Chloroplast Endoplasmic Reticulum Research Articles

Heterologous phospholipid:diacylglycerol acyltransferase channels eicosapentaenoic acid to triacylglycerol in Phaeodactylum tricornutum

Phaeodactylum tricornutum is a well-studied oleaginous model diatom with high triacylglycerol (TAG) and omega-3 eicosapentaenoic acid (EPA) contents, however, only a small portion of EPA is transferred from the polar lipids to TAG through phospholipid:diacylglycerol acyltransferase (PDAT). In this study, a Nannochloropsis oceanica PDAT (NoPDAT) was heterologously expressed in P. tricornutum to increase the EPA content in TAG. NoPDAT heterologous expression slightly disturbed the growth of the transgenic lines, but enhanced the TAG content by 16–18 %. Considering the localization of NoPDAT in P. tricornutum after heterologous expression together with our lipidomic results, NoPDAT is more likely to utilize phosphatidylcholine and phosphatidylethanolamine on the chloroplast endoplasmic reticulum membrane as the acyl donors. Especially, NoPDAT transferred the EPA acyl moiety from the sn-2 position of the two above-mentioned phospholipids to the sn-3 position of TAG, resulting in the increase of two major EPA-containing TAG molecules (16:1/16:0/20:5 and 16:1/16:1/20:5). Correspondingly, two major 16:0-containing TAG molecules (16,0/16:1/16:1 and 16:0/16:0/16:1) decreased in the transgenic strains. In conclusion, our attempt to express NoPDAT in P. tricornutum increases the EPA content in TAG by approximately one-fold, and demonstrates the versatility of diatom lipid metabolism and acyl editing.

Isolation of High-Quality Plastids from the Diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum, a model pennate diatom, carries a secondary plastid surrounded by four membranes. Its biological function remains mysterious, supposed to combine features of the primary chloroplast and the endomembrane system. Isolation of high-quality plastid from the diatom enables a more conclusive understanding of the special structure and metabolic pathways in the plastid. Due to the direct continuity between the chloroplast endoplasmic reticulum membrane (cERM) and the outer nuclear envelope together with the integration of cERM into the cellular endoplasmic reticulum (ER) system, the plastid isolation is still challenging. In this study, highly purified P. tricornutum plastids with the four-layered membrane are obtained by Percoll density gradient centrifugation. The isolated plastids are unlikely to contain any residue of nuclear and coatomer compartments, and they might contain a relatively small contamination of mitochondrion and ER debris.

Proteomes reveal the lipid metabolic network in the complex plastid of Phaeodactylum tricornutum.

Phaeodactylum tricornutum plastid is surrounded by four membranes, and its protein composition and function remain mysterious. In this study, the P. tricornutum plastid-enriched fraction was obtained and 2850 proteins were identified, including 92 plastid-encoded proteins, through label-free quantitative proteomic technology. Among them, 839 nuclear-encoded proteins were further determined to be plastidial proteins based on the BLAST alignments within Plant Proteome DataBase and subcellular localization prediction, in spite of the strong contamination by mitochondria-encoded proteins and putative plasma membrane proteins. According to our proteomic data, we reconstructed the metabolic pathways and highlighted the hybrid nature of this diatom plastid. Triacylglycerol (TAG) hydrolysis and glycolysis, as well as photosynthesis, glycan metabolism, and tocopherol and triterpene biosynthesis, occur in the plastid. In addition, the synthesis of long-chain acyl-CoAs, elongation, and desaturation of fatty acids (FAs), and synthesis of lipids including TAG are confined in the four-layered-membrane plastid based on the proteomic and GFP-fusion localization data. The whole process of generation of docosahexaenoic acid (22:6) from palmitic acid (16:0), via elongation and desaturation of FAs, occurs in the chloroplast endoplasmic reticulum membrane, the outermost membrane of the plastid. Desaturation that generates 16:4 from 16:0 occurs in the plastid stroma and outer envelope membrane. Quantitative analysis of glycerolipids between whole cells and isolated plastids shows similar composition, and the FA profile of TAG was not different. This study shows that the diatom plastid combines functions usually separated in photosynthetic eukaryotes, and differs from green alga and plant chloroplasts by undertaking the whole process of lipid biosynthesis.

