Kinetic properties of 137Cs uptake by the cesium-accumulating eustigmatophycean microalga.

Cesium-137 (137Cs) is one of the radioactive substances that was released into the environment as a result of the Fukushima nuclear disaster. Radiocesium exposure is of great concern due to its potential environmental implications. However, research on 137Cs removal using algae is still limited. This is the first report to describe the kinetic properties of 137Cs uptake by Vacuoliviride crystalliferum in the presence and absence of potassium. In this work, we studied the kinetic properties of 137Cs uptake using a freshwater microalga, V. crystalliferum (NIES 2860). We also analyzed the effects of temperature, light, and potassium (K) on the 137Cs uptake. Results showed that V. crystalliferum can remove up to 90% of 157 nM 137Cs within an hour. At 20 °C, the removal increased by up to 96%, compared to less than 10% at 5 °C. However, the removal was inhibited by nearly 90% in the dark compared to the removal in the light, implying that V. crystalliferum cells require energy to accumulate 137Cs. In the inhibition assay, K concentrations ranged from 0 to 500 µM and the inhibitory constant (Ki) for K was determined to be 16.7 µM. While in the uptake assay without potassium (- K), the Michaelis constant (Km) for Cs was 45 nM and increased to 283 nM by the addition of 20 µM potassium (+ K), indicating that V. crystalliferum had a high affinity for 137Cs. In addition, the maximum uptake velocity (Vmax) also increased from 6.75 to 21.10 nmol (mg Chl h)-1, implying the existence of Cs active transport system. In conclusion, V. crystalliferum is capable of removing radioactive 137Cs from the environment and the removal was favorable at both normal temperature and in the light.

Association of Phosphatidylinositol-Specific Phospholipase C with Calcium-Induced Biomineralization in the Coccolithophore Emiliania huxleyi.

Biomineralization by calcifying microalgae is a precisely controlled intracellular calcification process that produces delicate calcite scales (or coccoliths) in the coccolithophore Emiliania huxleyi (Haptophycea). Despite its importance in biogeochemical cycles and the marine environment globally, the underlying molecular mechanism of intracellular coccolith formation, which requires calcium, bicarbonate, and coccolith-polysaccharides, remains unclear. In E. huxleyi CCMP 371, we demonstrated that reducing the calcium concentration from 10 (ambient seawater) to 0.1 mM strongly restricted coccolith production, which was then recovered by adding 10 mM calcium, irrespective of inorganic phosphate conditions, indicating that coccolith production could be finely controlled by the calcium supply. Using this strain, we investigated the expression of differentially expressed genes (DEGs) to observe the cellular events induced by changes in calcium concentrations. Intriguingly, DEG analysis revealed that the phosphatidylinositol-specific phospholipase C (PI-PLC) gene was upregulated and coccolith production by cells was blocked by the PI-PLC inhibitor U73122 under conditions closely associated with calcium-induced calcification. These findings imply that PI-PLC plays an important role in the biomineralization process of the coccolithophore E. huxleyi.

Overexpression of Tisochrysis lutea Akd1 identifies a key cold-induced alkenone desaturase enzyme.

Alkenones are unusual long-chain neutral lipids that were first identified in oceanic sediments. Currently they are regarded as reliable palaeothermometers, since their unsaturation status changes depending on temperature. These molecules are synthesised by specific haptophyte algae and are stored in the lipid body as the main energy storage molecules. However, the molecular mechanisms that regulate the alkenone biosynthetic pathway, especially the low temperature-dependent desaturation reaction, have not been elucidated. Here, using an alkenone-producing haptophyte alga, Tisochrysis lutea, we show that the alkenone desaturation reaction is catalysed by a newly identified desaturase. We first isolated two candidate desaturase genes and found that one of these genes was drastically upregulated in response to cold stress. Gas chromatographic analysis revealed that the overexpression of this gene, named as Akd1 finally, increased the conversion of di-unsaturated C37-alkenone to tri-unsaturated molecule by alkenone desaturation, even at a high temperature when endogenous desaturation is efficiently suppressed. We anticipate that the Akd1 gene will be of great help for elucidating more detailed mechanisms of temperature response of alkenone desaturation, and identification of active species contributing alkenone production in metagenomic and/or metatranscriptomic studies in the field of oceanic biogeochemistry.

Cold-induced metabolic conversion of haptophyte di- to tri-unsaturated C37 alkenones used as palaeothermometer molecules.

