Chlorosome lipids from Chlorobium tepidum: characterization and quantification of polar lipids and wax esters

We have extracted polar lipids and waxes from isolated chlorosomes from the green sulfur bacterium Chlorobium tepidum and determined the fatty acid composition of each lipid class. Polar lipids amounted to 4.8 mol per 100 mol bacteriochlorophyll in the chlorosomes, while non-polar lipids (waxes) were present at a ratio of 5.9 mol per 100 mol bacteriochlorophyll. Glycolipids constitute 60 % of the polar lipids while phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and an aminoglycosphingolipid make up respectively 15, 3, 8 and 12 %. A novel glycolipid was identified as a rhamnose derivative of monogalactosyldiacylglycerol, while the other major glycolipid was monogalactosyldiacylglycerol. Tetradecanoic acid was the major fatty acid in the aminoglycosphingolipid, while the other polar lipids contained predominantly hexandecanoic acid. The chlorosome waxes are esters of unbranched fatty acids and fatty alcohols with 14 or 16 carbon atoms, joined to form molecules with between 28 and 32 carbon atoms. The stoichiometry between lipids and bacteriochlorophyll suggests that much of the chlorosome surface is covered by protein.     

The cuticular fatty acids of Calliphora vicina, Dendrolimus pini and Galleria mellonella larvae and their role in resistance to fungal infection

Epicuticular lipids in many terrestrial arthropods consist of vast numbers of polar and non-polar aliphatic compounds, which are mainly responsible for the water balance in these animals but can also affect conidia germination of entomopathogenic fungi. In this work the qualitative and quantitative profiles of cuticular fatty acids from three insect species differing in their susceptibility to fungal infection were studied. In an innovative approach, laser light scattering detection was coupled with HPLC in order to identify the non-chromophoric chemicals usually present in cuticular extracts. The acids identified contained from 5 to 20 carbon atoms in the alkyl chain and included unsaturated entities such as C(16:1), C(18:1), C(18:2), C(18:3) and C(20:1). There was a marked dominance of acids containing 16-18 carbon atoms. The relative contents of fatty acids in the extracted waxes varied from trace amounts to 44%. Cuticular fatty acids profile of Calliphora vicina (species resistant to fungal infection) significantly differs from profiles of Dendrolimus pini and Galleria mellonella (both species highly susceptible to fungal infection). The major difference is the presence of C(14:0), C(16:1) and C(20:0) in the cuticle of C. vicina. These three fatty acids are absent in the cuticle of D. pini while G. mellonella cuticle contains their traces. The concentrations of four fatty acids dominating in the G. mellonella larval cuticle (C(16:0), C(18:0), C(18:1) and C(18:2)) were found to fluctuate during the final larval instar and correlate with fluctuations in the susceptibility of larvae to fungal infection. The possible role of cuticular fatty acids in preventing fungal infection is discussed.     

Lipids of Pinus halepensis pollen

The total lipids of Pinus halepensis pollen were separated into individual classes of neutral and polar lipids and the components of each class were identified and determined quantitatively. Free fatty acids, waxes and triacylglycerols were found as the main constituents of neutral lipids and phosphatidylcholine and phosphatidylethanolamine of polar lipids. Glycerylether derivatives were detected in neutral and polar lipid fractions. Free and esterified volatile fatty acids were also found in pollen and its neutral lipid fraction.     

CUTICULAR LIPIDS OF TERRESTRIAL PLANTS AND ARTHROPODS: A COMPARISON OF THEIR STRUCTURE, COMPOSITION, AND WATERPROOFING FUNCTION

