Potential effects of UV radiation on photosynthetic structures of the bloom-forming cyanobacterium Cylindrospermopsis raciborskii CYRF-01

Abstract

Cyanobacteria are aquatic photosynthetic microorganisms. While of enormous ecological importance, they have also been linked to human and animal illnesses around the world as a consequence of toxin production by some species. Cylindrospermopsis raciborskii, a filamentous nitrogen-fixing cyanobacterium, has attracted considerable attention due to its potential toxicity and ecophysiological adaptability. We investigated whether C. raciborskii could be affected by ultraviolet (UV) radiation. Non-axenic cultures of C. raciborskii were exposed to three UV treatments (UVA, UVB, or UVA + UVB) over a 6 h period, during which cell concentration, viability and ultrastructure were analyzed. UVA and UVA + UVB treatments showed significant negative effects on cell concentration (decreases of 56.4 and 64.3%, respectively). This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe. Over 90% of UVA + UVB- and UVA-treated cells died. UVB did not alter cell concentration, but reduced cell viability in almost 50% of organisms. Transmission electron microscopy (TEM) revealed a drastic loss of thylakoids, membranes in which cyanobacteria photosystems are localized, after all treatments. Moreover, other photosynthetic- and metabolic-related structures, such as accessory pigments and polyphosphate granules, were damaged. Quantitative TEM analyses revealed a 95.8% reduction in cell area occupied by thylakoids after UVA treatment, and reduction of 77.6 and 81.3% after UVB and UVA + UVB treatments, respectively. Results demonstrated clear alterations in viability and photosynthetic structures of C. raciborskii induced by various UV radiation fractions. This study facilitates our understanding of the subcellular organization of this cyanobacterium species, identifies specific intracellular targets of UVA and UVB radiation and reinforces the importance of UV radiation as an environmental stressor.

Keywords: cell death; cell viability; cyanobacteria; thylakoid membranes; transmission electron microscopy; ultraviolet radiation.

FIGURE 1

Cell concentration evaluation after 6 h of exposure to ultraviolet radiation (UVA, UVB, and UVA + UVB). For each time point, the concentrations of UV-treated cultures (UVA, n = 3; UVB, n = 3 and UVA + UVB, n = 3) and their respective controls (total n = 9) were analyzed after fixation and cells counted by light microscopy using a hemocytometer. All cultures were in the exponential phase of growth. Data are expressed as mean ± SD (n = 3). Open symbols represent treatments whereas filled symbols represent control groups.

FIGURE 2

Cell viability of Cylindrospermopsis raciborskii exposed to UV radiation. In (A), live (green) and dead (red) cyanobacteria are observed. (B) After 6 h, all treatments induced significant cell death compared to untreated and 0 h groups (P < 0.0001 for all). Letters indicate a significant difference (P < 0.0001). In (C), both live and dead cells are seen on the same filament (after UVB treatment). Samples were stained with Syto 9 and propidium iodide (BacLight). Live and dead cells were viewed simultaneously, and 30 filaments/group were counted by fluorescence microscopy.

FIGURE 3

Different views of untreated C. raciborskii cells observed by transmission electron microscopy (TEM). In (A), a cross-section of a cell shows a general view of the cytoplasm, cell envelope and their organization. (B) At higher magnification, the structure of the cellular envelope is more clearly revealed. Note the cytoplasmic membrane (green), cell wall (red) and outer membrane (yellow). (C–E) Cytoplasmic structures such as air vesicles (V), lipid bodies (white arrowhead), polyhedral bodies (^∗), and polyphosphate granules (yellow arrowhead) are observed. In (F), the thylakoid membrane structure is seen at high magnification. Phycobilisomes (red arrowheads) are viewed as small electron-dense dots in association with thylakoid membranes.

FIGURE 4

A representative C. raciborskii filament seen by transmission electron microscopy (TEM). (A,Aii) A longitudinal section shows several cells tightly associated with each other by their envelopes (highlighted in blue in Aii). Thylakoid membranes with phycobilisomes (indicated by arrowheads in Ai) are clearly visible.

FIGURE 5

Cyanobacterium thylakoid membranes are drastically affected by UV radiation. While the cytoplasm of control (A), untreated Cylindrospermopsis raciborskii shows a high amount of thylakoid membranes, these structures were greatly reduced after exposure to UV radiation (B). Membranes are highlighted in red in (Ai) and (Bi). In (C), quantitative analyses show a significant reduction of thylakoid areas induced by all UV treatments. Letters indicate a significant difference (P < 0.05). Cells were fixed in a mixture of glutaraldehyde and paraformaldehyde and prepared for TEM. A total of 64 electron micrographs were evaluated and thylakoid areas measured using software Image J 1.41.

FIGURE 6

Electron micrograph of a representative degenerating cyanobacterium. In addition to a clear loss of thylakoid membranes, aggregates of dissolving phycobilisomes (red boxes) were observed in the cytoplasm. Note the loss of the integrity of the cell envelope (arrowheads).

FIGURE 7

Ultraviolet radiation induces disarrangement of C. raciborskii polyphosphate granules. Polyphosphate granules (boxed area in (A) and (B) and arrowheads in Ai and Bi) are seen as intact structures in control cells (A,Ai), and as disrupted structures in UV-treated cells (B,Bi). A polyphosphate granule is seen at an advanced stage of disintegration [(C), arrow]. A graph of the number of polyphosphate granules per cell for each group is shown in (D). A total of 64 electron micrographs were evaluated and the number of granules per section quantitated. A total of 273 granules were counted. Data are expressed as mean ± SD. Letters indicate significant differences between means (P < 0.05).