Field desorption mass spectrometry of lipids. I. The application of field desorption mass spectrometry to the investigation of natural waxes

Field desorption mass spectra have been recorded of n-dotriacontane, n-tetracosanoic acid and hexadecyl hexadecanoate as representatives of long straight-chain alcohols, acids and esters respectively. Molecular or quasimolecular ions were formed almost exclusively with fragmentation at a low level.Mixtures of long chain compounds have also been examined and the components characterized as their molecular ions. These included a fraction of higher α-ω diols from hydrolysed carnauba wax, unhydrolysed carnauba wax and a previously uninvestigated wax from a Livistonia sp.Results have shown that field desorption mass spectrometry has a most promising role in wax investigation by the ready characterization of constituents up to molecular weights of 2000 and greater.     

Content and Fatty Acid Composition of Steryl and Wax Esters in Germinating Spores of Polytrichum commune

Polytrichum commune spores contained 5.61 ± 0.52 mg steryl and wax esters, including volatile compounds, per 100 mg dry weight of spores. Volatile compounds were not found in 3-h-old sporelings. The content of the steryl and wax ester fraction, excluding the volatile compounds, is slightly increased during the first 6 h of germination. Thereafter, the content is decreased throughout the germination. Thus, 3-day-old sporelings contained 0.52 ± 0.05 mg steryl and wax esters per 100 mg dry weight of spores. In connection with protonema growth, steryl and wax esters were produced, and the 7-day-old cultures contained 5.09 ± 0.37 mg steryl and wax esters per 100 mg dry weight of spores. The main fatty acids of the steryl and wax ester fraction of dry spores and germinating spores as well as of protonemata were palmitic, oleic, linoleic and linolenic acids. Polyunsaturated C 20 acids were present only in trace or small amounts. Phytanic and phytenic acids were found in small amounts in dry spores, in 3- to 72-h-old sporelings, and in protonemata.     

植物超长链脂肪酸及角质层蜡质生物合成相关酶基因研究现状

超长链脂肪酸(very long chain fatty acids, VLCFAs)在生物体中具有广泛的生理功能, 它们参与种子甘油酯、生物膜膜脂及鞘脂的合成, 并为角质层蜡质的生物合成提供前体物质。角质层是覆盖在植物地上部分最表层的保护层, 由角质和蜡质组成, 其中蜡质又分为角质层表皮蜡和内部蜡, 在植物生长发育、适应外界环境方面起重要作用。VLCFAs的合成由脂肪酰-CoA延长酶催化, 该酶是由b-酮脂酰-CoA合酶、b-酮脂酰-CoA还原酶、b-羟脂酰-CoA脱水酶和反式烯脂酰-CoA还原酶组成的多酶体系。合成后的VLCFAs通过脱羰基与酰基还原作用进入角质层蜡质合成途径, 形成各种蜡质组分。文章就VLCFAs及角质层蜡质合成代谢途径中相关酶基因研究进展方面做了综述, 并对植物蜡质基因研究中存在的问题提出一些看法。     

角蜡蚧和日本龟蜡蚧蜡泌物的超微结构及化学成分分析

采用扫描电镜和气相色谱/质谱联用技术研究了角蜡Ceroplastes ceriferus (Fabricius) 和日本龟蜡蚧C. japonicus Green蜡泌物的超微结构及化学成分。结果表明,这2种蜡蚧分泌蜡质和形成蜡壳的过程基本相似。在1、2龄期分泌的蜡质为“干蜡”,蜡壳为星芒状,虫体周缘的蜡芒均为2大节,与其2个龄期发育相对应,每一个大节又分为若干小节。同时,虫体背面中央蜡质堆积成帽状,也分为均匀的多层。由此说明泌蜡过程具有节律性。虫体周缘与蜡芒对应的突起区上分布着密集的刻点状腺孔,每一个腺孔分泌1根蜡丝,这在以往玻片标本中是观察不到的。雌性第3龄幼虫和成虫期,虫体分泌“湿蜡”,形成龟背状蜡壳,泌蜡腺孔主要为三格腺和四格腺。在肛突区发现了密集的泌蜡腺孔,排列为纵条纹状。从角蜡蚧蜡泌物甲酯化处理样品中检测到14个组分,从直接测试 (未经甲酯化处理) 样品中检测到14个组分;而从日本龟蜡蚧则分别检测到10个组分和25个组分。它们的主要成分是一系列高级的长链饱和与不饱和的烃、脂肪酸、脂肪醇、酯类、醛类以及杂环、多环或大环状化合物。对它们可能的生物生态学功能进行了讨论。     

