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Appl Environ Microbiol. Apr 2005; 71(4): 2154–2157.
PMCID: PMC1082567

Differential Sunlight Sensitivity of Picophytoplankton from Surface Mediterranean Coastal Waters

Abstract

We tested the sensitivity of coastal picophytoplankton exposed to natural sunlight in short-term experiments. Cell abundance and cell-specific chlorophyll fluorescence were significantly reduced in Prochlorococcus spp. but not in Synechococcus, whereas picoeukaryotes had an intermediate response. These results are the first direct evidence of a differential sensitivity to sunlight of these ubiquitous marine members of unicellular phytoplankton.

Unicellular picophytoplankton, such as Synechococcus spp. and Prochlorococcus spp., are now recognized as ubiquitous members of marine plankton and play a significant role in the global carbon cycle. At least two ecotypes of Prochlorococcus, distinguished by their photophysiology and molecular phylogeny, coexist in the oceans (12). One ecotype is found near surface waters and is capable of coping with high-light conditions, whereas the other is not; consequently, the latter is found mainly in deep layers (12, 25). Yet, all Prochlorococcus isolates studied until now, including strains belonging to the high-light ecotype, appear to be sensitive to high levels of photosynthetically available radiation (PAR) (12). This finding raises an important issue, namely, to what extent natural populations of Prochlorococcus are negatively affected by solar radiation, particularly by UV radiation (UVR; 290 to 400 nm), if we consider that the waters where Prochlorococcus thrives are commonly very transparent to UVR.

Solar UVR is known to damage several cell targets and to reduce photosynthetic rates (24). Studies of large eukaryotic phytoplankton, however, have demonstrated a great difference in UV sensitivity among species (23). Differential tolerance to UVR among species is thought to depend on the efficiency of biochemical defenses. Once UV damage overrides these biochemical defenses, cells may cease to grow or may even lyse. In fact, high phytoplankton lysis rates have been documented for several marine waters (3, 6). Vaulot and Marie (22) and Partensky et al. (14) have suggested that UVR near the surface may negatively affect Prochlorococcus, but this has not been directly tested.

In surface coastal waters of the northwestern Mediterranean Sea at the Bay of Blanes, Synechococcus is more abundant than Prochlorococcus at all times of the year (19). Water temperature is a significant variable that explains the higher growth rates of Synechococcus in this bay during summer (1). Interestingly, in a nearby region of the northwestern Mediterranean Sea, the optimal growth temperature of Prochlorococcus (strain MED4) isolated from surface waters is 24°C (11). However, the population maximum of Prochlorococcus in the Bay of Blanes occurs at lower temperatures (19). We hypothesized that solar radiation, particularly UVR, may differentially affect Prochlorococcus and Synechococcus. Consequently, we performed a series of short-term experiments in the summer to test the sensitivities of surface populations of picophytoplankton from the Bay of Blanes exposed to sunlight.

Sampling for the experiments was done at one shallow (maximum depth, ~20 m) oligotrophic coastal station of the northwestern Mediterranean Sea located ~800 m offshore of Blanes, Spain (41°39.90′N, 2°48.03′E). Experiments 1 and 3 were done with samples collected from a 0.5-m depth on 5 and 11 August 2003, respectively, while experiment 2, performed on 7 August 2003, included samples collected from a 5-m depth. Surface samples were also collected monthly to monitor the dynamics of picophytoplankton. These samples were fixed with paraformaldehyde (1% final concentration) and glutaraldehyde (0.05% final concentration) and frozen in liquid nitrogen. Samples for the experiments were collected immediately after sunrise to avoid exposure of the organisms to high irradiances and prefiltered through a large 200-μm-mesh-size net bag to exclude large zooplankton. The experimental design used to test the effect of different wavebands was similar to the design used by Sommaruga et al. (21), except that in the present study spherical quartz glass bottles were used. The short-term experiments (4 h) were done under sunny conditions on the roof of the Institute of Marine Sciences, Barcelona, in a large water bath (200-liter) with black walls and circulating seawater to maintain in situ temperatures within 2°C. Inside the water bath, bottles were fixed to plastic racks to adjust their exposure depth (4 cm).

