RESUMO
Two parallel pathways compartmentalized in the chloroplast and the endoplasmic reticulum contribute to thylakoid lipid synthesis in plants, but how these two pathways are coordinated during thylakoid biogenesis and remodeling remains unknown. We report here the molecular characterization of a homologous ADIPOSE TRIGLYCERIDE LIPASE-LIKE gene, previously referred to as ATGLL. The ATGLL gene is ubiquitously expressed throughout development and rapidly upregulated in response to a wide range of environmental cues. We show that ATGLL is a chloroplast non-regioselective lipase with a hydrolytic activity preferentially towards 16:0 of diacylglycerol (DAG). Comprehensive lipid profiling and radiotracer labeling studies revealed a negative correlation of ATGLL expression and the relative contribution of the chloroplast lipid pathway to thylakoid lipid biosynthesis. Additionally, we show that genetic manipulation of ATGLL expression resulted in changes in triacylglycerol levels in leaves. We propose that ATGLL, through affecting the level of prokaryotic DAG in the chloroplast, plays important roles in balancing the two glycerolipid pathways and in maintaining lipid homeostasis in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Lipase Lipoproteica/metabolismo , Cloroplastos/metabolismo , Tilacoides/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Plantas/metabolismo , LipídeosRESUMO
Anoxygenic photosynthesis is diversified into two classes: chlorophototrophy based on a bacterial type-I or type-II reaction center (RC). Whereas the type-I RC contains both bacteriochlorophyll and chlorophyll, type-II RC-based phototrophy relies only on bacteriochlorophyll. However, type-II phototrophic bacteria theoretically have the potential to produce chlorophyll a by the addition of an enzyme, chlorophyll synthase, because the direct precursor for the enzyme, chlorophyllide a, is produced as an intermediate of BChl a biosynthesis. In this study, we attempted to modify the type-II proteobacterial phototroph Rhodovulum sulfidophilum to produce chlorophyll a by introducing chlorophyll synthase, which catalyzes the esterification of a diterpenoid group to chlorophyllide a thereby producing chlorophyll a. However, the resulting strain did not accumulate chlorophyll a, perhaps due to absence of endogenous chlorophyll a-binding proteins. We further heterologously incorporated genes encoding the type-I RC complex to provide a target for chlorophyll a. Heterologous expression of type-I RC subunits, chlorophyll synthase, and galactolipid synthase successfully afforded detectable accumulation of chlorophyll a in Rdv. sulfidophilum. This suggests that the type-I RC can work to accumulate chlorophyll a and that galactolipids are likely necessary for the type-I RC assembly. The evolutionary acquisition of type-I RCs could be related to prior or concomitant acquisition of galactolipids and chlorophylls.
RESUMO
The links between wound-response electrical signalling and the activation of jasmonate synthesis are unknown. We investigated damage-response remodelling of jasmonate precursor pools in the Arabidopsis thaliana leaf vasculature. Galactolipids and jasmonate precursors in primary veins from undamaged and wounded plants were analysed using MS-based metabolomics and NMR. In parallel, DAD1-LIKE LIPASEs (DALLs), which control the levels of jasmonate precursors in veins, were identified. A novel galactolipid containing the jasmonate precursor 12-oxo-phytodienoic acid (OPDA) was identified in veins: sn-2-O-(cis-12-oxo-phytodienoyl)-sn-3-O-(ß-galactopyranosyl) glyceride (sn-2-OPDA-MGMG). Lower levels of sn-1-OPDA-MGMG were also detected. Vascular OPDA-MGMGs, sn-2-18:3-MGMG and free OPDA pools were reduced rapidly in response to damage-activated electrical signals. Reduced function dall2 mutants failed to build resting vascular sn-2-OPDA-MGMG and OPDA pools and, upon wounding, dall2 produced less jasmonoyl-isoleucine (JA-Ile) than the wild-type. DALL3 acted to suppress excess JA-Ile production after wounding, whereas dall2 dall3 double mutants strongly reduce jasmonate signalling in leaves distal to wounds. LOX6 and DALL2 function to produce OPDA and the non-bilayer-forming lipid sn-2-OPDA-MGMG in the primary vasculature. Membrane depolarizations trigger rapid depletion of these molecules. We suggest that electrical signal-dependent lipid phase changes help to initiate vascular jasmonate synthesis in wounded leaves.
