitat, have developed mechanisms counteracting the damaging effects of UV. Besides repair of UV-induced damage of DNA by photoreactivation and excision repair (Britt 1995; Kim & Sancar 1995) and accumulation of carotenoids and detoxifying enzymes or radical quenchers and antioxidants that provide protection by scavenging harmful radicals or oxygen species (Mittler & Tel-Or 1991; Middleton & Teramura 1993), an important mechanism to prevent UV-induced photodamage is the syn-

222 thesis of photoprotective compounds (Garcia-Pichel & Castenholz 1991; Karentz et al. 1991c; Dunlap & Shick 1998; Sinha et al. 1998a). This review deals with the responses of algae and cyanobacteria to solar UV-B radiation and the role of photoprotective compounds in mitigating UV-B toxicity.

Effects of UV-B radiation on cyanobacteria Cyanobacteria are the largest and most widely distributed group of photosynthetic prokaryotes on Earth. Cyanobacterial populations occupy an important place in both aquatic and terrestrial ecosystems ranging from hot springs to the Antarctic and Arctic regions. The role of cyanobacteria in improving the fertility of rice paddy fields and other soils is well documented (Singh 1961; Roger & Kulasooriya 1980; Venkataraman 1981; Watanabe 1984; Sinha & Häder 1996a; Sinha et al. 1998b; Vaishampayan et al. 1998). Any substantial increase in the solar UV-B radiation might be detrimental to the cyanobacterial communities, which in turn may affect the productivity of higher plants (Sinha & Häder 1996a). All aquatic photosynthetic organisms depend on solar radiation as the primary source of energy in their natural environment. Light is one of the most important factors determining cyanobacterial growth in their natural habitats since cyanobacteria are predominantly photoautotrophic organisms. In addition, light is an environmental factor controlling orientation and habitat selection. The cyanobacterial populations in rice paddy fields, particularly in the tropics, are often exposed to high white light and UV-B irradiances (Sinha & Häder 1996a, b; Sinha et al. 1999a). Considering the vital role of cyanobacteria as a biofertilizer in rice and other crop production, the fluence rate of UV-B radiation impinging on the natural habitats, seems to be of major concern since UV-B radiation has been reported not only to impair motility and photoorientation (Donkor & Häder 1991) but also to affect a number of physiological and biochemical processes such as growth, survival, pigmentation, and total protein profile (Sinha et al. 1995a). Growth and survival of several rice-field cyanobacteria have been reported to be severely affected following UV-B irradiation for different durations. Growth ceases and survival is affected within 120–180 min of UV-B irradiation, depending upon the species. Strains such as Scytonema sp. and Nostoc commune, the filaments of which are embedded in mucilagenous sheath, have been reported

Figure 1. Effects of UV-B irradiation on the pigmentation of a rice-field cyanobacterium Anabaena sp.

to be more tolerant than Anabaena sp. and Nostoc sp., the filaments of which do not possess such covering (Sinha et al. 1995a). Many workers have suggested that the cellular constituents absorbing radiation between 280–315 nm are destroyed by UV-B radiation, which may further affect the cellular membrane permeability and protein damage eventually resulting in the death of the cell (Vincent & Roy 1993; Sinha et al. 1995a, 1997; Sinha & Häder 1996a). The studies on the effects of UV-B on pigmentation of various rice field cyanobacteria have revealed that the accessory pigment phycocyanin (λmax 620 nm) was bleached more rapidly and drastically than any other pigment such as Chl a (λmax 437 and 672 nm) or the carotenoids (λmax 485 nm) (Sinha et al. 1995a, b) (Figure 1). A decrease in the phycobiliprotein contents and disassembly of phycobilisomal complexes following UV-B irradiation have been reported in a number of cyanobacteria (Sinha et al. 1995b, c, 1997). Fluorescence emission spectra of phycobiliproteins after UV-B irradiation first show an increase followed by a shift towards shorter wavelength and finally a decrease in fluorescence (Figure 2), indicating impaired energy transfer from the accessory pigments to the photosynthetic reaction centers and subsequent bleaching of the pigments (Sinha et al. 1995b, c, 1997). Differentiation of vegetative cells into heterocysts has been reported to be severely affected by UV-B irradiation in a number of rice field cyanobacteria. Most probably the C:N ratio is altered following UV-B irradiation, which in turn affects the spacing pattern of heterocysts in a filament (Sinha et al. 1996). In addition, major heterocyst polypeptides of around 26, 54 and 55 kDa have been shown to be decreased in

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Figure 2. Fluorescence emission spectra of phycocyanin from Anabaena sp. after increasing UV-B exposure time, when excited at 620 nm.