The cosmopolitan marine haptophyte alga Emiliania huxleyi accumulates very long-chain (C37-C40) alkyl ketones with two to four trans-type carbon-carbon double bonds (alkenones). These compounds are used as biomarkers of haptophytes and as palaeothermometers for estimating sea-surface temperatures in biogeochemistry. However, the biosynthetic pathway of alkenones in algal cells remains enigmatic, although it is well known that the C37 tri-unsaturated alkenone (K37:3) becomes dominant at low temperatures, either by desaturation of K37:2 or by a separate pathway involving the elongation of tri-unsaturated alkenone precursors. Here, we present experimental evidence regarding K37:3 synthesis. Using the well-known cosmopolitan alkenone producer E. huxleyi, we labelled K37:2 with 13C by incubating cells with 13C-bicarbonate in the light at 25 °C under conditions of little if any K37:3 production. After stabilisation of the 13C-K37:2 level by depleting 13C-bicarbonate from the medium, the temperature was suddenly reduced to 15 °C. The 13C-K37:2 level rapidly decreased, and the 13C-K37:3 level increased, whereas the total 13C-K37 level-namely [K37:2 + K37:3]-remained constant. These 13C-pulse-chase-like experimental results indicate that 13C-K37:2 is converted directly to 13C-K37:3 by a desaturation reaction that is promoted by a cold signal. This clear-cut experimental evidence is indicative of the existence of a cold-signal-triggered desaturation reaction in alkenone biosynthesis.

Construction of a cyanobacterium synthesizing cyclopropane fatty acids.

Microalgae have received much attention as a next-generation source of biomass energy. However, most of the fatty acids (FAs) from microalgae are multiply unsaturated; thus, the biofuels derived from them are fluid, but vulnerable to oxidation. In this study, we attempted to synthesize cyclopropane FAs in the cyanobacterium Synechocystis sp. PCC 6803 by expressing the cfa gene for cyclopropane FA synthase from Escherichia coli with the aim of producing FAs that are fluid and stable in response to oxidization. We successfully synthesized cyclopropane FAs in Synechocystis with a yield of ~30% of total FAs. Growth of the transformants was altered, particularly at low temperatures, but photosynthesis and respiration were not significantly affected. C16:1(∆9) synthesis in the desA(-)/desD(-) strain by expression of the desC2 gene for sn-2 specific ∆9 desaturase positively affected growth at low temperatures via promotion of various cellular processes, with the exceptions of photosynthesis and respiration. Estimation of the apparent activities of desaturases suggested that some acyl-lipid desaturases might recognize the lipid side chain.

Characterization of a novel gene involved in cadmium accumulation screened from sponge-associated bacterial metagenome.

Metagenome research has brought much attention for the identification of important and novel genes of industrial and pharmaceutical value. Here, using a metagenome library constructed from bacteria associated with the marine sponge, Styllisa massa, a high-throughput screening technique using radioisotope was implemented to screen for cadmium (Cd) binding or accumulation genes. From a total of 3301 randomly selected clones, a clone 247-11C was identified as harboring an open reading frame (ORF) showing Cd accumulation characteristics. The ORF, termed as ORF5, was further analyzed by protein functional studies to reveal the presence of a protein, Cdae-1. Cdae-1, composed of a signal peptide and domain harboring an E(G/A)KCG pentapeptide motif, enhanced Cd accumulation when expressed in Escherichia coli. Although showing no direct binding to Cd in vitro, the presence of important amino acid residues related to Cd detoxification suggests that Cdae-1 may possess a different mechanism from known Cd binding proteins such as metallothioneins (MTs) and phytochelatins (PCs). In summary, using the advantage of bacterial metagenomes, our findings in this work suggest the first report on the identification of a unique protein involved in Cd accumulation from bacteria associated with a marine sponge.

Light intensity modulation by coccoliths of Emiliania huxleyi as a micro-photo-regulator.

In this study, we present experimental evidence showing that coccoliths have light-scattering anisotropy that contributes to a possible control of solar light exposure in the ocean. Changing the angle between the incident light and an applied magnetic field causes differences in the light-scattering intensities of a suspension of coccoliths isolated from Emiliania huxleyi. The magnetic field effect is induced by the diamagnetic torque force directing the coccolith radial plane perpendicular to the applied magnetic fields at 400 to 500 mT. The developed technique reveals the light-scattering anisotropies in the 3-µm-diameter floating coccoliths by orienting themselves in response to the magnetic fields. The detached coccolith scatters radially the light incident to its radial plane. The experimental results on magnetically oriented coccoliths show that an individual coccolith has a specific direction of light scattering, although the possible physiological effect of the coccolith remains for further study, focusing on the light-scattering anisotropies of coccoliths on living cells.