After exposure, the live samples were analyzed in a FACSCalibur flow cytometer (Becton Dickinson) in unstained aliquots (13). Milli-Q water was used as sheath fluid, and 1-μm-diameter yellow-green latex beads (Polysciences) were used as the internal fluorescence standard. The samples were run at the highest possible speed (~60 μl min−1) for 5 min, and the data were acquired in log mode. Abundances were calculated by the ratiometric method from the known amount of added beads, calibrated daily against TrueCount beads (Becton Dickinson).

Underwater UVR was measured with a multichannel filter radiometer (PUV-501; Biospherical Instruments) additionally equipped with natural fluorescence and temperature sensors. Hourly averaged total incident irradiance values were obtained from a meteorological station located 6 km away.

The experiments were performed at a time when Synechococcus abundance reached the population maximum (1.06 × 105 ml−1) and was ~35-fold higher than that of Prochlorococcus (Fig. (Fig.1).1). Picoeukaryotes were most abundant during the winter (data not shown) and were found at minimum values during the summer (Fig. (Fig.1).1). The maximum abundance of Prochlorococcus organisms (3.64 × 104 ml−1) occurred later in October during a period characterized by lower daily integrated total irradiance values (Fig. (Fig.1).1). The Synechococcus maximum was coincident with high water temperatures (25.2°C), and there was a significant correlation between both parameters for the study period (Pearson R = 0.706, P = 0.03). In contrast, the abundance of Prochlorococcus organisms was not significantly correlated with water temperature, and its maximum was observed at lower temperatures (18°C).

FIG. 1.
(A) Temporal changes in surface water temperature between May and November 2003 and daily integral of total incident irradiance; (B) abundances of surface populations of Prochlorococcus, Synechococcus, and picoeukaryotes in the Bay of Blanes. The arrow ...

In the first experiment, temperature stratification of the water column was weak; however, in the second and third experiments, there was a strong intrusion of cold water, and a clear thermocline was observed below a depth of 3 m (Fig. (Fig.2).2). Maximum fluorescence values were always found at the surface (Fig. (Fig.2).2). The coastal station was very transparent to UVR, with mean 1% attenuation depths of 13.1, 18.2, 27.6, and 50.1 m at the nominal wavelengths of 305, 320, 340, and 380 nm, respectively. At a 5-m depth, the intensities of UVR for the same wavelengths were 19, 26, 40, and 58% of those at the surface.

FIG. 2.
Depth profiles of temperature (left) and naturally induced fluorescence normalized to PAR (right) measured in the Bay of Blanes during three occasions in August 2003.

Prochlorococcus was strongly affected by solar radiation after 4 h of exposure (Fig. (Fig.3).3). Both PAR and UVR caused a significant decrease in cell abundance (one-way analysis of variance, P < 0.01), and there were significant differences among all treatments (post hoc Tukey test, P < 0.05). Samples from the 5-m depth tested in experiment 2 showed the same response to solar radiation as surface samples, but the effect was more pronounced: a decrease of 59% caused by PAR and a further loss of 21% caused by UVR (Fig. (Fig.3b).3b). Results from experiment 3 (Fig. (Fig.3c),3c), where the effects of UVB and UVA were assessed with surface samples, indicated that the loss was caused mainly by UVA (28%), followed by PAR (24%) and UVB (16%). The cell-specific chlorophyll fluorescence of Prochlorococcus was also affected, but there were differences among the three experiments concerning the contribution of PAR and UVR (Fig. 3d through f).

FIG. 3.
Cell abundance (left) and cell-specific fluorescence (right) of Prochlorococcus spp. after 4 h of exposure to different wavebands of natural solar radiation or kept in the dark on 5 August 2003 (0.5-m depth) (A and D), 7 August 2003 (5-m depth) (B and ...

In contrast to those for Prochlorococcus, the results for Synechococcus indicated no significant effects on either abundance or cell-specific fluorescence (Fig. (Fig.4),4), while in the case of picoeukaryotes, only a moderate negative effect of sunlight on their abundance was observed (Fig. (Fig.55).

FIG. 4.
Cell abundance (left) and cell-specific fluorescence (right) of Synechococcus spp. after 4 h of exposure to different wavebands of natural solar radiation or kept in the dark on 5 August 2003 (0.5-m depth) (A and D), 7 August 2003 (5-m depth) (B and E), ...
FIG. 5.
Cell abundance (left) and cell-specific fluorescence (right) of picoeukaryotes after 4 h of exposure to different wavebands of natural solar radiation or kept in the dark on 7 August 2003 (5-m depth) (A and C) and 11 August 2003 (0.5-m depth) (B and D). ...