concentration following UV-B irradiation, suggesting that the multilayered thick wall of heterocysts may be disrupted resulting in the inactivation of the nitrogenfixing enzyme nitrogenase by the penetrating oxygen. UV-B-induced membrane disruption leading to changes in membrane permeability and release of 14 Clabeled compounds have been observed in a number of rice-field cyanobacteria (Sinha et al. 1997). UVB-induced inactivation of the nitrogen fixing enzyme nitrogenase has been reported in many cyanobacteria (Kumar et al. 1996a; Sinha et al. 1996). Total protein profiles of several cyanobacteria show a linear decrease in protein content with increasing UV-B exposure time, indicating that cellular proteins are one of the main targets of UV-B. Ultraviolet radiation is known to damage proteins and enzymes, especially those rich in aromatic amino acids such as tryptophan, tyrosine, phenylalanine and histidine, all of which show strong absorption in the UV range from 270 to 290 nm (Kumar et al. 1996a; Sinha et al. 1996; Sinha & Häder 1998). The activity of the ribulose 1,5-bisphosphate carboxylase (Rubisco), the primary CO2 fixing enzyme, has been reported to be inhibited by UV-B irradiation in a number of rice-field cyanobacteria which may be due to protein destruction or enzyme inactivation (Sinha et al. 1997). The control of RuBISCO biosynthesis is strongly influenced by the prevailing light environment. During UV-B irradiation, proteins may undergo a number of modifications including photodegradation, increased aqueous solubility of membrane proteins, and fragmentation of the peptide chain, leading to inactivation of proteins (enzymes) and dis-

ruption of their structural entity (Sinha et al. 1997; Sinha & Häder 1998). Ultraviolet induced inhibition of 14 CO2 uptake in various rice-field cyanobacteria has been reported which could be due to the effect on the photosynthetic apparatus leading to the reduction in the supply of ATP and NADPH2 (Kumar et al. 1996a; Sinha et al. 1996). A disruption of the cell membrane and/or alteration in thylakoid integrity as a result of UV-B irradiation may partly or wholly destroy the component required for photosynthesis and may thus affect the rate of CO2 fixation (Sinha et al. 1996, 1997). In addition, ultraviolet-induced opening of the membrane-bound calcium channels has been demonstrated in the cyanobacterium Anabaena sp. (Richter et al. 1999). Cyanobacteria are common organisms in freshwater and terrestrial ecosystems of Antarctica and also in the north polar regions (Quesada & Vincent 1997). Cyanobacteria have colonized a wide variety of polar environments including soils, the surface and interior of rocks, lakes, ponds, streams, moss beds, melt pools on glaciers and ice shelves, and littoral marine sediments. In some of these habitats they dominate in terms of total biomass and productivity. Cyanobacteria are also a conspicuous element of mature microbial communities across the surface of Antarctic rock faces, as well as under and within translucent rocks (Vincent & Quesada 1994). Antarctic cyanobacteria achieve their greatest abundance in aquatic and wetland habitats. It is the benthic environment of lakes, ponds and streams that supports the largest standing stocks of cyanobacterial biomass in Antarctica. Cyanobacterial communities form crusts, films and spectacular mats up to several centimeters in thickness, often intensely colored by pigments. Mat forming cyanobacteria are especially wide spread in high latitude ponds and streams. In these environments the cyanobacteria often occupy shallow water habitats that are exposed to full sunlight. Cyanobacterial populations must therefore be capable of surviving frequent exposure to bright photosynthetically active radiation (PAR) as well as high levels of solar ultraviolet radiation (Quesada & Vincent 1997). It has been reported that even moderate levels of ultraviolet-B radiation can have a major physiological impact on Antarctic cyanobacteria, but there are substantial differences between closely related species in their ability to escape the damaging effects of this high energy waveband (Quesada & Vincent 1997). It has been concluded that the marine phototrophic organisms living in cold environments may be especially

224 prone to the damaging effects of ultraviolet radiation. These findings are especially relevant to the cold waters found in the north and south polar zones, where stratospheric ozone depletion and the associated increase in ambient UV-B radiation are proceeding most rapidly (Roos & Vincent 1998). Thus, in natural habitats avoidance of UV radiation seems to be of utmost importance for cyanobacterial growth and nitrogen fixation.

diated repair of the photosynthetic apparatus (Christopher & Mullet 1994). (5) A number of cyanobacteria have the ability to vary their phycobiliprotein composition (phycocyanin/phycoerythrin ratio), which allows regulation of the balance of wavelengths of light absorbed, a phenomenon known as chromatic adaptation (Tandeau de Marsac 1977). Below we discuss the role of MAAs and scytonemin in mitigating the harmful UV-B radiation effects in cyanobacteria in more detail.