1. Lipids deposited on the surface or embedded within the cuticle of terrestrial plants and arthropods are primarily responsible for the observed low rates of water loss through the cuticle. 2. These lipids are a mixture of long-chain compounds which include hydrocarbons (saturated, unsaturated, branched), wax esters, free fatty acids, alcohols, ketones, aldehydes, and cyclic compounds. 3. The cuticle of both plants and arthropods is a continuous, non-cellular multilayered membrane which overlies the epidermal cells. 4. In arthropods, horizontal division of the cuticle into layers is clearly visible. In plants, the layers comprising the cuticle are not sharply demarcated. 5. The substance responsible for the structural integrity of the plant cuticle (cutin) is composed of cross-esterified fatty acids; structural integrity in arthropod cuticle is provided by a chitin-protein complex. 6. Cuticular lipids are probably formed near the surface in both plants and arthropods; however, specific sites of synthesis are known for only a few species and little is known about their transport from these sites to the surface. The elaborate pore canal and wax canal system of arthropod cuticle is absent from plants. 7. The physical structure and arrangement of the lipid deposits on the cuticular surface that are so important in controlling water movement depend on both quantity and chemical composition, and are, in turn, specific to each species in relation to its environment. 8. Different lipid components are not equally efficient in reducing transpiration. Maximum waterproofing effectiveness is provided by long-chain, saturated, non-polar molecules containing few methyl branches. 9. Plants and arthropods can, within genetic constraints, alter the composition of their cuticular waxes to improve impermeability when conditions require increased water conservation. 10. None of the models proposed to explain the change in arthropod cuticular permeability which occurs at a species-specific temperature (‘transition temperature’) in many species is supported by the experimental data now available.     

Leaf cuticular waxes are arranged in chemically and mechanically distinct layers: evidence from Prunus laurocerasus L.

The composition and spatial arrangement of cuticular waxes on the leaves of Prunus laurocerasus were investigated. In the wax mixture, the triterpenoids ursolic acid and oleanolic acid as well as alkanes, fatty acids, aldehydes, primary alcohols and alcohol acetates were identified. The surface extraction of upper and lower leaf surfaces yielded 280 mg m ^{? 2} and 830 mg m ^{? 2}, respectively. Protocols for the mechanical removal of waxes from the outermost layers of the cuticle were devised and evaluated. With the most selective of these methods, 130 mg m ^{? 2} of cuticular waxes could be removed from the adaxial surface before a sharp, physically resistant boundary was reached. Compounds thus obtained are interpreted as ‘epicuticular waxes’ with respect to their localization in a distinct layer on the surface of the cutin matrix. The epicuticular wax film can be transferred onto glass and visualized by scanning electron microscopy. Prunus laurocerasus epicuticular waxes consisted entirely of aliphatic compounds, whereas the remaining intracuticular waxes comprised 63% of triterpenoids. The ecological relevance of this layered structure for recognition by phytotrophic fungi and herbivorous insects that probe the surface composition for sign stimuli is discussed.     

Role of cuticular lipids and water-soluble compounds in tick susceptibility to Metarhizium infection

The arthropod cuticle acts as a physiochemical barrier protecting the organism from pathogens' entry. Entomopathogenic fungi actively penetrate the cuticles of arthropod hosts and are therefore directly affected by cuticle composition. Previously we have observed that Metarhizium spp. developing on resistant ticks ultimately die without penetrating tick's cuticle, suggesting that the cuticles of resistant ticks have antifungal compounds. In the present study, lipids and water-soluble cuticular components were extracted from engorged female tick cuticles, of one susceptible and one resistant tick species to Metarhizium spp. While conidia exposed to lipids from the susceptible tick, Rhipicephalus annulatus, germinated and differentiated into appressorium, conidia exposed to lipids from the resistant tick, Hyalomma excavatum, were inhibited. Soluble cuticular component extracts from both susceptible and resistant ticks stimulated conidial germination but not appressorium differentiation. A comparative analysis of the fatty acid profile in lipid extract of each tick exhibited similar compositions, but the relative abundance of C16:0, C18:0, C18:1ω9C and C20:0 was 2–5 times higher in the extracts from resistant ticks. All of these fatty acids inhibited conidial germination in vitro at 1% and 0.1% w/v concentration, but C20:0 stimulated appressorium differentiation at low concentration. This is the first report demonstrating a possible link between the presence of antifungal compounds in a specific concentration in tick cuticle and tick resistance to infection.     

