Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms

Abstract

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls.

Figure 1.

Separation of the pigment protein complexes of C. meneghiniana by Suc density gradient centrifugation. A and B, LL pigment protein complexes. C and D, HL pigment protein complexes. A and C show the typical separation pattern when a low DM per Chl ratio of 10 was used. Higher detergent per Chl ratios (DM to Chl ratio of 20 or above) led to a dissociation of the oligomeric FCP complex into two distinct bands, FCPa and FCPb (B and D). [See online article for color version of this figure.]

Figure 2.

A, C, and E, Absorption spectra of thylakoid membranes (A), FCP (C), and PSI (E) complexes isolated from LL- and HL-grown C. meneghiniana. The FCP fractions were isolated after solubilization of thylakoids with a DM to Chl ratio of 10, and the PSI fractions were isolated after solubilization with a DM to Chl ratio of 10 and 40. Spectra are averages of at least four different samples. Note that the spectra were normalized to the Q_Y band of Chl a. B and D, Difference absorption spectra of HL minus LL thylakoids (B) and HL minus LL FCPs (D). The wavelength of the third maximum is indicated.

Figure 3.

Comparison of the absorption and the 77 K fluorescence excitation spectra of thylakoids (A), FCP (B), PSII (C), and PSI (D) from HL-cultivated C. meneghiniana. In B, the 77 K fluorescence excitation spectrum of the LL FCP is additionally presented. The inset shows the difference fluorescence excitation spectrum of the HL minus LL FCP in the range of 450 to 550 nm. For the fluorescence excitation spectrum, the fluorescence emission was set to the emission maximum of the Chl a fluorescence, which was located at 686 nm in the thylakoids, at 683 nm in the FCP, at 687 nm in PSII, and at 715 nm in PSI. Spectra are averages of at least four different samples.

Figure 4.

Comparison between the difference absorption spectrum of HL FCP complexes minus LL FCP complexes and the absorption spectrum of purified Ddx dissolved in MGDG. For the MGDG experiments, a ratio of MGDG to Ddx of 7.25 was chosen. The Ddx concentration in the MGDG experiment was 0.4 μm. Spectra were normalized to the third absorption peak.

Figure 5.

Comparison of the difference absorption spectrum of LL-adapted HL cells (DES 0) minus cells that were directly taken from the growth light of 160 to 180 μmol m⁻² s⁻¹ (DES 0.3) with the difference absorption spectrum of LL PSI (DES 0) minus HL PSI complexes (DES 0.3). Additionally, the difference absorption spectrum of Ddx dissolved in MGDG (DES 0) minus Ddx and Dtx dissolved in MGDG (DES 0.3) is depicted. For the MGDG experiments, Ddx or Ddx/Dtx concentrations of 0.4 μm were used. The MGDG per Ddx (Ddx/Dtx) ratio was 7.25. The wavelengths of the respective characteristic Ddx absorption maxima are depicted.

Figure 6.

A, Lipid composition of thylakoids and FCP complexes isolated from LL- or HL-grown C. meneghiniana cultures. FCP complexes were isolated with the different DM per Chl ratios of 10, 40, and 80. For separation by high-performance thin-layer chromatography, samples corresponding to a Chl a content of 2 μg were subjected to the plates. MGDG was separated with an eluent according to Ventrella et al. (2007), while the other lipids were separated with the eluent reported by Mock and Kroon (2002). B, Difference absorption spectra of HL FCP complexes minus LL FCP complexes. The FCP complexes were isolated with the different DM per Chl ratios mentioned above.

Figure 7.

TL glow curves of HL thylakoids from P. tricornutum that contained low concentrations (Ddx enriched, 130 mmol Dtx mol⁻¹ Chl a) or high concentrations (Dtx enriched, 710 mmol Dtx mol⁻¹ Chl a) of Dtx. The thylakoids were illuminated with 5,000 μmol m⁻² s⁻¹ PAR before the TL measurements were started. The TL glow curves represent average values of four independent measurements with three repetitions each. A Student’s t test calculation for the difference between means based on the maxima at 130°C revealed a statistical significance of P < 0.001. For further details, see “Materials and Methods” and “Results.”