Growth phase-dependent reorganization of cryptophyte photosystem I antennae.

Photosynthetic cryptophytes are eukaryotic algae that utilize membrane-embedded chlorophyll a/c binding proteins (CACs) and lumen-localized phycobiliproteins (PBPs) as their light-harvesting antennae. Cryptophytes go through logarithmic and stationary growth phases, and may adjust their light-harvesting capability according to their particular growth state. How cryptophytes change the type/arrangement of the photosynthetic antenna proteins to regulate their light-harvesting remains unknown. Here we solve four structures of cryptophyte photosystem I (PSI) bound with CACs that show the rearrangement of CACs at different growth phases. We identify a cryptophyte-unique protein, PsaQ, which harbors two chlorophyll molecules. PsaQ specifically binds to the lumenal region of PSI during logarithmic growth phase and may assist the association of PBPs with photosystems and energy transfer from PBPs to photosystems.

Consequences of light spectra for pigment composition and gene expression in the cryptophyte Rhodomonas salina.

Algae with a more diverse suite of pigments can, in principle, exploit a broader swath of the light spectrum through chromatic acclimation, the ability to maximize light capture via plasticity of pigment composition. We grew Rhodomonas salina in wide-spectrum, red, green, and blue environments and measured how pigment composition differed. We also measured expression of key light-capture and photosynthesis-related genes and performed a transcriptome-wide expression analysis. We observed the highest concentration of phycoerythrin in green light, consistent with chromatic acclimation. Other pigments showed trends inconsistent with chromatic acclimation, possibly due to feedback loops among pigments or high-energy light acclimation. Expression of some photosynthesis-related genes was sensitive to spectrum, although expression of most was not. The phycoerythrin α-subunit was expressed two-orders of magnitude greater than the ß-subunit even though the peptides are needed in an equimolar ratio. Expression of genes related to chlorophyll-binding and phycoerythrin concentration were correlated, indicating a potential synthesis relationship. Pigment concentrations and expression of related genes were generally uncorrelated, implying post-transcriptional regulation of pigments. Overall, most differentially expressed genes were not related to photosynthesis; thus, examining associations between light spectrum and other organismal functions, including sexual reproduction and glycolysis, may be important.

The preferential transport of NO3- by full-length Guillardia theta anion channelrhodopsin 1 is enhanced by its extended cytoplasmic domain.

Previous research of anion channelrhodopsins (ACRs) has been performed using cytoplasmic domain (CPD)-deleted constructs and therefore have overlooked the native functions of full-length ACRs and the potential functional role(s) of the CPD. In this study, we used the recombinant expression of full-length Guillardia theta ACR1 (GtACR1_full) for pH measurements in Pichia pastoris cell suspensions as an indirect method to assess its anion transport activity and for absorption spectroscopy and flash photolysis characterization of the purified protein. The results show that the CPD, which was predicted to be intrinsically disordered and possibly phosphorylated, enhanced NO3- transport compared to Cl- transport, which resulted in the preferential transport of NO3-. This correlated with the extended lifetime and large accumulation of the photocycle intermediate that is involved in the gate-open state. Considering that the depletion of a nitrogen source enhances the expression of GtACR1 in native algal cells, we suggest that NO3- transport could be the natural function of GtACR1_full in algal cells.

Isospectral intermediates in the photochemical reaction cycle of anion channelrhodopsin GtACR1.

The most effective tested optogenetic tools available for neuronal silencing are the light-gated anion channel proteins found in the cryptophyte alga Guillardia theta (GtACRs). Molecular mechanisms of GtACRs, including the photointermediates responsible for the open channel state, are of great interest for understanding their exceptional conductance. In this study, the photoreactions of GtACR1 and its D234N, A75E, and S97E mutants were investigated using multichannel time-resolved absorption spectroscopy. For each of the proteins, the analysis showed two early microsecond transitions between K-like and L-like forms and two late millisecond recovery steps. Spectral forms associated with potential molecular intermediates of the proteins were derived and their evolutions in time were analyzed. The results indicate the presence of isospectral intermediates in the photocycles and expand the range of potential intermediates responsible for the open channel state.

