Long-term light adaptation of light-harvesting and energy-transfer processes in the glaucophyte Cyanophora paradoxa under different light conditions.

In response to fluctuation in light intensity and quality, oxygenic photosynthetic organisms modify their light-harvesting and excitation energy-transfer processes to maintain optimal photosynthetic activity. Glaucophytes, which are a group of primary symbiotic algae, possess light-harvesting antennas called phycobilisomes (PBSs) consistent with cyanobacteria and red algae. However, compared with cyanobacteria and red algae, glaucophytes are poorly studied and there are few reports on the regulation of photosynthesis in the group. In this study, we examined the long-term light adaptation of light-harvesting functions in a glaucophyte, Cyanophora paradoxa, grown under different light conditions. Compared with cells grown under white light, the relative number of PBSs to photosystems (PSs) increased in blue-light-grown cells and decreased in green-, yellow-, and red-light-grown cells. Moreover, the PBS number increased with increment in the monochromatic light intensity. More energy was transferred from PBSs to PSII than to PSI under blue light, whereas energy transfer from PBSs to PSII was reduced under green and yellow lights, and energy transfer from the PBSs to both PSs decreased under red light. Decoupling of PBSs was induced by intense green, yellow, and red lights. Energy transfer from PSII to PSI (spillover) was observed, but the contribution of the spillover did not distinctly change depending on the culture light intensity and quality. These results suggest that the glaucophyte C. paradoxa modifies the light-harvesting abilities of both PSs and excitation energy-transfer processes between the light-harvesting antennas and both PSs during long-term light adaption.

An ancient glaucophyte c6-like cytochrome related to higher plant cytochrome c6A is imported into muroplasts.

Cytochrome c6 is a redox carrier in the thylakoid lumen of cyanobacteria and some eukaryotic algae. Although the isofunctional plastocyanin is present in land plants and the green alga Chlamydomonas reinhardtii, these organisms also possess a cytochrome c6-like protein designated as cytochrome c6A. Two other cytochrome c6-like groups, c6B and c6C, have been identified in cyanobacteria. In this study, we have identified a novel c6-like cytochrome called PetJ2, which is encoded in the nuclear genome of Cyanophora paradoxa, a member of the glaucophytes - the basal branch of the Archaeplastida. We propose that glaucophyte PetJ2 protein is related to cyanobacterial c6B and c6C cytochromes, and that cryptic green algal and land plant cytochromes c6A evolved from an ancestral archaeplastidial PetJ2 protein. In vitro import experiments with isolated muroplasts revealed that PetJ2 is imported into plastids. Although it harbors a twin-arginine motif in its thylakoid-targeting peptide, which is generally indicative of thylakoid import via the Tat import pathway, our import experiments with isolated muroplasts and the heterologous pea thylakoid import system revealed that PetJ2 uses the Sec pathway instead of the Tat import pathway.

Cyanophora paradoxa mitochondrial tRNAs play a double game.

Present-day mitochondria derive from a single endosymbiosis of an α-proteobacterium into a proto-eukaryotic cell. Since this monophyletic event, mitochondria have evolved considerably, and unique traits have been independently acquired in the different eukaryotic kingdoms. Mitochondrial genome expression and RNA metabolism have diverged greatly. Here, Cyanophora paradoxa, a freshwater alga considered as a living fossil among photosynthetic organisms, represents an exciting model for studying the evolution of mitochondrial gene expression. As expected, fully mature tRNAs are released from primary transcripts to function in mitochondrial translation. We also show that these tRNAs take part in an mRNA processing punctuation mechanism in a non-conventional manner, leading to mRNA-tRNA hybrids with a CCA triplet at their 3'-extremities. In this case, tRNAs are probably used as stabilizing structures impeding the degradation of mRNA by exonucleases. From our data we propose that the present-day tRNA-like elements (t-elements) found at the 3'-terminals of mitochondrial mRNAs in land plants originate from true tRNAs like those observed in the mitochondria of this basal photosynthetic glaucophyte.

High Sequence Divergence but Limited Architectural Rearrangements in Organelle Genomes of Cyanophora (Glaucophyta) Species.

