The First Symbiont-Free Genome Sequence of Marine Red Alga,Susabi-nori (Pyropia yezoensis)

Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis) using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb), which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35%) are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae.     

Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells

Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (K_m 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67^phox and p47^phox, ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47^phox, p67^phox, and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase.     

Cell wounding activates phospholipase D in primary mouse keratinocytes

Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D₃, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing.     

Lysophosphatidic acid stimulates phospholipase D activity and cell proliferation in PC-3 human prostate cancer cells

Phospholipase D (PLD) is activated in mammalian cells in response to a variety of growth factors and may play a role in cell proliferation. Lysophosphatidic acid (LPA) is a bioactive metabolite potentially generated as a result of PLD activation. Two human prostate cancer cell lines, PC-3 and LNCaP, express membrane PLD activity. The effects of LPA on PLD activity and proliferation were examined in PC-3 cells, which express hPLD1a/1b. Phorbol 12-myristate 13-acetate (PMA) induced a prolonged activation of PLD, as detected in both intact cells and membranes. LPA induced a transient activation of PLD that was maximal by 10 minutes. The EC₅₀ for LPA-induced PLD activation was approximately 1 μM. Pertussis toxin did not inhibit activation of PLD by LPA or PMA. Ro-31-8220 and bisindolylmaleimide I, inhibitors of protein kinase C, blocked activation by PLD by both PMA and LPA. PMA-induced activation of PLD did not appear to require translocation of PLDs from cytosol to membrane. LPA stimulated proliferation of PC-3 cells with an EC₅₀ of approximately 0.2 μM; this response was not inhibited by pertussis toxin. Perillyl alcohol, an anti-cancer drug, reversibly inhibited proliferation in response to either serum or LPA but did not inhibit activation of PLD by PMA or LPA. These data establish that LPA activates PLD and stimulates proliferation via G_i-independent pathways in a human prostate cancer cell line. J. Cell. Physiol. 174:261–272, 1998. © 1998 Wiley-Liss, Inc.     

Localization of Tie2 and phospholipase D in endothelial caveolae is involved in angiopoietin-1-induced MEK/ERK phosphorylation and migration in endothelial cells

Angiopoietin-1 (Ang1) and its receptor, Tie2, play critical roles in blood vessel formation. Ang1 triggers a variety of signaling events in endothelial cells leading to vasculogenic and angiogenic processes. However, the underlying mechanism for Ang1/Tie2 signaling is not fully understood. Here, we show that Tie2 and phospholipase D (PLD) are localized in the caveolae, specialized subdomains of the endothelial cell plasma membrane enriched with signaling molecules. Interestingly, Ang1 increased PLD activities in a dose- and time-dependent manner. Ang1-induced MEK/ERK activation was abrogated when PLD was inhibited, suggesting that PLD mediates Ang1-induced MEK/ERK activation. Moreover, PLD inhibitor, 1-butanol, inhibited Ang1-induced endothelial cell migration. Our results indicate that: (1) caveolae may be the platform for Tie2/PLD association in endothelial cells; (2) PLD is a new mediator of Ang1/Tie2-induced signaling pathway, and it participates in MAPK activation and endothelial cell migration.     

Development of a nuclear transformation system with a codon-optimized selection marker and reporter genes in Pyropia yezoensis (Rhodophyta)

Genetic transformation systems using reporter genes in whole plants have a wide variety of applications for molecular biological study including the visualization of expression patterns of particular genes and intracellular biological phenomena as well as the identification of novel genes. In this study, we assessed co-expression of each three codon-optimized reporter genes and a selectable marker in the nuclear transformation system of whole Pyropia yezoensis, a red marine alga. With the use of an endogenous promoter, both the codon-optimized hygromycin resistance gene and ß-glucuronidase gene (PyGUS) were co-expressed in P. yezoensis cells. A high level of GUS activity was observed in 60 % of the individuals in hygromycin-resistant lines. A histochemical GUS assay revealed that the PyGUS reporter gene was stably introduced and expressed throughout the algae's life cycle. In addition, two live cell reporters, humanized cyan fluorescent protein from Anemonia majano and luciferase from Gaussia princeps, were successfully expressed in whole P. yezoensis. The development of this transformation system involving three types of reporter genes provides opportunities for monitoring temporal changes in gene expression and for genetic screening in red marine algae.     

