Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales).

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

Improved tolerance of recombinant Chlamydomonas rainhardtii with putative 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase from Pyropia yezoensis to nitrogen starvation.

In a previous study, a putative 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) was highly expressed in a mutant strain of Pyropia yezoensis, which exhibited an improved growth rate compared to its wild strain. To investigate the functional role of the putative ACMSD (Pyacmsd) of P. yezoensis, the putative Pyacmsd was cloned and expressed in Chlamydomonas reinhardtii. Recombinant C. reinhardtii cells with Pyacmsd (Cr_Pyacmsd) exhibited enhanced tolerance compared to control C. reinhardtii cells (Cr_control) under nitrogen starvation. Notably, Cr_Pyacmsd cells showed accumulation of lipids in nitrogen-enriched conditions. These results demonstrate the role of Pyacmsd in the generation of acetyl-coenzyme A. Thus, it can be used to enhance the production of biofuel using microalgae such as C. reinhardtii and increase the tolerance of other biological systems to nitrogen-deficient conditions.

Prostaglandin production by the microalga with heterologous expression of cyclooxygenase.

Prostaglandins (PGs) are the physiologically active compounds synthesized from C20 polyunsaturated fatty acids (PUFAs) by cyclooxygenase (COX) and a series of PG synthases, and are utilized as pharmaceuticals. Currently, commercialized PGs are mainly produced by chemical synthesis under harsh conditions. By contrast, bioproduction of PGs can be an alternative, environmental-friendly, and inexpensive process with genetic engineering of model plants, although these conventional host organisms contain a limited quantity of PG precursors. In this study, we established an efficient PG production process using the genetically engineered microalga Fistulifera solaris which is rich in C20 PUFAs. A cox gene derived from the red alga Agarophyton vermiculophyllum was introduced into F. solaris. As a result, a transformant clone with high cox expression produced PGs (i.e., PGD2 , PGE2 , PGF2α , and 15-ketoPGF2α derived from arachidonic acid, and PGD3 , PGE3 , and PGF3α derived from eicosapentaenoic acid) as revealed by liquid chromatography/mass spectrometry. The total content of PGs was 1290.4 ng/g of dry cell weight, which was higher than that produced in the transgenic plant reported previously. The results obtained in this study indicate that the C20 PUFA-rich microalga functionally expressing COX is a promising host for PG bioproduction.

Establishment of a firefly luciferase reporter assay system in the unicellular red alga Cyanidioschyzon merolae.

The firefly luciferase (Luc) reporter assay is a powerful tool used to analyze promoter activities in living cells. In this report, we established a firefly Luc reporter assay system in the unicellular model red alga Cyanidioschyzon merolae. A nitrite reductase (NIR) promoter-Luc fusion gene was integrated into the URA5.3 genomic region to construct the C. merolae NIR-Luc strain. Luc activities in the NIR-Luc strain were increased, correlating with the accumulation of endogenous NIR transcripts in response to nitrogen depletion. Luc activity was also significantly increased by the overexpression of the MYB1 gene, which encodes a transcription factor responsible for NIR promoter activation. Thus, our results demonstrate the utility of the Luc reporter system in C. merolae.

Efficient extraction and preservation of thermotolerant phycocyanins from red alga Cyanidioschyzon merolae.

C-Phycocyanin (PC) is a protein used commercially as a natural blue pigment produced by cyanobacteria, cryptophytes, and rhodophytes. Although it is industrially synthesized from the cyanobacterium Arthrospira platensis, PC requires high levels of energy for its extraction, which involves freezing of cells. However, as a protein, PC is easily denatured at extreme temperatures. In this study, we extracted PC from the red alga Cyanidioschyzon merolae, denoted as CmPC, and found that this protein was tolerant to high temperatures and acidic pH. CmPC was extracted by suspending cells in water mixed with various salts and organic acids without freeze-drying or freeze-thaw. The stability of CmPC varied with salt concentration and was destabilized by organic acids. Our results indicate that C. merolae is a potential candidate for PC production with thermotolerant properties.

Anti-Herpes simplex virus (HSV-1) activity and antioxidant capacity of carrageenan-rich enzymatic extracts from Solieria filiformis (Gigartinales, Rhodophyta).