Assuntos
Arabidopsis , Oxilipinas , Ciclopentanos , Arabidopsis/fisiologiaRESUMO
Starch accounts for over 80% of the total dry weight in cereal endosperm and determines the kernel texture and nutritional quality. Amyloplasts, terminally differentiated plastids, are responsible for starch biosynthesis and storage. We screened a series of rice mutants with floury endosperm to clarify the mechanism underlying amyloplast development and starch synthesis. We identified the floury endosperm19 (flo19) mutant which shows opaque of the interior endosperm. Abnormal compound starch grains (SGs) were present in the endosperm cells of the mutant. Molecular cloning revealed that the FLO19 allele encodes a plastid-localized pyruvate dehydrogenase complex E1 component subunit α1 (ptPDC-E1-α1) that is expressed in all rice tissues. In vivo enzyme assays demonstrated that the flo19 mutant showed decreased activity of the plastidic pyruvate dehydrogenase complex. In addition, the amounts of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were much lower in the developing flo19 mutant endosperm, suggesting that FLO19 participates in fatty acid supply for galactolipid biosynthesis in amyloplasts. FLO19 overexpression significantly increased seed size and weight, but did not affect other important agronomic traits, such as panicle length, tiller number and seed setting rate. An analysis of single nucleotide polymorphism data from a panel of rice accessions identified that the pFLO19L haplotype was positively associated with grain length, implying a potential application in rice breeding. In summary, our study demonstrates that FLO19 is involved in galactolipid biosynthesis which is essential for amyloplast development and starch biosynthesis in rice.
Assuntos
Oryza , Grão Comestível , Endosperma/metabolismo , Galactolipídeos , Regulação da Expressão Gênica de Plantas , Mutação/genética , Melhoramento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastídeos/metabolismo , Complexo Piruvato Desidrogenase , Amido/metabolismoRESUMO
The thylakoid membranes of the chloroplast harbor the photosynthetic machinery that converts light into chemical energy. Chloroplast membranes are unique in their lipid makeup, which is dominated by the galactolipids mono- and digalactosyldiacylglycerol (MGDG and DGDG). The most abundant galactolipid, MGDG, is assembled through both plastid and endoplasmic reticulum (ER) pathways in Arabidopsis, resulting in distinguishable molecular lipid species. Phosphatidic acid (PA) is the first glycerolipid formed by the plastid galactolipid biosynthetic pathway. It is converted to substrate diacylglycerol (DAG) for MGDG Synthase (MGD1) which adds to it a galactose from UDP-Gal. The enzymatic reactions yielding these galactolipids have been well established. However, auxiliary or regulatory factors are largely unknown. We identified a predicted rhomboid-like protease 10 (RBL10), located in plastids of Arabidopsis thaliana, that affects galactolipid biosynthesis likely through intramembrane proteolysis. Plants with T-DNA disruptions in RBL10 have greatly decreased 16:3 (acyl carbons:double bonds) and increased 18:3 acyl chain abundance in MGDG of leaves. Additionally, rbl10-1 mutants show reduced [14 C]-acetate incorporation into MGDG during pulse-chase labeling, indicating a reduced flux through the plastid galactolipid biosynthesis pathway. While plastid MGDG biosynthesis is blocked in rbl10-1 mutants, they are capable of synthesizing PA, as well as producing normal amounts of MGDG by compensating with ER-derived lipid precursors. These findings link this predicted protease to the utilization of PA for plastid galactolipid biosynthesis potentially revealing a regulatory mechanism in chloroplasts.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Peptídeo Hidrolases/metabolismo , Ácidos Fosfatídicos/metabolismo , Plastídeos/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Vias Biossintéticas , Cloroplastos/metabolismo , Diglicerídeos/metabolismo , Retículo Endoplasmático/metabolismo , Galactolipídeos/metabolismo , Galactosiltransferases/metabolismo , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismoRESUMO
Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major components of thylakoid membranes and well-conserved from cyanobacteria to chloroplasts. However, cyanobacteria and chloroplasts synthesize these galactolipids using different pathways and enzymes, but they are believed to share a common ancestor. This fact implies that there was a replacement of the cyanobacterial galactolipid biosynthesis pathway during the evolution of a chloroplast. In this study, we first replaced the cyanobacterial MGDG biosynthesis pathway in a model cyanobacterium, Synechococcus elongatus PCC 7942, with the corresponding plant-type pathway. No obvious phenotype was observed under the optimum growth condition, and the content of membrane lipids was not largely altered in the transformants. We next replaced the cyanobacterial DGDG biosynthesis pathway with the corresponding plant-type pathway using the strain described above and isolated the strain harboring the replaced plant-type pathway instead of the whole galactolipid biosynthesis pathway. This transformant, SeGPT, can grow photoautotrophically, indicating that cyanobacterial galactolipid biosynthesis pathways can be functionally complemented by the corresponding plant-type pathways and that the lipid products MGDG and DGDG, and not biosynthesis pathways, are important. While SeGPT does not show strong growth retardation, the strain has low cellular chlorophyll content but it retained a similar oxygen evolution rate per chlorophyll content compared with the wild type. An increase in total membrane lipid content was observed in SeGPT, which was caused by a significant increase in DGDG content. SeGPT accumulated carotenoids from the xanthophyll groups. These results suggest that cyanobacteria have the capacity to accept other pathways to synthesize essential components of thylakoid membranes.
Assuntos
Galactolipídeos/biossíntese , Redes e Vias Metabólicas , Synechococcus/metabolismo , Carotenoides/metabolismo , Clorofila , Cucumis sativus , Lipídeos de Membrana/metabolismo , Organismos Geneticamente Modificados , Proteínas de Plantas/metabolismo , Synechococcus/genética , Xantofilas/metabolismoRESUMO
Galactolipids monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) constitute c. 50% and c. 30% of chloroplast membrane lipids, respectively. They are important for photosynthesis and stress tolerance. Mutations in DGD1, the major DGDG-synthesizing enzyme, severely reduce DGDG content and induce jasmonic acid (JA) overproduction, resulting in stunted growth. However, how DGDG reduction leads to JA overproduction is unknown. We introduced an inducible microRNA (ami-MGD1) into an Arabidopsis dgd1 mutant to reduce MGDG synthesis, thereby further diminishing galactolipid content, but partially restoring the MGDG : DGDG ratio. Galactolipid and Chl contents, expression of JA-biosynthesis and JA-responsive genes, photosystem II (PSII) maximum quantum efficiency, and chloroplast shape were investigated. Expression of JA-biosynthesis and JA-responsive genes were reduced in amiR-MGD1-transformed dgd1 plants. Stunted growth caused by JA overproduction was also partially rescued, but Chl reduction and PSII impairment remained similar to the original dgd1 mutant. Altered chloroplast shape, which is another defect observed in dgd1 but is not caused by JA overproduction, was also partially rescued. Our results reveal that an increased MGDG : DGDG ratio is the primary cause of JA overproduction. The ratio is also important for determining chloroplast shapes, whereas reduced Chl and photosynthesis are most likely a direct consequence of insufficient DGDG.
