The O2 photoevolution was more drastically reduced in Calothrix BI22 sp. than in Anabaena BI42 sp. (Figure 4).
3.4. Mycosporine Like Aminoacids
The HPLC analysis revealed the presence of two MAAs in Calothrix sp. BI22 (Figure 5). According to  these MAAs could be palythinol and mycosporine-2-glycine. Their retention times were 3.54 and 10.29 min espectively and their absorption maxima were 330 and 332 nm respectively. The content of these MAAs, 3.7 and 0.5 nmol∙g∙DW–1 respectively did not increase significantly with UV-B exposure. Instead, neither any peak nor an induction of any MAAs was observed in Anabaena sp. BI42 (Figure 5).
3.5. Nitrogenase Activity
Rates of nitrogen fixation (Table 1), measured as nitrogenase activity, were not significantly altered in Calothrix sp. BI22 due to UV-B radiation. Instead, Anabaena sp. BI42 showed a significant reduction at the very first hour of exposure (p < 0.05) which represents a 55% decline in the nitrogen fixation of this strain. When the culture of Anabaena sp. BI42 was irradiated for 3 hours, the nitrogenase activity drastically decreases (Table 1).
Figure 4. Photosynthetic parameters measured for both isolates Calothrix sp. BI22 and Anabaena sp. BI42 in the control or irradiated with UV-B for 1 or 3 h. The maximum photochemical efficiency of PSII reaction centers measured as the ratio FV/Fm and percentage of inhibition of O2 photoevolution after exposure for different times to UV-B. Results of FV/FM are reported as mean of three replicates ± standard error.
Figure 5. (a) HPLC chromatogram showing the retention times and wavelength of MAAs (palythinol and mycosporine-2-glycine) in Calothrix sp. BI22. (b1) Absorption spectrum showing the maximum absorbance for MAA palythinol (as purified by HPLC), at 332 nm and retention time 3.32 min. (b2) Absorption spectrum showing the maximum absorbance for MAA mycosporine-2-glycine, (as purified by HPLC), at 332 nm and its retention time was 10.08 min.
3.6. Lipid Peroxidation
The level of lipid membrane damage measured as TBARS was not significantly affected by UV-B exposure in Calothrix sp. BI22 (Table 1). Only after 3 hours of exposure, the level of damage became significantly different from the control values in Anabaena sp. BI42 (Table 1).
3.7. Proline Content
Quantification of proline showed that after 3 hours of exposure its accumulation became significantly different to control samples of Anabaena sp. BI42. However, proline content showed no significant difference during the experiments in Calothrix sp. BI22 (Table 1).
3.8. Antioxidant Enzymatic Activities
Both strains had changes in their antioxidant enzymes activities in response to UV-B exposure (Figure 6). SOD activity showed an opposite trend between the cyanobacterial strains; a significant decrease in Anabaena sp. BI42 and a significant increase in the enzymatic activity in Calothrix sp. BI22 after 3 hours of exposure to UV-B. On the other hand, in Anabaena BI42 and Calothrix sp. BI22, their catalase activity significantly increased after one hour of exposure and dropped down its activity after 3 hours of irradiation. There was no significant change in the APX activity in both strains (Figure 6).
Assays on SOD activity by non-denaturing PAGE gels showed that Anabaena sp. BI42 had two isoforms and Calothrix sp. BI22 one. There was no induction of any new isoform of this enzyme during the exposure to UV-B radiation (Figures 6(c) and (d)). The pre-incubation with inhibitors such as KCN and H2O2 showed that both strains have a Fe-dependent SOD isoform (data not shown) (Figure 7).
Cyanobacteria harvest light energy during the process of photosynthesis and assimilate it into carbon compounds. Any adverse effect of UVB may severely affect photosynthesis and related metabolic processes and overall growth performance of cyanobacteria. Although the survival of both cyanobacteria isolates tested decreased after exposure to UV-B irradiation, the responses were different. Previous reports stated that the differences could in part be explained on the basis of antioxidant defenses and DNA repair mechanisms present in each microorganism or simpler ones as morphological features such as presence of sheath [27,28]. For instance, Calothrix sp. BI22 has a muscilaginous sheath (data not shown) observed by optic microscopy whereas Anabaena sp. BI42 lacks it. It must be considered that in this work the UV lamp used provided almost exclusively UV-B light and PAR was not applied simultaneously during the irradiation period. Field conditions are quite different and so these results must not be extrapolated. For example, DNA-repair mechanisms included photoreactivation by photolyase after absorption of light energy at 400 nm or long wavelength UV-A .