Cyanophora is the glaucophyte model taxon. Following the sequencing of the nuclear genome of C. paradoxa, studies based on single organelle and nuclear molecular markers revealed previously unrecognized species diversity within this glaucophyte genus. Here, we present the complete plastid (ptDNA) and mitochondrial (mtDNA) genomes of C. kugrensii, C. sudae, and C. biloba. The respective sizes and coding capacities of both ptDNAs and mtDNAs are conserved among Cyanophora species with only minor differences due to specific gene duplications. Organelle phylogenomic analyses consistently recover the species C. kugrensii and C. paradoxa as a clade and C. sudae and C. biloba as a separate group. The phylogenetic affiliations of the four Cyanophora species are consistent with architectural similarities shared at the organelle genomic level. Genetic distance estimations from both organelle sequences are also consistent with phylogenetic and architecture evidence. Comparative analyses confirm that the Cyanophora mitochondrial genes accumulate substitutions at 3-fold higher rates than plastid counterparts, suggesting that mtDNA markers are more appropriate to investigate glaucophyte diversity and evolutionary events that occur at a population level. The study of complete organelle genomes is becoming the standard for species delimitation and is particularly relevant to study cryptic diversity in microbial groups.

Gene expression analysis of Cyanophora paradoxa reveals conserved abiotic stress responses between basal algae and flowering plants.

The glaucophyte Cyanophora paradoxa represents the most basal member of the kingdom Archaeplastida, but the function and expression of most of its genes are unknown. This information is needed to uncover how functional gene modules, that is groups of genes performing a given function, evolved in the plant kingdom. We have generated a gene expression atlas capturing responses of Cyanophora to various abiotic stresses. The data were included in the CoNekT-Plants database, enabling comparative transcriptomic analyses across two algae and six land plants. We demonstrate how the database can be used to study gene expression, co-expression networks and gene function in Cyanophora, and how conserved transcriptional programs can be identified. We identified gene modules involved in phycobilisome biosynthesis, response to high light and cell division. While we observed no correlation between the number of differentially expressed genes and the impact on growth of Cyanophora, we found that the response to stress involves a conserved, kingdom-wide transcriptional reprogramming, which is activated upon most stresses in algae and land plants. The Cyanophora stress gene expression atlas and the tools found in the https://conekt.plant.tools/ database thus provide a useful resource to reveal functionally related genes and stress responses in the plant kingdom.

Thylakoid membranes contain a non-selective channel permeable to small organic molecules.

The thylakoid lumen is a membrane-enclosed aqueous compartment. Growing evidence indicates that the thylakoid lumen is not only a sink for protons and inorganic ions translocated during photosynthetic reactions but also a place for metabolic activities, e.g. proteolysis of photodamaged proteins, to sustain efficient photosynthesis. However, the mechanism whereby organic molecules move across the thylakoid membranes to sustain these lumenal activities is not well understood. In a recent study of Cyanophora paradoxa chloroplasts (muroplasts), we fortuitously detected a conspicuous diffusion channel activity in the thylakoid membranes. Here, using proteoliposomes reconstituted with the thylakoid membranes from muroplasts and from two other phylogenetically distinct organisms, cyanobacterium Synechocystis sp. PCC 6803 and spinach, we demonstrated the existence of nonselective channels large enough for enabling permeation of small organic compounds (e.g. carbohydrates and amino acids with Mr < 1500) in the thylakoid membranes. Moreover, we purified, identified, and characterized a muroplast channel named here CpTPOR. Osmotic swelling experiments revealed that CpTPOR forms a nonselective pore with an estimated radius of ∼1.3 nm. A lipid bilayer experiment showed variable-conductance channel activity with a typical single-channel conductance of 1.8 nS in 1 m KCl with infrequent closing transitions. The CpTPOR amino acid sequence was moderately similar to that of a voltage-dependent anion-selective channel of the mitochondrial outer membrane, although CpTPOR exhibited no obvious selectivity for anions and no voltage-dependent gating. We propose that transmembrane diffusion pathways are ubiquitous in the thylakoid membranes, presumably enabling rapid transfer of various metabolites between the lumen and stroma.