Proteomic analysis of lipid body from the alkenone-producing marine haptophyte alga Tisochrysis lutea.

Lipid body (LB) is recognized as the cellular carbon and energy storage organelle in many organisms. LBs have been observed in the marine haptophyte alga Tisochrysis lutea that produces special lipids such as long-chain (C37 -C40) ketones (alkenones) with 2-4 trans-type double bonds. In this study, we succeeded in developing a modified method to isolate LB from T. lutea. Purity of isolated LBs was confirmed by the absence of chlorophyll auto-fluorescence and no contamination of the most abundant cellular protein ribulose-1,5-bisphosphate carboxylase/oxygenase. As alkenones predominated in the LB by GC-MS analysis, the LB can be more appropriately named as "alkenone body (AB)." Extracted AB-containing proteins were analyzed by the combination of 1DE (SDS-PAGE) and MS/MS for confident protein identification and annotated using BLAST tools at National Center for Biotechnology Information. Totally 514 proteins were identified at the maximum. The homology search identified three major proteins, V-ATPase, a hypothetical protein EMIHUDRAFT_465517 found in other alkenone-producing haptophytes, and a lipid raft-associated SPFH domain-containing protein. Our data suggest that AB of T. lutera is surrounded by a lipid membrane originating from either the ER or the ER-derived four layer-envelopes chloroplast and function as the storage site of alkenones and alkenes.

Quantitative Analysis of Carbon Flow into Photosynthetic Products Functioning as Carbon Storage in the Marine Coccolithophore, Emiliania huxleyi.

The bloom-forming coccolithophore Emiliania huxleyi (Haptophyta) is a dominant marine phytoplankton, cells of which are covered with calcareous plates (coccoliths). E. huxleyi produces unique lipids of C37-C40 long-chain ketones (alkenones) with two to four trans-unsaturated bonds, ß-glucan (but not α-glucan) and acid polysaccharide (AP) associated with the morphogenesis of CaCO3 crystals in coccoliths. Despite such unique features, there is no detailed information on the patterns of carbon allocation into these compounds. Therefore, we performed quantitative estimation of carbon flow into various macromolecular products by conducting (14)C-radiotracer experiments using NaH(14)CO3 as a substrate. Photosynthetic (14)C incorporation into low molecular-mass compounds (LMC), extracellular AP, alkenones, and total lipids except alkenones was estimated to be 35, 13, 17, and 25 % of total (14)C fixation in logarithmic growth phase cells and 33, 19, 18, and 18 % in stationary growth phase cells, respectively. However, less than 1 % of (14)C was incorporated into ß-glucan in both cells. (14)C-mannitol occupied ca. 5 % of total fixed (14)C as the most dominant LMC product. Levels of all (14)C compounds decreased in the dark. Therefore, alkenones and LMC (including mannitol), but not ß-glucan, function in carbon/energy storage in E. huxleyi, irrespective of the growth phase. Compared with other algae, the low carbon flux into ß-glucan is a unique feature of carbon metabolism in E. huxelyi.

n-Nonacosadienes from the marine haptophytes Emiliania huxleyi and Gephyrocapsa oceanica.

The hydrocarbons in cultures of marine haptophytes Emiliania huxleyi NIES837 and Gephyrocapsa oceanica NIES1315 were analyzed, and nonacosadienes and hentriacontadienes were detected as the major compounds in both strains. C29 and C31 monoenes and di-, tri- and tetra-unsaturated C33 alkenes were also detected as minor compounds but not C37 and C38 alkenes. The positions of the double bonds in the C29 and C31 alkenes were determined by mass spectrometry of their dimethyl disulfide (DMDS) adducts. Among the four C29 alkenes identified, the most abundant isomer was 2,20-nonacosadiene, and the other three compounds were 1,20-nonacosadiene, 3,20-nonacosadiene and 9-nonacosene, respectively. Hitherto, 2,20-nonacosadiene and 3,20-nonacosadiene were unknown to be natural products. The double bond at the n-9 (ω9) position in these C29 alkenes is hypothesized to be derived from precursors of unsaturated fatty acids possessing an n-9 double bond, such as (9Z)-9-octadecenoic acid. Nonacosadienes have the potential for being used as distinct haptophyte biomarkers.