Solieria filiformis has been reported to have molecules with various biological activities. In this study we used environmentally friendly extraction methods, such as enzyme-assisted extraction (EAE), as a first step to obtain bioactive compounds from this species. Five combinations of protease (PRO) and carbohydrase (AMG) were utilized (1:0, 0:1, 2:1, 1:1, 1:2 PRO:AMG) to obtain Water Soluble Enzymatic Hydrolysates (WSEHs). Extraction yields, biochemical and structural characterization, as well as in vitro activity against Herpes simplex virus type 1 (HSV-1) and antioxidant capacities were determined. All PRO:AMG combinations significantly improved yields. EAE yielded heterogeneous extracts rich in iota-carrageenan and phenols, as confirmed by FTIR spectra. The highest antiherpetic activity (EC50 4.5 ± 0.4 µg mL-1) was found in the WSEHs obtained under 2:1 PRO:AMG. At this combination high antioxidant capacity was also obtained for ABTS (2,2'-Azino-Bis-3-ethylbenzoThiazoline-6-Sulfonic acid) radical scavenging activity and Ferric Reducing Antioxidant Power (FRAP). These could probably play a synergistic role associated to the strong antiviral activity obtained. These results suggest that 2:1 PRO:AMG could be effective in promoting the hydrolytic breakdown of high MW polysaccharides, contributing to the improvement of WSEHs bioactivity. Although Solieria filiformis WSEHs showed promising results, further research, including separation and purification techniques are needed.

X-ray structure of C-phycocyanin from Galdieria phlegrea: Determinants of thermostability and comparison with a C-phycocyanin in the entire phycobilisome.

Galdieria phlegrea is a polyextremophilic red alga belonging to Cyanidiophyceae. Galdieria phlegrea C-phycocyanin (GpPC), an abundant light-harvesting pigment with an important role in energy capture and transfer to photosystems, is the C-phycocyanin (C-PC) with the highest thermal stability described so far. GpPC also presents interesting antioxidant and anticancer activities. The X-ray structure of the protein was here solved. GpPC is a [(αß)3]2 hexamer, with the phycocyanobilin chromophore attached to Cys84α, Cys82ß and Cys153ß. Details of geometry and interaction with solvent of the chromophores are reported. Comparison with the structure of a C-PC in the entire Porphyridium purpureum phycobilisome system reveals that linker polypeptides have a significant effect on the local structure of the chromophores environment. Comparative analyses with the structures of other purified C-PCs, which were carried out including re-refined models of G. sulphuraria C-PC, reveal that GpPC presents a significantly higher number of inter-trimer salt bridges. Notably, the higher number of salt bridges at the (αß)3/(αß)3 interface is not due to an increased number of charged residues in this region, but to subtle conformational variations of their side chains, which are the result of mutations of close polar and non-polar residues.

The lineage and diversity of putative amino acid sensor ACR proteins in plants.

Amino acid metabolic enzymes often contain a regulatory ACT domain, named for aspartate kinase, chorismate mutase, and TyrA (prephenate dehydrogenase). Arabidopsis encodes 12 putative amino acid sensor ACT repeat (ACR) proteins, all containing ACT repeats but no identifiable catalytic domain. Arabidopsis ACRs comprise three groups based on domain composition and sequence: group I and II ACRs contain four ACTs each, and group III ACRs contain two ACTs. Previously, all three groups had been documented only in Arabidopsis. Here, we extended this to algae and land plants, showing that all three groups of ACRs are present in most, if not all, land plants, whereas among algal ACRs, although quite diverse, only group III is conserved. The appearance of canonical group I and II ACRs thus accompanied the evolution of plants from living in water to living on land. Alignment of ACTs from plant ACRs revealed a conserved motif, DRPGLL, at the putative ligand-binding site. Notably, the unique features of the DRPGLL motifs in each ACT domain are conserved in ACRs from algae to land plants. The conservation of plant ACRs is reminiscent of that of human cellular arginine sensor for mTORC1 (CASTOR1), a member of a small protein family highly conserved in animals. CASTOR proteins also have four ACT domains, although the sequence identities between ACRs and CASTORs are very low. Thus, plant ACRs and animal CASTORs may have adapted the regulatory ACT domains from a more ancient metabolic enzyme, and then evolved independently.

Peptidyl-prolyl isomerase and the biological activities of recombinant protein cyclophilin from Pyropia yezoensis (PyCyp).