Assuntos
Cloroplastos , Galactolipídeos , Ciclopentanos , OxilipinasRESUMO
The brown alga Fucus vesiculosus is common to the intertidal zones of the Baltic Sea, where it is exposed to high fouling pressures by microorganisms. Our previous studies showed, repeatedly, the consistent antimicrobial activity of F. vesiculosus crude extracts against human pathogens, while untargeted metabolomics analyses have revealed a variety of metabolites. In this study, we applied the UPLC-QToF-MS/MS-based "bioactive molecular networking" (BMN) concept on the most bioactive n-hexane and n-butanol subextracts of Baltic F. vesiculosus coupled with in silico dereplication tools to identify the compounds responsible for antimicrobial activity. The first antimicrobial cluster identified by BMN was galactolipids. Our targeted isolation efforts for this class led to the isolation of six monogalactosyldiacylglycerol (MGDG) derivatives (1-6) and one digalactosyldiacylglycerol (DGDG, 7). The MGDGs 5 and 6 and the DGDG 7 exhibited activity against Staphylococcus aureus. The second compound class with high bioactivity was phlorotannins. In particular, phlorethol-type phlorotannins showed high correlations with antimicrobial activity based on the BMN approach, and two phlorotannins (8-9) were isolated. This study shows that antimicrobial components of F. vesiculosus reside in the algal cell walls and membranes and that BMN provides a complementary tool for the targeted isolation of bioactive metabolites.
Assuntos
Antibacterianos/metabolismo , Fucus/metabolismo , Animais , Metabolômica , Oceanos e MaresRESUMO
Aluminium (Al) is a key element that plays a major role in inhibiting plant growth and productivity under acidic soils. While lipids may be involved in plant tolerance/sensitivity to Al, the role of monogalactosyldiacylglycerol (MGDG) in Al response remains unknown. In this study, Arabidopsis MGDG synthase (AtMGD) mutants (mgd1, mgd2 and mgd3) and wild-type (Col-0) plants were treated with AlCl3; the effect of aluminium on root growth, aluminium distribution, plasma membrane integrity, lipid peroxidation, hydrogen peroxide content and membrane lipid compositions were analysed. Under Al stress, mgd mutants exhibited a more severe root growth inhibition, plasma membrane integrity damage and lipid peroxidation compared to Col-0. Al accumulation in root tips showed no difference between Col-0 and mutants under Al stress. Lipid analysis demonstrated that under Al treatment the MGDG content in all plants and MGDG/DGDG (digalactosyldiacylglycerol) remarkably reduced, especially in mutants impairing the stability and permeability of the plasma membrane. These results indicate that the Arabidopsis mgd mutants are hypersensitive to Al stress due to the reduction in MGDG content, and this is of great significance in the discovery of effective measures for plants to inhibit aluminium toxicity.
Assuntos
Alumínio/toxicidade , Arabidopsis/efeitos dos fármacos , Galactolipídeos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Poluentes do Solo/toxicidade , Alumínio/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Galactolipídeos/genética , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Mutação , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Poluentes do Solo/metabolismoRESUMO
Natural rubber is usually synthesized in the rubber particles present in the latex of rubber-producing plants such as the Pará rubber tree (Hevea brasiliensis) and rubber dandelion (Taraxacum kok-saghyz). Since the detailed lipid compositions of fresh latex and rubber particles of the plants are poorly known, the present study reports detailed compound lipid composition, focusing on phospholipids and galactolipids in the latex and rubber particles of the plants. In the fresh latex and rubber particles of both plants, phospholipids were much more dominant (85-99%) compared to galactolipids. Among the nine classes of phospholipids, phosphatidylcholines (PCs) were most abundant, at ~80%, in both plants. Among PCs, PC (36:4) and PC (34:2) were most abundant in the rubber tree and rubber dandelion, respectively. Two classes of galactolipids, monogalactosyl diacylglycerol and digalactosyl diacylglycerol, were detected as 12% and 1%, respectively, of total compound lipids in rubber tree, whereas their percentages in the rubber dandelion were negligible (< 1%). Overall, the compound lipid composition differed only slightly between the fresh latex and the rubber particles of both rubber plants. These results provide fundamental data on the lipid composition of rubber particles in two rubber-producing plants, which can serve as a basis for artificial rubber particle production in the future.