Identification of lipids in the cuticle of the parasitic nematode Anisakis simplex and the somatic tissues of the Atlantic cod Gadus morhua

The main aim of this work was to assign the cuticular lipids identified in a parasitic nematode and to distinguish those originating from its host. The hypothesis that long-chained fatty acids and sterols are imported by the parasite in the absence of certain enzymes was also tested. The organisms (Anisakis simplex and Gadus morhua) were extracted in petroleum ether and dichloromethane. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was used to identify unknown components, and electrospray ionization mass spectrometry (ESI/MS) to verify recognized groups of lipids. The lipid classes identified in the surface layer were free saturated and unsaturated fatty acids, triacylglycerols, sterols and non-polar sphingolipids (ceramides, sphingoid bases). The most abundant fraction consisted of fatty acids. The predominant saturated acids were tetradecanoic acid in the petroleum ether extract of A. simplex, hexadecanoic acid in the dichloromethane extract of A. simplex, and also the polyunsaturated octadecahexaenoic and octadecatrienoic acids in both extracts of the parasitic nematode. The mass spectrum revealed the presence of fatty acids with different numbers of carbons, and with odd and even numbers of unsaturated bonds. The MALDI-TOF mass spectrum also identified triacylglycerols (TAGs). The dominant short-chain TAGs were CoCoCy:₁, CoCoPg and Bu0:0B:₆. The majority of TAGs were found in the ether and dichloromethane extracts of A. simplex. Sterols were the least common class of lipids found in the nematode extracts; most likely, this is the fraction that is entirely incorporated from the host organism because of the parasite’s inability to synthesize them. MALDI-TOF also identified non-polar sphingolipids - ceramides and sphingoid bases. The signals due to N-octanoyl-d-erythro-octasphinganine (m/z 288.3) and N-tetranoyl-d-erythro-tetradecasphinganine (m/z 316.4) were dominant on the mass spectra; quite a large number of short-chain non-polar sphingolipids were also identified.     

Fractionation and lipase-catalyzed conversion of microalgal lipids to biodiesel

This study was conducted to evaluate the lipid fractionation and purification procedures of lipase-catalyzed conversion of neutral lipids to microalgal biodiesel. Microalgae lipids were efficiently recovered and purified by a combined extraction method and crude lipid extracts were separated into neutral lipids, glycolipids, and phospholipids by solid-phase extraction. The high purity of the neutral lipids fraction was confirmed by its low concentration of phosphorous (< 2.0 ppm). Transesterification was catalyzed by immobilized Candida antarctica lipase for 72 h with stepwise addition of methanol. The reaction displayed Michaelis–Menten kinetics and produced high yields of microalgal biodiesel (91.2% in the case of Dunaliella salina) with a high content of unsaturated fatty acids (81.5%). Neutral lipids were converted to biodiesel by three-step transesterification, while the removal of polar lipids maintained the activity of the immobilized lipase by reducing both reaction mixture viscosity and contamination risk.     

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.     

Non-polar and polar chemical profiling of six Casearia species (Salicaceae)

Six species of Casearia (C. decandra, C. grandiflora, C. javitensis, C. arborea, C. lasiophylla and C. ulmifolia) were chemically investigated in their non-polar and polar constituents. To this purpose, leaf extracts were analyzed by Gas- (GC) and Liquid-Chromatography (LC) coupled with Mass Spectrometry (MS). Twenty compounds, mainly identified as terpenes, fatty acids and hydrocarbons, and differentially expressed in the six species, were detected in the non-polar extracts. Fourteen compounds, among which glycosylated flavonoids and clerodane-type diterpenes, were tentatively identified by LC-MS/MS analysis in the leaf ethanol extracts.     

植物角质层蜡质的化学组成研究综述

角质层是植物与外界的第一接触面,而角质层蜡质则是由位于角质层外的外层蜡质和深嵌在角质层中的内层蜡质两部分构成。植物角质层蜡质成分极其复杂,具有重要的生理功能。综述了有关植物角质层蜡质的化学组成信息,探讨了目前植物角质层蜡质化学成分研究中存在的一些问题,展望了角质层蜡质成分的研究前景。     

Isolated thallus-associated compounds from the macroalga Fucus vesiculosus mediate bacterial surface colonization in the field similar to that on the natural alga