Characteristics of nuclear DNA of a Lepidoptera, the great wax moth Galleria mellonella, using reassociation kinetics

Reassociation kinetics of great wax moth--Galleria mellonella nuclear DNA short fragments was studied within the C0t values of 0.02 and 10(4). The least squares analysis of the experimental curve obtained revealed two components with C0t1/2 = 0.72 and C0t1/2 = 750 appropriately. The fraction of the component with C0t1/2 = 0.72 in the genome of great wax moth is 22% and that of the other one is 61%. There is also a rapid component which renatured at the C0t values less then 0,02. The fraction of this component in the genome is 17%. The great wax moth genome size determined kinetically is 4,83 X 10(8) base pairs. It was shown that the efficiency of DNA molecules network formation can qualify the genome studied as being a short or a long period of interspersion. On the basis that 80-90% of great wax moth DNA molecules participated in network formation which is inherent to the short period interspersion genomes, it was concluded that the great wax moth genome is of the Xenopus-like type.     

Lipids from roots of Onosma heterophylla

The lipids (waxes and fatty substances) extracted from roots of Onosma heterophylla are reported. The wax fraction consists of esters of palmitic acid and its homologues with higher alcohols. β-Sitosterol was detected in this fraction in minor quantities. GC/MS analysis of the fatty substances fraction showed that its main constituents were esters of saturated as well as mono-, di- and triunsaturated fatty acids. Paraffins were also detected in traces. The presence of methyl, ethyl and iso-propyl esters of fatty acids, discovered for the first time in the roots of a higher plant, are considered to be of particular importance in the biosynthesis of fatty substances.     

Biosynthesis and secretion of plant cuticular wax

The cuticle covers the aerial portions of land plants. It consists of amorphous intracuticular wax embedded in cutin polymer, and epicuticular wax crystalloids that coat the outer plant surface and impart a whitish appearance. Cuticular wax is mainly composed of long-chain aliphatic compounds derived from very long chain fatty acids. Wax biosynthesis begins with fatty acid synthesis in the plastid. Here we focus on fatty acid elongation (FAE) to very long chains (C24-C34), and the subsequent processing of these elongated products into alkanes, secondary alcohols, ketones, primary alcohols and wax esters. The identity of the gene products involved in these processes is starting to emerge. Other areas of this field remain enigmatic. For example, it is not known how the hydrophobic wax components are moved intracellularly, how they are exported out of the cell, or translocated through the hydrophilic cell wall. Two hypotheses are presented for intracellular wax transport: direct transfer of lipids from the endoplasmic reticulum to the plasma membrane, and Golgi mediated exocytosis. The potential roles of ABC transporters and non-specific lipid transfer proteins in wax export are also discussed. Biochemical-genetic and genomic approaches in Arabidopsis thaliana promise to be particularly useful in identifying and characterizing gene products involved in wax biosynthesis, secretion and function. The current review will, therefore, focus on Arabidopsis as a model for studying these processes.     

Increased levels of policosanol and very long-chain fatty acids in potato pulp fermented with Rhizopus oryzae

Significant amounts of policosanol and very long-chain fatty acids (VLFAs) ranging in carbon length from 22 to 30 were found in the lipophilic fraction obtained from potato pulp fermented with Rhizopus oryzae. It is believed that these compounds would have originally been present as suberin-related compounds, but not as wax, in the periderm of potato tubers and concentrated into potato pulp during the process of starch production. Moreover, the policosanol and VLFAs extracted from potato pulp with organic solvents were found to have increased after fermentation.     