Assuntos
Hevea/química , Látex/química , Lipídeos/química , Taraxacum/químicaRESUMO
KEY MESSAGE: Here we show that accumulation of galactose-containing lipids in plastid membranes in shoots and the other membranes in roots maintains Arabidopsis growth under acidic stress and acidic phosphate deficiency. Soil acidification and phosphate deficiency are closely related to each other in natural environments. In addition to the toxicity of high proton concentrations, acid soil can lead to imbalances of ion availability and nutritional deficiencies, including inorganic phosphate (Pi). Among plants, activation of non-phosphorus-containing galactolipid, digalactosyldiacylglycerol (DGDG), synthesis concomitant with phospholipid degradation, namely membrane lipid remodeling, is crucial for coping with Pi starvation. However, regulation mechanisms of membrane lipid composition during acidic stress have not been clarified. Here, we investigated lipid metabolism in Arabidopsis thaliana grown under acidic stress with or without Pi. Under Pi-sufficient acidic conditions, DGDG was increased in shoot membranes, and some Pi starvation-responsive genes that are involved in lipid remodeling were upregulated without reducing Pi content in leaves. In contrast, under acidic Pi deficiency, membrane lipid remodeling in roots was partially repressed at a lower external pH. Nevertheless, phenotypic comparison between wild type and the double mutant of MGD2/3, which are responsible for DGDG accumulation during Pi starvation, indicated that the complete absence of lipid remodeling in roots resulted in a loss of tolerance to Pi deficiency rather specifically under acidic conditions. This result suggested important physiological roles of galactolipid-enriched membranes under acidic Pi deficiency.
Assuntos
Arabidopsis/fisiologia , Galactolipídeos/metabolismo , Metabolismo dos Lipídeos , Lipídeos de Membrana/metabolismo , Fosfatos/deficiência , Fosfolipídeos/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Concentração de Íons de Hidrogênio , Fenótipo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Plastídeos/metabolismo , Estresse FisiológicoRESUMO
Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.
Assuntos
Proteínas de Arabidopsis/genética , Membrana Celular/genética , Galactolipídeos/biossíntese , Galactolipídeos/genética , Galactosiltransferases/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Galactolipídeos/metabolismo , Galactosiltransferases/metabolismo , Regulação da Expressão Gênica de Plantas , Lipídeos de Membrana/genética , Lipídeos de Membrana/metabolismo , Fotossíntese/genética , Transporte Proteico/genéticaRESUMO
Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U-13C]glucose, 13CO2, or D2O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid bodies. The metabolic origin of stress-induced triglyceride was found to be a continuous 8:2 ratio between de novo synthesized FA and acyl chain transfer from pre-stressed membrane lipids with little input from lipid remodeling. Membrane lipids were continually synthesized with associated acyl chain editing during nitrogen stress, in contrast to an overall decrease in total membrane lipid. The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions.
