Plastidial acyl carrier protein Δ9-desaturase modulates eicosapentaenoic acid biosynthesis and triacylglycerol accumulation in Phaeodactylum tricornutum

The unicellular marine diatom Phaeodactylum tricornutum accumulates up to 35% eicosapentaenoic acid (EPA, 20:5n3) and has been used as a model organism to study long chain polyunsaturated fatty acids (LC-PUFA) biosynthesis due to an excellent annotated genome sequence and established transformation system. In P. tricornutum, the majority of EPA accumulates in polar lipids, particularly in galactolipids such as mono- and di-galactosyldiacylglycerol. LC-PUFA biosynthesis is considered to start from oleic acid (18:1n9). EPA can be synthesized via a series of desaturation and elongation steps occurring at the endoplasmic reticulum and newly synthesized EPA is then imported into the plastids for incorporation into galactolipids via an unknown route. The basis for the flux of EPA is fundamental to understanding LC-PUFA biosynthesis in diatoms. We used P. tricornutum to study acyl modifying activities, upstream of 18:1n9, on subsequent LC-PUFA biosynthesis. We identified the gene coding for the plastidial acyl carrier protein Δ9-desaturase, a key enzyme in fatty acid modification and analyzed the impact of overexpression and knock out of this gene on glycerolipid metabolism. This revealed a previously unknown role of this soluble desaturase in EPA synthesis and production of triacylglycerol. This study provides further insight into the distinctive nature of lipid metabolism in the marine diatom P. tricornutum and suggests additional approaches for tailoring oil composition in microalgae.     

Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage

The plastids of ecologically and economically important algae from phyla such as stramenopiles, dinoflagellates and cryptophytes were acquired via a secondary endosymbiosis and are surrounded by three or four membranes. Nuclear‐encoded plastid‐localized proteins contain N‐terminal bipartite targeting peptides with the conserved amino acid sequence motif ‘ASAFAP’. Here we identify the plastid proteomes of two diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum, using a customized prediction tool (ASAFind) that identifies nuclear‐encoded plastid proteins in algae with secondary plastids of the red lineage based on the output of SignalP and the identification of conserved ‘ASAFAP’ motifs and transit peptides. We tested ASAFind against a large reference dataset of diatom proteins with experimentally confirmed subcellular localization and found that the tool accurately identified plastid‐localized proteins with both high sensitivity and high specificity. To identify nucleus‐encoded plastid proteins of T. pseudonana and P. tricornutum we generated optimized sets of gene models for both whole genomes, to increase the percentage of full‐length proteins compared with previous assembly model sets. ASAFind applied to these optimized sets revealed that about 8% of the proteins encoded in their nuclear genomes were predicted to be plastid localized and therefore represent the putative plastid proteomes of these algae.     

Targeting proteins to diatom plastids involves transport through an endoplasmic reticulum

Summary Diatoms and related algae, in contrast to higher plants, have a xanthophyll-dominated light harvesting complex and an endoplasmic reticulum (ER) network surrounding the plastid. We have previously demonstrated that polypeptide constituents of the light harvesting complex from the diatom Phaeodactylum tricornutum are nuclear encoded and synthesized as higher molecular weight precursors in the cytoplasm. The amino-termini of the precursor proteins, as deduced from their gene sequences, have features of a signal peptide. Here, we show that the precursor polypeptides can be cotranslationally imported and processed by an in vitro microsomal membrane system, suggesting that cytoplasmically synthesized proteins require a signal peptide to traverse an ER before entering the plastid. These results are discussed in the context of plastid evolution.     

Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif

Plastids of diatoms and related algae evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and its subsequent reduction into an organelle. As a result diatom plastids are surrounded by four membranes. Protein targeting of nucleus encoded plastid proteins across these membranes depends on N-terminal bipartite presequences consisting of a signal and a transit peptide-like domain. Diatoms and cryptophytes share a conserved amino acid motif of unknown function at the cleavage site of the signal peptides (ASAFAP), which is particularly important for successful plastid targeting. Screening genomic databases we found that in rare cases the very conserved phenylalanine within the motif may be replaced by tryptophan, tyrosine or leucine. To test such unusual presequences for functionality and to better understand the role of the motif and putative receptor proteins involved in targeting, we constructed presequence:GFP fusion proteins with or without modifications of the “ASAFAP”-motif and expressed them in the diatom Phaeodactylum tricornutum. In this comprehensive mutational analysis we found that only the aromatic amino acids phenylalanine, tryptophan, tyrosine and the bulky amino acid leucine at the +1 position of the predicted signal peptidase cleavage site allow plastid import, as expected from the sequence comparison of native plastid targeting presequences of P. tricornutum and the cryptophyte Guillardia theta. Deletions within the signal peptide domains also impaired plastid import, showing that the presence of F at the N-terminus of the transit peptide together with a cleavable signal peptide is crucial for plastid import. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. Gruber and S. Vugrinec contributed equally to this work.     

Identification of a plastid acyl-acyl carrier protein synthetase in Arabidopsis and its role in the activation and elongation of exogenous fatty acids

Plant cells are known to elongate exogenously provided fatty acid (FA), but the subcellular sites and mechanisms for this process are not currently understood. When Arabidopsis leaves were incubated with 14C-FAs with or=20 carbons) but not synthesis of 14C-unsaturated 18-carbon or 16-carbon FAs. Isolated pea chloroplasts were also able to elongate 14C-FAs (相似文献   

Changes in the fatty acid composition of symbiotic dinoflagellates from the hermatypic coral <Emphasis Type="Italic">Echinopora lamellosa</Emphasis> during adaptation to the irradiance level

The composition of fatty acids (FAs) of symbiotic dinoflagellates isolated from the hermatypic coral Echinoporal lamellosa adapted to the irradiance of 95, 30, 8, and 2% PAR was studied. Polar lipids and triacylglycerols (TAG) differed between them in FA composition. Polar lipids were enriched in unsaturated FAs, whereas TAG, in saturated FAs. Light exerted a substantial influence on the FA composition in both polar lipids and TAG. The elevation of irradiance resulted in the accumulation of 16:0 acid in both lipid groups and 16:1(n-7) acid in TAG. It seems likely that de novo synthesis of 16:0 acid occurred actively in the cells of symbiotic dinoflagellates in high light. Since these processes are energy-consuming ones, they utilize excessive energy. When light intensity declined, 18:4(n-3) and 20:5(n-3) acids accumulated in polar lipids, which was accompanied by the increase in the content of chlorophyll a in the cells of zooxanthellae, whereas the levels of 22:6(n-3) and 20:4(n-6) acids reduced. Although the relative content of particular FAs varied substantially in dependence of irradiance, the balance between the sum of saturated and unsaturated FAs changed insignificantly. We concluded that the role of photoadaptation could not be limited only to changes in the degree of lipid unsaturation and membrane fluidity. It is supposed that light-induced changes in the FA composition reflect the interrelation between photosynthesis and FA biosynthesis.     

Filling the Gap, Evolutionarily Conserved Omp85 in Plastids of Chromalveolates

Chromalveolates are a diverse group of protists that include many ecologically and medically relevant organisms such as diatoms and apicomplexan parasites. They possess plastids generally surrounded by four membranes, which evolved by engulfment of a red alga. Today, most plastid proteins must be imported, but many aspects of protein import into complex plastids are still cryptic. In particular, how proteins cross the third outermost membrane has remained unexplained. We identified a protein in the third outermost membrane of the diatom Phaeodactylum tricornutum with properties comparable to those of the Omp85 family. We demonstrate that the targeting route of P. tricornutum Omp85 parallels that of the translocation channel of the outer envelope membrane of chloroplasts, Toc75. In addition, the electrophysiological properties are similar to those of the Omp85 proteins involved in protein translocation. This supports the hypothesis that P. tricornutum Omp85 is involved in precursor protein translocation, which would close a gap in the fundamental understanding of the evolutionary origin and function of protein import in secondary plastids.     

A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis

Background

Diatoms are unicellular algae responsible for approximately 20% of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids.