The effects of stress on plant cuticular waxes

Plants are subject to a wide range of abiotic stresses, and their cuticular wax layer provides a protective barrier, which consists predominantly of long-chain hydrocarbon compounds, including alkanes, primary alcohols, aldehydes, secondary alcohols, ketones, esters and other derived compounds. This article discusses current knowledge relating to the effects of stress on cuticular waxes and the ways in which the wax provides protection against the deleterious effects of light, temperature, osmotic stress, physical damage, altitude and pollution. Topics covered here include biosynthesis, morphology, composition and function of cuticular waxes in relation to the effects of stress, and some recent findings concerning the effects of stress on regulation of wax biosynthesis are described.     

Analysis of the cuticular wax composition and ecophysiological studies in an arid plant - Ziziphus nummularia (Burm.f.) Wight & Arn.

Plants in arid regions are exposed to various abiotic stresses and the presence of the waxy cuticular layer acts as a defensive barrier, which consists mainly of long chain fatty acids, hydrocarbons and other derived compounds. Studies on the chemical composition and properties of cuticles of arid plants are scanty. The present study deals with the analysis of cuticular wax composition and effect of temperature on some ecophysiological parameters of an important arid plant Ziziphus nummularia. A total of 59 different wax compounds were detected from the leaf cuticle by capillary GC–MS. 4-Hydroxycyclohexanone, Heptacosane and 2,7-Dimethyloctane-3,5-dione were the dominant wax compounds in Z. nummularia. The variation of photosynthetic rate varied from 0.70 to 7.70 µmol CO2 m-2s-1 against the studied temperature range of 15–55 °C. The transpiration rate varies from 1.80 to 8.40 mmol H2O m-2s-1 within the temperature range of 15–55 °C. The quantum yield of photosystem II (Fv/Fm) also exhibited much variation due to the variation of temperature. The results clearly shows that Z. nummularia is highly adapted to restrict water loss and can tolerate high temperatures and can be considered as an appropriate species for vegetating the arid areas.     

Increased Levels of Policosanol and Very Long-Chain Fatty Acids in Potato Pulp Fermented with Rhizopus oryzae

Significant amounts of policosanol and very long-chain fatty acids (VLFAs) ranging in carbon length from 22 to 30 were found in the lipophilic fraction obtained from potato pulp fermented with Rhizopus oryzae. It is believed that these compounds would have originally been present as suberin-related compounds, but not as wax, in the periderm of potato tubers and concentrated into potato pulp during the process of starch production. Moreover, the policosanol and VLFAs extracted from potato pulp with organic solvents were found to have increased after fermentation.     

云南双蜡蚧蜡泌物的超微形态与化学成分

采用扫描电镜、红外光谱、气相色谱/质谱联用技术研究了云南双蜡蚧Dicyphococcus bigibbus Borchsenius（同翅目：蚧科）蜡泌物的超微结构和化学成分。结果发现：该虫的若虫与成虫分泌的蜡质从蜡腺排出初期均为湿蜡,在体外凝结形成质地稍硬而脆的玻璃状干蜡壳,蜡壳具有均匀脊和横层。幼期和雄性蜡壳小,船形,周缘带有蜡角,中央隆起。雌成虫蜡壳大,元宝形。蜡泌物的红外光谱吸收峰密集,峰型尖锐,种的特征明显。用红外光谱特征推断蜡泌物的主要基团和化学构成在GC/MS检测中得到印证。用甲酯化和非甲酯化两种处理方法检测证明,该虫蜡泌物主要成分是一系列高级的长链饱和与不饱和的烃、脂肪酸及酯类化合物。化合物的碳原子数从C₁₄～C₄₀,大部分为偶数个,特别是碳链长度在C₃₀～C₄₀的酯类化合物种类多,比例大。还将该蚧虫蜡泌物的特征与蚧科内近缘的蜡蚧属种类及瘤坚大球蚧、朝鲜毛球蚧、背刺毡蜡蚧、白蜡虫等作了对比,阐述了蜡泌物在蚧虫分类、系统学及防治中的意义。     

Ontogenetic and seasonal development of wax composition and cuticular transpiration of ivy (Hedera helix L.) sun and shade leaves