Outer Membrane Proteins Derived from Non-cyanobacterial Lineage Cover the Peptidoglycan of Cyanophora paradoxa Cyanelles and Serve as a Cyanelle Diffusion Channel.

The cyanelle is a primitive chloroplast that contains a peptidoglycan layer between its inner and outer membranes. Despite the fact that the envelope structure of the cyanelle is reminiscent of Gram-negative bacteria, the Cyanophora paradoxa genome appears to lack genes encoding homologs of putative peptidoglycan-associated outer membrane proteins and outer membrane channels. These are key components of Gram-negative bacterial membranes, maintaining structural stability and regulating permeability of outer membrane, respectively. Here, we discovered and characterized two dominant peptidoglycan-associated outer membrane proteins of the cyanelle (∼2 × 10(6) molecules per cyanelle). We named these proteins CppF and CppS (cyanelle peptidoglycan-associated proteins). They are homologous to each other and function as a diffusion channel that allows the permeation of compounds with Mr <1,000 as revealed by permeability measurements using proteoliposomes reconstituted with purified CppS and CppF. Unexpectedly, amino acid sequence analysis revealed no evolutionary linkage to cyanobacteria, showing only a moderate similarity to cell surface proteins of bacteria belonging to Planctomycetes phylum. Our findings suggest that the C. paradoxa cyanelle adopted non-cyanobacterial lineage proteins as its main outer membrane components, providing a physical link with the underlying peptidoglycan layer and functioning as a diffusion route for various small substances across the outer membrane.

Single-Pixel Densitometry Revealed the Presence of Peptidoglycan in the Intermembrane Space of the Moss Chloroplast Envelope in Conventional Electron Micrographs.

Chloroplasts are believed to be descendants of ancestral cyanobacteria that have a peptidoglycan layer between the outer and the inner membranes. In particular, cyanelles having peptidoglycan in Cyanophora paradoxa are considered as evidence for the endosymbiotic origin of chloroplasts. The moss Physcomitrella patens has a complete set of genes involved in the synthesis of peptidoglycan, but a peptidoglycan layer has not been observed by conventional electron microscopy to date. Recently, a new metabolic labeling technique using a fluorescent probe was applied to visualize putative peptidoglycan surrounding the chloroplasts. The exact localization of the peptidoglycan, however, has not been clearly identified. Here we examined conventional electron micrographs of two types of moss materials (mutants or ampicillin-treated plants), one presumably having peptidoglycan and the other presumably lacking peptidoglycan, and analyzed in detail, by single-pixel densitometry, the electron density of the chloroplast envelope membranes and the intermembrane space. Statistical analysis showed that the relative electron density within the intermembrane space with respect to that of the envelope membranes was significantly higher in the materials presumably having peptidoglycan than in the materials presumably devoid of peptidoglycan. We consider this difference as bona fide evidence for the presence of peptidoglycan between the outer and the inner envelope membranes in the wild-type chloroplasts of the moss, although its density is lower than that in bacteria and cyanelles. We will also discuss this low-density peptidoglycan in the light of the phylogenetic origin of peptidoglycan biosynthesis enzymes.

Alga-PrAS (Algal Protein Annotation Suite): A Database of Comprehensive Annotation in Algal Proteomes.

Algae are smaller organisms than land plants and offer clear advantages in research over terrestrial species in terms of rapid production, short generation time and varied commercial applications. Thus, studies investigating the practical development of effective algal production are important and will improve our understanding of both aquatic and terrestrial plants. In this study we estimated multiple physicochemical and secondary structural properties of protein sequences, the predicted presence of post-translational modification (PTM) sites, and subcellular localization using a total of 510,123 protein sequences from the proteomes of 31 algal and three plant species. Algal species were broadly selected from green and red algae, glaucophytes, oomycetes, diatoms and other microalgal groups. The results were deposited in the Algal Protein Annotation Suite database (Alga-PrAS; http://alga-pras.riken.jp/), which can be freely accessed online.

The flagellar apparatus of the glaucophyte Cyanophora cuspidata.

