Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae

Abstract

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1-100 nM were rapidly iron-depleted when ferric citrate-but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.

Fig. 1

Iron-dependent growth (a) and iron-dependent chlorophyll fluorescence (b) of O. tauri. The cells were precultured for one week in iron-free medium and then inoculated in media containing 0–10 μM ferric citrate (circles) or 0–10 μM ferric EDTA (squares). Selected curves are shown for the following concentrations of iron added to the media: 0 (empty symbols), 10 nM (grey symbols) and 100 nM (black symbols). Values obtained for other iron concentration are presented in Table S1. The cells were grown under a 12:12 light–dark regime, and the number and fluorescence of cells were measured everyday by flow cytometry in the middle of the day. Fluorescence was recorded at ≥670 nm (emission) with excitation at 488 nm (FL3). Data represents mean results from three experiments. Error bars are not shown for the sake of clarity, but SE values were ≤ 9 % for Fig. 1a and ≤ 11 % for Fig. 1b. Full data with SE values are presented in Table S1

Fig. 2

Effect of the concentration and source of iron on the growth of O.tauri, E. huxleyi and P. tricornutum (inter-specific competition). Cells of each species were grown separately for 1 week in iron-free medium and then inoculated together in media containing no iron (Fe 0) or different concentrations (1 nM–10 μM) of ferric citrate (C-XnM) or ferric EDTA (E-XnM). The number of cells of each species in the inoculum was inversely proportional to the estimated value of the cell surface. The cells were grown under a 12:12 light–dark regime, and the number of cells of each species was measured everyday by flow cytometry in the middle of the day. O. tauri: triangles; E. huxleyi: squares; P. tricornutum: circles. Data are from one representative experiment out of two independent experiments. Other conditions of growth are presented in Fig. S3

Fig. 3

Iron uptake from ferric citrate (closed symbols) or ferric EDTA (open symbols) during growth of O. tauri (triangles), E. huxleyi (squares) and P. tricornutum (circles). The cells of each species were precultured for 1 week in iron-free medium and then inoculated at 15 million cells/ml (O. tauri) or 1 million cells/ml (E. huxleyi and P. tricornutum) in a medium containing 0.1 μM ⁵⁵Fe-labeled ferric citrate or ferric EDTA (1:20). The cells were grown under a 12:12 light–dark regime. Aliquots of cells were harvested at different points in time during growth, washed three times with a buffer containing strong iron chelators, and the amount of cell-associated iron was determined by liquid scintillation. Results are expressed in p mol/million cells (E. huxleyi and P. tricornutum) or in p mol/10 million cells (O. tauri). Mean±SE from three experiments

Fig. 4

Regulation of iron uptake according to the day/night cycles. O. tauri (a) and E. huxleyi (b) cells were grown for 5 d under standard conditions (Mf medium + 0.1 μM ferric citrate) and a 12:12 light–dark regime in two growing chambers programmed in opposition of phase (day started at 10 a.m. in one chamber while night started at 10 a.m. in the second chamber). When the cells were in exponential growth phase, 50 ml of the cultures in both chambers were harvested every 3 h. The cells were washed once with iron-free medium, re-suspended in 1 ml of the same medium and distributed in two micro-centrifuge tubes (2 × 500 μl). ⁵⁵Fe (1 μM) was added as ferric citrate (1:20) (closed circles) in one tube and as ferrous ascorbate (1:100) (open circles) in the second tube. After 15 min incubation at 20 °C in the light, the cells were washed three times by centrifugation with the washing buffer containing strong iron chelators. Iron associated to the cells was counted by liquid scintillation. White parts of the graph shows the iron uptake rates during the day and dark parts of the graphs show the iron uptake rates during the night. Mean±SE from three experiments

Fig. 5

Copper-dependence of iron uptake. O. tauri (a) and E. huxleyi (b) cells were grown for 3 d under a 12:12 light–dark regime in Mf medium containing 0.1 μM ferric citrate and either 0.1 μM CuSO₄ (closed symbols) or 0.1 mM of the copper-chelating agent, bathocuproin sulfonate (open symbols). Cells were harvested 2 h after dawn, washed once with iron-free and copper-free Mf medium, and tested for iron uptake from 1 μM ferrous ascorbate (circles) or 1 μM ferric citrate (squares) in microtiter plates (see Sect. 2). Mean±SE from three experiments

Fig. 6

Autoradiography of dried gels after separation of whole cell extracts on blue native PAGE. O. tauri and E. huxleyi cells were grown for 5 d under standard conditions (Mf medium+0.1 μM ferric citrate) and a 12:12 light–dark regime. Cells in exponential growth phase were harvested in the middle of the day (“Day”) or in the middle of the night (“Night”), washed once by centrifugation with iron-free Mf medium and incubated in the same medium for 2.5 h (E. huxleyi) or 1.5 h (O. tauri) in the light at 20 °C with either 2 μM ⁵⁵ferrous ascorbate (1:100; “A”), 2 μM ⁵⁵ferric citrate (1:20; “C”) or 2 μM ⁵⁵ferric EDTA (1:20; “E”). Cells were then washed once by centrifugation with iron-free Mf medium (E. huxleyi) or (O. tauri) with a medium containing strong iron chelators (see Sect. 2), and whole cell extracts were prepared by sonication. After native PAGE (about 25 μg protein per lane), the gels were dried and autoradiographed