Localization and role of manganese superoxide dismutase in a marine diatom

Abstract

Superoxide dismutase (SOD) catalyzes the transformation of superoxide to molecular oxygen and hydrogen peroxide. Of the four known SOD isoforms, distinguished by their metal cofactor (iron, manganese [Mn], copper/zinc, nickel), MnSOD is the dominant form in the diatom Thalassiosira pseudonana. We cloned the MnSOD gene, sodA, using the expression vector pBAD, overexpressed the product in Escherichia coli, and purified the mature protein (TpMnSOD). This recombinant enzyme was used to generate a polyclonal antibody in rabbit that recognizes MnSOD in T. pseudonana. Based on quantitative immunoblots, we calculate that in vivo concentrations of TpMnSOD are approximately 0.9 amol cell(-1) using the recombinant protein as a standard. Immunogold staining indicates that TpMnSOD is localized in the chloroplasts, which is in contrast to most other eukaryotic algae (including chlorophytes and embryophytes) where MnSOD is localized exclusively in mitochondria. Based on the photosynthetic Mn complex in photosystem II, cellular Mn budgets cannot account for 50% to 80% of measured Mn within diatom cells. Our results reveal that chloroplastic MnSOD accounts for 10% to 20% of cellular Mn, depending on incident light intensity and cellular growth rate. Indeed, our analysis indicates that TpMnSOD accounts for 1.4% (+/-0.2%) of the total protein in the cell. The TpMnSOD has a rapid turnover rate with an apparent half-life of 6 to 8 h when grown under continuous light. TpMnSOD concentrations increase relative to chlorophyll, with an increase in incident light intensity to minimize photosynthetic oxidative stress. The employment of a Mn-based SOD, linked to photosynthetic stress in T. pseudonana, may contribute to the continued success of diatoms in the low iron regions of the modern ocean.

Figure 1.

Immunoblot of selected diatom species. Immunoblots showing the anti-TpMnSOD antibody we produced cross reacted with multiple diatom species. The phylum specificity of this antibody suggests that all diatoms have a MnSOD with similar structure. All lanes loaded with 30 μg total protein (except control loaded with 10 ng pure recombinant TpMnSOD): lane 1, Ditylum brightwellii CCMP358; lane 2, Navicula incerta CCMP542; lane 3, Nitzschia brevirostris CCMP551; lane 4, Stephanopyxis turris CCMP815; lane 5, T. pseudonana CCMP1010; lane 6, Skeletonema costatum CCMP1332; CON, control protein overexpressed and purified recombinant TpMnSOD. Note: T. pseudonana strain used in this immunoblot is CCMP1010 and different than CCMP1335, which is the one used for cloning and overexpression of sodA. Marker indicates molecular mass standards in kilodaltons.

Figure 2.

Immunoblot of T. pseudonana CCMP1335 cells and cells treated with 10 mg/mL cycloheximide (L = light, D = dark, Lc = light + cycloheximide, Dc = dark + cycloheximide) to inhibit protein synthesis. After 27 h, the protein is below detection in the cells grown under light with protein synthesis inhibited. This suggests that the turnover of TpMnSOD is related to processes that occur when cells are exposed to light. Conversely, cells exposed to continuous darkness show evidence of TpMnSOD throughout the experiment. Each lane is loaded with 8 μg of total protein extracts. Antibody was specifically raised against recombinant protein in control lane.

Figure 3.

Intracellular distribution of Mn for T. pseudonana CCMP1335. Total cellular Mn (Mn_tot) was estimated from the C_org-specific Mn quotas (micromoles Mn per moles C) of Sunda and Huntsman (1998; Fig. 7; Table II) and the C_org content of mid-log exponentially growing cells (0.89 pmol C_org cell⁻¹). Mn in PSII (Mn_PSII) is that modeled by Raven (1990). Mn in SOD was estimated as the average measured MnSOD concentration using the quantitative immunotechnique (moles of MnSOD per cell; Table II). Note that as C_org decreases in cells as light increases while moles of MnSOD per cell stays constant across light levels, the percent of Mn in MnSOD may increase with irradiance (see also Fig. 5). Error indicated is se (values are means, n = 2 ± se).

Figure 4.

Immunogold localization of MnSOD in T. pseudonana CCMP1335. A, Osmium tetroxide-stained electron micrograph of whole cell. B, A second different view of osmium tetroxide-stained cell. C, Immunogold labeling of the chloroplast with the anti-TpMnSOD antibody. D, Magnified view of delineated area in B. E, Immunogold labeling of the chloroplast in another cell of T. pseudonana. F, Magnified view of delineated area in E. Note the absence of labeling of mitochondrial and cytosolic regions. c, Chloroplast; p, pyrenoid; m, mitochondrion; n, nucleus; nc, nucleolus; v, vacuole. Arrows indicate black, electron-dense gold label corresponding to TpMnSOD. Faint and less electron-dense granules also apparent in the pyrenoid are crystalline formations of almost pure Rubisco (Falkowski and Raven, 2007). Scale bars are length as indicated.

Figure 5.

Comparison of growth rate, total cellular chlorophyll, and MnSOD per unit chlorophyll of T. pseudonana CCMP1335 cells grown at different continuous light intensities. Immunoblot images above the graph are of protein samples loaded according to equal chlorophyll concentrations. Growth rate (black circles and solid line) increases by 2-fold over these light levels. Concurrently, cellular chlorophyll (black squares, dotted line) decreases. Although MnSOD is constant per unit protein (data not shown), MnSOD per unit chlorophyll (black triangles, dashed and dotted line; western blot above image) increases. This supports the strong association of the relative contribution of MnSOD in the chloroplast to protecting the photosynthetic machinery, especially as the light-harvesting pigments decrease. Values are means, n = 2 ± sd.

Figure 6.

Diel expression of MnSOD in T. pseudonana CCMP1335. A, This figure demonstrates the quantum yield (F_v/F_m) of cells exposed to 12/12-h light/dark cycle under high light (800 μmol m⁻² s⁻¹, black triangles and dashed line) and control light (120 μmol m⁻² s⁻¹, black circles and solid line) over time (x axis). F_v/F_m decreases in the high light over the first 12 h when compared to the control and then recovered during and after the dark period. The dark period is represented by the shaded area. B, Immunoblot densitometric analysis shows significant recovery after the dark period of TpMnSOD in the high light (hatched bars) treatment as compared to the expression of TpMnSOD in the control light (solid bars) cultures (values are means, n = 2 ±sd). *, Significant differences between treatments (P = 0.0105).