Iron Deficiency Promotes the Lack of Photosynthetic Cytochrome c550 and Affects the Binding of the Luminal Extrinsic Subunits to Photosystem II in the Diatom Phaeodactylum tricornutum

Abstract

In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced Q_B and the S₂/S₃ states of the water-splitting machinery. However, electron transfer from Q_A to Q_B, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c₅₅₀ subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c₅₅₀ under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c₅₅₀, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.

Keywords: Phaeodactylum tricornutum; PsbV; cytochrome c550; electron transfer; iron limitation; photosystem II; pulse-amplitude modulation chlorophyll fluorescence; thermoluminescence.

Figure 1

(A) Growth curves for P. tricornutum cultures under different iron concentrations. Mean values ± SD of measurements from 10 independent cultures are presented. (B,D) Representative Western-blots of different photosynthetic proteins in samples of whole cells (B) and membrane extracts (D) from P. tricornutum grown under iron-replete (10 µM iron) and iron-deficient (0.1 or 0.04 µM iron) conditions. MW: molecular weight standard; Cc₅₅₀ and PsbO, the extrinsic cytochrome c₅₅₀ and PsbO subunits of PSII; D1, the intrinsic D1 subunit of PSII; RbcL, control with the RuBisCO large subunit. Numbers on the left are the MW values corresponding to the molecular weight standard. Protein extracts: 20 µg for RuBisCO large subunit; 10 µg for PsbO and D1; 2.5 µg for Cc₅₅₀. Membranes were incubated overnight with the following dilutions of the selected primary antibody: 1:1000 anti-Cc₅₅₀ and anti-RuBisCO large; 1:2000 anti-PsbO; 1:10,000 anti-D1. The complete Western-blots are shown in Figure S1 (Supplementary Material). (C) Intracellular concentrations of cytochrome c₆ (Cc₆) and Cc₅₅₀ measured by differential absorbance changes of P. tricornutum cells grown under different iron concentrations. Data represent mean values ± SD of at least four independent measurements. Double asterisks mark statistically different data groups (p < 0.01); ns = not statistically different.

Figure 2

Effect of iron deficiency on the PSII activity of P. tricornutum cells measured by the TL technique. (A) Representative standard TL glow curves, obtained after excitation of P. tricornutum cells with two flashes at 1 °C under different iron concentrations. Inset: TL glow curve of P. tricornutum cells in the presence of 10 or 0.04 µM iron and 20 µM DCMU. (B) Intensities obtained from the component analysis of TL curves from six independent experiments (see also Table 1). Double asterisks mark statistically different data groups (p < 0.01). See the Methods and Materials section for more details.

Figure 3

Photosystem II core of the diatom C. gracilis, PDB code, 6jlu [17]. (A) Membrane lateral view; (B) bottom luminal view (90° rotation). The main extrinsic subunits are highlighted: PsbO (blue), PsbV/cytochrome c₅₅₀ (red), PsbU (green), PsbQ′ (purple). The Figure was generated with the UCSF Chimera program (version 1.16; http://www.rbvi.ucsf.edu/chimera/).