Stramenopile-type Lipid Droplet Protein Functions as a Lipid Droplet Scaffold Protein in the Marine Diatom Phaeodactylum tricornutum.

Oleaginous microalgae gain great attention as feedstock for biofuels because of their substantial accumulation capacity of neutral lipids in the cytosolic compartment called lipid droplet (LD). Understanding the regulation mechanism of neutral lipid accumulation and degradation, which is mediated by LD-associated proteins, is an important issue in improving lipid productivity. However, LD-associated proteins vary among species and wait to be characterized in many microalgae. Stramenopile-type lipid droplet protein (StLDP) was previously identified as a primary LD protein in the marine diatom Phaeodactylum tricornutum. We produced a knockout mutant of StLDP by CRISPR/Cas9 genome editing. Also, we tried to complement this mutant by expressing recognition site-modified StLDP (RSM-StLDP) which is designed to avoid an attack by Cas9 nuclease expressing in the mutant. The RSM-StLDP:EGFP was localized to both LDs and the outer chloroplast-endoplasmic reticulum. Decrease of LD number per cell, increase of LD size, and no alteration of neutral lipid content in the mutant under nitrogen deficiency clearly indicate that StLDP acts as an LD scaffold protein. The number of LD per cell increased in the complemented strain compared to wild-type cells. The LD morphology in the mutant is probably over-rescued in the complemented strain by the strong function of the nitrate reductase promoter, which is also supported by high neutral lipid content in the complemented strain. The growth of stldp mutant showed a long lag phase relative to wild-type cells, suggesting that the low surface-to-volume ratio of fused LD decreased the efficiency of LD hydrolysis during the initial growth phase.

Δ6 Fatty Acid Elongase is Involved in Eicosapentaenoic Acid Biosynthesis Via the ω6 Pathway in the Marine Alga Nannochloropsis oceanica.

Nannochloropsis oceanica represents a promising sunlight-driven alga for producing eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17), a value-added very long-chain polyunsaturated fatty acid (VLC-PUFA). Here, we unraveled the function and roles of a Δ6 fatty acid elongase (NoΔ6-FAE) in N. oceanica. Heterologous expression of NoΔ6-FAE in yeast confirmed its function in elongating C18 Δ6-PUFAs rather than others. Subcellular localization experiments suggested that NoΔ6-FAE resides in the chloroplast endoplasmic reticulum. NoΔ6-FAE knockdown attenuated C20:3Δ8,11,14, C20:4Δ5,8,11,14, and EPA yet enhanced C18:3Δ6,9,12, leading to overall decreases in total fatty acids, triacylglycerol, diacylglycerol, free fatty acids, and polar membrane lipids. In contrast, NoΔ6-FAE overexpression in N. oceanica caused nearly opposite phenotypes. Moreover, N. oceanica lacked detectable C18:3Δ9,12,15, C18:4Δ6,9,12,15, and C20:4Δ8,11,14,17 even under NoΔ6-FAE knockdown or overexpression. Our results reveal the involvement of NoΔ6-FAE in EPA biosynthesis via the ω6 pathway in N. oceanica and highlight the potential of manipulating NoΔ6-FAE for improved lipid production.

Lipid production is more than doubled by manipulating a diacylglycerol acyltransferase in algae

AbstractOleaginous algae have the ability to synthesize a high level of triacylglycerol (TAG) and are considered as the next‐generation feedstock for biofuel production. Manipulating algal lipid biosynthetic pathways has potential to overproduce TAG and represents a feasible way toward bringing down algae‐derived biofuels production cost. Here we dissected functional roles and engineering potential of six diacylglycerol acyltransferase (DGAT) genes from the marine alga Phaeodactylum tricornutum. PtDGAT1, PtDGAT2B, and PtDGAT3, residing at the chloroplast endoplasmic reticulum (ER) readily for utilizing both chloroplast‐ and ER‐derived diacylglycerol, were transcriptionally correlated with TAG accumulation. Heterologous expression in yeast, in vitro assay and overexpression in P. tricornutum all supported that PtDGAT1 surpassed the other five PtDGATs in synthesizing TAG. Compared to wild type P. tricornutum, the PtDGAT1‐overexpressing strain produced more than doubled TAG and total lipids, which reached 57% and 73% of dry weight, respectively, record‐high levels ever achieved in this alga. Our results demonstrated the strategy of screening proper engineering targets and manipulating a single gene to pull carbon flux to lipids for TAG hyper‐accumulation without growth compromise. The engineered alga with a great trait improvement may serve as a potent lipid producer for production uses.