Involvement of phospholipase D in the low temperature acclimation-induced thermotolerance in grape berry

Both phospholipase D (PLD, EC 3.1.4.4) and salicylic acid (SA) play important roles in response to external stimulation and activating defense system in plants. However, roles of the two signals in plants during the development of thermotolerance induced by low temperature acclimation remain unclear. In the experiment presented in the paper, grape berries (Vitis vinifera L. cv. Chardonnay) were pretreated at 8 °C for 3 h and then transferred to 45 °C for heat stress. Compared with the control without low temperature pretreatment, membrane permeability and malondialdehyde (MDA) contents were reduced and the expression of HSP73 increased in the low temperature-pretreated berries under heat stress. During low temperature acclimation, PLD, SA and HSP73 could be activated. Meanwhile, the expression of HSP73 and the accumulation of free SA induced by low temperature can be inhibited by PLD activity inhibitor. All these results suggest that the activation of PLD is an early response to low temperature, and it is involved in the accumulation of free SA and the development of thermotolerance induced by low temperature acclimation.     

A Codon-Optimized Bacterial Antibiotic Gene Used as Selection Marker for Stable Nuclear Transformation in the Marine Red Alga Pyropia yezoensis

Marine macroalgae play an important role in marine coastal ecosystems and are widely used as sea vegetation foodstuffs and for industrial purposes. Therefore, there have been increased demands for useful species and varieties of these macroalgae. However, genetic transformation in macroalgae has not yet been established. We have developed a dominant selection marker for stable nuclear transformation in the red macroalga Pyropia yezoensis. We engineered the coding region of the aminoglycoside phosphotransferase gene aph7″ from Streptomyces hygroscopicus to adapt codon usage of the nuclear genes of P. yezoensis. We designated this codon-optimized aph7″ gene as PyAph7. After bombarding P. yezoensis cells with plasmids containing PyAph7 under the control of their endogenous promoter, 1.9 thalli (or individuals) of hygromycin-resistant strains were isolated from a 10-mm square piece of the bombarded thallus. These transformants were stably maintained throughout the asexual life cycle. Stable expression of PyAph7was verified using Southern blot analysis and genomic PCR and RT-PCR analyses. PyAph7 proved to be a new versatile tool for stable nuclear transformation in P. yezoensis.     

Isolation and evaluation of a strain of Pyropia yezoensis (Bangiales,Rhodophyta) resistant to red rot disease

Red rot disease (Pythium porphyrae) resistant strains of Pyropia yezoensis, AP1 and AP2, were isolated from live cells taken from lesions on infected P. yezoensis blades. The degree of resistance of these strains to red rot disease was evaluated over a range of environmental conditions including temperature (10, 15, and 20 °C), salinity (20, 26, and 32 ppt), and pH (7.5, 8.0, and 8.5). These conditions are within the range that red rot disease naturally occurs on Pyropia blades. P. yezoensis and Pyropia suborbiculata with low and high partial resistance to red rot disease, respectively, were used as controls. Infection with red rot disease occurred under all environmental conditions. The incidence, severity, and expansion of the disease increased with increasing temperature and decreasing salinity and pH. The resistance of the strains P. yezoensis-AP1 and P. yezoensis-AP2 was higher than that of P. yezoensis, but lower than that of P. suborbiculata. The degree of resistance was not significantly different between the AP1 and AP2 strains. These strains can therefore be considered to exhibit stable partial resistance to P. porphyrae, and as a good starting point for the development of more resistant strains that will prevent or reduce the incidence of red rot disease on Pyropia farms.     