Methodology/Principal Findings

The whole genome sequence of the diatom Phaeodactylum tricornutum has recently been completed. We identified and annotated genes for enzymes involved in carbohydrate pathways based on extensive EST support and comparison to the whole genome sequence of a second diatom, Thalassiosira pseudonana. Protein localization to mitochondria was predicted based on identified similarities to mitochondrial localization motifs in other eukaryotes, whereas protein localization to plastids was based on the presence of signal peptide motifs in combination with plastid localization motifs previously shown to be required in diatoms. We identified genes potentially involved in a C4-like photosynthesis in P. tricornutum and, on the basis of sequence-based putative localization of relevant proteins, discuss possible differences in carbon concentrating mechanisms and CO₂ fixation between the two diatoms. We also identified genes encoding enzymes involved in photorespiration with one interesting exception: glycerate kinase was not found in either P. tricornutum or T. pseudonana. Various Calvin cycle enzymes were found in up to five different isoforms, distributed between plastids, mitochondria and the cytosol. Diatoms store energy either as lipids or as chrysolaminaran (a β-1,3-glucan) outside of the plastids. We identified various β-glucanases and large membrane-bound glucan synthases. Interestingly most of the glucanases appear to contain C-terminal anchor domains that may attach the enzymes to membranes.

Conclusions/Significance

Here we present a detailed synthesis of carbohydrate metabolism in diatoms based on the genome sequences of Thalassiosira pseudonana and Phaeodactylum tricornutum. This model provides novel insights into acquisition of dissolved inorganic carbon and primary metabolic pathways of carbon in two different diatoms, which is of significance for an improved understanding of global carbon cycles.     

Distribution of unusual fatty acids in the triacylglycerols of sea buckthorn mesocarp oil

The structure of unusual fatty acid (FA) components of triacylglycerols (TAGs) of mature sea buckthorn (Hippophae rhamnoides L.) mesocarp oil was determined by GLC and MS, and the positional-species composition (PSC) of these TAGs was estimated using the methods of TAG chemical deacylation, TLC, GLC, and computer calculation. It was shown that the unusual FAs comprised n-cis-Δ9-hexadecenoic, n-cis-Δ9,12-hexadecadienoic (palmitolinoleic), and n-cis-Δ11-octadecenoic (cis-vaccenic) acids. The hexadecenoic acid predominated in the oil, and in its distribution in TAGs, it was similar to the total FAs differing from them only in some prevalence in the triunsaturated TAGs and in the TAGs with a shorter acyl chain, as well as in the sn-2 position of TAGs. Palmitolinoleic (16:2) acid comprised only 5% of total FAs, and it was exclusively concentrated in the sn-2 position of TAGs. As regards its distribution between various positional types and forms of TAGs, the 16:2 acid was similar to oleate and total FAs. As compared to the total TAGs, the TAGs with 16:2 acid were characterized by a lower FA chain length as well as by a highest unsaturation. The TAGs with vaccenic acid (V-TAGs) considerably exceeded O-TAGs, i.e., the TAGs containing oleic acid, another 18:1 positional isomer, both in their content in the total TAGs and in their unsaturation. In the composition of positional types and fractions of various unsaturation, O-TAGs were similar to the total TAGs, while V-TAGs were characterized by a very unusual structure, viz., a very high triunsaturated TAG level and an extremely low concentration of 1,3-disaturated-2-monounsaturated TAGs. In addition, oleic acid, like most other unsaturated FAs, was incorporated predominantly in the sn-2 position of TAGs, while vaccenic acid, being also unsaturated, was nevertheless by 90% concentrated in the sn-1,3 positions of V-TAGs. Unusual FAs were related to each other in the mechanism of their biosynthesis. In fact, hexadecenoic acid biosynthesis produced by palmitic acid desaturation, was, on the one hand, further desaturated forming palmitolinoleic acid, and, on the other hand, converted to vaccenic acid via C₂ elongation.     