Plasma Membrane-Type Aquaporins from Marine Diatoms Function as CO2/NH3 Channels and Provide Photoprotection.

Aquaporins (AQPs) are ubiquitous water channels that facilitate the transport of many small molecules and may play multiple vital roles in aquatic environments. In particular, mechanisms to maintain transmembrane fluxes of important small molecules have yet to be studied in marine photoautotrophic organisms. Here, we report the occurrence of multiple AQPs with differential cellular localizations in marine diatoms, an important group of oceanic primary producers. The AQPs play a role in mediating the permeability of membranes to CO2 and NH3 In silico surveys revealed the presence of five AQP orthologs in the pennate diatom Phaeodactylum tricornutum and two in the centric diatom Thalassiosira pseudonana GFP fusions of putative AQPs displayed clear localization to the plasma membrane (PtAGP1 and PtAQP2), the chloroplast endoplasmic reticulum (CER; PtAGP1 and PtAQP3), and the tonoplast (PtAQP5) in P. tricornutum In T. pseudonana, GFP-AQP fusion proteins were found on the vacuole membrane (TpAQP1) and CER (TpAQP2). Transcript levels of both PtAQP1 and PtAQP2 were highly induced by ammonia, while only PtAQP2 was induced by high (1%[v/v]) CO2 Constitutive overexpression of GFP-tagged PtAQP1 and PtAQP2 significantly increased CO2 and NH3 permeability in P. tricornutum, strongly indicating that these AQPs function in regulating CO2/NH3 permeability in the plasma membrane and/or CER. Cells carrying GFP-tagged PtAQP1 and PtAQP2 had higher nonphotochemical quenching under high light relative to that of wild-type cells, suggesting that these AQPs are involved in photoprotection. These AQPs may facilitate the efflux of NH3, preventing the uncoupling effect of high intracellular ammonia concentrations.

A type-I diacylglycerol acyltransferase modulates triacylglycerol biosynthesis and fatty acid composition in the oleaginous microalga, Nannochloropsis oceanica

BackgroundPhotosynthetic oleaginous microalgae are considered promising feedstocks for biofuels. The marine microalga, Nannochloropsis oceanica, has been attracting ever-increasing interest because of its fast growth, high triacylglycerol (TAG) content, and available genome sequence and genetic tools. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step of TAG biosynthesis in the acyl-CoA-dependent pathway. Previous studies have identified 13 putative DGAT-encoding genes in the genome of N. oceanica, but the functional role of DGAT genes, especially type-I DGAT (DGAT1), remains ambiguous.ResultsNannochloropsis oceanica IMET1 possesses two DGAT1 genes: NoDGAT1A and NoDGAT1B. Functional complementation demonstrated the capability of NoDGAT1A rather than NoDGAT1B to restore TAG synthesis in a TAG-deficient yeast strain. In vitro DGAT assays revealed that NoDGAT1A preferred saturated/monounsaturated acyl-CoAs and eukaryotic diacylglycerols (DAGs) for TAG synthesis, while NoDGAT1B had no detectable enzymatic activity. Assisted with green fluorescence protein (GFP) fusion, fluorescence microscopy analysis indicated the localization of NoDGAT1A in the chloroplast endoplasmic reticulum (cER) of N. oceanica. NoDGAT1A knockdown caused ~25% decline in TAG content upon nitrogen depletion, accompanied by the reduced C16:0, C18:0, and C18:1 in TAG sn-1/sn-3 positions and C18:1 in the TAG sn-2 position. NoDGAT1A overexpression, on the other hand, led to ~39% increase in TAG content upon nitrogen depletion, accompanied by the enhanced C16:0 and C18:1 in the TAG sn-1/sn-3 positions and C18:1 in the TAG sn-2 position. Interestingly, NoDGAT1A overexpression also promoted TAG accumulation (by ~2.4-fold) under nitrogen-replete conditions without compromising cell growth, and TAG yield of the overexpression line reached 0.49 g L−1 at the end of a 10-day batch culture, 47% greater than that of the control line.ConclusionsTaken together, our work demonstrates the functional role of NoDGAT1A and sheds light on the underlying mechanism for the biosynthesis of various TAG species in N. oceanica. NoDGAT1A resides likely in cER and prefers to transfer C16 and C18 saturated/monounsaturated fatty acids to eukaryotic DAGs for TAG assembly. This work also provides insights into the rational genetic engineering of microalgae by manipulating rate-limiting enzymes such as DGAT to modulate TAG biosynthesis and fatty acid composition for biofuel production.

