Functional characterisation of different fucoxanthin chlorophyll proteins (FCP) in diatoms
Zusammenfassung der Projektergebnisse
Diatoms are unicellular, photosynthetic organisms which play a major role in the marine ecosystems. Despite their importance, knowledge about their photosynthetic apparatus is scarce compared to e.g. higher plants. Like all photosynthetic organisms, diatoms possess light-harvesting or antenna complexes, proteins which carry the pigments involved in the collection of light energy. Three different groups of FCP polypeptides (Lhcf – main antenna proteins, Lhcr – photosystem I related proteins, Lhcx – related to protective proteins in green algae) can be distinguished on the level of genes. Despite this, usually only one or two main antenna complexes are purified. Thus, little was known about possible functional heterogeneities of these different polypeptides. In order to characterise single antenna proteins we transformed diatoms with different FCP genes modified by addition of tags in order to be able to separate specific FCPs against the background of all related proteins. Another approach included the improvement of biochemical preparation methods from wild type cells. Using these methods, a Fcp specifically serving photosystem I could be identified for the first time (Fcp4 from Cyclotella meneghiniana). In Phaeodactylum tricornutum tagging of FcpA (lhcf group) was used to identify this protein as part of the photosystem I antenna as well. In addition, this polypeptide is specifically associated in trimers with FcpE (Lhcf group), the most abundant Fcp polypeptide in P. tricornutum. FcpE forms trimers with several partners and if down-regulated, it is replaced in some of the trimers but not in the trimers with FcpB (Lhcf group), which constitute the main trimers in wild type. No specific photosystem II antenna proteins could be identified so far even by mass spectrometry, and results concerning different His-tagged lhcx proteins are still ambiguous. Trimeric FCPa complexes in C. meneghininana are built from Fcp6 (lhcx) and Fcp2 (lhcf). Oligomeric complexes (FCPb) of Fcp5 polypeptides (lhcf) are also abundant. Both these complexes were intensively characterised concerning excitation energy transfer between the pigments bound. Two differently bound subpopulations of fucoxanthin could be distinguished, absorbing more to the blue or the red, respectively. Both can be distinguished not only by their absorption, but also by the rate constants of transfer of energy to Chl a. The spectroscopic analyses by transient absorbance, absorbance anisotropy and resonance raman allowed to determine the precise stoichiometry of pigments per polypeptide and to create a model of the arrangement of the pigments. The FCPa trimers, which contain one of the lhcx proteins (Fcp6), are of special importance for the protection of the cells against high light intensities. We could demonstrate that the fluorescence yield of these trimers depends on all factors which trigger non-photochemical quenching (NPQ), a protection mechanism increasing heat dissipation compared to fluorescence. Diatoxanthin is one of the factors reducing the yield. Second, aggregation was demonstrated to be an important mechanism in NPQ, whereby aggregation of FCPa is enhanced when pH is lowered. This last finding explains for the first time on molecular level the fast transient, pH dependent NPQ seen in diatoms cells. Diatoxanthin then takes an additional role in quenching independent of the aggregation state of FCPa.
Projektbezogene Publikationen (Auswahl)
- Characterisation of a trimeric light-harvesting complex in the diatom Phaeodactylum tricornutum built of FcpA and FcpE proteins, J. Exp. Bot. 61(11): 3079-3087 (2010)
Joshi-Deo J, Schmidt M, Gruber A, Weisheit W, Mittag M, Kroth PG, Büchel C
- Oligomerization and pigmentation dependent excitation energy transfer in fucoxanthin-chlorophyll proteins, BBA-Bioenergetics 1797(5): 543-549 (2010)
Gildenhoff N, Amarie S, Gundermann K, Beer A, Büchel C, Wachtveitl J
- Pigment organization in fucoxanthin chlorophyll a/c2 proteins (FCP) based on resonance Raman spectroscopy and sequence analysis, BBA-Bioenergetics 1797(9): 1647-1656 (2010)
Premvardhan L, Robert B, Beer A, Büchel C
- The excitation energy transfer in the trimeric fucoxanthin-chlorophyll protein from Cyclotella meneghiniana analyzed by polarized transient absorption spectroscopy. Chem. Phys. 373(1-2): 104-109 (2010)
Gildenhoff N, Herz J, Gundermann K, Büchel C, Wachtveitl J