Glaucophytes are a kingdom-scale lineage of unicellular algae with uniquely underived plastids. The genus Cyanophora is of particular interest because it is the only glaucophyte that is a flagellate throughout its life cycle, making its morphology more directly comparable than other glaucophytes to other eukaryote flagellates. The ultrastructure of Cyanophora has already been studied, primarily in the 1960s and 1970s. However, the usefulness of that work has been undermined by its own limitations, subsequent misinterpretations, and a recent taxonomic revision of the genus. For example, Cyanophora's microtubular roots have been widely reported as cruciate, with rotationally symmetrical wide and thin roots, although the first ultrastructural work described it as having three wide and one narrow root. We examine Cyanophora cuspidata using scanning and transmission electron microscopy, and construct a model of its cytoskeleton using serial-section TEM. We confirm the earlier model, with asymmetric roots. We describe previously unknown and unsuspected features of its microtubular roots, including (i) a rearrangement of individual microtubules within the posterior right root, (ii) a splitting of the posterior left root into two subroots, and (iii) the convergence and termination of the narrow roots against wider ones in both the anterior and posterior subsystems of the flagellar apparatus. We also describe a large complement of nonmicrotubular components of the cytoskeleton, including a substantial connective between the posterior right root and the anterior basal body. Our work should serve as the starting point for a re-examination of both internal glaucophyte diversity and morphological evolution in eukaryotes.

Diverse origins of enzymes involved in the biosynthesis of chloroplast peptidoglycan.

Chloroplasts are believed to be descendants of ancestral cyanobacteria that had peptidoglycan layer between the outer and the inner membranes. Historically, the glaucophyte Cyanophora paradoxa and the rhizopod Paulinella chromatophora were believed to harbor symbiotic cyanobacteria having peptidoglycan, which were conventionally named "cyanelles". In addition, the complete set of genes involved in the synthesis of peptidoglycan has been found in the moss Physcomitrella patens and some plants and algae. The presence of peptidoglycan-like structures was demonstrated by a new metabolic labeling technique in P. patens. However, many green algae and all known red algae lack peptidoglycan-related genes. That is the reason why we questioned the origin of peptidoglycan-synthesizing enzymes in the chloroplasts of the green algae and plants. We performed phylogenetic analysis of ten enzymes involved in the synthesis of peptidoglycan exploiting the Gclust homolog clusters and additional genomic data. As expected, all the identified genes encoded in the chromatophore genome of P. chromatophora were closely related to cyanobacterial homologs. In the green algae and plants, only two genes, murA and mraY, were found to be closely related to cyanobacterial homologs. The origins of all other genes were diverse. Unfortunately, the origins of C. paradoxa genes were not clearly determined because of incompleteness of published genomic data. We discuss on the probable evolutionary scenarios to explain the mostly non-cyanobacterial origins of the biosynthetic enzymes of chloroplast peptidoglycan: A plausible one includes extensive multiple horizontal gene transfers during the early evolution of Viridiplantae.

Early evolution of the eukaryotic Ca2+ signaling machinery: conservation of the CatSper channel complex.

Calcium signaling is one of the most extensively employed signal transduction mechanisms in life. As life evolved into increasingly complex organisms, Ca(2+) acquired more extensive and varied functions. Here, we compare genes encoding proteins that govern Ca(2+) entry and exit across cells or organelles within organisms of early eukaryotic evolution into fungi, plants, and animals. Recent phylogenomics analyses reveal a complex Ca(2+) signaling machinery in the apusozoan protist Thecamonas trahens, a putative unicellular progenitor of Opisthokonta. We compare T. trahens Ca(2+) signaling to that in a marine bikont protist, Aurantiochytrium limacinum, and demonstrate the conservation of key Ca(2+) signaling molecules in the basally diverging alga Cyanophora paradoxa. Particularly, our findings reveal the conservation of the CatSper channel complex in Au. limacinum and C. paradoxa, suggesting that the CatSper complex likely originated from an ancestral Ca(2+) signaling machinery at the root of early eukaryotic evolution prior to the unikont/bikont split.

PlantRNA, a database for tRNAs of photosynthetic eukaryotes.

