Opposite Charge Movements Within the Photoactive Site Modulate Two-Step Channel Closing in GtACR1.

Guillardia theta anion channelrhodopsin 1 is a light-gated anion channel widely used as an optogenetic inhibitory tool. Our recently published crystal structure of its dark (closed) state revealed that the photoactive retinylidene chromophore is located midmembrane in a full-length intramolecular tunnel through the protein, the radius of which is less than that of a chloride ion. Here we show that acidic (glutamate) substitutions for residues within the inner half-tunnel enhance the fast channel closing and, for residues within the outer half-tunnel, enhance the slow channel closing. The magnitude of these effects was proportional to the distance of the mutated residue from the photoactive site. These data indicate that the local electrical field across the photoactive site controls fast and slow channel closing, involving outward and inward charge displacements. In the purified mutant proteins, we observed corresponding opposite changes in kinetics of the M photocycle intermediate. A correlation between fast closing and M rise and slow closing and M decay observed in the mutants suggests that the Schiff base proton is one of the displaced charges. Opposite signs of the effects indicate that deprotonation and reprotonation of the Schiff base take place on the same (outer) side of the membrane and explains opposite rectification of fast and slow channel closing. Оur comprehensive protein-wide acidic residue substitution screen shows that only mutations of the residues located in the intramolecular tunnel confer strong rectification, which confirms the prediction that the tunnel expands upon photoexcitation to form the anion pathway.

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.

Proteomic analysis of the phycobiliprotein antenna of the cryptophyte alga Guillardia theta cultured under different light intensities.

Plants and algae have developed various light-harvesting mechanisms for optimal delivery of excitation energy to the photosystems. Cryptophyte algae have evolved a novel soluble light-harvesting antenna utilizing phycobilin pigments to complement the membrane-intrinsic Chl a/c-binding LHC antenna. This new antenna consists of the plastid-encoded ß-subunit, a relic of the ancestral phycobilisome, and a novel nuclear-encoded α-subunit unique to cryptophytes. Together, these proteins form the active α1ß·α2ß-tetramer. In all cryptophyte algae investigated so far, the α-subunits have duplicated and diversified into a large gene family. Although there is transcriptional evidence for expression of all these genes, the X-ray structures determined to date suggest that only two of the α-subunit genes might be significantly expressed at the protein level. Using proteomics, we show that in phycoerythrin 545 (PE545) of Guillardia theta, the only cryptophyte with a sequenced genome, all 20 α-subunits are expressed when the algae grow under white light. The expression level of each protein depends on the intensity of the growth light, but there is no evidence for a specific light-dependent regulation of individual members of the α-subunit family under the growth conditions applied. GtcpeA10 seems to be a special member of the α-subunit family, because it consists of two similar N- and C-terminal domains, which likely are the result of a partial tandem gene duplication. The proteomics data of this study have been deposited to the ProteomeXchange Consortium and have the dataset identifiers PXD006301 and 10.6019/PXD006301.

A dinoflagellate Amylax triacantha with plastids of the cryptophyte origin: phylogeny, feeding mechanism, and growth and grazing responses.

The gonyaulacalean dinoflagellates Amylax spp. were recently found to contain plastids of the cryptophyte origin, more specifically of Teleaulax amphioxeia. However, not only how the dinoflagellates get the plastids of the cryptophyte origin is unknown but also their ecophysiology, including growth and feeding responses as functions of both light and prey concentration, remain unknown. Here, we report the establishment of Amylax triacantha in culture, its feeding mechanism, and its growth rate using the ciliate prey Mesodinium rubrum (= Myrionecta rubra) in light and dark, and growth and grazing responses to prey concentration and light intensity. The strain established in culture in this study was assigned to A. triacantha, based on morphological characteristics (particularly, a prominent apical horn and three antapical spines) and nuclear SSU and LSU rDNA sequences. Amylax triacantha grew well in laboratory culture when supplied with the marine mixotrophic ciliate M. rubrum as prey, reaching densities of over 7.5 × 10(3)  cells/ml. Amylax triacantha captured its prey using a tow filament, and then ingested the whole prey by direct engulfment through the sulcus. The dinoflagellate was able to grow heterotrophically in the dark, but the growth rate was approximately two times lower than in the light. Although mixotrophic growth rates of A. triacantha increased sharply with mean prey concentrations, with maximum growth rate being 0.68/d, phototrophic growth (i.e. growth in the absence of prey) was -0.08/d. The maximum ingestion rate was 2.54 ng C/Amylax/d (5.9 cells/Amylax/d). Growth rate also increased with increasing light intensity, but the effect was evident only when prey was supplied. Increased growth with increasing light intensity was accompanied by a corresponding increase in ingestion. In mixed cultures of two predators, A. triacantha and Dinophysis acuminata, with M. rubrum as prey, A. triacantha outgrew D. acuminata due to its approximately three times higher growth rate, suggesting that it can outcompete D. acuminata. Our results would help better understand the ecophysiology of dinoflagellates retaining foreign plastids.