Photoprotective mechanisms in cyanobacteria Since it is clear that microorganisms evolved and microbial mats were well established early in the Precambrian era (Rambler & Margulis 1980; Dillon & Castenholz 1999), some mechanism(s) must have been functioning to protect these organisms from the deleterious effects of UV radiation. There are at least five adaptation strategies by which cyanobacteria try to avoid high white light and ultraviolet radiation stress (Vincent & Roy 1993; Castenholz 1997; Quesada & Vincent 1997; Sinha et al. 1998a; Cockell & Knowland 1999): (1) Production of ultraviolet-absorbing substances such as mycosporine-like amino acids (MAAs) and scytonemin (Garcia-Pichel & Castenholz 1991; Garcia-Pichel et al. 1993; Büdel et al. 1997; Sinha et al. 1998a; Dillon & Castenholz 1999). (2) Escape from ultraviolet radiation by migration into habitats with reduced light exposure. Such strategies include phototactic, photokinetic and photophobic responses (Häder 1987a, b), vertical migration into deeper strata of mat communities (Bebout & Garcia-Pichel 1995) and sinking and floating behavior by a combination of gas vacuoles and ballast (Reynolds et al. 1987). This allows them to change their position in the water column as environmental conditions change and thus to always ensure a nearly constant external environment. (3) Production of quenching agents such as carotenoids (Burton & Ingold 1984) or systems such as superoxide dismutase that react with and thereby neutralize the highly toxic reactive oxygen species produced by ultraviolet-B radiation (Quesada & Vincent 1997). (4) Availability of a number of repair mechanisms such as photoreactivation and light-independent nucleotide excision repair of DNA (Britt 1995; Kim & Sancar 1995) and UV-A/blue-light me-

Mycosporine-like amino acids (MAAs) in cyanobacteria In the 1970s various substances were isolated and characterized which had a maximum absorption in the UV range. These substances were related to the mycosporines found in terrestrial fungi (Favre-Bonvin et al. 1987) and named mycosporine-like amino acids (MAAs). To date, a number of MAAs are documented of which mycosporine-glycine, palythine, palythene, palythinol, asterina-330, porphyra-334 and shinorine (Figure 3) have been characterized and well documented (Ito & Hirata 1977; Takano et al. 1978a, b, 1979; Tsujino et al. 1980; Dunlap & Chalker 1986; Gleason 1993). MAAs are water soluble substances characterized by a cyclohexenone or cyclohexenimine chromophore conjugated with the nitrogen substituent of an amino acid or its imino alcohol, having absorption maxima ranging from 310 to 360 nm (Figure 3) and an average molecular weight of around 300 (Sinha et al. 1998a; Cockell & Knowland 1999). A number of cyanobacteria isolated from freshwater, marine or terrestrial habitats contain MAAs (Garcia-Pichel & Castenholz 1993; Garcia-Pichel et al. 1993; Sinha et al. 1998a). Presence of MAAs has also been reported in Antarctic (Quesada & Vincent 1997) as well as in a community of halophilic cyanobacteria (Oren 1997). The occurrence of high concentrations of MAAs in organisms exposed to high levels of solar radiation has been described to provide protection as a UVabsorbing pigment (Garcia-Pichel et al. 1993; Sinha et al. 1998a), but there is no conclusive evidence for the exclusive role of MAAs as sunscreen, and it is possible that they play more than one role in the cellular metabolism of all or some organisms (Vincent & Roy 1993; Castenholz 1997; Oren 1997). It has been reported that MAAs may act as antioxidants to prevent

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Figure 3. Molecular structures, absorption maxima (λmax ) and extinction coefficients (ε) of some principal photoprotective compounds.

226 cellular damage resulting from UV-induced production of active oxygen species (Dunlap & Yamamoto 1995). Studies with cyanobacteria have shown that MAAs prevent 3 out of 10 photons from hitting cytoplasmic targets. Cells with high concentrations of MAAs are approximately 25% more resistant to ultraviolet radiation centered at 320 nm than those with no or low concentrations (Garcia-Pichel et al. 1993). This protection is unlikely to be effective for thin, solitary filaments, but may be especially important in mat communities or large phytoplankton. The MAAs in Nostoc commune have been reported to be extracellular and linked to oligosaccharides in the sheath (Böhm et al. 1995). These glycosylated MAAs represent perhaps the only known example of MAAs that are actively excreted and accumulated extracellularly and therefore act as a true screen (Ehling-Schulz et al. 1997). Experiments with a rice paddy field cyanobacterium, Anabaena sp., revealed the existence and induction by UV radiation of a single MAA, shinorine, a bisubstituted MAA containing both glycine and serine groups, with an absorption maximum at 334 nm and a fluorescence emission at 436 nm (Sinha et al. 1999b). There may be physiological limitations to the accumulation of osmotically active compounds such as MAAs within the cell, and it seems probable that the maximal specific content of MAAs in the cell is regulated by osmotic mechanisms which is reflected by the fact that field populations of halotolerant cyanobacteria contain unusually high concentration of MAAs (Oren 1997).

Scytonemin Scytonemin is a yellow-brown, lipid soluble dimeric pigment located in the extracellular polysaccharide sheath of some cyanobacteria. Scytonemin, which has an in vivo absorption maximum at 370 nm, has been proposed to serve as a UV sunscreen (Garcia-Pichel et al. 1992; Dillon & Castenholz 1999). It has a molecular mass of 544 Da and a structure based on indolic and phenolic subunits (Figure 3). Purified scytonemin has an absorption maximum at 386 nm, but it also absorbs significantly at 252, 278 and 300 nm (Proteau et al. 1993; Sinha et al. 1998a, 1999c). Strong evidence for the role of scytonemin as ultraviolet shielding compound has been presented in several cyanobacterial isolates and collected materials from various harsh habitats, mostly exposed to high irradiances (Garcia-Pichel & Castenholz 1991).