Previous studies have shown that the photosynthetic activity of surface populations of Prochlorococcus is strongly inhibited under high levels of PAR (12). Our study, however, provides the first direct evidence of the negative effects of PAR and UVR on the cell abundance and fluorescence of Prochlorococcus. Furthermore, the results for picoeukaryotes suggest an intermediate sensitivity of this group to solar radiation. However, because we have no information about the species composition of picoeukaryotes, it is difficult to make further conjectures, particularly since we know that there is a great difference in UV sensitivity among large eukaryotic phytoplankton species (23).

Our results are in agreement with several indirect lines of evidence suggesting that Prochlorococcus is more sensitive to sunlight, and particularly to UVR, than Synechococcus (2, 4, 5). Several factors probably contribute to the different levels of tolerance to sunlight in these picocyanobacteria. For example, DNA rich in A+T can be expected to be more susceptible to damage due to the formation of UV-induced cyclobutane thymine dimers, one of the most common types of DNA damage. Marine Synechococcus strains are characterized by a low A+T content, but most Prochlorococcus strains have high A+T contents (15). Interestingly, Prochlorococcus strain MED4 isolated from surface waters of the Mediterranean Sea has one of the highest A+T contents (69.2%) known for eubacteria (10).

An important negative physiological effect of PAR and UVR on oxygenic phototrophic organisms is the damage to photosystem II (PSII). It has been shown that Synechococcus sp. (strain PCC7942) attains rapid (within minutes) resistance to moderate UVB exposure by expressing the psbA II and III genes, which results in a rapid turnover of D1 protein (i.e., the protein where damage is mainly located) and, consequently, in an active PSII (7). In contrast to the molecular plasticity offered by the multiple copies of psbA genes found in all cyanobacteria examined to date (7), most Prochlorococcus strains have only one copy, similarly to organisms having chloroplasts (9). This difference may result in a reduced repair efficiency of PSII, as suggested before (15).

UVR and PAR are also known to have a significant bleaching and degradation effect on photosynthetic pigments (24). Our results showed that both wavebands, particularly UVA radiation, were effective in reducing the cell-specific chlorophyll fluorescence of Prochlorococcus but not of Synechococcus. In fact, since flow cytometric detection of Prochlorococcus is based on red-fluorescence detection, the changes in abundance reported in the left panels of Fig. Fig.33 are a consequence of the changes in cell-specific fluorescence reported in the right panels. These results may help to explain the common observation that red chlorophyll fluorescence, as detected by flow cytometry in surface populations of Prochlorococcus, is weak (15).

The seasonal dynamics of Prochlorococcus and Synechococcus observed in our study are found consistently in the coastal northwestern Mediterranean Sea (19). The late maximum abundance of Prochlorococcus organisms in fall seems to be a feature of other coastal areas as well (20). This dynamic is affected by several environmental factors (1, 14); however, one additional implication of our results is that the different sensitivities to sunlight of Prochlorococcus and Synechococcus contribute to the temporal segregation pattern observed. Certainly, our experimental design maximized the effects of sunlight by exposing these organisms close to the surface for 4 h, but considering the high UV transparency of this coastal area, we believe the disparity in sensitivity of these cyanobacteria may result in a different population development. Furthermore, the effect of solar radiation will be particularly pronounced when the water column is stratified. Indeed, Prochlorococcus collected at the base of the upper mixed layer (experiment 2) showed higher sensitivity to UVR when it was exposed close to the surface.

Recently, the genomes of different Prochlorococcus strains have been analyzed, revealing their genetic adaptations to different environmental changes such as changes in light quality and quantity (8, 16). Furthermore, a high degree of genetic variation in Prochlorococcus and Synechococcus spp. has been found in different marine areas (17, 18). Therefore, it remains to be tested whether surface populations of Prochlorococcus from environments where they outnumber Synechococcus populations (22) are as sensitive to sunlight as here observed.

Acknowledgments

We thank Ò. Gudayol for providing data on incident irradiance, C. Cardelús for logistics during the experiment, and M. Llabrés and S. Agustí for sharing information about their work.

This work was supported by FWF project 14153-BIO to R.S., MCyT project REN2001-2120/MAR and EU project EVK3-CT-2002-00078 (BASICS) to J.M.G., and by the scientific cooperation program between Austria and Spain (project ÖAD no. 17).

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