Ultrastructure of the Periplastidial Compartment of the Diatom Phaeodactylum tricornutum

Diatoms contain a secondary plastid that derives from a red algal symbiont. This organelle is limited by four membranes. The two outermost membranes are the chloroplast endoplasmic reticulum membrane (cERM), which is continuous with the host outer nuclear envelope, and the periplastidial membrane (PPM). The two innermost membranes correspond to the outer and inner envelope membranes (oEM and iEM) of the symbiont's chloroplast. Between the PPM and oEM lies a minimized symbiont cytoplasm, the periplastidial compartment (PPC). In Phaeodactylum tricornutum, PPC-resident proteins are localized in "blob-like-structures", which remain associated with plastids after cell disruption. We analyzed disrupted Phaeodactylum cells by focused ion beam scanning electron microscopy, revealing the presence of a vesicular network (VN) in the PPC, at a location consistent with blob-like structures. Presence of a VN in the PPC was confirmed in intact cells. Additionally, direct membrane contacts were observed between the PPM and nuclear inner envelope membrane at the level of the chloroplast-nucleus isthmus. This study provides insights into the PPC ultrastructure and opens perspectives on the function of this residual cytoplasm of red algal origin.

Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana

It is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in the genome sequences of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Subsequently, the subcellular localizations of the encoded proteins were determined. Nine and thirteen CA sequences were found in the genomes of P. tricornutum and T. pseudonana, respectively. Two of the β-CA genes in P. tricornutum corresponded to ptca1 and ptca2 identified previously. Immunostaining transmission electron microscopy of a PtCA1:YFP fusion expressed in the cells of P. tricornutum clearly showed the localization of PtCA1 within the central part of the pyrenoid structure in the chloroplast. Besides these two β-CA genes, P. tricornutum likely contains five α- and two γ-CA genes, whereas T. pseudonana has three α-, five γ-, four δ-, and one ζ-CA genes. Semi-quantitative reverse transcription PCR performed on mRNA from the two diatoms grown in changing light and CO(2) conditions revealed that levels of six putative α- and γ-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO(2) and 5% CO(2). However, mRNA levels of one putative α-CA gene, CA-VII in P. tricornutum, were reduced in the dark compared to that in the light. In T. pseudonana, mRNA accumulation levels of putative α-CA (CA-1), ζ-CA (CA-3) and δ-CA (CA-7) were analyzed and all levels found to be significantly reduced when cells were grown in 0.16% CO(2). Intercellular localizations of eight putative CAs were analyzed by expressing GFP fusion in P. tricornutum and T. pseudonana. In P. tricornutum, CA-I and II localized in the periplastidial compartment, CA-III, VI, VII were found in the chloroplast endoplasmic reticulum, and CA-VIII was localized in the mitochondria. On the other hand, T. pseudonana CA-1 localized in the stroma and CA-3 was found in the periplasm. These results suggest that CAs are constitutively present in the four chloroplastic membrane systems in P. tricornutum and that CO(2) responsive CAs occur in the pyrenoid of P. tricornutum, and in the stroma and periplasm of T. pseudonana.