This study investigated whether surface-associated compounds isolated from the macroalga Fucus vesiculosus had the potential to mediate microbial and/or macrobial epibiosis similar to that on the natural alga. To selectively yield thallus-associated compounds and avoid contamination by intracellular algal compounds, cell lysis was monitored by surface microscopy of algal cells and chemical profiling of algal surface extracts by coupled gas chromatography mass spectroscopy. The optimized extraction resulted in polar and non-polar algal surface extracts. The non-polar surface extract was immobilized in hydrogel, the polar surface extract was homogeneously perfused through the gel to ensure a temporally constant delivery of polar extract components. During a 7 day field trial, bacterial biofilms were formed on control gels and gels featuring polar and/or non-polar extract components. PERMANOVA revealed that bacterial community profiles on controls and on gels featuring polar or non-polar extract were significantly different from the profile on F. vesiculosus, while the profile on the gels bearing both polar and non-polar extracts was not. Moreover, the polar surface extracts inhibited the settlement of barnacle cyprids. Considering the pronounced effects of bacterial biofilms on invertebrate larval settlement, these results suggest that algal surface chemistry affects macrofouling not only directly but also indirectly, via its control of biofilm formation and composition.     

ABC-type transporters and cuticle assembly: Linking function to polarity in epidermis cells

The aerial organs of plants are covered with a cuticle, a continuous layer overlaying the outermost cell walls of the epidermis. The cuticle is composed of two major classes of the lipid biopolymers: cutin and waxes, collectively termed cuticular lipids. Biosynthesis and transport of cuticular lipids occur predominantly in the epidermis cells. In the transport pathway, cuticular lipids are exported from their site of biosynthesis in the ER/plastid to the extracellular space through the plasma membrane and cell wall. Growing evidence suggests that ATP-binding cassette (ABC) transporters are implicated in transport of cuticular lipids across the plasma membrane of epidermal cells. The Arabidopsis ABC-type transporter protein CER5 (WBC12) was reported to act as a wax monomers transporter. In recent works, our group and others showed that a CER5-related protein, DESPERADO (DSO/WBC11), is required for cutin and wax monomers transport through the plasma membrane of Arabidopsis epidermis cells. Unlike the cer5 mutant, DSO loss-of-function had a profound effect on plant growth and development, particularly dwarfism, postgenital organ fusions, and altered epidermal cell differentiation. The partially overlapping function of CER5 and DSO and the fact that these proteins are half-size ABC transporters suggest that they might form a hetero-dimeric complex while transporting wax components. An intriguing observation was the polar localization of DSO in the distal part of epidermis cells. This polar expression might be explained by DSO localization within lipid rafts, specific plasma membrane microdomains which are associated with polar protein expression. In this review we suggest possible mechanisms for cuticular lipids transport and a link between DSO function and polar expression. Furthermore, we also discuss the subsequent transport of cuticular constituents through the hydrophobic cell wall and the possible involvement of lipid transfer proteins in this process.Key words: ABC transporter, cuticular lipids, polar expression, plasma membrane, epidermis     

Infection process of Colletotrichum dematium on mulberry leaves: An unusual method of sporulation

Abstract

The pre-penetration and infection process of Colletotrichum dematium on mulberry leaf was investigated by scanning electron microscope. Conidia produced on germination appressoria directly or at the end of short germ tubes. Appressoria were formed mostly over cuticle, but sometimes over stomata also. At 72 h post-inoculation, an extensive network of sub-cuticular runner hyphae (RH) was produced. The RH were traceable by the cuticular bulgings on leaf surface. The RH emerged to leaf surface through ruptured cuticle to form secondary infection hyphae (SIH). The SIH re-entered the leaf tissue by sending penetration branches through stomata. Conidia were formed singly on short conidiophores from the RH and SIH, at short intervals. The conidia developed on RH were exposed to leaf surface through ruptured cuticle. Some times conidia were released through stomata also. The RH and SIH had thick knots from which hyphal branches and conidia were developed. Definite acervuli were not developed.     

