Towards time-resolved, coupled structure-function information on carotenoid excited state processes: X-ray and optical short-pulse double resonance spectroscopy

A novel soft X-ray and optical short-pulse double resonance spectroscopic technique tailor-made to elucidate processes involving the optically forbidden S1 (2(1)A(g)) state of carotenoids in native biological samples (e.g., photosynthetic antenna complexes) is described. The principle relies on probing the near carbon K-edge absorption of the optically excited sample with soft X-rays generated by a laser-induced plasma. A first application concerns location of the 2(1)A(g) state of beta-carotene in vitro. Further applications are proposed.     

Functions of the water soluble chlorophyll-binding protein in plants

Functional aspects of water soluble chlorophyll-binding protein (WSCP) in plants were investigated during the courses of leaf senescence, chlorophyll biogenesis, stress response and photoprotection. The cDNA sequence encoding WSCP from cauliflower was cloned into a binary vector to facilitate Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. The resultant transgenic tobacco plants overexpressed the CauWSCP gene under the control of a 35S-promoter. Analyses of protein and pigment contents indicate that WSCP overexpression does not enhance chlorophyll catabolism in vivo, thus rendering a role of WSCP in Chl degradation unlikely. Accumulation of higher levels of protochlorophyllide in WSCP overexpressor plants corroborates a proposed temporary storage and carrier function of WSCP for chlorophyll and late precursors. Although WSCP overexpressor plants did not show significant differences in non-photochemical quenching of chlorophyll fluorescence, they are characterized by significantly lower zeaxanthin accumulation and peroxidase activity at different light intensities, even at high light intensities of 700-900 μmol photons m⁻² s⁻¹. These results suggest a photoprotective function of the functional chlorophyll binding-WSCP tetramer by shielding of chlorophylls from molecular oxygen.     

Spectrally selective fluorescence imaging of Chlorobaculum tepidum reaction centers conjugated to chelator-modified silver nanowires

Photosynthesis Research - A polyhistidine tag (His-tag) present on Chlorobaculum tepidum reaction centers (RCs) was used to immobilize photosynthetic complexes on a silver nanowire (AgNW) modified...     

Channeling of eukaryotic diacylglycerol into the biosynthesis of plastidial phosphatidylglycerol

Plastidial glycolipids contain diacylglycerol (DAG) moieties, which are either synthesized in the plastids (prokaryotic lipids) or originate in the extraplastidial compartment (eukaryotic lipids) necessitating their transfer into plastids. In contrast, the only phospholipid in plastids, phosphatidylglycerol (PG), contains exclusively prokaryotic DAG backbones. PG contributes in several ways to the functions of chloroplasts, but it is not known to what extent its prokaryotic nature is required to fulfill these tasks. As a first step toward answering this question, we produced transgenic tobacco plants that contain eukaryotic PG in thylakoids. This was achieved by targeting a bacterial DAG kinase into chloroplasts in which the heterologous enzyme was also incorporated into the envelope fraction. From lipid analysis we conclude that the DAG kinase phosphorylated eukaryotic DAG forming phosphatidic acid, which was converted into PG. This resulted in PG with 2-3 times more eukaryotic than prokaryotic DAG backbones. In the newly formed PG the unique Delta3-trans-double bond, normally confined to 3-trans-hexadecenoic acid, was also found in sn-2-bound cis-unsaturated C18 fatty acids. In addition, a lipidomics technique allowed the characterization of phosphatidic acid, which is assumed to be derived from eukaryotic DAG precursors in the chloroplasts of the transgenic plants. The differences in lipid composition had only minor effects on measured functions of the photosynthetic apparatus, whereas the most obvious phenotype was a significant reduction in growth.     

Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in Photosystem II antenna size and stability

Xanthophylls (oxygen derivatives of carotenes) are essential components of the plant photosynthetic apparatus. Lutein, the most abundant xanthophyll, is attached primarily to the bulk antenna complex, light-harvesting complex (LHC) II. We have used mutations in Arabidopsis thaliana that selectively eliminate (and substitute) specific xanthophylls in order to study their function(s) in vivo. These include two lutein-deficient mutants, lut1 and lut2, the epoxy xanthophyll-deficient aba1 mutant and the lut2aba1 double mutant. Photosystem stoichiometry, antenna sizes and xanthophyll cycle activity have been related to alterations in nonphotochemical quenching of chlorophyll fluorescence (NPQ). Nondenaturing polyacrylamide gel electrophoresis indicates reduced stability of trimeric LHC II in the absence of lutein (and/or epoxy xanthophylls). Photosystem (antenna) size and stoichiometry is altered in all mutants relative to wild type (WT). Maximal ΔpH-dependent NPQ (qE) is reduced in the following order: WT>aba1>lut1≈lut2>lut2aba1, paralleling reduction in Photosystem (PS) II antenna size. Finally, light-activation of NPQ shows that zeaxanthin and antheraxanthin present constitutively in lut mutants are not qE active, and hence, the same can be inferred of the lutein they replace. Thus, a direct involvement of lutein in the mechanism of qE is unlikely. Rather, altered NPQ in xanthophyll biosynthetic mutants is explained by disturbed macro-organization of LHC II and reduced PS II-antenna size in the absence of the optimal, wild-type xanthophyll composition. These data suggest the evolutionary conservation of lutein content in plants was selected for due to its unique ability to optimize antenna structure, stability and macro-organization for efficient regulation of light-harvesting under natural environmental conditions.     