A novel protein in Photosystem II of a diatom Chaetoceros gracilis is one of the extrinsic proteins located on lumenal side and directly associates with PSII core components

The gene encoding a novel extrinsic protein (Psb31) found in Photosystem II (PSII) of a diatom, Chaetoceros gracilis, was cloned and sequenced. The deduced protein contained three characteristic leader sequences targeted for chloroplast endoplasmic reticulum membrane, chloroplast envelope membrane and thylakoid membrane, indicating that Psb31 is encoded in the nuclear genome and constitutes one of the extrinsic proteins located on the lumenal side. Homologous genes were found in a red alga and chromophytic algae but not in other organisms. Genes encoding the other four extrinsic proteins in C. gracilis PSII were also cloned and sequenced, and their leader sequences were characterized and compared. To search for the nearest neighbor relationship between Psb31 and the other PSII components, we crosslinked the PSII particles with the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and found that Psb31 directly associates with PSII core components through electrostatic interaction, suggesting that the novel Psb31 protein is one of the extrinsic proteins constituting the functional oxygen-evolving complex of C. gracilis PSII.

Aurearenophyceae classis nova, a New Class of Heterokontophyta Based on a New Marine Unicellular Alga Aurearena cruciata gen. et sp. nov. Inhabiting Sandy Beaches

A new heterokontophyte alga, Aurearena cruciata gen. et sp. nov., was isolated from sandy beaches in Japan. Isolates were characterized by light and electron microscopy, spectroscopy of pigment composition, and molecular phylogenetic analyses using 18S rDNA and rbcL. The alga usually possessed a cell wall but also retained two heterokont flagella beneath the cell wall. Each walled cell first produced only a single flagellate cell that subsequently divided into two flagellate cells. Electron-opaque vesicles, possibly associated with cell wall formation, were observed beneath the cell membrane. The chloroplast consisted of two compartments, each enclosed by a chloroplast envelope and the inner membrane of the chloroplast endoplasmic reticulum; these two compartments were surrounded by a common outer membrane of chloroplast endoplasmic reticulum. Molecular phylogenetic trees suggested that this alga was a new and independent member of the clade that included the Phaeophyceae and Xanthophyceae (PX clade). A new class, Aurearenophyceae classis nova was proposed for A. cruciata.

Re‐examination of ultrastructures of the stellate chloroplast organization in brown algae: Structure and development of pyrenoids

SUMMARYSome taxa of brown algae have a so‐called ‘stellate’ chloroplast arrangement composed of multiple chloroplasts arranged in a stellate configuration, or else a single chloroplast with radiating lobes. The fine structures of chloroplasts and pyrenoids have been studied, but the details of their membrane configurations as well as pyrenoid ontogeny have not been well understood. The ultrastructure of the single stellate chloroplast in Splachnidium rugosum and Scytothamnus australis were re‐examined in the present study, as well as the stellate arrangement of chloroplasts in Asteronema ferruginea and Asterocladon interjectum, using freeze‐substitution fixation. It was confirmed that the chloroplast envelope invaginated into the pyrenoid in Splachnidium rugosum, Scytothamnus australis and Asteronema ferruginea, but chloroplast endoplasmic reticulum (CER) remained on the surface of the chloroplast. The space between the invaginated chloroplast envelope and CER was filled with electron‐dense material. In Asteronema ferruginea, CER surrounding each pyrenoid was closely appressed to the neighboring CER over the pyrenoids, so that the chloroplasts formed a stellate configuration; however, in the apical cells chloroplasts formed two or more loose groups, or were completely dispersed. The pyrenoids of Asterocladon interjectum did not have any invagination of the chloroplast envelope, but a unique membranous sac surrounded the pyrenoid complex and occasionally other organelles (e.g. mitochondria). Immunolocalization of β‐1,3‐glucans showed that the membranous sac in Asterocladon interjectum did not contain photosynthetic products such as chrysolaminaran. Observations in the dividing cells of Splachnidium rugosum and Scytothamnus australis indicated that the pyrenoid in the center of the chloroplast enlarged and divided into two before or during chloroplast division.

Protein targeting in "secondary" or "complex" chloroplasts.