Cyclophilins are highly conserved proteins associated with peptidyl-prolyl cis-trans isomerase activity (PPIase). The present study was designed to analyze the biological activity of recombinant cyclophilin from the marine red algae Pyropia yezoensis (PyCyp). The cyclophilin gene from P. yezoensis was cloned into the pPROEX-HTA expression vector. The plasmid was transformed into BL21 Escherichia coli by high efficiency transformation. Recombinant protein was expressed using 0.1 mM IPTG and the fusion protein was purified by affinity column chromatography. The His-tag was removed by TEV protease. The recombinant protein was further purified on a HiPrep Sephacryl S-200 HR column and by reversed-phase high performance liquid chromatography with a Sep-pak plus C18 column. Purified cyclophilin was characterized by a variety of analytical methods and analyzed for its peptidyl-prolyl isomerase activity. Our recombinant PyCyp was shown to catalyze cis-trans isomerization. PyCyp was also evaluated for antimicrobial activity against both Gram-positive and Gram-negative bacteria cultures and showed significant antibacterial activity against tested pathogens. PyCyp was shown to permeabilize bacterial membranes as evidenced by increased fluorescence intensity in SYTOX Green uptake assays with Staphylococcus aureus. The radical scavenging activity of PyCyp increased in a dose-dependent manner, indicating significant antioxidant activity. This study provides information for the development of therapeutic proteins from marine algae.

Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: CtOYE and GsOYE.

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,ß-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.

Functional Characterization of Lycopene Cyclases Illustrates the Metabolic Pathway toward Lutein in Red Algal Seaweeds.

Carotenoids are essential phytonutrients synthesized by all photosynthetic organisms. Acyclic lycopene is the first branching point for carotenoid biosynthesis. Lycopene ß- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of its open ends and direct the metabolic flux into different downstream branches. Carotenoids of the ß,ß-branch (e.g., ß-carotene) are found in all photosynthetic organisms, but those of the ß,ε-branch (e.g., lutein) are generally absent in cyanobacteria, heterokonts, and some red algae. Although both LCYBs and LCYEs have been characterized from land plants, there are only a few reports on LCYs from cyanobacteria and algae. Here, we cloned four LCY genes from Porphyra umbilicalis and Pyropia yezoensis (susabi-nori) of Bangiales, the most primitive red algal order that synthesizes lutein. Our functional characterization in both Escherichia coli and Arabidopsis thaliana demonstrated that each species has a pair of LCYB and LCYE. Similar to LCYs from higher plants, red algal LCYBs cyclize both ends of lycopene, and their LCYEs only cyclize a single end. The characterization of LCYEs from red algae resolved the first bifurcation step toward ß-carotene and lutein biosynthesis. Our phylogenetic analysis suggests that LCYEs of the green lineage and the red algae originated separately during evolution.

Remodeling of excitation energy transfer in extremophilic red algal PSI-LHCI complex during light adaptation.

Photosynthetic PSI-LHCI complexes from an extremophilic red alga C. merolae grown under varying light regimes are characterized by decreasing size of LHCI antenna with increasing illumination intensity [1]. In this study we applied time-resolved fluorescence spectroscopy to characterize the kinetics of energy transfer processes in three types of PSI-LHCI supercomplexes isolated from the low (LL), medium (ML) and extreme high light (EHL) conditions. We show that the average rate of fluorescence decay is not correlated with the size of LHCI antenna and is twice faster in complexes isolated from ML-grown cells (~25-30 ps) than from both LL- and EHL-exposed cells (~50-55 ps). The difference is mainly due to a contribution of a long ~100-ps decay component detected only for the latter two PSI samples. We propose that the lack of this phase in ML complexes is caused by perfect coupling of this antenna to PSI core and lack of low-energy chlorophylls in LHCI. On the other hand, the presence of the slow, ~100-ps, fluorescence decay component in LL and EHL complexes may be due to the weak coupling between PSI core and LHCI antenna complex, and due to the presence of particularly low-energy or red chlorophylls in LHCI. Our study has revealed the remarkable functional flexibility of light harvesting strategies that have evolved in the extremophilic red algae in response to harsh or limiting light conditions involving accumulation of low energy chlorophylls that exert two distinct functions: as energy traps or as far-red absorbing light harvesting antenna, respectively.

On the nature of uncoupled chlorophylls in the extremophilic photosystem I-light harvesting I supercomplex.

