Kathi Gundermann

The fluorescence yield of the trimeric fucoxanthin–chlorophyll–protein FCPa in the diatom Cyclotella meneghiniana is dependent on the amount of bound diatoxanthin

The fluorescence yield of isolated fucoxanthin chlorophyll proteins, serving as light harvesting proteins in diatoms, was compared to the amount of diatoxanthin bound. Diatoxanthin was earlier shown to be involved in the xanthophyll cycle in diatoms as a functional analogue of zeaxanthin in higher plants. By growing cells under different light conditions, the amount of diatoxanthin in both the trimeric FCPa as well as the oligomeric FCPb of the diatom Cyclotella meneghiniana was increased. In the trimeric FCPa, the fluorescence yield decreased with increasing diatoxanthin content, whereas in the oligomeric FCPb fluorescence was generally lower, albeit constant. No pH dependence of fluorescence yield could be demonstrated except for artificially aggregated FCPa. Thus, diatoxanthin is able to quench fluorescence in FCPa, but the yield is also influenced by pH when the protein becomes aggregated.

Oligomerization and pigmentation dependent excitation energy transfer in fucoxanthin-chlorophyll proteins.

The ultrafast carotenoid to chlorophyll a energy transfer dynamics of the isolated fucoxanthin-chlorophyll proteins FCPa and FCPb from the diatom Cyclotella meneghiniana was investigated in a comprehensive study using transient absorption in the visible and near infrared spectral region as well as static fluorescence spectroscopy. The altered oligomerization state of both antenna systems results in a more efficient energy transfer for FCPa, which is also reflected in the different chlorophyll a fluorescence quantum yields. We therefore assume an increased quenching in the higher oligomers of FCPb. The influence of the carotenoid composition was investigated using FCPa and FCPb samples grown under different light conditions and excitation wavelengths at the blue (500nm) and red (550nm) wings of the carotenoid absorption. The different light conditions yield in altered amounts of the xanthophyll cycle pigments diadinoxanthin and diatoxanthin. Since no significant dynamic changes are observed for high light and low light samples, the contribution of the xanthophyll cycle pigments to the energy transfer is most likely negligible. On the contrary, the observed dynamics change drastically for the different excitation wavelengths. The analyses of the decay associated spectra of FCPb suggest an altered energy transfer pathway. For FCPa even an additional time constant was found after excitation at 500nm. It is assigned to the intrinsic lifetime of either the xanthophyll cycle carotenoids or more probable the blue absorbing fucoxanthins. Based on our studies we propose a detailed model explaining the different excitation energy transfer pathways in FCPa.

Factors determining the fluorescence yield of fucoxanthin-chlorophyll complexes (FCP) involved in non-photochemical quenching in diatoms.

Fucoxanthin-chlorophyll complexes (FCP) from the centric diatom Cyclotella meneghiniana were isolated and the trimeric FCPa complex was reconstituted into liposomes at different lipid to Chl a ratios. The fluorescence yield of the complexes in different environments was calculated from room temperature fluorescence emission spectra and compared to the aggregated state of FCPa. FCPa surrounded by high amounts of lipids resembled detergent solubilised complexes and with decreasing lipid levels, i.e. in a situation where protein contacts were increasingly favoured, the fluorescence yield of FCPa gradually decreased. In addition, the yield displayed a strong pH-dependency in case of lower lipid contents. The further reduction in fluorescence yield brought about by the conversion of diadinoxanthin to diatoxanthin was pH independent and only depended on the amount of diatoxanthin synthesised. The implications of these data for non-photochemical quenching in centric diatoms are discussed.

Identification of several sub-populations in the pool of light harvesting proteins in the pennate diatom Phaeodactylum tricornutum.

Diatoms are major contributors to the photosynthetic productivity of marine phytoplankton. In these organisms, fucoxanthin-chlorophyll proteins (FCPs) serve as light-harvesting proteins. We have explored the FCP complexes in Phaeodactylum tricornutum under low light (LL) and high light (HL) conditions. Sub-fractionating the pool of major FCPs yielded different populations of trimeric complexes. Only Lhcf and Lhc-like proteins were found in the trimers. Under LL, the first polypeptide fraction contained six different Lhcfs and was mainly composed of Lhcf10. It was characterised by the highest amount of fucoxanthin (Fx). The second was dominated by Lhcf10, Lhcf5 and Lhcf2, and had a lower Fx/Chl c ratio. Little Fx/Chl c also characterised the most abundant FCP complexes, found in fraction 3, composed mainly of Lhcf5. These FCPs bound Fx molecules with the strongest bathochromic shift. The last two fractions contained FCP complexes that were built mainly of Lhcf4, harbouring more Fx molecules that absorbed at shorter wavelengths. Under HL, the same main polypeptides were retrieved in the different fractions and spectroscopic features were almost identical except for a higher diadinoxanthin content. The total amount of Lhcf5 was reduced under HL, whereas the amount of the last two fractions and thereby Lhcf4 was increased. Lhcf11 was identified in different LL fractions, but not detected in any HL fraction, while two new Lhc-like proteins were only found under HL. This is the first report on different trimeric FCP complexes in pennate diatoms, which differ in polypeptide composition and pigmentation, and are differentially expressed by light.