All the algae with chlorophyll (Chl) c (haptophytes, cryptophytes, and heterokonts such as diatoms) acquired their chloroplasts by secondary endosymbiosis, where a nonphotosynthetic eukaryote host engulfed (or was invaded by) a red alga. This resulted in chloroplasts with four bounding membranes. The outermost membrane (chloroplast endoplasmic reticulum [ER]), is physically continuous with the rough ER, and in some algal species can be seen to have cytoplasmic ribosomes attached to its outer surface. All nuclear-encoded chloroplast proteins have an N-terminal ER targeting sequence, which is cleaved off during transit across this membrane. We know little about how proteins cross the next membrane and engage the import translocons of the envelope membranes. One way to study the targeting of proteins across the inner membranes is to make constructs lacking the ER signal sequence, translate them in vitro, and assay their import into pea chloroplasts.

The Pinguiophyceae classis nova, a new class of photosynthetic stramenopiles whose members produce large amounts of omega-3 fatty acids

SUMMARY The Pinguiophyceae class. nov., a new class of photo-synthetic stramenopiles (chromophytes), is described. The class includes five monotypic genera, Glossomastix, Phaeomonas, Pinguiochrysis (type genus), Pinguio-coccus and Polypodochrysis. These algae have an unusually high percentage of polyunsaturated fatty acids, especially 20:5 (n-3)(EPA, eicosapentaenoic acid). These fatty acids are the basis for choosing the Latin noun ‘Pingue’ (= fat, grease) as the root for the class name. Analyses of nuclear-encoded 18S rRNA and chloroplast-encoded rbcL gene sequence data showed that these algae formed a monophyletic group that could not be placed in any other class. Morphologically, the species are all single-celled microalgae from picoplanktonic size to over 40 urn in length. Each cell has one (or two) typical chloroplast(s) with a girdle lamella and a surrounding chloroplast endoplasmic reticulum. Pyrenoids occur within the chloroplast, varying from embedded to stalked, and membranes penetrate into the pyrenoid in all five genera. Phaeomonas has motile cells with two flagella, and the forward-directed flagellum bears mastigonemes (tripartite flagellar hairs). Two other genera (Glossomastix, Polypodochrysis) produce zoospores that possess only one smooth flagellum (no mastigonemes), and this flagellum apparently is the mature flagellum, a feature previously unknown in the photosynthetic stramenopiles. The major carotenoid pigments in the pinguiophytes are fucoxanthin, violaxanthin, zeaxanthin and P-carotene, as well as chlorophyll a and chlorophyll c-related pigment(s). These features support recognition of the Pinguiophyceae class. nov. as a unique group of algae.

ENDOMEMBRANE STRUCTURE AND THE CHLOROPLAST PROTEIN TARGETING PATHWAY IN HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE, CHROMISTA)

Chloroplasts in heterokont algae are surrounded by four membranes and probably originated from a red algal endosymbiont that was engulfed and retained by eukaryotic host. Understanding how nuclear‐encoded chloroplast proteins are translocated from the cytoplasm into the chloroplast across these membranes could give us some insights about how the endosymbiont was integrated into the host cell in the process of secondary symbiogenesis. In multiplastid heterokont algae such as raphidophytes, it has been unclear if the outermost of the four membranes surrounding the chloroplast (the chloroplast endoplasmic reticulum [CER] membrane) is continuous with the nuclear envelope and rough endoplasmic reticulum (ER). Here, we report detailed ultrastructural observations of the raphidophyte Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara that show that the CER membranes were continuous with ER membranes that had attached ribosomes, implying that the chloroplast with three envelope membranes is located within the ER lumen, that is, topologically the same structure as that of monoplastid heterokont algae. However, the CER membrane of H. akashiwo had very few, if any, ribosomes attached, unlike the CER membranes in other heterokont algae. To verify that proteins are first targeted to the ER, we assayed protein import into canine microsomes using a precursor for a nuclear‐encoded chloroplast protein, the fucoxanthin‐chlorophyll a/c protein of H. akashiwo. This demonstrated that the precursor has a functional signal sequence for ER targeting and is cotranslationally translocated into the ER, where a signal sequence of about 17 amino acids is removed. Based on these data, we hypothesize that in H. akashiwo, nuclear‐encoded chloroplast protein precursors that have been cotranslationally transported into the ER lumen are sorted in the ER and transported to the chloroplasts through the ER lumen.