Its role as a sunscreen was clearly demonstrated in the terrestrial cyanobacterium Chlorogloeopsis sp. (Garcia-Pichel et al. 1992). A desiccation-tolerant cyanobacterium, Lyngbya sp., collected from the bark of mango trees in India has been found to contain the yellow-brown sheath pigment, scytonemin (Sinha et al. 1999c). In cyanobacterial cultures, as much as 5% of the cellular dry weight may be accumulated as scytonemin. Naturally occurring cyanobacteria may have an even higher specific content (Castenholz 1997). The correlation between UV protection and scytonemin presence has been established under solar irradiance in a naturally occurring monospecific population of a cyanobacterium, Calothrix sp.; it was shown that high scytonemin content is required for uninhibited photosynthesis under high UV flux (Brenowitz & Castenholz 1997). Studies indicate that the incident UV-A radiation entering the cells may be reduced by around 90% due to the presence of scytonemin in the cyanobacterial sheaths (GarciaPichel & Castenholz 1991; Garcia-Pichel et al. 1992; Brenowitz & Castenholz 1997). Once synthesized, it remains highly stable and carries out its screening activity without further metabolic investment from the cell. Rapid photodegradation of scytonemin does not occur, which is evidenced by its long persistence in terrestrial cyanobacterial crusts or dried mats (GarciaPichel et al. 1992; Brenowitz & Castenholz 1997; Quesada et al. 1999). This strategy may be invaluable to several scytonemin containing cyanobacteria inhabiting harsh habitats, such as intertidal marine mats or terrestrial crusts, where they experience intermittent physiological inactivity (e.g., desiccation or freezing). During these metabolically inactive periods, other ultraviolet protective mechanisms such as active repair or biosynthesis of damaged cellular components are ineffective (Brenowitz and Castenholz 1997; EhlingSchulz et al. 1997; Quesada et al. 1999). It has been postulated that scytonemin may have evolved during the Precambrian and allowed colonization of exposed, shallow-water and terrestrial habitats by cyanobacteria or their oxygenic ancestors (Dillon & Castenholz 1999). In addition to the screening pigments described above, another UV protective agent with absorption maxima at 312 and 330 nm has been reported from the terrestrial cyanobacterium Nostoc commune, a species that also produces scytonemin (Scherer et al. 1988). A brown Nostoc sp. that produces three UV-absorbing compounds with absorption maxima at 256, 314 and 400 nm, has been reported to be resistant to high

227 visible and UV radiation (de Chazal & Smith 1994). The shielding role against UV-B-induced damage of certain cyanobacterial pigments (a brown-colored pigment from Scytonema hofmanii and a pink extract from Nostoc spongiaeforme) has been demonstrated (Kumar et al. 1996b).

Effects of UV-B radiation on phytoplankton Phytoplankton organisms are the basis of marine food webs and thus indirectly contribute to human nutrition. They also play an important role in the regulation of global climate. It has been estimated that about 50% of the total carbon fixation is attributed to marine ecosystems with most of it due to phytoplankton organisms (Häder et al. 1998). In addition to the contribution of phytoplankton to carbon fixation, there are specific effects of phytoplankton on climate. Some phytoplankton genera produce volatile substances, mainly dimethyl sulfide (DMS), providing a cooling effect on the atmosphere, since they are precursors of cloud condensation nuclei (Watson & Liss 1998). The cumulative effect of marine biota in the uptake of atmospheric CO2 and emission of DMS has been estimated to cool the atmosphere by up to 6 ◦ C (Watson & Liss 1998). The distribution of phytoplankton is not uniform in the oceans. The dependence on solar radiation as the primary source of energy restricts phytoplankton to depths at which the penetration of light supports photosynthesis. This range is defined as the euphotic zone. The horizontal distribution of phytoplankton is also highly variable. In large regions of the oceans a stable boundary exists between nutrient rich deep sea water and nutrient poor surface water. Thus, the accumulation of phytoplankton biomass is limited by nutrient availability. Regions near the coast and at the higher latitudes contain more phytoplankton biomass because of higher nutrient availability due to upwelling of nutrient-rich water from the deep sea and terrestrial influxes. Especially the Southern Ocean is highly productive (Valiela 1995). The life of phytoplankton depends on solar radiation that provides energy used via the photosynthetic process. Simultaneously, the excess UV-B radiation damages diverse targets within the organism. Inhibition of photosynthesis and growth, DNA damage, and finally cell death are among the common effects of UV-B on phytoplankton. High levels of visible radiation induce the formation of active oxygen species