Activation of phospholipase D by bradykinin and sphingosine 1-phosphate in A549 human lung adenocarcinoma cells via different GTP-binding proteins and protein kinase C delta signaling pathways

Phospholipase D (PLD) is involved in the signaling by many extracellular ligands, and its regulation appears to be quite complex. We investigated the signaling pathways initiated by bradykinin (BK) or sphingosine 1-phosphate (S1P) in A549 cells to define molecular mechanisms responsible for their additive effects on PLD activity. BK and S1P each elicited a sustained increase in phosphatidic acid content through a rapid and transient activation of PLD. The two pathways demonstrated rapid homologous downregulation, but heterologous desensitization was not observed. Action of both agonists required protein kinase C (PKC) activation and Ca(2+) influx but was mediated by different heterotrimeric G proteins. In membranes, inhibition of PKCdelta by rottlerin enhanced BK activation of PLD but inhibited that by S1P. Rottlerin inhibited activation of PLD in nuclei by both BK and S1P. By in situ immunofluorescence or cell fractionation followed by immunoblotting, PLD1 was concentrated primarily in nuclei, whereas the membrane fraction contained PLD2 and PLD1. Moreover, PKCdelta specifically phosphorylated recombinant PLD2, but not PLD1. BK and S1P similarly enhanced RhoA translocation to nuclei, whereas BK was less efficacious than S1P on RhoA relocalization to membranes. Effects of both agonists on the nuclear fraction, which contains only PLD1, are compatible with a RhoA- and PKCdelta-dependent process. In membranes, which contain both PLD1 and PLD2, the stimulatory effect of S1P on PLD activity can best be explained by RhoA- and PKCdelta-dependent activation of PLD1; in contrast, the effects of BK on RhoA translocation and enhancement of BK-stimulated PLD activity by PKC inhibition are both consistent with PLD2 serving as its primary target.     

Involvement of phospholipase D in sphingosine 1-phosphate-induced activation of phosphatidylinositol 3-kinase and Akt in Chinese hamster ovary cells overexpressing EDG3

Phospholipase D (PLD), phosphatidylinositol 3-kinase (PI3K), and Akt are known to be involved in cellular signaling related to proliferation and cell survival. In this report, we provide evidence that PLD links sphingosine 1-phosphate (S1P)-induced activation of the G protein-coupled EDG3 receptor to stimulation of PI3K and its downstream effector Akt in Chinese hamster ovary (CHO) cells. S1P stimulation of EDG3-overexpressing CHO cells but not vector-transfected cells induced activation of PLD, PI3K, and Akt in a time- and dose-dependent manner. Akt phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002 (2-(4-monrpholinyl)-8-phenyl-4H-1-benzopyran-4-one), indicating that Akt activation was dependent on PI3K. S1P-induced activation of PI3K and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid by serving as a phosphatidyl group acceptor in the transphosphatidylation reaction catalyzed by PLD, whereas both PI3K and Akt activation were not inhibited by 2-butanol without such reaction. Co-expression of wild-type PLD2 with myc-Akt resulted in increased Akt activation in response to S1P. In contrast, co-expression of a catalytically inactive mutant of PLD2 eliminated the S1P-induced Akt activation. The treatment of EDG3-expressing CHO cells with exogenous Streptomyces chromofuscus PLD, which caused an accumulation of phosphatidic acid, resulted in increases in PI3K activity and the phosphorylation of Akt, the latter of which was completely abolished by LY294002. Furthermore, S1P-induced membrane ruffling, which was dependent on PI3K and Rac, was inhibited by 1-butanol, but not by 2-butanol. These results demonstrate that PLD participates in the activation of PI3K and Akt stimulation of EDG3 receptor.     