Probing the carotenoid content of intact Cyclotella cells by resonance Raman spectroscopy

Abstract In this study, we demonstrate the selective in vivo detection of diadinoxanthin (DD) and diatoxanthin (DT) in intact Cyclotella cells using resonance Raman spectroscopy. In these cells, we were able to assess both the content of DD and DT carotenoids relative to chlorophyll and their conformation. In addition, the sensitivity and selectivity of the technique allow us to discriminate between different pools of DD on a structural basis, and to follow their fate as a function of the illumination conditions. We report that the additional DD observed when cells are grown in high-light conditions adopts a more twisted conformation than the lower levels of DD present when the cells are grown in low-light (LL) conditions. Thus, we conclude that this pool of DD is more tightly bound to a protein-binding site, which must differ from the one occupied by the DD present in LL conditions.

Structure and Functional Heterogeneity of Fucoxanthin-Chlorophyll Proteins in Diatoms

Fucoxanthin-chlorophyll proteins (FCPs) of diatoms are divided into three groups, the main light harvesting antennas Lhcf, the photosystem I-specific Lhcr, and Lhcx involved in photoprotection. All are closely related to higher plant light harvesting complexes (LHCs) when comparing sequences, albeit smaller and more hydrophobic. However, pigmentation differs from higher plant LHCs with around eight chlorophyll a, two chlorophyll c and six fucoxanthin per monomer. Fucoxanthin, with a carbonyl moiety conjugated to the polyene backbone, undergoes extreme bathochromic shifts upon protein binding, dividing the different fucoxanthins into more red, green and blue absorbing ones. Excitation energy transfer is extremely efficient, either directly from chlorophyll c to chlorophyll a or from fucoxanthin to chlorophyll a involving the S1/ICT state of fucoxanthin. Most Lhcf assemble into trimers, whereby only in centric diatoms Lhcx was found in trimers as well, and specific oligomeric FCP complexes are present. Whereas the arrangement of FCPs around the photosystems is largely unknown, spectroscopic measurements together with homology considerations allow for a first rough model of the pigment arrangement in trimeric and oligomeric FCP complexes. Blue fucoxanthin is bound analogously to lutein in LHCII, surrounded by the same four chlorophyll a, since binding sites are conserved. Additionally, chlorophyll a can be found in a604, a614, b605 and a611, although binding of the latter has to be different due to the lack of long wavelength absorption in FCPs. Chlorophyll c is most probably bound in b609 and a613. The red fucoxanthin cluster around helix 2, which has less sequence homology to LHCII. The green fucoxanthins are most probably located around the violaxanthin and b601 binding sites of LHCII, whereby the former is probably a mixed site for fucoxanthins and diadinoxanthin/diatoxanthin.

Exploring the mechanism(s) of energy dissipation in the light harvesting complex of the photosynthetic algae Cyclotella meneghiniana

Photosynthetic organisms have developed vital strategies which allow them to switch from a light-harvesting to an energy dissipative state at the level of the antenna system in order to survive the detrimental effects of excess light illumination. These mechanisms are particularly relevant in diatoms, which grow in highly fluctuating light environments and thus require fast and strong response to changing light conditions. We performed transient absorption spectroscopy on FCPa, the main light-harvesting antenna from the diatom Cyclotella meneghiniana, in the unquenched and quenched state. Our results show that in quenched FCPa two quenching channels are active and are characterized by differing rate constants and distinct spectroscopic signatures. One channel is associated with a faster quenching rate (16ns⁻¹) and virtually no difference in spectral shape compared to the bulk unquenched chlorophylls, while a second channel is associated with a slower quenching rate (2.7ns⁻¹) and exhibits an increased population of red-emitting states. We discuss the origin of the two processes in the context of the models proposed for the regulation of photosynthetic light-harvesting. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Light Harvesting, Energy Transfer and Photoprotection in the Fucoxanthin-Chlorophyll Proteins of Cyclotella meneghiniana

The excitation energy transfer and the protective role of diadinoxanthin and diatoxanthin in two different fucoxanthin-chlorophyll-proteins have been investigated using femtosecond transient absorption spectroscopy.

Coherent Effects in the Carbonyl Containing Carotenoid Fucoxanthin

Coherent effects in the isolated carbonyl containing carotenoid fucoxanthin in various solvents and fucoxanthin within the fucoxanthin-chlorophyll protein were investigated using femtosecond transient absorption spectroscopy.