Functional screening of a novel Δ15 fatty acid desaturase from the coccolithophorid Emiliania huxleyi.

The coccolithophorid Emiliania huxleyi is a bloom-forming marine phytoplankton thought to play a key role as a biological pump that transfers carbon from the surface to the bottom of the ocean, thus contributing to the global carbon cycle. This alga is also known to accumulate a variety of polyunsaturated fatty acids. At 25°C, E. huxleyi produces mainly 14:0, 18:4n-3, 18:5n-3 and 22:6n-3. When the cells were transferred from 25°C to 15°C, the amount of unsaturated fatty acids, i.e. 18:1n-9, 18:3n-3 and 18:5n-3, gradually increased. Among the predicted desaturase genes whose expression levels were up-regulated at low temperature, we identified a gene encoding novel ∆15 fatty acid desaturase, EhDES15, involved in the production of n-3 polyunsaturated fatty acids in E. huxleyi. This desaturase contains a putative transit sequence for localization in chloroplasts and a ∆6 desaturase-like domain, but it does not contain a cytochrome b5 domain nor typical His-boxes found in ∆15 desaturases. Heterologous expression of EhDES15 cDNA in cyanobacterium Synechocystis sp. PCC 6803 cells increased the level of n-3 fatty acid species, which are produced at low levels in wild-type cells grown at 30°C. The orthologous genes are only conserved in the genomes of prasinophytes and cryptophytes. The His-boxes conserved in orthologues varied from that of the canonical ∆15 desaturases. These results suggested the gene encodes a novel ∆15 desaturase responsible for the synthesis of 18:3n-3 from 18:2n-6 in E. huxleyi.

Difference in physiological responses of growth, photosynthesis and calcification of the coccolithophore Emiliania huxleyi to acidification by acid and CO2 enrichment.

Ocean acidification, one of the great global environmental issues at present, is expected to result in serious damage on marine calcareous organisms such as corals and calcifying algae, which potentially release huge amounts of CO2 from the ocean to the atmosphere. The coccolithophore, Emiliania huxleyi (Haptophyceae), which frequently produces blooms, has greatly contributed to the biological CO2 pump. This study was aimed at analyzing effects of how E. huxleyi responds to acidification. Acidification was performed by two methods, namely by just adding HCl under bubbling ordinary air at 8.2-8.4, 7.6-7.8 and 7.1-7.3 (acidification by HCl) and by bubbling with ordinary air or with increased CO2 concentration such as 406, 816 and 1,192 ppm that maintained pH of the medium at 8.0-8.3, 7.6-7.9 and 7.5-7.7 (acidification by CO2 enrichment). As a result, cell growth and cellular calcification of E. huxleyi were strongly damaged by acidification by HCl, but not by acidification by CO2 enrichment. The activities of photosystems such as F v/F m and ϕPSII were not affected by any acidification conditions while photosynthetic O2 evolution was slightly stimulated. A (45)Ca-radiotracer experiment revealed that Ca(2+)-uptake was strongly suppressed by acidification with HCl. This suppression recovered after increasing the dissolved inorganic carbon (DIC) concentration and further stimulated by an additional increase in DIC concentration. The production of storage and coccolith polysaccharides was increased by acidification by HCl and also highly stimulated by acidification with CO2 enrichment. The present study clearly showed that the coccolithophore, E. huxleyi, has an ability to respond positively to acidification with CO2 enrichment, but not just acidification.

Functional analysis of the N-terminal region of an essential histidine kinase, Hik2, in the cyanobacterium Synechocystis sp. PCC 6803.

Histidine kinases are sensory proteins involved in the perception of environmental changes. Here, we characterized one of three essential histidine kinases, Hik2, in the cyanobacterium Synechocystis sp. PCC 6803 by constructing a fused sensor, Hik2n-Hik7c, which has the signal input domain of Hik2 and the kinase domain of the phosphate-deficiency sensor Hik7. The coding region of the hik7 gene was replaced with the fused sensor to evaluate the signalling activity in vivo as the activity of alkaline phosphatase (AP), which is regulated by Hik7. Cells expressing Hik2n-Hik7c had weak AP activities under standard growth conditions. Saline stress by NaCl induced AP activity in a dose-dependent manner. Analysis of the effects of several salt compounds on induction of AP activity indicated that Hik2n-Hik7c responded to Cl- concentration. Amino acid substitution in the signal input domain of Hik2 resulted in loss of this responsiveness. These results suggest that the signal input domain of Hik2 responds to environmental Cl- concentration in Synechocystis.