PlantRNA database (http://plantrna.ibmp.cnrs.fr/) compiles transfer RNA (tRNA) gene sequences retrieved from fully annotated plant nuclear, plastidial and mitochondrial genomes. The set of annotated tRNA gene sequences has been manually curated for maximum quality and confidence. The novelty of this database resides in the inclusion of biological information relevant to the function of all the tRNAs entered in the library. This includes 5'- and 3'-flanking sequences, A and B box sequences, region of transcription initiation and poly(T) transcription termination stretches, tRNA intron sequences, aminoacyl-tRNA synthetases and enzymes responsible for tRNA maturation and modification. Finally, data on mitochondrial import of nuclear-encoded tRNAs as well as the bibliome for the respective tRNAs and tRNA-binding proteins are also included. The current annotation concerns complete genomes from 11 organisms: five flowering plants (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Medicago truncatula and Brachypodium distachyon), a moss (Physcomitrella patens), two green algae (Chlamydomonas reinhardtii and Ostreococcus tauri), one glaucophyte (Cyanophora paradoxa), one brown alga (Ectocarpus siliculosus) and a pennate diatom (Phaeodactylum tricornutum). The database will be regularly updated and implemented with new plant genome annotations so as to provide extensive information on tRNA biology to the research community.

Nucleotide substitution analyses of the glaucophyte Cyanophora suggest an ancestrally lower mutation rate in plastid vs mitochondrial DNA for the Archaeplastida.

A lot is known about the evolution and architecture of plastid, mitochondrial, and nuclear genomes, but surprisingly little is known about their relative rates of mutation. Most available relative-rate data come from seed plants, which, with few exceptions, have a mitochondrial mutation rate that is lower than those of the plastid and nucleus. But new findings from diverse plastid-bearing lineages have shown that for some eukaryotes the mitochondrial mutation rate is an order of magnitude greater than those of the plastid and nucleus. Here, we explore for the first time relative rates of mutation within the Glaucophyta-one of three main lineages that make up the Archaeplastida (or Plantae sensu lato). Nucleotide substitution analyses from distinct isolates of the unicellular glaucophyte Cyanophora paradoxa reveal 4-5-fold lower rates of mutation in the plastid and nucleus than the mitochondrion, which is similar to the mutational pattern observed in red algae and haptophytes, but opposite to that of seed plants. These data, together with data from previous reports, suggest that for much of the known photosynthetic eukaryotic diversity, plastid DNA mutations occur less frequently than those in mitochondrial DNA.

Molecular markers from different genomic compartments reveal cryptic diversity within glaucophyte species.

Glaucophytes are the least studied of the three major Archaeplastida (Plantae sensu lato) lineages. It has been largely recognized that comprehensive investigations of glaucophyte genetic and species diversity will shed light on the early evolution of photosynthetic eukaryotes. Here we used molecular phylogenetics and genetic distance estimations of diverse molecular markers to explore strain and species diversity within the glaucophyte genera Cyanophora and Glaucocystis. Single gene and concatenated maximum likelihood analyses of markers from three different genetic compartments consistently recovered similar intrageneric genetic groups. Distance analyses of plastid (psbA and rbcL) and mitochondrial (cob and cox1) genes, and the nuclear internal transcribed spacer (ITS) region, revealed substantial genetic divergence between some Cyanophora paradoxa and Glaucocystis nostochinearum strains. The genetic distances estimated between some glaucophyte strains currently considered the same species are similar or greater than divergence values calculated between different species in other unicellular algae, such as certain green algae and diatoms. The analyzed molecular markers are prospective candidates for future studies of species diversity in glaucophytes. Overall, our results unveil previously unrecognized cryptic diversity within Cyanophora and Glaucocystis species.

Phycobilisome model with novel skeleton-like structures in a glaucocystophyte Cyanophora paradoxa.