Femtosecond spectroscopy of native and carotenoidless purple-bacterial LH2 clarifies functions of carotenoids

EET between the two circular bacteriochlorophyll compartments B800 and B850 in native (containing the carotenoid rhodopin) and carotenoidless LH2 isolated from the photosynthetic purple sulfur bacterium Allochromatium minutissimum was investigated by femtosecond time-resolved transient absorption spectroscopy. Both samples were excited with 120-fs laser pulses at 800 nm, and spectral evolution was followed in the 720-955 nm range at different delay times. No dependence of transient absorption in the B800 band on the presence of the carotenoid rhodopin was found. Together with the likewise virtually unchanged absorption spectra in the bacteriochlorophyll Q_y region, these observations suggest that absence of rhodopin does not significantly alter the structure of the pigment-protein complex including interactions between bacteriochlorophylls. Apparently, rhodopin does also not accelerate B800 to B850 EET in LH2, contrary to what has been suggested previously. Moreover, “carotenoid-catalyzed internal conversion” can also be excluded for the bacteriochlorophylls in LH2 of A. minutissimum. Together with previous results obtained with two-photon fluorescence excitation spectroscopy, it can also be concluded that there is neither EET from rhodopin to B800 nor (back-)EET from B800 to rhodopin.     

Elucidation of structure-function relationships in photosynthetic light-harvesting antenna complexes by non-linear polarization spectroscopy in the frequency domain (NLPF)

Photosynthetically active pigments are usually organized into pigment-protein complexes. These include light-harvesting antenna complexes (LHCs) and reaction centers. Site energies of the bound pigments are determined by interactions with their environment, i.e., by pigment-protein as well as pigment-pigment interactions. Thus, resolution of spectral substructures of the pigment-protein complexes may provide valuable insight into structure-function relationships. By means of conventional (linear) and time-resolved spectroscopic techniques, however, it is often difficult to resolve the spectral substructures of complex pigment-protein assemblies. Nonlinear polarization spectroscopy in the frequency domain (NLPF) is shown to be a valuable technique in this regard. Based on initial experimental work with purple bacterial antenna complexes as well as model systems NLPF has been extended to analyse the substructure(s) of very complex spectra, including analyses of interactions between chlorophylls and "optically dark" states of carotenoids in LHCs. The paper reviews previous work and outlines perspectives regarding the application of NLPF spectroscopy to disentangle structure-function relationships in pigment-protein complexes.     

Evidence for a role of VIPP1 in the structural organization of the photosynthetic apparatus in Chlamydomonas

The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b(6)f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the Q(A)/Q(A)(-) redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids.     

Photophysical properties of Prochlorococcus marinus SS120 divinyl chlorophylls and phycoerythrin in vitro and in vivo

Prochlorococcus marinus SS120 is an ecologically important and biochemically intriguing marine cyanobacterium. In addition to divinyl chlorophylls (DV-Chls) a and b it possesses a particular form of phycoerythrin (PE), but no other phycobilins and therefore no complete phycobilisomes. Here, a spectroscopic characterisation of these DV-Chls and PE is provided. Comparison of fluorescence quantum yields, excited state lifetimes and absorption characteristics indicate similar light-harvesting properties of the DV-Chls as their monovinyl counterparts. PE, which is present only in tiny amounts, was purified and considerably enriched. A phycourobilin to phycoerythrobilin ratio of 3:1 chromophores per (alphabeta) PE monomer is suggested. The in vitro fluorescence lifetime of PE is 1.74 ns. In vivo time-resolved fluorescence measurements with synchrotron radiation were used to investigate the possible role of PE in light-harvesting. The fluorescence decay time for PE is about 550 ps, indicating an unusually slow excitation energy transfer. The decay time slowed to 1 ns after addition of glycerol to cell cultures. The contribution of PE to total light-harvesting capacity was estimated to be about one (alphabeta) PE monomer per 330 DV-Chl b molecules. Thus, the capacity of PE to function primarily as a photosynthetic light-harvesting pigment in P. marinus SS120 is low.