Comparison of photoacclimation in twelve freshwater photoautotrophs (chlorophyte, bacillaryophyte, cryptophyte and cyanophyte) isolated from a natural community.

Different representative of algae and cyanobacteria were isolated from a freshwater habitat and cultivated in laboratory to compare their photoacclimation capacity when exposed to a wide range of light intensity and to understand if this factor may modify natural community dominance. All species successfully acclimated to all light intensities and the response of phytoplankton to increased light intensity was similar and included a decrease of most photosynthetic pigments accompanied by an increase in photoprotective pigment content relative to Chl a. Most species also decreased their light absorption efficiency on a biovolume basis. This decrease not only resulted in a lower fraction of energy absorbed by the cell, but also to a lower transfer of energy to PSII and PSI. Furthermore, energy funnelled to PSII or PSI was also rearranged in favour of PSII. High light acclimated organisms also corresponded to high non-photochemical quenching and photosynthetic electron transport reduction state and to a low Φ'M. Thus photoacclimation processes work toward reducing the excitation pressure in high light environment through a reduction of light absorption efficiency, but also by lowering conversion efficiency. Interestingly, all species of our study followed that tendency despite being of different functional groups (colonial, flagellated, different sizes) and of different phylogeny demonstrating the great plasticity and adaptation ability of freshwater phytoplankton to their light environment. These adjustments may explain the decoupling between growth rate and photosynthesis observed above photosynthesis light saturation point for all species. Even if some species did reach higher growth rate in our conditions and thus, should dominate in natural environment with respect to light intensity, we cannot exclude that other environmental factors also influence the population dynamic and make the outcome harder to predict.

Non-photochemical quenching in cryptophyte alga Rhodomonas salina is located in chlorophyll a/c antennae.

Photosynthesis uses light as a source of energy but its excess can result in production of harmful oxygen radicals. To avoid any resulting damage, phototrophic organisms can employ a process known as non-photochemical quenching (NPQ), where excess light energy is safely dissipated as heat. The mechanism(s) of NPQ vary among different phototrophs. Here, we describe a new type of NPQ in the organism Rhodomonas salina, an alga belonging to the cryptophytes, part of the chromalveolate supergroup. Cryptophytes are exceptional among photosynthetic chromalveolates as they use both chlorophyll a/c proteins and phycobiliproteins for light harvesting. All our data demonstrates that NPQ in cryptophytes differs significantly from other chromalveolates - e.g. diatoms and it is also unique in comparison to NPQ in green algae and in higher plants: (1) there is no light induced xanthophyll cycle; (2) NPQ resembles the fast and flexible energetic quenching (qE) of higher plants, including its fast recovery; (3) a direct antennae protonation is involved in NPQ, similar to that found in higher plants. Further, fluorescence spectroscopy and biochemical characterization of isolated photosynthetic complexes suggest that NPQ in R. salina occurs in the chlorophyll a/c antennae but not in phycobiliproteins. All these results demonstrate that NPQ in cryptophytes represents a novel class of effective and flexible non-photochemical quenching.

Different phycobilin antenna organisations affect the balance between light use and growth rate in the cyanobacterium Microcystis aeruginosa and in the cryptophyte Cryptomonas ovata.

