Size and Fluorescence Properties of Algal Photosynthetic Antenna Proteins Estimated by Microscopy.

Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more "quenched" than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.

Antenna Protein Clustering In Vitro Unveiled by Fluorescence Correlation Spectroscopy.

Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.

Energy transfer pathways in the CAC light-harvesting complex of Rhodomonas salina.

Photosynthetic organisms had to evolve diverse mechanisms of light-harvesting to supply photosynthetic apparatus with enough energy. Cryptophytes represent one of the groups of photosynthetic organisms combining external and internal antenna systems. They contain one type of immobile phycobiliprotein located at the lumenal side of the thylakoid membrane, together with membrane-bound chlorophyll a/c antenna (CAC). Here we employ femtosecond transient absorption spectroscopy to study energy transfer pathways in the CAC proteins of cryptophyte Rhodomonas salina. The major CAC carotenoid, alloxanthin, is a cryptophyte-specific carotenoid, and it is the only naturally-occurring carotenoid with two triple bonds in its structure. In order to explore the energy transfer pathways within the CAC complex, three excitation wavelengths (505, 590, and 640 nm) were chosen to excite pigments in the CAC antenna. The excitation of Chl c at either 590 or 640 nm proves efficient energy transfer between Chl c and Chl a. The excitation of alloxanthin at 505 nm shows an active pathway from the S2 state with efficiency around 50%, feeding both Chl a and Chl c with approximately 1:1 branching ratio, yet, the S1-route is rather inefficient. The 57 ps energy transfer time to Chl a gives ~25% efficiency of the S1 channel. The low efficiency of the S1 route renders the overall carotenoid-Chl energy transfer efficiency low, pointing to the regulatory role of alloxanthin in the CAC antenna.

Modeling Dynamic Conformations of Organic Molecules: Alkyne Carotenoids in Solution.

Calculating the spectroscopic properties of complex conjugated organic molecules in their relaxed state is far from simple. An additional complexity arises for flexible molecules in solution, where the rotational energy barriers are low enough so that nonminimum conformations may become dynamically populated. These metastable conformations quickly relax during the minimization procedures preliminary to density functional theory calculations, and so accounting for their contribution to the experimentally observed properties is problematic. We describe a strategy for stabilizing these nonminimum conformations in silico, allowing their properties to be calculated. Diadinoxanthin and alloxanthin present atypical vibrational properties in solution, indicating the presence of several conformations. Performing energy calculations in vacuo and polarizable continuum model calculations in different solvents, we found three different conformations with values for the δ dihedral angle of the end ring ca. 0, 180, and 90° with respect to the plane of the conjugated chain. The latter conformation, a nonglobal minimum, is not stable during the minimization necessary for modeling its spectroscopic properties. To circumvent this classical problem, we used a Car-Parinello MD supermolecular approach, in which diadinoxanthin was solvated by water molecules so that metastable conformations were stabilized by hydrogen-bonding interactions. We progressively removed the number of solvating waters to find the minimum required for this stabilization. This strategy represents the first modeling of a carotenoid in a distorted conformation and provides an accurate interpretation of the experimental data.

Photoprotective strategies in the motile cryptophyte alga Rhodomonas salina-role of non-photochemical quenching, ions, photoinhibition, and cell motility.

We explored photoprotective strategies in a cryptophyte alga Rhodomonas salina. This cryptophytic alga represents phototrophs where chlorophyll a/c antennas in thylakoids are combined with additional light-harvesting system formed by phycobiliproteins in the chloroplast lumen. The fastest response to excessive irradiation is induction of non-photochemical quenching (NPQ). The maximal NPQ appears already after 20 s of excessive irradiation. This initial phase of NPQ is sensitive to Ca2+ channel inhibitor (diltiazem) and disappears, also, in the presence of non-actin, an ionophore for monovalent cations. The prolonged exposure to high light of R. salina cells causes photoinhibition of photosystem II (PSII) that can be further enhanced when Ca2+ fluxes are inhibited by diltiazem. The light-induced reduction in PSII photochemical activity is smaller when compared with immotile diatom Phaeodactylum tricornutum. We explain this as a result of their different photoprotective strategies. Besides the protective role of NPQ, the motile R. salina also minimizes high light exposure by increased cell velocity by almost 25% percent (25% from 82 to 104 µm/s). We suggest that motility of algal cells might have a photoprotective role at high light because algal cell rotation around longitudinal axes changes continual irradiation to periodically fluctuating light.

Isolation and characterization of CAC antenna proteins and photosystem I supercomplex from the cryptophytic alga Rhodomonas salina.

In the present paper, we report an improved method combining sucrose density gradient with ion-exchange chromatography for the isolation of pure chlorophyll a/c antenna proteins from the model cryptophytic alga Rhodomonas salina. Antennas were used for in vitro quenching experiments in the absence of xanthophylls, showing that protein aggregation is a plausible mechanism behind non-photochemical quenching in R. salina. From sucrose gradient, it was also possible to purify a functional photosystem I supercomplex, which was in turn characterized by steady-state and time-resolved fluorescence spectroscopy. R. salina photosystem I showed a remarkably fast photochemical trapping rate, similar to what recently reported for other red clade algae such as Chromera velia and Phaeodactylum tricornutum. The method reported therefore may also be suitable for other still partially unexplored algae, such as cryptophytes.

Antenna proton sensitivity determines photosynthetic light harvesting strategy.