New mechanistic insights into pre‐protein transport across the second outermost plastid membrane of diatoms

Chromalveolates like the diatom Phaeodactylum tricornutum arose through the uptake of a red alga by a phagotrophic protist, a process termed secondary endosymbiosis. In consequence, the plastids are surrounded by two additional membranes compared with primary plastids. This plastid morphology poses additional barriers for plastid‐destined proteins, which are mostly nucleus‐encoded. Recent investigations have focused on the postulated translocon of the second outermost membrane (periplastidal membrane, PPM). These studies identified a symbiont‐specific ERAD (endoplasmic reticulum‐associated degradation)‐like machinery (SELMA), which has been implicated in plastid pre‐protein import. Despite this recent progress, key factors for protein transport via SELMA are still unknown. As SELMA substrates presumably undergo ubiquitination, a corresponding ubiquitin ligase and an enzyme for the subsequent removal of ubiquitin need to reside in the space between the second and third membrane (periplastidal compartment, PPC). Here we characterize two proteins fulfilling these criteria. We show that ptE3P (P. t ricornutumE3 enzyme of the P PC), the ubiquitin ligase, and ptDUP (P. t ricornutumd e‐u biquitinating enzyme of the P PC), the de‐ubiquitinase, localize to the PPM and PPC, respectively. In addition, we demonstrate their retained functionality by in vitro data.     

Triacylglycerol accumulation of Phaeodactylum tricornutum with different supply of inorganic carbon

The diatom Phaeodactylum tricornutum produces large quantities of lipids, especially triacylglycerols (TAGs) under nitrogen or phosphorus limitation. In this study, production of lipids and TAGs during this process was compared under conditions with different inputs of inorganic carbon. With an abundant supply of inorganic carbon, considerable accumulation of biomass, lipids, and TAGs was identified after a nitrogen/phosphorus-limiting “induction incubation.” TAGs were still synthesized and accumulated even under inorganic carbon limitation with a cessation in the production of biomass and cellular lipids. This part of accumulated TAGs could be synthesized through recycling and transformation of other lipids such as glycolipids and phospholipids. Additionally, some alterations in the fatty acid profile following TAG accumulation were found. The content of the C16:0 fatty acid increased with decreases in C16:3 and C20:5, which could have been caused by enzymatic selectivity for these fatty acids during the process of TAG synthesis. It was concluded that nitrogen and phosphorus metabolism regulates the synthesis of TAG, while carbon metabolism promotes it by providing sufficient substrates.     

Isolation of mitochondrial and plastid ftsZ genes and analysis of the organelle targeting sequence in the diatom Chaetoceros neogracile (Diatoms,Bacillariophyceae)

Mitochondria and plastids multiply by division in eukaryotic cells. Recently, the eukaryotic homolog of the bacterial cell division protein FtsZ was identified and shown to play an important role in the organelle division process inside the inner membrane. To explore the evolution of FtsZ proteins, and to accumulate data on the protein import system in mitochondria and plastids of the red algal lineage, one mitochondrial and three plastid ftsZ genes were isolated from the diatom Chaetoceros neogracile, whose plastids were acquired by secondary endosymbiotic uptake of a red alga. Protein import into organelles depends on the N‐terminal organelle targeting sequences. N‐terminal bipartite presequences consisting of an endoplasmic reticulum signal peptide and a plastid transit peptide are required for protein import into diatom plastids. To characterize the organelle targeting peptides of C. neogracile, we observed the localization of each green fluorescent protein‐tagged predicted organelle targeting peptide in cultured tobacco cells and diatom cells. Our data suggested that each targeting sequences functioned both in tobacco cultured cells and diatom cells.     

Contribution of omega-3 fatty acid desaturase and 3-ketoacyl-ACP synthase II (KASII) genes in the modulation of glycerolipid fatty acid composition during cold acclimation in birch leaves