A phycoerythrin isolated from Rhodomonas salina induces apoptosis via ERK/Bak and JNK/Caspase-3 pathway in A549 cells.

Rhodomonas salina, Cryptophyta, Rhodomonas genus, is a valuable source for live feed in aquaculture and for the production of phycoerythrin (PE). In this study, PE was extracted from Rhodomonas salina and characterized as having a molecular weight of approximately 24 kDa, an absorbance at 545 nm, and a purity of up to 6.61 (which meets reagent grade requirements with an OD545/OD280 ratio >4). The effects of PE on anticancer activity and its underlying mechanisms were evaluated to assess the immunomodulatory potential on the human lung cancer A549 cell line. Biochemical assays and western blot analysis were applied to confirm the immune mechanisms. The results showed that after 24 h of exposure to PE, the proliferation of A549 cells was significantly and dose-dependently decreased. PE also caused the generation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential (MMP). The further results showed that PE can remarkably enhance the protein levels of cleaved caspase-3 and p53. Simultaneously, the BCL-2 family was also affected and had some changes, such as the dramatically enhance of Bim and Bak and the decrease of Bcl-2 level. However, it is interesting to note that there was no apparent alteration in Bax expression during the experiment. Furthermore, the biological mechanism for the potential of PE to induce apoptosis showed that the ERK/Bak and the JNK/caspase-3 signaling pathway were activated. This study provides evidence that the anticancer activity of PE in Rhodomonas salina may have potential for preventing cancer and serving as a novel immunostimulant in the pharmaceutical industry.

Molecular structures reveal the origin of spectral variation in cryptophyte light harvesting antenna proteins.

In addition to their membrane-bound chlorophyll a/c light-harvesting antenna, the cryptophyte algae have evolved a unique phycobiliprotein antenna system located in the thylakoid lumen. The basic unit of this antenna consists of two copies of an αß protomer where the α and ß subunits scaffold different combinations of a limited number of linear tetrapyrrole chromophores. While the ß subunit is highly conserved, encoded by a single plastid gene, the nuclear-encoded α subunits have evolved diversified multigene families. It is still unclear how this sequence diversity results in the spectral diversity of the mature proteins. By careful examination of three newly determined crystal structures in comparison with three previously obtained, we show how the α subunit amino acid sequences control chromophore conformations and hence spectral properties even when the chromophores are identical. Previously we have shown that α subunits control the quaternary structure of the mature αß.αß complex (either open or closed), however, each species appeared to only harbor a single quaternary form. Here we show that species of the Hemiselmis genus contain expressed α subunit genes that encode both distinct quaternary structures. Finally, we have discovered a common single-copy gene (expressed into protein) consisting of tandem copies of a small α subunit that could potentially scaffold pairs of light harvesting units. Together, our results show how the diversity of the multigene α subunit family produces a range of mature cryptophyte antenna proteins with differing spectral properties, and the potential for minor forms that could contribute to acclimation to varying light regimes.

Identification of the Channelrhodopsin Genes in the Green and Cryptophytic Algae from the White and Black Seas.

Due to the unique capability of modulating cell membrane potential upon photoactivation, channelrhodopsins of green (Chlorophyta) and cryptophytic (Cryptophyta) algae are widely employed in optogenetics, a modern method of light-dependent regulation of biological processes. To enable the search for new genes perspective for optogenetics, we have developed the PCR tests for the presence of genes of the cation and anion channelrhodopsins. Six isolates of green algae Haematococcus and Bracteacoccus from the White Sea region and 2 specimens of Rhodomonas sp. (Cryptophyta) from the regions of White and Black Seas were analyzed. Using our PCR test we have demonstrated the known Haematococcus rhodopsin genes and have discovered novel rhodopsin genes in the genus of Bracteacoccus. Two distantly homologous genes of anion channelrhodopsins were also identified in the cryptophytic Rhodomonas sp. from the White and Black Seas. These results indicate that the developed PCR tests might be useful tool for a broad-range screening of the Chlorophyta and Cryptophyta algae to identify unique channelrhodopsin genes.

Preference of Proteomonas sulcata anion channelrhodopsin for NO3- revealed using a pH electrode method.