Photoprotective non-photochemical quenching (NPQ) represents an effective way to dissipate the light energy absorbed in excess by most phototrophs. It is often claimed that NPQ formation/relaxation kinetics are determined by xanthophyll composition. We, however, found that, for the alveolate alga Chromera velia, this is not the case. In the present paper, we investigated the reasons for the constitutive high rate of quenching displayed by the alga by comparing its light harvesting strategies with those of a model phototroph, the land plant Spinacia oleracea. Experimental results and in silico studies support the idea that fast quenching is due not to xanthophylls, but to intrinsic properties of the Chromera light harvesting complex (CLH) protein, related to amino acid composition and protein folding. The pKa for CLH quenching was shifted by 0.5 units to a higher pH compared with higher plant antennas (light harvesting complex II; LHCII). We conclude that, whilst higher plant LHCIIs are better suited for light harvesting, CLHs are 'natural quenchers' ready to switch into a dissipative state. We propose that organisms with antenna proteins intrinsically more sensitive to protons, such as C. velia, carry a relatively high concentration of violaxanthin to improve their light harvesting. In contrast, higher plants need less violaxanthin per chlorophyll because LHCII proteins are more efficient light harvesters and instead require co-factors such as zeaxanthin and PsbS to accelerate and enhance quenching.

High light acclimation of Chromera velia points to photoprotective NPQ.

It has previously been shown that the long-term treatment of Arabidopsis thaliana with the chloroplast inhibitor lincomycin leads to photosynthetic membranes enriched in antennas, strongly reduced in photosystem II reaction centers (PSII) and with enhanced nonphotochemical quenching (NPQ) (Belgio et al. Biophys J 102:2761-2771, 2012). Here, a similar physiological response was found in the microalga Chromera velia grown under high light (HL). In comparison to cells acclimated to low light, HL cells displayed a severe re-organization of the photosynthetic membrane characterized by (1) a reduction of PSII but similar antenna content; (2) partial uncoupling of antennas from PSII; (3) enhanced NPQ. The decrease in the number of PSII represents a rather unusual acclimation response compared to other phototrophs, where a smaller PSII antenna size is more commonly found under high light. Despite the diminished PSII content, no net damage could be detected on the basis of the Photosynthesis versus irradiance curve and electron transport rates pointing at the excess capacity of PSII. We therefore concluded that the photoinhibition is minimized under high light by a lower PSII content and that cells are protected by NPQ in the antennas.

Extensive gain and loss of photosystem I subunits in chromerid algae, photosynthetic relatives of apicomplexans.

In oxygenic photosynthesis the initial photochemical processes are carried out by photosystem I (PSI) and II (PSII). Although subunit composition varies between cyanobacterial and plastid photosystems, the core structures of PSI and PSII are conserved throughout photosynthetic eukaryotes. So far, the photosynthetic complexes have been characterised in only a small number of organisms. We performed in silico and biochemical studies to explore the organization and evolution of the photosynthetic apparatus in the chromerids Chromera velia and Vitrella brassicaformis, autotrophic relatives of apicomplexans. We catalogued the presence and location of genes coding for conserved subunits of the photosystems as well as cytochrome b6f and ATP synthase in chromerids and other phototrophs and performed a phylogenetic analysis. We then characterised the photosynthetic complexes of Chromera and Vitrella using 2D gels combined with mass-spectrometry and further analysed the purified Chromera PSI. Our data suggest that the photosynthetic apparatus of chromerids underwent unique structural changes. Both photosystems (as well as cytochrome b6f and ATP synthase) lost several canonical subunits, while PSI gained one superoxide dismutase (Vitrella) or two superoxide dismutases and several unknown proteins (Chromera) as new regular subunits. We discuss these results in light of the extraordinarily efficient photosynthetic processes described in Chromera.

High photochemical trapping efficiency in Photosystem I from the red clade algae Chromera velia and Phaeodactylum tricornutum.

In the present work, we report the first comparative spectroscopic investigation between Photosystem I (PSI) complexes isolated from two red clade algae. Excitation energy transfer was measured in PSI from Chromera velia, an alga possessing a split PsaA protein, and from the model diatom Phaeodactylum tricornutum. In both cases, the estimated effective photochemical trapping time was in the 15-25ps range, i.e. twice as fast as higher plants. In contrast to green phototrophs, the trapping time was rather constant across the whole emission spectrum. The weak wavelength dependence was attributed to the limited presence of long-wavelength emitting chlorophylls, as verified by low temperature spectroscopy. As the trapping kinetics of C. velia PSI were barely distinguishable from those of P. tricornutum PSI, it was concluded that the scission of PsaA protein had no significant impact on the overall PSI functionality. In conclusion, the two red clade algae analysed here, carried amongst the most efficient charge separation so far reported for isolated Photosystems.

Violaxanthin inhibits nonphotochemical quenching in light-harvesting antenna of Chromera velia.

Non-photochemical quenching (NPQ) is a photoprotective mechanism in light-harvesting antennae. NPQ is triggered by chloroplast thylakoid lumen acidification and is accompanied by violaxanthin de-epoxidation to zeaxanthin, which further stimulates NPQ. In the present study, we show that violaxanthin can act in the opposite direction to zeaxanthin because an increase in the concentration of violaxanthin reduced NPQ in the light-harvesting antennae of Chromera velia. The correlation overlapped with a similar relationship between violaxanthin and NPQ as observed in isolated higher plant light-harvesting complex II. The data suggest that violaxanthin in C. velia can act as an inhibitor of NPQ, indicating that violaxanthin has to be removed from the vicinity of the protein to reach maximal NPQ.