Global searches for microalgae and aquatic plants that can eliminate radioactive cesium, iodine and strontium from the radio-polluted aquatic environment: a bioremediation strategy.

The Fukushima 1 Nuclear Power Plant accident in March 2011 released an enormously high level of radionuclides into the environment, a total estimation of 6.3 × 10¹7 Bq represented by mainly radioactive Cs, Sr, and I. Because these radionuclides are biophilic, an urgent risk has arisen due to biological intake and subsequent food web contamination in the ecosystem. Thus, urgent elimination of radionuclides from the environment is necessary to prevent substantial radiopollution of organisms. In this study, we selected microalgae and aquatic plants that can efficiently eliminate these radionuclides from the environment. The ability of aquatic plants and algae was assessed by determining the elimination rate of radioactive Cs, Sr and I from culture medium and the accumulation capacity of radionuclides into single cells or whole bodies. Among 188 strains examined from microalgae, aquatic plants and unidentified algal species, we identified six, three and eight strains that can accumulate high levels of radioactive Cs, Sr and I from the medium, respectively. Notably, a novel eustigmatophycean unicellular algal strain, nak 9, showed the highest ability to eliminate radioactive Cs from the medium by cellular accumulation. Our results provide an important strategy for decreasing radiopollution in Fukushima area.

Biofuels as a sustainable energy source: an update of the applications of proteomics in bioenergy crops and algae.

Sustainable energy is the need of the 21st century, not because of the numerous environmental and political reasons but because it is necessary to human civilization's energy future. Sustainable energy is loosely grouped into renewable energy, energy conservation, and sustainable transport disciplines. In this review, we deal with the renewable energy aspect focusing on the biomass from bioenergy crops to microalgae to produce biofuels to the utilization of high-throughput omics technologies, in particular proteomics in advancing our understanding and increasing biofuel production. We look at biofuel production by plant- and algal-based sources, and the role proteomics has played therein. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Changes in alkaline band formation and calcification of corticated charophyte Chara globularis.

Calcification by charophytes improves the quality of water, although most studies on calcification have only examined ecorticate species. We investigated the formation and relationship of alkalines and acids with regard to calcification on internodal cells in Chara corallina, an ecorticate species, and Chara globularis, a corticate species. We observed that alkaline and acidic areas with distinct banding patterns form on the internodal cells of C. corallina. The entire periphery of internodal cells was alkalized, and no distinct acidic area developed in C. globularis. By electron microscopy of these internodal cells, the calcified areas occurred primarily in alkaline areas with a banding pattern in C. coralline. However, phenomenon also occurred homogeneously inside of the entire cortex and cell wall in C. globularis. We also investigated the formation and relatiohship of alkalines and acids with regard to calcification on internodal cells of various ages from a single thallus of C. globularis. For internodal cells of C. globularis, a uniform calcified area lay between the cell wall and cortex on all cells, irrespective of age. In contrast, young cells bore an alkaline area that was uniform and widespread throughout their entire periphery, but the alkaline area in older cells was split into smaller segments in a banding pattern. Acidic areas were absent in young cells. These results indicate that the mechanisms by which alkaline and acid areas form differ in the presence and absence of cortex and between species.

Anaerobic coculture of microalgae with Thermosipho globiformans and Methanocaldococcus jannaschii at 68°C enhances generation of n-alkane-rich biofuels after pyrolysis.