Ion channel proteins are physiologically important molecules in living organisms. Their molecular functions have been investigated using electrophysiological methods, which enable quantitative, precise and advanced measurements and thus require complex instruments and experienced operators. For simpler and easier measurements, we measured the anion transport activity of light-gated anion channelrhodopsins (ACRs) using a pH electrode method, which has already been established for ion pump rhodopsins. Using that method, we successfully measured the anion transport activity and its dependence on the wavelength of light, i.e. its action spectra, and on the anion species, i.e. its selectivity or preference, of several ACRs expressed in yeast cells. In addition, we identified the strong anion transport activity and the preference for NO3- of an ACR from a marine cryptophyte algae Proteomonas sulcata, named PsuACR_353. Such a preference was discovered for the first time in microbial pump- or channel-type rhodopsins. Nitrate is one of the most stable forms of nitrogen and is used as a nitrogen source by most organisms including plants. Therefore, PsuACR_353 may play a role in NO3- transport and might take part in NO3--related cellular functions in nature. Measurements of a mutant protein revealed that a Thr residue in the 3rd transmembrane helix, which corresponds to Cys102 in GtACR1, contributed to the preference for NO3-. These findings will be helpful to understand the mechanisms of anion transport, selectivity and preference of PsuACR_353.

Mechanism of absorption wavelength shifts in anion channelrhodopsin-1 mutants.

Using a quantum mechanical/molecular mechanical approach, we show the mechanisms of how the protein environment of Guillardia theta anion channelrhodopsin-1 (GtACR1) can shift the absorption wavelength. The calculated absorption wavelengths for GtACR1 mutants, M105A, C133A, and C237A are in agreement with experimentally measured wavelengths. Among 192 mutant structures investigated, mutations at Thr101, Cys133, Pro208, and Cys237 are likely to increase the absorption wavelength. In particular, T101A GtACR1 was expressed in HEK293T cells. The measured absorption wavelength is 10 nm higher than that of wild type, consistent with the calculated wavelength. (i) Removal of a polar residue from the Schiff base moiety, (ii) addition of a polar or acidic residue to the ß-ionone ring moiety, and (iii) addition of a bulky residue to increase the planarity of the ß-ionone and Schiff base moieties are the basis of increasing the absorption wavelength.

Expression analysis of microbial rhodopsin-like genes in Guillardia theta.

The Cryptomonad Guillardia theta has 42 genes encoding microbial rhodopsin-like proteins in their genomes. Light-driven ion-pump activity has been reported for some rhodopsins based on heterologous E. coli or mammalian cell expression systems. However, neither their physiological roles nor the expression of those genes in native cells are known. To reveal their physiological roles, we investigated the expression patterns of these genes under various growth conditions. Nitrogen (N) deficiency induced color change in exponentially growing G. theta cells from brown to green. The 29 rhodopsin-like genes were expressed in native cells. We found that the expression of 6 genes was induced under N depletion, while that of another 6 genes was reduced under N depletion.

Phycoerythrin Association with Photosystem II in the Cryptophyte Alga Rhodomonas salina.

# Deceased. Cryptophyte algae belong to a special group of oxygenic photosynthetic organisms containing pigment combination unique for plastids - phycobiliproteins and chlorophyll a/c-containing antenna. Despite the progress in investigation of morphological and ecological features, as well as genome-based systematics of cryptophytes, their photosynthetic apparatus remains poorly understood. The ratio of the photosystems (PS)s I and II is unknown and information on participation of the two antennal complexes in functions of the two photosystems is inconsistent. In the present work we demonstrated for the first time that the cryptophyte alga Rhodomonas salina had the PSI to PSII ratio in thylakoid membranes equal to 1 : 4, whereas this ratio in cyanobacteria and higher plants was known to be 3 : 1 and 1 : 1, respectively. Furthermore, it was established that contrary to the case of cyanobacteria the phycobiliprotein antenna represented by phycoerythrin-545 (PE-545) in R. salina was associated only with the PSII, which indicated specific spatial organization of these protein pigments within the thylakoids that did not facilitate interaction with the PSI.

Tracing the fate of microplastic carbon in the aquatic food web by compound-specific isotope analysis.