Assuntos
Carbono/metabolismo , Ácidos Graxos/metabolismo , Marcação por Isótopo/métodos , Lipídeos de Membrana/metabolismo , Microalgas/metabolismo , Nitrogênio/deficiência , Triglicerídeos/metabolismo , Cromatografia Gasosa-Espectrometria de MassasRESUMO
This study demonstrates the bioefficacy and gives mechanistic insights into a plant galactolipid 1,2-di-O-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (dLGG) against metastatic melanoma using a syngeneic mouse model implanted with B16COX-2/Luc melanoma. dLGG-20 (p.o. dLGG 20 mg/kg) and anti-cancer drug CP-2 (i.p. cisplatin 2 mg/kg) treatment significantly inhibited lung metastasis of melanoma in mice 91 and 57%, respectively, as determined by bioluminescence intensity. Moreover, dLGG-20 and CP-2 treatment prolonged mouse mean survival time. dLGG-20 treatment significantly inhibited the expression levels of several molecular markers, that is, PCNA, MMP2, COX-2, VEGF, vimentin, snail, TGF-ß, ß-catenin, TNF-α, PD-1 and PD-L1 in mouse lung tissues compared to tumor control mice. Significant inhibition of macrophage and neutrophil infiltration and promotion of CD8 + Tc cell recruitment in the lung microenvironment was observed in dLGG-20-treated mice. A LC/MS-based comparative oxylipin metabolomics study showed that dLGG-20 treatment significantly induced (5.0- to 12.8-fold) the 12/15-LOX catalyzed oxylipin products in mouse serum including 17-HDHA from DHA, 15-HEPE from EPA, 8- and 12-HETEs from AA, and CYP450-derived 20-HETE from AA. CP-2 treatment increased 12/15-LOX derived 8-, 11- and 12-HETEs from AA, and CYP450 derived 11,12-EET from AA ad 9,10-DHOME from LA by 5.3- to 8.1-fold. Of note, dLGG and 17-HDHA were more effective than CP in preventing B16 melanoma cell-induced pulmonary vascular permeability in mice through inhibition of TNF-α production, up-regulation of tight junction proteins claudin1 and ZO-2 and deregulation of Src activation. In conclusion, this study shows the novel therapeutic effect of phytoagent dLGG and suggests its potential as a therapeutic agent for metastatic melanoma.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Permeabilidade Capilar/fisiologia , Glucosídeos/farmacologia , Glicerol/análogos & derivados , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Melanoma Experimental/secundário , Oxilipinas/sangue , Plantas/química , Artéria Pulmonar/fisiologia , Veias Pulmonares/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Transição Epitelial-Mesenquimal , Quinase 2 de Adesão Focal/metabolismo , Glicerol/farmacologia , Humanos , Masculino , Metabolômica , Camundongos Endogâmicos C57BL , Invasividade Neoplásica/prevenção & controle , Transdução de Sinais , Junções Íntimas/efeitos dos fármacos , Microambiente Tumoral , Fator de Necrose Tumoral alfa/fisiologia , Quinases da Família src/metabolismoRESUMO
Talaromyces thermophilus lipase (TTL) was found to hydrolyze monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) substrates presented in various forms to the enzyme. Different assay techniques were used for each substrate: pHstat with dioctanoyl galactolipid-bile salt mixed micelles, barostat with dilauroyl galactolipid monomolecular films spread at the air-water interface, and UV absorption using a novel MGDG substrate containing α-eleostearic acid as chromophore and coated on microtiter plates. The kinetic properties of TTL were compared to those of the homologous lipase from Thermomyces lanuginosus (TLL), guinea pig pancreatic lipase-related protein 2 and Fusarium solani cutinase. TTL was found to be the most active galactolipase, with a higher activity on micelles than on monomolecular films or surface-coated MGDG. Nevertheless, the UV absorption assay with coated MGDG was highly sensitive and allowed measuring significant activities with about 10â¯ng of enzymes, against 100â¯ng to 10⯵g with the pHstat. TTL showed longer lag times than TLL for reaching steady state kinetics of hydrolysis with monomolecular films or surface-coated MGDG. These findings and 3D-modelling of TTL based on the known structure of TLL pointed out to two phenylalanine to leucine substitutions in TTL, that could be responsible for its slower adsorption at lipid-water interface. TTL was found to be more active on MGDG than on DGDG using both galactolipid-bile salt mixed micelles and galactolipid monomolecular films. These later experiments suggest that the second galactose on galactolipid polar head impairs the enzyme adsorption on its aggregated substrate.