Photosystem I core-light-harvesting antenna supercomplexes (PSI-LHCI) were isolated from the extremophilic red alga Cyanidioschyzon merolae and studied by three fluorescence techniques in order to characterize chlorophylls (Chls) energetically uncoupled from the PSI reaction center (RC). Such Chls are observed in virtually all optical experiments of any PSI core and PSI-LHCI supercomplex preparations across various species and may influence the operation of PSI-based solar cells and other biohybrid systems. However, the nature of the uncoupled Chls (uChls) has never been explored deeply before. In this work, the amount of uChls was controlled by stirring the solution of C. merolae PSI-LHCI supercomplex samples at elevated temperature (~303 K) and was found to increase from <2% in control samples up to 47% in solutions stirred for 3.5 h. The fluorescence spectrum of uChls was found to be blue-shifted by ~20 nm (to ~680 nm) relative to the fluorescence band from Chls that are well coupled to PSI RC. This effect indicates that mechanical stirring leads to disappearance of some red Chls (emitting at above ~700 nm) that are present in the intact LHCI antenna associated with the PSI core. Comparative diffusion studies of control and stirred samples by fluorescence correlation spectroscopy together with biochemical analysis by SDS-PAGE and BN-PAGE indicate that energetically uncoupled Lhcr subunits are likely to be still physically attached to the PSI core, albeit with altered three-dimensional organization due to the mechanical stress.

Cyanidioschyzon merolae aurora kinase phosphorylates evolutionarily conserved sites on its target to regulate mitochondrial division.

The mitochondrion is an organelle that was derived from an endosymbiosis. Although regulation of mitochondrial growth by the host cell is necessary for the maintenance of mitochondria, it is unclear how this regulatory mechanism was acquired. To address this, we studied the primitive unicellular red alga Cyanidioschyzon merolae, which has the simplest eukaryotic genome and a single mitochondrion. Here we show that the C. merolae Aurora kinase ortholog CmAUR regulates mitochondrial division through phosphorylation of mitochondrial division ring components. One of the components, the Drp1 ortholog CmDnm1, has at least four sites phosphorylated by CmAUR. Depletion of the phosphorylation site conserved among eukaryotes induced defects such as mitochondrial distribution on one side of the cell. Taken together with the observation that human Aurora kinase phosphorylates Drp1 in vitro, we suggest that the phosphoregulation is conserved from the simplest eukaryotes to mammals, and was acquired at the primitive stage of endosymbiosis.

RSH enzyme diversity for (p)ppGpp metabolism in Phaeodactylum tricornutum and other diatoms.

The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae.

PI signal transduction and ubiquitination respond to dehydration stress in the red seaweed Gloiopeltis furcata under successive tidal cycles.

BACKGROUND: Intermittent dehydration caused by tidal changes is one of the most important abiotic factors that intertidal seaweeds must cope with in order to retain normal growth and reproduction. However, the underlying molecular mechanisms for the adaptation of red seaweeds to repeated dehydration-rehydration cycles remain poorly understood. RESULTS: We chose the red seaweed Gloiopeltis furcata as a model and simulated natural tidal changes with two consecutive dehydration-rehydration cycles occurring over 24 h in order to gain insight into key molecular pathways and regulation of genes which are associated with dehydration tolerance. Transcription sequencing assembled 32,681 uni-genes (GC content = 55.32%), of which 12,813 were annotated. Weighted gene co-expression network analysis (WGCNA) divided all transcripts into 20 modules, with Coral2 identified as the key module anchoring dehydration-induced genes. Pathways enriched analysis indicated that the ubiquitin-mediated proteolysis pathway (UPP) and phosphatidylinositol (PI) signaling system were crucial for a successful response in G. furcata. Network-establishing and quantitative reverse transcription PCR (qRT-PCR) suggested that genes encoding ubiquitin-protein ligase E3 (E3-1), SUMO-activating enzyme sub-unit 2 (SAE2), calmodulin (CaM) and inositol-1,3,4-trisphosphate 5/6-kinase (ITPK) were the hub genes which responded positively to two successive dehydration treatments. Network-based interactions with hub genes indicated that transcription factor (e.g. TFIID), RNA modification (e.g. DEAH) and osmotic adjustment (e.g. MIP, ABC1, Bam1) were related to these two pathways. CONCLUSIONS: RNA sequencing-based evidence from G. furcata enriched the informational database for intertidal red seaweeds which face periodic dehydration stress during the low tide period. This provided insights into an increased understanding of how ubiquitin-mediated proteolysis and the phosphatidylinositol signaling system help seaweeds responding to dehydration-rehydration cycles.

What Happened to the Phycobilisome?