ADP-Ribosylation Factors Do Not Activate Yeast Phospholipase Ds but Are Required for Sporulation

ADP-ribosylation factor (ARF) proteins in Saccharomyces cerevisiae are encoded by two genes, ARF1 and ARF2. The addition of the c-myc epitope at the C terminus of Arf1 resulted in a mutant (arf1-myc arf2) that supported vegetative growth and rescued cells from supersensitivity to fluoride, but homozygous diploids failed to sporulate. arf1-myc arf2 mutants completed both meiotic divisions but were unable to form spores. The SPO14 gene encodes a phospholipase D (PLD), whose activity is essential for mediating the formation of the prospore membrane, a prerequisite event for spore formation. Spo14 localized normally to the developing prospore membrane in arf1-myc arf2 mutants; however, the synthesis of the membrane was attenuated. This was not a consequence of reduced PLD catalytic activity, because the enzymatic activity of Spo14 was unaffected in meiotic arf1-myc arf2 mutants. Although potent activators of mammalian PLD1, Arf1 proteins did not influence the catalytic activities of either Spo14 or ScPld2, a second yeast PLD. These results demonstrate that ARF1 is required for sporulation, and the mitotic and meiotic functions of Arf proteins are not mediated by the activation of any known yeast PLD activities. The implications of these results are discussed with respect to current models of Arf signaling.     

Role of PLD−PKCζ signaling axis in p47phox phosphorylation for activation of NADPH oxidase by angiotensin II in pulmonary artery smooth muscle cells

We sought to determine the mechanism by which angiotensin II (ANGII) stimulates NADPH oxidase‐mediated superoxide (O₂^.?) production in bovine pulmonary artery smooth muscle cells (BPASMCs). ANGII‐induced increase in phospholipase D (PLD) and NADPH oxidase activities were inhibited upon pretreatment of the cells with chemical and genetic inhibitors of PLD2, but not PLD1. Immunoblot study revealed that ANGII treatment of the cells markedly increases protein kinase C‐α (PKC‐α), ‐δ, ‐ε, and ‐ζ levels in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PKC‐ζ, but not PKC‐α, ‐δ, and ‐ε, attenuated ANGII‐induced increase in NADPH oxidase activity without a discernible change in PLD activity. Transfection of the cells with p47phox small interfering RNA inhibited ANGII‐induced increase in NADPH oxidase activity without a significant change in PLD activity. Pretreatment of the cells with the chemical and genetic inhibitors of PLD2 and PKC‐ζ inhibited ANGII‐induced p47phox phosphorylation and subsequently translocation from cytosol to the cell membrane, and also inhibited its association with p22phox (a component of membrane‐associated NADPH oxidase). Overall, PLD?PKCζ?p47phox signaling axis plays a crucial role in ANGII‐induced increase in NADPH oxidase‐mediated O₂^.? production in the cells.     

Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: implications for epidermal growth factor signaling

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCalpha mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.     

Lipidomics reveals membrane lipid remodelling and release of potential lipid mediators during early stress responses in a murine melanoma cell line

Membranes are known to respond rapidly to various environmental perturbations by changing their composition and microdomain organization. In previous work we showed that a membrane fluidizer benzyl alcohol (BA) could mimic the effects of heat stress and enhance heat shock protein synthesis in different mammalian cells. Here we explore heat- and BA-induced stress further by characterizing stress-induced membrane lipid changes in mouse melanoma B16 cells. Lipidomic fingerprints revealed that membrane stress achieved either by heat or BA resulted in pronounced and highly specific alterations in lipid metabolism. The loss in polyenes with the concomitant increase in saturated lipid species was shown to be a consequence of the activation of phopholipases (mainly phopholipase A₂ and C). A phospholipase C–diacylglycerol lipase–monoacylglycerol lipase pathway was identified in B16 cells and contributed significantly to the production of several lipid mediators upon stress including the potent heat shock modulator, arachidonic acid. The accumulation of cholesterol, ceramide and saturated phosphoglyceride species with raft-forming properties observed upon both heat and BA treatments of B16 cells may explain the condensation of ordered plasma membrane domains previously detected by fluorescence microscopy and may serve as a signalling platform in stress responses or as a primary defence mechanism against the noxious effects of stresses.