Assuntos
Proteínas Fúngicas/química , Galactolipídeos/química , Lipase/química , Talaromyces/química , Ar/análise , Animais , Ácidos e Sais Biliares/química , Hidrolases de Éster Carboxílico/química , Ensaios Enzimáticos , Fusarium/química , Fusarium/enzimologia , Cobaias , Hidrólise , Cinética , Ácidos Linolênicos/química , Micelas , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Especificidade por Substrato , Propriedades de Superfície , Talaromyces/enzimologia , Raios Ultravioleta , Água/químicaRESUMO
Digalactosyldiacylglycerol (DGDG) is a major component of thylakoid membranes, occupying approximately 20% of the membrane system. This lipid composition is conserved from cyanobacteria to the chloroplasts of terrestrial plants, suggesting that DGDG is important for the function of photosynthetic membranes. Here we isolated the gene for DGDG synthase in the cyanobacterium Synechococcus elongatus PCC 7942 (7942dgdA) and found that this gene is essential for this species. 7942dgdA could be knocked out only when genes for cyanobacterial or plant DGDG synthases were expressed, indicating that the important factor was not the specific synthetic pathway but the lipid product. Lack of DGDG could not be compensated by the other membrane lipids in S. elongatus PCC 7942 or by glucosylgalactosyldiacylglycerol synthesized by the ß-GlcT gene of Chloroflexus aurantiacus. These results reveal that DGDG has an indispensable role in S. elongatus PCC 7942 and that the second galactose molecule is key. Conservation and distribution of the galactolipid synthetic pathway among oxygenic phototrophs is discussed. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.
Assuntos
Proteínas de Arabidopsis/genética , Galactolipídeos/genética , Galactosiltransferases/genética , Lipídeos de Membrana/metabolismo , Synechococcus/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas/genética , Cloroplastos/enzimologia , Galactolipídeos/metabolismo , Galactosiltransferases/metabolismo , Lipídeos de Membrana/genética , Fotossíntese/genética , Synechococcus/enzimologia , Tilacoides/enzimologiaRESUMO
In photosynthetic organisms, the photosynthetic membrane constitutes a scaffold for light-harvesting complexes and photosynthetic reaction centers. Three kinds of glycolipids, namely monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol, constitute approximately 80-90% of photosynthetic membrane lipids and are well conserved from tiny cyanobacteria to the leaves of huge trees. These glycolipids perform a wide variety of functions beyond biological membrane formation. In particular, the capability of adaptation to harsh environments through regulation of membrane glycolipid composition is essential for healthy growth and development of photosynthetic organisms. The genome analysis and functional genetics of the model seed plant Arabidopsis thaliana have yielded many new findings concerning the biosynthesis, regulation, and functions of glycolipids. Nevertheless, it remains to be clarified how the complex biosynthetic pathways and well-organized functions of glycolipids evolved in early and primitive photosynthetic organisms, such as cyanobacteria, to yield modern photosynthetic organisms like land plants. Recently, genome data for many photosynthetic organisms have been made available as the fruit of the rapid development of sequencing technology. We also have reported the draft genome sequence of the charophyte alga Klebsormidium flaccidum, which is an intermediate organism between green algae and land plants. Here, we performed a comprehensive phylogenic analysis of glycolipid biosynthesis genes in oxygenic photosynthetic organisms including K. flaccidum. Based on the results together with membrane lipid analysis of this alga, we discuss the evolution of glycolipid synthesis in photosynthetic organisms. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.