During the recent years, wide varieties of methodologies have been developed up to the level of commercial use to measure photosynthetic electron transport by modulated chlorophyll a-in vivo fluorescence. It is now widely accepted that the ratio between electron transport rates and new biomass (P (Fl)/B (C)) is not fixed and depends on many factors that are also taxonomically variable. In this study, the balance between photon absorption and biomass production has been measured in two phycobilin-containing phototrophs, namely, a cyanobacterium and a cryptophyte, which differ in their antenna organization. It is demonstrated that the different antenna organization exerts influence on the regulation of the primary photosynthetic reaction and the dissipation of excessively absorbed radiation. Although, growth rates and the quantum efficiency of biomass production of both phototrophs were comparable, the ratio P (Fl)/B (C) was twice as high in the cryptophyte in comparison to the cyanobacterium. It is assumed that this discrepancy is because of differences in the metabolic regulation of cell growth. In the cryptophyte, absorbed photosynthetic energy is used to convert assimilated carbon directly into proteins and lipids, whereas in the cyanobacterium, the photosynthetic energy is preferentially stored as carbohydrates.

Coherently wired light-harvesting in photosynthetic marine algae at ambient temperature.

Photosynthesis makes use of sunlight to convert carbon dioxide into useful biomass and is vital for life on Earth. Crucial components for the photosynthetic process are antenna proteins, which absorb light and transmit the resultant excitation energy between molecules to a reaction centre. The efficiency of these electronic energy transfers has inspired much work on antenna proteins isolated from photosynthetic organisms to uncover the basic mechanisms at play. Intriguingly, recent work has documented that light-absorbing molecules in some photosynthetic proteins capture and transfer energy according to quantum-mechanical probability laws instead of classical laws at temperatures up to 180 K. This contrasts with the long-held view that long-range quantum coherence between molecules cannot be sustained in complex biological systems, even at low temperatures. Here we present two-dimensional photon echo spectroscopy measurements on two evolutionarily related light-harvesting proteins isolated from marine cryptophyte algae, which reveal exceptionally long-lasting excitation oscillations with distinct correlations and anti-correlations even at ambient temperature. These observations provide compelling evidence for quantum-coherent sharing of electronic excitation across the 5-nm-wide proteins under biologically relevant conditions, suggesting that distant molecules within the photosynthetic proteins are 'wired' together by quantum coherence for more efficient light-harvesting in cryptophyte marine algae.

Rhodopsin-mediated photoreception in cryptophyte flagellates.

We show that phototaxis in cryptophytes is likely mediated by a two-rhodopsin-based photosensory mechanism similar to that recently demonstrated in the green alga Chlamydomonas reinhardtii, and for the first time, to our knowledge, report spectroscopic and charge movement properties of cryptophyte algal rhodopsins. The marine cryptophyte Guillardia theta exhibits positive phototaxis with maximum sensitivity at 450 nm and a secondary band above 500 nm. Variability of the relative sensitivities at these wavelengths and light-dependent inhibition of phototaxis in both bands by hydroxylamine suggest the involvement of two rhodopsin photoreceptors. In the related freshwater cryptophyte Cryptomonas sp. two photoreceptor currents similar to those mediated by the two sensory rhodopsins in green algae were recorded. Two cDNA sequences from G. theta and one from Cryptomonas encoding proteins homologous to type 1 opsins were identified. The photochemical reaction cycle of one Escherichia-coli-expressed rhodopsin from G. theta (GtR1) involves K-, M-, and O-like intermediates with relatively slow (approximately 80 ms) turnover time. GtR1 shows lack of light-driven proton pumping activity in E. coli cells, although carboxylated residues are at the positions of the Schiff base proton acceptor and donor as in proton pumping rhodopsins. The absorption spectrum, corresponding to the long-wavelength band of phototaxis sensitivity, makes this pigment a candidate for one of the G. theta sensory rhodopsins. A second rhodopsin from G. theta (GtR2) and the one from Cryptomonas have noncarboxylated residues at the donor position as in known sensory rhodopsins.