Increasing abundance of microplastics (MP) in marine and freshwaters is currently one of the greatest environmental concerns. Since plastics are fairly resistant to chemical decomposition, breakdown and reutilization of MP carbon complexes requires microbial activity. Currently, only a few microbial isolates have been shown to degrade MPs, and direct measurements of the fate of the MP carbon are still lacking. We used compound-specific isotope analysis to track the fate of fully labelled 13C-polyethylene (PE) MP carbon across the aquatic microbial-animal interface. Isotopic values of respired CO2 and membrane lipids showed that MP carbon was partly mineralized and partly used for cell growth. Microbial mineralization and assimilation of PE-MP carbon was most active when inoculated microbes were obtained from highly humic waters, which contain recalcitrant substrate sources. Mixotrophic algae (Cryptomonas sp.) and herbivorous zooplankton (Daphnia magna) used microbial mediated PE-MP carbon in their cell membrane fatty acids. Moreover, heteronanoflagellates and mixotrophic algae sequestered MP carbon for synthesizing essential ω-6 and ω-3 polyunsaturated fatty acids. Thus, this study demonstrates that aquatic micro-organisms can produce, biochemically upgrade, and trophically transfer nutritionally important biomolecules from PE-MP.

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.

Crystal structure of the first eukaryotic bilin reductase GtPEBB reveals a flipped binding mode of dihydrobiliverdin.

Phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes. The biosynthesis of phycoerythrobilin (PEB) is catalyzed by the subsequent action of two ferredoxin-dependent bilin reductases (FDBRs). Although 15,16-dihydrobiliverdin (DHBV):ferredoxin oxidoreductase (PebA) catalyzes the two-electron reduction of biliverdin IXα to 15,16-DHBV, PEB:ferredoxin oxidoreductase (PebB) reduces this intermediate further to PEB. Interestingly, marine viruses encode the FDBR PebS combining both activities within one enzyme. Although PebA and PebS share a canonical fold with similar substrate-binding pockets, the structural determinants for the stereo- and regiospecific modification of their tetrapyrrole substrates are incompletely understood, also because of the lack of a PebB structure. Here, we solved the X-ray crystal structures of both substrate-free and -bound PEBB from the cryptophyte Guillardia theta at 1.90 and 1.65 Å, respectively. The structures of PEBB exhibit the typical α/ß/α-sandwich fold. Interestingly, the open-chain tetrapyrrole substrate DHBV is bound in an unexpected flipped orientation within the canonical FDBR active site. Biochemical analyses of the WT enzyme and active site variants identified two central aspartate residues Asp-99 and Asp-219 as essential for catalytic activity. In addition, the conserved Arg-215 plays a critical role in substrate specificity, binding orientation, and active site integrity. Because these critical residues are conserved within certain FDBRs displaying A-ring reduction activity, we propose that they present a conserved mechanism for this reaction. The flipped substrate-binding mode indicates that two-electron reducing FDBRs utilize the same primary site within the binding pocket and that substrate orientation is the determinant for A- or D-ring regiospecificity.

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.

Genes functioned in kleptoplastids of Dinophysis are derived from haptophytes rather than from cryptophytes.

Toxic dinoflagellates belonging to the genus Dinophysis acquire plastids indirectly from cryptophytes through the consumption of the ciliate Mesodinium rubrum. Dinophysis acuminata harbours three genes encoding plastid-related proteins, which are thought to have originated from fucoxanthin dinoflagellates, haptophytes and cryptophytes via lateral gene transfer (LGT). Here, we investigate the origin of these plastid proteins via RNA sequencing of species related to D. fortii. We identified 58 gene products involved in porphyrin, chlorophyll, isoprenoid and carotenoid biosyntheses as well as in photosynthesis. Phylogenetic analysis revealed that the genes associated with chlorophyll and carotenoid biosyntheses and photosynthesis originated from fucoxanthin dinoflagellates, haptophytes, chlorarachniophytes, cyanobacteria and cryptophytes. Furthermore, nine genes were laterally transferred from fucoxanthin dinoflagellates, whose plastids were derived from haptophytes. Notably, transcription levels of different plastid protein isoforms varied significantly. Based on these findings, we put forth a novel hypothesis regarding the evolution of Dinophysis plastids that ancestral Dinophysis species acquired plastids from haptophytes or fucoxanthin dinoflagellates, whereas LGT from cryptophytes occurred more recently. Therefore, the evolutionary convergence of genes following LGT may be unlikely in most cases.