that affect cell integrity. UV-B reduces the content of photosynthetic pigments in phytoplankton and leads to lower photosynthetic rates (Gerber & Häder 1995). Apart from the photosynthetic pigments a major target of UV damage is the electron transport chain of photosystem II (Bornman 1989). Functional relationships between the wavelength of radiation and the photoinhibition of photosynthesis (biological weighing functions, BWFs) have been determined experimentally (Cullen et al. 1992; Helbling et al. 1992; Neale et al. 1998a; Ghetti et al. 1999). The inhibitory effect of UV radiation increases exponentially with decreasing wavelength in the UV-A and the UV-B portion of the spectrum. However, due to its higher proportion in the solar spectrum, the inhibitory effect of UV-A radiation has been estimated to be higher than the UV-B effect, in some cases even for the conditions of ozone depletion (Cullen et al. 1992; Arrigo 1994). The shape of a BWF may be strongly influenced by the physiological state of the organisms, e.g. the induction of tolerance mechanisms. For example, the inhibitory effect of UV radiation in the wavelength range of MAA absorption can be greatly diminished if high concentrations of these UV-absorbing compounds are present in the organisms (Neale et al. 1998a). The peak absorption of DNA lies in the UV-C range that is absorbed in the upper layers of the atmosphere and does not reach the ozone layer. However, the absorption of UV-B radiation by DNA is sufficient to induce severe damage to the DNA of phytoplankton cells. Absorbed quanta of UV can induce changes in the molecular structure of the DNA (Karentz et al. 1991a). The main effect of UV-B radiation is the formation of dimers between two adjacent pyrimidine bases, cis-syn cyclobutan dimers and pyrimidine (6–4) pyrimidone photoproducts. These DNA lesions interfere with DNA transcription and replication and can lead to misreadings of the genetic code causing mutations and death. There are big differences in the susceptibility of phytoplankton species to DNA damage that seem to be correlated with cell size and shape. Taking into account that DNA is among the main lethal targets of UV-B radiation, the susceptibility of a species to UV-B induced DNA damage is a good indicator of overall UV-B sensitivity (Karentz et al. 1991a). Investigations of the effects of ozone depletion on phytoplankton productivity in the Southern ocean, where the increase in UV-B radiation is most pronounced, show large differences. Smith et al. (1992) estimated a reduction in primary production of 6–12%

228 in the marginal ice zone of the Bellinghausen Sea. Another study simulating the effect on aquatic primary production using model-derived radiation conditions and BWFs, only results in a reduction of less than 1% integrated over the Southern Ocean (Arrigo 1994). The large difference between the estimates reflects the complexity of large scale estimates on UV-B effects. A differential impact of UV-B radiation may be partially a result of interactions with other factors, such as the extent of vertical mixing. By vertical mixing organisms from deeper levels are brought to the surface where they become photoinhibited while inhibited organisms from near surface are transported to deeper levels where availability of light becomes limiting. Thus the overall decrease in photosynthesis becomes larger (Neale et al. 1998b). In addition, solar radiation and UV-B radiation is known to inhibit the ability of phytoplankton to move and orient within the water column (Tirlapur et al. 1993). Photoprotective mechanisms in phytoplankton The recent effects of UV-B are not new phenomena but have accompanied the whole evolutionary process of phytoplankton. Therefore, phytoplankton organisms have developed certain tolerance mechanisms to avoid harmful UV radiation. These include (a) vertical migration within the water column to avoid exposure to excessive doses of harmful radiation. Many species of phytoplankton actively move up and down in the water column, controlled by gravitactic and phototactic orientation (Häder 1988). This mechanism allows the organisms to adjust the impinging radiation to a level that is suitable for photosynthesis and to avoid excess doses of visible as well as UV radiation (Häder 1988; Tirlapur et al. 1993), (b) repair of DNA damage (Karentz et al. 1991b). There are three mechanisms known that can repair damaged portions of DNA: – Photoenzymatic repair (PER) that involves the enzyme DNA photolyase that monomerizes cyclobutane dimers in the presence of visible or UV-A light, – Nucleotide excision repair (NER) has a broader spectrum of action and involves the recognition of damaged DNA portions and the excision and resynthesis of the damaged strand by DNA polymerase, and – Recombinational repair (postreplication repair) may resolve DNA damage that has been bypassed by the replication machinery (Karentz et al. 1991a, b).

The capacity and kinetics of DNA repair differ greatly between phytoplankton species. This includes different relative importance of either PER and NER. The removal of cyclobutane dimers by PER in the presence of PAR or UV-A radiation may enhance the not lightdependent NER of other DNA lesions (Karentz et al. 1991a, b), and (c) the production of UV-absorbing compounds, MAAs (Carreto et al. 1990a, b; Dunlap et al. 1995; Helbling et al. 1996; Vernet & Whitehead 1996; Xiong et al. 1997).

MAAs in phytoplankton Several phytoplankton organisms from different regions and taxonomic groups have been found to contain MAAs. Most of the research has focused on marine phytoplankton, but there are a few reports on the occurrence of MAAs in freshwater algae (Xiong et al. 1999). So far MAAs have been reported to occur predominantly in species of the Dinophyceae, Bacillariophyceae and Haptophyceae. In addition to the reports from experiments with phytoplankton in culture, there are numerous reports on UV-absorbing properties of ocean waters that are assumed to be caused by the presence of MAAs (Helbling et al. 1994; Vernet et al. 1989). A UV-absorbing compound was found in a phytoplankton bloom in the Icelandic Basin, which showed an absorption spectrum and chromatographic behavior similar but not identical to scytonemin. However, this pigment has not yet been chemically characterized, and therefore, there is no evidence for the presence of scytonemin or similar pigments in phytoplankton organisms (Llewellyn & Mantoura 1997). Besides MAAs, sporopollenin, a biopolymer of variable chemical composition, has been proposed to play a possible role in screening UV radiation in some freshwater phytoplankton species (Xiong et al. 1997). The assumption that MAAs act as protectants against UV radiation has been derived from the fact that the distribution of MAAs in marine organisms often shows a correlation with depth and thus with the dosage of UV or PAR radiation (Dunlap & Shick 1998). In some phytoplankton species the accumulation of MAAs is induced by UV radiation (Carreto et al. 1990a, b) which supports the theoretical assumption that the presence of UV-absorbing compounds provides screening to the constituents of a cell (Garcia-Pichel 1996). Direct evidence for their protective function in phytoplankton has been demonstrated

