Potential UV-B effects on charophytes
The glasshouse experiment showed that 5 wk of daily repeated exposure to UVbe doses of 5.4 and 9.3 kJ m−2 d−1 caused significant growth reductions in C. aspera. This is consistent with growth reductions in response to UV-B exposure as found in plant species from terrestrial and marine environments (Caldwell et al., 1998; Searles et al., 2001; Van de Poll et al., 2001). Despite the fact that the ambient dose of 3.8 kJ m2 d−1 could not be taken into account, the significant dose–response relation indicated that also low doses of UV-B radiation affect plant performance. DNA damage corresponded to the pattern of growth reduction as increased plant CPD concentrations occurred at increasing UV-B doses. The presence of CPDs in the DNA affects DNA transcription and replication (Sauerbier & Hercules, 1978; Draper & Hays, 2000). Because the average growth reduction correlated significantly to the CPD levels in the plants (Pearson coefficient −0.57, P < 0.001), it is likely that UV-B induced DNA damage influenced plant growth in this study. Studies on higher plants, marine phytoplankton and marine macro algae also reported that reduced growth corresponded to increased levels of CPDs (Mazza et al., 1999; Buma et al., 2000; Van de Poll et al., 2001).
Several light-dependent and independent ways to repair DNA damage have been reported in plants (Britt, 1995). Light dependent repair by photolyase enzymes was shown to be the most efficient CPD repair mechanisms in higher plants and marine macrophytes (Quaite et al., 1994; Pakker et al., 2000a,b). Since first no significant CPD repair was found in between two subsequent UV-B treatments, and second CPDs were present at all times, the DNA repair systems in the charophycean algae apparently were unable to repair all damage. This unbalanced induction and repair causes a gradual accumulation of CPDs in the DNA, as was previously observed for marine macro algae (Van de Poll et al., 2002).
While damage to DNA and subsequent growth reduction occurred, no adequate protection by increased UV-B absorption was found. Only a small increase was found in UV-B and UV-A absorbing compounds in Chara aspera due to UV-A exposure, but in general the absorbance in the UV radiation region was low (this study; De Bakker et al., 2001; Rae et al., 2001). In higher plants induction of flavonoids has been reported, while in marine organisms mycosporine-like amino acids (MAAs) act as effective sunscreen (Cockell & Knowland, 1999), protecting underlying tissue from UV radiation. Neither types of secondary metabolites have been found in Chara aspera (data not shown) or in charophycean algae in general (Wegner-Hambloch, 1983; De Bakker et al., 2001), indicating that these organisms have a limited capacity to produce protective compounds under UV exposure.
UV-B penetration in freshwater systems
The glasshouse study showed that Charophycean algae are potentially sensitive to UV-B radiation. But do they have to cope with solar UV-B radiation under natural conditions?
While previous freshwater studies on UV-B radiation mainly focussed on factors affecting attenuation (e.g. Scully & Lean, 1994; Morris et al., 1995; Huovinen et al., 2003), the (potential) ecological effects for submerged vegetation have only occasionally received attention (Rae et al., 2001). The direct impact of solar UV-B radiation was studied on CPD levels in charophyte vegetation. Since the relation between UV-B exposure and growth is difficult to determine in plants growing in natural aquatic systems, UV-B induced DNA damage is a good alternative to study solar UV-B exposure and its effects on aquatic vegetation. Furthermore, this type of DNA damage is a highly specific indicator for UV-B stress, because it reflects exposure to short wavelength radiation only, as CPDs are only slightly induced by UV-A radiation and not by PAR (Quaite et al., 1992).
The accumulated CPDs in the DNA dosimeters correlated significantly to the Setlow weighted UV-B doses calculated from spectroradiometrical analysis above water and below water. This was also found in several earlier studies from different marine locations around the world (Boelen et al., 1999; van de Poll et al., 2002). The results were comparable for both methods, except for the Buiten Muy II where resuspension of the sediment during the spectroradiometer measurements might have disturbed light penetration. Therefore, the DNA dosimeters are useful tools to estimate potential UV-B exposure of plants in freshwater systems. Both methods based on incident irradiance and daily UV-B doses showed that UV-B radiation attenuated quickly in the water column. Consequently, UV-B radiation did not reach the charophycean algae. The attenuation coefficients of UV-B radiation of Dutch phytoplankton dominated freshwater systems measured by De Lange (2000) were often lower than our measurements, leading to deeper penetration of UV-B. Therefore, our results contrast with expectations that enhanced transparency due to presence of submerged charophytes would result in deeper penetration of UV-B radiation in those shallow freshwater systems.
The additional evidence that no direct damage of UV-B induced CPDs in charophycean DNA had occurred supports the findings that charophycean algae were not exposed to solar UV-B radiation in either system under natural conditions. The study of Rae et al. (2001) on attenuation of and sensitivity to UV-B radiation in a New Zealand lake supports our findings. They showed that the 1% depth of solar UV-B irradiance (305 nm) only reached the most upper limit of the vegetation of Chara fibrosa, so no significant exposure occurred to that charophycean alga either.
This picture may alter when temporal dynamics is accounted for. The water table in Lake Veluwe is controlled to an approximately constant water level in winter and summer. Therefore it is not expected that charophycean algae are exposed to UV-B radiation at any moment during the year. But, in the Buiten Muy at Texel, the water table fluctuates during the year, being lowest at the end of the summer. Figure 4 displays the water levels for nine subsequent years. When assuming constant attenuation of UV-B radiation in time and constant vegetation height, charophytes will potentially be exposed to UV-B radiation for a certain period almost each summer depending on the weather conditions. This occurs in eight out of the nine monitored years. This coincides with field observations in shallow freshwater that show that charophycean algae have a more compressed growth form compared with deeper freshwater systems.
Figure 4. Water table in the Buiten Muy from 1994 to 2002. Black line represents the water level; the grey line the depth where 1% UV-B penetrates, assuming constant attenuation of UV-B in time. The soil level and the top of the Littorella vegetation are shown by the horizontal lines for Buiten Muy II. The water table in the Buiten Muy I is c. 13 cm higher than in Buiten Muy I. Data were provided by Staatsbosbeheer.
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In some years, the Buiten Muy may even dry out and the water table will drop below the soil surface (in four out of the nine years). Charophycean algae die and have to re-establish after such dry periods from oospores. Some charophycean algae are also able to regenerate from bulbils; vegetative reproduction structures formed near the rhizoids. In a glasshouse study De Bakker et al. (2001) showed that exposure to UV-B alters the reproduction strategy of Chara aspera leading to an increase in vegetative bulbils, and a decrease of generative oospores. This shift in reproduction strategy may increase the survival chances of the algae, because re-establishment success from bulbils is higher than from oospores (Van den Berg et al., 2001). Field observations support this idea. Since, higher regeneration rates have been found in shallow freshwater (Nat, personal comment). Therefore it seems that UV-B exposure triggers a mechanism that enhances the survival chances of charophytes after dry periods.
However, note that from the limited data available it is unclear what actual level of UV-B causes significant effects on plant performance or on reproduction strategies (e.g. a threshold value). This is a topic that clearly needs further study.
In conclusion, charophytes are sensitive to UV-B radiation. Increasing levels of UV-B radiation led to DNA damage and growth reduction, while no increase in protective UV-B absorbing compounds was found. However, by contrast to the glasshouse experiment, charophycean algae are not frequently exposed to UV-B radiation under field conditions. Nevertheless, in wetlands with fluctuating water tables plants may be regularly exposed to UV-B radiation during certain periods of the year.