The laboratory studies presented here show that both photosynthesis and nitrogen fixation in Anabaena sp. BI42 are affected by short exposure times to UV-B, while in Calothrix sp. BI22 only photosynthesis decreased.
Table 1. Effect of different times of exposure to UV-B on nitrogenasa activity, proline content and lipid peroxidation in Anabaena sp.BI42 and Calothrix sp.BI22.
Figure 6. Superoxide dismutase (SOD) in vitro activities in Anabaena sp. BI42 (a) and Calothrix sp. BI22 (b) after different times of exposure to UV-B. Results are reported as mean of three replicates ± standard error. Identification of SOD in-gel activities in Anabaena sp. BI42 (c) and Calothrix sp. BI22 (d) for the control and cells irradiated with UV-B for 1 and 3 h.
Figure 7. APX and CAT activities in Anabaena sp. BI42 (a) and Calothrix sp. BI22 (b) exposed to UV-B for 0, 1 and 3 h. Results are reported as mean of three replicates ± standard error.
4.1. Pigmentation and Photosynthesis
Several studies as this one suggest that phycobiliproteins, chlorophyll and carotenoids are negatively influenced by UV-B radiation .
The increase in fluorescence emission peaks with the UV-B treatments (Figure 3) was not accompanied by a shift of the peak to shorter wavelengths as has been reported for larger exposure to UV-B . However, the observed slight decay in fluorescence intensity was probably due to less efficiency in light capture in UV-B irradiated cells and loss in efectivity of energy transfer to the photosynthetic reaction center.
Photosynthetic oxygen production declined with artificial UV-B radiation in both isolates investigated. It could be possible that structural changes within the photosynthetic apparatus were induced by short exposure times which affected the energy transfer within the antenna complex. Photosynthesis was more affected in Calothrix sp. BI22, as showed by the fluorescence spectra and O2 photoevolution. Photosynthetic quantum yield that was determined by pulse amplitude-modulated fluorometry can provide information on both photosynthesis and overall acclimation status . Quantum yields of PSII fluorescence showed a decline upon UV-B exposure in Calothrix sp. BI22 (Figure 4). This is consistent with previous studies  where accumulation of un-repaired damage over time in cultures exposed to UVR is suggested. A possible explanation is that essential proteins of the PSII like D1 are damaged during exposure [32-34] and probably this explains what was observed in Anabaena sp. BI42. Phycobiliproteins fluorescence may also be contributing to photosynthetic quantum yield as both cyanobacteria possessed different phycobiliproteins .
4.2. Nitrogen Fixation
The results of nitrogenase activity (Table 1) showed that in Anabaena sp. BI42 this process was drastically inhibited after 3 hours of exposure (near 100%). This is in accordance to previous studies on other strains of Anabaena sp., but using higher doses of UV-B [29,35] (Newton et al., 1979, Lesser, 2008). In Calothrix sp. BI22, the N2 fixing activity was not affected by UV-B exposure in any treatment. Nitrogen fixation requires ATP and reductant for its activity that come from photosynthesis. Even though photosynthesis (Fv/Fm and O2 photoevolution) was altered in Calothrix sp. BI22, there is evidence that cyanobacteria contain sufficient endogenous content of reductant and ATP to support the nitrogenase activity . However,  showed that nitrogenase inactivation by UV-B is caused by direct damage to nitrogenase polypeptide. They also proved that restoration of the activity depends on de novo synthesis of the enzyme rather than the cellular content of ATP and reductant.
4.3. Mycosporine Like Aminoacids
Synthesis of UV-absorbing compounds, such as mycosporine amino acids (MAAs), is an important mechanism preventing UV photodamage. The degree of protection by MAAs depends on the type of aminoacid, location (cytoplasm or extracellular glycan) and species . Calothrix sp. BI42 had two types of MAAs, palythynol (88%) and mycosporine-2-glycine (12%). The same kind of MAAs had been found in Nostoc punctiforme in colonies exposed to high solar radiation . MAA-Gly is reported to be located in the extracellular glycan ; . Apart from the benefits from being in the outer membrane, MAA-Gly is reported to give additional protection because it can act as a radical quencher . The experimental conditions used in this study did not induce any increase or variation in Calothrix’s MAAs content. In other studies MAAs synthesis was also influenced by the irradiation conditions  and higher doses or longer times of irradiation increased MAAs content . A constituve level of MAAs can appear in all growth conditions of cyanobacteria as observed in Calothrix sp. BI42. This cyanobacterium grows in aggregates which is said to substantially increase the screening efficiency of MAAs . Anabaena sp. BI22 did not show the presence of any MAAs nor any induction of them with exposure to the UV-B doses assayed. Other surveys reported that from 22 cyanobacterial isolates, only 13 were found to contain MAAs . However,  found in an Anabaena sp. isolated from an Indian rice paddy-field the presence of shinorine. None of the strains tested showed the presence of scytonemin (data not shown) which is said to be induced by UV-A irradiation and other type of stresses .