Nucleomorph Small RNAs in Cryptophyte and Chlorarachniophyte Algae.

The regulation of gene expression and RNA maturation underlies fundamental processes such as cell homeostasis, development, and stress acclimation. The biogenesis and modification of RNA is tightly controlled by an array of regulatory RNAs and nucleic acid-binding proteins. While the role of small RNAs (sRNAs) in gene expression has been studied in-depth in select model organisms, little is known about sRNA biology across the eukaryotic tree of life. We used deep sequencing to explore the repertoires of sRNAs encoded by the miniaturized, endosymbiotically derived "nucleomorph" genomes of two single-celled algae, the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. A total of 32.3 and 35.3 million reads were generated from G. theta and B. natans, respectively. In G. theta, we identified nucleomorph U1, U2, and U4 spliceosomal small nuclear RNAs (snRNAs) as well as 11 C/D box small nucleolar RNAs (snoRNAs), five of which have potential plant and animal homologs. The snoRNAs are predicted to perform 2'-O methylation of rRNA (but not snRNA). In B. natans, we found the previously undetected 5S rRNA as well as six orphan sRNAs. Analysis of chlorarachniophyte snRNAs shed light on the removal of the miniature 18-21 nt introns found in B. natans nucleomorph genes. Neither of the nucleomorph genomes appears to encode RNA pseudouridylation machinery, and U5 snRNA cannot be found in the cryptophyte G. theta. Considering the central roles of U5 snRNA and RNA modifications in other organisms, cytoplasm-to-nucleomorph RNA shuttling in cryptophyte algae is a distinct possibility.

Crystal structure of a natural light-gated anion channelrhodopsin.

The anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 Å reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.

High-efficiency optogenetic silencing with soma-targeted anion-conducting channelrhodopsins.

Optogenetic silencing allows time-resolved functional interrogation of defined neuronal populations. However, the limitations of inhibitory optogenetic tools impose stringent constraints on experimental paradigms. The high light power requirement of light-driven ion pumps and their effects on intracellular ion homeostasis pose unique challenges, particularly in experiments that demand inhibition of a widespread neuronal population in vivo. Guillardia theta anion-conducting channelrhodopsins (GtACRs) are promising in this regard, due to their high single-channel conductance and favorable photon-ion stoichiometry. However, GtACRs show poor membrane targeting in mammalian cells, and the activity of such channels can cause transient excitation in the axon due to an excitatory chloride reversal potential in this compartment. Here, we address these problems by enhancing membrane targeting and subcellular compartmentalization of GtACRs. The resulting soma-targeted GtACRs show improved photocurrents, reduced axonal excitation and high light sensitivity, allowing highly efficient inhibition of neuronal activity in the mammalian brain.

Implications for the impairment of the rapid channel closing of Proteomonas sulcata anion channelrhodopsin 1 at high Cl- concentrations.

Natural anion channelrhodopsins (ACRs) have recently received increased attention because of their effectiveness in optogenetic manipulation for neuronal silencing. In this study, we focused on Proteomonas sulcata ACR1 (PsuACR1), which has rapid channel closing kinetics and a rapid recovery to the initial state of its anion channel function that is useful for rapid optogenetic control. To reveal the anion concentration dependency of the channel function, we investigated the photochemical properties of PsuACR1 using spectroscopic techniques. Recombinant PsuACR1 exhibited a Cl- dependent spectral red-shift from 531 nm at 0.1 mM to 535 nm at 1000 mM, suggesting that it binds Cl- in the initial state with a Kd of 5.5 mM. Flash-photolysis experiments revealed that the photocycle was significantly changed at high Cl- concentrations, which led not only to suppression of the accumulation of the M-intermediate involved in the Cl- non-conducting state but also to a drastic change in the equilibrium state of the other photo-intermediates. Because of this, the Cl- conducting state is protracted by one order of magnitude, which implies an impairment of the rapid channel closing of PsuACR1 in the presence of high concentrations of Cl-.