Functional characterization of isolated plastids from two marine diatoms

A protocol for the isolation of intact plastids from two marine centric diatoms, Odontella sinensis (Greville) Grunow and Coscinodiscus granii Gough, has been worked out. The cells were broken in a Yeda Press, and the intact plastids were purified by centrifugation in Percoll gradients. Electron microscopy indicates that at least one of the four envelope membranes is present in the isolated plastids. The plastids are photosynthetically active as proven by CO2 fixation which was measured by light-dependent oxygen evolution. Rates up to 50 μmol O2 · (mg Chl)−1 · h−1, i.e. about 40% of the in vivo rate of photosynthesis were obtained. The inhibition of CO2 fixation by external phosphate and the ability of the plastids to reduce added 3-phosphoglycerate photosynthetically indicate the presence of a phosphate translocator in the envelope of the diatom plastids. Light-dependent O2 evolution upon addition of nitrite indicates the presence of nitrite reductase in these plastids. Purified envelope membranes of Odontella plastids analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis contain polypeptides similar to those of the envelope of higher-plant chloroplasts. However, there are additional bands present, which in part may be constituents of the two additional envelope membranes (“chloroplast endoplasmic reticulum”) and in part may represent additional components of the inner membranes.

The fine structure of the male gamete of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae). I. General structure of the cell

The ultrastructure of male gametes of Ectocarpus siliculosus is described and discussed in relation to the information available on other brown algal zoids. In general, the fine structure and the organelle associations agree with those of other brown algal cells. In particular, the male gametes typically contain a single chloroplast with a pyrenoid and a prominent concave eyespot, into which the swelling of the posterior flagellum (the photoreceptor) fits. The continuity of the chloroplast endoplasmic reticulum and nuclear membrane, as well as the presence of a perinuclear Golgi consisting of several dictyosomes, is shown. The fine structure of microbodies, lipid bodies and different types of vesicles, including physodes and coated vesicles, is also discussed. Several specializations of cellular elements and their interactions are unique and have not been reported before. These features include the connection between the nuclear envelope, the Golgi and the flagellar bases mediated by fibres resembling intermediate filaments, and the organization of the Golgi as well as the association of mitochondria and a microtubular flagellar root.

The fine structure of the male gamete of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae). I. General structure of the cell

The ultrastructure of male gametes of Ectocarpus siliculosus is described and discussed in relation to the information available on other brown algal zoids. In general, the fine structure and the organelle associations agree with those of other brown algal cells. In particular, the male gametes typically contain a single chloroplast with a pyrenoid and a prominent concave eyespot, into which the swelling of the posterior flagellum (the photoreceptor) fits. The continuity of the chloroplast endoplasmic reticulum and nuclear membrane, as well as the presence of a perinuclear Golgi consisting of several dictyosomes, is shown. The fine structure of microbodies, lipid bodies and different types of vesicles, including physodes and coated vesicles, is also discussed. Several specializations of cellular elements and their interactions are unique and have not been reported before. These features include the connection between the nuclear envelope, the Golgi and the flagellar bases mediated by fibres resembling intermediate filaments, and the organization of the Golgi as well as the association of mitochondria and a microtubular flagellar root.

Phylogenetic position of the Chromista plastids based on small subunit rRNA coding regions.

The kingdom Chromista contains eukaryotic organisms with tubular mastigonemes on the leading flagellum of their bi-flagellated stages, and plastids within a chloroplast endoplasmic reticulum (CER). The complex series of events leading to the formation of the CER is hypothesized to have occurred only once. Thus, all organisms with plastid-CER connections are believed to be monophyletic and derived from a single secondary endosymbiotic event. Analyses of sequence data from the 16s rRNA gene from three of the four Chromista pigmented classes indicate that these algae are not monophyletic. The validity of the kingdom Chromista and the number of secondary plastid endosymbioses are questioned.