Changes in Glycolipid and Phospholipid Compositions in Cotyledons of Germinating Soybeans

This investigation was conducted to observe changes in the compositions of fatty acids, glycolipids (GL) and phospholipids (PL) in cotyledons of soybean seeds which were germinated either in the dark or the light at 28°C for 8 days. The patterns of changes in lipid composition depended on the germinating conditions tested. In general, non-polar lipids were metabolized at a faster rate than polar lipids. Changes in lipid contents in cotyledons were also observed more clearly with the polar lipids than with the non-polar ones, especially in the light-grown seedlings. The major component of lipid, GL in chloroplasts, appeared rapidly at an earlier stage in the cotyledons of light-grown seedlings. During germination of soybean seeds, acyl sterylglucoside in cotyledons decreased rapidly, but monogalactosyl diglyceride and digalactosyl diglyceride (DGD) increased in the light-grown seedlings, whereas sterylglucoside and DGD increased in the dark-grown seedlings.

The major PL present immediately after immersion were phosphatidyl ethanolamine (PE), phosphatidyl choline (PC) and phosphatidyl inositol (PI). During germination under both conditions, light and dark, PE in cotyledons decreased with PC or PI, while phosphatidic acid increased rapidly, and phosphatidyl glycerol and diphosphatidyl glycerol also increased slightly. These changes in glycolipid and phospholipid compositions during germination seem to occur from the formation of photosynthetic tissues and the metabolic interconversion of phospholipids.     

Fine structure of plant cuticles in relation to water permeability: The fine structure of the cuticle of Clivia miniata reg. leaves

The fine structure of the upper cuticular membrane (CM) of Clivia miniata leaves was investigated using electron microscopy. The CM is made up of a thin (130 nm) lamellated cuticle proper (CP) and a thick (up to 7 m over periclinal walls) cuticular layer (CL) of marbled appearance. Evidence is presented to show that the electron lucent lamellae of the CP do not simply represent layers of soluble cuticular lipids (SCL). Instead, the lamellation is probably due to layers of cutin differing in polarity. It is argued that the SCL in the Cp are the main barrier to water. Thickening of the CM during leaf development takes place by interposition of cutin between the CM and the cellin wall. The cutin of young, expanding leaves has a high affinity for KMnO₄ and is therefore relatively polar. As leaves mature, the external CL underneath the CP becomes non-polar, as only little contrast can be obtained with permanganate as the post fixative.Abbreviations CM cuticular membrane - CP cuticle proper - CL cuticular layer - SCL soluble cuticular lipids (cuticular waxes)     

Lipid biochemistry of echinochloa crus-galli during anaerobic germination

Seven day old seedlings of Echinochloa crus-galli var. oryzicola (Vasing) had a higher total lipid content when germinated under N₂ than in air, although ungerminated seeds contained more lipid than either seedling. The triacylglycerol pool was not depleted under anaerobiosis as it was in air and only air-grown seedlings showed a net increase in free fatty acids and polar lipids. Concentrations of most of the individual acids of the total fatty acid profile declined during germination in air and in the free acid and polar lipid fractions of these seedlings the relative proportion of polyunsaturated fatty acids increased. Compared to air-grown seedlings, ungerminated seeds and N₂-grown seedlings had a similar qualitative and quantitative lipid composition. Our results show that mobilization of storage lipids was apparently severely inhibited under anoxia. The importance of lipid metabolism to the germination and growth of Echinochloa during anoxia is discussed in terms of maintaining membrane integrity and serving (indirectly) to reoxidize pyridine nucleotides.     

Nutrient-mediated germination of Beauveria bassiana conidia on the integument of the bark beetle Dendroctonus ponderosae (Coleoptera:Scolytidae)

Field-collected adult mountain pine beetles, Dendroctonus ponderosae, were inoculated with the white muscardine fungus, Beauveria bassiana, and thoroughly examined externally with a scanning electron microscope. Germinating conidia were found at a very low incidence, and only on antennal clubs. However, mortality of inoculated controls was high, and B. bassiana was confirmed as the causative agent. Germination on the cuticle was greatly increased by sonication of adult beetles. The hypothesis that hemolymph released through sonication-damaged membranes provided a nutrient stimulus that enhanced conidial germination was tested by placing hemolymph or yeast-malt extract broth on the cuticle of otherwise nontreated beetles, which were then inoculated with conidia. Significant germination occurred on the cuticle of these beetles. Therefore, a limiting factor for conidial germination on the sclerotized cuticle of D. ponderosae is probably a lack of sufficient nutrients.