4.4. Antioxidant Potential
Anabaena sp. BI42, after 3 hours of exposure showed a significant increase of lipid membrane damage measured as TBARS (Table 1). This provides indirect evidence of increased photoxidative damage stress by ROS after UV-B exposure in this cyanobacterium as has been reported before [27,46]. There was no significant change in lipid peroxidation in Calothrix sp. BI22.
Proline is an iminoacid essential to primary metabolism whose accumulation has been related to unfavourable conditions in plants  and cyanobacteria [48,49]. According to  intracellular proline detoxiﬁes harmful ROS directly rather than improving key antioxidant enzymes. Our results are in accordance with this, because Anabaena showed an increase in TBARS and in proline content after 3 hours of exposure. No effect in the proline content was seen in Calothrix sp. BI22 (Table 1).
During oxidative stress ROS triggered the activity of several antioxidative enzymes such as SOD (EC 184.108.40.206), CAT (EC 220.127.116.11), and APX (EC 18.104.22.168). High activity of these enzymes in cyanobacteria could be linked to stress tolerance efficiency .
As has been stated by , cyanobacteria can have iron-containing SOD (cytosolic) and manganese-containing SOD (thylacoid-bound) isoforms. The strains tested have both a Fe-SOD isoform and no other isoform was induced due to the experimental conditions (Figures 6(c) and (d)). SOD activities have been shown to change dramatically in response to conditions that favour the formation of superoxide . Calothrix sp. BI22 showed a significant increase of its activity after the maximum exposure time (Figure 6(b)).  reported that is the first ROS generated as a consequence of UV-B stress. Like in other studies [54,55], the result obtained supports the fact that SOD plays an important role in protecting Calothrix sp. BI42 from UV-B stress. However, Anabaena sp. BI42 showed a decrease in the same conditions (Figure 5(a)). It has also been reported that only UV-B produced the decrease of SOD activity in microalgae [56,57] found that in plants SOD activity can increase or diminish according to the species, UV-B dose and the presence or not of concomitant PAR or UVA radiation.
CAT activity had a significant increase in both strains after one hour of exposure to UV-B (Figures 7(a) and (b)). After 3 hours of irradiation, levels of CAT activity fell down reaching the values observed in the Control treatment in both strains. This result has been reported before [54,58] and inactivation of CAT due to UV-B is as consequence of photoinactivation and degradation  of the haem group in CAT. According to , UV-B inhibition of CAT could be a survival strategy in order to prevent accumulation of H2O2 in the cell.
UV-B irradiation did not significantly affect the APX activity in both strains (Figures 7(a) and (b)). It seems probable that in this moderate UV-B irradiation study, APX is not involved in the protective mechanisms against it in both cyanobacterial strains.
Many effects reported in the literature indicate species-specific responses to UVR. In this case, the two strains studied under laboratory conditions had different potential survival rates that can be partially explained by the synthesis of UV absorbing compounds and their antioxidant potential.
Two cyanobacteria strains isolated from the same rice field had different responses to UV-B irradiation doses comparable to the natural solar UV radiation reaching Uruguay’s latitude during rice growing season. Both photosynthesis and nitrogen fixation were affected by moderate UV-B radiation in the Anabaena isolate. Calothrix sp. BI22 seems to have a better and wide suite of protective mechanisms which include constitutive adaptations like presence of a sheath or MAAs. It also showed a better antioxidant response to UV-B which may be involved in the successful scavenging of ROS and protecttion of physiological processes of this cyanobacterium.
All in all these results indicate that Calothrix sp. BI22 may be a better candidate than Anabaena sp. BI42 to be used as an inoculant in Uruguayan rice paddy fields when considering UV-B thriving strategies.
Financial assistance was from Consejo Sectorial de Investigación Científica (CSIC-Universidad de la República). We thank Dr. D.-P. Häder for sending us his papers and providing MAAs standards and Dr. J. Monza and P. Díaz for their suggestions.