Temperate and boreal tree species respond to low positive temperatures (LT) or a shortening of the photoperiod (SD) by inducing cold acclimation. One of the metabolic consequences of cold acclimation is an increase in fatty acid (FA) desaturation in membrane lipids, which allows functional membrane fluidity to be maintained at LT. The molecular mechanisms of FA desaturation were investigated in leaves of birch seedlings (Betula pendula) during cold acclimation. Four genes involved in FA biosynthesis were isolated: a 3-ketoacyl-ACP synthase II gene (BpKASII) involved in the elongation of palmitoyl-ACP to stearoyl-ACP, and three omega-3 FA desaturase genes (BpFAD3, BpFAD7, and BpFAD8) involved in the desaturation of linoleic acid (18:2) to alpha-linolenic acid (18:3). BpFAD7 was the main omega-3 FAD gene expressed in birch leaves, and it was down-regulated by LT under SD conditions. LT induced the expression of BpFAD3 and BpFAD8 and a synchronous increase in 18:3 occurred in glycerolipids. Changes in the photoperiod did not affect the LT-induced increase in 18:3 in chloroplast lipids (MGDG, DGDG, PG), but it modulated the LT response detected in extra-chloroplastic lipids (PC, PE, PI, PS). A decrease in the proportion of the 16-carbon FAs in lipids occurred at LT, possibly in relation to the regulation of BpKASII expression at LT. These results suggest that LT affects the whole FA biosynthesis pathway. They support a co-ordinated action of microsomal (BpFAD3) and chloroplast enzymes (BpFAD7, BpFAD8) in determining the level of 18:3 in extra-chloroplastic membranes, and they highlight the importance of dynamic lipid trafficking.     

Diatom Vacuolar 1,6‐β‐Transglycosylases can Functionally Complement the Respective Yeast Mutants

Diatoms are unicellular photoautotrophic algae, which can be found in any aquatic habitat. The main storage carbohydrate of diatoms is chrysolaminarin, a nonlinear β‐glucan, consisting of a linear 1,3‐β‐chain with 1,6‐β‐branches, which is stored in cytoplasmic vacuoles. The metabolic pathways of chrysolaminarin synthesis in diatoms are poorly investigated, therefore we studied two potential 1,6‐β‐transglycosylases (TGS) of the diatom Phaeodactylum tricornutum which are similar to yeast Kre6 proteins and which potentially are involved in the branching of 1,3‐β‐glucan chains by adding d ‐glucose as 1,6‐side chains. We genetically fused the full‐length diatom TGS proteins to GFP and expressed these constructs in P. tricornutum, demonstrating that the enzymes are apparently located in the vacuoles, which indicates that branching of chrysolaminarin may occur in these organelles. Furthermore, we demonstrated the functionality of the diatom enzymes by expressing TGS1 and 2 proteins in yeast, which resulted in a partial complementation of growth deficiencies of a transglycosylase‐deficient ?kre6 yeast strain.     

Subcellular proteomics for determining iron-limited remodeling of plastids in the model diatom Thalassiosira pseudonana (Bacillariophyta)

Diatoms are important primary producers in the world's oceans, yet their growth is constrained in large regions by low bioavailable iron (Fe). Low-Fe stress-induced limitation of primary production is due to requirements for Fe in components of essential metabolic pathways including photosynthesis and other chloroplast plastid functions. Studies have shown that under low-Fe stress, diatoms alter plastid-specific processes, including components of electron transport. These physiological changes suggest changes of protein content and in protein abundances within the diatom plastid. While in silico predictions provide putative information on plastid-localized proteins, knowledge of diatom plastid proteins remains limited in comparison to well-studied model photosynthetic organisms. To address this, we employed shotgun proteomics to investigate the proteome of subcellular plastid-enriched fractions from Thalassiosira pseudonana to gain a better understanding of how the plastid proteome is remodeled in response to Fe limitation. Using mass spectrometry-based peptide identification and quantification, we analyzed T. pseudonana grown under Fe-replete and -limiting conditions. Through these analyses, we inferred the relative quantities of each protein, revealing that Fe limitation regulates major metabolic pathways in the plastid, including the Calvin cycle. Additionally, we observed changes in the expression of light-harvesting proteins. In silico localization predictions of proteins identified in this plastid-enriched proteome allowed for an in-depth comparison of theoretical versus observed plastid-localization, providing evidence for the potential of additional protein import pathways into the diatom plastid.     