Phycobilisome (PBS) is a photosynthetic antenna supercomplex consisting of a central core subcomplex with several peripheral rods radiating from the core. Subunit structure of PBS was studied in a glaucocystophyte Cyanophora paradoxa strain NIES 547. Subunit composition of PBS was identified by N-terminal sequencing and genes for the subunits were determined by homology search of databases. They included rod linker proteins CpcK1 and CpcK2, rod-core linker proteins CpcG1 and CpcG2, and core linker proteins ApcC1 and ApcC2. Subfractionation by native polyacrylamide gel electrophoresis provided evidence for novel subcomplexes (ApcE/CpcK1/CpcG2/ApcA/ApcB/CpcD and ApcE/CpcK2/CpcG1/ApcA/ApcB), which connect rod and core subcomplexes. These skeleton-like structures may serve as a scaffold of the whole PBS assembly. Different roles of ApcC1 and ApcC2 were also suggested. Based on these findings, structural models for PBS were proposed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Proteomic analysis of the Cyanophora paradoxa muroplast provides clues on early events in plastid endosymbiosis.

Glaucophytes represent the first lineage of photosynthetic eukaryotes of primary endosymbiotic origin that diverged after plastid establishment. The muroplast of Cyanophora paradoxa represents a primitive plastid that resembles its cyanobacterial ancestor in pigment composition and the presence of a peptidoglycan wall. To attain insights into the evolutionary history of cyanobiont integration and plastid development, it would thus be highly desirable to obtain knowledge on the composition of the glaucophyte plastid proteome. Here, we provide the first proteomic analysis of the muroplast of C. paradoxa. Mass spectrometric analysis of the muroplast proteome identified 510 proteins with high confidence. The protein repertoire of the muroplast revealed novel paths for reduced carbon flow and export to the cytosol through a sugar phosphate transporter of chlamydial origin. We propose that C. paradoxa possesses a primordial plastid mirroring the situation in the early protoalga.

Antiproliferative activity of Cyanophora paradoxa pigments in melanoma, breast and lung cancer cells.

The glaucophyte Cyanophora paradoxa (Cp) was chemically investigated to identify pigments efficiently inhibiting malignant melanoma, mammary carcinoma and lung adenocarcinoma cells growth. Cp water and ethanol extracts significantly inhibited the growth of the three cancer cell lines in vitro, at 100 µg · mL(-1). Flash chromatography of the Cp ethanol extract, devoid of c-phycocyanin and allophycocyanin, enabled the collection of eight fractions, four of which strongly inhibited cancer cells growth at 100 µg · mL(-1). Particularly, two fractions inhibited more than 90% of the melanoma cells growth, one inducing apoptosis in the three cancer cells lines. The detailed analysis of Cp pigment composition resulted in the discrimination of 17 molecules, ten of which were unequivocally identified by high resolution mass spectrometry. Pheophorbide a, ß-cryptoxanthin and zeaxanthin were the three main pigments or derivatives responsible for the strong cytotoxicity of Cp fractions in cancer cells. These data point to Cyanophora paradoxa as a new microalgal source to purify potent anticancer pigments, and demonstrate for the first time the strong antiproliferative activity of zeaxanthin and ß-cryptoxanthin in melanoma cells.

Coordination mechanism of cell and cyanelle division in the glaucophyte alga Cyanophora sudae.

In unicellular algae with a single chloroplast, two mechanisms coordinate cell and chloroplast division: the S phase-specific expression of chloroplast division genes and the permission of cell cycle progression from prophase to metaphase by the onset of chloroplast division. This study investigated whether a similar mechanism exists in a unicellular alga with multiple chloroplasts using the glaucophyte alga Cyanophora sudae, which contains four chloroplasts (cyanelles). Cells with eight cyanelles appeared after the S phase arrest with a topoisomerase inhibitor camptothecin, suggesting that the mechanism of S phase-specific expression of cyanelle division genes was conserved in this alga. Inhibition of peptidoglycan synthesis by ß-lactam antibiotic ampicillin arrested cells in the S-G2 phase, and inhibition of septum invagination with cephalexin resulted in cells with two nuclei and one cyanelle, despite inhibition of cyanelle division. This indicates that even in the unicellular alga with four chloroplasts, the cell cycle progresses to the M phase following the progression of chloroplast division to a certain division stage. These results suggested that C. sudae has two mechanisms for coordinating cell and cyanelle division, similar to the unicellular algae with a single chloroplast.