Assuntos
Galactolipídeos/genética , Glicolipídeos/genética , Fotossíntese/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Cianobactérias/genética , Cianobactérias/crescimento & desenvolvimento , Evolução Molecular , Galactolipídeos/biossíntese , Genoma de Planta , Glicolipídeos/biossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Filogenia , Sementes/genética , Sementes/crescimento & desenvolvimentoRESUMO
Ribosomal protein S6 kinase (S6K) functions as a key component in the target of rapamycin (TOR) pathway involved in multiple processes in eukaryotes. The role and regulation of TOR-S6K in lipid metabolism remained unknown in plants. Here we provide genetic and pharmacological evidence that TOR-Raptor2-S6K1 is important for thylakoid galactolipid biosynthesis and thylakoid grana modeling in rice (Oryza sativa L.). Genetic suppression of S6K1 caused pale yellow-green leaves, defective thylakoid grana architecture. S6K1 directly interacts with Raptor2, a core component in TOR signaling, and S6K1 activity is regulated by Raptor2 and TOR. Plants with suppressed Raptor2 expression or reduced TOR activity by inhibitors mimicked the S6K1-deficient phenotype. A significant reduction in galactolipid content was found in the s6k1, raptor2 mutant or TOR-inhibited plants, which was accompanied by decreased transcript levels of the set of genes such as lipid phosphate phosphatase α5 (LPPα5), MGDG synthase 1 (MGD1), and DGDG synthase 1 (DGD1) involved in galactolipid synthesis, compared to the control plants. Moreover, loss of LPPα5 exhibited a similar phenotype with pale yellow-green leaves. These results suggest that TOR-Raptor2-S6K1 is important for modulating thylakoid membrane lipid biosynthesis, homeostasis, thus enhancing thylakoid grana architecture and normal photosynthesis ability in rice.
Assuntos
Galactolipídeos/biossíntese , Regulação da Expressão Gênica de Plantas , Oryza/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Serina-Treonina Quinases TOR/genética , Tilacoides/genética , Agrobacterium tumefaciens/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Genótipo , Oryza/metabolismo , Oryza/ultraestrutura , Fenótipo , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Plântula/genética , Plântula/metabolismo , Plântula/ultraestrutura , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Tilacoides/metabolismo , Tilacoides/ultraestrutura , Transcrição GênicaRESUMO
Photosynthetic organelles in plants and algae are characterized by the high abundance of glycolipids, including the galactolipids mono- and digalactosyldiacylglycerol (MGDG, DGDG) and the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). Glycolipids are crucial to maintain an optimal efficiency of photosynthesis. During phosphate limitation, the amounts of DGDG and SQDG increase in the plastids of plants, and DGDG is exported to extraplastidial membranes to replace phospholipids. Algae often use betaine lipids as surrogate for phospholipids. Glucuronosyldiacylglycerol (GlcADG) is a further glycolipid that accumulates under phosphate deprived conditions. In contrast to plants, a number of eukaryotic algae contain very long chain polyunsaturated fatty acids of 20 or more carbon atoms in their glycolipids. The pathways and genes for galactolipid and sulfolipid synthesis are largely conserved between plants, Chlorophyta, Rhodophyta and algae with complex plastids derived from secondary or tertiary endosymbiosis. However, the relative contribution of the endoplasmic reticulum- and plastid-derived lipid pathways for glycolipid synthesis varies between plants and algae. The genes for glycolipid synthesis encode precursor proteins imported into the photosynthetic organelles. While most eukaryotic algae contain the plant-like galactolipid (MGD1, DGD1) and sulfolipid (SQD1, SQD2) synthases, the red alga Cyanidioschyzon harbors a cyanobacterium-type DGDG synthase (DgdA), and the amoeba Paulinella, derived from a more recent endosymbiosis event, contains cyanobacterium-type enzymes for MGDG and DGDG synthesis (MgdA, MgdE, DgdA).
Assuntos
Galactolipídeos/metabolismo , Glicolipídeos/metabolismo , Microalgas/metabolismo , Plantas/metabolismo , Galactolipídeos/química , Glicolipídeos/química , Estrutura MolecularRESUMO
Cyanobacteria carry out oxygenic photosynthesis and share many features with chloroplasts, including thylakoid membranes, which are mainly composed of membrane lipids and protein complexes that mediate photosynthetic electron transport. Although the functions of the various thylakoid protein complexes have been well characterized, the details underlying the biogenesis of thylakoid membranes remain unclear. Galactolipids are the major constituents of the thylakoid membrane system, and all the genes involved in galactolipid biosynthesis were recently identified. In this chapter, I summarize recent advances in our understanding of the factors involved in thylakoid development, including regulatory proteins and enzymes that mediate lipid biosynthesis.