Xanthophyll synthesis in diatoms: quantification of putative intermediates and comparison of pigment conversion kinetics with rate constants derived from a model

Recently, we reported the presence of the violaxanthin-antheraxanthin-zeaxanthin cycle in diatoms, and showed that violaxanthin is the putative precursor of both diadinoxanthin and fucoxanthin in the diatom Phaeodactylum tricornutum Bohlin (M. Lohr and C. Wilhelm, 1999, Proc. Natl. Acad. Sci. USA 96: 8784–8789). In the present study, two possible intermediates in the synthesis of violaxanthin from β-carotene were identified in P. tricornutum, namely β-cryptoxanthin and β-cryptoxanthin epoxide. In low light, the latter pigment prevails, but in high light β-cryptoxanthin accumulates, probably as the result of an increased activity of the xantophyll-cycle de-epoxidase. The apparent kinetics of several xanthophyll conversion steps were determined for P. tricornutum and Cyclotella meneghiniana Kützing. The experimentally determined conversion rates were used to evaluate the hypothetical pathway of xanthophyll synthesis in diatoms. For this purpose a mathematical model was developed which allows the calculation of theoretical rates of pigment conversion for microalgae under steady-state growth conditions. A comparison between measured and calculated conversion rates agreed well with the proposal of a sequential synthesis of fucoxanthin via violaxanthin and diadinoxanthin. The postulation of zeaxanthin as an obligatory intermediate in the synthesis of violaxanthin, however, resulted in large discrepancies between the measured and calculated rates of its epoxidation. Instead of zeaxanthin, β-cryptoxanthin epoxide may be involved in the biosynthesis of violaxanthin in diatoms. Received: 16 March 2000 / Accepted: 30 June 2000     

Triacylglycerol accumulation and profiling in the model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum (Baccilariophyceae) during starvation

Although substantial economic barriers exist, marine diatoms such as Thalassiosira pseudonana and Phaeodactylum tricornutum hold promise as feedstock for biodiesel because of their ability to manufacture and store triacylglycerols (TAGs). The recent sequencing of these two marine diatom genomes by the United States Department of Energy Joint Genome Institute and the development of improved systems for genetic manipulation should allow a more systematic approach to understanding and maximizing TAG production. However, in order to best utilize these genomes and genetic tools, we must first gain a deeper understanding of the nutrient-mediated regulation of TAG anabolism. By determining both the yield and molecular species distribution of TAGs we will, in the future, be able to fully characterize the effects of genetic manipulation. Here, we lay the groundwork for understanding TAG production in T. pseudonana and P. tricornutum, as a function of nitrate and silicate depletion. Diatoms were starved of either nitrate or silicate, and TAGs were extracted with hexane from lyophilized samples taken at various time intervals following starvation. The timing of TAG production and the relative abundance of TAGs were estimated by fluorescence spectroscopy using Nile red and the total yield per biomass determined by gravimetric assay. TAGs were analyzed using thin layer chromatography, gas chromatography–mass spectrometry, and electrospray ionization mass spectrometry to identify the major TAG species produced during the growth curve. Under our conditions, the TAG yield from T. pseudonana is about 14–18% of total dry weight. The TAG yield from P. tricornutum is about 14% of total dry weight. Silicate-starved T. pseudonana accumulated an average of 24% more TAGs than those starved for nitrate; however, the chemotypes of the TAGs produced were generally similar regardless of the starvation condition employed.     

Proteomics to reveal metabolic network shifts towards lipid accumulation following nitrogen deprivation in the diatom Phaeodactylum tricornutum

The marine diatom Phaeodactylum tricornutum is attracting considerable interest as a candidate for biofuel production due to its fast growth and high lipid content. Nitrogen deficiency can increase the lipid content in certain microalgae species, including P. tricornutum. However, the molecular basis of such changes remains unclear without analyzing metabolism at the proteomic level. We attempted to systematically analyze protein expression level changes of P. tricornutum upon N deprivation. We observed translational level changes that could overall redirect the metabolic network from carbon flux towards lipid accumulation. N deprivation led to an increase in the expression of genes involved in nitrogen assimilation and fatty acid biosynthesis and a concomitant decrease in photosynthesis and lipid catabolism enzymes. These molecular level changes are consistent with the observed physiological changes, e.g., in photosynthesis rate and saturated lipid content. Our results provide information at the proteomic level of the key enzymes involved in carbon flux towards lipid accumulation in P. tricornutum and suggest candidates for genetic manipulation in microalgae breeding for biodiesel production.