229 where high amounts of intracellular MAAs diminish the inhibitory effect of UV radiation on photosynthesis (Lesser 1996; Neale et al. 1998a). The accumulation of MAAs may also protect microalgae from inhibition of motility by UV-B radiation (our unpublished data). The effectivity of screening by UV-absorbing compounds largely depends on cell size. Due to the longer optical path length internal sunscreens are more effective in large cells. However, smaller cells may increase the effectivity of screening if they form dense populations that provide mutual shading from deleterious UV-B radiation. However, in this case it is doubtful that the presence of UV-screening substances will provide a competitive advantage to the cells producing them since other species may also benefit from the screening of UV radiation (Garcia-Pichel 1996). The same applies to cases in which the UV-absorbing substances are released to the surrounding medium like in the dinoflagellate Lingulodinium polyedra (Vernet & Whitehead 1996). The synthesis of MAAs is strongly influenced by radiation intensity as well as by the spectral composition. In the dinoflagellate Alexandrium excavatum isolated from the continental shelf near Buenos Aires, the transfer from low (20 µE m−2 s−1 ) to high (200 µE m−2 s−1 ) PAR led to a change in MAA composition and an overall increase in UV absorption within a time scale of a few hours (Carreto et al. 1989, 1990a). Accumulation of MAAs in this organism also depends on the spectral composition of the light; blue light is more effective than green and red light, and UV-A radiation strongly enhances MAA accumulation (Carreto et al. 1990a). Exposure to sunlight leads to a strong increase of MAAs content in Alexandrium excavatum (Carreto et al. 1990b). In the dinoflagellate Prorocentrum micans, after 21 days of growth in the presence of UV radiation the organisms contained higher concentrations of MAAs in comparison to those grown in the absence of UV radiation (Lesser 1996). The ubiquitous haptophyte species, Phaeocystis pouchetii, contains UV-absorbing compounds only in the colonial stage of its life cycle, and the concentration has been found to be higher in isolates from the Antarctic region than in isolates from temperate regions (Marchant et al. 1991). In contrast to temperate Phaeocystis populations, in the Antarctic isolate the synthesis of the UV-absorbing compounds was enhanced under sublethal UV-B stress (Marchant et al. 1991). In some freshwater phytoplankton species a dramatic increase in MAAs content was found after artificial UV-B irradiation which further increased af-

Figure 4. The ratio of UV peak absorption (ranging from 325 to 337 nm) to Chl a absorption (665 nm) in methanolic extracts from Gyrodinium dorsum exposed to simulated solar radiation under different cut-off filters (GG and WG series, Schott & Gen., Germany) for up to 72 h.

ter exposure to natural sunlight (Xiong et al. 1997). In long-term experiments with natural Antarctic phytoplankton assemblages MAA concentrations have been shown to increase after exposure to PAR with UV but not to PAR without UV (Villafañe et al. 1995). The spectral effect of radiation on MAA synthesis differs largely between species. In Phaeocystis antarctica the induction of MAA synthesis is induced predominantly by short wavelength UV-A but also by UV-B radiation, while some diatom species respond predominantly to long wavelength UV-A and short wavelength visible radiation (Riegger & Robinson 1997). In the dinoflagellate Gyrodinium dorsum the accumulation of MAAs is stimulated by PAR and UV radiation, but the most prominent induction is caused by radiation below 345 nm (Figure 4). There are numerous studies revealing that synthesis of MAAs is influenced by the irradiation conditions. However, in most of these studies the spectral resolution is limited to broad wavelength ranges with treatments such as PAR only or PAR plus UV. So, it is evident that MAAs are accumulated in response to radiation, but a true action spectrum for MAA synthesis is not yet available.

