Pre-purification of diatom pigment protein complexes provides insight into the heterogeneity of FCP complexes

Abstract

Background: Although our knowledge about diatom photosynthesis has made huge progress over the last years, many aspects about their photosynthetic apparatus are still enigmatic. According to published data, the spatial organization as well as the biochemical composition of diatom thylakoid membranes is significantly different from that of higher plants.

Results: In this study the pigment protein complexes of the diatom Thalassiosira pseudonana were isolated by anion exchange chromatography. A step gradient was used for the elution process, yielding five well-separated pigment protein fractions which were characterized in detail. The isolation of photosystem (PS) core complex fractions, which contained fucoxanthin chlorophyll proteins (FCPs), enabled the differentiation between different FCP complexes: FCP complexes which were more closely associated with the PSI and PSII core complexes and FCP complexes which built-up the peripheral antenna. Analysis by mass spectrometry showed that the FCP complexes associated with the PSI and PSII core complexes contained various Lhcf proteins, including Lhcf1, Lhcf2, Lhcf4, Lhcf5, Lhcf6, Lhcf8 and Lhcf9 proteins, while the peripheral FCP complexes were exclusively composed of Lhcf8 and Lhcf9. Lhcr proteins, namely Lhcr1, Lhcr3 and Lhcr14, were identified in fractions containing subunits of the PSI core complex. Lhcx1, Lhcx2 and Lhcx5 proteins co-eluted with PSII protein subunits. The first fraction contained an additional Lhcx protein, Lhcx6_1, and was furthermore characterized by high concentrations of photoprotective xanthophyll cycle pigments.

Conclusion: The results of the present study corroborate existing data, like the observation of a PSI-specific antenna complex in diatoms composed of Lhcr proteins. They complement other data, like e.g. on the protein composition of the 21 kDa FCP band or the Lhcf composition of FCPa and FCPb complexes. They also provide interesting new information, like the presence of the enzyme diadinoxanthin de-epoxidase in the Lhcx-containing PSII fraction, which might be relevant for the process of non-photochemical quenching. Finally, the high negative charge of the main FCP fraction may play a role in the organization and structure of the native diatom thylakoid membrane. Thus, the results present an important contribution to our understanding of the complex nature of the diatom antenna system.

Keywords: Anion exchange chromatography; Fucoxanthin chlorophyll protein; Lhcx; Mass spectrometry; Photosystem I; Photosystem II.

Fig. 1

Elution profile of the pigment protein complexes of T. pseudonana separated by anion exchange chromatography (AEC). Figure 1 depicts the protein absorption at 280 nm and the stepwise increase of the KCl concentration. Before the separation, isolated thylakoids were solubilized with a β-DM per Chl ratio of 20. Solubilized thylakoids with a total amount of 200 to 500 μg Chl were loaded onto the AEC column. The numbers of the peaks denote the fractions that were collected and further characterized. Figure 1 shows a typical elution profile. For more information see the Methods section

Fig. 2

Absorption spectra of the different AEC fractions. The absorption spectra were normalized to the Q_Y band of Chl a. For the measurements the Chl concentration of the isolated pigment protein complexes was adjusted in such a way that the absorption in the blue part of the spectrum did not exceed absorption values of 1. Figure 2a shows the absorption spectrum in the wavelength range from 350 to 750 nm, Fig. 2b presents a detailed view of the red absorption maximum of Chl a. Figure 2 shows typical absorption spectra. For additional information see the Methods section

Fig. 3

77 K fluorescence spectra of the five AEC fractions. The spectra were normalized to the fluorescence emission maximum (Fig. 3a) or the excitation maximum of the Chl a fluorescence (Fig. 3b). For the 77 K fluorescence measurements the pigment protein complexes were adjusted to an optical density of 0.1 in the red part of the spectrum and then diluted with glycerol until a final glycerol concentration of 60% was obtained. Figure 3a shows the fluorescence emission spectra with a constant excitation at 440 nm, for the excitation spectra depicted in Fig. 3b the constant emission wavelength was set to the maximum of the emission spectrum. Fig. 3 shows typical emission and excitation spectra. For further details see the Methods section

Fig. 4

Pigment composition of the different AEC fractions and thylakoid membranes of T. pseudonana. The pigment composition is depicted as mM pigment M^− 1 Chl a. Figure 4 shows the mean values of three independent measurements with the respective standard deviations. For further information see the Methods section

Fig. 5

Representative gel image of the protein composition of the five AEC fractions determined by SDS-PAGE. Numbers 1 to 5 in Fig. 5 correspond to the respective fractions depicted in Fig. 1. Lanes 2 to 5 are derived from the original gel depicted in Additional file 7A, lane 1 is derived from the original gel shown in Additional file 7B. Proteins were stained with colloidal Coomassie Brilliant Blue. M denotes the molecular weight markers. For detailed information on the nature of the protein bands see section ‘Protein composition of the separated pigment protein complexes’. MS data for the 18 and 21 kDa FCP bands of lanes 2 to 5 (i.e. AEC fractions 2 to 5) are provided in Additional file 5. MS data for the complete analysis of photosynthetic proteins of fractions 1 to 4) can be found in Additional file 6