We tested different alga-bacterium-archaeon consortia to investigate the production of oil-like mixtures, expecting that n-alkane-rich biofuels might be synthesized after pyrolysis. Thermosipho globiformans and Methanocaldococcus jannaschii were cocultured at 68°C with microalgae for 9 days under two anaerobic conditions, followed by pyrolysis at 300°C for 4 days. Arthrospira platensis (Cyanobacteria), Dunaliella tertiolecta (Chlorophyta), Emiliania huxleyi (Haptophyta), and Euglena gracilis (Euglenophyta) served as microalgal raw materials. D. tertiolecta, E. huxleyi, and E. gracilis cocultured with the bacterium and archaeon inhibited their growth and CH(4) production. E. huxleyi had the strongest inhibitory effect. Biofuel generation was enhanced by reducing impurities containing alkanenitriles during pyrolysis. The composition and amounts of n-alkanes produced by pyrolysis were closely related to the lipid contents and composition of the microalgae. Pyrolysis of A. platensis and D. tertiolecta containing mainly phospholipids and glycolipids generated short-carbon-chain n-alkanes (n-tridecane to n-nonadecane) and considerable amounts of isoprenoids. E. gracilis also produced mainly short n-alkanes. In contrast, E. huxleyi containing long-chain (31 and 33 carbon atoms) alkenes and very long-chain (37 to 39 carbon atoms) alkenones, in addition to phospholipids and glycolipids, generated a high yield of n-alkanes of various lengths (n-tridecane to n-pentatriacontane). The gas chromatography-mass spectrometry (GC-MS) profiles of these n-alkanes were similar to those of native petroleum crude oils despite containing a considerable amount of n-hentriacontane. The ratio of phytane to n-octadecane was also similar to that of native crude oils.

Characterization of the subdomains in the N-terminal region of histidine kinase Hik33 in the cyanobacterium Synechocystis sp. PCC 6803.

Histidine kinase Hik33 responds to a variety of stress conditions and regulates the expression of stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. However, the mechanisms of response and regulation remain unknown. Generally, a histidine kinase perceives a specific signal via its N-terminal region. Hik33 has two transmembrane helices, a periplasmic loop, and HAMP and PAS domains in its N-terminal region, all of which might be involved in signal perception. To investigate the functions of these subdomains in vivo, we expressed a chimeric histidine kinase (Hik33n-SphSc) by fusing the N-terminal region of Hik33 with the C-terminal region of a sensory histidine kinase that is activated under phosphate-deficient conditions, SphS. Hik33n-SphSc responded to several stimuli that are perceived by intact Hik33 and regulated expression of the phoA gene for alkaline phosphatase, which is normally regulated under phosphate-deficient conditions by SphS. We introduced genes for modified versions of Hik33n-SphSc into Synechocystis and monitored expression of phoA under standard and stress conditions. Hik33n-SphSc lacking either the transmembrane helices or both the HAMP and PAS domains had no kinase activity, whereas Hik33n-SphSc lacking the HAMP or the PAS domain enhanced expression of phoA. Moreover, variants of Hik33n-SphSc, in which the membrane-localizing region was replaced by those of other histidine kinases, also responded to stress conditions. Thus, transmembrane helices, regardless of sequence, appear to be essential for the function of Hik33, while the HAMP and PAS domains play important roles in regulating kinase activity in vivo.

Enzymological evidence for the function of a plastid-located pyruvate carboxylase in the Haptophyte alga Emiliania huxleyi: a novel pathway for the production of C4 compounds.

Pyruvate carboxylase (PYC) catalyzes the ß-carboxylation of pyruvate to yield oxaloacetate (OAA). We previously isolated a cDNA encoding a putative PYC (EhPYC1) from the haptophyte alga Emiliania huxleyi and then proposed that EhPYC1 contributes to active anaplerotic ß-carboxylation during photosynthesis although PYC activity was not detected in the cell extracts. Involvement of PYC in photosynthetic carbon metabolism is unique, since PYC generally functions in non-photosynthetic organisms. In the present study, we demonstrate that EhPYC1 is highly sensitive to endogenous proteases and therefore is easily degraded in cell extracts. By avoiding proteolytic degradation, PYC activity can be detected in the cell extracts of E. huxleyi. The activity of a recombinant His-tagged EhPYC1 expressed in Streptomyces lividans was inhibited by l-malate in a mixed non-competitive manner. Immunofluorescence labeling showed that EhPYC1 is located in the plastid. This result agrees with the prediction that a bipartite plastid-targeting signal is present that functions to deliver proteins into the four-membrane plastid of haptophyte algae. This is the first finding of a plastid-located PYC. These results indicate that E. huxleyi possesses a unique pathway to produce OAA catalyzed by PYC, and the pathway may provide carbon skeletons for amino acid biosynthesis in the plastid. A database search indicates that PYC genes are widespread in green algae, diatoms and brown algae, suggesting the crucial role of PYC in various aquatic phototrophs.