Effects of UV-B radiation on macroalgae Coastal areas all over the world are inhabited by macroalgae (Lüning 1985). As macroalgae grow from supralittoral to sublittoral zones they are exposed to varying levels of solar radiation. The absorption of solar radiation in the water column begins in the lower wavelength bands. Algae growing in the supralittoral

230 or in the eulittoral are exposed to solar radiation including UV, while algae growing in the sublittoral are exposed mainly to PAR. Many algae show a distinct zonation at their growing site (Hanelt 1996; Franklin & Forster 1997; Bischof et al. 1998). The influence of increasing solar radiation affects the macroalgae directly and indirectly. A direct influence is damage of DNA (Pakker & Breeman 1997), pigment composition and the photosynthetic apparatus (Aguilera et al. 1999). An indirect effect can be caused by changes in the environment of the algae like desiccation, changes in temperature and the release of toxic substances by the influence of UV irradiation on chemicals in the water. In their natural environment photoinhibition depends on the growth site of the algae. Macroalgae growing at the surface or in the intertidal show much higher photoinhibition compared to macroalgae growing in the subtidal or in crevices. In the field, as well as under laboratory conditions, the influence of UV-B radiation on photosynthetic quantum yield, oxygen evolution and respiration as well as the recovery has been investigated by several workers (Hanelt et al. 1993; Larkum & Wood 1993; Häder et al. 1996; Häder 1997; Pérez-Rodríguez et al. 1998; Gröniger et al. 1999). Algae growing in transparent waters show a higher photoinhibition than algae growing in turbid waters (Hanelt et al. 1993). A reduced photosynthetic activity of algae around midday was shown under field as well as laboratory conditions (Häder 1997). Depending on the stage in their life cycle different photoinhibition and recovery levels were found in Laminaria saccharina (Hanelt et al. 1997). Laboratory studies on Dasycladus vermicularis exposed to PAR with or without UV radiation led to a complete loss in oxygen evolution after 24 h of PAR + UV-A + UV-B radiation (Pérez-Rodríguez et al. 1998). In Mastocarpus stellatus a drastic decline in the ratio of the photosynthetic quantum yield was shown when exposed for 3 days to a solar simulator (Figure 5). The decrease is mainly caused by PAR while the effect of additional UV-A or UV-B does not affect the algae as drastically as the PAR. The algae show a recovery up to about 50% for PAR with or without UV-A, while the thalli irradiated with PAR + UV-A + UV-B recover only to 40% (Gröniger et al. 1999).

Figure 5. Ratio of the yield of dark-adapted (0 h) control sample and a dark-adapted sample after 72 h of exposure and 42 h of recovery in Mastocarpus stellatus. The thalli were exposed to PAR (395 nm cut-off filter), PAR + UV-A (320 nm cut-off filter) or PAR + UV-A + UV-B (295 nm cut-off filter) from a solar simulator in a 12 h/12 h light/dark cycle. For recovery they were placed in low light conditions in a 12 h/12 h light/dark cycle.

Photoprotective substances in macroalgae UV-absorbing substances in macroalgae were first reported in 1961 (Tsujino & Saito 1961). In the last few years qualitative and quantitative studies were carried out to survey the distribution of MAAs among macroalgae. MAAs are distributed in macroalgae from polar to tropical habitats. Karentz et al. (1991c) reported the presence of MAAs in macroalgae from the Antarctic. A survey of Rhodophyta, Phaeophyta and Chlorophyta from tropical to polar habitats gives a broad overview of the distribution of MAAs in macroalgae (Häder & Figueroa 1997; Karsten et al. 1998a, b, c). The percentage of investigated species containing MAAs is much higher in red algae compared to brown and green algae. Also the number of different MAAs as well as the total amount of MAA is higher in Rhodophyta compared to Phaeophyta or Chlorophyta. A decreasing amount of MAAs is found with increasing depth in the water column as well as in species from higher latitudes. Deep water (more than 2 m; depending on water turbidity) algal species do not contain MAAs (Maegawa et al. 1993; Karsten et al. 1998c). Therefore, the MAA content in algae varies between classes and with growing depths. Differential results in MAA analysis are also influenced exposure to solar radiation. Basically three patterns of MAAs distribution are found among macroalgae; (a) high initial MAA content and no increase during light treatment, (b) low

231

Figure 6. Absorption spectra of the MAAs of Gracilaria cornea after increasing exposure time to UV-B.

MAA content with an increase during light treatment, and (c) no initial MAA and no induction during light treatment. The red macroalga, Chondrus crispus, harvested from the subtidal zone shows an increase in the number and amount of MAAs (Karsten et al. 1998a). Initial high levels but no significant increase in the MAAs content was found in the upper intertidal Rhodophyta, Porphyra umbilicalis (Gröniger et al. 1999). Similarly, no in vivo induction of MAAs was recorded after exposure to either UV alone or in combination with PAR in the marine red alga, Gracilaria cornea, which possesses a very high amount of naturally occurring MAAs having an absorption maximum at 334 nm. The MAAs were highly stable against UVB irradiation (Figure 6) and heat treatment (Sinha et al. 2000). In the Chlorophyta Dasycladus vermicularis UV-absorbing substances were found, but their absorption spectra and retention times did not resemble those of known MAAs (Pérez-Rodríguez et al. 1998). The intertidal rhodophytes, Mastocarpus stellatus and Ceramium rubrum were exposed to a solar simulator using different cut-off filters to produce PAR only or PAR + UV-A or PAR + UV-A + UV-B. Small changes in the MAAs content were observed in both algae (Figure 7). As both algae were collected from the intertidal zone in Helgoland in July 1998 their initial content of MAAs at the beginning of the experiment might have been sufficient for protection to solar radiation or the maximum accumulation for these species had already been reached. In Polyides rotundus, exposed under the same conditions, no initial MAAs were found and no MAAs were induced during the experiment. P. rotundus, growing strictly in the sub-

Figure 7. Contents of MAAs in Mastocarpus stellatus and Ceramium rubrum after three days of exposure (12/12 h light/dark cycle) to indicated irradiation treatments. The initial value is set to 100% (n = 3; average standard deviation ± 25%).

tidal, does not produce MAAs as protection against harmful radiation. Bleaching of the thalli started on the second day of exposure. P. rotundus might not have the possibility to produce MAAs as it is not necessary under normal growing conditions.