The phycobilisome (PBS) is the major light-harvesting complex of photosynthesis in cyanobacteria, red algae, and glaucophyte algae. In spite of the fact that it is very well structured to absorb light and transfer it efficiently to photosynthetic reaction centers, it has been completely lost in the green algae and plants. It is difficult to see how selection alone could account for such a major loss. An alternative scenario takes into account the role of chance, enabled by (contingent on) the evolution of an alternative antenna system early in the diversification of the three lineages from the first photosynthetic eukaryote.

Insight into transketolase of Pyropia haitanensis under desiccation stress based on integrative analysis of omics and transformation.

BACKGROUND: Pyropia haitanensis, distributes in the intertidal zone, can tolerate water losses exceeding 90%. However, the mechanisms enabling P. haitanensis to survive harsh conditions remain uncharacterized. To elucidate the mechanism underlying P. haitanensis desiccation tolerance, we completed an integrated analysis of its transcriptome and proteome as well as transgenic Chlamydomonas reinhardtii carrying a P. haitanensis gene. RESULTS: P. haitanensis rapidly adjusted its physiological activities to compensate for water losses up to 60%, after which, photosynthesis, antioxidant systems, chaperones, and cytoskeleton were activated to response to severe desiccation stress. The integrative analysis suggested that transketolase (TKL) was affected by all desiccation treatments. Transgenic C. reinhardtii cells overexpressed PhTKL grew better than the wild-type cells in response to osmotic stress. CONCLUSION: P. haitanensis quickly establishes acclimatory homeostasis regarding its transcriptome and proteome to ensure its thalli can recover after being rehydrated. Additionally, PhTKL is vital for P. haitanensis desiccation tolerance. The present data may provide new insights for the breeding of algae and plants exhibiting enhanced desiccation tolerance.

Putative trehalose biosynthesis proteins function as differential floridoside-6-phosphate synthases to participate in the abiotic stress response in the red alga Pyropia haitanensis.

BACKGROUND: The heteroside floridoside is a primary photosynthetic product that is known to contribute to osmotic acclimation in almost all orders of Rhodophyta. However, the encoding genes and enzymes responsible for the synthesis of floridoside and its isomeric form, L- or D-isofloridoside, are poorly studied. RESULTS: Here, four putative trehalose-6-phosphate synthase (TPS) genes, designated as PhTPS1, PhTPS2, PhTPS3, and PhTPS4, were cloned and characterized from the red alga Pyropia haitanensis (Bangiophyceae). The deduced amino acid sequence is similar to the annotated TPS proteins of other organisms, especially the UDP-galactose substrate binding sites of PhTPS1, 2, which are highly conserved. Of these, PhTPS1, 4 are involved in the biosynthesis of floridoside and isofloridoside, with isofloridoside being the main product. PhTPS3 is an isofloridoside phosphate synthase, while PhTPS2 exhibits no activity. When challenged by desiccation, high temperature, and salt stress, PhTPS members were expressed to different degrees, but the responses to thermal stress and desiccation were stronger. CONCLUSIONS: Thus, in P. haitanensis, PhTPSs encode the enzymatical activity of floridoside and isofloridoside phosphate synthase and are crucial for the abiotic stress defense response.

Organellar DNA Polymerases in Complex Plastid-Bearing Algae.

DNA replication in plastids and mitochondria is generally regulated by nucleus-encoded proteins. In plants and red algae, a nucleus-encoded enzyme called POP (plant and protist organellar DNA polymerase) is involved in DNA replication in both organelles by virtue of its dual localization. POPs are family A DNA polymerases, which include bacterial DNA polymerase I (PolI). POP homologs have been found in a wide range of eukaryotes, including plants, algae, and non-photosynthetic protists. However, the phylogeny and subcellular localizations of POPs remain unclear in many algae, especially in secondary and tertiary plastid-bearing groups. In this study, we report that chlorarachniophytes possess two evolutionarily distinct POPs, and fluorescent protein-tagging experiments demonstrate that they are targeted to the secondary plastids and mitochondria, respectively. The timing of DNA replication is different between the two organelles in the chlorarachniophyte Bigelowiella natans, and this seems to be correlated to the transcription of respective POP genes. Dinoflagellates also carry two distinct POP genes, possibly for their plastids and mitochondria, whereas haptophytes and ochrophytes have only one. Therefore, unlike plants, some algal groups are likely to have evolved multiple DNA polymerases for various organelles. This study provides a new insight into the evolution of organellar DNA replication in complex plastid-bearing organisms.