The ontogenetic and seasonal development of wax composition and cuticular transpiration of sun and shade leaves of ivy (Hedera helix L.) was analysed by investigating leaves varying in age between 4 and 202 d. It was discovered that the total amount of solvent-extractable wax was composed of two distinct fractions, separable by column chromatography: (i) a less polar or apolar monomeric wax fraction consisting of the typical linear, long-chain aliphatics usually described as cuticular wax components and (ii) a polar, oligomeric wax fraction consisting of primary alcohols and acids mostly esterified to C₁₂-, C₁₄- and C₁₆-ω-hydroxyfatty acids. The apolar wax fraction, which could be analysed directly by gas chromatography coupled with mass spectrometry (GC-MS), exhibited pronounced seasonal changes in composition. Wax amounts in the apolar fraction reached a maximum after about 30 d and gradually decreased again during the remaining period of the season investigated. In contrast, the polar wax fraction, which was analysable by GC-MS only after transesterification, rapidly increased early in the season, reaching a plateau after 40 d, and then remained constant during the rest of the season. Thus, total amounts of solvent-extractable cuticular waxes, which can be determined gravimetrically, will only be detected by GC-MS after fractionation and transesterification, a methodological approach rarely applied in the past in cuticular wax analysis. Additionally, investigation of the cutin polymer matrix after depolymerisation through transesterification, revealed that only those primary alcohols and acids forming an essential part of the apolar and the polar wax fractions were esterified during the investigated season and incorporated in increasing amounts into the cutin polymer matrix (matrix-bound wax fraction). Thus, it can be concluded that a complete analysis of cuticular wax of ivy and its seasonal development can only be achieved if all the relevant fractions (i) the less polar or apolar, (ii) the polar and (iii) the wax fraction bound to the cutin polymer matrix are investigated. Cuticular transpiration rapidly decreased within the first 30 d and essentially remained constant during the rest of the season. Thus, changes in cuticular water permeability were closely correlated with the most prominent changes in wax amounts and composition occurring during the first 30 d of ontogenetic leaf development. However, during the remainder of the year, up to 202 d, cuticular transport properties remained constant, although significant quantitative and qualitative changes in cuticular wax composition continued to occur. Thus, our study clearly demonstrated that there will be no simple relationship between chemical composition of cuticular waxes and transport properties of isolated ivy leaf cuticles. Received: 2 March 1998 / Accepted: 26 June 1998     

Temporal and spatial patterns of wax secretion and related behavior in the division of labour of the honeybee (Apis mellifera capensis)

Summary Current ethograms for honeybee behavior are hypothesized to consist of age-related cohorts of workers having a high probability of performing a small set of related tasks in a restricted portion of the nest. Wax secretion and wax working related activities were assayed in this light. It is shown that wax secretion is significantly affected by worker age and that bees between 3 and 21 days old form such a cohort. Comb building festoons, previously thought to be the site of wax secretion, represent only a small fraction of newly secreted wax in the nest. Wax secretion remains constant relative to age in the cohort but varies significantly with season as does bee participation in festooning behavior. Wax secretion and wax working are both definable in terms of time and space in the nest, the relative probability of activity changing with season. Secretion itself is constrained by the cyclical activity of the underlying wax gland complex.     

柑橘真棉蚧蜡泌物的超微形态、红外光谱与化学成分特征

采用扫描电镜、红外光谱、气相色谱/质谱联用技术研究了柑橘真棉蚧Eupulvinariacitricola(Kuwana)(半翅目:蚧科)蜡泌物的超微形态、红外光谱和卵囊蜡质的化学成分特征。结果发现:该蚧雌虫背面腺体分泌湿蜡,在体表面形成薄的半透明蜡壳,背面蜡壳分为6个小区,表面由湿蜡凝结成片状和蜡块状构造。雌成虫产卵期由腹面多格腺分泌丝状蜡结成卵囊。雄若虫也分泌湿蜡,雄蛹的蜡茧薄,表面有浅的凹凸花纹。用雌成虫卵囊、背面蜡壳和雄茧的蜡质作红外光谱分析,它们的谱图具有共同的基本特征,吸收峰群分为5个区域:2900cm-1附近的3个强吸收峰组成的峰群是>CH2和—CH3的特征吸收峰;1800cm-1~1500cm-1之间的吸收峰群是羰基(>C=O)和C=C键的伸缩振动;1500cm-1~1400cm-1区域是>CH2和—CH3基团的吸收峰;1400cm-1~1000cm-1之间为饱和碳链—C—C—C—骨架振动。730cm-1附近为环状化合物的基团吸收峰群。由此确定它们的组成都是长碳链结构的脂肪酸、脂肪醇、烃类、酯类或带有环状结构的化合物。其主要区别在第2、3、4区吸收峰的形式、强度和数量,反映出基团的种类和数量不同。雌成虫卵囊蜡质甲酯化后GC/MS检测出7个组分(表1),全部为长链脂肪酸。未经甲酯化的蜡质检测出4类16个组分(表2),可分为长链烃、酸、醇、酯。第一类为饱和长链烃,5个组分,占总组分的38.38%;第二类是长链脂肪酸,包括直链饱和酸和不饱和酸共6个组分,占31.59%;第三类是直链饱和醇,占2.87%;第四类是酯类化合物,4个组分,占总组分的27.16%。     