Evolutionary significance of photoprotective compounds Sagan (1973) first considered the possibility that organic molecules may have acted as a UV screen in the aqueous environments of the early Earth. The nature and evolutionary origin of the first specific photoprotective compounds on the Achaean Earth is unknown. But according to one assumption early aromatic containing reaction centers were some of the earliest UV-screens that over the period altered from a nonproductive dissipative UV-screen to a light harvesting role in photosynthesis (Mulkidjanian & Junge 1997). MAAs play a vital role as osmotic regulators in some cyanobacteria (Oren 1997) and such alternative roles may have given rise to the first UV-screening MAAs (Cockell 1998). MAAs evolution as specific UV-protectants may represent an early innovation in dealing with Achaean UV-B flux. Certain MAAs such as mycosporine-glycine specifically absorb in the UVB range of the spectrum. It has been postulated that later, as oxygen levels increased, UV-A screening MAAs became more important since many of the effects of UV-A are mediated through oxygen free radicals (Garcia-Pichel 1998) and thus the role of UV-

232 A as a damaging agent in the biosphere increased. In these compounds, the nitrogen atom replacing the ketone function has a greater mesomeric effect on the benzene ring and the absorbance is shifted towards the UV-A. Most probably, a mutation in the earliest UV-B screening compounds resulted in a UV-A screen which became physiologically advantageous. Since MAAs have been reported in several eukaryotic algae, it is likely that they were passed to the eukaryotic algae by cyanobacteria in the plastidic line (Cockell & Knowland 1999 and references therein). Dillon & Castenholz (1999) suggests that the protection against UV radiation provided by scytonemin may have been an important ecophysiological factor in cyanobacterial evolutionary history. Scytonemin would have facilitated the ecological expansion of cyanobacterial mat communities into exposed shallow-water and terrestrial habitats during the early to mid-Precambrian despite the high levels of UV radiation impinging on the Earth’s surface at that time.

Database A database on photoprotective compounds in cyanobacteria, phytoplankton and macroalgae has been constructed (http://www.biologie.uni-erlangen.de/botanik1/index.html). It contains information on the algae, the reference, the MAAs found in the algae, the collection site and depths. Further information on the different MAAs like absorption maximum and extinction coefficient as well as experimental procedures are available. The database allows to obtain information on the different algae and to compare the results from different growth sites or collection dates (Gröniger et al. 2000).

the inhibitory effects of UV-B. Different sensitivity of species and the subsequent changes in community structures may lead to changes in steady state conditions that still sustain a high primary production. Changes in species composition might have consequences propagating within aquatic food webs that are at present difficult to predict. The ecological significance of photoprotective compounds in diverse organisms as screening agents against UV-induced damage has yet to be elucidated. Not much is known about the spatial distribution of MAAs within the cells. The reports of specific distributions of absorption due to MAAs in the water column indicate a role of these compounds in protection against UV radiation, but there are many reports showing that the presence of MAAs in an organism is no proof that this organism is UV-tolerant (Xiong et al. 1997). Higher MAAs concentrations in Prorocentrum micans, adapted to UV radiation did not completely mitigate the detrimental effects of UV on photosynthesis (Lesser 1996). Literature survey reveals that MAAs may serve at least three different functions: (a) they may protect the cells from UV photodamage by playing a sunscreen role, (b) they may to a certain extent transfer radiant energy to the photosynthetic reaction centers, which is supported by the fact that the emitted fluorescence spectrum peaks at a wavelength near the Soret band of chlorophyll absorption (Sivalingam et al. 1976; Sinha et al. 1999b), and (c) that they may aid in osmotic regulation (Oren 1997). Thus, irrespective of the fact that the UV protective potentials of MAAs and scytonemin are a primary or secondary function or they are synthesized/accumulated due to UV radiation, the presence of these compounds in an organism may provide protection to the internal organelles and components from the full impact of deleterious UV radiation.

Conclusions Acknowledgements Increases in the level of UV-B radiation are likely to induce changes in community structure since there are great differences in susceptibility of species to UV-induced damage. Species with larger cells, having the ability to accumulate UV-screening substances or with more effective repair mechanisms will likely be favored. There is ample evidence that the change in the radiation climate originating from the depletion in stratospheric ozone has significant adverse effects on aquatic organisms. However, the action of defense mechanisms in organisms partially mitigates

The work outlined in this review was partially supported by the European Union (DG XII, Environmental Programme, ENV4-CT97-0580). We thank M. Schuster for excellent technical assistance.

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