Ether-soluble Neutral Portion of Cellulose Cigarette Smoke Condensate

Higher alcohols with a carbon length ranging from 16 to 30 found in the lipophilic fraction from potato pulp were shown to be present as ferulate and in a free form, but not as wax. Thin-layer chromatography of the neutral lipids in potato pulp indicated a few spots with scavenging activity toward the 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable radical, the major active component being characterized as alkyl ferulate which showed almost the same level of activity as γ-oryzanol.     

Presence of higher alcohols as ferulates in potato pulp and its radical-scavenging activity

Higher alcohols with a carbon length ranging from 16 to 30 found in the lipophilic fraction from potato pulp were shown to be present as ferulate and in a free form, but not as wax. Thin-layer chromatography of the neutral lipids in potato pulp indicated a few spots with scavenging activity toward the 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable radical, the major active component being characterized as alkyl ferulate which showed almost the same level of activity as gamma-oryzanol.     

Cuticular hydrocarbons and wax esters of the ectoparasitoid Habrobracon hebetor: Ontogenetic,reproductive, and nutritional effects

Hydrocarbon and wax ester components of cuticular lipids of the braconid parasitoid Habrobracon hebetor Say reared at 25 degrees C on larvae of a pyralid moth have been identified by GC-MS and analyzed with respect to adult age, mating status, and diet. The hydrocarbons range in carbon number from C(21) to C(45) and consist of a homologous series of n-alkanes, 11-, 13-, and 15-methyl alkanes, 13,17-dimethyl alkanes, and Z-5, Z-7, and Z-9-alkenes. The wax esters found in the cuticular lipid fraction are a series of homologous compounds with the acid portion being short chain, unbranched, even carbon number acids from C(8) to C(20) (predominately C(8) to C(16)). The alcohol portions of the esters are secondary alcohols with carbon number from C(22) to C(25) (predominately C(23) and C(25)) with the hydroxyl function located at C(6), C(7), C(8), and C(9). Gender, age, and nutritional states were significant factors for variation in several of the individual esters, but mating status did not affect wax ester composition. Ontogenetic examinations indicated that prepupal, and early pupal cuticular lipids contain only hydrocarbons. Low levels of wax esters are detectable in late stage pupae, and somewhat greater quantities of wax esters are present on newly eclosed adults. When pharate adults emerge from the cocoon, however, their cuticular lipids consist of approximately equal amounts of hydrocarbons and wax esters, and 6d post emergence from the cocoon, wax esters are the predominant lipid component.     

Characterization of the components of plant cuticles in relation to the penetration of 2,4-D

Quantitative comparisons were made of the components of the cuticles of leaves of plantain, fat hen, dandelion, dock, chickweed and forget-me-not. Hydrocarbons and triterpenoids were prominent in the surface wax of plantain; esters and alcohols in the other surface waxes. Polar compounds predominated in the cuticular waxes. Cuticle development in plantain, dandelion and chickweed was similar, but the cutins differed in the relative proportions of hydroxy-fatty and fatty acid components. Sorption of 2,4-D by the cuticular membrane was inversely related to the amount of cuticular wax. Hydrocarbons and an aldehyde fraction isolated from surface wax most effectively reduced the penetration of water in a model system.